HEMATOPOIETIC STEM CELL COMPOSITIONS, METHODS OF MAKING AND

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims priority to, and the benefit of, U.S. Provisional Application No. 62/745,110 filed on October 12, 2018; U.S. Provisional Application No. 62/783,122 filed on December 20, 2018; and U.S. Provisional Application No. 62/815,340 filed on March 7, 2019. The contents of each of these applications are incorporated by reference herein, in their entirety and for all purposes.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[02] The contents of the file named“POTH-032_001WO_SeqListing_ST25.txt”, which was created on October 12, 2019, and is 45.2 KB in size are hereby incorporated by reference in their entirety.

FIELD OF THE DISCLOSURE

[03] The disclosure is directed to molecular biology, and more, specifically, to compositions and methods for the selection and expansion of hematopoietic stem cells (HSCs).

BACKGROUND OF THE INVENTION

[04] There has been a long-felt but unmet need in the art for a method of selecting and expanding hematopoietic stern cells (HSCs) in culture, and in particular, for producing a population of HSCs fit for use in an adoptive cell therapy. The disclosure provides compositions and methods for selecting and expanding HSCs to produce an enriched population of HSCs with superior performance for use in adoptive cell therapies.

SUMMARY OF THE INVENTION

[05] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, and wherein at least 70% of the population of

SUBSTITUTE SHEET (RULE 26) modified HSCs express one or more cell-surface marker(s) comprising CD34. The present disclosure also provides a pharmaceutical composition comprising the composition and a pharmaceutically-aeceptable carrier. The transgene can be any exogenous nucleic acid sequence or can encode for any exogenous amino acid sequence.

[06] The present disclosure also provides a composition comprising a population of modified HSCs, wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, and wherein at least 70% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+. The present disclosure also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier. The transgene can be any exogenous nucleic acid sequence or can encode for any exogenous ammo acid sequence.

[07] In one aspect, at least 90% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene. In one aspect at least 95% of the plural ity of HSCs of the population comprise the transgene or the sequence encoding the transgene.

[08] In one aspect, at least 85% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34. In another aspect, about 75% to about 95% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 In one aspect, at least 85% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+. In another aspect, about 75% to about 95% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+.

[09] In one aspect, at least 45% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and do not express one or more cell-surface marker(s) comprising CD38. In another aspect, about 65% to about 75% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and do not express one or more cell-surface marker(s) comprising CD38. In one aspect, at least 45% of the population of modified HSC comprise the cell-surface marker phenotype CD34+ and CD38-. In another aspect, at least about 65% to about 75% of the population of modified HSCs comprise the cell- surface marker phenotype CD34+ and CD38-.

[010] In one aspect, at least 0.2% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38. In another aspect, about 0.2% to about 2% of the population of

SUBSTITUTE SHEET (RULE 26) modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38. In one aspect, at least 0.2% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38- and CD90+. In another aspect, about 0.2% to about 2% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38- and CD9Q+.

[Oil] In one aspect, at least 0.2% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA. In another aspect, about 0.2% to about 2% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA. In one aspect, at least 0.2% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+ and CD45RA-. In another aspect, about 0.2% to about 2% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+ and CD45RA-.

[012] In one aspect, at least 0.4% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD45RA. In another aspect, about 0.4% to about 3% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD45RA. In one aspect, at least 0.4% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD90+ and CD45RA-. In another aspect, about 0.4% to about 3% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD90+ and CD45RA-.

[013] In one aspect, at least 0.04% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34, CD90 and CD49f and do not express one or more cell- surface markerfs) comprising CD38 and CD45RA. In another aspect, about 0.04% to about 1% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34, CD90 and CD49f and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA. In one aspect, at least 0.04% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+. In another aspect, about 0.04% to about 1% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+.

SUBSTITUTE SHEET (RULE 26) [014] The present disclosure also provides a method of producing a modified HSC or a population of modified HSCs comprising: a) introducing a transgene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction of the transgene into an HSC or a plurality of HSCs to produce a modified HSC or a plurality of modified HSCs; and b) culturing the HSC or the plurality of HSCs from step a) m a culture media comprising an expansion agent, wherein the expansion agent comprises a HD AC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor, a pyrimido-indole derivative, or a combination thereof, thereby producing a modified HSC or a population of modified HSCs. Step a) can occur prior to step b), step b) can occur prior to the step a) or step a) and step b) can occur concurrently. The transgene can be any exogenous nucleic acid sequence or can encode for any exogenous amino acid sequence.

[015] The present disclosure also provides a method of producing a modified HSC or a population of modified HSCs comprising: a) introducing a transgene comprising a selection gene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction of the transgene into an HSC or a plurality of HSCs to produce a modified HSC or a plurality of modified HSCs; and b) culturing the HSC or the plurality' of HSCs from step a) in a culture media comprising a selection agent and an expansion agent, wherein the expansion agent comprises a HDAC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor, a pyrimido-indole derivative, or a combination thereof, thereby producing a a modified HSC or population of modified HSCs. Step a) can occur prior to step b), step b) can occur prior to the step a) or step a) and step b) can occur concurrently. The transgene can be any exogenous nucleic acid sequence or can encode for any exogenous amino acid sequence.

[016] The expansion agent can comprise at least two of a HDAC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrimido-indole derivative. The expansion agent can comprise a HDAC inhibitor and a HMT inhibitor. In one aspect, the expansion agent comprise valproic acid and UNC0638. The expansion agent can comprise at least three of a HDAC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrimido-indole derivative. The expansion agent can comprise a HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimido-indole derivative. In one aspect, the expansion agent comprises StemRegenin 1 (SRI), UM171 and UNC0638.

SUBSTITUTE SHEET (RULE 26) [017] The plurality of HSCs in step b) can be cultured in a culture media comprising the expansion agent or the selection agent and expansion agent for at least 24 hours. The plurality of HSCs in step b) can be cultured in a culture media comprising the expansion agent or the selection agent and expansion agent for at least 4 days. The plurality of HSCs in step b) can be cultured in a culture media comprising expansion agent or the selection agent and expansion agent for at least 7 days. The plurality of HSCs in step b) can be cultured m a culture media comprising expansion agent or the selection agent and expansion agent for at least 11 days. The plurality of HSCs in step b) can be cultured in a culture media comprising expansion agent or the selection agent and expansion agent for about 24 hours to about 11 days. The plurality' of HSCs in step b) can be cultured in a culture media comprising expansion agent or the selection agent and expansion agent for about 24 hours to about 7 days.

[Q18] The present disclosure provides a method of producing a modified HSC or a population of modified HSCs, comprising: a) introducing a transgene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction of the transgene into an HSC or a plurality of HSCs to produce a modified HSC or a plurality of modified HSCs; b) culturing the HSC or the plurality of HSCs from step a) in a culture media comprising a HD AC inhibitor, a HMT inhibitor, or a combination thereof; c) removing the HD AC inhibitor from the culture media; and d) culturing the HSC or the plurality of HSCs in a culture media comprising a HMT inhibitor following step c; e) removing the HMT inhibitor from the culture media following step d); and f) culturing the HSC or the plurality of HSCs in in a culture media comprising an expansion agent following step e, wherein the expansion agent comprises a HD AC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrirmdo-indole derivative, thereby producing a modified HSC or a population of modified HSCs. Step a) can occur prior to step b), step b) can occur prior to the step a) or step a) and step b) can occur concurrently. The transgene can be any exogenous nucleic acid sequence or can encode for any exogenous amino acid sequence.

[019] The present disclosure also provides a method of producing a modified HSC or a population of modified HSCs, comprising: a) introducing a transgene comprising a selection gene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction of the transgene into an HSC or a plurality of HSCs to produce a modified HSC or a plurality of modified HSCs; b) culturing the HSC or the plurality' of HSCs

SUBSTITUTE SHEET (RULE 26) from step a) in a culture media comprising a HD AC inhibitor, a HMT inhibitor, or a combination thereof; c) removing the HD AC inhibitor from the culture media; and d) culturing the HSC or the plurality of HSCs in a culture media comprising a HMT inhibitor following step c; e) removing the HMT inhibitor from the culture media following step d); and f) culturing the HSC or the plurality of HSCs in m a culture media comprising a selection agent and an expansion agent following step e, wherein the expansion agent comprises a HD AC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrimido-mdole derivative, thereby producing a modified HSC or a population of modified HSCs. Step a) can occur prior to step b), step b) can occur prior to the step a) or step a) and step b) can occur concurrently.

[020] The HSC or plurality of HSCs in step b) can be cultured in a culture media comprising the HD AC inhibitor and the HMT inhibitor for at least 12 hours. The HSC or plurality of HSCs in step b) can be cultured in a culture media comprising the HD AC inhibitor and the HMT inhibitor for at least 24 hours. The HSC or plurality of HSCs in step b) can be cultured in a culture media comprising the HD AC inhibitor and the HMT inhibitor for about 3 hours to about 30 hours.

[021] The HSC or plurality of HSCs in step d) can be cultured in a culture media comprising a HMT inhibitor for at least 48 hours following step c. The HSC or plurality of HSCs in step d) can be cultured in a culture media comprising a HMT inhibitor for at least 3 days following step c. The HSC or plurality of HSCs in step d) can be cultured in a culture media comprising a HMT inhibitor for about 1 day to about 5 days following step c.

[022] The HMT inhibitor in step b) and the HMT inhibitor in step d) can be the same or different. The HMT inhibitor in step b) can comprise UNC0638 The HMT inhibitor in step d) can comprise UNC0638. The HMT inhibitor in step b) and step d) can comprise UNC0638 The culture media can comprise about 0.5 mM to about 2 mM of UNC0638 in step b), in step d) or in both step b) and step d). The culture media can comprise about 1 mM of UNC0638 in step b), in step d) or in both step b) and step d).

[023] The HD AC inhibitor can comprise valproic acid or sodium phenylbutyrate. In a preferred aspect, the HD AC inhibitor is valproic acid. The culture media can comprise about 0.25 mM to about 1.25 mM of valproic acid. The culture media can comprise about 0.5 mM or about 0.75 mM of valproic acid. The culture media can comprise about 0.5 mM to about 2 mM

SUBSTITUTE SHEET (RULE 26) of sodium phenylbutyrate. The culture media can comprise about 1.5 mM of sodium phenylbutyrate.

[024] The culture media m step b) comprising the combination of the HD AC inhibitor and the HMT inhibitor can further comprise a second HD AC inhibitor, a second HMT inhibitor, StemRegenin 1 (SRI), UM171, or a combination thereof.

[025] The plurality of HSCs in step f) can be cultured in a culture media comprising the expansion agent or the selection agent and expansion agent for at least 5 days following step e. The plurality of HSCs in step f) can be cultured in a culture media comprising the expansion agent or the selection agent and expansion agent for at least 7 days following step e. The plurality of HSCs in step f) can be cultured in a culture media comprising the expansion agent or the selection agent and expansion agent for about 4 days to about 9 days following step e.

[Q26] The HMT inhibitor in step f) can comprise UNC0638. The culture media in step f) can comprise about 0.5 mM to about 2 m.M of UNC0638. The culture media in step f) can comprise about 1 mM of UNC0638. The aryl hydrocarbon receptor inhibitor in step f) can comprise StemRegenin 1 (SRI). The culture media in step f) can comprise about 0.5 iiM to about 2 mM of SRI . The culture media in step f) can comprise about 1 m.M of SRl . The pyrimido-indole derivative m step f) can comprise UM171. The culture media in step f) can comprise about 25 nM to about 50 nM of UM171. The culture media in step f) can comprise about 35 nM of UM171.

[027] The expansion agent can further comprise valproic acid. The culture media in step f) can comprise about 0.25 mM to about 1.25 mM of valproic acid. The culture media in step f) can comprise about 1 mM of valproic acid. The expansion agent can further comprise nicotinamide. The culture media in step f) can comprise about 2.5 mM to about 10 mM of nicotinamide. The culture media in step f) can comprise about 5 mM of nicotinamide. The expansion agent can further comprise gareinoi. The culture media in step f) can comprise about 5 mM to about 15 mM of gareinoi. The culture media in step f) can comprise about 10 mM of gareinoi. The expansion agent can further comprise sodium phenylbutyrate. The culture media in step f) can comprise about 1 mM to about 2 rnM of sodium phenylbutyrate. The culture media in step f) can comprise about 1.5 mM of sodium phenylbutyrate.

SUBSTITUTE SHEET (RULE 26) [028] The transgene can comprise a selection gene or comprise a sequence encoding for a selection marker. The selection marker can be a nucleic acid molecule or a protein. In one aspect, the transgene can comprise a dihydrofolate reductase (DHFR) resistance gene or the transgene can comprise a sequence encoding a DHFR mutein enzyme. Step f) can further comprise a selection agent. The selection agent can comprise methotrexate or pralatrexate. The culture media in step f) can comprise about 100 nM to about 500 nM of methotrexate. The culture media in step f) can comprise about 250 nM of methotrexate. The culture media in step f) can comprise about 50 nM to about 250 nM of pralatrexate. The culture media in step f) can comprise about 125 nM of pralatrexate. The selection agent can comprise pralatrexate and dipyridimole. The culture media in step f) can comprise about 50 nM to about 250 nM of pralatrexate and about 1 mM to about 10 mM of dipyridimole. The culture media in step f) can comprise about 125 nM of pralatrexate and about 5 mM of dipyridimole.

[029] Culturing the plurality of HSCs with an expansion agent can occur prior to culturing the plurality of HSCs with a selection agent, culturing the plurality of HSCs with a selection agent can occur prior to culturing the plurality of HSCs with an expansion agent, or culturing the plurality of HSCs with an expansion agent and culturing the plurality of HSCs with a selection agent can occur concurrently.

[030] The culture conditions m steps e) - f) can in at least two-fold expansion of the plurality of HSCs compared to a plurality of HSCs cultured under culture conditions sufficient for cell proliferation and for introduction of the transgene wherein said conditions do not comprise a selection agent and expansion agent. The culture conditions in steps e) - f) result in at least five fold expansion of the plurality of HSCs compared to a plurality of HSCs cultured under culture conditions sufficient for cell proliferation and for introduction of the transgene wherein said conditions do not comprise a selection agent and expansion agent. The culture conditions in steps e) - f) result in about two-fold to about five-fold expansion of the plurality of HSCs compared to a plurality of HSCs cultured under culture conditions sufficient for cell proliferation and for introduction of the transgene wherein said conditions do not comprise a selection agent and expansion agent.

[031] The present disclosure also provides a method of producing and/or expanding a modified HSC or a population of modified HSCs, comprising: a) introducing a transgene comprising a DHFR resistance gene or a sequence encoding the transgene under culture

SUBSTITUTE SHEET (RULE 26) conditions sufficient for cell proliferation and for introduction of the transgene into an HSC or a plurality of HSCs to produce a HSC or a plurality of modified HSCs; b) culturing the HSC or the plurality of HSCs from step a) in a culture media comprising valproic acid and UNC0638 for at least 24 hours; c) removing the valproic acid from the culture media; d) culturing the HSC or the plurality of HSCs in a culture media comprising UNC0638 for at least 3 days following step c; e) removing the UNC0638 from the culture media, following step d); and f) culturing the HSC or the plurality of HSCs m a culture media comprising a selection agent and an expansion agent for at least 7 days following step e, wherein the selection agent comprises methotrexate or praiatrexate and wherein the expansion agent comprises SRI, UM171 and UNC0638, thereby producing and/or expanding a modified HSC or a population of modified HSCs

[032] The transgene can comprise a sequence encoding for a therapeutic agent. The therapeutic agent can be a protein or a RNA that provides a therapeutic benefit when

administered to a cell or a subject. The therapeutic agent can be a therapeutic protein or a therapeutic RNA. The therapeutic agent can be human beta-giobin (HBB), T87Q human beta- giobin (HBB T87Q), BAF chromatin remodeling complex subunit (BCL11A) shRNA, insulin like growth factor 2 binding protein 1 (IGF2BP1), interleukin 2 receptor gamma (IL2RG), alpha gaiaetosidase A (GLA), alpha-L-klurondase (IDUA), iduronate 2-sulfatase (IDS), cystinosm lysosomal cysteine transporter (CTNS). The transgene can comprise a sequence encoding a chimeric antigen receptor (CAR) The transgene can comprise a sequence encoding a non- naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical. In one aspect, the transgene can comprise a sequence for a CAR and a sequence for a CSR. The transgene can comprise a sequence encoding for an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, w'herein the inducible proapoptotic polypeptide does not comprise a non-human sequence. The transgene can he integrated into the genome of the HSC. The integration can be stable or transient.

[033] The transgene can be introduced to the HSC by any means known in the art. In one aspect, introducing the transgene comprises introducing a transposon comprising the transgene

SUBSTITUTE SHEET (RULE 26) or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into an HSC or a plurality of HSCs to produce a modified HSC or a plurality of modified HSCs. The transposon can he integrated into the genome of the HSC by the transposase. The integration can be stable or transient. The sequence encoding the transposase can comprise an amino acid or a nucleic acid sequence encoding a transposase protein. The nucleic acid sequence encoding a transposase protein can comprise an RNA sequence or comprise a DNA sequence.

[034] In one aspect, when introducing the transgene comprises introducing a transposon comprising the transgene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase, the culture conditions in steps b) - d) result in an increase in frequency of transposition and at least a one-fold increase in the yield of transposed cells in the plurality of modified HSCs compared to a plurality of modified HSCs cultured under culture conditions sufficient for cell proliferation and for transposition wherein said conditions do not comprise a HD AC inhibitor and a HMT inhibitor. The culture conditions in steps b) - d) result in an increase in frequency of transposition and at least a two-fold increase m the yield of transposed HSCs in the plurality of modified HSCs compared to a plurality of modified cells cultured under culture conditions sufficient for cell proliferation and for transposition wherein said conditions do not comprise a HD AC inhibitor and a HMT inhibitor. The culture conditions in steps b) - d) result in an increase in frequency of transposition and about one-fold to about a four-fold increase in the yield of transposed cells in the plurality of modified HSCs compared to a plurality of modified HSCs cultured under culture conditions sufficient for cell proliferation and for transposition wherein said conditions do not comprise a HD AC inhibitor and a HMT inhibitor.

[035] The present invention provides a plurality of modified cells produced by any of the methods disclosed herein. The present invention provides compositions comprising a plurality of modified cells produced by any of the methods disclosed herein. The present invention provides pharmaceutical compositions comprising a plurality of modified cells produced by any of the methods disclosed herein and and a pharmaceutically-acceptable carrier.

[036] The present invention provides a modified cell population wherein at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of modified cells in the population comprise a genome-integrated transposon.

SUBSTITUTE SHEET (RULE 26) [037] The present invention provides compositions comprising a modified HSC population wherem at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of modified HSC in the population comprise a genome- integrated transgene. The present invention provides pharmaceutical compositions comprising a modified HSC population wherein at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of modified HSCs in the population comprise a genome-integrated transgene a and a pharmaeeutically-aceeptabie carrier.

[038] The present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically-effective amount of the compositions or pharmaceutical compositions disclosed herein. The present invention also provides the compositions or pharmaceutical compositions disclosed herein for use in treating a disease or disorder. The pharmaceutical composition can comprise a plurality of autologous HSCs. The pharmaceutical composition can comprise a plurality of allogeneic HSCs.

[Q39] The present composition comprising at least one promoter, a sequence encoding a therapeutic agent and a sequence encoding a selectable marker, wherein the promoter comprises an EF1 alpha promoter (EF1 alpha), a CMV promoter, an MND promoter, an SV40 promoter, a PGK1 promoter, an hPGK promoter, a Ube promoter, an SPTA1 promoter, an Ank-l promoter, a Gly-A promoter, a CAG promoter, an HI promoter, a U6 promoter, or a combination thereof; and wherein the therapeutic agent comprises human beta-globm (HBB), human beta-globin comprising a T87Q mutation (T87Q HBB), BAT chromatin remodeling complex subunit (BCL11A) shRNA, insulin like growth factor 2 binding protein 1 (JGF2BP1), interleukin 2 receptor gamma (TL2RG), alpha galactosidase A (GLA), alpha-L-idurondase (IDIJA), iduronate 2-sulfatase (IDS), cystmosin lysosomal cysteine transporter (CTNS).

[040] The at least one promoter comprises a sequence capable of expressing a transgene in a cell. The composition can further comprise a sequence encoding an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. The sequence encoding the selectable marker can comprise a nucleic acid sequence encoding a DHFR enzyme or a nucleic acid sequence encoding a DHFR mutein enzyme.

SUBSTITUTE SHEET (RULE 26) [041] In one aspect, the composition comprises an SPTA1 promoter and an hPGK promoter, wherein the therapeutic agent comprises HBB or T87Q FIBB, and wherein the selectable marker comprises a DHFR enzyme or a DHFR mutem enzyme.

[042] In one aspect, the composition comprises an Ank-l promoter and an MND promoter, wherein the therapeutic agent comprises BCL11 A shRNA, and wherein the selectable marker comprises a DHFR enzyme or a DHFR mutein enzyme.

[043] In one aspect, the composition comprises an EF1 alpha promoter, wherein the therapeutic agent comprises IL2RG, and wherein the selectable marker comprises a DHFR enzyme or a DHFR mutein enzyme.

[Q44] In one aspect, the composition comprises an MND promoter, wherein the therapeutic agent comprises IL2RG, and wherein the selectable marker comprises a DHFR enzyme or a DHFR mutein enzyme.

[045] In one aspect, the composition comprises an SPTA1 promoter and an MND promoter, wherein the therapeutic agent comprises IGF2BP1, and wherein the selectable marker comprises a DHFR enzyme or a DHFR mutein enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

[046] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawmg(s) will be provided by the Office upon request and payment of the necessary fee.

[047] Fig. 1 is a schematic diagram depicting the selection and expansion of isolated hematopoietic stem cells (HSCs) from mobilized peripheral blood.

[048] Fig. 2 is a schematic diagram showing HSC expansion.

[049] Fig. 3 is a bar graph depicting the fold expansion of different hematopoietic cell subsets over 7 days of cell culture.

[050] Fig. 4 is a schematic diagram of the piggyBac (PB) transposon vector construct used for HSC selection.

[051] Fig. 5A is a schematic timeline of the piggyBac (PB) transposition by nucleofection, expansion treatment (SR1/UM171 /UNC0638 or VP A) and methotrexate (MTX) selection treatment of mPB CD34+ cells.

SUBSTITUTE SHEET (RULE 26) [052] Fig. 5B is a representative flow cytometry scatterplot of cells collected from Day 4, showing the population of cells with GFP expression (cells with DHFR transposition).

[053] Fig. 6 is a series of flow cytometry scatterplots showing the GFP expression profiles of hematopoietic cell subsets on Day 1 1 of cell culture.

[054] Fig. 7 is a series of bar graphs showing the quantification of the flow cytometry results from Fig. 6.

[055] Fig. 8A is a bar graph showing the absolute number of hematopoietic cells from the flow cytometry' results in each hematopoietic cell subset following treatment with VP A or SR1/UMC171/UNC0638 (m the presence or absence of MTX). The absolute number of cells from a Day 0 control is also shown.

[056] Fig. 8B is a bar graph showing the absolute number of hematopoietic cells from the flow' cytometry' results depicting the absolute number of cells in the CD34+/38-/45RA-/90+/49f+ subset of hematopoietic stem cells.

[057] Fig. 9 is a schematic depiction of the PB-HBB-PGK-iC9-T2A-DHFR construct map.

[058] Fig. 10A show's a schematic diagram of a construct for erythroid-specific expression of the human therapeutic beta-globin and the constitutive expression of GFP and DHFR (PB-HBB- PGK-GFP-T2A-DHFR construct) (SEQ ID NO: 14936)

[059] Fig 10B is a schematic timeline of the piggyBae transposition of a therapeutic vector containing T87Q beta-globin, followed by expansion with a combination of SRI, UM171 and UNC0638 treatment and selection with methotrexate.

[060] Fig. I OC shows flow cytometry scatterplots depicting the frequency of GFP+ cells at day 4, before selection and expansion, and at Day 11, after 7 days of selection and expansion.

[061] Fig. 1 1 A show's a schematic diagram of a construct for erythroid-specific expression of a BCL1 la targeted shRNA and the constitutive expression of GFP and DHFR

(nano.PB.Ank.BCLl lAshrRNA.EFla.GFP-T2A-DHFR construct).

[062] Fig. 1 IB shows a schematic diagram of a construct for erythroid-specific expression of the Insulin growth factor 2 binding protein 1 (IGF2BP1) and the constitutive expression of GFP and DHFR (nano.PB.SPTAl-IGF2BPl . EF 1 a. GFP-T2 A-DHFR construct).

[063] Fig. 11C is a schematic timeline of the piggyBae transposition of each therapeutic vector, followed by expansion with a combination of SRI, UM171 and UNC0638 (SUIT) and selection with methotrexate.

SUBSTITUTE SHEET (RULE 26) [064] Fig. 1 ID shows flow cytometr scalterplots depicting the frequency of GFP+ cells at day 4, before selection and expansion, and at Day 11, after 7 days of selection and expansion. This protocol yielded a high selection purity of CD34+ cells expressing a therapeutic vector.

[065] Fig. 12A is a schematic diagram depicting the treatment scheme of human mobilized peripheral blood CD34+ cells with transposition, transposition boosting, selection, and multiple expansion reagents, followed by characterization via both m vitro and in vivo assays.

[066] Fig. 12B is a bar graph quantitating the absolute number of transposed (GFP+) ceils yielded at Day 11 after expansion and selection.

[067] Fig. 12C is a bar graph showing the absolute numbers of GFP+ and GFP- cells within the CD34+/38-/90+/45RA-/49f+ hematopoietic stem cell subset as determined using flow cytometry.

[Q68] Fig. 12D is a box plot showing the output of a long-term culture initiating cell (LTC-IC) assay.

[Q69] Fig. 12E show¾ the engraftment of uncultured mobilized peripheral blood CD34 cells compared to cytokine, VP A, VUU and SUIT expansion conditions as assessed in the bone marrow of NSG mice 20 weeks post transplant.

[070] Fig. 13 A shows a schematic diagram of a construct for erythroid-specific expression of a BCLi 1 A targeted shRNA and the constitutive expression of GFP and DHFR (PB-Ank- BCL11 ashRN A-MND. GFP-T2 A-DHFR construct).

[071] Fig 13B is a schematic diagram of the culture conditions tested. VU is treated with the recovery treatment, SUU expansion and PTX selection.

[072] Fig 13C is a bar graph depicting the absolute number of transposed (GFP+) phenotypic HSCs as determined using the CD34+/CD90+/CD45RA- subset.

[073] Fig. 14A is a schematic timeline of the piggyBac transposition by nucleofection, expansion treatment (SR1/UM171/UNC0638), and selection treatment via either methotrexate, pralatrexate or pralatrexate + dipyridamole.

[074] Fig. 14B is a representative flow cytometry scatterplots of the GFP expression at Day 4, before selection, and Day 11, after selection. Selection efficiency is increased with pralatrexate compared to methotrexate, and further increased with the addition of dipyridamole.

[075] Fig. 15A is a schematic diagram of a construct for constitutive expression of Interleukin 2 Receptor subunit gamma, GFP and DFFFR (PB-EF 1 a-IL2RG-T2 A-GFP-T2A-DHFR).

SUBSTITUTE SHEET (RULE 26) [076] Fig. 15B is a flow' cytometry scatterplot depicting the frequency of GFP+ ceils 7 days after electroporation (4.97%).

[077] All documents cited herein, including any cross referenced or related patent or application is hereby incorporated herein by reference m its entirety for all purposes, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term m a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

DETAILED DESCRIPTION OF THE INVENTION

[078] The present disclosure provides a composition comprising a population of modified hematopoieti c stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-aeceptable carrier.

[079] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-aeceptable carrier.

SUBSTITUTE SHEET (RULE 26) [080] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 70% to about 99%, about 75% to about 95% or about 85% to about 95% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically- acceptable carrier.

[081] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[082] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[083] The present disclosure provides a composition comprising a population of modified hematopoietic stem ceils (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population

SUBSTITUTE SHEET (RULE 26) comprise the transgene or the sequence encoding the transgene, wherein at least about 70% to about 99%, about 75% to about 95% or about 85% to about 95% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaliy-acceptabie carrier.

[084] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and do not express one or more cell-surface marker(s) comprising CD38 The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaliy-acceptabie carrier.

[085] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99 9% or

100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and do not express one or more cell-surface marker(s) comprising CD38. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaliy-acceptabie carrier.

[086] The present disclosure provides a composition comprising a population of modified hematopoietic stem ceils (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population

SUBSTITUTE SHEET (RULE 26) comprise the transgene or the sequence encoding the transgene, wherein at least about 45% to about 90%, about 50% to about 80% or about 65% to about 75% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and do not express one or more cell-surface marker(s) comprising CDS 8. The present invention also provides a

pharmaceutical composition comprising the composition and a pharmaceuticaliy-acceptable carrier.

[087] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+ and CD38-. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaliy-acceptable carrier.

[Q88] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99 9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+ and CD38-. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaliy-acceptable carrier.

[089] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 45% to

SUBSTITUTE SHEET (RULE 26) about 90%, about 50% to about 80% or about 65% to about 75% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+ and CD38-. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically- acceptable carrier.

[090] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least

50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least

85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[091] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0 1 %, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0 6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least

45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or

100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising

SUBSTITUTE SHEET (RULE 26) CD38. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable earner.

[092] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[093] The present disclosure provides a composition comprising a population of modified hematopoieti c stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1 %, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least

50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least

85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

96%, at least 97%, at least 98%, at least 99%, at least 99 5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38- and CD90+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[094] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least

SUBSTITUTE SHEET (RULE 26) 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least

45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or

100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38- and CD90+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable earner.

[095] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38- and CD90+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[096] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.1 %, at least 0.2%, at least 0.3%, at least 0.4%, at least 0 5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0 9%, at least 1 %, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[097] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%,

SUBSTITUTE SHEET (RULE 26) at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.1 %, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least

45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or

100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CDS 8 and CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[098] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[099] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least

15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least

50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least

85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

SUBSTITUTE SHEET (RULE 26) 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs compose the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA-. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaJly-acceptable carrier.

[0100] The present disclosure provides a composition comprising a population of modified hematopoietic stem ceils (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least

45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or

100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA-. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutical! y-aeceptable carrier.

[0101] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99 9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA-. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaJly-acceptable carrier.

[0102] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least

SUBSTITUTE SHEET (RULE 26) 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell- surface marker(s) comprising CD34, CD90 and CD49f and do not express one or more cell- surface marker(s) comprising CD38 and CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutieally-acceptahle carrier.

[Q1Q3] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0 06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0 1 %, at least 0.2%, at least 0.3%, at least 0.4%, at least 0 5%, at least 0.6%, at least 0.7%, at least 0 8%, at least 0.9%, at least 1 %, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell-surface marker) s) comprising CD34, CD90 and CD49f and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[0104] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at

SUBSTITUTE SHEET (RULE 26) least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2% or about 0.04% to about 1% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34, CD90 and CD49f and do not express one or more cell-surface marker(s) comprising CDS 8 and CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaliy-acceptable carrier.

[0105] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1 5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least

70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least

93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least

99.5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceuticaliy- acceptable carrier.

[0106] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least

SUBSTITUTE SHEET (RULE 26) 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[0107] The present disclosure provides a composition comprising a population of modified hematopoietic stem ceils (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2% or about 0.04% to about 1% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[OIOS] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0 07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0 2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0 6%, at least 0.7%, at least 0.8%, at least 0 9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least

35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least

70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least

93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least

99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell- surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[0109] The present disclosure provides a composition comprising a population of modified hematopoietic stem ceils (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%,

SUBSTITUTE SHEET (RULE 26) at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least

65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least

92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least

99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell- surface marker(s) comprising CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[0110] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99 9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.2% to about 5%, about 0.2% to about 3% or about 0.4% to about 3% of the population of modified HSCs express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD45RA. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically- acceptable carrier.

[0111] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein a plurality of HSCs of the population comprise a transgene or a sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least

SUBSTITUTE SHEET (RULE 26) 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD90+ and CD45RA-. The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutiealiy-aceeptabie carrier.

[0112] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD90+ and CD45RA- The present invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically- acceptable carrier.

[0113] The present disclosure provides a composition comprising a population of modified hematopoietic stem cells (HSCs), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of HSCs of the population comprise the transgene or the sequence encoding the transgene, wherein at least about 0.2% to about 5%, about 0.2% to about 3% or about 0.4% to about 3% of the population of modified HSCs comprise the cell-surface marker phenotype CD34+, CD90+ and CD45RA-. The present

SUBSTITUTE SHEET (RULE 26) invention also provides a pharmaceutical composition comprising the composition and a pharmaceutically-acceptable carrier.

[0114] The present disclosure provides composition comprising at least one promoter, a sequence encoding a therapeutic agent and a sequence encoding a selectable marker, wherein the promoter is an EF1 alpha promoter (EF1 alpha), a CMV promoter, an MM) promoter, an SV40 promoter, a PGK1 promoter, an hPGK promoter, a Ubc promoter, an SPTA1 promoter, an Ank- 1 promoter, a Gly-A promoter, a CAG promoter, an HI promoter, a U6 promoter, or a combination thereof; and wherein the therapeutic agent is human beta-globin (HBB), T87Q human beta-globin (HBB T87Q), BAF chromatin remodeling complex subunit (BCL11 A) shRNA, insulin like growth factor 2 binding protein 1 (IGF2BPI), interleukin 2 receptor gamma (IL2RG), alpha galactosidase A (GLA), alpha-L-idurondase (IDUA), iduronate 2-sulfatase (IDS), cystinosin lysosomal cysteine transporter (CTNS). The composition can be all or part of a transposon, a plasmid, a vector, or a cassette. The composition can be a linear nucleic acid molecule or a circular nucleic acid molecule. The at least one promoter can comprise a sequence capable of expressing a transgene in a cell.

[0115] The present disclosure provides a composition comprising an hPGK promoter, wherein the therapeutic agent is human beta-globin (HBB) or human beta-globin comprising a T87Q mutation (HBB T87Q), and wherein the selectable marker is a DHFR enzyme or a DHFR mutein enzyme. In one aspect, the hPGK promoter comprises the nucleic acid sequence of SEQ ID NO: 14696, wherein the human beta-globin (HBB) or T87Q human beta-globin (HBB T87Q) comprises the nucleic acid sequence of SEQ ID NO: 14969 or SEQ ID NO: 14478, and wherein the sequence encoding the DHFR enzyme or DHFR mutein enzyme comprises the nucleic acid sequence of SEQ ID NO: 14976, SEQ ID NO: 14709, or SEQ ID NO: 14901. The composition can the nucleic acid sequence of SEQ ID NO: 14936.

[0116] The present disclosure provides a composition comprising an Ank-l promoter and an MND promoter, wherein the therapeutic agent is BCLl 1 A shRNA, and wherein the selectable marker is a DHFR enzyme or a DHFR mutein enzyme. In one aspect, the Ank-l promoter comprises the nucleic acid sequence of SEQ ID NO: 14701, and wherein the MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14699, and wherein the BAF chromatin remodeling complex subunit (BCLl 1 A) shRNA comprises the nucleic acid sequence of SEQ ID NO: 14713, and wherein sequence encoding the DHFR enzyme or DHFR mutein enzyme

SUBSTITUTE SHEET (RULE 26) comprises the nucleic acid sequence of SEQ ID NO: 14976, SEQ ID NO: 14709 or SEQ ID NO: 14901. The composition can compose the nucleic acid sequence of SEQ ID NO: 14712 or SEQ ID NO: 14714.

[0117] The present disclosure provides a composition comprising an EF1 alpha promoter, wherein the therapeutic agent is IL2RG, and wherein the selectable marker is a DHFR enzyme or a DHER mutein enzyme. In one aspect, the EF1 alpha promoter comprises the nucleic acid sequence of SEQ ID NO: 14697, SEQ ID NO: 14698, or SEQ ID NO: 14706, wherein the interleukin 2 receptor gamma (IL2RG) comprises the nucleic acid sequence of SEQ ID NO: 14718, and wherein sequence encoding the DHFR enzyme or DHFR mutein enzyme comprises the nucleic acid sequence of SEQ ID NO: 14976, SEQ ID NO: 14709 or SEQ ID NO: 14901.

The composition can comprise the nucleic acid sequence of SEQ ID NO: 14703.

[0118] The present disclosure provides a composition comprising an MND promoter, wherein the therapeutic agent is IL.2RG, and wherein the selectable marker is a DHER enzyme or a DHFR mutein enzyme. In one aspect, the MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14699, wherein the interleukin 2 receptor gamma (IL2RG) comprises the nucleic acid sequence of SEQ ID NO: 14718, and wherein sequence encoding the DHFR enzyme or DHFR mutein enzyme comprises the nucleic acid sequence of SEQ ID NO: 14976, SEQ ID NO:

14709 or SEQ ID NO: 14901. The composition can comprise the nucleic acid sequence of SEQ ID NO: 14717

[0119] The present disclosure provides a composition comprising an SPTA1 promoter and an MND promoter, wherein the therapeutic agent is IGF2BP1, and wherein the selectable marker is a DHFR enzyme or a DHFR mutein enzyme. In one aspect the SPTA1 promoter comprises the nucleic acid sequence of SEQ ID NO: 14700, wherein the MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14699, wherein the IGF2BP1 comprises the nucleic acid sequence of SEQ ID NO: 14721 , and wherein sequence encoding the DHFR enzyme or DHFR mutein enzyme comprises the nucleic acid sequence of SEQ ID NO: 14976, SEQ ID NO: 14709 or SEQ ID NO: 14901. The composition can comprise the nucleic acid sequence of SEQ ID NO: 14720.

[0120] The composition further comprises a sequence encoding an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (h) a linker, and fc) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. The sequence encoding the inducible proapoptotic polypeptide can comprise the nucleic acid

SUBSTITUTE SHEET (RULE 26) sequence of SEQ ID NO: 14715 or SEQ ID NO: 14670. The inducible proapoptotic polypeptide can comprise the amino acid sequence of SEQ ID NO: 14647.

[0121] The EF1 alpha promoter comprises the nucleic acid sequence of SEQ ID NO: 14697, SEQ ID NO: 14698, or SEQ ID NO: 14706. The hPGK promoter comprises the nucleic acid sequence of SEQ ID NO: 14696. The MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14699. The SPTA1 promoter comprises the nucleic acid sequence of SEQ ID NO: 14700. The ank-1 promoter comprises the nucleic acid sequence of SEQ ID NO: 14701. The Gly-A promoter comprises the nucleic acid sequence of SEQ ID NO: 14702.

[0122] The HBB comprises the amino acid sequence of SEQ ID NO: 14724. The sequence encoding the HBB comprises the nucleic acid sequence of SEQ ID NO: 14969. The HBB T87Q comprises the amino acid sequence of SEQ ID NO: 14477. The sequence encoding HBB T87Q comprises the nucleic acid sequence of SEQ ID NO: 14478. The BCL11A shRNA comprises the nucleic acid sequence of SEQ ID NO: 14713. The IGF2BP1 comprises the amino acid sequence of SEQ ID NO: 14722. The nucleic acid encoding the IGF2BPI comprises the nucleic acid sequence of SEQ ID NO: 14721. The IL2RG comprises the amino acid sequence of SEQ ID NO: 14723. The nucleic acid encoding the IL2RG comprises the nucleic acid sequence of SEQ ID NO: 14718 The GLA comprises the ammo acid sequence of SEQ ID NO: 5974 or SEQ ID NO: 5975. The nucleic acid encoding the GLA comprises the nucleic acid sequence of SEQ ID NO: 14970. The IDUA comprises the amino acid sequence of any one of SEQ ID NO: 6715 - SEQ ID NO: 6720. The nucleic acid encoding the IDUA comprises the nucleic acid sequence of SEQ ID NO: 14971 The IDS comprises the ammo acid sequence of any of SEQ ID NO: 6709 - SEQ ID NO: 6714. The nucleic acid encoding the IDS comprises the nucleic acid sequence of SEQ ID NO: 14972. The CTNS comprises the amino acid sequence of any of SEQ ID NO: 3672 - SEQ ID NO: 3679. The nucleic acid encoding the CTNS comprises the nucleic acid sequence of SEQ ID NO: 14973. The nucleic acid encoding the selectable marker comprises a nucleic acid sequence encoding a DTIFR enzyme or a nucleic acid sequence encoding a DHFR mutein enzyme. The nucleic acid encoding the DTIFR enzyme comprises the nucleic acid sequence of SEQ ID NO: 14976. The DTIFR enzyme comprises the ammo acid sequence of SEQ ID NO: 14476. The amino acid sequence of the DHFR enzyme further comprises a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DTIFR enzyme comprises a

SUBSTITUTE SHEET (RULE 26) substitution of a Phenylalanine (F) or a Leucine (L) at position 80. The ammo acid sequence of the DHFR enzyme comprises a substitution of a Leucine (L) or a Valine (V) at position 113.

[0123] The amino acid sequence of the DHFR enzyme comprises a substitution of a Valine (V) or an Aspartic Acid (D) at position 153. The DHFR mutein enzyme comprises the amino acid sequence of SEQ ID NO: 14725. The sequence encoding the DHFR mutein enzyme comprises the nucleic acid sequence of SEQ ID NO: 14709 or SEQ ID NO: 14901.

[0124] The present disclosure provides methods for expansion of a population of cells. The expansion of a population of cells includes the expansion of a plurality of modified cells.

Preferably, the cell is an HSC or the population of ceils is a population of HSCs.

[0125] The present disclosure also provides methods for selecting a population of cells. The selection of a population of cells includes the selection of a plurality of modified cells comprising a selection marker. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0126] The present disclosure also provides methods for expansion and selection of a population of cells. The expansion and selection of a population of cells includes the expansion of a plurality' of modifi ed cells. The sel ection of a population of cells includes the selection of a plurality of modified cells comprising a selection marker. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0127] The present disclosure provides a method of producing a population of modified hematopoietic stem ceils (HSCs), comprising: a) introducing a transgene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction of the transgene into a plurality of HSCs to produce a plurality of modified HSCs; and b) culturing the plurality of HSCs from step a) in a culture media comprising a HDAC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor, a pynmido-mdole derivative, or a combination thereof, thereby producing a population of modified HSCs.

[0128] The present disclosure provides a method of producing a population of modified hematopoietic stem cells (HSCs), comprising: a) introducing a transgene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction into a plurality of HSCs to produce a plurality of modified HSCs; and b) culturing the plurality of HSCs from step a) in a culture media comprising at least two of a HDAC inhibitor, a HMT

SUBSTITUTE SHEET (RULE 26) inhibitor, a aryl hydrocarbon receptor inhibitor or a pynmido-mdole derivative, thereby producing a population of modified HSCs.

[0129] The present disclosure provides a method of producing a population of modified hematopoietic stem cells (HSCs), comprising: a) introducing a transgene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction into a plurality of HSCs to produce a plurality of modified HSCs; and b) culturing the plurality of HSCs from step a) in a culture media comprising at least three of a HD AC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrimido-indole derivative, thereby producing a population of modified HSCs.

[0130] The present disclosure provides a method of producing a population of modified hematopoietic stem cells (HSCs), comprising: a) introducing a transgene or a sequence encoding the transgene under culture conditions sufficient for cell proliferation and for introduction into a plurality of HSCs to produce a plurality of modified HSCs; and b) culturing the plurality of HSCs from step a) in a culture media comprising each of a HD AC inhibitor, a HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pynmido-mdole derivative, thereby producing a population of modified HSCs.

[0131] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposa.se under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for at least about 3 days, wherein the expansion agent comprises at least two of an HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrimido- indole derivative, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0132] The present disclosure provides a method of expanding a population of modified ceils, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for at least about 3 days, wherein the expansion agent

SUBSTITUTE SHEET (RULE 26) comprises each of an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimi do- indole derivative, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0133] The HMT inhibitor, aryl hydrocarbon receptor inhibitor, pyrimido-indole derivative, or a combination thereof, can be present in the culture media (e.g., in contact with the cell or plurality of cells) for at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days. In a preferred aspect, at least 7 days.

[0134] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days, wherein the expansion agent comprises at least two of an HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrimido-indole derivative, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0135] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days, wherein the expansion agent comprises each of an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimido- indole derivative, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0136] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a dihydrofolate reductase (DHFR) resistance gene or a transposon comprising a sequence encoding a DHFR mutein enzyme or a sequence

SUBSTITUTE SHEET (RULE 26) encodmg the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of ceils to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises at least two of SRI, UM171 or UNC0638, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0137] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises each of SRI, UMI 71 and UNC0638, thereby expanding a population of modified cells.

[0138] The HMT inhibitor, aryl hydrocarbon receptor inhibitor, pyrimido-indole derivative, or a combination thereof, can be present in the culture media (e.g., in contact with the cell or plurality of cells) for about 3 days to about 14 days; about 3 days to about 13 days; about 3 days to about 12 days; about 3 days to about 12 days; about 3 days to about 1 1 days; about 3 days to about 10 days; about 3 days to about 9 days; about 3 days to about 8 days; about 3 days to about 7 days; about 4 days to about 9 days; about 4 days to about 8 days; about 4 days to about 7 days; or about 5 days to about 7 days. In a preferred aspect, about 5 days to about 7 days.

[0139] The HMT inhibitor can be UNC0638, BIX01294, UNC0642, A-366, UNC0224, UNC0631, UNC0646, BRD477Q, UNC0631 , chaetocm, EPZ005687, EPZ6438, GSK126, GKS343, Ell, UNCI 99, EPZ004777, EPZ5676, LEY-507, AZ505 or A-893. In a preferred aspect, the HMT inhibitor is UNC0638. The term UNC0638 also includes UNC0638 hydrate.

[0140] UNC0638 can be present in the culture media in an amount from about 0.5 mM to about 2 mM; about 0.5 mM to about 1.5 mM; about 0.5 mM to about 1 mM; or about 0.75 mM to about 1.25 mM In some aspects, UNC0638 is present in the culture media at about 0.75 mM; about 1 mM or about 1.25 mM. In a preferred aspect, UNC0638 is present in the culture media at about

SUBSTITUTE SHEET (RULE 26) 1.0 mM. in another preferred aspect UNC0638 is present in the culture media at about 1.0 mM for about 4 days to about 1 1 days. In another preferred aspect, UNC0638 is present in the culture media at about 1.0 mM for at least 7 days.

[0141] The aryl hydrocarbon receptor inhibitor can be StemRegenin 1 (SRI), a pha- naphthoflavone, beta-naphthoflavone, brevifolincarboxylic acid, 6,2',4'-Trimethoxyflavone, D,L- Sulforaphane, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PDM2, salicylamide, 1 ,3-dichloro- 5-[(!E)-2-(4-methoxyphenyl)ethenyl]-benzene, CH 223191 , methylindoles, methoxyindoles, indole-3-carbinol, mexiletine, hydroxytamoxifen, raloxifene, 2,3,7,8-tetrachlorodibenzo-p- dioxin, laquinimod, aminoflavone (NSC686288), 0379931 13, diindolylmethane (DIM), tranilast, fiutamide or omeprazole. In a preferred aspect, the aryl hydrocarbon receptor inhibitor is SRI .

[0142] SRI can be present in the culture media in an amount from about 0.5 mM to about 2 mM; about 0.5 m.M to about 1.5 mM; about 0.5 mM to about 1 mM; or about 0.75 mM to about 1.25 mM. In some aspects, SRI is present m the culture media at about 0.75 mM; about 1 mM or about 1.25 mM In a preferred aspect, SRI is present in the culture media at about 1.0 mM. In another preferred aspect, SRI is present in the culture media at about 1.0 mM for about 4 days to about 1 1 days. In another preferred aspect, SRI is present in the culture media at about 1.0 mM for at least 7 days.

[0143] The pyrimi do-indole derivative can be a pyrimido-[4,5-b]-indole derivative. In a preferred aspect, the pyrimido-indole derivative inhibitor is UM171 and has the following

chemical structure:

[0144] UM171 can be present in the culture media in an amount from about 25 nM to about 75 nM; or about 25 nM to about 50 nM. In a preferred aspect, UM171 is present in the culture media at about 35 nM. In another preferred aspect, UM171 is present in the culture media at

SUBSTITUTE SHEET (RULE 26) about 35 nM for about 4 days to about 11 days. In another preferred aspect, UM171 is present in the culture media at about 35 nM for at least 7 days.

[0145] When the culture media comprises a combination of SRI, UM171 and UNC0638 (termed SUU herein), the SRI is present from about 0.75 mM; about 1 mM or about 1.25 mM; the UM171 is present from about 25 nM to about 50 nM; the UNC0638 is present from about 0.75 mM; about 1 mM or about 1.25 mM. In a preferred aspect, the SRI is present m the culture media at about 1 mM, the UM171 is present in the culture media at about 35 nM and UNC0638 is present in the culture media at about 1.0 mM for about 4 days to about 11 days. In a preferred aspect, the SRI is present in the culture media at about 1 mM, the UM171 is present in the culture media at about 35 nM and UNC0638 is present in the culture media at least 7 days.

[0146] Hematopoietic stem cells (HSCs) or a plurality of HSCs cultured in a culture media comprising SRI and UMI7I demonstrate phentotypic expansion from about 2 fold to about 10 fold, about 4 fold to about 10 fold, about 6 fold to about 10 fold, or about 8 fold to about 10 fold. Addition of UNC0638 to SRI and UM171 phentotypic expansion about 0.5 fold, about 1 fold, about 1.5 fold or about 2 fold. The SUU combination demonstrates phentotypic expansion from 2 fold to about 15 fold, about 4 fold to about 15 fold, about 6 fold to about 15 fold, or about 8 fold to about 15 fold or about 10 fold to about 1 5 fold, with an average phentotypic expansion of about 12 fold. The SUU combination significantly expands (more than about 2 fold, about 4 fold, about 6 fold, about 8 fold, or about 10 fold) cobblestone forming cells compared to cytokine culture only. HSCs cultured m SUU engraft well as demonstrated by in vivo engraftment assays.

[0147] The expansion agent can comprise valproic acid (VP A). The culture media can comprise about 0.25 mM to about 1.25 mM of VP A. The culture media can comprise about 1 mM of VP A. HSCs or a plurality of HSCs cultured in a culture media comprising valproic acid demonstrate phentotypic expansion from about 300 fold to about 350 fold with an average phenotypic expansion of about 335 fold but does not have a significant expansion effect on cobblestone forming cells compared to culture in the presence of cytokines alone. HSCs or a plurality of HSCs cultured in valproic acid demonstrate engraftment, either alone or in combination with UNC0638 and/or UMT71.

[0148] The expansion agent can further comprise nicotinamide. The culture media can comprise about 2.5 mM to about 10 mM of nicotinamide. The culture media can comprise about

SUBSTITUTE SHEET (RULE 26) 5 mM of nicotinamide. HSCs or a plurality of HSCs cultured in a culture media comprising SUU and nicotinamide show at least a one-fold, at least a two-fold or at least a three-fold increase in phentotypic expansion when compared to culturing in a culture media comprising SUU alone. HSCs or a plurality of HSCs cultured in a culture media comprising SUU and nicotinamide show about a one-fold to about a four-fold increase, about a two-fold to about a 4 fold increase or about a one-fold to about a three-fold increase in phentotypic expansion when compared to culturing in a culture media comprising SUU alone.

[0149] The expansion agent can further comprise garcinol. The culture media can comprise about 5 pM to about 15 mM of garcinol. The culture media can comprise about 10 mM of garcinol. HSCs or a plurality of HSCs cultured m a culture media comprising S UU and garcinol show at least a 0.5 fold, at least a 1 fold, at least a 1.5 fold or at least a 2 fold increase in phentotypic expansion when compared to culturing in a culture media comprising SUU alone. However, the addition of garcinol didn’t improve function over SUU alone.

[0150] The expansion agent can further comprise NaPB. The culture media can comprise about 1 mM to about 2 mM of NaPB. The culture media can comprise about 1.5 mM of NaPB. HSCs or a plurality of HSCs cultured in a culture media comprising NaPB demonstrate

phentotypic expansion.

[0151] Additional expansion agents may comprise dmPGE2, 5-azacytidine, 4-HPR, hlGFBP2, hANGPTLS, PCI-34051, GW9662 and N-acetylcysteme.

[0152] The present disclosure provides a method of expanding a population of modified ceils, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for at least about 3 days, wherein the expansion agent comprises at least two of SRI , UM171 and UNC0638, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0153] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality

SUBSTITUTE SHEET (RULE 26) of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for at least about 3 days, wherein the expansion agent comprises each of SRI, UM171 and UNC0638, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0154] In one aspect, the selection gene is a dihydrofolate reductase (DHFR) resistance gene.

In one aspect, the selection agent is methotrexate, pralatrexate, pyrimethamine, dapsone, raltitrexed, trimetrexate, metoprine, iclaprim, aminopterin, lometrexol, nolatrexed, brodimoprim, trimethoprim, pemetrexed, proguanil, piritrexim, or cycioguaml. In one aspect, when the selection gene is DHFR or a sequence encoding a DHFR mutein enzyme, the selection agent is methotrexate, pralatrexate, pyrimethamine, dapsone, raltitrexed, trimetrexate, metoprine, iclaprim, aminopterin, lometrexol, nolatrexed, brodimoprim, trimethoprim, pemetrexed, proguanil, piritrexim, or cycioguaml. In one aspect, when the selection gene is DHFR or a sequence encoding a DHFR mutein enzyme, the selection agent is methotrexate ( M l X) or pralatrexate (PTX) The DHFR mutein enzyme can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 14677. The DHFR mutein enzyme is encoded by a nucleic acid sequence comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO; 14678. The ammo acid sequence of the DHFR mutein enzyme can further comprises a mutation at one or more of positions 80, 113, or 153. In some embodiments, the amino acid sequence of the DHFR mutein enzyme comprises one or more of a substitution of a Phenylalanine (F) or a Leucine (L) at position 80, a substitution of a Leucine (L) or a Valine (V) at position 113, and a substitution of a Valine (V) or an Aspartic Acid (D) at position 153. In one aspect, when the transposon comprises a sequence encoding a DHF mutein enzyme the selection agent is methotrexate or pralatrexate.

[0155] The methotrexate can be present in the culture media in an amount from about 100 nM to about 500 nM. In a preferred aspect, the culture media can comprise about 250 nM of methotrexate. The pralatrexate can be present m the culture media m an amount from about 50 nM to about 250 nM. In a preferred aspect, the culture media can comprise about 125 nM of pralatrexate.

[0156] In some aspects, the selection agent is pralatrexate and dipyridamole (DP). The culture media can comprise about 50 nM to about 250 nM of pralatrexate and about 1 mM to about 10

SUBSTITUTE SHEET (RULE 26) mM of dipyridamole. In a preferred aspect, the culture media can comprise about 125 nM of praiatrexate and about 5 mM of dipyndimole.

[0157] In some aspects, culturing the plurality of modified cells with an expansion agent occurs prior to culturing the plurality of modified cells with a selection agent. In some aspects, culturing the plurality' of modified cells with a selection agent occurs prior to culturing the plurality of modified cells with an expansion agent. In some aspects, culturing the plurality of modified cells with an expansion agent and culturing the plurality of modified cells with a selection agent occur concurrently.

[0158] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DTIFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for at least about 3 days, wherein the selection agent comprises methotrexate or praiatrexate and wherein the expansion agent comprises at least two of SRI , UM171 and UNC0638, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0159] The present disclosure provides a method of expanding a population of modified ceils, comprising: a) introducing a transposon comprising a DKFR resistance gene or a transposon comprising a sequence encoding a DTIFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified ceils in a culture media comprising a selection agent and an expansion agent for at least about 3 days, wherein the selection agent comprises methotrexate or praiatrexate and wherein the expansion agent comprises each of SRI, UM171 and UNC0638, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0160] The disclosed culture methods for expanding a population of modified ceils can be compared to culture methods utilizing culture media not comprising a selection agent, an expansion agent, or a combination thereof.

SUBSTITUTE SHEET (RULE 26) [0161] in preferred aspects; the culture methods comprising a culture media comprising a selection agent, an expansion agent, or a combination thereof result in at least a one-fold, at least a two-fold, at least a three-fold, at least a four-fold, at least a five-fold; at least a six-fold; at least a seven-fold; at least an eight-fold; at least a nine-fold or at least a ten-fold expansion of the plurality of modified ceils compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent, an expansion agent, or a combination thereof. Preferably, the ceil is an HSC or the population of ceils is a population of HSCs.

[0162] The present disclosure provides a method of expanding a population of modified ceils, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days, wherein the expansion agent comprises at least two of an HMT inhibitor, a aryl hydrocarbon receptor inhibitor or a pyrimido-indole derivative, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0163] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days, wherein the expansion agent comprises each of an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimido-indole derivative, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0164] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon

SUBSTITUTE SHEET (RULE 26) comprising a sequence encoding a DHFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises at least two of SRI, UM171 or UNC0638, wherein said culturing results m at least two-fold expansion of the plurality of modified cells compared to a plurality of modified ceils cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0165] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises each of SRI , UM171 and UNC0638, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0166] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days, wherein the expansion agent comprises at least two of an HMT inhibitor, a ary! hydrocarbon receptor inhibitor or a pyrimido-indole derivative, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified ceils cultured under culture

SUBSTITUTE SHEET (RULE 26) conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0167] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days, wherein the expansion agent comprises each of an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimido- indole derivative, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0168] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises at least two of SRI , UM171 or UNC0638, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0169] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; and b) culturing the plurality of modified cells in a culture media comprising a selection

SUBSTITUTE SHEET (RULE 26) agent and an expansion agent for about 4 days to about 9 days, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises each of SRI, UM171 and UNC0638, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified ceils cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0170] In some aspects of the present disclosure, when the transgene or a sequence encoding the transgene is introduced into a plurality of HSCs using a transposon comprising a sequence encoding the transgene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into the plurality of HSCs to produce a plurality of modified HSCs, the present invention provides methods for increasing frequency of transposition in the plurality of cells.

[0171] The present disclosure provides a method of increasing the frequency of transposition in a cell comprising a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into the cell to produce a modified cell and b) culturing the modified cell in a culture media comprising a histone deacetylase (HD AC) inhibitor, a histone methyltransferase (HMT) inhibitor, or a combination thereof, thereby increasing the frequency of transposition in the cell. Preferably, the ceil is an HSC,

[0172] The present disclosure provides a method of increasing the frequency of transposition in a population of cells comprising a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells and b) culturing the plurality of modified cells in a culture media comprising a histone deacetylase (HD AC) inhibitor, a histone methyltransferase (HMT) inhibitor, or a combination thereof, thereby increasing the frequency of transposition in a population of cells. Preferably, the population of cells is a population of HSCs.

[0173] The cell or plurality of cells can be cultured m a culture media comprising the HD AC inhibitor, the HMT inhibitor, or a combination thereof, before, after or concurrently with introducing the transposon or a sequence encoding the transposon and the transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and

SUBSTITUTE SHEET (RULE 26) for transposition into the cell or plurality of cells to produce the modified cell or plurality of modified cells.

[0174] A modified cell comprises a genome-integrated transposon. A plurality of modified cells comprise a plurality of cells comprising a genome-integrated transposon. The integration can be stable or transient.

[0175] The HD AC inhibitor, the HMT inhibitor, or a combination thereof can be present in the culture media (e.g., in contact with the cell or plurality of cells) for at least about 1 minute, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, or any number of minutes in between.

[0176] The HD AC inhibitor, the HMT inhibitor, or a combination thereof can be present in the culture media (e.g., in contact with the cell or plurality of cells) for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 34 hours, at least about 36 hours, or any number of hours in between.

[0177] The HD AC inhibitor, the HMT inhibitor, or a combination thereof can be present in the culture media (e.g., in contact with the cell or plurality of cells) for at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days.

[0178] The HD AC inhibitor can be present in the culture media (e.g., in contact with the ceil or plurality of cells) for at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, or any number of hours in between.

[0179] The HMT inhibitor can be present m the culture media (e.g., in contact with the cell or plurality of cells) in the presence of the HD AC inhibitor for at least about 3 hours, at least about

SUBSTITUTE SHEET (RULE 26) 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, or at least about 4 days.

[0180] The HMT inhibitor can be present in the culture media (e.g., in contact with the cell or plurality of cells) in the absence of the HD AC inhibitor for at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, or at least about 4 days.

[0181] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for at least one hour; at least about 3 hours, at least about 6 hours, about 12 hours, about 18 hours, at least about 24 hours.

[0182] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for at least one hour; at least about 3 hours, at least about 6 hours, about 12 hours, about 18 hours, at least about 24 hours following the introduction of the transposon or a sequence encoding the transposon and the transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a cell or a plurality of cells to produce the modified cell or the plurality' of modified cells.

[0183] The methods of increasing the frequency of transposition can further comprise a recovery period following the introducing step, wherein the modified cell or plurality of modified cells and the HD AC inhibitor, HMT inhibitor, or combination thereof are not in contact. That is, the modified cell or plurality of modified cells can be cultured in a culture media not comprising the HD AC inhibitor, HMT inhibitor, or combination thereof as part of the recovery period following the introduction of the transposon or sequence encoding the transposon. The recovery period can have a duration of at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, or any number of minutes in between. The recovery period can have a duration of least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, or any number of hours m between.

SUBSTITUTE SHEET (RULE 26) [0184] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for at least one hour; at least about 3 hours, at least about 6 hours, about 12 hours, about 18 hours, at least about 24 hours following the in troduction of the transposon or a sequence encoding the transposon and the transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a cell or a plurality of cells to produce the modified cell or plurality of modified cells and then the HD AC inhibitor is removed from the cell culture media. The HD AC inhibitor can be removed by any means known in the art (e.g., washing the ceils and adding culture media not comprising an HD AC inhibitor).

[0185] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (and in contact with the plurality of cells) for at least one hour; at least about 3 hours, at least about 6 hours, about 12 hours, about 18 hours, at least about 24 hours following the introduction of the transposon or a sequence encoding the transposon and the transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce the plurality' of modifi ed cells and then the HD AC inhibitor is removed from the cell culture media. The HD AC inhibitor can be removed by any means known in the art (e.g., washing the cells and adding culture media not comprising an HD AC inhibitor). Following removal of the HD AC inhibitor the modified cell or plurality of modified cells are cultured in a culture media comprising an HMT inhibitor (e.g., without an HD AC inhibitor present) for at least about 3 hours, at least about 6 hours, about 12 hours, about 18 hours, at least about 1 day, at least about 2 days, at least about 3 days, or at least about 4 days. In some aspects, the HMT inhibitor is removed from the cell culture media at the same time as removal of the HD AC inhibitor (e.g., washing the cells) and new culture media is added comprising an HMT inhibitor and not comprising an HD AC inhibitor.

[0186] The HD AC inhibitor, the HMT inhibitor, or a combination thereof can be present in the culture media (e.g., in contact with the ceil or plurality of cells) for about 30 minutes to about 36 hours; about 1 hour to about 34 hours; about 2 hours to about 32 hours; about 3 hours to about 30 hours; about 4 hours to about 28 hours; about 6 hours to about 26 hours; about 6 hours to about 24 hours; about 8 hours to about 24 hours; about 10 hours to about 24 hours; about 12 hours to about 24 hours; about 14 hours to about 24 hours; about 16 hours to about 24 hours; about 18 hours to about 24 hours; or about 18 hours to about 24 hours.

SUBSTITUTE SHEET (RULE 26) [0187] The HD AC inhibitor, the HMT inhibitor, or a combination thereof can be present in the culture media (e.g., in contact with the cell or plurality of cells) for about 1 day to about 2 days; about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, about 1 day to about 10 days, about 1 day to about 11 days, about 1 day to about 12 days, about 1 day to about 13 days, about 1 day to about 14 days.

[0188] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for about 30 minutes to about 36 hours; about 1 hour to about 34 hours; about 2 hours to about 32 hours; about 3 hours to about 30 hours; about 4 hours to about 28 hours; about 6 hours to about 26 hours; about 6 hours to about 24 hours; about 8 hours to about 24 hours; about 10 hours to about 24 hours; about 12 hours to about 24 hours; about 14 hours to about 24 hours; about 16 hours to about 24 hours; about 18 hours to about 24 hours; or about 18 hours to about 24 hours.

[0189] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for about 1 hour to about 24 hours; about 2 hours to about 24 hours; about 3 hours to about 24 hours; about 4 hours to about 24 hours; about 5 hours to about 24 hours; about 6 hours to about 24 hours; about 8 hours to about 24 hours; about 10 hours to about 24 hours; about 12 hours to about 24 hours; about 14 hours to about 24 hours; about 16 hours to about 24 hours; about 18 hours to about 24 hours; or about 18 hours to about 24 hours.

[0190] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for about 1 hour to about 24 hours; about 2 hours to about 24 hours; about 3 hours to about 24 hours; about 4 hours to about 24 hours; about 5 hours to about 24 hours; about 6 hours to about 24 hours; about 8 hours to about 24 hours; about 10 hours to about 24 hours; about 12 hours to about 24 hours; about 14 hours to about 24 hours; about 16 hours to about 24 hours; about 18 hours to about 24 hours; or about 18 hours to about 24 hours following the introduction of the transposon or a sequence encoding the transposon and the transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a cell or a plurality of cells to produce the modified cell or plurality of modified cells.

SUBSTITUTE SHEET (RULE 26) [0191] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for about 1 hour to about 24 hours; about 2 hours to about 24 hours; about 3 hours to about 24 hours; about 4 hours to about 24 hours; about 5 hours to about 24 hours; about 6 hours to about 24 hours; about 8 hours to about 24 hours; about 10 hours to about 24 hours; about 12 hours to about 24 hours; about 14 hours to about 24 hours; about 16 hours to about 24 hours; about 18 hours to about 24 hours; or about 18 hours to about 24 hours following the introduction of the transposon or a sequence encoding the transposon and the transposase or a sequence encoding the transposase under culture conditions sufficient for ceil proliferation and for transposition into a cell or a plurality of cells to produce the modified cell or plurality of modified cells and then the HD AC inhibitor is removed from the cell culture media. The HD AC inhibitor can be removed by my means known in the art (e.g., washing the cells and adding culture media not comprising an HD AC inhibitor).

[0192] The combination of the HD AC inhibitor and the HMT inhibitor can both be present in the culture media (e.g., in contact with the cell or plurality of cells) for about 1 hour to about 24 hours; about 2 hours to about 24 hours; about 3 hours to about 24 hours; about 4 hours to about 24 hours; about 5 hours to about 24 hours; about 6 hours to about 24 hours; about 8 hours to about 24 hours; about 10 hours to about 24 hours; about 12 hours to about 24 hours; about 14 hours to about 24 hours; about 16 hours to about 24 hours; about 18 hours to about 24 hours; or about 18 hours to about 24 hours following the introduction of the transposon or a sequence encoding the transposon and the transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a cell or a plurality of cells to produce the modified cell or the plurality of modified cells and then the HD AC inhibitor is removed from the cell culture media. The HD AC inhibitor can be removed by any means known in the art (e.g., washing the cells and adding culture media not comprising an HD AC inhibitor). Following removal of the HD AC inhibitor the modified cell or the plurality of modified cells are cultured in a culture media comprising an HMT inhibitor (e.g., without an HD AC inhibitor present) for about one day to about 7 days; about one day to about 6 days; about one day to about 5 days; about one day to about 4 days. In some aspects, the HMT inhibitor is removed from the cell culture media at the same time as removal of the HD AC inhibitor (e.g..

SUBSTITUTE SHEET (RULE 26) washing the cells) and new culture media is added comprising an HMT inhibitor and not comprising an HD AC inhibitor.

[0193] The present disclosure provides a method of increasing the frequency of transposition in a cell or a population of cells, comprising: a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a cell to produce a modified cell or into a plurality of cells to produce a plurality of modified cells; b) culturing the modified cell or the plurality of modified cells in a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 24 hours; c) removing the HD AC inhibitor from the culture media; d) culturing the modified cell or the plurality of modified cells in a culture media comprising a HMT inhibitor for about 3 days following step c; thereby increasing the frequency of transposition in a cell or a population of cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0194] The present disclosure provides a method of increasing the frequency of transposition in a cell or a population of cells, comprising: a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a cell to produce a modified cell or a plurality of cells to produce a plurality of modified cells; b) culturing the modified cell or the plurality of modified cells in a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 3 hours to about 30 hours; c) removing the HDAC inhibitor from the culture media; d) culturing the modified cell or the plurality of modified cells in a culture media comprising a HMT inhibitor for about 1 day to about 5 days following step c; thereby increasing the frequency of transposition in a cell or a population of cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0195] The disclosed culture methods for increasing the frequency of transposition can be compared to culture methods utilizing culture media not comprising a HDAC inhibitor, a HMT inhibitor, or a combination thereof. The disclosed culture methods for increasing the frequency of transposition can be compared to identical culture conditions sufficient for cell proliferation and for transposition into a cell but utilizing culture media not comprising a HDAC inhibitor, a HMT inhibitor, or a combination thereof.

SUBSTITUTE SHEET (RULE 26) [0196] in preferred aspects; the culture methods comprising a culture media comprising a HD AC inhibitor, a HMT inhibitor, or a combination thereof result in an increase in frequency of transposition and at least a one-fold; at least a two-fold; at least a three-fold; at least a four-fold increase in the yield of transposed cells in the plurality of modified cells when compared to culture conditions sufficient for cell proliferation and for transposition into the cell utilizing culture media not comprising a HD AC inhibitor, a HMT inhibitor, or a combination thereof.

[0197] Culture conditions sufficient for cell proliferation and transposition comprise culture in the presence of one or more cytokines. In one aspect, the one or more cytokines can comprise, consist essential of, or consist of human recombinant stem cell factor (hrSCF), human recombinant thrombopoietin (hrTPO), human recombinant FMS-like tyrosine kinase 3 ligand (hrFLTSL). In one aspect the culture conditions sufficient for cell proliferation comprise culture in the presence of each of hrSCF, hrTPO and hrFLTSL The culture media can comprise about 50 ng ml to about 200 ng/ml of hrSCF. The culture media can comprise about lOOng/m! of hrSCF. The culture media can comprise about 50 ng/ml to about 200 ng/ml of hrTPO. The culture media can comprise about 100 ng/ml of hrTPO. The culture media can comprise about 50 ng/ml to about 200 ng/ml of hrFLT3L. The culture media can comprise lOOng/ml of hrFLT3L.

[0198] The present disclosure provides a method of increasing the frequency of transposition in a population of cells, comprising: a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposa.se under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells in a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 24 hours; c) removing the FID AC inhibitor from the culture media; d) culturing the plurality of modified cells in a culture media comprising a HMT inhibitor for about 3 days following step c; wherein said culturing results in an increase in frequency of transposition and at least a one-fold increase in the yield of transposed cells m the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a HD AC inhibitor and FIMT inhibitor. The culture conditions not comprising a HD AC inhibitor and HMT inhibitor are sufficient for cell proliferation and for transposition.

[0199] The present disclosure provides a method of increasing the frequency of transposition in a population of cells, comprising: a) introducing a transposon or a sequence encoding the

SUBSTITUTE SHEET (RULE 26) transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells m a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 3 hours to about 30 hours; c) removing the HD AC inhibitor from the culture media; d) culturing the plurality of modified cells in a culture media comprising a HMT inhibitor for about 1 day to about 5 days following step c; wherein said culturing results in an increase in frequency of transposition and at least a one-fold increase in the yield of transposed cells in the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a HD AC inhibitor and HMT inhibitor. The culture conditions not comprising a HD AC inhibitor and HMT inhibitor are sufficient for cell proliferation and for transposition.

[0200] The present methods can be used to increase the frequency of transposition in a naturally poor transposer cell. The term“a naturally poor transposer cell” as used herein means a cell that has a transposition frequency of less than or equal to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, or 15% when utilizing standard transposon in a standard nucleofection or electroporation assay and without the addition of any transposition enhancing or boosting agents. The term“a naturally high transposer cell” as used herein means a cell that has a transposition frequency of equal to or greater than about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% when utilizing standard transposon in a standard nucleofection or electroporation assay and without the addition of any transposition enhancing or boosting agents. In one aspect, the present methods increase the frequency of transposition from less than or equal to about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% to equal to greater than about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

[0201] For example, a cell transposed with a vector comprising a transposon comprising an EFIa!pha promoter expressing GFP and DHFR is“a naturally poor transposer cell” with a transposition frequency less than or equal to about 10% when utilizing standard transposon in a standard nucleofection or electroporation assay and without the addition of any transposition enhancing or boosting agents. A cell transposed with a vector comprising a transposon comprising an EFIalpha promoter expressing GFP and DHFR is“a naturally high transposer

SUBSTITUTE SHEET (RULE 26) cell” with a transposition frequency of equal to greater than about 15%. In one aspect, the present methods increase the frequency of transposition of the transposon comprising an EFIalpha promoter expressing GFP and DHFR from less than or equal to about 10% to equal to or greater than about 15%.

[0202] Without being bound by any theory, the HD AC inhibitor, the HMT inhibitor, or a combination thereof can open the chromatin of a cell or ceils in a plurality of cells and/or increase access of the transposon and transpose as to the genomic DNA of a ceil or ceils in a plurality of cells.

[0203] The HD AC inhibitor, the HMT inhibitor, or a combination thereof can be present in the culture media in an amount from about 0.5 mM to about 2 mM; about 1 0 mM to about 1 mM; about 2.0 mM to about 1.0 mM; or about 5.0 mM to about 1.0 mM.

[0204] The HD AC inhibitor can be a pan-HDAC inhibitor, a class I HDAC inhibitor, a class II HD AC inhibitor or a class I and class IT inhibitor. Non-limiting examples of pan-HDAC inhibitors include Trichostatm A (TSA), Vorinostat, CAY10433 (targets class I and II), or sodium phenylbutyrate (targets class I and Ila). Non-limiting examples of class I HDAC inhibitors (targeting HDAC 1 , 2, 3, and 8) include MS-275, CAY10398, or Entinostat. Non- limiting examples of class II HDAC inhibitors (targeting HDAC 4, 5, 6, 7, 9, and 10) include MC-1568, Scnptaid, or CAY 10603. Valproic acid (VP A) can inhibits multiple histone deacetylases from both Class I and Class II (but not HDAC6 or HDAC 10) and has high potency for Class I HDACs.

[0205] The HDAC inhibitor can be valproic acid, sodium phenylbutyrate (NaPB), tnchostatin A, vorinostat, CAY10433, MS-275, CAY10398, entinostat, MC-1568, scriptaid, or CAY10603. In a preferred aspect, the HDAC inhibitor is valproic acid.

[0206] VP A can be present in the culture media in an amount from about 0.1 mM to about 2 mM; about 0.25 mM to about 1 mM; about 0.25 to about 0.75 mM; about 0.25 to about 0.5 mM or about 0.5 mM to about 0.75 mM. In some aspects, VP A is present in the culture media at about 0.25 mM; about 0.5 mM; about 0.75 mM or about 1 mM. In a preferred aspect, VP A is present in the culture media at about 0.5 mM or about 0.75 mM. In another preferred aspect,

VP A is present m the culture media at about 0.5 mM for about 24 hours. In another preferred aspect, VTA is present in the culture media at about 0.75 mM for about 24 hours.

SUBSTITUTE SHEET (RULE 26) [0207] NaPB can be present in the culture media in an amount from about 0.5 mM to about 3 mM; about 1.0 mM to about 2.0 mM; or about 1.0 mM to about 1.5 mM. In a one aspect, NaPB is present in the culture media at about 1.5 mM. In another aspect, NaPB is present in the culture media at about 1.5 mM for about 1 day to about 7 days.

[0208] The HMT inhibitor can be a selective inhibitor of G9a/GLP histone methyltransferases, which methylate lysine 9 of histone 3 (H3K9). Non-limiting examples of G9a/GLP inhibitors include BIX01294, UNC0642, A-366, UNC0224, UNC0631, UNC0646, BRD4770, or

UNC0631. Non-limiting examples of histone lysine methyltransferases include chaetocin, EPZ005687, EPZ6438, GSK126, GKS343, Ell, UNCI 99, EPZ004777, EPZ5676, LEY-507, AZ505, or A-893.

[0209] The HMT inhibitor can be 2-Cyclohexyl-N-(l-isopropylpiperidin-4-yl)-6-methoxy-7- (3-(pyrrolidin~i~y!)propoxy) quinazolin-4-amine (UNC0638), BIX01294, UNC0642, A-366, UNC0224, UNC0631, UNC0646, BRD4770, UNC0631 , chaetocin, EPZ005687, EPZ6438, GSK126, GKS343, Ell, UNCI 99, EPZ004777, EPZ5676, LLY-507, AZ505 or A-893. In a preferred aspect, the HMT inhibitor is UNC0638. The term UNC0638 also includes UNC0638 hydrate.

[0210] UNC0638 can be present in the culture media in an amount from about 0.5 mM to about 2 mM: about 0.5 mM ΐo about 1.5 mM: about 0.5 mM ΐo about 1 mM; or about 0.75 mM ΐo about 1.25 mM. In some aspects, UNC0638 is present in the culture media at about 0.75 mM; about 1 mM or about 1.25 mM In a preferred aspect, UNC0638 is present in the culture media at about 1.0 mM. In another preferred aspect, UNC0638 is present in the culture media at about 1.0 mM for about 4 days to about 11 days.

[0211] The culture media for increasing the frequency of transposition comprising a HD AC inhibitor, HMT inhibitor, or a combination thereof, can further comprise a DNA

methyltransferase inhibitor, aryl hydrocarbon receptor inhibitor, a pyrimido-indole derivative, a second HD AC inhibitor, a second HMT inhibitor, or a combination thereof.

[0212] The DNA methyltransferase inhibitor can be 5-azacytidine. The 5-azacytidine can be present in the culture media in an amount from about 0.1 mM to about 1 mM or about 0.5 mM to about 1 mM. In a one aspect, the 5-azacytidine is present m the culture media at about 0.1 mM, about 0.5 mM or about 1 mM for about 24 hours.

SUBSTITUTE SHEET (RULE 26) [0213] The aryl hydrocarbon receptor inhibitor can be StemRegenin 1 (SRI), alpha- naphthoflavone, beta-naphthoflavone, brevifolincarboxylic acid, 6,2',4'-Trimethoxyfiavone, D,L- Sulforaphane, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PDM2, salicylamide, l,3-dichloro- 5-[(lE)-2-(4-methoxyphenyl)ethenyl]-benzene, CH 223191 , methylmdoles, methoxyindoles, indole-3-carbinol, mexiletine, hydroxytamoxifen, raloxifene, 2,3,7,8-tetrachlorodibenzo-p- dioxm, laquimmod, aminoflavone (NSC686288), CB7993113, diindolyimethane (DIM), tranilast, flutamide or omeprazole. In a preferred aspect, the aryl hydrocarbon receptor inhibitor is SRI . SRI can be present in the culture media in an amount from about 0.5 mM to about 2 mM; about 0.5 mM to about 1.5 mM; about 0.5 mM to about 1 mM; or about 0.75 mM to about 1.25 mM. In some aspects, SRI is present in the culture media at about 0.75 mM; about 1 mM or about 1.25 m.M. In a preferred aspect, SRI is present in the culture media at about 1.0 mM.

[0214] The pyrimido-indole derivative can be a pyrimido-[4,5-b]-indole derivative in a preferred aspect, the pyrimido-indole derivative inhibitor is UM171 and has the following

chemical structure: can be present in the culture rnedia in an amount from about 25 nM to about 75 nM; or about 25 nM to about 50 nM. In a preferred aspect, UM171 is present in the culture media at about 35 nM.

[0215] In some aspects, the culture media for increasing the frequency of transposition in a cell or plurality of cells as disclosed herein can include the non-limiting agents; valproic acid;

UNC0638; SRI; UM171 ; valproic acid and UNC0638; valproic acid and SRI; valproic acid and UM171; UNC0638 and SRI ; UNC0638 and UM171 ; SRI and UM171 ; valproic acid, UNC0638 and SRI; valproic acid, UNC0638 and UM171 ; UNC0638, SRI and UM171 ; valproic acid, SRI , UM171; or valproic acid, UNC0638, SRI and UM171. In a preferred aspect, the culture media for increasing the frequency of transposition m a cell or plurality ⁷ of cells as disclosed herein comprises a combination of VP A and UNC0638.

SUBSTITUTE SHEET (RULE 26) [0216] When the culture media comprises a combination VP A and UNC0638, the VP A is present from about 0.1 mM to about 2 mM; about 0.25 mM to about 1 mM; about 0.25 to about 0.75 mM; about 0.25 to about 0.5 mM or about 0.5 mM to about 0.75 mM and the UNC0638 is present from about 0.5 mM to about 2 mM; about 0.5 mM to about 1.5 mM; about 0.5 mM to about 1 mM; or about 0.75 mM to about 1.25 mM. In one aspect, the VP A is present in the culture media at about 0.5 mM or about 0.75 mM and UNC0638 is present in the culture media at about 0.75 mM; about 1 mM or about 1.25 mM. In a preferred aspect, the VTA is present in the culture media at about 0.5 mM or about 0.75 mM and UNC0638 is present m the culture media at about 1.0 mM for about 4 days to about 11 days.

[0217] The present disclosure provides a method of increasing the frequency of transposition in a population of cells, comprising: a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells in a culture media comprising valproic acid and UNC0638 for about 24 hours; c) removing the valproic acid from the culture media; and d) culturing the plurality of modified cells in a culture media comprising UNC0638 for about 3 days following step c, thereby increasing the frequency of transposition in a population of cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0218] The present disclosure provides a method of increasing the frequency of transposition in a population of cells, comprising: a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality' of modified cells in a culture media comprising valproic acid and UNC0638 for about 3 hours to about 30 hours; c) removing the valproic acid from the culture media; and d) culturing the plural ity of modified cells in a culture media comprising UNC0638 for about 1 day to about 5 days following step c, thereby increasing the frequency of transposition in a population of cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0219] The present disclosure provides a method of increasing the frequency of transposition in a population of cells, comprising: a) introducing a transposon or a sequence encoding the

SUBSTITUTE SHEET (RULE 26) transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells m a culture media comprising valproic acid and UNC0638 for about 24 hours; c) removing the valproic acid from the culture media; and d) culturing the plurality of modified cells in a culture media comprising UNC0638 for about 3 days following step c, wherein said culturing results in an increase in frequency of transposition and at least a one-fold increase in the yield of transposed cells in the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising valproic acid and UNC0638. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0220] The present disclosure provides a method of increasing the frequency of transposition in a population of cells, comprising: a) introducing a transposon or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality' of modified cells in a culture media comprising valproic acid and UNC0638 for about 3 hours to about 30 hours; c) removing the valproic acid from the culture media; and d) culturing the plurality of modified cells in a culture media comprising UNC0638 for about 1 day to about 5 days following step c, wherein said culturing results in an increase in frequency of transposition and at least a one-fold increase in the yield of transposed cells in the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising valproic acid and UNC0638. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0221] The present disclosure provides methods for increasing the frequency of transposition in a cell or population of cells and further comprises expanding that population of cells. The method of increasing the frequency of transposition in a cell or plurality of cells and expansion of a population of cells includes the expansion of a plurality of modified ceils. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0222] The present disclosure also provides methods for increasing the frequency of transposition in a cell or plurality of cells and further comprises expanding and selecting that population of cells. The method of increasing the frequency of transposition in a cell or plurality of cells and expansion of a population of cells includes the expansion and selection of a plurality

SUBSTITUTE SHEET (RULE 26) of modified cells. The selection of a population of cells includes the selection of a plurality of modified cells comprising a selection marker. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0223] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells in a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 24 hours; c) removing the HD AC inhibitor from the culture media; d) culturing the plurality of modified cells in a culture media comprising a HMT inhibitor for about 3 days following step c; e) removing the HMT inhibitor from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days following step e, wherein the expansion agent comprises an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimido-indole derivative, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0224] The present disclosure provides a method of expanding a population of modified ceils, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutem enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells in a culture media comprising valproic acid and UNC0638 for about 24 hours; c) removing the valproic acid from the culture media; d) culturing the plurality of modified cells in a culture media comprising UNC0638 for about 3 days following step c; e) removing the UNC0638 from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days following step e, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises SRI, UM171 and UNC0638, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

SUBSTITUTE SHEET (RULE 26) [0225] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells m a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 3 hours to about 30 hours; c) removing the HD AC inhibitor from the culture media; d) culturing the plurality of modified cells in a culture media comprising a HMT inhibitor for about 1 day to about 5 days following step c; e) removing the HMT inhibitor from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days following step e, wherein the expansion agent comprises an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimido-indole derivative, thereby expanding a population of modified cells. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0226] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutem enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells in a culture media comprising valproic acid and UNC0638 for about 3 hours to about 30 hours; c) removing the valproic acid from the culture media; d) culturing the plurality of modified cells in a culture media comprising UNC0638 for about 1 day to about 5 days following step c; e) removing the UNC0638 from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days following step e, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises SRI, UM171 and UNC0638, thereby expanding a population of modified cells.

[0227] The present disclosure provides a method of expanding a population of modified ceils, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality

SUBSTITUTE SHEET (RULE 26) of modified cells; b) culturing the plurality of modified cells in a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 24 hours; c) removing the HD AC inhibitor from the culture media; d) culturing the plurality of modified cells in a culture media comprising a HMT inhibitor for about 3 days following step c; e) removing the HMT inhibitor from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days following step e, wherein the expansion agent comprises an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrinudo-indole derivative, wdierein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0228] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells in a culture media comprising valproic acid and UNC0638 for about 24 hours; c) removing the valproic acid from the culture media; d) culturing the plurality of modified cells in a culture media comprising UNC0638 for about 3 days following step c; e) removing the UNCQ638 from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 7 days following step e, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises SRI, UM171 and UNC0638, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0229] The present disclosure provides a method of expanding a population of modified ceils, comprising: a) introducing a transposon comprising a selection gene or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality

SUBSTITUTE SHEET (RULE 26) of modified cells; b) culturing the plurality of modified cells in a culture media comprising a HD AC inhibitor and a HMT inhibitor for about 3 hours to about 30 hours; c) removing the HD AC inhibitor from the culture media; d) culturing the plurality of modified cells in a culture media comprising a HMT inhibitor for about 1 day to about 5 days following step c; e) removing the HMT inhibitor from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days following step e, wherein the expansion agent comprises an HMT inhibitor, a aryl hydrocarbon receptor inhibitor and a pyrimido-indole derivative, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent

[0230] The present disclosure provides a method of expanding a population of modified cells, comprising: a) introducing a transposon comprising a DHFR resistance gene or a transposon comprising a sequence encoding a DHFR mutein enzyme or a sequence encoding the transposon and a transposase or a sequence encoding the transposase under culture conditions sufficient for cell proliferation and for transposition into a plurality of cells to produce a plurality of modified cells; b) culturing the plurality of modified cells in a culture media comprising valproic acid and UNC0638 for about 3 hours to about 30 hours; c) removing the valproic acid from the culture media; d) culturing the plurality of modified cells in a culture media comprising UNC0638 for about 1 day to about 5 days following step c; e) removing the UNC0638 from the culture media; and f) culturing the plurality of modified cells in a culture media comprising a selection agent and an expansion agent for about 4 days to about 9 days following step e, wherein the selection agent comprises methotrexate or pralatrexate and wherein the expansion agent comprises SRI , UM171 and UNC0638, wherein said culturing results in at least two-fold expansion of the plurality of modified cells compared to a plurality of modified cells cultured under culture conditions not comprising a selection agent and expansion agent. Preferably, the cell is an HSC or the population of cells is a population of HSCs.

[0231] in the methods of the disclosure, the transposon can be integrated into the genome of the cell by the transposase. The integration can be transient or stable. The transposon or the sequence encoding the transposon can be comprised within a composition; the transposase or a sequence encoding a transposase can be comprised within a composition; or the transposon or

SUBSTITUTE SHEET (RULE 26) the sequence encoding the transposon and the transposase or a sequence encoding a transposase can he comprised within a composition.

[0232] The sequence encoding the transposase can comprise an amino acid or a nucleic acid sequence encoding a transposase protein. The nucleic acid sequence encoding a transposase protein can comprise an RNA sequence. The nucleic acid sequence encoding a transposase protein can comprise a DNA sequence.

[0233] The transposon can be a piggyBac ^® (PB) transposon, a piggy-Bac ^® like transposon, a piggyBat transposon, a Sleeping Beauty transposon, a Helraiser transposon, a Tol2 transposon or a TcBuster transposon. The transposase can be a piggyBac ^® transposase, a piggy-Bac ^® like transposase, a Super piggyBac ^® (SPB) transposase, a piggyBat transposase, a Sleeping Beauty transposase, a hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, a Tol2 transposase, a TcBuster transposase or a hyperactive TcBuster transposase. The Helitron transposase can be a Helibatl transposase.

[Q234] When the transposon is a piggyBac ^® transposon, the transposase can be a piggyBac ^® transposase or a Super piggyBac ^® transposase. When the transposon is a piggy-Bac ^® like transposon, the transposase can be a piggy-Bac ^® like transposase. When the transposon is a Sleeping Beauty transposon, the transposase can be a Sleeping Beauty transposase. When the transposon is a Helraiser transposon, the transposase can be a Helitron transposase. When the transposon is a Tol2 transposon, the transposase can be a Tol2 transposase. When the transposon is a TcBuster transposon, the transposase can be a TcBuster transposase or a hyperactive TcBuster transposase.

[0235] The piggyBac ^® transposase comprises, consists essential of, or consists of, an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to the amino acid sequence of SEQ ID NO: 14487. The piggyBac ^® transposase comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14487. The piggyBac ^® transposase comprises an ammo acid substitution at one or more of positions 30, 165, 282 or 538 of SEQ ID NO: 14487. The piggyBac ^® transposase comprises an ammo acid substitution at two or more of positions 30, 165, 282 or 538 of SEQ ID NO: 14487. The piggyBac ^® transposase comprises an amino acid substitution at three or more of positions 30, 165, 282 or 538 of SEQ ID NO: 14487. The piggyBac ^® transposase comprises an amino acid substitution at each of positions 30, 165, 282 or 538 of SEQ ID NO: 14487. The amino acid

SUBSTITUTE SHEET (RULE 26) substitution at position 30 of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I) (I30V). The amino acid substitution at position 165 of SEQ ID NO: 14487 is a substitution of a serine (S) for a glycine (G) (G165S). The ammo acid substitution at position 282 of SEQ ID NO: 14487 is a substitution of a valine (V) for a methionine (M) (M282V). The ammo acid substitution at position 538 of SEQ ID NO: 14487 is a substitution of a lysine (K) for an asparagine (N) (N538K).

[0236] The piggyBac ^® transposase comprises, consists essential of, or consists of an amino acid substitution at each of positions 30, 165, 282 or 538 of SEQ ID NO: 14487. The Super piggyBac ^® transposase comprises, consists essential of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to the amino acid sequence of SEQ ID NO: 14484. The Super piggyBac ^® transposase comprises, consists essential of, or consists of, the amino acid sequence SEQ ID NO: 14484.

[0237] The TcBuster transposase comprises, consists essential of, or consists of, an ammo acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to the amino acid sequence of SEQ ID NO: 14735. The TcBuster transposase comprises, consists essential of, or consists of, the ammo acid sequence of SEQ ID NO: 14735.

[0238] The piggyBat transposase comprises, consists essential of, or consists of, an ammo acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to the amino acid sequence of SEQ ID NO: 14965. The piggyBat transposase comprises, consists essential of, or consists of, an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to the amino acid sequence of SEQ ID NO: 14967. The piggyBat transposase comprises, consists essential of, or consists of, an ammo acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage m between identical to the ammo acid sequence of SEQ ID NO: 14968. The nucleic acid encoding the piggyBat transposase comprises, consists essential of, or consists of, an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage m between identical to the ammo acid sequence of SEQ ID NO: 14966. The nucleic acid encoding the piggyBat transposon composes, consists essential of, or consists of, an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage m between identical to the ammo acid sequence of SEQ ID NO: 14963. The nucleic acid encoding the piggyBat transposon comprises, consists essential of, or consists of, an amino acid sequence

SUBSTITUTE SHEET (RULE 26) at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to the amino acid sequence of SEQ ID NO: 14964.

[0239] Transposons and transposes suitable for use in the compositions and methods of the present disclosure are described in more detail herein.

[0240] The transposon can comprise, consist essential of, or consist of at least one exogenous sequence (transgene sequence). The transposon can comprise, consist essential of, or consist of at least two exogenous sequences (transgene sequences). The transposon can comprise, consist essential of, or consist of at least three exogenous sequences (transgene sequences). The transposon can comprise, consist essential of, or consist of at least four exogenous sequences (transgene sequences). The transposon can comprise, consist essential of, or consist of at least five exogenous sequences (transgene sequences).

[0241] The exogenous sequence can comprise, consist essential of, or consist of a sequence encoding a chimeric antigen receptor (CAE).

[Q242] The exogenous sequence can comprise, consist essential of, or consist of a sequence encoding a therapeutic agent. The therapeutic agent can be a therapeutic protein. The therapeutic agent can be a therapeutic RNA. The therapeutic RNA can be iRNA, siRNA, or shRNA

[0243] The therapeutic agent can be human beta-globin (HBB), human beta-globin comprising a T87Q mutation (HBB T87Q), BAF chromatin remodeling complex subunit (BCL1 1 A) shRNA, insulin like growth factor 2 binding protein 1 (IGF2BP1), interleukin 2 receptor gamma

(IL2RG), alpha gaiactosidase A (GLA), alpha-L-idurondase (IDUA), iduronate 2-sulfatase (IDS), cystinosin lysosomal cysteine transporter (CTNS).

[0244] The exogenous sequence can comprise, consist essential of, or consist of a sequence encoding a selection marker. A selection marker is a gene introduced into a cell, especially a cell in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used to indicate the success of a transposition, transduction, transfection or other procedure meant to introduce exogenous or foreign DNA into a cell. A non-limiting example of a selectable marker is an antibiotic resistance gene (an antibiotic resistance marker is a gene that produces a protein that provides ceils expressing this protein with resistance to an antibiotic). The selection marker can be a DHFR resistance gene. The selection marker can be a sequence encoding a DHFR mutein enzyme. The DHFR mutein enzyme can comprise, consist essentially of, or

SUBSTITUTE SHEET (RULE 26) consist of the ammo acid sequence of SEQ ID NO: 14677. The DHFR mutein enzyme is encoded by a nucleic acid sequence comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 14678. The amino acid sequence of the DHFR mutein enzyme can further comprises a mutation at one or more of positions 80, 113, or 153. In some embodiments, the amino acid sequence of the DHFR mutein enzyme comprises one or more of a substitution of a Phenylalanine (F) or a Leucine (L) at position 80, a substitution of a Leucine (L) or a Valine (V) at position 113, and a substitution of a Valine (V) or an Aspartic Acid (D) at position 153.

[0245] The exogenous sequence can comprise, consist essential of, or consist of a sequence encoding an inducible proapoptotic polypeptide. The inducible proapoptotic polypeptide can be an inducible caspase polypeptide. The inducible proapoptotic polypeptide can comprise, consist essential of, or consist of (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible caspase polypeptide does not comprise a non-human sequence. In a one aspect, the non-human sequence is a restriction site.

[0246] In a one aspect, the ligand binding region inducible caspase polypeptide comprises a FK506 binding protein 12 (FKBP12) polypeptide. The amino acid sequence of the FK506 binding protein 12 (FKBP12) polypeptide can comprise, consist essential of, or consist of a modification at position 36 of the sequence. The modification can be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). The FKBP12 polypeptide comprises, consists essential of, or consists of, the ammo acid sequence of SEQ ID NO: 14494. The FKBP12 polypeptide can be encoded by a nucleic acid sequence comprising, consisting essential of, or consisting of, the nucleic acid sequence of SEQ ID NO: 14495.

[0247] The linker region of the inducible proapoptotic polypeptide comprises, consists essential of, or consists of, the ammo acid sequence of SEQ ID NO: 14496. The linker region of the inducible proapoptotic polypeptide can be encoded by a nucleic acid sequence comprising, consisting essential of, or consisting of, the nucleic acid sequence of SEQ ID NO: 14497.

[0248] The truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence. The truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 of the sequence. The truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide

SUBSTITUTE SHEET (RULE 26) comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14498. The truncated caspase 9 polypeptide of the inducible proapoptotic polypeptide can he encoded by a nucleic acid sequence comprising, consisting essential of, or consisting of, the nucleic acid sequence of SEQ ID NO: 14499.

[0249] The inducible proapoptotic polypeptide comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14503. The inducible proapoptotic polypeptide can be encoded by a nucleic acid sequence comprising, consisting essential of, or consisting of, the nucleic acid sequence of SEQ ID NO: 14636.

[0250] The transposon or the exogenous sequence can further comprise, consist essential of, or consist of at least one sequence encoding a self-cleavmg peptide. The at least one self-cleaving peptide can comprise, consist essential of, or consist of T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG- P2A peptide. The GSG-T2A peptide comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14638. The T2A peptide comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14637. The E2A peptide comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14639. The GSG- E2A peptide comprises, consists essential of, or consists of, the ammo acid sequence of SEQ ID NO: 14640. The F2A peptide comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14641. The GSG-F2A peptide comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14642 The P2A peptide comprises, consists essential of, or consists of, the amino acid sequence of SEQ ID NO: 14643. The GSG- P2A peptide comprises, consists essential of, or consists of, the ammo acid sequence of SEQ ID NO: 14644

[0251] The miRE sh49 BCL1 1 A of the disclosure is encoded by a sequence comprising:

gtacaagtaatagggccctacgagcttgctgtttgaatgaggcttcagtactttaca gaatcgttgcctgcacatcttggaaacacttgctggg attacttcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtga gcgccgcacagaacactcatggatttagtgaag ccacagatgtaaatccatgagtgttctgtgcgttgcctactgcctcggacttcaaggggc tacaattggagcaattatcttgtttactaaaactg

[0252] The HBB T87Q of the disclosure comprises an ammo acid sequence comprising SEQ ID NO: 14934. The GLA of the disclosure comprises an amino acid sequence comprising any one of SEQ ID NO: 5974 - SEQ ID NO: 5975. The IDUA of the disclosure comprises an amino

5UB5TITUTE SHEET (RULE 26) acid sequence comprising any one of SEQ ID NO: 6715 - SEQ ID NO: 6720. The IDS of the disclosure comprises an ammo acid sequence comprising any one of SEQ ID NO: 6709 - SEQ ID NO: 6714. The CTNS of the disclosure comprises an amino acid sequence comprising any ^¬ one of SEQ ID NO: 3672 - SEQ ID NO: 3679.

[0253] The IGF2BP1 of the disclosure is encoded by a nucleic acid sequence comprising SEQ ID NO: 14721.

The IGF2BP1 of the disclosure comprises an ammo acid sequence comprising SEQ ID NO: 14722.

The IL2RG of the disclosure is encoded by a nucleic acid sequence comprising SEQ ID NO: 14718.

The IL2RG of the disclosure comprises an amino acid sequence comprising SEQ ID NO: 14723.

[0254] Exogenous sequences and transgenes suitable for use in the compositions and methods of the present disclosure are described in more detail herein.

[0255] The present disclosure provides a population of modified cells produced by any of the methods disclosed herein. The present disclosure also provides a composition comprising, consisting essential of, or consisting of a population of modified cells produced by any of the methods disclosed herein. The present disclosure also provides a pharmaceutical composition comprising, consisting essential of, or consisting of a population of modified cells produced by any of the methods disclosed herein and a pharmaceutically-acceptable carrier

[0256] The present disclosure provides a modified cell population wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99 5%, at least 99.9% or 100% of the plurality of modified cells in the population comprise a genome-integrated transposon. The present disclosure also provides a composition comprising, consisting essential of, or consisting of a modified cell population wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of modified cells in the population comprise a genome-integrated transposon. The present disclosure also provides a pharmaceutical composition comprising, consisting essential of, or consisting of a modified cell population wherein at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% of the plurality of modified cells in the population comprise

SUBSTITUTE SHEET (RULE 26) a genome-integrated transposon and a pharmaceutically-acceptable earner. The genome- integrated transposon can comprise at least one of, at least two of, at least three of, at least four of, at least five of a sequence comprising a selection marker, a sequence encoding a chimeric antigen receptor (CAR), a sequence encoding a therapeutic agent, a sequence encoding an inducible proapoptotic polypeptide or a sequence encoding a self-cleaving peptide.

[0257] The population of cells or plurality of cells can comprise, consist essential of, or consist of somatic cells, germlme cells, stem cells, or a combination thereof. A cell in the population of cells or plurality of cells can be a somatic cell, a germline cell or a stem cell.

[0258] The stem cells within the population of stem cells or plurality of stem cells can be induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), embryonic stem cells, adult tissue stem cells, or a combination thereof. A stem cell within the population of stem cells or plurality of stem cells can be an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), an embryonic stem cell or an adult tissue stem cell.

[Q259] The population of cells or plurality of cells can comprise, consist essential of, or consist of mammalian cells. A cell in the population of cells or plurality of cells can be a mammalian cell. The population of cells or plurality of cells can comprise, consist essential of or consist of human cells, non-human cells, or a combination thereof. A cell in the population of cells or plurality of cells can be a human cell. A cell in the population of cells or plurality of cells can be a non-human cell (not a human cell). Preferably, the population of cells or plurality of cells can comprise, consist essential of, or consist of human cells. Preferably, a cell in the population of cells or plurality of cells is a human cell.

[0260] The population of cells or plurality of cells can comprise, consist essential of, or consist of autologous cells. A cell in the population of cells or plurality of cells can be an autologous cell. The population of cells or plurality of cells can comprise, consist essential of, or consist of allogeneic cells. A cell in the population of cells or plurality of cells can be an allogeneic cell.

[0261] The population of cells or plurality of cells can be in vivo, ex vivo, in vitro or m situ. A cell in the population of cells or plurality of cells can be in vivo, ex vivo, in vitro or in situ.

[0262] The population of cells or plurality of cells can comprise, consist essential of, or consist of immune cells, neural cells, endothelial cells, epithelial cells, muscle cells, bone cells, hematopoeitic cells, or any combination thereof. A cell in the population of cells or plurality of cells can be an immune cell. A cell in the population of cells or plurality of cells can be a neural

SUBSTITUTE SHEET (RULE 26) cell. A cell in the population of cells or plurality of cells can be an endothelial cell. A cell in the population of cells or plurality of cells can be an epithelial cell. A cell in the population of cells or plurality of ceils can be a muscle cell. A cell in the population of ceils or plurality of ceils can be a bone ceil. A cell m the population of cells or plurality of cells can be hematopoetic cell.

[0263] The immune cells within the population of immune cells or plurality of immune cells can be T-cells, Natural Killer (NK) cells, Natural Killer (NK)-like cells, hematopoeitic progenitor cells, or B-celis. An immune cell in the population of immune cells or plurality of immune cells can be a T-cell, a Natural Killer (NK) cell, a Natural Killer (NK)-like cell, a hematopoeitic progenitor cell, or a B-cell. The T-cell can be a stem memory T-cell (TSCM), a TscM-like cell, a peripheral blood (PB) derived T cell, an umbilical cord blood (UBC) derived T- ce!i, a helper T-cell, a cytotoxic T-cell, a regulator}' T-cell or a gd T-cell.

[0264] A hematopoeitic stem cell (HSC) or HSC descendent cell can be isolated, derived or obtained from the peripheral blood, the umbilical cord blood, the bone marrow} a peritoneal dialysis effluent, an adult stem cell, or an induced pluripotent stem cell (iPSC) of a subject. The peripheral blood can comprise mobilized peripheral blood of a subject.

[Q265] The cells in a population of cells or a plurality of cells can be isolated from, derived from or obtained from a healthy subject. The cells in a population of cells or a plurality of cells cannot be isolated from, derived from or obtained from a healthy subject. The cells in a population of cells or a plurality of cells cannot be isolated from, derived from or obtained from a non-healthy subject. The cells in a population of cells or a plurality of cells cannot be isolated from, derived from or obtained from a subject with a disease or disorder. In one aspect, the disease or disorder is a proliferation disorder. In one aspect, the proliferation disorder is cancer.

[0266] The cells in a population of cells or a plurality of cells isolated from, derived from or obtained from a non-healthy subject can be tumor cells or cancer cells. The cells in a population of cells or a plurality of cells isolated from, derived from or obtained from a non-healthy subject can comprise, consist essential of, or consist of a genetic or epigenetic marker of a disease or disorder. In one aspect, the genetic or epigenetic marker causes the disease or disorder. In one aspect, the genetic or epigenetic marker predicts a risk of occurrence, a severity , or a prognosis of the disease or disorder. In one aspect, the disease or disorder is a proliferation disorder. In one aspect, the proliferation disorder is cancer.

SUBSTITUTE SHEET (RULE 26) [0267] Cells and/or populations of cells suitable for use in the compositions and methods of the present disclosure are described in more detail herein.

[0268] The transgene or transposon can be introduced to the cell or the plurality of cells by any means known in the art. The transposon can be introduced to the cell or the plurality of cells via nucieofection or electroporation. Conditions sufficient for transposition comprise a

nucieofection or an electroporation.

[0269] The nucieofection or the electroporation can comprise at least one of an application of one or more pulses of electricity at a specified voltage, a buffer, and one or more supplemental factor(s). The buffer can comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucieofection buffer or any combination thereof. The one or more supplemental factor(s) comprise: (a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof; (b) a salt, a mineral, a metabolite or any combination thereof; (c) a cell medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and/or (e) a reagent that modifies or stabilizes one or more nucleic acids.

[0270] The recombinant human cytokine, the chemokine, the interleukin or any combination thereof can comprise, consist essential of, or consist of IL2, IL7, IL12, IL15, IL21 , IL1 , IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, ILl 1 , IL13, IL14, IL16, IL17, IL18, IL19, IL2G, IL22, IL23, 11,25, IL26, IL27, IL28, IL29, IL30, IL31, 11,32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha/TL-IFl , IL-1 beta/IL-I F2, IL-12 p70, IL-12/IL-35 p35, IL-13, 11,-17/11,-17,4, IL-17A/F Heterodimer, IL-17F, IL-18/IL-1F4, IL-23, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1 ), Lymphotoxin-alpha/TNF-beta, TGF-beta, TNF-alpha,

TRANCE/TNFSFl 1/RANK L, or any combination thereof.

[0271] The salt, the mineral, the metabolite or any combination thereof can comprise, consist essential of, or consist of HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS/FCS, Human serum, serum-substitute, anti-biotics, pH adjusters, Earle’s Salts, 2-MercaptoethanoJ, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgClz, NazHPOr, NAH2PO4, Sodium lactobionate, Mamtol, Sodium succinate, Sodmm

Chloride, CINa, Glucose, Ca(N03j2, Tris/HCl, K2HPO4, KH2PO4, Polyethylemmme, Poly-

SUBSTITUTE SHEET (RULE 26) ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone,

Pop313, Crown- 5, or any combination thereof.

[0272] The cell medium can comprise, consist essential of, or consist of PBS, BBSS,

OptiMEM, DMEM, RPM1 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CIS OpTimizer T Ceil Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium,

ImmunoCult-XF T Cell Expansion Medium or any combination thereof.

[0273] The inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof can comprise, consist essential of, or consist of inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1

Interferons, pro- inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG- 1, IPS-1, FADD, RIP I, TRAF3, AIM2, ASC, Caspasel, Pro-11. I B. PI3K, Akt, Wnt3A, glycogen synthase kinase-3 b (GSK-3 b), TWS119, Bafilomycin, Chloroquine, Quinacrine, AC-YVAD- CMK, Z-VAD-FMK, Z-IETD-FMK or any combination thereof.

[Q274] The reagent that modifies or stabilizes one or more nucleic acids can comprise, consist essential of, or consist of a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPOy a net neutral charge DNA binding peptide with or without a NLS sequence, a TREX1 enzyme or any combination thereof.

[0275] Methods of introducing exogenous sequences into cells and/or populations of cells, including nucleofection and electroporation, suitable for use in the compositions and methods of the present disclosure are described in more detail herein.

[0276] The disclosure provides methods of transplantation comprising transplanting a therapeutically effective amount of a population of cells of the disclosure, a composition comprising a population of cells of the disclosure or a pharmaceutical composition comprising a population of cells of the disclosure to a subject in need thereof. The disclosure also provides a population of cells of the disclosure or a pharmaceutical composition comprising a population of cells of the disclosure for use in transplantation.

[0277] The disclosure provides methods of treating a subject for a disease or disorder, comprising administering to the subject a therapeutically effective amount of a population of cells of the disclosure, a composition comprising a population of ceils of the disclosure or a pharmaceutical composition comprising a population of ceils of the disclosure. The disclosure

SUBSTITUTE SHEET (RULE 26) also provides a population of cells of the disclosure or a pharmaceutical composition comprising a population of cells of the disclosure for use in treating a disease or disorder.

[0278] The disclosure provides methods of treating a subject for a disease or disorder, comprising administering to the subject a therapeutically effective amount of a population of modified cells of the disclosure, a composition comprising a population of modified cells of the disclosure or a pharmaceutical composition comprising a population of modified ceils of the disclosure. The disclosure also provides a population of modified ceils of the disclosure or a pharmaceutical composition comprising a population of modified ceils of the disclosure for use in treating a disease or disorder.

[Q279] The population of cells of the disclosure, the composition comprising the population of cells of the disclosure or the pharmaceutical composition comprising the population of cells of the di sclosure comprise a plurality of autologous cells. The population of cells of the disclosure, the composition comprising the populati on of cells of the disclosure or the pharmaceutical composition compri sing the population of cells of the disclosure comprise a plurality of allogeneic cells.

[Q28Q] The methods of treating a disease or disorder, methods of transplantation or methods of adoptive immunotherapy described herein can further comprise, consist essentially of or consist of administering a myeioablative agent. The myeloablative agent comprises low dose and/or local irradiation. The myeloablative agent comprises busulphan, treosulphan, meiphalan, thiotepa or a combination thereof. The myeloablative agent can be administered prior to, after or concurrently with administration with the pharmaceutical compositions comprising the modified HSCs of the present invention.

[0281] The methods of treating a disease or disorder, methods of transplantation or methods of adoptive immunotherapy described herein can further comprise, consist essentially of or consist of administering an activating composition to induce or increase proliferation of the plurality modified HSCs in vivo. The activating composition can be administered prior to, after or concurrently with administration with the pharmaceutical compositions comprising the modified HSCs of the present invention.

[0282] A therapeutically -effective amount can be a single dose. The therapeutically-effective amount can be a single dose over a lifetime of the subject.

SUBSTITUTE SHEET (RULE 26) [0283] The compositions of the disclosure can be used to treat a disease or disorder including, but not limited to: Osteopetrosis, Parkinson’s Disease, Hunter Syndrome, Sickle Cell Disease, Severe Combined Immunodeficiency, Alpha-mannosidosis, Sideroblastic anemia, Autosomal Recessive Hyper IgE Syndrome, Primary Myelofibrosis, Cutaneous vasculitis, X-linked protoporphyria, Fucosidosis, Maroteaux Lamy syndrome, WAS Related Disorders, Chronic Granulomatous, Thalassemia Major, Hereditary Angioedema, Hereditary' Lymphedema, Hyper IgM Syndrome, Friedrich’s Ataxia, Charcot Marie Tooth Disease, Phenylketonuria,

Methylmalonic Acidemia, Adrenoleukodystrophy, Kugelberg Welander Syndrome, Retinitis Pigmentosa, Hydrocephalus, Hereditary Sensory- and Autonomic Neuropathy Type IV,

Mucopolysaccharidosis Type III, Corneal Dystrophies, Erythropoietic Protoporphyria, Fabry Disease, Werdnig-Hoffman Disease, Hypoposphatasia, Coats Disease, Fan coni Anemia, Niemann Pick Disease, Crigler-Najjar Syndrome, Hemophilia A, Hemophilia B,

Leukodystrophy, Sandhoff Disease, Usher Syndrome, Wolman Disease, Dupuytren’s

Contracture, Wolfram Syndrome, X-Linked Myotubular Myopathy, Canavan Disease, Ehler’s Danios Syndrome, Epidermolysis Bullosa, Osteogenesis Imperfecta, Short Bowel Syndrome, Giant Axonal Neuropathy, Paroxysmal Nocturnal Hemoglobinuria, Phelan-McDermid

Syndrome, Retinoschisis, Beta-Thalassemia, Hypophosphatasia, Propionic Acidemia,

Cholesteryl Ester Storage Disease, Cystmosis, Glycogen Storage Disease Type II Pornpe Disease, Mucopolysaccharidoses (MPS I H-S Hurler-Scheie), Mucopolysaccharidoses (Type II (Hunter syndrome)), and Mucopolysaccharidoses (Type IV (Morquio))

[0284] The compositions of the disclosure may be used to treat a disease or disorder by use of a therapeutic transgene encoding for an exogenous nucleic acid sequence or exogenous ammo acid sequence. For certain diseases or disorders the therapeutic transgene can include [Disease (therapeutic transge): Beta-Thalassemia (HBB T87Q, BCL11A shRNA, 1GF2BP1), Sickle Cell Disease (HBB T87Q, BCL1 1 A shRNA, IGF2BP1), Hemophilia A (Factor VIII), Hemophilia B (Factor IX), X-linked Severe Combined Immunodeficiency (Interleukin 2 receptor gamma (IL2RG)), Hypophosphatasia (Tissue Non-specific Alkaline Phosphatase (TNAP)),

Osteopetrosis (TCIRG1), Glycogen Storage Disease Type II (Pornpe Disease) (Alpha

Glueosidase (GAA)), A!pha-Galactosidase A Deficiency (Fabry disease) (Alpha-galactosidase A (GLA)), Mucopolysaccharidosis Type I (MPS I) (Alpha-L-iduronidase (IDIJA)),

Mucopolysaccharidosis Type II (NO’S II) (Iduronate 2-suIfatase (IDS)), Mucopolysaccharidosis

SUBSTITUTE SHEET (RULE 26) Type IIIA (MPS IIIA) (sulfoglycosamine-sulfohydrolase (SGSH)), Mucopolysaccharidosis Type MB (MPS DIB) (N-alpha-acetylglucosaminidase (NAGLU)), Mucopolysaccharidosis Type IV A (MPS IVA) (Morquio) (N-acetylgalactosamine-6-sulfate sulfatase (GALNS)),

Mucopolysaccharidosis Type IV B (MPS 1VB) Beta-galactosidase (GLB1 (Beta-galactosidase (GLB1)), Cholesteryl Ester Storage Disease (CESD) (Lysosomal acid lipase (LIP A)), Cystinosis (Cystinosin lysosomal cystine transporter (CTOS)), X-iinked chronic granulomatous disease (X- CGD) (CYBB), Wiskott-Aldrich Syndrome (WAS) (WAS), X-linked Adrenoleukodystrophy (X- ALD) (ABCD1), Metachromatic leukopdystrophy (MLD) (ARSA), Phenylketonuria (PAH), Methylmalonic academia (MMUT), Propionic Acidemia (PCCA, PCCB), Retinitis Pigmentosa (RPE65), Usher Syndrome (MY07A), and Gaucher Disease (GBA).

[0285] Administration of the population of cells can be systemic. Administration of the population of cells can be intravenous, local, mtra-tumorai, intraspinal, intracerebroventricular, intraocular or intraosseous. In some aspects, the administration is direct to the cerebral spinal fluid (CSF).

[0286] Methods of using the compositions and methods of the present disclosure are described in more detail herein.

[0287] A modified cell is a genetically modified cell. A modified HSC is a genetically modified HSC,

[0288] The transgene can comprise a selection gene, a therapeutic gene, an inducible proapoptotic gene, or a combination thereof. The transgene can comprise a sequence encoding a selection marker, a sequence encoding a therapeutic agent, a sequence encoding an inducible proapoptotic polypeptide, or a combination thereof. A modified HSC or a plurality of modified HSCs can comprise a sequence encoding a selection marker, a sequence encoding a therapeutic agent, a sequence encoding an inducible proapoptotic polypeptide, or a combination thereof.

[0289] The transgene can encode a selection marker. The transgene can comprise a sequence that facilitates positive selection of the modified HSC or a plurality of modified HSCs. The transgene can encode a protein that facilitates positive selection of the modified HSC or a plurality of modified HSCs. The transgene can comprise a protein that is active in dividing cells and not active m non-dividing ceils. The transgene can encode a protein that is active m dividing cells and not active in non-dividing cells. The transgene can comprise a metabolic marker. The transgene can encode a metabolic marker. The transgene sequence can comprise a mutation,

SUBSTITUTE SHEET (RULE 26) wherein the mutation facilitates positive selection of the modified HSC or plurality of modified HSCs. The transgene can comprise a DHFR resistance gene. The transgene can comprise a DKFR mutein enzyme. The DKFR mutein enzyme can comprise the amino acid sequence of SEQ ID NO: 14475. The amino acid sequence of the DHFR mutein enzyme can comprise a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme can comprise a substitution of a Phenylalanine (F) or a Leucine (L) at position 80. The amino acid sequence of the DHFR mutein enzyme can comprise a substitution of a Leucine (L) or a Valine (V) at position 113. The amino acid sequence of the DHFR mutein enzyme can comprise a substitution of a Valine (V) or an Aspartic Acid (D) at position 153.

[0290] The transgene can comprise a sequence encoding a therapeutic agent or protein and a promoter sequence encoding a promoter capable of expressing the therapeutic protein in an HSC or a plurality of HSCs. The modified HSC or the plurality of modified HSCs can further comprise a sequence encoding a therapeutic agent or protein and a promoter sequence encoding a promoter capable of expressing the therapeutic protein in an HSC or in a plurality of HSCs.

The therapeutic protein can be expressed and secreted from a modified HSC or a plurality of modified HSCs. The therapeutic protein can be expressed and not secreted from a modified HSC or a plurality of modified HSCs.

[0291] The therapeutic protein can comprise a human beta globm. The human beta globin can comprise a substitution of Glutamine (Q) for Threonine (T) at position 87 The human beta globin can comprise the ammo acid sequence of SEQ ID NO: 14477 The human beta globm comprises a sequence encoded by the nucleic acid sequence of SEQ ID NO: 14478. The promoter can be a constitutive promoter, an inducible promoter or a conditional promoter. The promoter can be a cell type specific or a tissue specific promoter. The promoter can be an erythroid promoter.

[0292] The transgene can comprise a sequence encoding an inducible proapoptotic

polypeptide. The inducible proapoptotic polypeptide can comprise (a) a ligand binding region,

(b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible caspase polypeptide does not comprise a non-human sequence. The transgene can comprise a cell-surface protein.

The transgene can encode a cell-surface protein. The cell-surface protein can be a detectable label. The detectable label can be a fluorophore.

SUBSTITUTE SHEET (RULE 26) [0293] The introduction of the transgene can modify an endogenous sequence of the HSC or the plurality of HSCs, wherein the modification comprises at least one of an insertion, a deletion, a substitution, an inversion, a transposition, a frames hift, or a translocation of a nucleic acid or an amino acid encoded by the nucleic acid. The introduction of the transgene can modify an endogenous sequence of the HSC or the plurality' of HSCs, wherein the modification comprises an insertion. The insertion can comprise the transgene. The transgene can facilitate positive selection of the modified HSC or the plurality of modified HSCs. The introduction of the transgene can modify an endogenous sequence encoding a DHFR enzyme of the HSC or the plurality of HSCs, wherein the DHFR enzyme is modified to comprise the amino acid sequence of SEQ ID NO: 14476. The amino acid sequence can further comprise a mutation at one or more of positions 80, 113, or 153. The amino acid sequence can comprise a substitution of a Phenylalanine (F) or a Leucine (L) at position 80. The amino acid sequence can comprise a substitution of a Leucine (L) or a Valine (V) at position 1 13. The amino acid sequence can comprise a substitution of a Valme (V) or an Aspartic Acid (D) at position 153.

[0294] The HSC or the plurality of HSCs can be modified by any means known in the art. The transgene can be introduced into an HSC or a plurality' of HSCs using any means known in the art.

[0295] The HSC or the plurality of HSCs can be modified using a gene editing composition. The transgene can be introduced into an HSC or a plurality of HSCs using a gene editing composition. The gene editing composition can comprise a dCas9-Clo051 fusion protein. The HSC or the plurality of HSCs can be modified by homologous recombination or by non- homologous recombination. The gene editing composition comprises a nuclease protein or a nuclease domain thereof. The gene editing composition comprises a sequence encoding a nuclease protein or a sequence encoding a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof comprises a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or the nuclease domain thereof comprises one or more of CRISPR/Cas protein, a nuclease- inactivated Cas (dCas) protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZEN), and an endonuclease. The nuclease or the nuclease domain thereof comprises a nuclease- inactivated Cas (dCas) protein and an endonuclease. The nuclease or the nuclease domain thereof comprises a nuclease-inactivated Cas9 (dCas9) protein and an endonuclease, wherein the

SUBSTITUTE SHEET (RULE 26) endonuclease comprises a CIo05l nuclease or a nuclease domain thereof. The gene editing composition comprises a fusion protein. The fusion protein comprises a nuclease-inactivated Cas9 (dCas9) protein and a Cfo051 nuclease or a C!oQ51 nuclease domain. The gene editing composition further comprises a guide sequence. The guide sequence comprises an RNA sequence. The fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 14479. The fusion protein is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 14480. The fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 14481. Thefusion protein is encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 14482.

[0296] A transgene can be introduced into the HSC or the plurality ofHSCs by a transposon comprising the transgene or a sequence encoding the transgene and a transposase or a sequence encoding the transposase. The transposon can be integrated into the genome of at least one HSC or a plurality of HSCs by a transposase. The transposon can be stably integrated or transiently integrated.

[0297] A hematopoeitic stem cell (HSC) or HSC descendent cell can be isolated, derived or obtained from the peripheral blood, the umbilical cord blood, the bone marrow, a peritoneal dialysis effluent, an adult stem cell, or an induced pluripotent stem cell (iPSC) of a subject. The peripheral blood can comprise mobilized peripheral blood of a subject. The subject can be a mammal. The subject can be a human subject or non-human subject.

[0298] An HSC or a plurality of HSCs can comprise an edited genome. The edited genome comprises a sequence having at least one substitution, insertion, deletion, inversion,

transposition, frameshift or a combination thereof. A sequence of the genome of an HSC or a plurality of HSCs is edited. The sequence of the genome can be edited to correct a genetic defect or to improve a function or an activity of an endogenous gene or a product of the endogenous gene. The genetic defect can be a genetic defect that results m beta-thalassemia or sickle-cell anemia. The endogenous gene can comprise human beta globm.

[0299] Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood ceils from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood and umbilical cord blood.

SUBSTITUTE SHEET (RULE 26) [0300] HSCs of the disclosure may be isolated or derived from a primary or cultured stem cell. HSCs of the disclosure may be isolated or derived from an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced p!uripotent stem cell fiPSC).

[0301] HSCs produced by the methods of the disclosure may retain features of“primitive” stem cells that, while isolated or derived from an adult stem cell and while committed to a single lineage, share characteristics of embryonic stem cells. For example, the“primitive” HSCs produced by the methods of the disclosure retain their“sternness” following division and do not differentiate. Consequently, as an adoptive cell therapy, the“primitive” HSCs produced by the methods of the disclosure not only replenish their numbers, but expand m vivo,“primitive”

HSCs produced by the methods of the disclosure may be therapeutically-effective when administered as a single dose.

[Q3Q2] The compositions and methods of the disclosure further provide for the administration of an enriched plurality of HSCs. At least one HSC of the enriched plural ity of HSCs is modified to comprise a DHFR gene or a sequence encoding a DHFR gene that confers resistance to methotrexate (MTX). In some embodiments, each HSC of the enriched plurality' of HSCs is modified to comprise a DHFR gene or a sequence encoding a DHFR gene that confers resistance to methotrexate (MTX). HSCs of the disclosure may be pre-modified by genomic editing or further modified by gene editing. HSCs of the disclosure may be engineered to express a therapeutic protein, which, may be secreted into the recipient’s blood stream, bone marrow, or specific stem cell niche.

[0303] Pharmaceutical compositions of the disclosure comprise HSCs produced by the methods of the disclosure. HSCs produced by the methods of the disclosure may be referred to herein as“therapeutic HSCs.” Therapeutic HSCs comprise autologous or allogeneic HSCs for use as an adoptive cell therapy. Therapeutic HSCs or a plurality of therapeutic HSCs comprise or consist of a modified HSC or a plurality of modified HSCs that are expanded by contacting the HSC expansion compositions of the disclosure.

[0304] Therapeutic HSCs may be isolated or derived from any human source.

[0305] Therapeutic HSCs may be genetically modified to express or to secrete one or more ions, small molecules, peptides, or proteins to affect the activity of another cell or cell type (e.g. a cancer cell, a stem cell or progenitor cell (an osteoclast, a mesenchymal stem cell, a neural progenitor ceil or glial cell), or an immune cell) or to condition a particular biological niche or

SUBSTITUTE SHEET (RULE 26) microenvironment (an extracellular matrix, an injury site, a stem cell niche) to create more favorable conditions for engraftment of the therapeutic HSCs.

[0306] Therapeutic HSCs may be genetically modified to contain an inducible proapoptotic polypeptide of the disclosure (i.e. a safety switch) m the event that, for example, that one or more of the therapeutic HSCs is incompatible with the subject’s immune system or undergoes a malignant transformation. In some embodiments, therapeutic HSCs are administered to a subject to tolerize the subject’s immune system to a subsequent transplant of a cell, tissue, graft or organ derived from the same donor as the therapeutic HSCs. Once therapeutic HSCs tolerize the subject’s immune system, the immune system will be hyporeactive to the subsequent transplant and should not reject the subsequent transplant.

[0307] In some embodiments of the disclosure, an HSC of the disclosure is isolated or derived from a mammal. In some embodiments, an HSC of the disclosure is isolated or derived from a human.

[0308] In some embodiments, an HSC of the disclosure is isolated or derived from an individual of any combination of X and Y chromosomes. In some embodiments, an HSC of the disclosure is isolated or derived from an individual having XX chromosomes. In some embodiments, an HSC of the disclosure is isolated or derived from an individual having XY chromosomes.

[0309] In some embodiments, an HSC of the disclosure is allogeneic with respect to an intended subject of a cell therapy comprising the HSC. In some embodiments, an HSC of the disclosure is autologous with respect to a donor of the HSC and/or an intended subject of a cell therapy comprising the HSC.

[0310] In some embodiments, an HSC of the disclosure is isolated or derived from a healthy donor. In some embodiments, an HSC isolated or derived from a healthy donor does not comprise a genetic or epigenetic marker or determinant of a disease, including, but not limited to a blood-borne disease, a disease affecting bone marrow or blood cells, a clotting or

cardiovascular disorder, an inflammatory disorder, a proliferative disorder, a cancer (liquid or solid), or an infectious disease (e.g. a viral or microbial disorder communicable through contact with bone marrow or blood cells). In some embodiments, a heathy donor is neither

immunocompromised nor autoimmune. In some embodiments, a healthy donor neither has nor has been diagnosed with a disease, including, but not limited to a blood-borne disease, a disease

SUBSTITUTE SHEET (RULE 26) affecting bone marrow or blood cells, a clotting or cardiovascular disorder, an inflammator disorder, a proliferative disorder, a cancer (liquid or solid), or an infectious disease (e.g. a viral or microbial disorder communicable through contact with bone marrow or blood cells).

[0311J In some embodiments, an HSC of the disclosure is isolated or derived from a human of any age, including a neonate, an infant, a child, a young adult (e.g. aged between 12 and 18 years, inclusive of the endpoints), an adult (e.g. aged between 19 and 65 years, inclusive of the endpoints), a senior adult (e.g. aged between 66 and 90 years, inclusive of the endpoints), an elderly adult (e.g. at least 91 years of age), or a centenarian (e.g. at least 100 years of age).

[0312] In some embodiments, an HSC of the disclosure is derived or differentiated from an induced pluripotent stem cell (iPSC). In some embodiments, an HSC of the disclosure is derived or differentiated from an embryonic stem cell (primary or cultured).

[0313] In some embodiments of the discl osure, a pharmaceutical composition comprises an HSC produced by the methods of the discl osure or a composition comprising an HSC produced by the methods of the disclosure. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically-acceptable carrier.

[0314] The disclosure provides a use of pharmaceutical composition of the disclosure in the treatment of a disease, comprising providing a therapeutically effective amount of the pharmaceutical composition to a subject in need thereof. In some embodiments, a therapeutically effective amount of the pharmaceutical composition comprises or consists of a single dose. In some embodiments, a therapeutically effective amount of the pharmaceutical composition comprises or consists of a single dose over the lifetime of the subject. In some embodiments, a therapeutically effective amount of the pharmaceutical composition comprises or consists of at least one single dose. In some embodiments, the use further comprises administering an activation composition to increase in vivo proliferation of an HSC of the pharmaceutical composition to activate or to reactivate the cell therapy . In some embodiments, including those wherein at least one HSC, a portion of the HSCs or each HSC of the pharmaceutical composition comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide, the use further comprises administering an induction agent to induce apoptosis in those HSCs comprising the inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide. The induction agent may be administered to reduce the activity of the pharmaceutical composition if a less aggressive treatment is required, to end the treatment if the

SUBSTITUTE SHEET (RULE 26) therapy is no longer required, or to terminate the HSCs should the subject experience an adverse event.

[0315] The disclosure provides a method of treating a disease, comprising administering a therapeutically-effective amount of the pharmaceutical composition to a subject in need thereof. In some embodiments, a therapeutically effective amount of the pharmaceutical composition comprises or consists of a single dose. In some embodiments, a therapeutically effective amount of the pharmaceutical composition comprises or consists of a single dose over the lifetime of the subject. In some embodiments, a therapeutically effective amount of the pharmaceutical composition comprises or consists of at least one single dose. In some embodiments, the method further comprises administering an activation composition to increase in vivo proliferation of an HSC of the pharmaceutical composition to activate or to reactivate the cell therapy. In some embodiments, including those wherein at least one HSC, a portion of the HSCs or each HSC of the pharmaceutical composition comprises an inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide, the method further comprises administering an induction agent to induce apoptosis in those HSCs comprising the inducible caspase polypeptide or a sequence encoding an inducible caspase polypeptide. The induction agent may be administered to reduce the activity of the pharmaceutical composition if a less aggressive treatment is required, to end the treatment if the therapy is no longer required, or to terminate the HSCs should the subject experience an adverse event.

[0316] In some embodiments of the disclosure, a pharmaceutical composition is provided or administered to a subject systemically. In some embodiments, pharmaceutical composition is provided or administered to a subject via an intravenous route. In some embodiments, pharmaceutical composition is provided or administered to a subject via an injection or an infusion. In some embodiments, pharmaceutical composition is provided or administered to a subject via an intravenous injection. In some embodiments, pharmaceutical composition is provided or administered to a subject via an intravenous infusion.

[0317] In some embodiments of the disclosure, a pharmaceutical composition is provided or administered to a subject locally. In some embodiments, pharmaceutical composition is provided or administered to a subject via an intraosseous route. In some embodiments, pharmaceutical composition is provided or administered to a subject via an injection or an infusion. In some embodiments, pharmaceutical composition is provided or administered to a subject via an

SUBSTITUTE SHEET (RULE 26) intraosseous injection. In some embodiments, pharmaceutical composition is provided or administered to a subject via an intraosseous infusion.

[0318] The transposon can comprise, consist essential of, or consist of at least one exogenous sequence (transgene sequence). The transposon can comprise, consist essential of, or consist of at least two exogenous sequences (transgene sequences). The transposon can comprise, consist essential of, or consist of at least three exogenous sequences (transgene sequences). The transposon can comprise, consist essential of, or consist of at least four exogenous sequences (transgene sequences). The transposon can comprise, consist essential of, or consist of at least five exogenous sequences (transgene sequences).

[0319] The exogenous sequence can comprise, consist essential of, or consist of a sequence encoding a chimeric antigen receptor (CAE).

[Q32Q] The exogenous sequence can comprise, consist essential of, or consist of a sequence encoding a therapeutic agent. The therapeutic agent can be a therapeutic protein. The therapeutic agent can be a therapeutic RNA. The therapeutic RNA can be iRNA, siRNA, or shRNA.

[0321] The therapeutic agent can be human beta-globin (HBB), T87Q human beta-globin (HBB T87Q), BAF chromatin remodeling complex subunit (BCL1 1 A) shRNA, insulin like growth factor 2 binding protein 1 (IGF2BP1), interleukin 2 receptor gamma (IL2RG), alpha gaiactosidase A (GLA), alpha-L-idurondase (IDU.A), iduronate 2-sulfatase (IDS), eystinosm lysosomal cysteine transporter (CTNS).

[0322] The compositions of the disclosure can be used to treat a disease or disorder including, but not limited to: Osteopetrosis, Parkinson’s Disease, Hunter Syndrome, Sickle Cell Disease, Severe Combined Immunodeficiency, Alpha-mannosidosis, Sideroblastic anemia, Autosomal Recessive Hyper IgE Syndrome, Primary Myelofibrosis, Cutaneous vasculitis, X-linked protoporphyria, Fucosidosis, Maroteaux Lamy syndrome, WAS Related Disorders, Chrome Granulomatous, Thalassemia Major, Plereditaiy Angioedema, Hereditary Lymphedemia, Hyper IgM Syndrome, Friedrich’s Ataxia, Charcot Marie Tooth Disease, Phenylketonuria,

Methylmalonic Acidemia, Adrenoleukodystrophy, Kugeiberg Welander Syndrome, Retinitis Pigmentosa, Hydrocephalus, Hereditary Sensory and Autonomic Neuropathy Type IV, Mucopolysaccharidosis Type III, Corneal Dystrophies, Erythropoietic Protoporphyria, Fabry Disease, Werdnig-Hoffman Disease, Hypoposphatasia, Coats Disease, Fanconi Anemia,

SUBSTITUTE SHEET (RULE 26) Niemann Pick Disease, Crigler-Najjar Syndrome, Hemophilia A, Hemophilia B,

Leukodystrophy, Sandhoff Disease, Usher Syndrome, Wolman Disease, Dupuytren’s

Contracture, Wolfram Syndrome, X-Lmked Myotubular Myopathy, Canavan Disease, Elder’ s Danlos Syndrome, Epidermolysis Bullosa, Osteogenesis Imperfecta, Short Bowel Syndrome, Giant Axonal Neuropathy, Paroxysmal Nocturnal Hemoglobinuria, Phelan-McDermid

Syndrome, Retinoschisis, Beta-Thalassemia, Hypophosphatasia, Propionic Acidemia,

Cholesteryl Ester Storage Disease, Cystinosis, Glycogen Storage Disease Type II Pompe Disease, Mucopolysaccharidoses (MPS I H-S Hurler-Scheie), Mucopolysaccharidoses (Type II (Hunter syndrome)), and Mucopolysaccharidoses (Type IV (Morquio)).

[Q323] The compositions of the disclosure may be used to treat a disease or disorder by use of a therapeutic transgene encoding for an exogenous nucleic acid sequence or exogenous amino acid sequence. For certain diseases or disorders the therapeutic transgene can include [Disease (therapeutic transge): Beta-Thalassemia (HBB T87Q, BCL11A shRNA, IGF2BP1), Sickle Cell Disease (HBB T87Q, BCLI 1 A shRNA, IGF2BP1), Hemophilia A (Factor VIII), Hemophilia B (Factor IX), X-linked Severe Combined Immunodeficiency (Interleukin 2 receptor gamma (IL2RG)), Hypophosphatasia (Tissue Non-specific Alkaline Phosphatase (TNAP)),

Osteopetrosis (TCIRG1), Glycogen Storage Disease Type II (Pompe Disease) (Alpha

Glucosidase (GAA)), Alpha-Galactosidase A Deficiency (Fabry disease) (Alpha-galactosidase A (GLA)), Mucopolysaccharidosis Type I (NIPS I) (Alpha-L-iduronidase (IDUA)),

Mucopolysaccharidosis Type II (MPS II) (Iduronate 2-sulfatase (IDS)), Mucopolysaccharidosis Type IIIA (MPS IIIA) (sulfoglycosamine-sulfohydrolase (SGSH)), Mucopolysaccharidosis Type IIIB (MPS IIIB) (N-alpha-acetylglucosaminidase (NAGLU)), Mucopolysaccharidosis Type IV A (MPS IV A) (Morquio) (N-aeetylgalactosamine-6-sulfate suifatase (GALNS)),

Mucopolysaccharidosis Type IV B (MPS IVB) Beta-galactosidase (GLB1 (Beta-ga!actosidase (GLB1)), Cholestery l Ester Storage Disease (CESD) (Lysosomal acid lipase (LIP A)), Cystinosis (Cystmosin lysosomal cystine transporter (CTNS)), X-linked chronic granulomatous disease (X- CGD) (CYBB), Wiskott-Aldrich Syndrome (WAS) (WAS), X-linked Adrenoleukodystrophy (X- ALD) (ABCD1), Metachromatic leukopdystrophy (MED) (ARSA), Phenylketonuria (PAH), Methylmalonic academia (MMLJT), Propionic Acidemia (PCCA, PCCB), Retinitis Pigmentosa (RPE65), Usher Syndrome (MY07A), and Gaucher Disease (GBA).

SUBSTITUTE SHEET (RULE 26) [0324] in some embodiments the selection agent is methotrexate, pralatrexate, pyrimethamine, dapsone, raltitrexed, trimetrexate, metoprine, iclaprim, ammoptenn, lometrexol, nolatrexed, brodimoprim, trimethoprim, pemetrexed, proguanil, piritrexim, and cycloguanil.

[0325] In some embodiments of the disclosure, at least one HSC of the plurality of therapeutic HSCs comprises a genetic modification. In some embodiments, a portion of the HSCs of the plurality of therapeutic HSCs comprise a genetic modification. In some embodiments, the portion comprises at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage m between of the plurality of therapeutic HSCs. In some embodiments, each HSC of the plurality of therapeutic HSCs comprise a genetic modification.

[0326] In some embodiments of the methods of the disclosure, including those wherein at least one HSC of the plurality of therapeutic HSCs comprises a genetic modification, the genetic modification is a single strand break, a double strand break, a sequence deletion, a sequence insertion, a sequence substitution or any combination thereof. In some embodiments, the sequence deletion, the sequence insertion, the sequence substitution or the combination thereof comprise(s) a sequence encoding an intron, an exon, a promoter, an enhancer, a transcriptional repressor, a CpG site or any combination thereof.

[0327] HSCs of the disclosure may be modified by a gene editing composition comprising a DNA localization domain and an effector domain.

[0328] In some embodiments, the DNA localization domain may comprise a DNA binding domain of Cas9, an inactivated Cas9, a short Cas9, a short and inactivated Cas9, a TALEN or a Zinc-finger protein.

[0329] In some embodiments, the effector comprises an endonuclease. Preferably, the endonuclease is a type IIS endonuclease. In some embodiments, the type IIS endonuclease is one or more of Acil, Mnll, Alwl, Bbvl, Bed, BceAI, BsmAl, BsmFI, BspCNI, Bsrl, BtsCI, Hgal, Hphl, HpyAV, Mboll, My II, P!el, SfaNI, Acul, BciVI, BfuAI, BmgBI, Bmrl, Bpml, BpuEI, Bsal, BseRI, Bsgl, Bsml, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Btsl, Earl, Ecil, Mmel, NmeAIII, BbvCI, BpulOI, BspQI, Sapl, Bael, BsaXI, CspCI, Bfil, Mboll, Acc36I, Fokl or Clo051. In some embodiments, the type IIS endonuclease is Clo051.

[0330] In some embodiments of the disclosure, a genetic modification is introduced to an HSC of the disclosure by a composition comprising a DNA binding domain and an endonuclease

SUBSTITUTE SHEET (RULE 26) domain. In some embodiments, the DNA binding domain comprises a guide RNA. In some embodiments, the DNA binding domain composes a sequence isolated or derived from a Cas9, a Transcription Activator-Like Effector Nuclease (TALEN), a Centromere and Promoter Factor 1 (Cpfl) or a zinc- finger nuclease (ZFN). In some embodiments, the Cas9 is a catalytically- inactive Cas9 (dCas9) or a short and cataiytieaily-inactive Cas9 (dsCas9).

[0331] In some embodiments, the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphyloccocus pyogenes. In some embodiments, the dCas9 comprises a dCas9 with substitutions at positions 10 and 840 of the amino acid sequence of the dCas9 which inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A. In some embodiments, the“X” residue at position 1 of the dCas9 sequence is a methionine (M). In some embodiments, the amino acid sequence of the dCas9 comprises the sequence of:

i XDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE

61 ATRLKRTARR RYTRRKNRI C YLQEI FSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLAIAH MI KFRGHFLI EGDLNPDNSD 18 1 VDKLFIQLVQ TYNQLFEENP INAS GVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN 24 1 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MI KRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 361 GYI DGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGS I PHQIHLGELH 42 1 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKS E ETITPWNFEE 4 8 1 VVDKGASAQS FI ERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 54 1 SGEQKKAIVD LLFKTNRKVT VKOLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 72 1 HEHIANLAGS PAIKKGILQT VKWDELVKV MGRHKPENIV I EMARENQTT QKGQKNSRER 7 8 1 MKRIEEGIKE LGSQI LKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDA 84 1 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEWKKMK NYWRQLLNAK LI TQRKFDNL 901 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 961 KLVSDFRKDF QFYKVREINN YHHAHDAY ^’ LN AWGTALIKK YPKLESEFVY GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEI RKR PLI ETN GETG EIVWDKGRDF 1 08 1 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDS PTVA 1 14 1 YSVLWAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLI IKLPK 1201 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 12 61 QHKHYLDEI I EQISEFSKRV I LADANI,DKV LSAYNKHRDK PI REQAENI I HLFTLTNLGA 132 1 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD (SEQ ID NO: 14 4 94 } .

SUBSTITUTE SHEET (RULE 26) in some embodiments, the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphylococcus aureus. In some embodiments, the dCas9 comprises a dCas9 with substitutions at positions 10 and 580 of the ammo acid sequence of the dCas9 which inactivate the catalytic site. In some embodiments, these substitutions are D10A and N580A. In some embodiments, the dCas9 is a small and inactive Cas9 (dSaCas9). In some embodiments, the amino acid sequence of the dSaCas9 comprises the sequence of:

1 rnkrnyilglA igitsvgygi idyetrdvid agvrlfkean vennegrrsk rgarrlkrrr

61 rhriqrvkkl Ifdynlltdh selsginpye arvkglsqkl seeefsaall hlakrrgvhn

121 vneveedtgn elstkeqisr nskaleekyv aelqlerlkk dgevrgsinr fktsdyvkea

181 kqllkvqkay hqldqsfidt yidlletrrt yyegpgegsp fgwkdikewy emlmghctyf

241 peelrsvkya ynadlynaln dlnnlvitrd enekleyyek fqilenvfkq kkkptlkqia

301 keilvneedi kgyrvtstgk pef fcnl vyh dlkditarke iienaelldq iaklltlyqs

361 sediqeeltn Inseltqeei eqisnlkgyt gthnlslkai nllldel ht ndnqiaifnr

421 lklvpkkvdl sqqkeipttl vddfilspvv krsfiqsikv inaiikkygl pndiiielar

481 eknskdaqkm inemqkrnrq tnerieelir ttgkenakyl iekiklhdmq egkclyslea

541 ipledllnnp fnyevdhiip rsvsfdnsfn nkvlvkqeeA skkgnrtpfq ylsssdskis

601 yetfkkhiln lakgkgris k tkkeylleer dlnrfsvqkd finrnlvdcr yatrglmnll

661 rsyfrvnnld vkvksinggf ts flrrkwkf kkernkgykh haedalllan adfi fke kk

721 ldkakkvmen qmfeekqaes mpeieteqey keifitphqi khikdfkdyk yshrvdkkpn

781 relindtlys trkddkgntl ivnnlnglyd kdndklkkli nkspekllmy hhdpqtyqkl

841 klimeqygde knplykyyee tgnyltkysk kdngpvikki kyygnklnah Iditddypns

901 rnkvvkl slk pyrfdvyldn gvykfvtvkn Idvlkkenyy evnskcyeea kklkklsnqa

961 efias fynnd likingelyr vigvnndlln rievnmidit yreylenmnd krppriikti

1021 asktqsikky stdilgnlye vkskkhpqii kkg ( SEQ ID NO: 14495 ) .

[0333] In some embodiments, the endonuclease domain comprises a sequence isolated or derived from a Cas9, a Transcription Activator-Like Effector Nuclease (TALEN), or a type IIS endonuclease. In some embodiments, the type IIS endonuclease is Acil, Mnll, Alwl, Bbvl, Bed, BceAI, BsmAI, BsmEI, BspCNI, Bsrl, BtsCI, Hgal, Hphl, HpyAV, Mboll, My II, Plel, SfaNI, Acul, BciVI, BfuAI, BmgBI, Bmrl, Bprnl, BpuEI, Bsai, BseRI, Bsgl, Bsrnl, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, Btsl, Earl, fed. Mmel, NmeAIII, BbvCI, BpulOI, BspQI, Sapl, Bael, BsaXI, CspCI, Bfil, Mboll, Acc36I, Fokl or Clo051. In some embodiments, the type IIS endonuclease is Clo051. In some embodiments, the DNA binding domain and the endonuclease domain are covalently or non-covaiently linked. In some embodiments, the DNA binding domain and the endonuclease domain are covalently linked as a fusion protein.

5UB5TITUTE SHEET (RULE 26) [0334] in some embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCasti and CloOSl . An exemplary CloOSl nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of:

EGIKSNISLLKDELRGQ1SHISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHL G GSRKPDGIVYSTTLEDNFGnVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVNPNK WWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAEKIRSGE MTIEELERAMFNN SEFILK Y (SEQ ID NO: 14496).

[0335] An exemplary dCas9-Clo051 nuclease domain may comprise, consist essentially of or consist of, the amino acid sequence of (CloOSl sequence underlined, linker bold italics, dCas9 sequence in italics):

MAPKKKRKVEGIKSNISLLKDELRGOISfflSHEYLSLIDLAFDSKONRLFEMKVLELLV N

E Y GFKGRHLGGSRKPDGI VY S TTLEDNF TK A Y SEGY SLPIS O ADEMERYVREN SN

RDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEOLRRLSMTTGVNGSAVNWNLL

LGAEKIRSGEMTIEELERAIviFNNSEFILKYGGG yD graiG/^/Gmy G ^ irD ZgF

SKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLOEI FSNEMAK

VDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA DLRLIYL

ALAHMIKFFiGHFLIEGDLNPDNSD VDKLFIQL VO TTNQLFEENPINASG VDAKAILSARLSKSR

RLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIG

DQYADLFIAAKNLSDAILLSDILRVNTFJTKAPLSASMIKRYDFJJHQDLTLLKALV RQQLPEKY

KEIFFDQSKNG YA GYIDGGASQEEFYKFJKPILEKMDGTEEI VKLNREDIJMKQRTFDNGSI

PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR KSEETIT

PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVIJ’KHSIXYEYFTVYNELTKVK YVTEGMRK

PAFLSGEQKKAIVDLLFKTNRKVrVKQLKEDYFKKIECFDSVFJSGVEDRFNASLGT YHDLLKJ

IKDKDFLDNEF.NED1LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY TGWGR1S

RKLINGTRDKQSGKTILDFLKSDGFANRNFMQLTHDDSLTFKEDIQKAQVSGQGDSL HEHIAN

LAGSPA1KKGILOTVKWDELVKVMGRHKPENIVIEMARENOTTOKGOKNSRERMKR1 EEGIK

ELGSQILKEHPVEFTTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQS FLKDDSI

DNKVLrRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLlTQRKFDNLTKAERGGLS ELDK

AGFIKRQLVETRQnKHVAQILDSRMNTKYDENDKLlREVKVniKSKLVSDFRKDFQF YKVRE lNNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQElGKATAKYFF YS

NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAIYRKVLSMPQVNIVKK TEVQTG

SUBSTITUTE SHEET (RULE 26) GFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELL GI

HMERSSFEKNPIDFLEAKGYKEFKKDLIIKLPKYSLFELENGRKRMLASAGELOKGN ELALPS

KYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL DKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTnDRKRYTSTKEVLDATLIHQSiTGL YETRI DLSQLGGDGSPKKKRKVSS (SEQ ID NO: 14497).

[0336] For more detail regarding genomic editing tools, see WO 2016/205554, the contents of which are incorporated by reference herein in their entirety).

[0337] Compositions comprising a DNA localization domain and an effector domain may be contained in a transposon. Compositions comprising a DNA localization domain and an effector domain, including those contained in a vector, may be further contained in a vector for expression and/or for delivery' to an HSC of the disclosure.

[Q338] The disclosure provides a nanotransposon comprising: (a) a sequence encoding a transposon insert, comprising a sequence encoding a first inverted terminal repeat (ITR), a sequence encoding a second inverted terminal repeat (ITR), and an intra-XTR sequence; (h) a sequence encoding a backbone, wherein the sequence encoding the backbone comprises a sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints, and a sequence encoding a selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, and (c) an inter-ITR sequence. In some embodiments, the inter-ITR sequence of (c) comprises the sequence of (b). In some embodiments, the intra-ITR sequence of (a) comprises the sequence of (b).

[0339] In some embodiments of the nanotransposons of the disclosure, the sequence encoding the backbone comprises between 1 and 600 nucleotides, inclusive of the endpoints. In some embodiments, the sequence encoding the backbone consists of between 1 and 50 nucleotides, between 50 and 100 nucleotides, between 100 and 150 nucleotides, between 150 and 200 nucleotides, between 200 and 250 nucleotides, between 250 and 300 nucleotides, between 300 and 350 nucleotides, between 350 and 400 nucleotides, between 400 and 450 nucleotides, between 450 and 500 nucleotides, between 500 and 550 nucleotides, between 550 and 600 nucleotides, each range inclusive of the endpoints.

[0340] In some embodiments of the nanotransposons of the disclosure, the inter-ITR sequence comprises between 1 and 1000 nucleotides, inclusive of the endpoints. In some embodiments, the inter-ITR sequence consists of between 1 and 50 nucleotides, between 50 and 100

SUBSTITUTE SHEET (RULE 26) nucleotides, between 100 and 150 nucleotides, between 150 and 200 nucleotides, between 200 and 250 nucleotides, between 250 and 300 nucleotides, between 300 and 350 nucleotides, between 350 and 400 nucleotides, between 400 and 450 nucleotides, between 450 and 500 nucleotides, between 500 and 550 nucleotides, between 550 and 600 nucleotides, between 600 and 650 nucleotides, between 650 and 700 nucleotides, between 700 and 750 nucleotides, between 750 and 800 nucleotides, between 800 and 850 nucleotides, between 850 and 900 nucleotides, between 900 and 950 nucleotides, or between 950 and 1000 nucleotides, each range inclusive of the endpoints.

[0341] In some embodiments of the nanotransposons of the disclosure, including the short nanotransposons (SNTs) of the disclosure, the inter-ITR sequence comprises between 1 and 200 nucleotides, inclusive of the endpoints. In some embodiments, the inter-ITR sequence consists of between 1 and 10 nucleotides, between 10 and 20 nucleotides, between 20 and 30 nucleotides, between 30 and 40 nucleotides, between 40 and 50 nucleotides, between 50 and 60 nucleotides, between 60 and 70 nucleotides, between 70 and 80 nucleotides, between 80 and 90 nucleotides, or between 90 and 100 nucleotides, each range inclusive of the endpoints.

[0342] In some embodiments of the nanotransposons of the disclosure, the selectabl e marker having between 1 and 200 nucleotides, inclusive of the endpoints, comprises a sequence encoding a sucrose-selectable marker. In some embodiments, the sequence encoding a sucrose- selectable marker comprises a sequence encoding an RNA-OUT sequence. In some

embodiments, the sequence encoding an RNA-OUT sequence comprises or consists of 137 base pairs (bp). In some embodiments, the selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, comprises a sequence encoding a fluorescent marker. In some embodiments, the selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, comprises a sequence encoding a cell surface marker.

[0343] In some embodiments of the nanotransposons of the disclosure, the sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints, comprises a sequence encoding a mini origin of replication. In some embodiments, the sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints, comprises a sequence encoding an R6K origin of replication. In some embodiments, the R6K origin of replication comprises an R6K gamma origin of replication. In some embodiments, the R6K origin of replication comprises an R6K mini origin of replication. In

SUBSTITUTE SHEET (RULE 26) some embodiments, the R6K origin of replication comprises an R6K gamma mini origin of replication in some embodiments, the R6K gamma mini origin of replication comprises or consists of 281 base pairs (bp).

[0344] In some embodiments of the nanotransposons of the disclosure, the sequence encoding the backbone does not comprise a recombination site, an excision site, a ligation site or a combination thereof. In some embodiments, neither the nanotransposon nor the sequence encoding the backbone comprises a product of a recombination site, an excision site, a ligation site or a combination thereof. In some embodiments, neither the nanotransposon nor the sequence encoding the backbone is derived from a recombination she, an excision site, a ligation site or a combination thereof.

[0345] In some embodiments of the nanotransposons of the disclosure, a recombination site comprises a sequence resulting from a recombination event. In some embodiments, a

recombination site comprises a sequence that is a product of a recombination event. In some embodiments, the recombination event comprises an activity of a recombinase (e.g., a recombinase site).

[Q346] In some embodiments of the nanotransposons of the disclosure, the sequence encoding the backbone does not further comprise a sequence encoding foreign DNA.

[0347] In some embodiments of the nanotransposons of the disclosure, the inter-ITR sequence does not comprise a recombination site, an excision site, a ligation site or a combination thereof. In some embodiments, the inter-ITR sequence does not comprise a product of a recombination event, an excision event, a ligation event or a combination thereof In some embodiments, the inter-ITR sequence is not derived from a recombination event, an excision event, a ligation event or a combination thereof.

[0348] In some embodiments of the nanotransposons of the disclosure, the inter-ITR sequence comprises a sequence encoding foreign DNA.

[0349] In some embodiments of the nanotransposons of the disclosure, the mtra-ITR sequence comprises at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell. In some embodiments, the mammalian cell is a human cell.

[0350] In some embodiments of the nanotransposons of the disclosure, the mtra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of

SUBSTITUTE SHEET (RULE 26) expressing an exogenous sequence in a mammalian cell and a second sequence encoding an insulator.

[0351] in some embodiments of the nanotransposons of the disclosure, the mtra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, a polyadenosme (poly A) sequence and a second sequence encoding an insulator.

[0352] In some embodiments of the nanotransposons of the disclosure, the mtra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, at least one exogenous sequence, a polyadenosine (poly A) sequence and a second sequence encoding an insulator.

[0353] In some embodiments of the nanotransposons of the disclosure, the sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell is capable of expressing an exogenous sequence in a human cell. In some embodiments, the sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell comprises a sequence encoding a constitutive promoter. In some embodiments, the sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell comprises a sequence encoding an inducible promoter. In some embodiments, the intra-ITR sequence comprises a first sequence encoding a first promoter capable of expressing an exogenous sequence in a mammalian cell and a second sequence encoding a second promoter capable of expressing an exogenous sequence in mammalian cell, wherein the first promoter is a constitutive promoter, wherein the second promoter is an inducible promoter, and wherein the first sequence encoding the first promoter and the second sequence encoding the second promoter are oriented in opposite directions. In some embodiments, the sequence encoding a promoter capable of expressing an exogenous sequence m a mammalian cell comprises a sequence encoding a cell-type or tissue-type specific promoter. In some embodiments, the sequence encoding a promoter capable of expressing an exogenous sequence m a mammalian cell comprises a sequence encoding an elongation factor-1 alpha (EF1 alpha) promoter, a sequence encoding a cytomegalovirus (CMV) promoter, a sequence encoding an MND promoter, a sequence encoding an simian vacuolating virus 40 (SV40) promoter, a sequence encoding a phosphoglycerate kinase 1 (PGK1) promoter, a sequence encoding a human phosphoglycerate kinase l(hPGK) promoter, a sequence encoding a ubiquitin c (Ubc) promoter,

SUBSTITUTE SHEET (RULE 26) a sequence encoding an SPTA1 promoter, a sequence encoding an ankryin-l (Ank-l) promoter, a sequence encoding a Gly-A promoter, a sequence encoding a CAG promoter, a sequence encoding an HI promoter, or a sequence encoding a U6 promoter.

[0354] In some embodiments of the nanotransposons of the disclosure, the polyadenosine (poly A) sequence is isolated or derived from a viral poly A sequence. In some embodiments, the polyadenosine (poly A) sequence is isolated or derived from an (SV40) poly A sequence.

[0355] In some embodiments of the nanotransposons of the disclosure, the at least one exogenous sequence comprises an inducible proapoptotic polypeptide. In some embodiments, the inducible caspase polypeptide comprises (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non- human sequence. In some embodiments, the inducible caspase polypeptide comprises (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence.

[Q356] In some embodiments, the sequence encoding the hPGK promoter comprises or consists of the nucleic acid sequence:

[0357] Ttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggt tccgggaaacgcagcggcgcc gaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgct acccttgtgggccccccggcgacgcttcctgct ccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaa gccgcacgtctcactagtaccctcgcaga cggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcgg ctgctcagcagggcgcgccgagagc agcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttc ctgcccgcgcggtgttccgcattctgca agcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctc cccag (SEQ ID NO: 14696).

[0358] In some embodiments, the sequence encoding the EFla promoter comprises or consists of the nucleic acid sequence:

[0359] ggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttgggg ggaggggtcggcaattgaa ccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctcc gcctttttcccgagggtgggggagaaccgt atataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacac aggtaagtgccgtgtgtggttcccgcgggcctg gcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacg tgattcttgatcccgagcttcgggttggaagtggg tgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcc tggcctgggcgctggggccgccgcgtgcga atctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaat ttttgatgacctgctgcgacgctttttttctggcaaga tagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcggg cggcgacggggcccgtgcgtcccagcgcaca tgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaa gctggccggcctgctctggtgcctggcc

SUBSTITUTE SHEET (RULE 26) tcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttg cgtgagcggaaagatggccgcttcccgg ccctgctgcagggagctcaaaatggaggacgcggegctcgggagagcgggcgggtgagtc acceacacaaaggaaaagggcctttcc gtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcga ttagttctcgagcttttggagtacgtcgtcttta ggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaa gttaggccagcttggcacttgatgtaattctcc ttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggtt caaagtttttttcttccatttcaggtgtcgtga (SEQ ID NO: 14697).

[0360] In some embodiments, the sequence encoding the EFla promoter comprises or consists of the nucleic acid sequence:

[0361] ggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggg gtcggcaattgaaccggtg cctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgccttt ttcccgagggtgggggagaaccgtatataa gtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacag (SEQ ID NO: 14698).

[0362] In some embodiments, the sequence encoding the MND promoter comprises or consists of the nucleic acid sequence:

[0363] gatccatcgattagtccaatttgttaaagacaggatatcagtggtccaggctctagtttt gactcaacaatatcaccagctgaagc ctatagagtacgagccatagatagaataaaagattttatttagtctccagaaaaaggggg gaatgaaagaccccacctgtaggtttggcaag ctaggatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtgg taagcagttcctgccccggctcagggcca agaacagttggaacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgc cccggctcagggccaagaacagatggtccc cagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgcccc aaggacctgaaatgaccctgtgccttatttgaa ctaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataa aagagcccacaacccctcactcggcgcg

(SEQ ID NO: 14699).

[0364] In some embodiments, the sequence encoding the SPTA1 promoter comprises or consists of the nucleic acid sequence:

[0365] ccagactttcaagaagagaatgtaaaggactctcagtttttctatgtgagatccaagaac aggggaaaccacaaatgtagaatc aaaatcttctcttgtagatcatggaagttagattattcaagctgcatttacactggaatg aagtttggcatggatagccccactggcttgtagaaa ggtctaagagcatcccactattttctttttgcttgtttgtttcatttttgtaagttagtc taaaataactatgcctatggaaaaagtctacctctttttgttc tccattaaaattaatacatacagtaggatgatttataccatatatatcagaagtcagcca tattttcaacttttttgtgacatcattggtttgtcaaata aggtatcctaatttttgttttacaaaatattaatgaacacatcatataactttttttgtt ccttccatttaaatgattaagagtacaactaaatcagattttt aaagaceaaattgttgaggttaggaataaaatettattttgtgaaagactctetagaaaa agccaggataattcgttgacttaaagactgtgaaa aatctgcagttgagttggatgaagttgttgagctgtatttcctataaatatatggggttt tttttctaatttttcatccaaaaaccttactttcattttttaa gtcttattgagtaactttgaaagccagagcctcccaaaactgctgagtcacccagtatct gtaaaacttagcagttgcctcagctgagtatgtct tctaaagataatgtcgattgtgtatggctgatgggattctaggaccaagcaagaggtttt tttttttcccccacatacttaacgtttctatatttctatt

SUBSTITUTE SHEET (RULE 26) tgaattcgactggacagttccatttgaattatttctctctctctctctctctctctgaca cattttatcttgccaggttctaaaccc (SEQ ID NO: 14700).

[0366] In some embodiments, the sequence encoding the Ank-1 promoter comprises or consists of the nucleic acid sequence:

[0367] Cgcgttcgaaggggcaaccaggggtccgcgccggccgaggcctggggagcggggcctcct ggggttgggggaggagg tgctcttgtaatctgcggtggtccccaggcgggcgccacccctccgcccgcccgtgccgg gagcgcccggcccgacagcaagcgcctct ggggccgataaggccctcgggggcctggcccgcacgtcacaggccccgcagaggctgcgg tgagtccgccagccccagctgctcctc ctcaagcccccaaggcccttcggcggcaattcccaccgg (SEQ ID NO: 14701).

[0368] In some embodiments, the sequence encoding the Gly-A promoter comprises or consists of the nucleic acid sequence:

[0369] ttgcatctcatgtctcttacattgctgtgtggctcaccatgagtttgggagtctttcaga acctcagaacactcaaatgatttaaattt ctcaaatacattcatttcacatataggaagtcactttcatttggaccactgggtcttgac attagaaatgagaaggtccatggctccacaacagc tacctcagcctggcacgtgccctggcctcagagattcacagtccagttctttgtccagtt gggtggctcctgtctaccaccttaccatgcccac ttaactgatgcaaagttaatatcacaagtagcaacctgttccttgcagtgaaaattttac ttaccactttcatagccccaagatatccatgtatcttt attaacaggcgcttaacaacttgcatcatttaaaatgcctcccctgcctatcagctgatg atggccgcaggaaggtgggcctggaagataac agctagcaggctaaggtcagacactgacacttgc (SEQ ID NO: 14702).

[0370] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises an inducible proapoptotic polypeptide, the ligand binding region comprises a FK506 binding protein 12 (FKBP12) polypeptide. In some embodiments, the ammo acid sequence of the ligand binding region comprises a FK506 binding protein 12 (FKBP12) polypeptide. In some embodiments, the FK506 binding protein 12

(FKBP12) polypeptide comprises a modification at position 36 of the sequence. In some embodiments, the modification comprises a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). In some embodiments, the FKBP12 polypeptide is encoded by an amino acid sequence comprising

GVQVETISPGDGRTFPKRGQTCVYHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG

WEEGVAQMSVGQRA LllSPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 14635). In some embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising

GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGG

CCAGACTTGCGICGIGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACA

SUBSTITUTE SHEET (RULE 26) GCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATC

CGAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGA CATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTC

ATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG ID NO: 14636).

[0371] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises an inducible proapoptotic polypeptide, the linker region is encoded by an amino acid comprising GGQGS (SEQ ID NO: 14637) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO: 14638). In some embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[0372] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises an inducible proapoptotic polypeptide, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an arginine (R) at position 87 of the sequence. In some embodiments, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence. In some embodiments, the truncated caspase 9 polypeptide is encoded by an amino acid comprising

GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF

SSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN P EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFE Q WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 14639). In some embodiments, the truncated caspase 9 polypeptide is encoded by a nucleic acid sequence comprising

GGATTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTA

CATCCTGAGCATGGAACCCTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTG

' ^' AGAGAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTG C

GGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCC

AAGAAAATGGTGGTGGGCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGA

TTGCTGCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCC C

GGAGCAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACAT

CTTCAACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCA

SUBSTITUTE SHEET (RULE 26) GGCCTGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTG

AGGACGAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGA

CTGAGGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGTGACATT

TTCGTGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGATCCCAAGTCAGGG

AGCTGGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCATTCAGAGGA

CCTGCAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCATCTACA

AACAGATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTTCC

(SEQ ID NO: 14640).

[0373] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises an inducible proapoptotic polypeptide, the inducible proapoptotic polypeptide is encoded by an amino acid sequence comprising

GVT ⁾ VETJSPGDGRTFPK RGQTCWHYTGMLEDGKKVDSSRDRNKPFKFML.GKQEVIRG

WEEGVAQMSVGQRAKLnSPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFGD

VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFS SLHF

MVEVKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGC PVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPG5NPEPDA T PFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHS E DLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 14641 ). In some embodiments, the inducible proapoptotic polypeptide is encoded by a nucleic acid sequence comprising

ggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaaaggggc cagacttgcgtcgtgcattacaccgggatgctg gaggacgggaagaaagtggacagctccagggatcgcaacaagcccttcaagttcatgctg ggaaagcaggaagtgatccgaggatggg aggaaggcgtggcacagatgtcagtcggccagcgggccaaactgaccattagccctgact acgcttatggagcaacaggccacccagg gatcattccccctcatgccaccctggtcttcgatgtggaactgctgaagctggagggagg aggaggatccggatttggggacgtgggggc cctggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaaccctgcgg ccactgtctgatcattaacaatgtgaacttctg cagagaaagcggactgcgaacacggactggctccaatattgactgtgagaagctgcggag aaggttctctagtctgcactttatggtcgaa gtgaaaggggatctgaccgccaagaaaatggtgctggccctgctggagctggctcagcag gaccatggagctctggattgctgcgtggtc gtgatcctgtcccacgggtgccaggcttctcatctgcagttccccggagcagtgtacgga acagacggctgtcctgtcagcgtggagaaga tcgtcaacatcttcaacggcacttcttgccctagtctggggggaaagccaaaactgttct ttatccaggcctgtggcggggaacagaaagat cacggcttcgaggtggccagcaccagccctgaggacgaatcaccagggagcaaccctgaa ccagatgcaactccattccaggagggac tgaggacctttgaccagctggatgctatctcaagcctgcccactcctagtgacattttcg tgtcttacagtaccttcccaggctttgtctcatggc

SUBSTITUTE SHEET (RULE 26) gcgatcccaagtcagggagctggtacgtggagacactggacgacatctttgaacagtggg cccattcagaggacctgcagagcctgctgc tgcgagtggcaaacgctgtctctgtgaagggcatctacaaacagatgcccgggtgcttca attttctgagaaagaaactgttctttaagacttc c (SEQ ID NO: 14642).

[0374] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises an inducible proapoptotic polypeptide, the exogenous sequence further comprises a sequence encoding a selectable marker. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a detectable marker. In some embodiments, the detectable marker comprises a fluorescent marker or a cell-surface marker. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a protein that is active in dividing cells and not active in non- dividing cells. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a metabolic marker. In some embodiments, the sequence encoding the selectable marker comprises a sequence encoding a dihydrofolate reductase (DHFR) mutein enzyme. In some embodiments, the DHFR mutein enzyme comprises or consists of the amino acid sequence of:

1 MVGSLNCIVA VSQNMGI GKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL

61 VIMGKKTWFS I PEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL

12 1 TEQPELANKV DMVWIVGGSS VYKEAMNHPG HLKLFVTRIM QDFESDTFFP

18 1 EIDLEKYKLL PEYPGVLSDV QEEKGI KYKF EVYEKND (SEQ ID NO: 14725 ) . In Some embodiments, the DHFR mutein enzyme is encoded by a the nucleic acid sequence comprising or consisting of

atggtcgggtctctgaattgtatcgtcgccgtgagtcagaacatgggcattgggaag aatggcgatttcccatggccacctctgcgcaacga gtcccgatactttcagcggatgacaactacctcctctgtggaagggaaacagaatctggt catcatgggaaagaaaacttggttcagcattcc agagaagaaccggcccctgaaaggcagaatcaatctggtgctgtcccgagaactgaagga gccaccacagggagctcactttctgagcc ggtccctggacgatgcactgaagctgacagaacagcctgagctggccaacaaagtcgata tggtgtggatcgtcgggggaagttcagtgt ataaggaggccatgaatcaccccggccatctgaaactgttcgtcacacggatcatgcagg actttgagagcgatactttctttcctgaaattga cctggagaagtacaaactgctgcccgaatatcctggcgtgctgtccgatgtccaggaaga gaaaggcatcaaatacaagttcgaggtctat gagaagaatgac ( SEQ ID NO: 14901) In some embodiments, the amino acid sequence of the DHFR mutein enzyme further comprises a mutation at one or more of positions 80, 113, or 153. In some embodiments, the amino acid sequence of the DHFR mutein enzyme comprises one or more of a substitution of a Phenylalanine (F) or a Leucine (L) at position 80, a substitution of a

SUBSTITUTE SHEET (RULE 26) Leucine (L) or a Valine (V) at position 113, and a substitution of a Valine (V) or an Aspartic Acid (D) at position 153.

[0375] in some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises an inducible proapoptotic polypeptide and/or the exogenous sequence comprises a sequence encoding a selectable marker, the exogenous sequence further comprises a sequence encoding a non-naturally occurring antigen receptor, and/or a sequence encoding a therapeutic polypeptide. In some embodiments, the non-naturally occurring antigen receptor comprises a T cell Receptor (TCR). In some embodiments, a sequence encoding the TCR comprises one or more of an insertion, a deletion, a substitution, an invertion, a transposition or a frameshift compared to a corresponding wild type sequence. In some embodiments, a sequence encoding the TCR comprises a chimeric or recombinant sequence. In some embodiments, the non-naturally occurring antigen receptor comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises: (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In some embodiments, the ectodomain of (a) of the CAR further comprises a signal peptide. In some embodiments, the ectodomain of (a) of the CAR further comprises a hinge between the antigen recognition region and the transmembrane domain. In some embodiments, the endodomain comprises a human (T)3z endodomain. In some embodiments, the at least one costimulatory domain comprises a human 4-1 BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. In some

embodiments, the at least one costimulatory domain comprises a human CD28 and/or a 4- IBB costimulatory domain. In some embodiments, the antigen recognition region comprises one or more of a scFv, a VHH, a VH, and a Centynn.

[0376] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises an inducible proapoptotic polypeptide and/or the exogenous sequence comprises a sequence encoding a selectable marker, the exogenous sequence further comprises a sequence encoding a transposase.

[0377] In some embodiments of the nanotransposons of the disclosure, the mtra-ITR sequence comprises a sequence encoding a selectable marker, an exogenous sequence, a sequence encoding an inducible caspase polypeptide, and at least one sequence encoding a self-cleaving peptide. In some embodiments, the at least one sequence encoding a self-cleaving peptide is

SUBSTITUTE SHEET (RULE 26) positioned between one or more of: (a) the sequence encoding a selectable marker and the exogenous sequence, (b) the sequence encoding a selectable marker and the inducible caspase polypeptide, and (c) the exogenous sequence and the inducible caspase polypeptide. In some embodiments, a first sequence encoding a self-cleaving peptide is positioned between the sequence encoding a selectable marker and the exogenous sequence and a second sequence encoding a self-cleaving peptide is positioned between the exogenous sequence and the inducible caspase polypeptide.

[0378] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises one or more of an inducible proapoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) are recognized by a piggyBae transposase or a piggyBae-!ike transposase. In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) are recognized by a piggyBae transposase. In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) are recognized by a piggyBac- like transposase. In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprise a TTAA, a TTAT or a TTAX recognition sequence. In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprise a TTAA, a TTAT or a TTAX recognition sequence and a sequence having at least 50% identity to a sequence isolated or derived from a piggyBae transposase or a piggyBac-like transposase. In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprise at least 2 nucleotides (nts), 3 nts, 4 nts, 5 nts, 6 nts, 7 nts, 8 nts, 9 nts, 10 nts, 11 nts, 12 nts, 13 nts, 14 nts, 1 5 nts, 16 nts, 17 nts, 18 nts, 19 nts, or 20 nts.

[0379] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises one or more of an inducible proapoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) are recognized by a piggyBae transposase or a piggyBac-like transposase.

SUBSTITUTE SHEET (RULE 26) In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprises the sequence of

ICCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 14727) or a sequence having at least 70% identity to the sequence of

CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 14727). In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprises the sequence of

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA

CGCATG (SEQ ID NO: 14728). In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprises the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 14727) and comprises the sequence of

CCCT AG AA AGATAAT CAT ATT GT GACGT ACGTT A A AGAT AAT C ATGC GT AAAATT GA

CGCATG (SEQ ID NO: 14728). In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprises the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 14727) and comprises the sequence of

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAAATTGA

CGCATG (SEQ ID NO: 14729). In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) comprises the sequence of CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID

14727) and comprises the sequence of

TTAACCCTAGAAA GAT AATCATATTGTGACGT ACGTT AAAGATAATCATGTGTAAAA

TTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGA T

ATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTA T

TTATGTTTATTTATTTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAA

CAAAACTTTTA (SEQ ID NO: 14730).

[0380] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises one or more of an inducible proapoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted

SUBSTITUTE SHEET (RULE 26) terminal repeat (ITR) are recognized by a piggyBac transposase or a piggyBac-like transposase. In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) is recognized by a piggyBac transposase having an amino acid sequence of at least 20% identity to the amino acid sequence of

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 S El LDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTKMCRNI YD PLLCFKLFFT DEIISEIVKK TNAEI SLKRR ESMTGATFRD TNEDEI YAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLFMDDK3 IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEO LLGFRGRCPF RMYI PNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GT SMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD OMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCE (SEQ ID

: 14487} . In some embodiments, the sequence encoding a first inverted terminal repeat

(ITR) or the sequence encoding a second inverted terminal repeat (ITR) is recognized by a piggyBac transposase having the ammo acid sequence of

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG

61 S El LDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNI YD PLLCFKLFFT DEI I SEIVKW TNAEI SLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL I RCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLT I DEQ LLGFRGRCPF RMYI PNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ

361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GT SMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCE (SEQ ID

NO : 14487) . In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) is recognized by a piggyBac transposase having an ammo acid sequence of at least 20% identity to the amino acid sequence of

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG

61 S El LDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG

121 PTRMCRNI YD PLLCFKLFFT DEI I SEIVKW TNAEI SLKRR ESMTSATFRD TNEDEI YAFF

SUBSTITUTE SHEET (RULE 26) 181 GILVMTAVRK DNHMSTDDLF DRS LSMVYVS VMSRDRFDFL I RCLRMDDKS I RPTLRENDV

241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGPCPF RVYIPNKPSK YGIKILMMCD

301 SGTKYMINGM PYLGRGTQTK GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAK LLQ

361 EPYKLTIVGT VRSNKREI PE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC

421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN

481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TS S FMRKRLE APTLKRYLRD NISNILPKEV

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV I CREHNI DMC QSCF (SEO ID NO:

14484 ) .In some embodiments, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) is recognized by a piggyBac transposase having the amino acid sequence of

1 MGSSLDDEHI ! LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG

61 S El LDEQNVI EQPGSSLASN RI LTLPQRT I RGKNKHCWST SKSTRRSRVS ALNIVRSQRG

121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF

181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL I RCLRMDDKS I RPTLRENDV

241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD

301 SGTKYMINGM PYLGRGTQTK GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ

361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC

421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN

481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV I CREHNI DMC QSCF (SEQ ID NO: 14484 ; .

[0381] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises one or more of an inducible proapoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) are recognized by a Sleeping Beauty transposase. In some embodiments, the Sleeping Beauty transposase is a hyperactive Sleeping Beauty transposase (SB100X).

[0382] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises one or more of an inducible proapoptotic polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) are recognized by a Helitron transposase.

[0383] In some embodiments of the nanotransposons of the disclosure, including those wherein the at least one exogenous sequence comprises one or more of an inducible proapoptotic

SUBSTITUTE SHEET (RULE 26) polypeptide, a sequence encoding a selectable marker, and an exogenous sequence, the sequence encoding a first inverted terminal repeat (ITR) or the sequence encoding a second inverted terminal repeat (ITR) are recognized by a Tol2 transposase.

[0384] The disclosure provides a cell comprising a nanotransposon of the disclosure. In some embodiments, the cell further comprises a transposase composition. In some embodiments, the transposase composition comprises a transposase or a sequence encoding the transposase that is capable of recognizing the first ITR or the second ITR of the nanotransposon. In some embodiments, the transposase composition comprises a nanotransposon comprising the sequence encoding the transposase. In some embodiments, the cell comprises a first nanotransposon comprising an exogenous sequence and a second nanotransposon comprising a sequence encoding a transposase. In some embodiments, the cell is an allogeneic cell.

[0385] The disclosure provides a composition comprising the nanotransposon of the disclosure.

[0386] The disclosure provides a composition comprising the cell of the disclosure. In some embodiments, the cell comprises a nanotransposon of the disclosure. In some embodiments, the cell is not further modified. In some embodiments, the cell is allogeneic.

[Q387] The disclosure provides a composition comprising the cell of the disclosure. In some embodiments, the cell comprises a nanotransposon of the disclosure. In some embodiments, the cell is not further modified. In some embodiments, the cell is autologous.

[0388] The disclosure provides a composition comprising a plurality of cells of the disclosure. In some embodiments, at least one cell of the plurality of cells comprises a nanotransposon of the disclosure. In some embodiments, a portion of the plurality of cells comprises a

nanotransposon of the disclosure. In some embodiments, the portion comprises at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,

90%, 95%, 97%, 99% or any percentage in between of the plurality of cells. In some

embodiments, each cell of the plurality of cells comprises a nanotransposon of the disclosure. In some embodiments, the plurality of cells does not comprise a modified cell of the disclosure. In some embodiments, at least one cell of the plurality of cells is not further modified. In some embodiments, none of the plurality of ceils is not further modified. In some embodiments, plurality of ceils is allogeneic. In some embodiments, an allogeneic plurality of cells are produced according to the methods of the disclosure. In some embodiments, plurality of cells is

SUBSTITUTE SHEET (RULE 26) autologous. In some embodiments, an autologous plurality of cells are produced according to the methods of the disclosure.

[0389] The disclosure provides a modified cell comprising: (a) a nanotransposon of the disclosure; (b) a sequence encoding an inducible proapoptotic polypeptide; and wherein the cell is a T cell, (c) a modification of an endogenous sequence encoding a T cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR. In some embodiments, the cell further comprises: (d) a non- naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E), and (e) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

[Q39Q] The disclosure provides a modified cell comprising: (a) a nanotransposon of the disclosure; (b) a sequence encoding an inducible proapoptotic polypeptide; (c) a non-naturally occurring sequence comprising an HLA class I histocompatibility antigen, alpha chain E (HLA- E), and (e) a modification of an endogenous sequence encoding Beta-2-Microglobulin (B2M), wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

[0391] In some embodiments of the modified cells of the disclosure, the non-naturally occurring sequence comprising a HLA-E further comprises a sequence encoding a B2M signal peptide. In some embodiments, the non-naturally occurring sequence comprising an HLA-E further comprises a linker, wherein the linker is positioned between the sequence encoding the sequence encoding a B2M polypeptide and the sequence encoding the HLA-E. In some embodiments, the non-naturally occurring sequence comprising an HLA-E further comprises a sequence encoding a peptide and a sequence encoding a B2M polypeptide. In some

embodiments, the non-naturally occurring sequence comprising an HLA-E further comprises a first linker positioned between the sequence encoding the B2M signal peptide and the sequence encoding the peptide, and a second linker positioned between the sequence encoding the B2M polypeptide and the sequence encoding the HLA-E.

[0392] In some embodiments of the cells, unmodified cells and modified cells of the disclosure, the cell is a mammalian cell.

SUBSTITUTE SHEET (RULE 26) [0393] in some embodiments of the cells, unmodified cells and modified cells of the disclosure, the cell is a human cell.

[0394] in some embodiments of the cells, unmodified cells and modified cells of the disclosure, the cell is a stem cell.

[0395] In some embodiments of the cells, unmodified cells and modified ceils of the disclosure, the cell is a differentiated cell.

[0396] In some embodiments of the cells, unmodified cells and modified ceils of the disclosure, the cell is a somatic cell.

[0397] In some embodiments of the cells, unmodified cells and modified ceils of the disclosure, the cell is an immune cell or an immune cell precursor. In some embodiments, the immune cell is a lymphoid progenitor cell, a natural killer (NK) cell, a cytokine induced killer (CIK) cell, a T lymphocyte (T cell), a B lymphocyte (B-cell) or an antigen presenting cell (APC). In some embodiments, the immune cell is a T cell, an early memory T cell, a stem cell- like T cell, a stem memory T cell (Tscm), or a central memory T cell (Tcm). In some

embodiments, the immune cell precursor is a hematopoietic stem cell (HSC). In some embodiments, the cell is an antigen presenting cell (APC).

[0398] In some embodiments of the cells, unmodified cells and modified ceils of the disclosure, the cell further comprises a gene editing composition. In some embodiments, the gene editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. In some embodiments, the gene editing composition comprises a sequence encoding a nuclease protein or a sequence encoding a nuclease domain thereof. In some embodiments, the e sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof comprises a DNA sequence, an RNA sequence, or a combination thereof. In some embodiments, the nuclease or the nuclease domain thereof comprises one or more of a CRISPR/Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZEN), and an endonuclease. In some embodiments, the CRISPR'Cas protein comprises a nuclease-inactivated Cas (dCas) protein.

[0399] In some embodiments of the cells, unmodified cells and modified cells of the disclosure, the cell further comprises a gene editing composition. In some embodiments, the gene editing composition comprises a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof. In some embodiments, the nuclease or

SUBSTITUTE SHEET (RULE 26) the nuclease domain thereof comprises a nuclease-inactivated Cas (dCas) protein and an endonuclease. In some embodiments, the endonuclease comprises a Clo05l nuclease or a nuclease domain thereof. In some embodiments, the gene editing composition comprises a fusion protein. In some embodiments, the fusion protein comprises a nuclease-inactivated Cas9 (dCas9) protein and a Ck>051 nuclease or a Clo051 nuclease domain. In some embodiments, the gene editing composition further comprises a guide sequence. In some embodiments, the guide sequence comprises an RNA sequence. In some embodiments, the fusion protein comprises or consists of the ammo acid sequence:

MAPKKKRKVEGI KSNI SLLKDELRGQI SHI SHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDG IVYSTTLEDNFGI IVDTKAYSEGYSLPI SQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFI SGS FKGKF EEQLRRLSMTTGVNGSAVNWNLLLGAEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDK KYSIGLAI GTNSVGWA VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI CYLQEI FSNEMAKVDDS FFHRLEES FLVEEDKKHERHPI FGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMI KFRGHFLIEGD LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEK KNGLFGNLIALSLGLTP NFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMI KRYDE HHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFI KPI LEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS GEQKKAIVDLLFKTNRK VTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQ VS GQGDSLHEHIANLAGS PAI KKGI L0TVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRI EEGI K ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD DSIDNKVLTRSDKNRGK SDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI TKHVAQILDSRMNTKY DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALI KKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKK TEVQTGGFSKES ILPKRN SDKLIARKKDWDPKKYGGFDS PTVAYSVLWAKVEKGKSKKLKSVKELLGI TIMERS S E ^' EKNPI DFLEAKGYKEVKKDLI I KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEI IEQI SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFD

TTIDP.KRYTSTKEVLDATLIHQS ITGLYETRI DLSQLGGDGS PKKKRKVS S (SEQ ID NO: 14731) or a nucleic acid comprising or consisting of the sequence:

1 atggcaccaa agaagaaaag aaaagtggag ggcatcaagt caaacatcag cctgctgaaa 61 gacgaactgc ggggacaq t: tagtcacatc agtcacgagt acctqtcact gattgatetg 121 gccttcgaca gcaagcagaa tagactgttt gagatgaaag tgctggaact gctggtcaac 181 gag ta gget tcaagggcag acatctgggc qqq teta gga aacctgacgg ca eq q tac 241 agtaccacac tgqaagacaa etteggaatc attgtegata ccaaggctt ttccgagggc 301 tactctctgc caattaq tea q gca gatgag atggaaaggt acq q cqcqa aaactcaaat 361 agggaegagg aagtcaaccc caataagtgg tgggagaatt teagegagga agtgaagaaa 421 tac ac eg tctttatctc aggeaget c aaaqqqaagt ttgaggaaca gc tqcqqaqa

SUBSTITUTE SHEET (RULE 26) 481 ctgtccatga ctaccqqqq t gaaeggatet gctgtcaacq tqgtcaatct gctgctgggc 541 qcagaaaaga t caggtccgg ggagatqaca attgaggaac tggaaegege catgttcaac 601 aat tc tqaq t ttatcctgaa gtatggagqc gggggaagcg ataagaaata ctcca teqqa 661 ctggccattg gcaccaattc cgtgggctgg gctgtcatca cagacgagt a caaggtgcca 721 agcaagaagt tcaagq tee ta qgggaacacc gatcgccaca q ta tcaaga a aaatctgatt 781 qqagccctgc tgttc.gactc aggcqaqact gctgaagcaa cccgactgaa qcggactgct 841 aggcqccqa t atacccggag aaaaaatcgg a ctgctacc tgcaggaaat tt tcaqcaac 901 gagatggcca agqtqgacga tagtttcttt caccgcctgg aggaatcatt cctggtggag 961 gaag taaga aacacqaqcg q ca tcccatc tttggcaaca t tq tgg a ega agtegettat 1021 cacgagaagt accctactat ctatcatctg aggaagaaac tggtggactc caccgataag 1081 gcagacctgc gcctgatcta t ctggccctg gctcacatga tcaagttccg ggggcatttt 1141 ctgatcgagg gaqatctqaa ccctgacaat tctgatgtgg acaagctgtt catccagctg 1201 gtccagacat acaatcagct gtttgaggaa aacccaatta atgcctcagg cgtggacgca 1261 aaggccatcc tgagcgccag actgtccaaa tcta g g cge.e. tggaaaacct qatcqctcag 1321 ctgccaggag agaagaaaaa cggcctgttt gggaatctga ttgcactgtc cctgggcctg 1381 acaccc.aact tcaaqtctaa ttttgatctg gccgaggacq etaagetgea gctgtccaaa 1441 gacacttatg acgatgacct ggataacctg ctggctcaga teggegatea gtacgcagac 1501 ctqttcctgg ccgctaagaa tctgaqtqac gccatcctgc tgteagatat tctqcgcgtg 1561 aacacagaga ttactaaggc cccactgagt gcttcaatga tcaaaagat tgaegageae 1621 catcaggatc tgaccctqct gaaggctctg gtgaggcaqc aqctgcccga gaaatacaag 1681 qaaatcttct ttgatcagag caagaatgga tacgccggct atattgaegg cqgggctt cc

1741 cagqaqqaq t tctacaagtt catcaagccc a 11 ctggaaa agatggaegg caccqaqqaa 1801 c.tgctggtga agetgaateg ggaggacctg ctgagaaaac agaggacatt tgataacgga

1861 agcatccctc accagat ea tctgggcgaa ctgcacgcca tcctqcgacg gcaggaggac 1921 ttctacccat tt c.tgaagga taaccqcqag aaaat egaaa agatcctgac cttcagaat c 1981 ccc tac ta tag tggggcctct ggcacgggqa aa tagtagat ttgcctgga t gacaaqaaag 2041 tcagaggaaa ctatcacccc ctggaac.ttc gaggaagtqq tegataaagg c.gctagcgca 2101 cagtccttca ttgaaaqqa t q a ca aatttt gacaagaacc tq ccaaatga gaaggtgctg 2161 cccaaacaca gcctgctgta egaatattte acagtgtata aegagetgae taaagtgaag 2221 tacg tcaccg aagggatgcg caagcccgca ttcctgtccg gagageagaa gaaaqcca tc 2281 gtggacctgc tgtttaaqac aaatcggaaa gtgactgtca aacagctgaa ggaagactat 2341 ttcaagaaaa ttgagtgttt cgattcagtg gaaatcagcg gegtegagga caggtttaac 2401 gcctccctgg ggacctacca cgatctqctq aagatcatca aggataagga cttcctggac 2461 aacgaggaaa atgaggacat cctggaggac attgtgctga cactgactct gtttgaggat 2521 c.gc.gaaatga tcqaqqaacg actgaagact tatgcccatc tqttcgatga caaagtgatg 2581 aagcagctga aaagaaggcg ctacaccgga tggggacgcc tgagccgaaa actgatcaat 2641 qqqattagag acaagcagag cggaaaaact a cctggact ttctgaagtc cqatqgcttc 2701 gccaacagga acttcatgca gctgattcac gatgactctc tgaccttcaa ggaggacatc

2761 cagaaagcac aggtg tctqg ccagggggac agtctgcacg aqca ta tege aaacctggcc 2821 ggcagccccg ccatcaagaa agggattctg cagaccgtga aggtggtgga cqaactggt c 2881 aagq teaag g ga cgacacaa acctgagaac a tcgtga ttg agatggcccg cgaaaa tcag 2941 ac.aactcaga agqqccagaa aaacagtcga gaacggatqa agagaat ega ggaaggeate

3001 aaggagctgg ggtcacaqat cctgaaggag catcctgtgg aaaacactca gctgcagaat 3061 qaqaaactgt at c.tgtacta tctgcaqaat ggacgggata tgtacgtgga ccaggagctg 3121 gatat taaaca ga ctgagtga ttatgacg tq qa tgcca teg tccctcagag ct tcctqaag 3181 gatgactcca ttqacaacaa ggtgc.tgacc aggtccgaca agaaccgcgg c.aaatcagat 3241 aatgtgccaa gegaggaagt q g tcaagaaa atgaagaac t actqqagqca gctgctgaat 3301 gccaagctga tcaca cagcg gaaatttqat aacctga eta aggeagaaag aqqaqgcctg 3361 tctgagctgg acaaggccgg cttcatcaag cggcagctgg tggagacaag acagatcact 3421 aagcacgtcg ctcaqattct g g a ta ge.a ga atgaacacaa aqtacgatga aaacgacaag 3481 ctgatcaggg aggtgaaag t cattactctg aaatccaagc tggtgtctga ctttagaaag 3.541 gattataccag t tttataaagt caggqaqatc aa ca a cta e.e. accatgctca tqacqcatac 3601 ctgaacgcag tggtcgggac cgccctgatt aagaaatacc ccaagctgga gtccgagttc 3661 gtgtacggag actataaagt g ta cgatgtc cggaagatqa tcgccaaate. tgagcaggaa 3721 attggcaagg ccaccgctaa gtatttcttt tacagtaaca tcatgaattt ctttaagacc 3781 qaaatcacac tg gcaaatgg ggagatcaqa aaa a g g ce.te. tgattgagac caacqgggag

5UB5TITUTE SHEET (RULE 26) 3841 acaggagaaa tcqtqtggga caagggaagg ga1111gcta ccq tq cgca a agtcctgtcc 3901 atgccccaag tgaatattgt caagaaaact gaagtgcaga ccgggggatt ctctaaggag 3961 agtattctgc ctaagcgaaa ctctgataaa ctqa tcgccc ggaagaaaga ctqqqacccc 4021 aagaagtatg gcqqqttcga ctct ccaaca gtggcttaca gtgtcctggt ggtcgcaaag 4081 gtggaaaagg ggaaq tccaa gaaactgaag tctgtcaaaq aq c cq c tggg aatcactatt 4141 atggaaegea gctccttcga gaagaatcct atcgattttc tggaagccaa gggctataaa 4201 gagg tgaaga aagacctgat cattaaqc tq cca a a a tact ca ctg111ga gc tggaaaa c 4261 ggacgaaagc gaatgctggc aagcgccgga gaactgcaga agggcaatga gctggccctg 4321 ccctccaaat acqtqaac t t cctgtatctg gctagccact acqaqa a a ct gaaggggtcc 4381 cctgaggata acgaacagaa gcagctgttt gtggagcagc acaaacatta tctggacgag 4441 atcattgaac agatttcaga gttcagcaag agagtgatcc tggctgacgc aaatctggat 4501 aaagtcctga gcqcatacaa gacaaaccaa tccqqq a qca ggccgaaaat 4561 atcatt cat c tgttcaccct gacaaacctg ggcgcccctg cagccttcaa gtattttgac 4621 accacaatcg at egg gag atacacttct a ccaaag agg tgctggatgc taccctqatc 4681 caccagagta ttaccggcct gtatgagaca cgcatcgacc tgt cacagct gggaggegat 4741 gggagcccca agaaaaagcg gaagqtqtct agttaa (SEQ ID NO: 147 3 2 ) , In some embodiments, the fusion protein comprises or consists of the amino acid sequence:

1 MPKKKRKVEG IKSNI SLLKD ELRGQI SHI S HEYLSLI DLA FDSKQNRLFE MKVLELLVNE 61 YGFKGRHLGG SRKPDGIVYS TTLEDNFGII VDTKAYSEGY SLPI SQADEM ERYVREN SNR 121 DEEVNPNKWW ENFSEEVKKY YFVFISGSFK GKFEEQLRRL SMTTGVNGSA VNWNLLLGA 181 EKIRSGEMTI EELERAMFNN SEFILKYGGG GSDKKYSIGL AI GTNSVGWA VITDEYKVPS 241 KKFKVLGNTD RHSIKKNLIG ALLFDSGETA EATRLKRTAR RRYTRRKNRI CYLQEI FSNE 301 MAKVDDSFFH RLEES FLVEE DKKHERHPIF GNIVDEVAYH EKYPTI YHLR KKLVDSTDKA 361 DLRLIYLALA HMIKFRGHFL I EGDLNPDNS DVDKLFIQLV QTYNQLFEEN PINAS GVDAK 421 AI LSARDSKS RRLENLIAQL PGEKKNGLFG NLIALSLGLT PNFKSNFDLA EDAKLQLS KD 481 TYDDDLDNLL AQIGDOYADL FLAAKNLSDA ILLSDILRVN TEITKAPLSA SMIKRYDEHH 541 ODLTLLKALV RQQLPEKYKE I FFDOSKNGY AGYIDGGASQ EEFYKFIKPI LEKMDGTEEL 601 LVKLNREDLL RKQRTFDNGS I PHQIHLGEL HAI LRRQEDF YPFLKDNREK IEKILTFRIP 661 YYVGPLARGN SRFAWMTRK3 EETITPWNFE EWDKGASAQ S FI ERMTNFD KNLPNEKVLP 721 KHSLLYEYFT VYNELTKVKY VTEGMRKPAF LSGEQKKAIV DLLFKTNRKV TVKOLKEDYF 781 KKI ECFDSVE ISGVEDRFNA SLGTYHDLLK IIKDKDFLDN EENEDI LEDI VLTLTLFEDR 841 EMI EERLKTY AHLFDDKVMK QLKRRRYTGW GRLSRKLING IRDKQSGKTI LDFLKSDGFA 901 NRNFMQLIHD DSLTFKEBIQ KAQVSGQGDS LHEHIANLAG SPAIKKGILQ TVKWDELVK 961 VMGRHKPENI VI EMARENQT TQKGQKNSRE RMKRI EEGI K ELGSQILKEH EVENTQLQNE 1021 KLYLYYLQNG RDMYVDQELD INRLSDYDVD AIVPQSFLKD DS IDNKVLTR SDKNRGKSDN 1081 VPSEEWKKM KNYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK 1141 HVAQILDSRM NTKYDENDKL I REVKVITLK SKLVSDFRKD FQFYKVREIN NYHHAHDAYL 12 01 NAWGTAL I K KYPKLESEFV YGDYKVYDVR KMIAKSEQEI GKATAKYFFY SNIMNFFKTE 1261 ITLANGEIRK RPLIETNGET GEIVWDKGRD FATVRKVLSM PQVNIVKKTE VQTGGFSKES 1321 ILPKRNSDKL IARKKDWDPK KYGGFDSPTV AYSVLWAKV EKGKSKKLKS VKELLGITIM 1381 ERSSFEKNPI DFLEAKGYKE VKKDLIIKLP KYSLFELENG RKRMLASAGE LQKGNELALP 1441 SKYVNFLYLA SHYEKLKGS P EDNEQKQLFV EQHKHYLDEI IEQISEFSKR VI LADANLDK

SUBSTITUTE SHEET (RULE 26) 1501 VLSAYNKHRD KPIREQAENI IHLFTLTNLG APAAFKYFDT TI DRKRYTST KEVLDATLIH 1561 QSITGLYETR IDLSQLGGDG SPKKKRKV ( ( SEQ ID NO: 14733) OG a nucleic acid comprising or consisting of the sequence:

1 atgcctaaga agaageggaa ggtggaaggc atcaaaagca aca tctccct cctgaaagac

61 qaactccggg ggcagattag ccacattagt cacgaat ace tctccctcat cgacctggct 121 ttcqa tagca agcagaacag gctctttgaq a tqaaagtgc tggaactgct eg tcaa tqag 181 tacgggttca agggtcgaca cct eggegga tctaggaaac cagacggcat cgtgtatagt 241 accacactgg aagacaac11 tgggatcatt gtggatacca aggea tactc tgagggttat 301 agtctgccca tttcacaggc egaegagatq gaa eggt eg tgcgcgagaa ctcaaataga 361 gatgaggaag tcaaccctaa caagtggtgg gagaaettet ctgaggaagt gaagaaatac 421 tacttcgtct ttatcagcgg q tccttcaag ggtaaatttq aggaacagct caggagactg 481 agcatgact ccggcgtgaa tggcagcgcc gtcaacgtgg tcaatctgct cctgggcgct 541 gaaaagattc ggagcggaga gatgaccatc gaagagctgg agagggcaat gtttaataat 601 agcgagttta tcctgaaata cggtggcggt ggatccgata aaaagtattc ta t tqqccca 661 gccatcggca ctaattccq t tggatgggct gtcataaccg atgaa ta ca a agtaccttca 721 aagaaattta aggtgttggg gaacacagac egteattega ttaaaaagaa tettateggt 781 gccctccta t tcgatagtgg egaaaeggea gagg cga ctc gcctgaaacg aaccqctcgg 841 agaaggtata cacgtcgcaa gaaccgaata tgttacttac aagaaatttt tagcaatgag 901 atggccaaag ttgaegat tc tttctttcac cgtttggaag aq tee t tcct tgtegaagag 961 gacaagaaac atga eggea ccccatcttt ggaaacatag tagatgaggt ggcatatcat 1021 gaaaaq tacc caaegattta tcacctcaqa aaaaagctag ttgactcaac tgataaaqeg 1081 gacctgaggt taatetaett gget c.ttgcc catatgataa agttccgtgg gcactttctc 1141 attgagggtg atetaaa tee gga easeteg gatgtcgaca aactg ttea t ccagttagta 1201 caaacctata at cagttgtt tgaaqaqaac cctataaatg caagtggcgt ggatgegaag 1261 geta t tet ta gcgcccgcct ctctaaatcc eqaeggetag aaaacctgat cgcacaa t ta 1321 cccggagaga agaaaaatgg gttgttcggt aaccttataq cgctctcact aggcctgaca

1381 ccaaatttta ag tcgaac11 ega ettaget gaagatgcca aa t tqcagct tagtaaggac

1441 aegtaega tg aegatetega caatctactq gcacaaattg gagatcagta tgcqqactta 1501 tttttggctg ccaaaaacct tagegatgea atcctcctat ctgacatact gagagttaat 1561 actgagatta ccaaggcqcc g tta teeget tcaatgatca aaaqq ta eg tgaacatcac 1621 caagacttga cacttctcaa ggccctagtc cgt cagcaac tgcctgagaa atataaggaa 1681 atattctttg atcagtcgaa aaacgggtac gcaggttata ttgaeggegg agcqaqtcaa 1741 gaggaattet acaagtttat caaacccata ttagagaaga tggatgggac ggaagagttg 1801 cttgtaaaac tcaatcqcqa agatctactg cgaaagcaqc qqactttcga caacggtagc 1861 attccacatc aaatccact t aggegaattg catgctatac ttagaaggea ggaggatttt 1921 tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa tcctaacctt tcqcatacct 1981 tactatgtgg gacccctggc ccgaggg ac tctcggttcg catggatgac aagaaagtcc 2041 gaagaaacga ttactcca tg qaattttgag gaagttgteg a taaaqgtgc gtcagctcaa 2101 tcgttcatcg agaggatgac caactttqac aagaatttac cgaacgaaaa agtattgcct 2161 aagcacag 11 ta etttaega gtatttcaca q tq ta ca atg a ctcacgaa ag t taaq tat 2221 gtcactgagg gcatgcgtaa aceegcc11.1. etaageggag aacagaagaa agcaatagta 2281 gatctgttat tcaagaccaa ccgcaaagtg acagttaagc aa t tqaaaga ggactacttt 2341 aagaaaattg aatgettega ttctgtcgag atctccgggg tagaagateg atttaatgcg 2401 tcac t tgg ta cgtatcatga cctcctaaaq a fcaattaaag ataaggaett cc tgga taa c 2461 gaagagaatg aagata tet t agaagatata gtgttgactc ttaccctctt tgaagategg 2521 gaaatgattg aggaaagact aaaaacatac get c cctgt tegaegataa ggttatgaaa 2581 cagttaaaga ggegtegeta tacgggctqq gga ega ttgt egcggaaact tatcaacggg 2641 ataagagaca agcaaagtgg taaaactatt ctcgattttc taaagagcga egget tegee 2701 aat ggaact ttatgeaget gatccatgat gactctttaa ccttcaaaga ggatatacaa 2761 aaggcacagg tttccggaca aggggactca ttgcacgaac atattgegaa tcttgctggt 2821 tcgccagcca tcaaaaaggg catactccaq acagtcaaag tagtggatga gctaqttaag 2881 gtcatgggac gtcacaaacc ggaaaacatt gtaategaga tggeaegega aaatcaaacg

2941 actcagaagg ggcaaaaaaa cagtcgagag eggatgaaga qaatagaaga gggtattaaa

3001 gaactgggca gccagatctt aaaggageat cctgtggaaa atacccaatt gcagaacgag

SUBSTITUTE SHEET (RULE 26) 3 061 aaactttacc tctattacct acaaaatgga agggacatqt atgttgatca ggaactggac 3121 ataaaccgtt tatetgatta egaegtegat gccattgtac cccaatcctt tttgaaggac 3 18 1 gat teaa eg a caataaagt gcttacacqc teg ga a aga accgagggaa aaq tqacaa t 324 1 gttccaagcg aggaagtegt aaagaaaatg aagaactatt ggeggeaget cctaaatgcg 3301 aaactgataa cgcaaaqaaa q ttcgataac ttaactaaag ctqaqagggg tggcttgtct 3361 gaacttgaca aggeeggatt tattaaacgt cagct cgtgg aaacccgcca aatcacaaag 342 1 catq t tq ca c aqatactaga ttcccgaa tq a.a. ta ega aat egaegagaa ega taaq c tq 34 8 1 attegggaag tcaaagtaat cact ttaaag tcaaaattgg tgteggaett cagaaaggat 354 1 111 caa c t ataaaq t tag qgagataaat aactaccacc a tq eg ca ega egettatett 3 601 aatgeegteg tagggaccgc actcattaag aaatacccga agetagaaag tgagtttgtg 3 661 tatggtgatt acaaagttta tgacgtccgt aagatgateg cgaaaagcga acaggagata 7^1 ggcaaggcta caqccaaata cttcttttat tctaacatta tqaatttctt t g cggaa 37 8 1 atcactctgg caaacggaga gatacgcaaa cgacctttaa ttgaaaccaa tggggagaca

3 84 1 qqtgaaa eg ta tgggat a gggccqqqac ttegega egg tgagaaaagt tttqtccatg 3 901 cccc cTtca acatagtaaa gaaaactgag gtgcagaccg gagggttttc aaagga cg 3 961 attcttccaa aaaggaatag tga t agctc atcgctcqta aaaaggactg ggacccgaaa 4 02 1 aagtacggtg gettegatag ccctacagtt gcctattctg tcctagtagt ggcaaaagtt

4 08 1 qaqaaqggaa aatccaag a actgaaqtca gtcaaagaat tattggggat aacqattatg 4141 gagegetegt cttttgaaaa gaaccccatc gacttccttg aggegaaagg ttacaaggaa 4 2 01 gtaaaaaagg atctcataat taaa ct cca aagtataqtc tgtttgagtt agaaaatggc 42 61 cqaaaacgga tgttggctag egeeggagag ett caaaagg ggaacgaact cqcactaccg 4 32 1 tetaaa acg tq a a 1 eet gtatttagcq tccca ttacg agaagttgaa agq t tcacct 4 38 1 gaagataacg aacagaagca actttttgtt gagcagcaca aacattatct egaegaaate 4 44 1 atagagcaaa tttcggaa t t cagtaagaga gtcatcc tag c tga tq cca a tctggacaaa 4 501 qtattaagcg cat caacaa gcacagggat aaacccatac gtgageagge ggaaaatatt 4 561 atcca 1 q t ttactcttac caacctcgqc qctccagccg cattcaagta 111 tqaca. ca 4 62 1 aegatagate gcaaacgata cacttctacc aaggaggtgc tagaegegae actgattcac 4 68 1 caatccatca cggga t ta ta tga a actegg atagatttgt cacaqcttgg gggtgacgga 4 74 1 tcccccaaga agaagaggaa agtctga (SEQ ID NO: 1 47 3 4) .

[0400] In some embodiments of the ceils, unmodified cells and modified cells of the disclosure, a nanotransposon comprises the gene editing composition comprising a guide sequence and a sequence encoding a fusion protein comprising a sequence encoding an inactivated Cas9 (dCas9) and a sequence encoding a Clo051 nuclease or a nuclease domain thereof.

[0401] In some embodiments of the cells, unmodified cells and modified cells of the disclosure, the cell expresses the gene editing composition transiently.

[0402] In some embodiments of the cells, unmodified cells and modified cells of the disclosure, the cell is a T cell and the guide RNA comprises a sequence complementary to a target sequence encoding an endogenous TCR In some embodiments, the guide RNA comprises a sequence complementary to a target sequence encoding a B2M polypeptide.

[0403] In some embodiments of the cells, unmodified cells and modified cells of the disclosure, the guide RNA comprises a sequence complementary' to a target sequence within a safe harbor site of a genomic DN A sequence.

SUBSTITUTE SHEET (RULE 26) [0404] in some embodiments of the cells, unmodified cells and modified cells of the disclosure, the CloOSl nuclease or a nuclease domain thereof induces a single or double strand break in a target sequence. In some embodiments, a donor sequence, a donor plasmid, or a donor nanotransposon intra-ITR sequence integrated at a position of single or double strand break and/or at a position of cellular repair within a target sequence.

[0405] The disclosure provides a composition comprising a modified cell according to the disclosure. In some embodiments, the composition further comprises a pharmaceutically- aceeptabie carrier.

[0406] The disclosure provides a composition comprising a plurality of modified cells according to the disclosure. In some embodiments, the composition further comprises a pharmaceuticaliy-aeceptable carrier.

[Q4Q7] The disclosure provides a composition of the disclosure for use in the treatment of a disease or disorder.

[0408] The disclosure provides the use of a composition of the disclosure for the treatment of a disease or disorder.

[Q4Q9] The disclosure provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeuticaliy-effective amount of a composition of the disclosure. In some embodiments, the subject does not develop graft vs. host (GvH) and/or host vs. graft (HvG) following administration of the composition. In some embodiments, the administration is systemic. In some embodiments, the composition is administered by an intravenous route. In some embodiments, the composition is administered by an intravenous injection or an intravenous infusion.

[0410] The disclosure provides a method of treating a disease or disorder comprising administering to a subject in need thereof a therapeuticaliy-effective amount of a composition of the disclosure. In some embodiments, the subject does not develop graft vs. host (GvH) and/or host vs. graft (H vG) following administration of the composition. In some embodiments, the administration is local. In some embodiments, the composition is administered by an intra- tumoral route, an mtraspinal route, an mtracerebroventricular route, an intraocular route or an intraosseous route. In some embodiments, the composition is administered by an intra-tumoral injection or infusion, an intraspinal injection or infusion, an mtracerebroventricular injection or infusion, an intraocular injection or infusion or an intraosseous injection or infusion.

SUBSTITUTE SHEET (RULE 26) [0411] in some embodiments of the methods of treating a disease or disorder of the disclosure, the therapeutically effective dose is a single dose and wherein the allogeneic cells of the composition engraft and/or persist for a sufficient time to treat the disease or disorder. In some embodiments, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of doses in between that are manufactured

simultaneously.

[0412] In some embodiments of the methods of treating a disease or disorder of the disclosure, the therapeutically effective dose is a single dose and wherein the autologous cells of the composition engraft and/or persist for a sufficient time to treat the disease or disorder. In some embodiments, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of doses in between that are manufactured

simultaneously.

[0413] A construct for constitutive expression of Interleukin 2 Receptor subunit gamma, GFP, and DHFR (nano.PB.EFla.IL2RG-T2A-GFP~T2A~DHFR ) is encoded by a sequence comprising:

ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAA TT

GACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATA T

T AAGTTTT ATTATATTTAC ACTTAC AT ACTA ATAATA AATTC AACAA AC AATTT ATTT

ATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAAC

AAAACTTTTAtcgaatacctecagcccgggggatgcagagggacagcccccccccaa agcccccagggatgtaattacgtccc tcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccc cgcatccccgagccggcagcgtgcgg ggacageecgggcaeggggaaggtggeacgggatcgettectctgaaegcttctcgetge tctttgageetgcagacaectggggggat. acggggaaaagttgactgtgcctttcgatcgaaccatggacagttogc/r/g-caaag /ggato grtr c g g gg /c/r/g-c agctaatggaccttctaggtcltgaaaggagtgggaattggctccggtgcccgtcagtgg gcagagcgcacalcgcccacagtccccg agaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcgggglaa actgggaaagtgatglcgtgtactgg ctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaac gttctttttcgcaacgggttgccgccagaa cacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggccctt gcgtgccttgaattacttccacctggctgcag tacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgc gcttaaggagccccttcgcctcgtgcttgag ttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcct gtctcgctgctttcgataagtctctagccat ttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgc gggccaagatctgcacactggtatttcggtttttg gggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcc tgcgagcgcggccaccgagaatcg

SUBSTITUTE SHEET (RULE 26) gacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgta tcgccccgccctgggcggcaaggctggc ccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggag ctcaaaatggaggacgcggcgctcgg gagagcgggcgggtgagtcacccacacaaaggaaaagggccttccgtcctcagccgtcgc ttcatgtgactccacggagtaceggg cgccgtccaggcacctcgattagttctcgagctittggagtacgtcgtcttiaggttggg gggaggggttttatgcgatggagtticcccac actgagtgggtggagactgaagttaggccagctlggcacttgatgtaattctccttggaa tttgccclttttgagtttggatcttggttcattct caagcctcagacagtggttcaaagttttttctccatttcaggtgtcgtgagaatctaata cgactcactatagggtgtgctgtctcatcatttt ggcaaagattggccaccaagcttaccgccatgctgaagcccagcctgccttttaccagcc tgctgttcctgcagctgcctctgcttggc gtgggcctgaataccaccatcctgacacctaacggcaacgaggatacaaccgccgacttc ttcctgaccaccatgcctaccgatag cctgagcgtgtccacactgccactgcctgaggtgcagtgcttcgtgttcaacgtcgagta catgaactgcacctggaacagctccag cgagccccagcctaccaatctgacactgcactattggtacaagaacagcgacaacgacaa ggtgcagaagtgcagccactacct gttcagcgaggaaatcaccagcggctgccagctgcagaagaaagagatccacctgtacca gaccttcgtggtgcagctccaggat cctagagagcctagaaggcaggccacacagatgctgaaactgcagaacctcgtgatcccc tgggctcccgaaaacctgactctgc acaagctgagcgagagccagctggaactgaactggaacaaccggttcctgaatcactgcc tggaacatctggtgcagtaccggac egactgggatcactettggaeagagcagagcgtggactaceggcacaagttcagcctgcc atetgtggaeggccagaagcggtae acctttagagtgcggagccggttcaaccctctgtgtggatctgctcagcattggagcgag tggtcacacccaatccactggggcagc aacaccagcaaagagaaccccttcctgttcgccctggaagccgtggttatcagcgtgggc tctatgggcctgatcatctccctgctgt gcgtgtacttctggctggaaagaaccatgcctcggatccccactctgaagaacctggaag atctcgtgaccgagtaccacggcaac tteagtgcttggagcggcgtgtcaaaaggactggecgaaagcctgeagectgactacage gagagactgtgtctggtgtctgagat ccctcctaaaggcggcgctctcggagaaggacctggtgcctctccatgcaatcagcacag cccttattgggcccctccttgctacac cctgaagcctgaaacaggcagcggcgaaggcagaggctctctgcttacatgtggcgacgt cgaagagaatcccggaccta gg gagc aagggcgaggagctgltcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagtlcagcgtglccgg cgagggcgagggcgatgccacctacggcaagctgaccctgaagitcatctgcaccaccgg caagcigcccgtgccctggcccaccct cgtgaccaccctgacciacggcgtgcagtgcitcagccgciaccccgaccacatgaagca gcacgacitcitcaagtccgccatgccc gaaeeciacgtccaggagcgcaccatcitcttcaaggacgacggcaactacaagacccgc gccgaggtgaagticgagggcgaca ccctggtgaaccgcatcgagctgaagggcaicgacUcaaggaggacggcaacatcciggg gcacaagciggagtacaaciacaa cagccacaacgtctatatcatggccsacaagcagaagaacggcatcaaggtgaacttcaa gatccgccacaacatcsassacssc ascstscasctceccsaccactaccascasaacacccccatcssceacssccccstscts ctscccsacaaccactacctsascac ccastccsccctsascaaasaccccaaceasaascsisatcacatsstcctsctssasit cstsaccsccsccsssatcactcicss ca/ggacg gcfff/ac ggaaggaagaggeagcctgctgacatgtggcgaegiggaggagaaccctggcecaatggt gggcagcc tgaattgtatcgtggccgtgtcccagaacatgggcatcggcaagaatggcgattttcctt ggccccctctgagaaatgagtccagat actttcagaggatgaccacaaccagctccgtggagggcaagcagaacctggtcatcatgg gcaagaagacatggttctctatccc

SUBSTITUTE SHEET (RULE 26) agagaagaaccgccccctgaagggccggatcaatctggtgctgagcagggagctgaagga gccaccccagggagcacactttct

caagtacaagttcgaggtgtatgagaagaacgattgataacatatgcctttaattaaaca ctagttctatagtgtcacctaaattcccttagt gagggttaatggccgtaggccgccagaattgggt

cattttatgtttcaggttcagggggaggtgtgggaggttttttcggactctaggacctgc gcatgcgcttggcgtaatcatggtcatagctgtttc ctgttttccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaagcgttcag aggaaagcgatcccgtgccaccttccccgtg cccgggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcg gagccccgggcggctcgctgctgcc ccctagcgggggagggacgtaattacatccctgggggctttgggggggggctgtccctct caccgcggtggagctccagcttttgttcgaa

tcgagacctctagctagcccgcctaatgagcgggcttttttttggcttgttgtccac aaccgttaaaccttaaaagctttaaaagccttatatattc ttttttttcttataaaacttaaaaccttagaggctatttaagttgctgatttatattaat tttattgttcaaacatgagagcttagtacgtgaaacatgag agcttagtacgttagccatgagagcttagtacgttagccatgagggtttagttcgttaaa catgagagcttagtacgttaaacatgagagcttag tacgtactatcaacaggttgaactgctgatccacgttgtggtagaattggtaaagagagt cgtgtaaaatatcgagttcgcacatcttgttgtct gattattgatttttggcgaaaccatttgatcatatgacaagatgtgtatctaccttaact taatgattttgataaaaatcattacctagggaagactc

(SEQ ID NO: 14703)

[0414] The 3’ITR of the nano.PB.EFla.IL2RG-T2A-GFP-T2A-DHFR construct is encoded by a sequence comprising:

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA

CGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATT A

AGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTA T

GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

04151 The Insulator of the nano.PB.EFla.IL2RG-T2A-GFP-T2A-DHFR construct is encoded by a sequence comprising:

gagggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggg gcagcagcgagccgcccggggctccgc

SUBSTITUTE SHEET (RULE 26) tccggtceggcgctecceccgcatceccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggaicgctttec tctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0416] The EFla Promoter of the nano.PB.EF 1 a.IL2RG-T2A-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

agctttgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggac cttctaggtcttgaaaggagtgggaattggctc cggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagg ggtcggcaattgaaccggtgcctag agaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttccc gagggtgggggagaaccgtatataagtg cagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtg ccgtgtgtggttcccgcgggcctggcctctt tacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattct tgatcccgagcttcgggttggaagtgggtggg agagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggc ctgggcgctggggccgccgcgtgcgaatc tggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttt tgatgacctgctgcgacgctttttttctggcaagat agtettgtaaatgcgggecaagatctgcacactggtatttcggtttggggcegcgggegg cgacggggcccgtgcgtcccagcgcac atgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctca agctggccggcctgctctggtgcctg gcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccag ttgcgtgagcggaaagatggccgcttc ccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtg agtcacccacacaaaggaaaagg gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccagg cacctcgattagttctcgagcttttggagtac gtcgtcttaggttggggggaggggtttatgcgatggagtttccccacacigagtgggtgg agacigaagttaggccagcttggcactig atgtaattctccttggaatttgcccttttgagtttggatcttggttcattctcaagcctc agacagtggtcaaagttMttcttccatttcaggtg tcgtga (italicized) (SEQ ID NO: 14706)

[0417] The IL2RG of the nano.PB.EF 1 a.IL2RG-T2 A-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

atgctgaagcccagcctgccttttaccagcctgctgttcctgcagctgcctctgctt ggcgtgggcctgaataccaccatcctgacac ctaacggcaacgaggatacaaccgccgacttcttcctgaccaccatgcctaccgatagcc tgagcgtgtccacactgccactgcct gaggtgcagtgcttcgtgttcaacgtcgagtacatgaactgcacctggaacagctccagc gagccccagcctaccaatctgacact gcactattggtacaagaacagcgacaacgacaaggtgcagaagtgcagccactacctgtt cagcgaggaaatcaccagcggctg ccagctgcagaagaaagagatccacctgtaccagaccttcgtggtgcagctccaggatcc tagagagcctagaaggcaggccac acagatgctgaaactgcagaacctcgtgatcccctgggctcccgaaaacctgactctgca caagctgagcgagagccagctggaa ctgaactggaacaaccggttcctgaatcactgcctggaacatctggtgcagtaccggacc gactgggatcactcttggacagagca gagcgtggactaccggcacaagttcagcctgccatctgtggacggccagaagcggtacac ctttagagtgcggagccggttcaac cctctgtgtggatctgctcagcattggagcgagtggtcacacccaatccactggggcagc aacaccagcaaagagaaccccttcct gttcgccctggaagccgtggttatcagcgtgggctctatgggcctgatcatctccctgct gtgcgtgtacttctggctggaaagaacc

SUBSTITUTE SHEET (RULE 26) atgcctcggatccccactctgaagaacctggaagatctcgtgaccgagtaccacggcaac ttcagtgcttggagcggcgtgtcaaa aggactggccgaaagcctgcagcctgactacagcgagagactgtgtctggtgtctgagat ccctcctaaaggcggcgctctcgga gaaggacctggtgcctctccatgcaatcagcacagcccttattgggcccctccttgctac accctgaagcctgaaaca (bold) (SEQ ID NO: 14707).

[0418] The EGFP of the nano.PB.EFla.IL2RG-T2A-GFP-T2A-DHFR construct is encoded by a sequence comprising:

qtggtgqgcqqgg^cgq Q c tcqccgg tgKf lccqtcctggtcgqgctggqcggcgqcgtqqqcggccqcqqgttcqg cgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctg caccaccggcaagctgcccgtgccctg gcccaccctcgtgaccaccctgacctacggcgtecagtgcttcagccgctaccccgacca catgaagcagcacgacttcttcaagtcc gccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactac aagacccgcgccgaggtgaagttcga ss2csacaccct22tsaacc2catcsasct2cia222catc2acttcaassa22acssca acatcctsssscacaasctssa2tac aactacaacagccacaacgictataicatggccgacaagcagaagaacggcaicaaggtg aacitcaagaiccgccacaacaicga ggacggcagc2tgcagcicgccgaccactaccagcagaacacccccatcggcgacggccc cgtgctgct2cccgacaaccactac ctsagcacccagtccgccctsaecaaagaccccaacgamascstsatcacatggtcctsc tggagttcetsaccgccsccsssatc actctcggcatggacgagctgtacaag (italicized and underlined) (SEQ ID NO: 14708)

[0419] The DHFR of the nano.PB.EF! a.IL2RG-T2A-GFP-T2A-DHFR construct is encoded by a sequence comprising:

laatggcgattttccttggccccctctgaga tggttctctatcccagagaagaaccgccccctgaagggccggatcaatctggtgctgagc agggagctgaaggagccaccccagg gagcacactttctgtccaggtctctggacgatgccctgaagctgaccgagcagcctgagc tggccaacaaggtggacatggtgtgg atcgtgggcggctctagcgtgtataaggaggccatgaatcaccctggccacctgaagctg ttcgtgacacggatcatgcaggacttt gagtccgataccttctttccagag;

gaacgattga (bold underlined) (SEQ ID NO: 14709) B.EF 1 a IL2RG-T2A-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

taagatacattgatgagtttggacaaaccacaaetagaatgcagtgaaaaaaatgcttta tttgtgaaatttgtgatgctatgctttatttgt aaccattataagctgcaataaacaagt (bold italicized) (SEQ ID NO: 14710).

The 5’ITR of the nano.PB.EF 1 a.lL2RG-T2A-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

SUBSTITUTE SHEET (RULE 26)

asaatscat2C2tcaattttacscas ctatctttctass2 (bold italicized underlined) (SEQ ID NO: 14711).

[0422] A vector for erythroid-specific expression of a BCL! 1 A targeted shRNA and the constitutive expression of GFP and DHFR (nano.PB-Ank-BCLl lashRNA-MND.GFP-T2A-

DHFR construct) is encoded by a sequence comprising:

ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGT

AAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAA T

AGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAA

TTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATA A

AGTAACAAAACTTTTAtcgaatacctgcagcccgggggatgcagagggacagccccc ccccaaagcccccagggatgta attacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggtccg gcgctccccccgcatccccgagccggca gcgtgcggggacagcccgggcacggggaaggtggcacgggatcgcttcctctgaacgctc tcgctgctctttgagcctgcagacacct

& tecm& a R&cteX8CCtttcg&t£gc0tcgaaggggcaaccaggggtccgcgccggcc gaggcctggggag cggggcctcctggggtgggggaggaggtgctcttgtaatctgcggtggtccccaggcggg cgccacccctccgcccgcccgtgccgg gagcgcccggcccgacagcaagcgcctctggggccgataaggccctcgggggcctggccc gcacgicacaggccccgcagaggc tgcggtgagtccgccagccccagctgctcctcctcaagcccccaaggcccttcggcggca attcccaccggggt&ccgtaca&gtmt igggccctacgagcttgctgtttgaatgaggcttcagtactttacagaatcgttgcctgc acatcttggaaacacttgctgggattact icgacltettaaeceaaeagaaggetcgagaaggtatattgetgttgacagigagcgceg eacagaacaeteatggatttagtgaa gccacagatgtaaatccatgagtgttctgtgcgttgcctactgcctcggacttcaagggg ctacaattggagcaattatcttgtttact aaaactgaataccttgctatctctttgatacatttttacaaagctgaattaaaatggtat aaattaaatcactgcgtcgacatcgaacc atggacaetGATCCATCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGC

TCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGAT

AGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCT

GTAGGTTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGG

CCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGT

TGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCG

GCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTA

GAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTT

ATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCG A

GCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGgggccaccaagcttgtccga attctaatacga

SUBSTITUTE SHEET (RULE 26) ctcactatagggtetectgtctcatcattttggcaaagattggccaccaagcttaccecc a gg/gagcaagggcgaggagc/gttoaccg gggtggtgcecatcctggtcgagctggacggegacgtaaacggccacaagttcagcgtgt ccggcgagggegagggcgatgccacc tacggcaagctgaecctgaagttcaictgcaccaceggcaagctgeccgigccciggccc acccicgtgaccaccetgacctacggcg tgcagtgctteagccgctaccecgaccacatgaagcagcaegacttcttcaagtccgcca tgeccgaaggctacgtccaggagcgca ccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcg acaccctggtgaaccgcatcgagctg aagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactac aacagccacaacgtctatatcatggc cgacaagcagaagaacggc tcaaggigaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgacc acia ccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgag cacccagtccgccctgagcaaagac cccaacgagaagcgtgatcacatggtcctgctggagttcgtgaccgccgccgggatcact ctcggcatggacgagctgtacaagsaa

tgaatcaceetggccacetgaagctgttcgtgaeacggateatgcaggactttgagt cegataecttcttteeagagategacetg

ICC M& agaagaacgattgataacatatgcctttaattaaacactagttctatagtgtcacctaaa ttccctttagtgagggttaatggccgtaggccgc caga .\tgg^.ccagacaigataagatacattgatgagttggacaaaccacaactagaatgcag tgaaaaaaatgctttatttgtgaaa ttgtgatgctattgcttatttgtaaccattataagctgcaataaacaagttaacaacaac aattgcatiicattt atgtticsLggttc&gggggp ggtgtgggaggtttttcggactctaggacctgcgcatgcgcttggcgtaatcatggtcat agctgtttcctgttttccccgtatccccccaggt gtctgcaggctcaaagagcagcgagaagcgttcagaggaaagcgatcccgtgccaccttc cccgtgcccgggctgtccccgcacgctgc cggctcggggatgcggggggagcgccggaccggagcggagccccgggcggctcgctgctg ccccctagcgggggagggacgtaat tacatccctgggggctttgggggggggctgtccctctcaccgcggtggagctccagcttt tgttcgaatggggccccccctcgagggtatc

cecctcacessaectccaasceeceactsasatetcctaaatecacaeeeaceeatt eecectatttaeaaaeaeasascaatatt aaigagcgggcittittttggctEgttgtccacaaccgtiaaacctiaaaagctitaaaa gcctiatatatEcttttitttcttataaaacttaaaaccti agaggctatttaagttgctgatttatattaattttattgttcaaacatgagagcttagta cgtgaaacatgagagcttagtacgttagccatgagag cttagtacgttagccatgagggtttagttcgttaaacatgagagcttagtacgttaaaca tgagagcttagtacgtactatcaacaggttgaact

SUBSTITUTE SHEET (RULE 26) gctgatccacgttgtggtagaattggtaaagagagtcgtgtaaaatatcgagttcgcaca tcttgttgtctgattattgatttttggcgaaaccatt tgatcatatgacaagatgtgtatctaccttaacttaatgattttgataaaaatcattacc tagggaagactc (SEQ ID NO: 14712).

[0424] The 3 TTR of the nanoPB. Ank-BCLl 1 ashRNA. MND. GFP-T2A-DHFR construct is encoded by a sequence comprising:

[0425] CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAA AATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAG ATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTT ATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGT

AAC AAAAC TTTTA (ALL CAPS) (SEQ ID NO: 14704).

[Q426] The Insulator of the nanoPB. Ank-BCLl lashRNA.MND.GFP-T2A-DHFR construct is encoded by a sequence comprising:

gagggacagcccccccccaaagcccccagggatgtaatacgtccctcccccgctagg gggcagcagcgagccgcccggggctccgc tccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggatcgctttcc tctgaacgettctcgctgctetttgagcctgcagacacctggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0427] The Ank rin-1 Promoter of the nanoPB. Ank-BCLl 1 ashRNA.MND. GFP-T2A-DHFR construct is encoded by a sequence comprising:

cgcgtcgaaggggcaaccaggggtecgcgccggccgaggcctggggagcggggcctc ctggggtgggggaggaggtgctcttgt aatctgcggtggtccccaggcgggcgccacccctccgcccgcccgtgccgggagcgcccg gcccgacagcaagcgcctctggggc cgataaggccctcgggggcctggcccgcacgtcacaggccccgcagaggctgcggtgagt ccgccagccccagctgclcctcctca agcccccaaggcccttcggcggcaattcccaccgg (italicized) (SEQ ID NO: 14701).

[0428] The miRE sh49 BCL11A of the nanoPB. Ank-BCLl 1 ashRNA.MND GFP-T2A-DHFR construct is encoded by a sequence comprising:

gtacaagtaatagggccctacgagcttgctgtttgaatgaggcttcagtactttaca gaatcgttgcctgcacatcttggaaacactt gctgggattacttcgacttcttaacccaacagaaggctcgagaaggtatattgctgttga cagtgagcgccgcacagaacactcat ggatttagtgaagccaeagaigiaaateeatgagtgttetgtgcgttgeetaetgccteg gaettcaaggggciacaattggagcaa ttatcttgtttactaaaactgaataccttgctatctctttgatacatttttacaaagctg aattaaaatggtataaattaaatcactgc

(bold) (SEQ ID NO: 14713).

[0429] The BBBMND PROMOTER of the nanoPB. Ank-BCLl 1 ashRNA.MND. GFP-T2A- DHFR construct is encoded by a sequence comprising:

GATCCATCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGT

TTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAGAAT

SUBSTITUTE SHEET (RULE 26) AAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGG

TTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAA

CAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAA

CAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCA

GGGCCAAGAAC AGA TGGTCCCC AGATGCGGTCCCGCCC TCAGCAGT GT CTAGAGAA

CCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGA

ACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCA A

TAAAAGAGCCCACAACCCCTCACTCGGCGCG (ALL CAPS underlined) (SEQ ID NO:

14699).

[0430] The EGFP of the nanoPB. Ank-BCLl 1 ashRNA.MND.GFP-T2A-DHFR construct is encoded by a sequence comprising:

atggteaecaageecmsmectsttcaccgeeeteetecccatccteetceaectseacgg ceacetaaacssccacaagttcag cgtgiccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagitcatctg caccaccggcaagctgcccgtgccctg gcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgacca catgaagcagcacgacttcttcaagtcc gccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactac aagacccgcgccgaggtgaagttcga gggcgacaccctggtgaaccgcatcgagctgaagggcatcga£tcaaggaggacggc:a acaicctggggcacaagc:tggagtac aactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtg aacttcaagatccgccacaacatcga ssacsscaecstscasctcsccsaccactaccascaeaacacccccatcsscsacssccc cstectsctscccsacaaccactac cigagcacccagiccgcccigagcaaagaccccaacgagaagcgtgaicacaiggiccig ctggagtcgtgaccgccgccgggatc actcicggcatggacgagctgtacaag (italicized underlined) (SEQ ID NO: 14708).

The DHFR of the nanoPB.Ank-BCLl lashRNA.MND.GFP-T2A-DHFR construct is encoded by a sequence comprising:

atggtgggcagcctgaattgtatcgtggccgtgtcccagaacatgggcatcggcaagaat ggcga

tggttctctatcccagagaagaaccgccccctgaagggccggatcaatctggtgctgagc agggagctgaaggagccaccccagg gagcacactttctgtccaggtctctggacgatgccctgaagctgaccgagcagcctgagc tggccaacaaggtggacatggtetgg

gagaagggcatcaagtacaagttcgaggtgtatgagaagaacgattgataa (bold underlined) (SEQ ID NO: 14709).

SUBSTITUTE SHEET (RULE 26) [0432] The SV40 Pofy(A } of the nanoPB . Ank-BCL 11 ashRN A.MND . GFP-T2 A-DHFR construct is encoded by a sequence comprising:

aaccattataagctgcaataaacaagt (bold italicized) (SEQ ID NO: 14710).

[0433] The 577 ? of the nanoPB . Ank-BCL 11 ashRN A.MND. GFP-T2A-DHFR construct is encoded by a sequence comprising:

[0434] A vector for erythroid-specific expression of a BCL! 1 A targeted shRNA and the expression of an iC9 safety switch (nanoPB. Ank-BCLl l ashRNA.MND.iC9-T2A-DHFR ) is encoded by a sequence comprising:

[0435] ttaaCCCT AGA AAGAT A ATC AT ATT GT GAC GT A CGTT AAA GAT A AT CAT GCGT A A A ATT GACGC AT GTGTTTT AT CGGTCTGT AT AT C GAGGTTT ATTTATT A ATTT G A AT

AGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAA

TTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATA A

AGTAACAAAACTTTTAtcgaatacctgcagccc! :eatgcagagggacagcccccccccaaagcccccagggatgta attacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggtccg gcgctccccccgcatccccgagccggca gcgtgcggggacagcccgggcacggggaaggtggcacgggatcgcttcctctgaacgctc tcgctgctcttgagcctgcagacacct ggggggatacggggaaaagtgactgtgccttcgategcgtfcgaaggggcaaccaggggf ccgcg ^ggccgaggcc/ggggag cggggcctcctggggttgggggaggaggtgctcttgtaatctgcggtggtccccaggcgg gcgccacccctccgcccgcccgtgccgg gagcgcccggcccgacagcaagcgcctctggggccgalaaggccctcgggggcctggccc gcacgtcacaggccccgcagaggc tgcggtgagtccgccagccccagctgctcctcctcaagcccccaciggccctcggcggca atcccaccggggi2icc ^! gt&c&' &' ^! gt&&t agggccctacgagcttgctgtttgaatgaggcttcagtactttacagaatcgttgcctgc acatcttggaaacacttgctgggattact tcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtgagcgccg cacagaacactcatggatttagtgaa gccacagatgtaaatccatgagtgttctgtgcgttgcctactgcctcggacttcaagggg ctacaattggagcaattatcttgtttact aaaactgaataccttgctatctctttgatacatttttacaaagctgaattaaaatggtat aaattaaatcactgcgtcgacatcgaacc atggacagttGATCCATCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCA

SUBSTITUTE SHEET (RULE 26) ACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAG

CAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCA

GGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGT

AAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGT

CCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAG

GACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGC

TTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC

ACTCGGCGCGgggccaccaagcttgtccgaattctaatacgactcactatagggtga agctgccacca/gggggfr aggfgg aaacaatctctccgggggatgggcggacattccctaaaaggggccagacctgcgtggtgc attacaccggcatgctggaagatggca agaaggtggacagcagccgggacagaaacaagcccttcaagttcatgctgggcaagcaag aagtgatcagaggctgggaagagg gcgtcgcccagatgtctgttggacagagagccaagctgacaatcagccccgattacgcct atggcgccacaggacaccctggcatca tcctccacatgccacactggtgttcgacgtggaactgctgaagctggaaggcggcggagg atctggctttggagaigtgggagccctg gaaagcctgagaggcaatgccgatctggcctacatcctgagcatggaaccttgcggixac tgcctgatatcaacaacgtgaactctg tagagagagcggcctgcggaccagaaccggcagcaatatcgattgcgagaagctgcggcg gagattcagcagcctgcacttcatgg iggaagtgaagggcgacctgaccgccaagaaaatggigctggcictgciggaactggccc agcaagaicatggcgccciggaitgci gigtggtcgigatcctgtcicacggctgtcaggccagccaccttcaattccciggcgccg igtatggcacagatggctgtccigtgtccgtg gaaaagatcgtgaacatcticaacggcaccagcigtcctagccicggcggaaagcccaag ctgitcttcatccaagcctgtggcggcg agcagaaggatcacggattgaggtggccagcacaagccccgaggatgagtctcctggaag caaccctgagcctgacgccacacct

Uccaagagggcctgagaacctcgaccagctggacgciatcagciccctgcctacacc tagcgacaiciicgigtcciacagcacaiic cccggcitgtgtctggcgggaccctaagtctggcictggtacgtggaaaccciggacgac atctitgagcagigggctcacagcgag gacclccagictctgctgctgagagtggccaatgccgigtccgtgaagggcatctacaag cagatgcctggctgctlcaacttcclgcgg aagaage/gfflffcaagiaccagegaaggaagaggcagcctgctgacatgtggcgacgt ggaggagaaccctggcccaatggtgggc agcctgaattgtatcgtggccgtgtcccagaacatgggcatcggcaagaatggcgatttt ccttggccccctctgagaaatgagtcc ag .atactttcagaggatga .ccacaaccagctccgtggagggcaagcaga .acctggt .catcatgggcaagaaga .catggt .tctcta

aiaccttctttccagagaicgaectggagaaHtacaagcii

lattgataacatatgccttaattaaacactagttctatagtgtcacctaaattcc ctitagtg&gggttaatggccgtaggccgccagaatigggiceagacaigataagt iiacattgatgagtitggaeaaaccacaaeiaga

SUBSTITUTE SHEET (RULE 26) taggaectgcgcatgegcttggcgtaaicatggtc atagctgtttcctgttttccccgtatccccccaggtgtctgcaggctcaaagagcagcga gaagcgttcagaggaaagcgatcccgtgccac cttccccgtgcccgggctgtccccgcacgctgccggctcggggatgcggggggagcgccg gaccggagcggagccccgggcggctc gctgctgccccctagcgggggagggacgtaattacatccctgggggctttgggggggggc tgtccctctcaccgcggtggagctccagct ttgttegaattggggceeccectcgagggtatcgat gatatctaiaacaagaaaatatatatataataagttatcacgtaastagaacatg

aagggcgcctcgagacctctagctagcccgcctaatgagcgggcttttttttggctt gttgtccacaaccgttaaaccttaaaagctttaaaag ccttatatattcttttttttcttataaaacttaaaaccttagaggctatttaagttgctg atttatataattttattgttcaaacatgagagcttagtacgt gaaacatgagagcttagtacgttagccatgagagcttagtacgtagccatgagggtttag ttcgttaaacatgagagcttagtacgttaaaca tgagagcttagtacgtactatcaacaggttgaactgctgatccacgttgtggtagaattg gtaaagagagtcgtgtaaaatatcgagttcgcac atcttgttgtctgattattgatttttggcgaaaccatttgatcatatgacaagatgtgta tctaccttaacttaatgattttgataaaaatcattacctag ggaagactc (SEQ ID NO: 14714).

[0436] The 3’ITR of the nanoPB.Ank-BCLl lashRNA.MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA

CGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATT A

AGTTTTATTATATTTAC ACTT ACATACTAAT A ATAAATTC A AC AAAC A ATTT ATTTAT

GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0437] The Insulator 1 of the nanoPB.Ank-BCLl lashRNA.MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

gagggaeagcccccecceaaagcccecagggatgtaattacgtcccteccecgctag ggggcageagcgagecgcccggggctecgc tccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggatcgctttcc tctgaaegcttctcgetgctctttgagcetgcagacaectggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0438] Th eAnkyrin-1 Promoter of the nanoPB. Ank-BCLl 1 ashRNA.MND. iC9-T2 A-DHFR construct is encoded by a sequence composing:

cgcgttcgaaggggcaaccaggggtccgcgccggccgaggcctggggagcggggcct cctggggttgggggaggaggtgctcttgt aatctgcggtggtccccaggcgggcgccacccctccgcccgcccgtgccgggagcgcccg gcccgacagcaagcgcctctggggc

SUBSTITUTE SHEET (RULE 26) cgataaggccctcgggggcctggcccgcacgtcacaggccccgcagaggctgcggtgagt ccgccagccccagctgctcctcctca agcccccaaggcccttcggcggcaattcccaccgg (italicized) (SEQ ID NO: 14701).

[0439] The miRE sh49 BCL11A of the nanoPB. Ank-BCLl lashRNA.MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

gtacaagtaatagggccctacgagcttgctgtttgaatgaggcttcagtactttaca gaatcgttgcctgcacatcttggaaacactt g ^c tggg ^a tt ^ac tcgacttcttaacccaacagaaggctcgagaaggtatattgctgttgacagtga gcgccgcacagaacactcat ggatttagtgaagccacagatgtaaatccatgagtgttctgtgcgttgcctactgcctcg gacttcaaggggctacaattggagcaa ttatcttgtttactaaaactgaataccttgctatctctttgatacatttttacaaagctg aattaaaatggtataaattaaatcactgc

(bold) (SEQ ID NO: 14713).

[0440] The BBBMND PROMOTER of the nanoPB. Ank-BCLl 1 ashRNA.MND iC9-T2 A- DHFR construct is encoded by a sequence comprising:

GATCCATCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCT

AGTTTTGAGTCAACAATATCAGCAGCTGAAGCGTATAGAGTACGAGCCATAGAT

AGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCA

CCTGTAGGTTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAAT

ATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA

AGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAG

TTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCC

CTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTG

AAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGT

TCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCG

GCGCG (bold ALL CAPS) (SEQ ID NO: 1444699).

[0441] The iC9 of the nanoPB. Ank-BCLl 1 ashRNA.MND iC9-T2A-DHFR construct is encoded by a sequence comprising:

atgggggtccaggtggaaacaatctciccgggggatgggcggacattccciaaaagg ggccagaccigcgtggtgcattacaccggc atsctssaasatescaaeaassteeacascascceeeacasaaacaascccttcaaettc atectssscaascaaeaasteatca sassctsssaasassscstcscccasaistcisttssacaeasasccaascteacaatca sccccsatacscctatssceccaca esacacccisscatcaitcctccacatsccacactsststtcsacstssa cisctsaascissaasscsscssassatcisscttss asatstsssasccctesaaascctsasasscaatsccsatcisscctacatcctsascat ssaaccttscssccactsccisattatc aacaacstsaacttctstasasasascsscctscssaccasaaccsscascaatatcsat tscsasciasctscsscssasaitco scascctscacitcaisstssaastsaassscs cctsaccsccaasaaaatsstsctssctctscissaactsscccascaas ic

SUBSTITUTE SHEET (RULE 26) atggcgccctssatsctstgtggtcstgatcctgtctcacssctgtcassccagccacct tcaattccctsscgccstgtatsscacaga tsscistcctststccatssaaaasatcstsaacatcttcaacsscaccasctatcctas cctceecssacuiscccaascisttcttcai ccaaRccWggcggcgagcagaagmtcacggatt ggtggceagcacaagceccgaggatga&ctcctggaagcaacect sasccisacsccacacctttccaasasssccteaeaaccttcsaccascissacsctaic asciccctscciacacctascsacaictt cgtgtcctacagcacattccccggctttgtgtcttggcgggaccctaagtctggctcttg gtacgtggaaaccctggacgacatctttgagc agtgggctcacagcgaggctcctccagtctctgctgctgagagtggccaatgccgtgtcc gtsaagggcatctacaagcagatgcctgg ctgcttcaacttcctgcggaagaagctgtttttcaagaccagc (italicized underlined) (SEQ ID NO: 14715).

[04421 The DHFR of the nanoPB. Ank-BCLl lashRNA.MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

14709)

[0443] The SV40 Pofy(A) of the nanoPB. Ank-BCLl 1 ashRNA.MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

tgctttatttgtaaccanataagctgcaataaacaagttaacaaeaacaattgcancant tatgttcaggttcagggggaggtgtggga ggtttttt (bold italicized) (SEQ ID NO: 14710).

[0444] The Insulator 2 of the nanoPB. Ank-BCLl lashRNA.MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

ttttccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaagcgttcagagg aaagcgatcccgtgccaccttccccgtgccc gggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggag ccccgggcggctcgctgctgccccct agcgggggagggacgtaattacatccctgggggctttgggggggggctgtccctc (underlined) (SEQ ID NO: 14716).

[0445] The 577ft of the nanoPB. Ank-BCLl 1 ashRN A.MND. iC9-T2 A-DHFR construct is encoded by a sequence comprising:

SUBSTITUTE SHEET (RULE 26)

asaatscatecstcaaitttacscasactatctttctasse (bold italicized underlined) (SEQ ID NO: 1471 1).

[0446] A vector for constitutive expression of Interleukin 2 Receptor subunit gamma, iC9 and

DHFR (nanoPB.MND.IL2RG-T2A-iC9-T2A-DHFR) is encoded by a sequence comprising: ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGT

AAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAA T

AGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAA

TTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATA A

AGTAACAAAACTTTTAtcgaatacctgcaccccgggggatgcagagggacacccccc ccccaaaecccccagggatgta atacgtcectececcgctagggggcagcagcgagccgcceggggctcegctccggtcegg cgetceccccgeatccecgagccggca gcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgct tctcgctgctctttgagcctgcagacacct ggggggatacggggaaaagttgactgtgccttcgatcgcaccggtgaaccatggacagtT GAT C CAT C GATTAGT C C

CGGCGCGgggecaecaagcttgtcctgc aggagggtcgacgaattctaatacgactcactatagggtgtgctgtctcatcattttggc aaagatggccaccaagctaccgccatgctga agcccagcctgccttttaccagcctgctsttcctgcagctgcctctgcttsgcgtggscc tgaataccaccatcctgacacctaacgg caacgaggatacaaccgccsacttcttcctsaccaccatgcctaccgatascctgagcgt gtccacactgccactgcctgagstsc aetgcttcetettcaacgtceaetacatsaactgcaccteeaacasctccaecgascccc agcctaccaatcteacactecactatt ggtacaagaacagcgacaacgacaaggtgcagaagtgcagccactacctgttcagcgagg aaatcaccagcggctgccagctg cagaagaaagagatccacctgtaccagaccttcgtggtgcagctccaggatcctagagag cctagaaggcaggccacacagatg ctgaaactgcagaacctcgtgatcccctgggctcccgaaaacctgactctgcacaagctg agcgagagccagctggaactgaact ggaacaaccggttcctgaatcactgcctggaacatctggtgcagtaccggaccgactggg atcactcttggacagagcagagcgt ggactaccggcacaagttcagcctgccatctgtggacggccagaagcggtacacctttag agtgcggagccggttcaaccctctgt

SUBSTITUTE SHEET (RULE 26) gtggatctgctcagcattggagcgagtggtcacacccaatccactggggcagcaacacca gcaaagagaaccccttcctgttcgcc ctggaagccgtggttatcagcgtgggctctatgggcctgatcatctccctgctgtgcgtg tacttctggctggaaagaaccatgcctc ggatccccactctgaagaacctggaagatctcgtgaccgagtaccacggcaacttcagtg cttggagcggcgtgtcaaaaggact ggccgaaagcctgcagcctgactacagcgagagactgtgtctggtgtctgagatccctcc taaaggcggcgctctcggagaagga cctggtgcctctccatgcaatcagcacagcccttattgggcccctccttgctacaccctg aagcctgaaacaggcagcggcgaaggc agaggctctctgcttacatgtggcgacgtcgaagagaatcccggaccta/gggggfccag g/ggaaacaa/cfcft ggggga/gggcg gacqtccctaaaaggggccaggcc Lgtggtg xittqc ggg gctggqagatggcqqgqqggtgggcqi_gcagccgggqc agaaacaagcccttcaagttcatgctgggcaagcaagaagtgatcagaggctgggaagag ggcgtcgcccagatgtctgttggaca gagagccaagctgacaatcagccccgattacgcctatggcgccacaggacaccctggcat cattcctccacatgccacactggtgttc giicgtggqactgctgqagctggiuiggcggcggqggcactggcmggcigi gtgggggccctggqqqgcctgg ggcaqtgccg atctggcctacatcctgagcatggaaccttgcggccactgcctgattatcaacaacgtga acttctgtagagagagcggcctgcggacc agaaccggcagcaatatcgatigcgagaagctgcggcggagattcagcagcctgcactca tggtggaagigaagggcgacctgac cgccaagaaaatggtgctggctcigctggaaciggcccagcaagatcatggcgccctgga ttgctgtgiggtcgtgatcctgtctcacg gctgtcaggccagccacctcaattccctggcgccgtgtatggcacagatggcigtcctgt gtccgtggaaaagatcgtgaacatcttca acggcaccagctgtcctagcctcggcggaaagcccaagctgttcttcatccaagcctgtg gcggcgagcagaaggatcacggatUg aggtggccagcacaagccccgaggaigagtctcctggaagcaacccigagcctgacgcca cacctitccaagagggcctgagaacc ttcgaccagctggacgctaicagctccctgcctacacctagcgacatcttcgtgtcctac agcacattccccggctttgtgtcttggcggga ccctaagtctggctcttggtacgtggaaaccctggacgacatcttgagcagtgggctcac agcgaggacctccagtctctgctgcAgag agtggccaaigccglgtccgigaagggcatcicicaagcagatgcctggcigcttcaaci iccigcggaagaagctgUUicaagacca gcgaaggaagaggcagcctgctgacatgtggcgacgtggaggagaaccctggcceaatgg tgggcagcctgaattgtatcgtggccg tgtcccagaacatgggcatcggcaagaatggcgattttccttggccccctctgagaaatg agtccagatactttcagaggatgacca caaccagctccgtggagggcaagcagaacctggtcatcatgggcaagaagacatggttct ctatcccagagaagaaccgccccct gaagggccggatcaatctggtgctgagcagggagctgaaggagccaccccagggagcaca ctttctgtccaggtctctggacgat

tgagaagaacgattgataacatatgccttaataaacactagttctatagtgtcacctaaa ttcccttagtgagggttaatggccgtagg

ggggg ggtgtgggaggtttttc ggactctaggacctgcgcatgcgcttggcgtaatcatggtcatagctgt

cccaggtgtctgcaggctcaaagagcagcgagaagcgttcagaggaaagcgatcccg tgccaccttccccgtgcccgggctgtccccgc

5UB5TITUTE SHEET (RULE 26) acgetgceggctcggggatgcggggggagcgccggaecggagcggagcccegggcggctc gctgetgceccctagcgggggaggg acgtaattacatccetgggggcttgggggggggctgtecctcteaccgeggtggagctcc agctttgtcgaattggggceccecctcga

igcetaagggcgcetcgagacctctagctag cccgcetaatgagcgggcttttttttggcttgttgtccacaaecgttaaaccttaaaagc ttiaaaagecttatatattcttttttttcttataaaactta aaaecttagaggetatttaagttgetgatttatattaattttattgttcaaacatgagag ettagtacgtgaaacatgagagetagtacgttagcea tgagagcttagtacgttagccatgagggtttagttcgttaaacatgagagettagtacgt taaacatgagagcttagtacgtactateaacaggt tgaaetgctgatceacgttgtggtagaattggtaaagagagtcgtgtaaaatatcgagtt cgcacatcttgttgtctgatiattgatttttggcgaa accatttgatcatatgacaagatgtgtatctacettaacttaatgattttgataaaaatc attacctagggaagactc ( SEQ ID NO :

14717).

[0448] The 3TTR of the nanoPB.M D.XL2RG~T2A-iC9~T2A-DHFR construct is encoded by a sequence comprising:

CCCT AGAA AGATAAT CAT ATT GT GACGT ACGTT A A A GAT A AT C ATGC GT AAA ATT GA

CGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATT A AGTTTTATTATATTTAC ACTT ACATACTAAT A ATAAATTC A ACAA AC A ATTT ATTTAT

3TTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0449] The Insulator 1 of the nanoPB.MND.IL2RG-T2A-iC9-T2A-DHFR construct is encoded by a sequence comprising:

gagggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggg gcagcagcgagccgcccggggctccgc tccggtccggcgctccccccecatccccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggatcgcttcc tctgaaegcttctcgetgctcttgagcetgcagacaectggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0450] The BBBMND of the nanoPB.MND.IL2RG-T2A-iC9-T2A-DHFR construct is encoded by a sequence comprising:

SUBSTITUTE SHEET (RULE 26) AAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCA

GTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGC

CCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCT

GAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTG

TTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTC

GGCGCG (bold ALL CAPS) (SEQ ID NO: 14699).

[0451] The IL2RG of the nanoPB.MND.IL2RG-T2A-iC9-T2A-DHFR construct is encoded by a sequence comprising:

atgctgaagcccagcctgccttttaccagcctgctgttcctgcagctgcctctgctt ggcgtgggcctgaataccaccatcctgacac ctaacggcaacgaggatacaaccgccgacttcttcctgaccaccatgcctaccgatagcc tgagcgtgtccacactgccactgcct gaggtgcagtgcttcgtgttcaacgtcgagtacatgaactgcacctggaacagctccagc gagccccagcctaccaatctgacact gcactattggtacaagaacagcgacaacgacaaggtgcagaagtgcagccactacctgtt cagcgaggaaatcaccagcggctg ccagctgcagaagaaagagatccacctgtaccagaccttcgtggtgcagctccaggatcc tagagagcctagaaggcaggccac aeagatgetgaaaeigcagaacetegtgatcceeigggctcecgaaaacctgaetetgca caagetgagegagagecagctggaa ctgaactggaacaaccggttcctgaatcactgcctggaacatctggtgcagtaccggacc gactgggatcactcttggacagagca gagcgtggactaccggcacaagttcagcctgccatctgtggacggccagaagcggtacac ctttagagtgcggagccggttcaac cctctgtgtggatctgctcagcattggagcgagtggtcacacccaatccactggggcagc aacaccagcaaagagaaccccttcct gttcgccctggaagccgtggttatcagcgtgggctctatgggcctgatcatctccctgct gtgcgtgtacttctggctggaaagaacc atgcctcggatccccactctgaagaacctggaagatctcgtgaccgagtaccacggcaac ttcagtgcttggagcggcgtgtcaaa aggactggccgaaagcctgcagcctgactacagcgagagactgtgtctggtgtctgagat ccctcctaaaggcggcgctctcgga gaaggacctggtgcctctccatgcaatcagcacagcccttattgggcccctccttgctac accctgaagcctgaaaca (bold)

(SEQ ID NO: 14718).

[0452] The iC9 of the nanoPB.MND.IL2RG-T2A-iC9-T2A-DHFR construct is encoded by a sequence comprising:

atzzz ccaz&A aacaaictcicc zz imzcz cattccciaaaa mcca ccizc ztzc^tacacczzc atsctssaasatescaaeaassteeacascascceeeacasaaacaascccttcaaettc atectssscaascaaeaasteatca sassctsssaasassscstcscccasaistcisttssacaeaeasccaascteacaatca sccccsatacscctatssceccaca esacacccisscatcaitcctccacatsccacactsststtcsacstssa cisctsaascissaasscsscssassatcisscttss asatstsssasccctesaaascctsasasscaatsccsatcisscctacatcctsascat ssaaccttscssccactsccisattatc aacaacstsaacttctstasasasascsscctscssaccasaaccsscascaatatcsat tscsasciasctscsscssasaitco scascctscacitcaisstssaastsaassscs cctsaccsccaasaaaatsstsctssctctscissaactsscccascaas ic

SUBSTITUTE SHEET (RULE 26) atggcgccctssatsctstgtggtcstgatcctgtctcacssctgtcassccagccacct tcaattccctsscgccstgtatsscacaga tsscistcctststccatssaaaasatcstsaacatcttcaacsscaccasctatcctas cctceecssacuiscccaascisttcttcai ccaaRccWggcggcgagcagaagmtcacggatt ggtggceagcacaagceccgaggatga&ctcctggaagcaacect sasccisacsccacacctttccaasasssccteaeaaccttcsaccascissacsctaic asciccctscciacacctascsacaictt cgtgtcctacagcacattccccggctttgtgtcttggcgggaccctaagtctggctcttg gtacgtggaaaccctggacgacatctttgagc agtgggctcacagcgaggctcctccagtctctgctgctgagagtggccaatgccgtgtcc gtsaagggcatctacaagcagatgcctgg ctgcttcaacttcctgcggaagaagctgtttttcaagaccagc (italicized underlined) (SEQ ID NO: 14715).

[0453] The DHFR of the nanoPB . MND . IL2RG- T2 A- iC9 - T2 A-DHFR construct is encoded by a sequence comprising:

14709)

[0454] The SV40 Poly (A) of the nanoPB. MND. IL2RG-T2A-iC9-T2A-DHFR construct is encoded by a sequence comprising:

tgctttatttgtaaccanataagctgcaataaacaagttaacaaeaacaattgcancant tatgttcaggttcagggggaggtgtggga ggtttttt (bold italicized) (SEQ ID NO: 14710).

[0455] The Insulator 1 of the nanoPB. MND. IL2RG-T2A-iC9-T2A-DHFR construct is encoded by a sequence comprising:

ttttccccgtatccecccaggtgtetgcaggetcaaagageagcgagaagcgtcagagga aagcgatcccgtgccacctccecgtgecc gggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggag ccccgggcggctcgctgctgccccct agcgggggagggacgtaattacatccctgggggctttgggggggggctgtccctc (underlined) (SEQ ID NO: 14719).

[0456] The 577ft of the nanoPB.MND.IL2RG-T2A-iC9-T2A-DHFR construct is encoded by a sequence comprising:

SUBSTITUTE SHEET (RULE 26)

asaatscatecstcaaitttacscasactatctttctasse (bold italicized underlined) (SEQ ID NO: 1471 1).

[0457] A vector for erythroid-specific expression of Insulin growth factor 2 binding protein 1

(IGF2BP1 ) and the expression of an iC9 safety switch (nanoPB.SPTA.IGF2BPl .MND.iC9- T2A-DHFR) is encoded by a sequence comprising:

ttaaCCCT AGAAAGAT AAT CAT ATT GT GAC GTACGTT AAAGAT AATC ATGC GT

AAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAA T

AGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAA

TTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATA A

AGTAACAAAACTTTTAtcgaatacctgcagcccgggggatgcagagggacagccccc ccccaaagcccccagggatgta attacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggtccg gcgctccccccgcatccccgagccggca geeegg¾taceeee¾aaagtgactgtgccttcgatcg6Y;agac7//caagaagagaa /g aaagga67cfcagffltf67a/g gaga/ ccaagaacaggggaaaccacaaatgtagaatcaaaatcttctcttgtagatcatggaagt tagattattcaagctgcatttacactgga atgaagtttggcatggatagccccactggcttgtagaaaggtctaagagcatcccactat ittctttttgcttgtitgtttcattttigtaagttag tctaaaataactatgcctatggaaaaagtctacctctttttgttctccattaaaattaat acatacagtaggatgatttataccatatatatca gaagtcagccatattttcaacttttttgtgacatcattggttgtcaaataaggtatccta attttgttttacaaaatattaatgaacacatcat ataactttttttgttccttccatttaaatgattaagagtacaactaaatcagatttttaa agaccaaattgttgaggttaggaataaaatcttatt ttgtgaaagactctctagaaaaagccaggataattcgttgacttaaagactgtgaaaaat ctgcagttgagtggatgaagttgttgagcl gtaiticciataaaiataiggggiitiittciaaiitiicaiccaaaaaccitactitca itttaagictiaitgagtaacitigaaagccagagc ctcccaaaactgctgaglcacccagtatctgtaaaacltagcagttgcclcagctgagla tgtcttctaaagataatgtcgattgtgiatgg ctgatgggatlctaggaccaagcaagaggtUttlttlttcccccacatacltaacglttc tataUtclatttgaaltcgactggacagttccatl tgaattattlctctctctclctctctctclctgacacatutatcttgccaggttctaaac ccggtaccgccatgaacaagctttacatcggcaa cctcaacgagagcgtgacccccgcggacttggagaaagtgtttgcggagcacaagatctc ctacagcggccagttcttggtcaaat ccggctacgccttcgtggactgcccggacgagcactgggcgatgaaggccatcgaaactt tctccgggaaagtagaattacaagg aaaacgcttagagattgaacattcggtgcccaaaaaacaaaggagccggaaaattcaaat ccgaaatattccaccccagctccga tgggaagtactggacagcctgctggctcagtatggtacagtagagaactgtgagcaagtg aacaccgagagtgagacggcagtg gtgaatgtcacctattccaaccgggagcagaccaggcaagccatcatgaagctgaatggc caccagttggagaaccatgccctga aggtctcctacatccccgatgagcagatagcacagggacctgagaatgggcgccgagggg gctttggctctcggggtcagccccg ccagggctcacctgtggcagcgggggccccagccaagcagcagcaagtggacatccccct tcggctcctggtgcccacccagtat gtgggtg ^cca t ^a tggcaaggagggggccaccatccgcaacatcacaaaacagacccagtccaagat agacgtgcataggaag

SUBSTITUTE SHEET (RULE 26) gagaacgcaggtgcagctgaaaaagccatcagtgtgcactccacccctgagggctgctcc tccgcttgtaagatgatcttggagat tatgcataaagaggctaaggacaccaaaacggctgacgaggttcccctgaagatcctggc ccataataactttgtagggcgtctca ttggcaaggaaggacggaacctgaagaaggtagagcaagataccgagacaaaaatcacca tctcctcgttgcaagaccttaccc tttacaaccctgagaggaccatcactgtgaagggggccatcgagaattgttgcagggccg agcaggaaataatgaagaaagttcg ggaggcctatgagaatgatgtggctgccatgagcctgcagtctcacctgatccctggcct gaacctggctgctgtaggtcttttccca gcttcatccagcgcagtcccgccgcctcccagcagcgttactggggctgctccctatagc tcctttatgcaggctcccgagcaggag atggtgcaggtgtttatccccgcccaggcagtgggcgccatcatcggcaagaaggggcag cacatcaaacagctctcccggtttgc cagcgcctccatcaagattgcaccacccgaaacacctgactccaaagttcgtatggttat catcactggaccgccagaggcccaat tcaaggctcagggaagaatctatggcaaactcaaggaggagaacttctttggtcccaagg aggaagtgaagctggagacccaca tacgtgtgccagcatcagcagctggccgggtcattggcaaaggtggaaaaacggtgaacg agttgcagaatttgacggcagctga ggtggtagtaecaagagaccagacccetgatgagaaegaecaggtcatcgtgaaaateat eggacafffctatgccagteagatg gctcaacggaagatccgagacatcctggcccaggttaagcagcagcatcagaagggacag agtaaccaggcccaggcacggag gaagtgagtceacGATCCATCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCC A

GGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATA

GATAGAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCA

CCTGTAGGTTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAATATG

GGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACA

GTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCC

CGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTC

T AGAG A AC C A T C AG A TGTTT CC AGGGT GCCCC A A GG ACCTG A A AT G A CCCT GT GCC

TTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCC C

GAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGaccggteccacca/gggg gfccagg/g gaaacaatctctccgggggatgggcgmcattccctaaaaggggccaeacctgcgtggtgc altacaccggcatgctggaagatggc aagaaggtggacagcagccgggacagaaacaagcccltcaagtcatgctgggcaagcaag aagtgaicagaggctgggaagag ggcgtcgcccagatgicigitggacagagagccaagctgacaatcagccccgatacgcci atggcgccacaggacaccciggcaic attcctccacatgccaeactggtgttcgacgtggaactgetgaagctggaaggcggegga ggatctggetttggagatgtgggagccet ssaaascctmsasscaatsccgatctsscetacatcctgascateeaaccttscgsceac tgcctsattatcaacaacstsaacttct giagagagagcggectgcggaccagaaccggcagcaatatcgattgcgagaagetgcggc ggagattcagcagectgcactteatg gtggaagtgaagggcgacetgaccgccaagaaaatggtgetggctctgctggaactggce cagcaagateatggcgccctggattgc tgtgtggtcgtgatcetgtctcacggetgteaggccagceaccttcaaticcctggcgce gtgtatggeacagatggcigtcctgtgtecgt ggaaaagatcgtgaacatcttcaacggcaccagctgtcctagcctcggcggaaagcceaa gctgttctcatccaagcctgtggcggc

SUBSTITUTE SHEET (RULE 26) sascasaassatcacssatttsasstssccascacaasccccsassatsastctcctssa ascaaccctsascctsacsccacac ciitccaasaeeeccteasaaccticsaccaectssacsctaicasctccciscctacac ciasceacatcttcstetcctacaecacat iccccggcttgtglcttggcgggaccciaagtclggctcitggtacgiggaaacccigga cgacalcttgagcag gggcicaagctgit

£ffcaagaccagicacagcgaggacctccagtctctgctgctgagagtggccaatg ccgtgtccgtgaagggcatctacaagcagatgcct ggctgcttcaacttcctgcggaaggaaggaagaggcagcctgctgacatgtggcgacgtg gaggagaaccctggcccaatggtgggca gcctgaattgtatcgtggccgtgtcccagaacatgggcatcggcaagaatggcgattttc cttggccccctctgagaaatgagtcca gatactttcagaggatgaccacaaccagctccgtggagggcaagcagaacctggtcatca tgggcaagaagacatggttctctat cccagagaagaaccgccccctgaagggccggatcaatctggtgctgagcagggagctgaa ggagccaccccagggagcacact ttctgtccaggtctctggacgatgccctgaagctgaccgagcagcctgagctggccaaca aggtggacatggtgtggatcgtgggc

o

gg¾g; O Oq ggg ca itjKcRaRaKgitacaagttcgaggtgtatgagaagaacgattgataaeatatgecttta attaaacactagttctatagtgtcaectaaatccett tagtgagggttaatggccgtaggccgccagaattgggtc cagacatgataagatacattgotgagttggacaaaccacaactagaaig

tgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttcggactct &ggacctgcgcaXgcgcttggcgtaatcatggtcata gctgtttcctgtttccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaag cgttcagaggaaagcgatcccgtgccacct ccccgtgcccgggctgtccccgcacgctgccggctcggggatgcggggggagcgccggac cggagcggagccccgggcggctcgc tgctgccccctagcgggggagggacgtaattacatccctgggggcttgggggggggctgt ccctctcaccgcggtggagctccagctt gttcgaattggggccccccctcgagggtatcgatgatatctataacaagaaaatatatat ataataagttatcacgtaagtagaacatgaaata acaatataattatcgtatgagttaaatcttaaaagtcacgtaaaagataatcatgcgtca ttttgactcacgcggtcgttatagttcaaaatcagtg acacttaccgcattgacaagcacgcctcacgggagctccaagcggcgactgagatgtcct aaatgcacagcgacggattcgcgctatttag aaagagagagcaatatttcaagaatgcatgcgtcaattttacgcagactatctttctagg gttaatctagctagccttaagggcgcctcgagac ctctagctagcccgcctaatgagcgggcttttttttggcttgttgtccacaaccgttaaa ccttaaaagctttaaaagccttatatattcttttttttctt ataaaacttaaaaccttagaggctatttaagttgctgatttatataattttattgttcaa acatgagagcttagtacgtgaaacatgagagcttagt acgttagccatgagagcttagtacgttagccatgagggtttagttcgttaaacatgagag cttagtacgttaaacatgagagcttagtacgtact atcaacaggttgaactgctgatccacgttgtggtagaattggtaaagagagtcgtgtaaa atatcgagttcgcacatcttgttgtctgattattga tttttggcgaaaccatttgatcatatgacaagatgtgtatctaccttaacttaatgattt tgataaaaatcattacctagggaagactc (SEQ ID NO: 14720).

[0459] The 3TTR of the nanoPB.SPTAIGF2BPl .MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA

SUBSTITUTE SHEET (RULE 26) GCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTA

AGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTA T

GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0460] The Insulator 1 of the nanoPB.SPTA.IGF2BPl.MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

gagggacagcccccecccaaagcccecagggatgtaatacgtcccteccccgctagg gggcagcagcgagccgcccggggctccgc tccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggatcgctttcc tctgaaegcttctcgetgctcttgagcetgcagacaectggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0461] The SPTA1 Promoter of the nanoPB.SPTA.IGF2BP! MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

ccagactttcaagaagagaatgtaaaggactctcagtttttctatgtgagatccaag aacaggggaaaccacaaatgtagaatcaaaa tcttctcttgtagatcatggaagttagattattcaagctgcatttacactggaatgaagt ttggcatggatagccccactggcttgtagaaag gtctaagagcatcccactattttcttttgcttgttgttcatttttgtaagttagtctaaa ataactatgcctatggaaaaagtctacctcttttg ttctccattaaaattaatacatacagtaggatgatttataccatatatatcagaagtcag ccatattttcaactttttgtgacatcatggtttg tcaaataaggtatcctaatttttgttttacaaaatattaatgaacacatcatataacttt tttgttccttccatttaaatgattaagagtacaact aaatcagattttaaagaccaaattgttgaggttaggaataaaatcttatttgtgaaagac tctctagaaaaagccaggataattcgttg acttaaagactgtgaaaaatctgcagttgagttggatgaagttgttgagctgtatttcct ataaatatatggggtttttttctaattttcatcc aaaaaccltacttcatttlttaagtcttaltgagtaactttgaaagccagagcctcccaa aactgctgaglcacccagtatctgtaaaaclt agcagttgcctcagctgagtatglcttctaaagataatgtcgatlgtgtatggctgatgg gattctaggaccaagcaagaggttlttltttttc ccccacalacttaacgttlctalatttctatltgaattcgactggacagttccatttgaa ttatltctctctctctclctctctctclgacacattttal cttgccaggttctaaaccc (italicized) (SEQ ID NO: 14700).

[0462] The IGF2BP1 of the nanoPB.SPTA.IGF2BPl .MND.iC9-T2A~DHFR construct is encoded by a sequence comprising:

atgaacaagctttacatcggcaacctcaacgagagcgtgacccccgcggacttggag aaastgtttecggagcacaagatctcct acagcggccagttcttggtcaaatccggctacgccttcstggactgcccggacgagcact gggcgatgaaggccatcgaaacttc ggtgcccaaaaaaeaaaggagecggaaaattcaaate cgaaatattccaccccagctccgatgggaagtactggacagcctgctggctcagtatggt acagtagagaactgtgagcaagtga acaccgagagtgagacggcagtggtgaatgtcacctattccaaccgggagcagaccaggc aagccatcatgaagctgaatggcc accagttggagaaccatgccctgaaggtctcctacatccccgatgagcagatagcacagg gacctgagaatgggcgccgagggg gctttggctctcggggteagceccgccagggctcacctgtggcagcgggggceccagcca agcageagcaagtggacatcccect

SUBSTITUTE SHEET (RULE 26) tcggctcctggtgcccacccagtatgtgggtgccattattggcaaggagggggccaccat ccgcaacatcacaaaacagacccag tccaagatagacgtgcataggaaggagaacgcaggtgcagctgaaaaagccatcagtgtg cactccacccctgagggctgctcct ccgcttgtaagatgatcttggagattatgcataaagaggctaaggacaccaaaacggctg acgaggttcccctgaagatcctggcc cataataactttgtagggcgtctcattggcaaggaaggacggaacctgaagaaggtagag caagataccgagacaaaaatcacc atctcctcgttgcaagaccttaccctttacaaccctgagaggaccatcactgtgaagggg gccatcgagaattgttgcagggccga gcaggaaataatgaagaaagttcgggaggcctatgagaatgatgtggctgccatgagcct gcagtctcacctgatccctggcctga acctggctgctgtaggtcttttcccagcttcatccagcgcagtcccgccgcctcccagca gcgttactggggctgctccctatagctcc tttatgeaggctccegageaggagatggtgcaggtgtttatcccegeceaggeagtgggc gccateatcggcaagaaggggcagc acatcaaacagctctcccggtttgccagcgcctccatcaagattgcaccacccgaaacac ctgactccaaagttcgtatggttatca tcactggaccgccagaggcccaattcaaggctcagggaagaatctatggcaaactcaagg aggagaacttctttggtcccaagga ggaagtgaagetggagaeceacatacgtgtgccageateagcagctggecgggtcattgg caaaggtggaaaaacggtgaacg agttgcagaatttgacggcagctgaggtggtagtaccaagagaccagacccctgatgaga acgaccaggtcatcgtgaaaatcat cggacatttctatgccagtcagatggctcaacggaagatccgagacatcctggcccaggt taagcagcagcatcagaagggacag agtaaecaggcceaggeacggaggaagtga (bold) (SEQ ID NO: 14721)

[0463] The bhhMND PROMOTER of the nanoPB . SPT ATGF2BP 1. MND. iC9-T2 A-DHFR construct is encoded by a sequence comprising:

GATCCATCGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGT

TTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAGAAT

AAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGG

TTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAA

CAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAA

CAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCA

GGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAA

CCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGA

ACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCA A

TAAAAGAGCCCACAACCCCTCACTCGGCGCG (bold ALL CAPS) (SEQ ID NO: 14699).

[0464] The iC9 of the nanoPB . S PT A. IGF2BP 1. MND . iC9- T2 A-DHFR construct is encoded by a sequence comprising:

atssssstccasstssaaacaatctctccsssssatssscssacattccctaaaass ssccasacctscstsstscattacaccssc atgctssaasatsscaagaasstssacascagccgggacagaaacaagcccttcaasttc atgctssscaascaagaastsatca saggctsssaasagggcstcgcccagatstctgtssacagagasccaagctgacaatcas ccccsattacgcctatsscgccaca

SUBSTITUTE SHEET (RULE 26) ggacaccctggcatcaitcctccacatgccacactggtgttcgacgtggaactgctgaag ctggaaggcggcggaggatctggcttigg agatgtgggagccctggaaagcctgagaggcaatgcegatctggcctaeatcctgagcat ggaaccttgeggccacigccigattate aacaaegtgaacttctgiagagagageggcctgeggaccagaaceggcagcaataicgat tgegagaagctgcggcggagaitca gcagcctgcacitcatggtggaagtgaagggcgacctgaccgccaagaaaatggtgctgg ctetgctggaaetggcccagcaagatc atggcgccctggattgctgtgtggtcgtgatcctgtctcacggctgtcaggccagccacc ttcaattccctggcgccgtgtatggcacaga tggctgtcctgtgtccgtggaaaagatcgtgaacatcttcaacggcaccagctgtcctag cctcggcggaaagcccaagctgttcttcat ccaagcctgtggcggcgagcagaaggatcacggatttgaggtggccagcacaagccccga ggatgagtctcctggaagcaaccct gagcctgacgccacacctttccaagagggcctgagaaccttcgaccagctggacgctatc agctccctgcctacacctagcgacatctt cgtgtcctacagcacaticcccggctttgtgtcttggcgggaccctaagtciggctcttg gtacgtggaaaccctggacgacatctttgagc agtgggctcaagctgtttcaagaccagc (italicized underlined) (SEQ ID NO: 14715).

The D1TF of the nanoPB.SPTA.IGF2BPl MND.iC9-T2A-DHFR construct is encoded by a sequence comprising:

¾¾tg¾gtecagataetttc¾gaggatgacc¾eaaec¾gctcegtggagggeaag c¾gaacetggteatcatgggc¾¾gaagac¾

_. f££tigtcl¾tec ggaga g _. gcgcccec aagggccgggtc ¾tetggtgctoaocagggaocfg S§ _J SCc¾ecj;e¾gg

jtggaes

gagaagggc (bold underlined) (SEQ ID NO:

14709).

[0466] The SV40 Poly (A } of the nanoPB.SPTA.IGF2BPl MND. i C9-T2 A-DHFR construct is encoded by a sequence comprising:

ggtitttt (bold italicized) (SEQ ID NO: 14710).

[0467] The Insulator 2 of the nanoPB.SPTA.IGF2BPi .MND . iC9-T2 A-DHFR construct is encoded by a sequence comprising:

Tttceccgtatccccecaggtgtctgeaggctcaaagagcagegagaagcgtteagagga aagcgatcecgtgccacetteccegtgcc egggctgteccegcacgctgceggctcggggatgcggggggagcgccggaecggagcgga gccecgggcggctcgctgetgcecc cta ^g cgggfi&agggacgfaattacatccctgggggctttgggggggggc ccctclimderlmed) (SEQ ID NO:

14716).

SUBSTITUTE SHEET (RULE 26) [0468] The 577ft of the nanoPB.SPTA.IGF2BPl .MND. iC9-T2 A-DHFR construct is encoded by a sequence comprising:

gatatctataacaagaaaataiatatataaiaagtiatcacgtaagtagaacatgaa ataacaataiaaitaicgtatgagtiaaatctiaaaagtc acgtaaaagataatcatgcgtcattttgactcacgcggtcgttatagttcaaaatcagtg acacttaccgcattgacaagcacgcctcacggg agctccaagcggcgactgagatgtcctaaatgcacagcgacggattcgcgctatttagaa agagagagcaatatttcaagaatgcatgcgt caattttacgcagactatctttctaggg (bold italicized underlined) (SEQ ID NO: 14711).

[0469] A vector for the expression of HBB T87Q, iCasp9, and DHFR (PB-HBB-PGK-iCasp9- T2A-DHFR construct) is encoded by a sequence comprising:

[0470] ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGT A AAATT GACGC AT GT GTTTT ATCGGT CT GT AT AT CGAGGTTT ATTTATT A ATTT GAAT AGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAA TTTATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAA

AGTAACAAAACTTTTActgaatacctgcagcccgggggatgcagagggacagccccc ccccaaagcccccagggatgta attacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggtccg gcgctccccccgcatccccgagccggca gcgtgcggggacagcccgggcacggggaaggtggcacgggatcgcttcctctgaacgctc tcgctgctctttgagcctgcagacacct ggggggatacggggaaaagttgactgtgccttcgatcgagcgctgacgtcgcggccgctc ccgcgatacgcgctaggtattgaataag aaaaatgaagttaaggtggttgatggtaacactatgctaataactgcagagccagaagca ccataagggacatgataagggagc eagcagacctetgateicttcetgaatgetaatciiaaacatcetgaggaagaatgggac ttecaiiiggggtgggectatgataggg taataagacagtagtgaatatcaagctacaaaaagceccettteaaattcttctcagtec taacttttcataetaageccagtecttcc aaagcagactgtgaaagagtgatagttccgggagactagcactgcagattccgggtcact gtgagtgggggaggcagggaagaa gggcteaeaggacagteaaaeeatgeeeecigttttteetiettcaagtagaccietata agaeaaeagagacaactaaggeigagt ggccaggcgaggagaaaccatctcgccgtaaaacatggaaggaacacttcaggggaaagg tggtatctctaagcaagagaact gagtggagtcaaggetgagagatgeaggataagcaaatgggtagtgaaaagacattcatg aggaeageiaaaaeaaiaagtaa tgtaaaatacagcatagcaaaactttaacctccaaatcaagcctctacttgaatcctttt ctgagggatgaataaggcataggcatc aggggctgttgccaatgtgcattagctgtttgcagcctcaccttctttcatggagtttaa gatatagtgtattttcccaaggtttgaact agctctcatttctttatgtttaaatgcactgacctcccacattccctttagtaaaatatt cagaaataatttaaatacatcattgcaa tgaaaataaatgttttttattaggcagaatccagatgctcaaggcccttcataatatccc ccagtttagtagttggacttagggaaca aaggaacctttaatagaaattggacagcaagaaagcgagcttagtgatacttgtgggcca gggcattagccacaccagccaccac tttctgataggcagcctgcactggtggggtgaattctttgccaaagtgatgggccagcac acagaccagcacgttgcccaggagct gtgggaggaagataagaggtatgaacatgattagcaaaagggcctagcttggactcagaa taatccagccttatcccaaceataa aataaaagcagaatggtagctggattgtagctgctattagcaatatgaaacctcttacat cagttacaatttatatgcagaaatattt

SUBSTITUTE SHEET (RULE 26) atatgcagaaatattgctattgccttaacccagaaattatcactgttattctttagaatg gtgcaaagaggcatgatacattgtatcat tattgccctgaaagaaagagattagggaaagtattagaaataagataaacaaaaaagtat attaaaagaagaaagcattttttaa aattacaaatgcaaaattaccctgatttggtcaatatgtgtaccctgttacttctcccct tcctatgacatgaacttaaccatagaaaa gaaggggaaagaaaacatcaagggtcccatagactcaccctgaagttctcagggtccacg tgcagcttgtcacagtgcagctcac tcagctgggcaaaggtgcccttgaggttgtccaggtgagccaggccatcactaaaggcac cgagcactttcttgccatgagccttca ccttagggttgcccataacagcatcaggagtggacagatccccaaaggactcaaagaacc tctgggtccaagggtagaccacca gcagcctaagggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacc caagagtcttctctgtctccacatg cccagtttctattggtctccttaaacctgtcttgtaaccttgataccaacctgcccaggg cctcaccaccaacttcatccacgttcacct tgccccacagggcagtaacggcagacttctcctcaggagtcaggtgcaccatggtgtctg tttgaggttgctagtgaacacagttgt gtcagaagcaaatgtaagcaatagatggctctgccctgacttgtatgcccagccctggct cctgccctccctgctcctgggagtaga ttggccaaccctagggtgtggctccacagggtgaggtctaagtgatgacagccgtacctg tccttggctcttctggcactggcttagg ^a gtggacttcaaaccctcagccctccctctaagatatatctcttggccccatacc atcagtacaaattgctactaaaaacatcctcct ttgcaagtgtatttacctagaatatgtcacattctgtctcaggcatccattttctttatg atgccgtttgaggtggagttttagtcaggtg gtcagcttctccttttttttgccatctgccctgtaagcatcctgctggggacccagatag gagtcatcactctaggctgagaacatctg ggcacacaccctaagcctcagcatgactcatcatgactcagcattgctgtgcttgagcca gaaggtttgcttagaaggttacacaga accagaaggcgggggtggggcactgaccccgacaggggcctggccagaactgctcatgct tggactatgggaggtcactaatgg agacacacagaaatgtaacaggaactaaggaaaaactgaagcttatttaatcagagatga ggatgctggaagggatagaggga gctgagcttgtaaaaagtatagtaatcattcagcaaatggttttgaagcacctgctggat gctaaacactattttcagtgcttgaatc ataaataagaataaaacatgtatcttattccccacaagagtccaagtaaaaaataacagt taattataatgtgctctgtcccccagg etggagigeagtggcacgatgteageicactgeaaectcegeeieecgggggaciagtct egagggeiggitagaaggttetaeig gaggagggtcccagcccattgctaaattaacatcaggctctgagactggcagtatatctc taacagtggttgatgctatcttctggaa cttgcctgctacattgagaccactgacccatacataggaagcccatagctctgtcctgaa ctgttaggccactggtccagagagtgt gcatctcctttgatcctcataataaccctatgagatagacacaattattactcttacttt atagatgatgatcctgaaaacataggagt caaggcacttgcccctagctgggggtataggggagcagtcccatgtagtagtagaatgaa aaatgctgctatgctgtgcctccccc acctttcccatgtctgccctctactcatggtctatctctcctggctcctgggagtcatgg actccacccagcaccaccaacctgaccta accacctatctgagcctgccagcctataacccatctgggccctgatagctggtggccagc cctgaccccaccccaccctccctggaa cctctgatagacacatctggcacaccagctcgcaaagtcaccgtgagggtcttgtgtttg ctgagtcaaaattccttgaaatccaagt ccttagagactcctgctcccaaatttacagtcatagacttcttcatggctgtctccttta tccacagaatgattcctttgcttcattgccc catccatctgatcctcctcatcagtgcagcacagggcccatgagcagtagctgcagagtc tcacataggtctggcactgcctctgac atgtccgaccttaggcaaatgcttgactcttctgccctcgagaagcttatcgatggctag tgcatgcaaatctgacactcagtgggcc tgggtgaaggtgagaattttattgctgaatgagagcctctggggacatcttgccagtcaa tgagtctcaggttcaatttccttctcagt

SUBSTITUTE SHEET (RULE 26) cttggagtaacagaagctcatgcatttaataaacggaaattttgtattgaaatgagagcc attggaaatcatttactccagactccta cttataaaaagagaaactgaggctcagagaagggtggggactttctcagtatgacatgga aatgatcaggcttggattcaaagctc ctgactttctgtctagtgtatgtgcagtgagccccttttcctctaactgaaagaaggaaa aaaaaatggaacccaaaatattctacat agtttccatgtcacagccagggctgggcagtctcctgttatttcttttaaaataaatata tcatttaaatgcataaataagcaaaccct gctcgggaatgggagggagagtctctggagtccaccccttctcggccctggctctgcaga tagtgctatcaaagccctgacagagc cctgcccattgctgggccttggagtgagtcagcctagtagagaggcagggcaagccatct catagctgctgagtgggagagagaa aagggctcattgtctataaactcaggtcatggctattcttattctcacactaagaaaaag aatgagatgtctacatataccctgcgtc ccctcttgtgtactggggtccccaagagctctctaaaagtgatggcaaagtcattgcgct agatgccatcccatctattataaacctg catttgtctccacacaccagtcatggacaataaccctcctcccaggtccacgtgcttgtc tttgtataatactcaagtaatttcggaaa atgtattctttcaatcttgttctgttattcctgtttcaatggcttagtagaaaaagtaca tacttgttttcccataaattgacaatagaca atttcacatcaatgtctatatgggtcgttgtgtttgctgtgtttgcaaaaactcacaata actttatattgttactactctaagaaagtta caacatggtgaatacaagagaaagctattacaagtccagaaaataaaagttatcatcttg aggccatttaattaacatatgtcgcgag

tccgggaaacgcagcggcgccgaccctgggtctcgcacatcttcacgtccgtcgcag cgtcacccggatcttcgccgctacccttgtg ggccccccggcgacgcttccigctccgcccctaagtcgggaaggttccttgcggttcgcg gcgtgccggacgtgacaaacggaagcc gcacg#cfc ^* actogtoc ^* cctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggc tgtggccaatagcggc tgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgt ggggcggtagtgtgggccctgttcct gcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccct cgttgaccgaatcaccgacctctctccccag gggccaccaagcttgtcctgcaggagggtcgacgcctctagagccacc atgggcgtgcaggtcgagactatcagccccggagatggg agaacatttccaaaaagagggcagactgcgtcgtccactacactggcaigctggaggacg ggaaaaaagtcgatagcagtcggga icggaataagccattcaagittatgctggggaagcaggaggtcaiccgggggigggaaga gggggicgcicagaigtcagigggcca gcgggcaaagcigacaaiciccccagactaigcatacggcgctaccgggcatcciggaat iatcccaccccatgccaccctggtgittg acgtggaactgctgaagctggaagggggcggggggagtggctitggggatgtgggcgccc iggagagtctgagagggaacgctgat etggcttacattctgagcatggaaccatgcggccactgcctgatcattaacaatgtgaae tttgtagagagtcagggctgcgaactcga acagggagcaacatcgattgtgaaaagctgaggcgccggttctctagtctgcactttatg gtcgaggtgaagggagacctgacagca aagaagatggtgctggccctgctggagctggcccagcaggaccacggggcactggactgt tgcgtcgtcgtgatcctgtctcacggct gtcaggcaagtcacctgcaatccccggcgccgtctacggcactgatgggtgccccgtctc cgtggaaaagattgtgaatatctttaatg gaacatcatgcccatctctggggggaaagccaaaactgttcttatccaggcttgtggagg ggaacagaaggatcacggctttgaagt cgcctccacaagtccagaagatgaaagcccaggcagtaatcccgagcctgatgcaacacc cttccaggaggggctgcggacctttg atcagctggatgcaatticttcecigcccacaecaicagatatttttgtgagctacagta cifitcetggetiegtetcctggagggaccctaa aagcgggagctggtacgtggagacactggacgatattttcgaacagtgggcacattctga ggacctgcaatctctgctgctgcgggtcg

SUBSTITUTE SHEET (RULE 26)

ccggggagggcagagggtcactgctgacatgcggcgacgtggaggagaaccctggaccaa tggtgggcagcctgaattgtatcgtg gccgtgtcccagaacatgggcatcggcaagaatggc gat¾

ggaggcc¾tg¾¾tc¾ecctggecacctg¾¾getgttegtg¾eacgg¾tc¾t ge¾gg¾etttg¾gtccg¾tacettctttcc¾g¾g¾t

gcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttt tcactgcattctagttgtggtttgtccaaactca

/caa g/afcffatoa/gtogtgtcgaaattctagcttggcgtaatcatggtcatagctgttcct gtgtgaacgagccggaagcataaagtgta ggcctactggccgaattcgtttatcgtcgagggtaccgatcacatatgccttaattaaac actagttctatagtgtcacctaaatcccttagt gagggttaatggccgtaggccgccagaattgggtccagacatgataagatacattgatga gtttggacaaaccacaactagaatgcagtga aaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagc tgcaataaacaagttaacaacaacaattgcattcatttta tgtttcaggttcagggggaggtgtgggaggttttttcggactctaggacctgcgcatgcg cttggcgtaatcatggtcatagctgtttcctgtttt

gctgtccccgcaegctgccggcteggggatgeggggggagegccggaccggageggagce ecgggcggctcgetgctgccceetag cgggggagggacgtaattacatccctgggggcttgggggggggctgtccctctcaccgcg gtggagctccagctttgttcgaaltgggg

[0471] The 3’ITR of the PB-RBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

[0472] CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAA AATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAG

ATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATT T

ATTTATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAG T

AACAAAACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0473] The Insulator 1 of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

SUBSTITUTE SHEET (RULE 26) [0474] gagggacageccecccecaaagcecccagggatgtaattacgtcceicceccgetagggg gcagcagegagccgeccgg ggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagc ccgggcacggggaaggtggcacggg atcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctggggggatac ggggaaaa (underlined) (SEQ ID NO: 14705).

[0475] The T87Q beta-globin cassete with LCR of the PB-HBB~PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

ctaggtatfgaataagaaaaatgaagttaaggtggttgafggtaacactatgetaat aaetgcagagccagaagcaecafaaggg acatgataagggageeagcagacetetgatctctteetgaatgctaatcttaaacateet gaggaagaatgggaettecatttgggg tgggcctatgatagggtaataagacagtagtgaatatcaagctacaaaaagecccettte aaattcttcteagtcctaactttteata ctaageceagtccttceaaagcagaetgtgaaagagtgatagtteegggagactagcact geagattccgggtcaetgtgagtggg ggaggcagggaagaagggetcacaggaeagtcaaaecatgecccetgtttttccttctte aagtagacctctataagacaaeagag acaaetaaggctgagtggccaggegaggagaaaeeatctcgecgtaaaacatggaaggaa cactteaggggaaaggtggtatct ctaagcaagagaactgagtggagtcaaggetgagagatgeaggataageaaatgggtagt gaaaagacattcatgaggaeagc taaaaeaataagtaatgtaaaatacageatageaaaactttaacctecaaatcaagcetc tacttgaatccttttetgagggatgaa taaggcataggcatcaggggetgttgecaatgtgcattagctgtttgcagectcacettc tttcatggagtttaagatatagtgtatttt cccaaggtttgaactagetcttcatttctttatgttttaaatgcactgaectcecacatt cectttttagtaaaatatteagaaataattta aatacatcattgeaatgaaaataaatgttttttattaggcagaatccagatgetcaaggc cettcataatatcceecagtttagtagtt ggaettagggaaeaaaggaaectttaatagaaattggacagcaagaaagcgagcttagtg atacttgtgggceagggcattagcc acaecagccaecactttctgataggcagectgcactggtggggtgaattctttgecaaag tgatgggecagcaeacagaccagcae gttgcceaggagetgtgggaggaagataagaggtatgaacatgattagcaaaagggccta gettggaetcagaataatceageet iatcecaaeeataaaataaaagcagaaiggtagctggaiigtagetgetaitageaatat gaaaecteiiacateagttacaatttat atgcagaaatatltatatgeagaaataitgetattgecttaaeecagaaattatcacigt tattetiiagaatggtgcaaagaggeaig aiacattgiaieaitattgeccigaaagaaagagattagggaaagtattagaaataagat aaacaaaaaagtatattaaaagaag aaagcattttttaaaaitaeaaatgcaaaattaceetgaittggteaatatgtgtaecct gttaettetcccettcetaigaeatgaaett aaceatagaaaagaaggggaaagaaaacatcaagggieecatagaeieaecctgaagiie teagggtccacgtgeagcttgtca cagtgcagcteactcagctgggeaaaggtgeceiigaggttgteeaggtgagecaggcca teactaaaggcaeegageacttteii gccatgagccttcaccttagggttgcccataacagcateaggagtggaeagatccceaaa ggactcaaagaacctetgggtccaa gggtagaccaecagcagcctaagggtgggaaaatagaceaataggcagagagagtcagtg cctatcagaaacccaagagtcttc tctgtctceacatgcccagtttctattggtctccttaaacctgtcttgtaacettgatac caacctgcccagggcctcaccaccaacttc aiceacglteacettgccccaeagggcagiaacggeagacltcicetcaggagtcaggtg eaceatggtgtetgtttgaggttgetag tgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctgecctgacttgtatg eccagccctggctcctgccetccctgct

SUBSTITUTE SHEET (RULE 26) cetgggagtagattggccaaccctagggtgtggctccacagggigaggictaagigaiga cagccgtacctgtccttggcicttctg gcactggcttaggagttggacttcaaaccctcagccctccctctaagatatatctcttgg ccccataccatcagtacaaattgctacta aaaacatcctcctttgcaagtgtatttacctagaatatgtcacattctgtctcaggcatc cattttctttatgatgccgtttgaggtggag ttttagtcaggtggtcagcttctccttttttttgccatctgccctgtaagcatcctgctg gggacccagataggagtcatcactctaggc tgagaacatctgggcacacaccctaagcctcagcatgactcatcatgactcagcattgct gtgcttgagccagaaggtltgcttaga aggttaeacagaaccagaaggcgggggtggggcaetgacccegaeaggggcctggecaga aetgcteatgettggactatggga ggtcactaatggagacaeaeagaaatgtaaeaggaactaaggaaaaactgaagcttattt aatcagagatgaggatgetggaag ggatagagggagctgagettgtaaaaagtatagtaatcattcageaaatggttttgaagc aectgctggatgctaaaeactatttte agtgcttgaatcataaataagaataaaaeatgtatcttattceecacaagagtecaagta aaaaataaeagttaattataatgtgct ctgteceecaggctggagtgcagtggeacgatgteagctcaetgcaacetccgceteceg ggggaetagtctcgagggctggttag aaggttctactggaggagggtcecagcceattgctaaattaacatcaggctetgagaetg gcagtatatetctaacagtggttgatg ctatettctggaacttgeetgctacattgagaccactgacecatacataggaagcceata gctctgtcetgaactgttaggecactgg tccagagagtgtgcatctcctttgatcctcataataaccetatgagatagaeacaattat tactcttactttatagatgatgatcctgaa aaeataggagteaaggcacttgcecctagctgggggtataggggagcagtcecatgtagt agtagaatgaaaaatgctgctatgct gtgcctcccecacctttcccatgtctgccctetactcatggtctatctctcctggetcct gggagtcatggactecacccagcaccacc aaectgacctaaecacctatctgagcctgccagcctataacecatctgggcectgatagc tggtggccagecctgaccccaecceac cctccctggaacctctgatagacacatctggcacaccagctcgcaaagteaccgtgaggg tcttgtgtttgctgagtcaaaattcctt gaaatccaagtccttagagactectgctcccaaatttacagtcatagacttettcatggc tgtctectttatccaeagaatgattecttt gcttcattgecccatccatctgatcctccteatcagtgcagcacagggcccatgagcagt agctgcagagtctcacataggtetggc actgceietgacatgtcegaecttaggcaaatgeiigaciettctgceetcgagaagcit atcgaiggciagtgeatgeaaatctgac aeteagtgggectgggtgaaggtgagaattit it etga tgaga ectciggggaeateitgecagteaatgagteteaggitea atllccitcieagtcttggagtaacagaagcteatgeaiitaataaaeggaaatiiigta ttgaaatgagagceatiggaaateatiia etecagaeiectacttataaaaagagaaactgaggctea gagaagggtggggacttteieagta tga eatggaaa tgatcaggctt ggatlcaaagcteetgaetttctgtciagtgtatgtgcagtgageeccttttcetctaac tgaaagaaggaaaaaaaaaiggaaecc aaaatattctacatagtttccatgtcacagccagggctgggcagtctcctgttatttctt ttaaaataaatatatcatttaaatgcataa ataagcaaaccctgctcgggaatgggagggagagtctctggagtccaccccttctcggcc ctggctctgcagatagtgctatcaaa gccctgacagagccctgcccattgctgggccttggagtgagtcagcctagtagagaggca gggcaagccatctcatagctgctga gtgggagagagaaaagggctcattgtctataaactcaggtcatggctattcttattctca cactaagaaaaagaatgagatgtctac atataccctgcgtcccctcttgtgtactggggtccccaagagctctctaaaagtgatggc aaagtcattgcgctagatgccatcccat ctattataaacctgcatttgtctccacacaccagtcatggacaataaccctcctcccagg tccacgtgcttgtctttgtataatactcaa gtaatttcggaaaatgtattctttcaatcttgttctgttattcctgtttcaatggcttag tagaaaaagtacatacttgttttcccataaat

SUBSTITUTE SHEET (RULE 26)

ctaagaaagttacaacatggtgaatacaagagaaagctattacaagtccagaaaataaaa gttatcatcttgaggccatt (bold) (SEQ ID NO: 14935).

[0476] The human-PGK promoter of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

ttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggt tccgggaaacgcagcggcgccgaccctg ggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttg tgggccccccggcgacgcttcctgctccgcc cctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgc acgtctcactagtaccc (italicized) (SEQ ID NO: 14696).

[0477] The iCasp9 of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

[0478] The DHFR of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

g¾gc¾cactttctgtccaggtctctggaeg¾tgccctgaagctg¾ccgagcagcct g¾gctggcc¾¾e¾¾ggtgg¾c¾tggtgtgg

5UB5TITUTE SHEET (RULE 26) atcgtgggcggctctagcgtgtataaggaggccatgaatcaccctggccacctgaagctg ttcgtgacacggatcatgcaggacttt gagtccgataccttctttccagagatcgacctgg¾g¾¾gt¾c¾¾gctgctgccc gagtatcctggcgtgctgtctgatgtgcaggag gagaagggcatcaagtacaagttcgaggtgtatgagaagaacgat (bold underlined) (SEQ ID NO: 14709).

The SV40Pofy(A) of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

acaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttca caaataaagcatttttttcactgcattctagttg tggtttgtccaaactcatcaatgtcitcttatcatgtc (italicized underlined) (SEQ ID NO: 14710).

[0479] The Insulator 2 of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

ttttccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaagcgtcagagga aagcgatcccgtgccacctccccgtgccc gggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggag ccccgggcggctcgctgctgccccct agcgggggagggacgtaattacatccctgggggctttgggggggggctgtccctc (underlined) (SEQ ID NO: 14716).

[0480] The 5TTR of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

tatttcaas tscatscstcaattttacscasactatctttctasss (bold italicized underlined) (SEQ ID NO:

14711). A piggyBac transposon vector construct for expression of HBB, GFP, and DHFR (PB.HBB.PGK.GFP-T2A-DHFR) is encoded by a sequence comprising:

ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATT

G ACGC AT GT GTTTT AT CGGT CT GT AT AT CG AGGTTT A TTT ATTA A TTT G A AT AG A T A T

TAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATT T

ATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAAC

AAAACTTTTAtcgaatacctgcagcccgggggatgcagagggacagcccccccccaa agcccceagggatgtaattacgtccc tcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccc cgcatccccgagccggcagcgtgcgg ggacagcccgggcacggggaaggtggcacgggatcgcttcctctgaacgcttctcgctgc tctttgagcctgcagacacctggggggat acggggaaaagttgactgtgcctttcgatcgagcgctgacgtcgcggccgctcccgcgat acgcgctaggtattgaataagaaaaatg aagttaaggtggttgatggtaacactatgctaataactgcagagccagaagcaccataag ggacatgataagggagccagcaga cctctgatctcttcctgaatgctaatcttaaacatcctgaggaagaatgggacttccatt tggggtgggcctatgatagggtaataag acagtagtgaatatcaagctacaaaaagccccctttcaaattcttctcagtcctaacttt tcatactaagcccagtccttccaaagca

SUBSTITUTE SHEET (RULE 26) gactgtgaaagagtgatagttccgggagactagcactgcagattccgggtcactgtgagt gggggaggcagggaagaagggctc acaggacagtcaaaccatgccccctgtttttccttcttcaagtagacctctataagacaa cagagacaactaaggctgagtggcca ggcgaggagaaaccatctcgccgtaaaacatggaaggaacacttcaggggaaaggtggta tctctaagcaagagaactgagtg gagtcaaggctgagagatgcaggataagcaaatgggtagtgaaaagacattcatgaggac agctaaaacaataagtaatgtaa aatacagcatagcaaaactttaacctccaaatcaagcctctacttgaatccttttctgag ggatgaataaggcataggcatcagggg ctgttgccaatgtgcattagctgtttgcagcctcaccttctttcatggagtttaagatat agtgtattttcccaaggtttgaactagctctt catttctttatgttttaaatgcactgacctcccacattccctttttagtaaaatattcag aaataatttaaatacatcattgcaatgaaaa taaatgttttttattaggcagaatccagatgctcaaggcccttcataatatcccccagtt tagtagttggacttagggaacaaaggaa cctttaatagaaattggacagcaagaaagcgagcttagtgatacttgtgggccagggcat tagccacaccagccaccactttctga taggcagcctgcactggtggggtgaattctttgccaaagtgatgggccagcacacagacc agcacgttgcccaggagctgtggga ggaagataagaggtatgaacatgattagcaaaagggcctagcttggactcagaataatcc agccttatcccaaccataaaataaa agcagaatggtagctggattgtagctgctattagcaatatgaaacctcttacatcagtta caatttatatgcagaaatatttatatgca gaaatattgctattgccttaacccagaaattatcactgttattctttagaatggtgcaaa gaggcatgatacattgtatcattattgccc tgaaagaaagagattagggaaagtattagaaataagataaacaaaaaagtatattaaaag aagaaagcattttttaaaattaca aatgcaaaattaccctgatttggtcaatatgtgtaccctgttacttctccccttcctatg acatgaacttaaccatagaaaagaaggg gaaagaaaacateaagggtcccatagactcaccetgaagttetcagggtccaegtgcagc ttgtcaeagtgeagctcacteagctg ggcaaaggtgcccttgaggttgtccaggtgagccaggccatcactaaaggcaccgagcac tttcttgccatgagccttcaccttagg gttgeccataacageatcaggagtggacagatccecaaaggactcaaagaacetctgggt ccaagggtagaceaccagcagect aagggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagt cttctctgtctccacatgcccagttt ctattggtctccttaaacctgtcttgtaaccttgataccaacctgcccagggcctcacca ccaacttcatccacgttcaccttgcccca eagggeagtaaeggcagaciictecteaggagtcaggigeaecatggtgtcigtttgagg ttgctagtgaacacagttgigieagaa gcaaatgtaagcaatagatggctctgccctgacttgtatgcccagccctggctcctgccc tccctgctcctgggagtagattggccaa ccctagggtgtggctccacagggtgaggtctaagtgatgacagccgtacctgtccttggc tcttctggcactggcttaggagtggac ttcaaaccctcagccctccctctaagatatatctcttggccccataccatcagtacaaat tgctactaaaaacatcctcctttgcaagt gtatttacctagaatatgtcacattctgtctcaggcatccattttctttatgatgccgtt tgaggtggagttttagtcaggtggtcagctt ctcctttttttgccatctgccctgtaagcatcctgctggggacccagataggagtcatca ctctaggctgagaacatctgggcacac accctaagcctcagcatgactcatcatgactcagcattgctgtgcttgagccagaaggtt tgcttagaaggttacacagaaccagaa ggcgggggtggggcactgaccccgacaggggcctggccagaactgctcatgcttggacta tgggaggtcactaatggagacaca cagaaatgtaacaggaactaaggaaaaactgaagcttatttaatcagagatgaggatgct ggaagggatagagggagctgagct tgtaaaaagtatagtaatcattcagcaaatggttttgaagcacctgctggatgctaaaca ctattttcagtgcttgaatcataaataa gaataaaacatgtatcttattccccacaagagtccaagtaaaaaataacagttaattata atgtgctctgtcccccaggctggagtg

SUBSTITUTE SHEET (RULE 26) cagtggcacgatgtcagctcactgcaacctccgcctcccgggggactagtctcgagggct ggttagaaggttctactggaggaggg tcccagcccattgctaaattaacatcaggctctgagactggcagtatatctctaacagtg gttgatgctatcttctggaacttgcctgc tacattgagaccactgacccatacataggaagcccatagctctgtcctgaactgttaggc cactggtccagagagtgtgcatctcctt tgatcctcataataaccctatgagatagacacaattattactcttactttatagatgatg atcctgaaaacataggagtcaaggcact tgcccctagctgggggtataggggagcagtcccatgtagtagtagaatgaaaaatgctgc tatgctgtgcctcccccacctttccca tgtctgccctctactcatggtctatctctcctggctcctgggagtcatggactccaccca gcaccaccaacctgacctaaccacctatc tgagcctgccagcctataacccatctgggccctgatagctggtggccagccctgacccca ccccaccctccctggaacctctgatag acacatctggcacaccagctcgcaaagtcaccgtgagggtcttgtgtttgctgagtcaaa attccttgaaatccaagtccttagaga ctcctgctcccaaatttacagtcatagacttcttcatggctgtctcctttatccacagaa tgattcctttgcttcattgccccatccatctg atcctcctcatcagtgcagcacagggcccatgagcagtagctgcagagtctcacataggt ctggcactgcctctgacatgtccgacc ttaggcaaatgcttgactcttctgccctcgagaagcttatcgatggctagtgcatgcaaa tctgacactcagtgggcctgggtgaag gtgagaattttattgctgaatgagagcctctggggacatcttgccagtcaatgagtctca ggttcaatttccttctcagtcttggagta acagaagctcatgcatttaataaacggaaattttgtattgaaatgagagccattggaaat catttactccagactcctacttataaaa agagaaactgaggctcagagaagggtggggactttctcagtatgacatggaaatgatcag gcttggattcaaagctcctgactttc tgtctagtgtatgtgcagtgagccccttttcctctaactgaaagaaggaaaaaaaaatgg aacccaaaatattctacatagtttccat gtcacagccagggctgggcagtctcctgttatttcttttaaaataaatatatcatttaaa tgcataaataagcaaaccctgctcggga atgggagggagagtctctggagtccaecccttctcggcectggctctgeagatagtgeta tcaaagccetgacagagccetgcccat tgctgggccttggagtgagtcagcctagtagagaggcagggcaagccatctcatagctgc tgagtgggagagagaaaagggctc attgtctataaactcaggtcatggctattcttattctcacactaagaaaaagaatgagat gtctacatataccctgcgtcccctcttgt gtactggggtccccaagagctctctaaaagtgatggcaaagtcattgcgctagatgccat cccatctattataaacctgcatttgtct ccacacaccagtcatggacaataaccctcctcccaggtccacgtgcttgtctttgtataa tactcaagtaatttcggaaaatgtattct ttcaatcttgttctgttattcctgtttcaatggcttagtagaaaaagtacatacttgttt tcccataaattgacaatagacaatttcacat caatgtctatatgggtcgttgtgtttgctgtgtttgcaaaaactcacaataactttatat tgttactactctaagaaagttacaacatgg tgaatacaagagaaagctattacaagtccagaaaataaaagttatcatcttgaggccatt taattaacatatgtcgcgagtactcaccg gtgccagacactgggggaccc ttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggt tecgggaa acgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccg gatcttcgccgctacccttgtgggcccccc ggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccg gacgtgacaaacggaagccgcacgtctc actagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgg gctgtggccaatagcggctgctcag cagggcgcgecgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcgg tagtgtgggccctgttectgcccg cgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttg accgaatcaccgacctctctccccagggg ccaccaagcttgtcctgcaggagggtcgacgcctctagagccacca/ g/g gc gggcgagg gc/g/fc‘accgggg/gg/gccc

SUBSTITUTE SHEET (RULE 26) tcctggtcsasctggacggcsacstaaacggccacaaettcascgtstccggcgagggcs assscsatgccacctacsscaasctg accctsaasttcatctgcaccaccsscaasctgcccgtsccctsscccaccctcstgacc accctgacctacsscgtscastscttcag ccsctaccccgaccacatgaagcagcacgacttcttcaastccsccatscccgaaggcta cgtccaggagcgcaccatcttcttcaag gacsacsscaactacaasacccscgccgaggtsaasttcgagggcsacaccctggtgaac cscatcgagctgaassscatcsact tcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacg tctatatcatggccgacaagcagaa gaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagct cgccgaccactaccagcagaacacc cccatcfrgcgacggccccgtgctgctfrcccgacaaccactacctgagcacccagtccf rccctgagcaaagaccccaacgagciagc gcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacg agctgtacaag gaaggaagaggeagcc

cc¾cctga gg| tc&f c gggtc tgc ^ cW& gcggt¾cg|tc g£¾g¾ggtcgacj;tggg g tggg&ct g. tgccc&a&f gctggg-gtg t tef&gg£g.g^g&t§jggSgg.fgggggc¾g tcg ggtgtgt&g&gg tg&gt tgataatgataaagatctacgcgttacttaaacactagttctatagtgtcacctaaattc cctttagtgagggttaatggccgtaggccgccaga

gaigciaiigctitatitgtaaceatiaiaagetgcaataaaeaagtia&c&a mp;acaacaatigcatic&itiatgtitcaggtcagggggaggt gtgggaggtttttcggactctaggacctecgcatgcgcttggcgtaatcatggtcatagc tgtttcctgtttccccgtatccccccaggtgtct geaggcteaaagagcagegagaagegteagaggaaagegatcccgtgeeacettcecegt gcccgggetgtecccgeaegetgccgg ctcggggatgcggggggagcgccggaccggagcggagccccgggcggctcgctgctgccc cctagcgggggagggacgtaattaca tccctgggggctttgggggggggctgtccctctcaccgcggtggagctccagcttttgtt cgaatggggccccccctcgagggtatcgatg

ctcacsssasctccaascsscsactsasai2tcctaaatscacascsacssattcsc sctatttasaaasaeaeascaatatttcaa

ctegggttaacgagacctctagctagcccgcctaatgagcgggctttttttggcttgttg tccacaaccgttaaaccttaaaagctttaaaagccttatatattcttttttttcttataa aacttaaaaccttagaggctatttaagttgctgatttatatt aattttattgttcaaacatgagagcttagtacgtgaaacatgagagcttagtacgttagc catgagagcttagtacgtiagccatgagggtttag ttcgttaaacatgagagcttagtacgtiaaacatgagagcttagtacgtactatcaacag gttgaactgctgatccacgttgtggtagaattggt aaagagagtcgtgtaaaatatcgagttcgcacatcttgttgtctgattattgatttttgg cgaaaccatttgatcatatgacaagatgtgtatctac cttaacttaatgattttgataaaaatcattacctagggaagactc (SEQ ID NO: 14936).

SUBSTITUTE SHEET (RULE 26) [0482] The 3 TTR of the PB.HBB.PGK. GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA

CGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATT A

AGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTA T

GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0483] The Insulator 1 of the PB.HBB.PGK. GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

gagggacagcccccccccaaagcccccagggatgtaatacgtccctcccccgctagg gggcagcagcgagccgcccggggctccgc tccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggatcgctttcc tctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0484] The HUB cassette in opposite orientation of the PB.HBB.PGK. GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

ctaggtattgaafaagaaaaatgaagttaaggtggttgatggtaacactatgefaat aaetgeagagccagaagcaecataaggg acatgataagggagccagcagacctctgatctcttcctgaatgctaatcttaaacatcct gaggaagaatgggacttccatttgg tgggcctatgatagggtaataagacagtagtgaatatcaagctacaaaaagccccctttc aaattcttctcagtcctaacttttcata ctaagcccagtccttccaaagcagactgtgaaagagtgatagttccgggagactagcact gcagattccgggtcactgtgagtggg ggaggcagggaagaagggctcacaggacagtcaaaccatgccccctgtttttccttcttc aagtagacctctataagacaacagag acaactaaggctgagtggccaggcgaggagaaaccatctcgccgtaaaacatggaaggaa cacttcaggggaaaggtggtatct ctaagcaagagaactgagtggagtcaaggctgagagatgcaggataagcaaatgggtagt gaaaagacattcatgaggacagc taaaacaataagtaatgtaaaatacagcatagcaaaactttaacctccaaatcaagcctc tacttgaatccttttctgagggatgaa taaggcataggcatcaggggctgttgccaatgtgcattagctgtttgcagcctcaccttc tttcatggagtttaagatatagtgtatttt cccaaggtttgaactagctcttcatttctttatgttttaaatgcactgacctcccacatt ccctttttagtaaaatattcagaaataattta aatacatcattgcaatgaaaataaatgttttttattaggcagaatccagatgctcaaggc ccttcataatatcccccagtttagtagtt ggaeiiagggaaeaaaggaaectttaatagaaaiiggaeagcaagaaagegagcttagtg atacttgtgggeeagggeattagcc acaccagccaccactttctgataggcagcctgcactggtggggtgaattctttgccaaag tgatgggccagcacacagaccagcac gttgcccaggagctgtgggaggaagataagaggtatgaacatgattagcaaaagggccta gcttggactcagaataatccagcct

atgcagaaatatttatatgcagaaatattgctattgccttaacccagaaattatcac tgttattctttagaatggtgcaaagaggcatg atacattgtatcattattgccctgaaagaaagagattagggaaagtattagaaataagat aaacaaaaaagtatattaaaagaag

SUBSTITUTE SHEET (RULE 26) aaagcattttttaaaattacaaatgcaaaattaccctgatttggtcaatatgtgtaccct gttacttctccccttcctatgacatgaactt aaccatagaaaagaaggggaaagaaaacatcaagggtcccatagactcaccctgaagttc tcagggtccacgtgcagcttgtca cagtgcagctcactcagctgggcaaaggtgcccttgaggttgtccaggtgagccaggcca tcactaaaggcaccgagcactttctt gccatgagccttcaccttagggttgcccataacagcatcaggagtggacagatccccaaa ggactcaaagaacctctgggtccaa gggtagaccaccagcagcctaagggtgggaaaatagaccaataggcagagagagtcagtg cctatcagaaacccaagagtcttc tctgtctccacatgcccagtttctattggtctccttaaacctgtcttgtaaccttgatac caacctgcccagggcctcaccaccaacttc atccacgttcaccttgccccacagggcagtaacggcagacttctcctcaggagtcaggtg caccatggtgtctgtttgaggttgctag tgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctgccctgacttgtatg cccagccctggctcctgccctccctgct cetgggagtagattggceaaecetagggtgtggetccacagggtgaggtctaagtgatga cagccgtaectgtccttggetcttctg gcactggcttaggagttggacttcaaaccctcagccctccctctaagatatatctcttgg ccccataccatcagtacaaattgctacta aaaacatcctcctttgcaagtgtatttacctagaatatgtcacattctgtctcaggcatc cattttctttatgatgccgtttgaggtggag ttttagtcaggtggtcagcttctccttttttttgccatctgccctgtaagcatcctgctg gggacccagataggagtcatcactctaggc tgagaacatctgggcacacaccctaagcctcagcatgactcatcatgactcagcattgct gtgcttgagccagaaggtttgcttaga aggttacacagaaccagaaggcgggggtggggcactgaccccgacaggggcctggccaga actgctcatgcttggactatggga ggtcactaatggagacaeaeagaaatgtaaeaggaaciaaggaaaaacigaagcttaiii aatcagagaigaggatgeiggaag ggatagagggagctgagcttgtaaaaagtatagtaatcattcagcaaatggttttgaagc acctgctggatgctaaacactattttc agtgcttgaatcataaataagaataaaacatgtatcttattccccacaagagtccaagta aaaaataacagttaattataatgtgct ctgtcccccaggctggagtgcagtggcacgatgtcagctcactgcaacctccgcctcccg ggggactagtctcgagggctggttag aaggttctactggaggagggtcccagcccattgctaaattaacatcaggctctgagactg gcagtatatctctaacagtggttgatg ctatcttctggaacttgcctgctacattgagaccactgacccatacataggaagcccata gctctgtcctgaactgttaggccactgg tccagagagtgtgcatctcctttgatcctcataataaccctatgagatagacacaattat tactcttactttatagatgatgatcctgaa aacataggagtcaaggcacttgcccctagctgggggtataggggagcagtcccatgtagt agtagaatgaaaaatgctgctatgct gtgcctcccccacctttcccatgtctgccctctactcatggtctatctctcctggctcct gggagtcatggactccacccagcaccacc aacctgacctaaccacctatctgagcctgccagcctataacccatctgggccctgatagc tggtggccagccctgaccccaccccac cctccctggaacctctgatagacacatctggcacaccagctcgcaaagtcaccgtgaggg tcttgtgtttgctgagtcaaaattcctt gaaatccaagtccttagagactcctgctcccaaatttacagtcatagacttcttcatggc tgtctcctttatccacagaatgattccttt gcttcattgccccatccatctgatcctcctcatcagtgcagcacagggcccatgagcagt agctgcagagtctcacataggtctggc actgcctctgacatgtccgaccttaggcaaatgcttgactcttctgccctcgagaagctt atcgatggctagtgcatgcaaatctgac actcagtgggcctgggtgaaggtgagaattttattgctgaatgagagcctctggggacat cttgccagtcaatgagtctcaggttca atttccttctcagtcttggagtaacagaagctcatgcatttaataaacggaaattttgta ttgaaatgagagccattggaaatcattta ctccagactcctacttataaaaagagaaactgaggctcagagaagggtggggactttctc agtatgacatggaaatgatcaggctt

SUBSTITUTE SHEET (RULE 26) ggattcaaagctcctgactttctgtctagtgtatgtgcagtgagccccttttcctctaac tgaaagaaggaaaaaaaaatggaaccc aaaatattctacatagtttccatgtcacagccagggctgggcagtctcctgttatttctt ttaaaataaatatatcatttaaatgcataa ataagcaaaccctgctcgggaatgggagggagagtctctggagtccaccccttctcggcc ctggctctgcagatagtgctatcaaa gccctgacagagccctgcccattgctgggccttggagtgagtcagcctagtagagaggca gggcaagccatctcatagctgctga gigggagagagaaaagggetcattgteiaiaaacicaggteatggctattcttatteica cactaagaaaaagaatgagatgtetac atataccctgcgtcccctcttgtgtactggggtccccaagagctctctaaaagtgatggc aaagtcattgcgctagatgccatcccat ctattataaacctgcatttgtctccacacaccagtcatggacaataaccctcctcccagg tccacgtgcttgtctttgtataatactcaa gtaatttcggaaaatgtattctttcaatcttgttctgttattcctgtttcaatggcttag tagaaaaagtacatacttgttttcccataaat tgacaatagacaatttcacatcaatgtctatatgggtcgttgtgtttgctgtgtttgcaa aaactcacaataactttatattgttactact ctaagaaagttaeaacatggtgaatacaagagaaagctattacaagtccagaaaataaaa gttatcatcttgaggccat (bold)

(SEQ ID NO: 14935)

[0485] The hPGK promoter of the PB.HBB.PGK.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

ttgcgccmccaaggcagccctgggtttgcgcagggaegcggctgctctgggcgtggt tccgggaaacgcagcggcgccgaccctg ggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttg tgggccccccggcgacgcttcctgctccgcc cctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgc acgtctcactagtaccctcgcagacgg acagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctg ctcagcagggcgcgccgagagca gcggccgggaaggggcggtgcgggaggcggggtgtggggcgglagtgtgggccctgttcc tgcccgcgcggtgtccgcattctgca agcctccggagcgcacgtcggcagtcggciccctcgtigaccgaatcaccgacctcicic cccag (italicized) (SEQ ID NO: 14696).

[0486] The GFP of the PB.HBB.PGK.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

atggigagcaagggcgaggagctgUcaccgggglggigcccatcctggtcgagctgg acggcgacgiaaacggccacaagttcag cgtgtccggcgagggcgagggcgatgccacctacggcaagcigaccctgaagttcatcig caccaccggcaagcigcccgtgccctg gcccacccicgtgaccaccctgacctac<z<zcQigcagigcitcagccgciac cccgaccacaigaagcagcacgacitcitcaagtcc eccatecccsaassciacstccassascscaccatcitcttcaassaceaceecaactac aaeacccececcsassteaasttcsa ssscsacaccctseisaaccscatcsasctsaassscatcsacttcaassassacsscaa catcctsssscacaasctssastac aactacaacaeccacaacetctataicateeccmcaaecasaaeaaceecaicaaestea acitcaaeatcceccacaacatcsci ssacsscascsiscasctcscceaccactaccascasaacacccccatceecsacssccc catsctsctsccceacaaccaciac ctsascacccaatccsccctsascaaasaccccaacsasaascscsatcacatsstccts ctssasttcetsaccsccsccsssat cactctcggcatggacgagctgtacaag (italicized and underlined) (SEQ ID NO: 14708).

SUBSTITUTE SHEET (RULE 26) [0487] The DHFR of the PB . HBB . PGK. GFP- T2 A-DHF R construct is encoded by a nucleic acid sequence comprising:

atcgtgggcggctctagcgtgtataaggaggccatgaatcaccctggccacctgaagctg ttcgtgacacggatcatgcaggacttt £aasssc4ic¾agtaca¾gttcy aggtgt¾tgagaa«aacgattg _. a (bold underlined) (SEQ ID NO: 14709). [Q488] The SV40 Poly (A) of the PB.HBB.PGK.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

aaccaitataagctgcaataaacaagt (bold italicized) (SEQ ID NO: 14710).

[0489] The Insulator 2 of the PB.HBB.PGK.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

ttttccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaagcgttcagagg aaagcgatcccgtgccacctccccgtgccc gggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggag ccccgggcggctcgctgctgccccct agcgggggagggacgtaattacatccctgggggctttgggggggggctgtccct (underlined) (SEQ ID NO: 14716).

[0490] The 5717? of the PB.HBB.PGK.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

cctcaceeeasctccaaecescsacteaeatetcctaaatscacaecsacssattcscsc tatttaeaaaeasasascaatattca aeaatecatscstcaattttacecae ctatctttctasss (bold italicized underlined) (SEQ ID NO: 14711).

[0491] A piggyBac transposon vector construct for expression of GFP and DHFR

(PB.EFla.GFP-T2A-DHFR) is encoded by a sequence comprising:

ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATT

GACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATA T

TAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATT T

ATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAAC

AAAACTTTTAtcgaatacctgcagcccgggggatgcagagggacagcccccccccaa agcccccagggatgtaattacgtccc

SUBSTITUTE SHEET (RULE 26) tcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccc cgcatccccgagccggcagcgtgcgg ggacagcccgggcacggggaa^gtggcacgggatcgctttoctctgaacgctctcgctgc icttgagcctgcagacacctggggggat acgggg &aagitgactgtgcctttcgatcgaaccatgg&cagitagetttgcaaagat ggataaagttttaaacagagaggaatctttgc agctaatggaccttctaggtcttgaaaggagtgggaattggctccggtgcccgtcagtgg gcagagcgcacatcgcccacagtccccg agaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaa actgggaaagtgatgtcgtgtactgg ctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaac gttctttttcgcaacgggtttgccgccagaa cacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggccctt gcgtgccttgaattacttccacctggctgcag tacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgc gcttaaggagccccttcgcctcgtgcttgag ttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcct gtctcgctgctttcgataagtctctagccat ttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgc gggccaagatctgcacactggtatttcggtttttg ggg¥gcgggcggcgacggggcccgtgcgtcecagcgcacatgttcggcgaggcggggcc tgcgagcgcggccaccgagaatcg gacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgta tcgccccgccctgggcggcaaggctggc ccggtcggcaccagttgc· pgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcgg cgctcgg

cgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggtt ggggggaggggttttatgcgatggagtttccccac actgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaa tttgcccttttgagtttggatctggttcattct caagcctcagacagtggttcaaagttttUcUccattcaggigtcgtgagaaXlcX& aX&cgaclcacXaiagggXgXgcigXcicalcaxXXi ggcdd&gaXimiccdccd&gctiaccmcatggtgagcaagggcgaggagctg ticaccggggiggigcccatcctggtcgagctgga cggcgacgtaaacggccacaagtcagcgtgiccggcgagggcgagggcgalgccacctac ggcaagctgaccctgaagiicatct gcaccaccggcaagctgcccglgccctggcccaccctcgtgaccaccctgacctacggcg tgcagtgcttcagccgctaccccgacc acatgaagcagcacgaciictcaagtccgccatgcccgaaggctacgiccaggagcgcac caictcitcaaggacgacggcaact acaagacccgcgccgaggfgaagltcgagggcgacaccclggtgaaccgcatcgagclga agggcatcgacttcaaggaggacg gcaacaicciggggcacaagctggagiacaactacaacaeccacaacgtciatatcatge ccgacaagcaeaagaacegcatcaa ggigaactcaagaiccgccacaacaicgaggacggcagcgtgcagcicgccgaccaciac cagcagaacacccccatcggcgac ggccccgtgcigcigcccgacaaccaciaccigagcacccagiccgcccigagcaaagac cccaacgagaagcgtgaicacatggt cctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagma ggaava cagcctsctmcatgtssc gacgtggaggagaaccctggcccaatggtgggcagcctgaattgtatcgtggccgtgtcc cagaacatgggcatcggcaagaatgg

ggagctgaaggagc£acc€£agggagcacacttt£tgtccaggtct£tgga £gatgcc€tgaag£tgaccgagcag€ctgagctg

SUBSTITUTE SHEET (RULE 26) cgtgacacggatcatgcaggactttgagtccgataccttctttccagagatcgacctgga gaagtacaagctgctgcccgagtatcc tggcgtgctgtctgatgtgcaggaggagaagggcatcaagtacaagttcgaggtgtatga gaagaacgattgataacatatgcctt aattaaacactagttctatagtgtcacctaaattcccttagtgagggttaatggccgtag gccgccagaatgggtccagacatgataagat

tggae¾¾¾ccacaaet¾g¾¾tgc

ggactctaggacctgcgcatgcgcttggcgtaatcatggtcatagctgtttcctgttttc cccgtatccccccaggtgtctgcaggctcaaaga gcagcgagaagcgttcagaggaaagcgatcccgtgccaccttccccgtgcccgggctgtc cccgcacgctgccggctcggggatgcgg ggggagcgccggaccggagcggagccccgggcggctcgctgctgccccctagcgggggag ggacgtaattacatccctgggggcttt cggggggggctgtccctctcaccgcggtcgagctccagcttttgttcgaatggggccccc cctcgagggtatcgatgfflffflfctotoacffla

ffla^acgcagaefiifcflitofagggttaacgagacctctagctagcccgcctaatga gcgggcttttttttggcttgtgtccacaaccgttaa accttaaaagctttaaaagccttatatattcttttttttcttataaaacttaaaacctta gaggctatttaagttgctgatttatattaattttattgttcaaa catgagagcttagtacgtgaaacatgagagcttagtacgttagccatgagagcttagtac gttagccatgagggtttagttcgttaaacatgag agcttagtacgttaaacatgagagcttagtacgtactatcaacaggttgaactgctgatc cacgttgtggtagaattggtaaagagagtcgtgt aaaatatcgagttcgcacatcttgttgtctgattattgattttggcgaaaccatttgatc atatgacaagatgtgtatctaccttaacttaatgatttt gataaaaatcattacctagggaagactc (SEQ ID NO: 14937).

[0492] The 3 TTR of the PB.EF 1 a.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA

CGCATGTGTTTTATCGGTCTGTATATCGA GGTTT ATTT ATT AATTTGAATAGATATTA

AGTTTTATTATATTTAC ACTT ACATACTAAT A ATAAATTC A AC AA AC A ATTT ATTTAT

GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0493] The Insulator of the PB.EFla.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

gagggaeagcccccecceaaagcccecagggatgtaattacgtcccteccecgctagggg gcageagcgagecgcccggggctecgc tccggtccggcgetcecccegcatccccgagceggcagcgtgcggggacagccegggeac ggggaaggtggcacgggategctttcc tctgaaegcttctcgetgctctttgagcetgcagacaectggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

SUBSTITUTE SHEET (RULE 26) The EFla Promoter of the PB.EF1 a.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

agctttgcaaagatggataaagtttaaacagagaggaatctttgcagctaatggacc ttctaggtcttgaaaggagtgggaattggctc cggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagg ggtcggcaattgaaccggtgcct g agaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttccc gagggtgggggagaaccgtatataagtg cagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtg ccgtgtgtggttcccgcgggcctggcctctt tacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattct tgatcccgagcttcgggttggaagtgggtggg agagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggc ctgggcgctggggccgccgcgtgcgaatc tggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttt tgatgacctgctgcgacgctttttttctggcaagat agtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggc ggcgacggggcccgtgcgtcccagcgcac atgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctca agctggccggcctgctctggtgcctg gcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccag ttgcgtgagcggaaagatggccgcttc ccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtg agtcacccacacaaaggaaaagg gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccagg cacctcgattagttctcgagcttttggagtac gtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggt ggagactgaagttaggccagcttggcacttg atgtaattctccttggaatttgcccttttgagtttggatcttggttcattctcaagcctc agacagtggtcaaagttMttcttccatttcaggtg tcgtga (italicized) (SEQ ID NO: 14706).

[0495] The GFP of the PB.EFla.GFP-T2A-DRFR construct is encoded by a nucleic acid sequence comprising:

atggtgagcaagggcgaggagcigttcaccggggtggtgcccaicciggicgagclg gacggcgacgtaaacggccacaagitcag cgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctg caccaccggcaagctgcccgtgccctg gcccacccicgtgaccaccctgacciacggcgtgcagigctcagccgctaccccgaccac atgaagcagcacgactctcaagtcc gccalgcccgaaggctacglccaggagcgcaccatcttcttcaaggacgacggcaactac aagacccgcgccgagglgaagftcga gggcgacaccctggtgaaccgcatcgagctgaagggcategacticaaggaggacggcaa caicctggggcacaagctggagiac aaciacaacagccacaacgtciatatcaiggccgacaagcagaagaacggcatcaaggig aacticaagatccgccacaacatcga ggacggeagcgtgcagctcgccgaccactaccageagaacacceccatcggcgacggccc cgigctgctgcccgacaaccactac ctgagcacccagiccgccctgagcaaagaccccaacgagaagcgtgatcacatggtcctg ctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaag (italicized underlined) (SEQ ID NO: 14708).

[0496] The DHFR of the PB EF 1 a. GFP-T2 A-DF1FR construct is encoded by a nucleic acid sequence comprising:

atggtgggcagcctgaattgtatcgtggccgtgtcccagaacatgggcatcggcaag aatggcgattttccttggccccctctgaga

SUBSTITUTE SHEET (RULE 26) aatgagtccagatactttcagaggatgaccacaaccagctccgtggagggcaagcagaac ctggtcatcatgggcaagaagaca tggttctctatcccagagaagaaccgccccctgaagggccggatcaatctggtgctgagc agggagctgaaggagccaccccagg gagcac¾ctttctgtcc¾ggtctctgg¾cgatgcectg¾¾gctgaccg¾gc¾g cctgagctggecaacaaggtggacatggtgtgg gagtccgataccttctttccagagatcgacctggagaagtacaagctgctgcccgagtat cctggcgtgctgtctgatgtgcaggag

(bold underlined) (SEQ ID NO: 14709).

The SV40 Poly (A) of the PB.EFla.GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

gtgtgggaggtttttt (bold underlined) (SEQ ID NO: 14710).

The 5’ITR of the PB.EFla.GFP-T2A-DHFR construct is encoded bv a nucleic acid sequence comprising:

asaatscatscstcaattttacscasactatctttctasss (bold italicized underlined) (SEQ ID NO: 1471 1 ).

[0499] A vector for the expression of HBB T87Q, iCasp9, and DHFR (PB-HBB-PGK-iCasp9-

T2A-DHFR construct) is encoded by a sequence comprising:

ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATT GACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATAT T AAGTTTT ATTATATTTAC ACTTAC AT ACTA ATAATA AATTC AACAA AC AATTT ATTT

ATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAAC

AAAACTTTTActgaatacctgcageccgggggatgcagagggacageccceccecaa agcceccagggatgtaatacgtccc tcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccc cgcatccccgagccggcagcgtgcgg ggacagcccgggcacggggaaggtggcacgggatcgcttcctctgaacgcttctcgctgc tctttgagcctgcagacacctggggggat acggggaaaagttgactgtgcctttcgatcgagcgctgacgtcgcggccgctcccgcgat acgcgctaggtattgaataagaaaaatg aagttaaggtggttgatggtaacactatgctaataactgcagagccagaagcaccataag ggacatgataagggagccagcaga cctctgatctcttcctgaatgctaatcttaaacatcctgaggaagaatgggacttccatt tggggtgggcctatgatagggtaataag acagtagtgaatatcaagctacaaaaagccccctttcaaattcttctcagtcctaacttt tcatactaagcccagtccttccaaagca gactgtgaaagagtgatagttccgggagactagcactgcagattccgggtcactgtgagt gggggaggcagg

SUBSTITUTE SHEET (RULE 26) acaggacagtcaaaccatgccccctgtttttccttcttcaagtagacctctataagacaa cagagacaactaaggctgagtggcca ggcgaggagaaaccatctcgcegtaaaacaiggaaggaaeactteaggggaaaggtggia ictetaagcaagagaactgagtg gagtcaaggctgagagatgcaggataagcaaatgggtagtgaaaagacattcatgaggac agctaaaacaataagtaatgtaa aatacagcatagcaaaactttaacctccaaatcaagcctctacttgaatccttttctgag ggatgaataaggcataggcatcagggg ctgttgccaatgtgcattagctgtttgcagcctcaccttctttcatggagtttaagatat agtgtattttcccaaggtttgaactagctctt catttctttatgttttaaatgcactgacctcccacattccctttttagtaaaatattcag aaataatttaaatacatcattgcaatgaaaa taaatgttttttattaggcagaatccagatgctcaaggcccttcataatatcccccagtt tagtagttggacttagggaacaaaggaa cctttaatagaaattggacagcaagaaagcgagcttagtgatacttgtgggccagggcat tagccacaccagccaccactttctga taggcagcctgcactggtggggtgaattctttgccaaagtgatgggccagcacacagacc agcacgttgcccaggagctgtggga ggaagataagaggtatgaacatgattagcaaaagggcctagcttggactcagaataatcc agccttatcccaaccataaaataaa agcagaatggtagctggattgtagctgctattagcaatatgaaacctcttacatcagtta caatttatatgcagaaatatttatatgca gaaatattgctattgccttaacccagaaattatcactgttattctttagaatggtgcaaa gaggcatgatacattgtatcattattgccc tgaaagaaagagattagggaaagtattagaaataagataaaeaaaaaagtatattaaaag aagaaagcattttttaaaattaca aatgcaaaattaccctgatttggtcaatatgtgtaccctgttacttctccccttcctatg acatgaacttaaccatagaaaagaaggg gaaagaaaacatcaagggtcccatagactcaccctgaagttctcagggtecacgtgcage ttgteacagtgcagcteactcagctg ggcaaaggtgcccttgaggttgtccaggtgagccaggccatcactaaaggcaccgagcac tttcttgccatgagccttcaccttagg gttgcccataacagcatcaggagtggacagatccccaaaggactcaaagaacctctgggt ccaagggtagaccaccagcagcct aagggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagt ctctctgtctccacatgcccagttt ctattggtctccttaaacctgtcttgtaaccttgataccaacctgcccagggcctcacca ccaacttcatccacgttcaccttgcccca cagggcagtaacggcagacttctcctcaggagtcaggtgcaccatggtgtctgtttgagg ttgctagtgaacacagttgtgtcagaa gcaaatgtaagcaatagatggctctgccctgacttgtatgcccagccctggctcctgccc tccctgctcctgggagtagattggccaa ccctagggtgtggctccacagggtgaggtctaagtgatgacagccgtacctgtccttggc tcttctggcactggcttaggagttggac ttcaaaccctcagccctccctctaagatatatctcttggccccataccatcagtacaaat tgctactaaaaacatcctcctttgcaagt gtatttacctagaatatgtcacattctgtctcaggcatccattttctttatgatgccgtt tgaggtggagttttagtcaggtggtcagctt ctccttttttttgccatctgccctgtaagcatcctgctggggacccagataggagtcatc actctaggctgagaacatctgggcacac accctaagcctcagcatgactcatcatgactcagcattgctgtgcttgagccagaaggtt tgcttagaaggttacacagaaccagaa ggcgggggtggggcactgaeccegacaggggcctggccagaactgetcatgetiggacia tgggaggtcactaatggagacaca cagaaatgtaacaggaactaaggaaaaactgaagcttatttaatcagagatgaggatgct ggaagggatagagggagctgagct tgtaaaaagtatagtaatcattcagcaaatggttttgaagcacctgctggatgctaaaca ctattttcagtgcttgaatcataaataa gaataaaacatgtatcttattccccacaagagtccaagtaaaaaataacagttaattata atgtgctctgtcccccaggctggagtg ^ca gtggcacgatgtcagctcactgcaacctccgcctcccgggggactagtctcgag ggctggttagaaggttctactggaggaggg

SUBSTITUTE SHEET (RULE 26) tcccagcccattgctaaattaacatcaggctctgagactggcagtatatctctaacagtg gttgatgctatcttctggaacttgcctgc tacattgagaccactgacccatacataggaagcccatagctctgtcctgaactgttaggc cactggtccagagagtgtgcatctcctt tgatcctcataataaccctatgagatagacacaattattactcttactttatagatgatg atcctgaaaacataggagtcaaggcact tgcccctagctgggggtataggggagcagtcccatgtagtagtagaatgaaaaatgctgc tatgctgtgcctcccccacctttccca tgtctgccctctactcatggtctatctctcctggctcctgggagtcatggactccaccca gcaccaccaacctgacctaaccacctatc tgagcctgccagcctataacccatctgggccctgatagctggtggccagccctgacccca ccccaccctccctggaacctctgatag acacatctggcacaccagctcgcaaagtcaccgtgagggtcttgtgtttgctgagtcaaa attccttgaaatccaagtccttagaga ctcctgctcccaaatttacagtcatagacttcttcatggctgtctcctttatccacagaa tgattcctttgcttcattgccccatccatctg atcctcctcatcagtgcagcacagggcccatgagcagtagctgcagagtctcacataggt ctggcactgcctctgacatgtccgacc ttaggcaaatgcttgactcttctgccctcgagaagcttatcgatggctagtgcatgcaaa tctgacactcagtgggcctgggtgaag gtgagaattttattgctgaatgagagcctctggggacatcttgccagtcaatgagtctca ggttcaatttccttctcagtcttggagta acagaagctcatgcatttaataaacggaaattttgtattgaaatgagagccattggaaat catttactccagactcctacttataaaa agagaaactgaggctcagagaagggtggggactttcteagtatgaeatggaaatgateag gettggattcaaagctcetgactttc tgtctagtgtatgtgcagtgagccccttttcctctaactgaaagaaggaaaaaaaaatgg aacccaaaatattctacatagtttccat gtcacagccagggctgggcagtctcctgttatttcttttaaaataaatatatcatttaaa tgcataaataagcaaaccctgctcggga atgggagggagagtctctggagtccaccccttctcggccctggctctgcagatagtgcta tcaaagccctgacagagccctgcccat tgctgggccttggagtgagtcagcctagtagagaggcagggcaagccatctcatagctgc tgagtgggagagagaaaagggctc attgtctataaactcaggtcatggctattcttattctcacactaagaaaaagaatgagat gtctacatataccctgcgtcccctcttgt gtactggggtccccaagagctctctaaaagtgatggcaaagtcattgcgctagatgccat cccatctattataaacctgcatttgtct ccacacaccagtcatggacaataaccctcctcccaggtccacgtgcttgtctttgtataa tactcaagtaatttcggaaaatgtattct ttcaatcttgttctgttattcctgtttcaatggcttagtagaaaaagtacatacttgttt tcccataaattgacaatagacaatttcacat caatgtctatatgggtcgttgtgtttgctgtgtttgcaaaaactcacaataactttatat tgttactactctaagaaagttacaacatgg tgaatacaagagaaagctattacaagtccagaaaataaaagttatcatcttgaggccatt taattaacatatgtcgcgagtactcaccg gtgcc&gac&ctggggg&cccttgcgccttttccaaggcagccctggg tttgcgcagggacgcggctgctctgggcgtggttccgggaa acgcagcggcgccgaccctgggtctcgcacattctlcacgtccgttcgcagcgtcacccg gatcttcgccgctacccttgtgggcccccc ggcgaegcttcctgetcegcccctaagtegggaaggitccttgcggitcgcggcgtgceg gacgtgacaaacggaagecgcacgicte actogtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgg gctgtggccaatagcggctgctcagca gggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggta gtgtgggccctgttcctgcccgcgc ggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgacc gaatcaccgacctctctccccaggggccacc aagcitgtcctgcaggagggtcgacgcctctagagccaccatgggcgigciggicgiigi iciaic gccccggag igggaga caiii ccaaaaagagggcagacttgcgtcgtccactacactggcatgctggaggacgggaaaaaa gtcgatagcagtcgggatcggaata

SUBSTITUTE SHEET (RULE 26)

ggcagagggtcactgctgacatgcggci

assatgaccacaac aaccgccc ggg££S zagccaccccagggagcacactttctgtcca atgtctggacgatgcc agaagaaegat†gataatgataaagatetacgcgttaegtatagccactttaaaagaa aaggg caacffg tf «gc ge ? /gg ttacaaataaagcaataecatcacaaalttcacaaataaagcatltuttcactgcaltcl azttsteelttslccaaaclcatcaaletatctt c /gfcgtgtcgaaattetagcttggcgtaatcatggtcatagctgttectgtgtgaacgag ccggaagcataaagtglaggcctacttgg ccgaattcgtttatcgtcgagggtaccgatcacatatgcctttaattaaacactagttct atagtgtcacctaaattccctttagtgagggttaatg gccgtaggccgccagaattgggtccagacatgataagatacattgatgagtttggacaaa ccacaactagaatgcagtgaaaaaaatgcttt atttgtgaaatttgtgatgctatgctttatttgtaaccatataagctgcaataaacaagt taacaacaacaattgcattcattttatgtttcaggttc agggggaggtgtgggaggtttttcggacictaggaceigcgcatgcgeitggcgtaatca tggicatagctgittcctgttttccecgtatcce cccaggtgtctgcaggctcaaagagcagcgagaagcgttcagaggaaagcgatcccgtgc caccttccccgtgcccgggctgtccccgc acgctgccggctcggggatgcggggggagcgccggaccggagcggagccccgggcggctc gctgctgccccctagcgggggaggg acgtaattacatccctgggggcttgggggggggctgtccctctcaccgcggtggagctcc agctttgtcgaattggggccccccctcga

SUBSTITUTE SHEET (RULE 26)

The 3’ITR of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA CGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTA AGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTAT GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0501] The Insulator 1 of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

gagggacagcccecccccaaagcecccagggatgtaatacgtccetcccccgctagg gggcagcagegagccgcccggggctccge tecggteeggcgctccceccgeatecccgagccggeagcgtgeggggacageccgggcac ggggaaggtggcacgggatcgetttcc tctgaacgcttctcgctgctctttgagcctgcagaeacetggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0502] The T87Q beta-g!obin cassette with LCR of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

ctaggtatigaataagaaaaatgaagttaaggtggttgaiggtaacactatgetaat aaetgeagagccagaagcaecaiaaggg acatgataagggagccagcagacctctgatctcttcctgaatgctaatcttaaacatcct gaggaagaatgggacttccatttgg tgggcctatgatagggtaataagacagtagtgaatatcaagctacaaaaagccccctttc aaattcttctcagtcctaacttttcata ctaagcccagtccttccaaagcagactgtgaaagagtgatagttccgggagactagcact gcagattccgggtcactgtgagtggg ggaggcagggaagaagggctcacaggacagtcaaaccatgccccctgtttttccttcttc aagtagacctctataagacaacagag acaactaaggctgagtggccaggcgaggagaaaccatctcgccgtaaaacatggaaggaa cacttcaggggaaaggtggtatct ctaagcaagagaactgagtggagtcaaggctgagagatgcaggataagcaaatgggtagt gaaaagacattcatgaggacagc taaaacaataagtaatgtaaaatacagcatagcaaaactttaacctccaaatcaagcctc tacttgaatccttttctgagggatgaa taaggcataggcatcaggggctgttgccaatgtgcattagctgtttgcagcctcaccttc tttcatggagtttaagatatagtgtatttt cccaaggtttgaactagctcttcatttctttatgttttaaatgcactgacctcccacatt ccctttttagtaaaatattcagaaataattta aatacatcattgcaatgaaaataaatgttttttattaggcagaatccagatgctcaaggc ccttcataatatcccccagtttagtagtt ggacttagggaacaaaggaacctttaatagaaattggacagcaagaaagcgagcttagtg atacttgtgggccagggcattagcc acaccagccaccactttctgataggcagcctgcactggtggggtgaattctttgccaaag tgatgggccagcacacagaccagcac gttgcccaggagctgtgggaggaagataagaggtatgaacatgattagcaaaagggccta gcttggactcagaataatccagcct

SUBSTITUTE SHEET (RULE 26) atgcagaaatatttatatgcagaaatattgctattgccttaacccagaaattatcactgt tattctttagaatggtgcaaagaggcatg atacattgtatcattattgccctgaaagaaagagattagggaaagtattagaaataagat aaacaaaaaagtatattaaaagaag aaagcattttttaaaattacaaatgcaaaattaccctgatttggtcaatatgtgtaccct gttacttctccccttcctatgacatgaactt aaccatagaaaagaaggggaaagaaaacatcaagggtcccatagactcaccctgaagttc tcagggtccacgtgcagcttgtca cagtgcagctcactcagctgggcaaaggtgcccttgaggttgtccaggtgagccaggcca tcactaaaggcaccgagcactttctt gccatgagccttcaccttagggttgcccataacagcatcaggagtggacagatccccaaa ggactcaaagaacctctgggtccaa gggtagaccaccagcagcctaagggtgggaaaatagaccaataggcagagagagtcagtg cctatcagaaacccaagagtcttc tctgtctccacatgcccagtttctattggtctccttaaacctgtcttgtaaccttgatac caacctgcccagggcctcaccaccaacttc atccacgttcaccttgccccacagggcagtaacggcagacttctcctcaggagtcaggtg caccatggtgtctgtttgaggttgctag tgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctgccctgacttgtatg cccagccctggctcctgccctccctgct cetgggagtagattggceaaecetagggtgtggetccacagggtgaggtctaagtgatga cagccgtaectgtccttggetcttctg gcactggcttaggagttggacttcaaaccctcagccctccctctaagatatatctcttgg ccccataccatcagtacaaattgctacta aaaacatcctcctttgcaagtgtatttacctagaatatgtcacattctgtctcaggcatc cattttctttatgatgccgtttgaggtggag ttttagtcaggtggtcagcttctccttttttttgccatctgccctgtaagcatcctgctg gggacccagataggagtcatcactctaggc tgagaacatctgggcacacaccctaagcctcagcatgactcatcatgactcagcattgct gtgcttgagccagaaggtttgcttaga aggttacacagaaccagaaggcgggggtggggcactgaccccgacaggggcctggccaga actgctcatgcttggactatggga ggtcactaatggagacaeacagaaatgtaacaggaactaaggaaaaactgaagcttattt aatcagagatgaggatgctggaag ggatagagggagctgagcttgtaaaaagtatagtaatcattcagcaaatggttttgaagc acctgctggatgctaaacactattttc agtgcttgaatcataaataagaataaaacatgtatcttattccccacaagagtccaagta aaaaataacagttaattataatgtgct ctgtcccccaggctggagtgcagtggcacgatgtcagctcactgcaacctccgcctcccg ggggactagtctcgagggctggttag aaggttctactggaggagggtcccagcccattgctaaattaacatcaggctctgagactg gcagtatatctctaacagtggttgatg ctatcttctggaacttgcctgctacattgagaccactgacccatacataggaagcccata gctctgtcctgaactgttaggccactgg tccagagagtgtgcatctcctttgatcctcataataaccctatgagatagacacaattat tactcttactttatagatgatgatcctgaa aacataggagtcaaggcacttgcccctagctgggggtataggggagcagtcccatgtagt agtagaatgaaaaatgctgctatgct gtgcctcccccacctttcccatgtctgccctctactcatggtctatctctcctggctcct gggagtcatggactccacccagcaccacc aacctgacctaaccacctatctgagcctgccagcctataacccatctgggccctgatagc tggtggccagccctgaccccaccccac cctccctggaacctctgatagacacatctggcacaccagctcgcaaagtcaccgtgaggg tcttgtgtttgctgagtcaaaattcctt gaaatccaagtccttagagactcctgctcccaaatttacagtcatagacttcttcatggc tgtctcctttatccacagaatgattccttt gcttcattgccccatccatctgatcctcctcatcagtgcagcacagggcccatgagcagt agctgcagagtctcacataggtctggc actgcctctgacatgtccgaccttaggcaaatgcttgactcttctgccctcgagaagctt atcgatggctagtgcatgcaaatctgac actcagtgggcctgggtgaaggtgagaattttattgctgaatgagagcctctggggacat cttgccagtcaatgagtctcaggttca

SUBSTITUTE SHEET (RULE 26) atttccttctcagtcttggagtaacagaagctcatgcatttaataaacggaaatttgtat tgaaatgagagccattggaaatcatta ctccagactcctacttataaaaagagaaactgaggctcagagaagggtggggactttctc agtatgacatggaaatgatcaggctt ggattcaaagctcctgactttctgtctagtgtatgtgcagtgagccccttttcctctaac tgaaagaaggaaaaaaaaatggaaccc aaaatattctacatagtttccatgtcacagccagggctgggcagtctcctgttatttctt ttaaaataaatatatcatttaaatgcataa ataagcaaaccctgctcgggaatgggagggagagtctctggagtccaccccttctcggcc ctggctctgcagatagtgctatcaaa gccctgacagagccctgcccattgctgggccttggagtgagtcagcctagtagagaggca gggcaagccatctcatagctgctga gtgggagagagaaaagggeteattgtcfafaaactcaggteatggetattettattctca eaetaagaaaaagaatgagatgtetac atataccctgcgtcccctcttgtgtactggggtccccaagagctctctaaaagtgatggc aaagtcattgcgctagatgccatcccat ctattataaacctgcatttgtctccacacaccagtcatggacaataaccctcctcccagg tccacgtgcttgtctttgtataatactcaa gtaatttcggaaaatgtattctttcaatcttgttctgttattcctgtttcaatggcttag tagaaaaagtacatacttgttttcccataaat tgacaatagacaatttcacatcaatgtctatatgggtcgttgtgtttgctgtgtttgcaa aaactcacaataactttatattgttactact ctaagaaagltaeaaeatggtgaataeaagagaaagetattacaagtccagaaaataaaa giiatcatcttgaggeeatt (bold)

(SEQ ID NO: 14935)

[0503] The human-PGK promoter of the PB-HBB-PGK-iCasp9-T2 A-DHFR construct is encoded by a sequence comprising:

ttgcgcctttccaaggcagccctgggttgcgcagggacgcggctgctctgggcgtgg tccgggaaacgcagcggcgccgaecctg ggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttg tgggccccccggcgacgcttcctgctccgcc cctaagtcgggaaggttecttgcggttcgcggcgtgccggacgtgacaaacggaagccgc acgtctcactaglaccc (italicized) (SEQ ID NO: 14696)

[0504] The iCasp9 of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

atgggcgtgcaggtcgagactatcagccccggagatgggagaacatttccaaaaaga gggcagacttgcgtcgtccactacactggc atgctggaggacgggaaaaaagtcgatagcagtcgggatcggaataagccattcaagttt atgctggggaagcaggaggtcatccg ggggigggi giigggggicgcicagdtgtciigigggccisgcgggciu gctgdcmiiciccccagactaigcaiacggcgciaccg ggcatcctggaattatcccaccccatgccaccctggtgttgacgtggaactgctgaagct ggaagggggcggggggagtggctttgg ggatgtgggcgccctggagagtctgagagggaacgctgatctggcttacattctgagcat ggaaccatgcggccactgcctgatcatta acaatgtgaacttttgtagagagtcagggctgcgaactcgaacagggagcaacatcgatt gtgaaaagctgaggcgccggttctctag tctgcactttatggtcgaggtgaagggagacctgacagcaaagaagatggtgctggccct gctggagctggcccagcaggaccacg gggcactggactgttgcgtcgtcgtgatcctgtctcacggctgtcaggcaagtcacctgc aattccccggcgccgtctacggcactgatg ggtgccccgtctccgtggaaaagattgtgaatatcttaatggaacatcatgcccatctct ggggggaaagccaaaactgtcttatcca ggcttgtggaggggaacagaaggatcacggcttgaagtcgcctccacaagtccagaagat gaaagcccaggcagtaatcccgagc

SUBSTITUTE SHEET (RULE 26)

[0505] The DHFR of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

atggtgggcagcctgaattgtatcgtggccgtgtcccagaacatgggcatcggcaagaat ggcgattttccttggccccctctgaga aatgagtccagatactttcagaggatgaccacaaccagctccgtggagggcaagcagaac ctggtcatcatgggcaagaagaca tggttctctateecagagaagaaccgccceetgaagggccggatcaatetggtgctgage agggagctgaaggagccaeeceagg

The SV40Poly(A) of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

acaacttsittattscascttataatssttacaaataaascaatascatcacaaatttca caaataaascattttttcactscattctastts tsstltetccaaactcatcaatgtatcltatcatetc (italicized underlined) (SEQ ID NO: 14710).

[0506] The Insulator 2 of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

tttcceegtatcceeceaggtgtetgcaggetcaaagageagcgagaagcgtcagaggaa agcgatecegtgccaccttceecgtgecc gggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggag ccccgggcggctcgctgctgccccct agcgggggagggacgtaattacatccctgggggcttgggggggggctgtccctc (underlined) (SEQ ID NO: 14716).

[0507] The 5’ITR of the PB-HBB-PGK-iCasp9-T2A-DHFR construct is encoded by a sequence comprising:

[0508] atatctataacaagaaaatatatatataataaettatcacstaagtasaacatsaaataa caatataattatcstatsastta

tattcaasaatscatscstcaatttacscasactatctttctasss (bold italicized underlined) (SEQ ID NO: 14711).

[0509] A construct for constitutive expression of Interleukin 2 Receptor subunit gamma, GFP, and DHFR (Nano-PB-V5-JctR-Ank-miRE-sh49-BCLl lA-EFla-GFP-T2A-DHFR) is encoded

SUBSTITUTE SHEET (RULE 26) by a sequence comprising:

ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAA TT

GACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATA T

TAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATT T

ATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAAC

AAAACTTTTAtcgaatacctgcagcccgggggatgcagagggacagcccccccccaa agcccccagggatgtaattacgtccc tcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccc cgcatccccgagccggcagcgtgcgg ggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctg ctctttgagcctgcagacacctggggggat acggggaaaagttgactgtgcctttcgatcgcgffcgflaggggcaaccagggg/ccgcg cgccgaggcc^ggggaecggggccrccr ggggttgggggaggaggtgctcttgtaatctgcggtggtccccaggcgggcgccacccct ccgcccgcccgtgccgggagcgcccgg cccgacagcaagcgcctctggggccgataaggccctcgggggcctggcccgcacgtcaca ggccccgcagaggctgcggtgagtc cgccagccccagctgctcctcctcaagcccccaaggcccttcggcggcaattcccaccgg ggt&cogtaaiagtmtagggccctsic gagcttgctgtttgaatgaggcttcagtactttacagaatcgttgcctgcacatcttgga aacacttgctgggattacttcgacttctta acccaacagaaggctcgagaaggtatattgctgttgacagtgagcgccgcacagaacact catggatttagtgaagccacagatg taaatccatgagtgttctgtgcgttgcctactgcctcggacttcaaggggctacaattgg agcaattatcttgtttactaaaactgaat accttgctatctctttgatacatttttacaaagctgaattaaaatggtataaattaaatc actgcgtcgacatcgaaccatggacagtt4

GCTnGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCT TCT

AGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACAT CG

CCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGA A

GGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCmTTCCCGAG

GGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTmTCGCAACGG G

TTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTT AC

GGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCT TGAT

CCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTrAAGGAGCC CC

TTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAAT CT

GGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGAIAAGTCTCIAGCCATTIAAAATT TTTG

ATGACCTGCTGCGACGGTmTn'CTGGCAAGATAGTCTTGTAAATGCGGGCGAAGATC TG

CACACmGTATTTCGGTTnTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGC

GCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGT A

GTCTCAAGCTGGCCGGCCTGGTCTGGTGCCTGGCCTCGGGCCGCGGTGTATCGCCCC GC

CGTGGGCGGGAAGGCTGGCCCGGTCGGGACCAGTTGCGTGAGGGGAAAGATGGCCGC T

SUBSTITUTE SHEET (RULE 26) TCCCGGCCCTGCTGCAGGGAGGi’CAAAATGGAGGACGCGGGGCTGGGGAGAA CGGGCG

GGTGAGTCACCCACACAAAGGAAAAGGGCCrrrCCGTGCTCAGGCGTCGGriCATGT GAC

TCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGA'ITAGn'CTCGAGCTITTGGAG TACG

TCGTCTr GGTTGGGGGGM}GGGTTTIATGCGATG(}AGTTTCCCCACACT(MGTGGGTG

GAGAGTGAAGTTAGGCCAGGTTGGCACTTGATGTAAnCTGCTTGGAATTTGCCCTmT G

A GTTTGGA TCTTGGTTCA TTCTCAA GCCTCA GA CA GTGGTTCAAA GTTTTTTTCTTCCA ITT

C4GG7 ^’ GICGJ ^’ G igaattctaatacgactcactatagggtgtgctgtctcatcattttggcaaagattggcc accaagcttaccgcc i gKtgqKcqqgggcgqggqgc tcaccggggtggtgcccMcctggtcgqgctggqcgKCgacgtaqqcggccqc gtcqgcg tgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgca ccaccggcaagctgcccgtgccctggc ccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccaca tgaagcagcacgacttcttcaagtccgc catgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaa gacccgcgccgaggtgaagttcgagg gcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaaca tcctggggcacaagctggagtacaa ctacaacagccacaacgtciatatcatggccgacaagcagaagaacggcatcaaggtgaa cttcaagatccgccacaacatcgagg acggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccg tgctgctgcccgacaaccactacctg agcacccagtccgccctgagcaaagaccccaacgagaagcgtgatcacatggtcctgcig gagttcgigaccgccgccgggatcact crcggca^gg cgagctetoc-aaggaa ^gg aa ^g a ^gg ca ^g cct ^g cteacat ^g t ^gg c ^g ac ^g t ^gg a ^gg a ^g aaccct ^gg cccaatggtggg

gataccttctttccagagatcgacctggagaagtacaagctgctgcccgagtatcct ggcgtgctgtctgatgtgcaggaggagaa gggcatcaagtacaagttcgaggtgtatgagaagaacgatgataacatatgccttaataa acactagtctatagtgtcacctaaattc ccttagtgagggtaatggccgtaggccgccagaattgggtccagacatga aagafacetfgai^a^ ii&gacaaaccacaacfti^e

agctgttcetattttecccgiatecceccaggtgtctgcaggctcaaagagcagcaa gaagcgttcagaggaaagegateccgtgecaect tccccgtgcccgggctatccccgcacgctgccggctcggggatgcgaggggagcgccaaa ccggaacggagccccgggcggctcg ctactgccccctagcgggggaaggacgtaatacatccctgggggcttgggggggggctgt ccctctcaccgcggtggagctccagctt ttgttcgaattggggeccecccicgagggtatcgat( 4 TA TCTA TAA CAA GAAAA TA TA TA TA TALA TAAGTT A TCACGTAAGTAGAACATGAAA TAACAA TATAATTA TCGTA TGAGTTAAA TCTTAAAAG

5UB5TITUTE SHEET (RULE 26) TCACGTAAAAGA TAA TCATGCGTCA TTTTGACTCACGCGGTCGTTA TAGTTCAAAA TCA

GTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGA

TG TCCTAAA TGCA CA G CGA CG GA TTCGCGCTA ITT A C 4GAGCAATATTTCA

GAA TGCA TGCGTCAA TTTTA CGCA GA CTA TCTTTCTA G ’ taatctagctagcc ttaagggcgcc tcea gacctctagctagcccgcctaatgagcgggcttttttttggcttgttgtccacaaccgtt aaaccttaaaagctttaaaagccttatatattctttttt ttcttataaaacttaaaaccttagaggctatttaagttgctgatttatattaattttatt gttcaaacatgagagcttagtacgtgaaacatgagagct tagtacgttagccatgagagcttagtacgttagccatgagggtttagttcgttaaacatg agagcttagtacgttaaacatgagagcttagtacg tactatcaacaggttgaactgctgatccacgttgtggtagaattggtaaagagagtcgtg taaaatatcgagttcgcacatcttgttgtctgatta ttgatttttggcgaaaccatttgatcatatgacaagatgtgtatctaccttaacttaatg attttgataaaaatcattacctagggaagactc (SEQ ID NO: 14975).

[0510] The 3 TTR of the Nano-PB-V5-JctR-Ank-miRE-sh49-BCLl 1 A-EF 1 a-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

CCCT AGAA AGATAAT CAT ATT GT GACGT ACGTT A A AGAT AAT C ATGC GT AAAATT GA CGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTA AGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTAT GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0511] The Insulator 1 of the Nano-PB-V5-JctR-Ank-miRE-sh49-BCLl 1 A-EF1 a-GFP-T2A- DHFR construct is encoded by a sequence comprising:

gagggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctag ggggcagcagcgagccgcccggggctccgc tccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggatcgctttcc tctgaacgcttctcgctgctcttgagcctgcagacacctggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0512] The Ank promoter of the Nano-PB-V5-JctR-Ank-miRE-sh49-BCLl 1 A-EF I a-GFP- T2A-DHFR construct is encoded by a sequence comprising:

cgcgttcgaaggggcaaccaggggtccgcgcgccgaggcctggggagcggggcctcc tggggttgggggaggagglgctcttgtaa tctgcggiggtccccaggcgggcgecacccctccgcecgcccgtgcegggagcgcceggc ccgaeagcaagcgcetctggggecg ataaggccctcgggggcctggcccgcacgtcacaggccccgcagaggctgcggtgagtcc gccagccccagctgctcctcctcaag cccccaaggcccttcggcggcaattcccaccgg (italicized) (SEQ ID NO: 14701 ).

[0513] The miR-E sh49 BCL11 A of the Nano-PB-V5- JctR- Ank-miRE-sh49-BCL 1 1 A-EF 1 a- GFP-T2A-DHFR construct is encoded by a sequence comprising:

gtacaagtaatagggccctacgagcttgctgtttgaatgaggcttcagtactttaca gaatcgttgcctgcacatcttggaaacactt

SUBSTITUTE SHEET (RULE 26) gctgggaiiacttcgacttciiaacccaacagaaggcicgagaaggiaiaiigctgiiga cagtgagcgccgcacagaacactcai ggatttagtgaagccacagatgtaaatccatgagtgttctgtgcgttgcctactgcctcg gacttcaaggggctacaattggagcaa ttatcttgtttactaaaactgaataccttgctatctctttgatacattttacaaagctga attaaaatggtataaataaatcactgc

(bold) (SEQ ID NO: 14713).

[0514] The IT i alpha of the Nano-PB-V5-JctR-Ank-miRE-sh49-BCLl 1 A-EF 1 a-GFP-T2 A- DHFR construct is encoded by a sequence comprising:

AGCI T1 'GCAAAGAl'GGAlAAAGl "I TlAAACAGAGAGGAATCTTl GCAGCTAATGGACCT1 'C TA GGTCTTGAAA GGA GTGGGAA TTGGCTC CGGTGCCCGTCA GTGGGCAGA GCGCA CA TC GCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGA AGGTGGCGCGGGGTAAACl GGGAAAGJ GATGTCGTGTACl GGCTCCGCCTTl Ί TCCCGA GGGTGGGGGA GAA CC GTA TA TAA GTGCA GTA GTCGCCGTGAA CGTTCTTTTTCGCAACGG GTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTA

CGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTC TTGA

TCCCGAGGTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGGTTAAGGAGC CC

CTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAA T

CTGGTGGCACCTTCGCGCCTGTCTCGCTGCnTCGATAAGTCTCTAGCCATTTAAAAT TTTT

GATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAG ATCT

GCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA G

CGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGG

TAGTCTCAAGCTGGCCGGCCTGCTCTGGTGGGTGGCCTCGCGCCGCCGTGTATCGCC CC

GCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCC G

CTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGG G

CGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCAT GTG

ACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGA GTA

CGTCGTCTTTAGGITGGGGGGAGGGGTnTATGCGATGGAGnTCCCCACACTGAGTGG G

JGGAGAGGGAAGTTAGGCCAGCTTGGCACTTGAIGTAATTCTCCTTGGAArTTGCCC TTTT

TGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCT TCCAT

riGA GG IGTCGTGA (ALL CAPS italicized) (SEQ ID NO: 14706).

[0515] The eGFP of the Nano-PB-V 5-JctR- Ank-miRE-sh49-BCLl 1 A-EF1 a-GFP-T2A-DHFR construct is encoded by a sequence composing:

atz aKcaazzzcxazzazct&tcacczzzztSQitzcccatcctgztczazctzzaczz czacsitaaaczzccacaaxttcax

SUBSTITUTE SHEET (RULE 26) cgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctg caccaccggcaagcigcccgtgccctg gcccaccctcgtgaccaccetgacctacggcgigcagtgcitcagecgctacccegacca caigaagcagcacgacttcttcaagicc gccatgcecgaaggctacgtccaggagcgcaccaicttcttcaaggacgacggcaaetac aagacecgcgccgaggtgaagttcga gggcgacaccctggtgaaccgcatcgagctgaagggcaicg cttcaagg ggacggcaacatcctggggc caagciggagtac aactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtg aacttcaagatccgccacaacatcga ggacggcagcgtgcagclcgccgaccactaccagcagaacacccccatcggcgacggccc cgtgctgctgcccgacaaccactac ctgagcacccagtccgccctgagcaaagaccccaacgagaagcgtgatcacatggtcctg ctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaag (italicized underlined) (SEQ ID NO: 14708).

[0516] The DHFR of the Nano-PB-V5-JctR-Ank-miRE-sh49-BCLl 1 A-EF 1 a-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

_. f££tigtcl¾tecggg¾gg¾ tgcgcccec gSccgggtc ¾tetggtgcto¾oc ^¾ocf § S§ _J SCc¾ecj;e¾gg gagcacacttctffltocaggtctct¾a^ocggiS^gct¾^ugcj^accgagcajBcc tsgggtggccaacaassgtggacatssgtgtgg g _. fgg£gg _. gg£tciaj¾cgt&f t§ _J gsagggi;gtga l€gecgtggceacj;tgaa&c tcgtg¾eacg¾atcat ¾gggettt gggtec&ataccttetttcj; g fggg£gl^agaa ¾caa ^o e ct ccg &f gctggegtget .t&g.tete£g.gg gagaagggeatc¾agtae¾¾gitcg¾ggigi¾tgagaagaacgattgataa (bold underlined) (SEQ ID NO: 14709).

[0517] The SV40 poly A signal of the Nano-PB-V5-JctR-Ank-miRE-sh49-BCLl 1 A-EF1 a- GFP-T2 A-DHFR construct is encoded by a sequence comprising:

[0518] The Insulator 2 of the Nano-PB-V5-JctR-Ank-miRE-sh49-BCL 1 1 A-EF 1 a-GFP-T2A- DHFR construct is encoded by a sequence comprising:

ttttccccgtatccecccaggtgtetgcaggetcaaagageagcgagaagcgttcagagg aaagcgatcccgtgccaccticcecgtgecc gggctgtccecgcaegctgecggcteggggatgcggggggagegccggaccggageggag ccecgggcggctcgctgctgecccet agcgggggagggacgtaattacatccctgggggctttgggggggggctgtccctc (underlined) (SEQ ID NO: 14716).

[0519] The 5’-lTR of the Nano-PB- V5- JctR-Ank-miRE-sh49-BCL 11 A-EF 1 a-GFP-T2A-

DHFR construct is encoded by a sequence comprising:

A A CAA TA TAA TTA TCGTA TGA GTTA A A TCTTAAAA G TCA CGTAAAA GA TAA TCA TGCG T

SUBSTITUTE SHEET (RULE 26) CA TTTTGA CTCA CGCGGTCGTTA TA GTTCAAAA TCA GTGA CA CTTA CCG CA TTGA CAA

GCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGG

A TTCGCG CTA TTTAGAAAG. 1AGCAATATTTCAAGAATGCATGCGTCAATTTTACG

(bold italicized underlined) (SEQ ID NO: 1471 1).

[0520] A vector for the expression of IGF2BP1 , GFP, and DHFR (PB-V5-JctR-SPTAl - IGF2BPl-EFla-GFP-T2A-DHFR) is encoded by a sequence comprising:

ttaaCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAA TT

GACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATA T

TAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATT T

ATGTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAAC

AAAACTTTTAtcgaatacctgcagcccgggggatgcagagggacagcccccccccaa agcccccagggatgtaattacgtccc tcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccc cgcatccccgagccggcagcgtgcgg ggacagcccgggcacggggaaggtggcacgggatcgctttcct gaacgctctcgctgctctttaagcctgcagacacctggggggat acggggaaaagttgactgtgcctttcgatcg ccagactttcaagaagagaatgtaaaggactctcagtttttctatgtgagatccaagaac aggggaaaccacaaatgtagaatcaaaatcttctctgtagatcatggaagttagatatca agctgcatttacactggaatgaagttg gcatggatagccccactggcttgtagaaaggtctaagagcatcccactatttctttttgc ttgttgttcattttgtaagttagtctaaaata actatgcctatggaaaaagtctacctctttttgttctccattaaaattaatacatacagt aggatgatttataccatatatatcagaagtcagc catattttcaacttttttgtgacatcattggtttgtcaaataaggtatcctaatttttgt tttacaaaatattaatgaacacatcatataacttttttt gttcctccalttaaatgaltaagagtacaactaaatcagaittttaaagaccaaatgttg aggttaggaataaaatcltatlttgtgaaag actclctagaaaaagccaggalaattcgttgacltaaagactglgaaaaalctgcagttg agtlggatgaagttgtgagctglatttccta taaatalatggggttitUttctaattittcatccaaaaaccUactticaUtUtaagtcit aUgagtaacUtgaaagccagagcctcccaaa actgclgagtcacccagtatctgtaaaacttagcagtgcctcagclgagtatgtcttcta aagataatgtcgattglgtatggclgatggg attctaggaccaagcaagaggtttttUttttcccccacatacttaacgtttctatatUct atttgaatlcgactggacagttccatttgaattatt ictctctctcictctctctctctgacacattttatctgccaggttctaaacccggtaccg ccatgaacaagcttacatcggcaacctcaac gasascgtgacccccscggacttssagaaaststttscgsascacaagatctcctacagc ggccagttcttgstcaaatccsscta cgccttcgtggactgcccggacgagcactgggcgatgaaggccatcgaaactttctccgg gaaagtagaattacaaggaaaacgc ttagagattgaacattcggtgcccaaaaaacaaaggagccggaaaattcaaatccgaaat attccaccccagctccgatgggaag tactggacagcctgctggctcagtatggtacagtagagaactgtgagcaagtgaacaccg agagtgagacggcagtggtgaatgt cacctattccaaccgggagcagaccaggcaagccatcatgaagctgaatggccaccagtt ggagaaccatgccctgaaggtctcc tacatccccgatgagcagatagcacagggacctgagaatgggcgccgagggggctttggc tctcggggtcagccccgccagggct cacctgtggcagcgggggccccagccaagcagcagcaagtggacatcccccttcggctcc tggtgcccacccagtatgtgggtgc

SUBSTITUTE SHEET (RULE 26) cattattggcaaggagggggccaccatccgcaacatcacaaaacagacccagtccaagat agacgtgcataggaaggagaacg caggtgcagctgaaaaagccatcagtgtgcactccacccctgagggctgctcctccgctt gtaagatgatcttggagattatgcata aagaggctaaggacaccaaaacggctgacgaggttcccctgaagatcctggcccataata actttgtagggcgtctcattggcaa ggaaggacggaacctgaagaaggtagagcaagataccgagacaaaaatcaccatctcctc gttgcaagaccttaccctttacaac cctgagaggaccatcactgtgaagggggccatcgagaattgttgcagggccgagcaggaa ataatgaagaaagttcgggaggcc tatgagaatgatgtggctgccatgagcctgcagtctcacctgatccctggcctgaacctg gctgctgtaggtcttttcccagcttcatc cagcgcagtcccgccgcctcccagcagcgttactggggctgctccctatagctcctttat gcaggctcccgagcaggagatggtgca ggtgtttatccccgcccaggcagtgggcgccatcatcggcaagaaggggcagcacatcaa acagctctcccggtttgccagcgcct ccatcaagattgcaccacccgaaacacctgactccaaagttcgtatggttatcatcactg gaccgccagaggcccaattcaaggct cagggaagaatctatggcaaactcaaggaggagaacttctttggtcccaaggaggaagtg aagctggagacccacatacgtgtg ccagcatcagcagctggccgggtcattggcaaaggtggaaaaacggtgaacgagttgcag aatttgacggcagctgaggtggta gtaccaagagaccagacccctgatgagaacgaccaggtcatcgtgaaaatcatcggacat ttctatgccagtcagatggctcaac ggaagatccgagacatcctggcccaggttaagcagcagcatcagaagggacagagtaacc aggcccaggcacggaggaagtg ai cgac&tcemcc&tmeic&atagctttgcaaagatssataaagttttaa acagagaggaatctttgcagctaatggaccttctaggt cttgaaaggagigggaattggctccggtgcccgtcagtgggcagagcgcacatcgcccac agtccccgagaagtggggggagggg tcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcg tgiactggctccgcctitttcccgagggt gggggagaaccgtatataagtgcagtagicgccgtgaacgtcmttcgcaacgggtitgcc gccagaacacaggtaagtgccgtgtg tggttcccgceeecctggcctctttacseettateecccttecgteccttsaattacttc caccteectecagtacsteattctteatcccsa gcUcgggUggaagigggigggagagtcgaggccUgcgctaaggagcccctlcgccicgtg citgagUgaggcctggcctgggcg ciggggccgccgcgigcgaaiciggiggcaccitcgcgcctgtcicgctgciUcgataag iciciagccattaaaatiitigatgaccigc tgcgacgcttt!itctggcaagatagtcitgtaaatgcgggccaagaictgcacactggi atttcggtmtggggccgcgggcggcgacg gggcccgtgcgtcccagcgcacatgUcggcgaggcggggcctgcgagcgcggccaccgag aaicggacgggggtagtctcaagc tggccggcclgctclggtgcciggcclcgcgccgccglgtatcgccccgcccigggcggc aaggclggcccgglcggcaccagtlgcg

Igagcggaa gatggccgcttcccggccctgctgcagggagclcaaaatggaggacgcggcgctcgggag agcgggcgggtgagt cacccacacaaaggaaaagggcciUccgicctcagccgicgctcatgtgactccacggag taccgggcgccgtccaggcacctcg attagttctcgagcttiggagtacgtcgtctttaggtiggggggaggggttttatgcg tggagtttccccacactgagtgggtggagact gaagttaggceagcttggeacttgatgtaatictccttggaaittgcccttttigagttt ggaicttggttcaitcicaagcctcagacagtggit c g/ff/fflc/tecagte-agg g/cg/gagaattctaataceactcactataeeetgtgctgtctcatcatttgecaaaeat ggccaccaa gcttaccgccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggt cgagctggacggcgacgtaaacggc cacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctg aagttcatctgcaccaccggcaagc tgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagcc gctaccccgaccacatgaagcagcac

SUBSTITUTE SHEET (RULE 26) gacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaag gacgacggcaactacaagacccgcgc cgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgactt caaggaggacggcaacatcctggg gcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaa gaacggcatcaaggtgaacttcaa

iggaaggaagaggcagcctgctgacatgtggcgacgtgga

cctaaattccctttagtgagggttaatggccgtaggccgccagaatgggtcCAGi

aggactctaggacctgcgcatgcgcttggcgtaatcatggtcatagctgtttcctgtttt ccccgtatccccccaggtgtctg caggctcaaagagcagcgagaagcgttcagaggaaagcgatcccgtgccaccttccccgt gcccgggctgtccccgcacgctgccggc tcggggatgcggggggagcgccggaccggagcggagccccgggcggctcgctgctgcccc ctagcgggggagggacgtaattacat ccctgggggctttgggggggggctgtccctctcaccgcggtggagctccagctttgttcg aattggggccccccctcgagggtatcgalg

aastcacetaaaasataatcatscstcattsactcacscestcsttataettcaaaatca stsacacttaccscaiteacaascacec ctcacsssasctccaascsscsactsasatstcctaaatgcacascsacggattcgcsct atttasaaasaeaeascaatatttcaa gaafgcafecg caafl!tfflcgcagflcftifctffcfflgggttaatctagctagccttaagggcgcct cgagacctctagctagcccgcctaat gagcgggcittittitggcttgttgiccacaaccgtiaaaccttaaaagcittaaaagcc tiatatatictrtttttictiataaaacttaaaacctiaga ggctatttaagttgctgatttatattaattttattgttcaaacatgagagcttagtacgt gaaacatgagagcttagtacgttagccatgagagctia gtacgttagccatgagggttiagttcgttaaacatgagagctiagtacgttaaacatgag agcttagtacgtactatcaacaggtgaactgctg atccacgttgtggtagaattggtaaagagagtcgtgtaaaatatcgagttcgcacatctt gttgtctgattattgatttttggcgaaaccatttgat catatgacaagatgtgtatctaccttaacttaatgattttgataaaaatcattacctagg gaagactc (SEQ ID NO: 14977).

SUBSTITUTE SHEET (RULE 26) The 31TR of the PB- V5- JctR-SPTAl -IGF2BP1 -EF 1 a-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAAATTGA

CGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATT A

AGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTA T

GTTTATTTATTTATTAAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAA

AACTTTTA (ALL CAPS) (SEQ ID NO: 14704).

[0522] The Insulator 1 of the PB-V5-JctR-SPTAl-IGF2BPl-EFla-GFP-T2A-DHFR construct is encoded by a sequence comprising:

gagggacagcccccccccaaagcccccagggatgtaatacgtccctcccccgctagg gggcagcagcgagccgcccggggctccgc tccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcac ggggaaggtggcacgggatcgctttcc tctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaa (underlined) (SEQ ID NO: 14705).

[0523] The SPTAlPromoter of the PB-V5-JctR-SPTAl-IGF2BPl -EFla-GFP-T2A-DHFR construct is encoded by a sequence comprising:

ccagactttcaagaagagaatgtaaaggactctcagtttttctatgtgagatccaag aacaggggaaaccacaaatgtagaatcaaaa tcttctcttgtagatcatggaagttagattattcaagctgcatttacactggaatgaagt ttggcatggatagccccactggcttgtagaaag gtctaagagcatcccactatttctitttgcttgtttgtttcattMtgtaagttagtctaa aataactatgcctatggaaaaagtctacctctttttg ttctccattaaaattaatacatacagtaggatgatttataccatatatatcagaagtcag ccatattttcaacttttttgtgacatcattggtttg tcaaaiaaggtatcctaatltttgttUacaaaatatiaatgaacacalcatalaacttit tltgttccticcalttaaatgaltaagagtacaact aaalcagatttttaaagaccaaattgttgaggttaggaataaaalcttattttgtgaaag actctctagaaaaagccaggataattegttg actlaaagactgtgaaaaalctgcagltgagttggalgaagttgtlgagctgtalttcct ataaalatatggggtlttltUctaaitttlcatcc aaaaacctaciUcattttaagtciiatgagiaacttgaaagccagagccicccaaaactg cigagtcacccagiaicigtaaaact agcagUgccicagctgagiaigtcticiaaagataaigtcgattgtgtaiggcigatggg aitctaggaccaagcaagaggttttUitic ccccacatacttaacgttctatatltclatttgaaltcgaciggacagttccatltgaat tatttctclctctctctctctctctctgacacatttat ctgccaggttctaaaccc (italicized) (SEQ ID NO: 14700).

[0524] The IGF2BP1 of the PB-V5-JctR-SPTAl-IGF2BPl -EF 1 a-GFP-T2 A-DHFR construct is encoded by a sequence comprising:

atgaacaagctttacatcggcaacctcaacgagagcgtgacccccgcggacttggag aaagtgtttgcggagcacaagatctcct acagcggccagttcttggtcaaatccggctacgccttcgtggactgcccggacgagcact gggcgatgaaggccatcgaaactttc tccgggaaagtagaattacaaggaaaacgcttagagattgaacattcggtgcccaaaaaa caaaggagccggaaaattcaaatc cgaaatattccaccccagctccgatgggaagtactggacagcctgctggctcagtatggt acagtagagaactgtgagcaagtga

SUBSTITUTE SHEET (RULE 26) acaccgagagtgagacggcagtggtgaatgtcacctattccaaccgggagcagaccaggc aagccatcatgaagctgaatggcc accagttggagaaccatgccctgaaggtctcctacatccccgatgagcagatagcacagg gacctgagaatgggcgccgagggg gctttggctctcggggtcagccccgccagggctcacctgtggcagcgggggccccagcca agcagcagcaagtggacatccccct tcggctcctggtgcccacccagtatgtgggtgccattattggcaaggagggggccaccat ccgcaacatcacaaaacagacccag tccaagatagacgtgcataggaaggagaacgcaggtgcagctgaaaaagccatcagtgtg cactccacccctgagggctgctcct ccgcttgtaagatgatcttggagattatgcataaagaggctaaggacaccaaaacggctg acgaggttcccctgaagatcctggcc cataataactttgtagggcgtctcattggcaaggaaggacggaacctgaagaaggtagag caagataccgagacaaaaatcacc atctcctcgttgcaagaccttaccctttacaaccctgagaggaccatcactgtgaagggg gccatcgagaattgttgcagggccga gcaggaaataatgaagaaagttcgggaggcctatgagaatgatgtggctgccatgagcct gcagtctcacctgatccctggcctga acctggctgctgtaggtcttttcccagcttcatccagcgcagtcccgccgcctcccagca gcgttactggggctgctccctatagctcc tttatgcaggctcccgagcaggagatggtgcaggtgtttatccccgcccaggcagtgggc gccatcatcggcaagaaggggcagc acatcaaacagctctcccggtttgccagcgcctccatcaagattgcaccacccgaaacac ctgactccaaagttcgtatggttatca tcactggaccgccagaggcccaattcaaggctcagggaagaatctatggcaaactcaagg aggagaacttctttggtcccaagga ggaagtgaagctggagacccacatacgtgtgccagcatcagcagctggccgggtcattgg caaaggtggaaaaacggtgaacg agttgcagaatttgacggcagctgaggtggtagtaccaagagaccagacccctgatgaga acgaccaggtcatcgtgaaaatcat cggacatttctatgccagtcagatggctcaacggaagatccgagacatcctggcccaggt taagcagcagcatcagaagggacag agtaaccaggcccaggcacggaggaagtga (bold) (SEQ ID NO: 14721 ).

[0525] The EF la Promoter of the PB-V5-JctR-SPTAl -IGF2BP 1 -EF 1 a-GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

agciUgcaaagaiggataaagUitaaacagagaggaatciUgcagctaaiggacctc taggictgaaaggagigggaaiiggcic cggtgcccgicagtgggcagagcgcacatcgcccacagtccccgagaagtiggggggagg ggtcggcaattgaaccggtgcctag agaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgccttMcccga gggtgggggagaaccgtaiataagtg cagtagicgccgtgaacgttctuttcgcaacgggtiigccgccagaacacaggtaagigc cgtgtgtggiicccgcgggcciggccicti lacgggttalggcccttgcglgccltgaattacttccacclggctgcaglacgtgaltct gatcccgagcUcgggttggaaglggglggg agagttcgaggccUgcgcltaaggagccccttcgcclcgtgcttgagttgaggcctggcc tgggcgctggggccgccgcgtgcgaatc tggtggcaccttcgegcctgtctcgctgetttcgataagtetctagccatitaaaatttt gatgacctgctgcgacgcttittitctggcaagat agtcttgtaaatgcgggccaagatctgcacaetggtatttcggtttttggggccgcgggc ggcgacggggcecgtgcgtcceagcgcac atgttcggcgaggcggggcctgcgagcgcggccaccgagaateggacgggggtagtctca agctggccggcctgctctggtgectg gcctcgcgcegccgtgtatcgecccgccctgggcggcaaggctggcccggtcggcaccag ttgegtgagcggaaagatggcegcttc ccggccctgctgcagggagctcaaaaiggaggacgcggcgctcggg gagcgggcgggtgagtcacccacacaaaggaaaagg gccittccgtcctcagccgtcgcttcatgtgaciccacggagiaccgggcgccgtccagg cacctcgattagttctcgagctttiggagtac

SUBSTITUTE SHEET (RULE 26) gte&ct tggggf&afrfrggttttatgcgatggagtttccccacactgagtgggtgfrafra ctxaazttaggccazcttzgcacttz atetaattctecttssaatttseccttttteasttssatcttsstteattctcaaecctc aeacastssttcaaastttttttcttccatttcasetg (italicized underlined) (SEQ ID NO: 14706).

[0526] The GFP of the PB-V5-JctR-SPTAl-IGF2BPl -EF 1 a-GFP-T2 A-DHFR construct is encoded by a nucleic acid sequence comprising:

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg gacggcgacgtaaacggccacaagttc agcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatc tgcaccaccggcaagctgcccgtgc cctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccg accacatgaagcagcacgacttcttca agtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggca actacaagacccgcgccgaggtga

¾g £gicgaggacggcagcgtgcagctcgccgaccactaccagcagaac¾cccccatcggc gacggccccgtgctgctgcccg acaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgtgatc acatggtcctgctggagttcgtgac cgccgccgggatcactctcggcatggacgagctgtacaag (italicized underlined) (SEQ ID NO: 14708).

[0527] The DHFR of the PB-V5-JctR-SPTAl-IGF2BPl-EFla-GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

¾¾tg¾gtecagataetttc¾gaggatgacc¾eaaec¾gctcegtggagggeaag c¾gaacetggteatcatgggc¾¾gaagac¾ gagcacactttctgtccaggtctctggacgatgccctgaagctgaccgagcagcctgagc tggccaacaaggtggacatggtgtgg atcgtgggcggctctagcgtgtataaggaggccatgaatcaccctggccacctgaagctg ttcgtgacacggatcatgcaggacttt gagtccgataccttctttccagagatcgacctggagaagtacaagctgctgcccgagtat cctggcgtgctgtctgatgtgcaggag gagaagggcatcaagtacaagttcgaggtgtatgagaagaacgattga (bold italicized) (SEQ ID NO: 14709).

[0528] The SV40 PoIy(A) of the PB-V5-JctR-SPTAl-IGF2BPl-EFla-GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAG TGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCA TTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTC AGGTTCAGGGGGAGGTGTGGGAGGTTTTTT (bold all caps) (SEQ ID NO: 14710).

[0529] The 5ΊTK of the PB-V5-JctR-SPTAl-IGF2BPl-EFla-GFP-T2A-DHFR construct is encoded by a nucleic acid sequence comprising:

SUBSTITUTE SHEET (RULE 26)

asaatscatecstcaaitttacscasactatctttctasse (bold italicized underlined) (SEQ ID NO: 1471 1).

[0530] In some embodiments of the methods of the disclosure, a modified HSCor modified HSC descendent ceil of the disclosure may be produced by introducing a transgene into an HSC or an HSC descendent cell of the disclosure. The introducing step may comprise delivery of a nucleic acid sequence and/or a genomic editing construct via a non-transposition delivery system.

[0531] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises one or more of topical delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical deliver}', some delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery . In some

embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo , in vivo, in vitro or in situ by mechanical transfection comprises cell squeezing, cell bombardment, or gene gun techniques. In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection comprises liposomal delivery, delivery by micelles, and delivery by polymerosomes.

[0532] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises a non-viral vector. In some embodiments, the non-viral vector comprises a nucleic acid. In some embodiments, the non-viral vector comprises plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), Doggy Bone™ DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA

5UB5TITUTE SHEET (RULE 26) oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises a transposon of the disclosure.

[0533] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises a viral vector. In some embodiments, the viral vector is a non- integrating non-chromosomal vector. Exemplary non-integrating non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes viruses. In some embodiments, the viral vector is an integrating chromosomal vector. Integrating chromosomal vectors include, but are not limited to, adeno-associated vectors (AAV),

Lentiviruses, and gamma-retroviruses.

[0534] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises a combination of vectors. Exemplary, non-limiting vector combinations include: viral and non-viral vectors, a plurality' of non-viral vectors, or a plurality' of viral vectors. Exemplary but non-limiting vectors combinations include: a combination of a DNA-derived and an RNA-derived vector, a combination of an RNA and a reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

[0535] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ stably integrates a nucleic acid sequence, transiently integrates a nucleic acid sequence, produces site-specific integration a nucleic acid sequence, or produces a biased integration of a nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

[0536] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, m vivo, in vitro or m situ stably integrates a nucleic acid sequence. In some embodiments, the stable chromosomal integration can be a random integration, a site-specific integration, or a biased integration. In some embodiments, the site- specific integration can be non-assisted or assisted. In some embodiments, the assisted site- specific integration is co-delivered with a site-directed nuclease. In some embodiments, the site-

5UB5TITUTE SHEET (RULE 26) directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In some embodiments, the transgene with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining. In some embodiments the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material m a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

[Q537] In some embodiments, the site-specific transgene integration occurs at a site that disrupts expression of a target gene. In some embodiments, disrupti on of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-specific integration include but are not limited to TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allo-rejection.

[0538] In some embodiments, the site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In some embodiments, enhancement of target gene expression occurs by site-specific integration at nitrons, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0539] In some embodiments of the methods of the disclosure, enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. In some embodiments, enzymes create single-strand breaks. In some embodiments, enzymes create double-strand breaks. In some embodiments, examples of break-inducing enzymes include but are not limited to: transposases, mtegrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVER™, and CPF1. In some embodiments, break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, as a nucleoprotein complex with a guide RNA (gRNA).

SUBSTITUTE SHEET (RULE 26) [0540] in some embodiments of the methods of the disclosure, the site-specific transgene integration is controlled by a vector-mediated integration site bias. In some embodiments vector- mediated integration site bias is controlled by the chosen lentiviral vector. In some embodiments vector-mediated integration site bias is controlled by the chosen gamma-retroviral vector.

[0541] In some embodiments of the methods of the disclosure, the site-specific transgene integration site is a non-stable chromosomal insertion. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

[0542] In some embodiments of the methods of the disclosure, the genome modification is a non-stable integration of a transgene. In some embodiments, the non-stable integration can be a transient non-chromosomal integration, a semi-stable non chromosomal integration, a semi- persistent non-chromosomal insertion, or a non-stable chromosomal insertion. In some embodiments, the transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic.

[0543] In some embodiments, the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

[Q544] In some embodiments of the methods of the disclosure, the genome modification is a semi-stable or persistent non-chromosomal integration of a transgene. In some embodiments, a DNA vector encodes a Scaffold/matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.

[0545] In some embodiments of the methods of the disclosure, the genome modification is a non-stable chromosomal integration of a transgene. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

[0546] In some embodiments of the methods of the disclosure, the modification to the genome by transgene insertion can occur via host cell-directed double-strand breakage repair (homology- directed repair) by homologous recombination (HR), microhomology -mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinant enzyme-mediated modification. In some embodiments, the modification to the genome by transgene insertion can occur via CRISPR-Cas9, TALEN, ZFNs, Cas-CLOVER, and cpfl .

SUBSTITUTE SHEET (RULE 26) [0547] Poly(histidine) (i.e., poly(L-histidine)), is a pH-sensitive polymer due to the imidazole ring providing an electron lone pair on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties through protonation-deprotonation. The various embodiments enable intracellular delivery of gene editing tools by complexing with poly (histi din e)-based micelles. In particular, the various embodiments provide triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block may be poly (ethylene oxide) (PEO), and the charged block may be poly(L-histidine). An example tri block copolymer that may be used in various embodiments is a PEQ-b-PLA-b-PHIS, with variable numbers of repeating units in each block varying by design. The gene editing tools may be various molecules that are recognized as capable of modifying, repairing, adding and/or silencing genes m various cells. The correct and efficient repair of double-strand breaks (DSBs) in DNA is critical to maintaining genome stability ⁷ in cells. Structural damage to DNA may occur randomly and unpredictably in the genome due to any of a number of intracellular factors (e.g., nucleases, reactive oxygen species, etc.) as well as external forces (e.g., ionizing radiation, ultraviolet (UV) radiation, etc.). In particular, correct and efficient repair of double-strand breaks (DSBs) in DNA is critical to maintaining genome stability. Accordingly, cells naturally possess a number of DNA repair mechanisms, which can be leveraged to alter DNA sequences through controlled DSBs at specific sites. Genetic modification tools may therefore be composed of programmable, sequence-specific DNA-binding modules associated with a nonspecific DNA nuclease, introducing DSBs into the genome. For example CRISPR, mostly found in bacteria, are loci containing short direct repeats, and are part of the acquired prokaryotic immune system, conferring resistance to exogenous sequences such as plasmids and phages. RNA-guided endonucleases are programmable genetic engineering tools that are adapted from the

CRISPR/CRISPR-associated protein 9 (Cas9) system, which is a component of prokaryotic innate immunity.

[0548] Diblock copolymers that may be used as intermediates for making triblock copolymers of the embodiment micelles may have hydrophilic biocompatible poly(ethylene oxide) (PEO), which is chemically synonymous with PEG, coupled to various hydrophobic aliphatic

poly(anhydrides), poly(nucleic acids), poly(esters), poly (ortho esters), poly(peptides),

poly(phosphazenes) and poly(saccharides), including but not limited by poly(lactide) (PLA), poiy(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(e-caprolactone) (PCL), and

SUBSTITUTE SHEET (RULE 26) poly (trimethylene carbonate) (PTMC). Polymeric micelles comprised of 100% PEGylated surfaces possess improved in vitro chemical stability, augmented in vivo bioavailablity, and prolonged blood circulatory half-lives. For example, aliphatic polyesters, constituting the polymeric micelle's membrane portions, are degraded by hydrolysis of their ester linkages in physiological conditions such as m the human body. Because of their biodegradable nature, aliphatic polyesters have received a great deal of attention for use as implantable biomaterials in drug delivery devices, bioresorbable sutures, adhesion barriers, and as scaffolds for injury repair via tissue engineering.

[0549] In various embodiments, molecules required for gene editing (i.e., gene editing tools) may be delivered to cells using one or more micelle formed from self-assembled triblock copolymers containing poly(histidine). The term "gene editing" as used herein refers to the insertion, deletion or replacement of nucleic acids in genomic DNA so as to add, disrupt or modify the function of the product that is encoded by a gene. Various gene editing systems require, at a minimum, the introduction of a cutting enzyme (e.g., a nuclease or recombinase) that cuts genomic DNA to disrupt or activate gene function.

[0550] Further, in gene editing systems that involve inserting new or existing

nudeotides/nucleic acids, insertion tools (e.g. DNA template vectors, transposable elements (transposons or retrotransposons) must be delivered to the cell in addition to the cutting enzyme (e.g. a nuclease, recombinase, integra.se or transposase). Examples of such insertion tools for a recombma.se may include a DNA vector. Other gene editing systems require the delivery of an integrase along with an insertion vector, a transposase along with a transposon/retrotransposon, etc. In some embodiments, an example recombinase that may be used as a cutting enzyme is the CRE recombinase. In various embodiments, example integrases that may be used in insertion tools include viral based enzymes taken from any of a number of viruses including, but not limited to, AAV, gamma retrovirus, and lentivirus. Example transposons/retrotransposons that may be used in insertion tools include, but are not limited to, the piggyBac transposon, Sleeping Beauty transposon, and the LI retrotransposon.

[0551] In certain embodiments of the methods of the disclosure, the transgene is delivered in vivo. In certain embodiments of the methods of the disclosure, in vivo transgene delivery can occur by: topical delivery , adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery , magnetofection or by

SUBSTITUTE SHEET (RULE 26) nanoparticle-mediated delivery. In certain embodiments of the methods of the disclosure, in vivo transgene deliver} by transfection can occur by liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In certain embodiments of the methods of the disclosure, m vi vo mechanical transgene delivery can occur by cell squeezing, bombardment, and gene gun. In certain embodiments of the methods of the disclosure, in vivo nanoparticle-mediated transgene deliver} ⁷ can occur by liposomal delivery, delivery by micelles, and delivery by polymerosomes. In various embodiments, nucleases that may be used as cutting enzymes include, but are not limited to, Cas9, transcription activator-like effector nucleases (TALENs) and zinc finger nucleases.

[0552] In various embodiments, the gene editing systems described herein, particularly proteins and/or nucleic acids, may be comp!exed with nanoparticles that are poly(histidine)-based micelles. In particular, at certain pHs, poly(histidine)-containing triblock copolymers may assemble into a micelle with positively charged poly(histidine) units on the surface, thereby enabling complexing with the negatively-charged gene editing molecule(s). Using these nanoparticles to bind and release proteins and/or nucleic acids in a pH-dependent manner may provide an efficient and selective mechanism to perform a desired gene modification. In particular, this micelle-based delivery system provides substantial flexibility with respect to the charged materials, as well as a large payload capacity, and targeted release of the nanoparticle payload. In one example, site-specific cleavage of the double stranded DNA may be enabled by delivery of a nuclease using the poly(lustidine)-based micelles.

[0553] The various embodiments enable intracellular delivery of gene editing tools by complexing with poly(hisddine)-based micelles. In particular, the various embodiments provide triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block may be polyt ethylene oxide) (PEO), and the charged block may be poly(L-histidine). An example tri-block copolymer that may be used in various embodiments is a PEO-b-PLA-b-PHIS, with variable numbers of repeating units in each block varying by design. Without wishing to be bound by a particular theory, it is believed that believed that in the micelles that are formed by the various embodiment triblock copolymers, the hydrophobic blocks aggregate to form a core, leaving the hydrophilic blocks and poly(histidme) blocks on the ends to form one or more surrounding layer.

SUBSTITUTE SHEET (RULE 26) [0554] in certain embodiments of the methods of the disclosure, non-viral vectors are used for transgene deliver} . In certain embodiments, the non-viral vector is a nucleic acid. In certain embodiments, the nucleic acid non-viral vector is plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mKNA (ssRNA), and double-stranded mRNA (dsRNA). In certain embodiments, the non-viral vector is a transposon. In certain embodiments, the transposon is piggyBac™.

[0555] In certain embodiments of the methods of the disclosure, transgene deliver} ⁷ can occur via viral vector. In certain embodiments, the viral vector is a non-integrating non-chromosomal vectors. Non-integrating non-chromosomal vectors can include adeno-associated virus (AAV), adenovirus, and herpes viruses. In certain embodiments, the viral vector is an integrating chromosomal vectors. Integrating chromosomal vectors can include adeno-associated vectors (AAV), Lentiviruses, and gamma-retroviruses.

[Q556] In certain embodiments of the methods of the disclosure, transgene deliver} ⁷ can occur by a combination of vectors. Exemplar _} ⁷ but non-limiting vector combinations can include: viral plus non-viral vectors, more than one non-viral vector, or more than one viral vector. Exemplary but non-limiting vectors combinations can include: DNA-derived plus RNA-derived vectors, RNA plus reverse transcriptase, a transposon and a transposase, a non-viral vectors plus an endonuclease, and a viral vector plus an endonuclease.

[0557] In certain embodiments of the methods of the disclosure, the genome modification can be a stable integration of a transgene, a transient integration of a transgene, a site-specific integration of a transgene, or a biased integration of a transgene.

[0558] In certain embodiments of the methods of the disclosure, the genome modification can be a stable chromosomal integration of a transgene. In certain embodiments, the stable chromosomal integration can be a random integration, a site-specific integration, or a biased integration. In certain embodiments, the site-specific integration can be non-assisted or assisted. In certain embodiments, the assisted site-specific integration is co-delivered with a site-directed nuclease. In certain embodiments, the site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain homology to upstream and downstream regions of the site of genomic integration. In certain embodiments, the transgene with homologous nucleotide extensions enable genomic integration by homologous recombination,

SUBSTITUTE SHEET (RULE 26) microhomology-mediated end joining, or nonhomofogous end-joining in certain embodiments the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokme (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

[0559] In certain embodiments, the site-specific transgene integration occurs at a site that disrupts expression of a target gene. In certain embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In certain embodiments, exemplary target genes targeted by site-specific integration include but are not limited to TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allo-rejection.

[0560] In certain embodiments, the site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In certain embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0561] In certain embodiments of the methods of the disclosure, enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. In certain embodiments, enzymes create single-strand breaks. In certain embodiments, enzymes create double-strand breaks. In certain embodiments, examples of break-inducing enzy mes include but are not limited to: transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), zmc finger nucleases (ZFN), Cas- CLOVER™, and cpfl . In certain embodiments, break-inducing enzymes can be delivered to the cell encoded m DNA, encoded m mRNA, as a protein, as a nucleoprotein complex with a guide RNA (gRNA).

[0562] In certain embodiments of the methods of the disclosure, the site-specific transgene integration is controlled by a vector-mediated integration site bias. In certain embodiments

SUBSTITUTE SHEET (RULE 26) vector-mediated integration site bias is controlled by the chosen lentiviral vector. In certain embodiments vector-mediated integration site bias is controlled by the chosen gamma-retroviral vector.

[0563] In certain embodiments of the methods of the disclosure, the site-specific transgene integration site is a non-stable chromosomal insertion. In certain embodiments, the integrated transgene may become silenced, removed, excised, or further modified. In certain embodiments of the methods of the disclosure, the genome modification is a non-stable integration of a transgene. In certain embodiments, the non-stable integration can be a transient non- chromosomal integration, a semi-stable non chromosomal integration, a semi-persistent non- chromosomal insertion, or a non-stable chromosomal insertion. In certain embodiments, the transient non-ehromosoma! insertion can be epi-ehromosoma! or cytoplasmic. In certain embodiments, the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

[Q564] In certain embodiments of the methods of the disclosure, the genome modification is a semi-stable or persistent non-chromosomal integration of a transgene. In certain embodiments, a DNA vector encodes a Scaffold/matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.

[0565] In certain embodiments of the methods of the disclosure, the genome modification is a non-stable chromosomal integration of a transgene. In certain embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

[0566] In certain embodiments of the methods of the disclosure, the modification to the genome by transgene insertion can occur via host cell-directed double-strand breakage repair (homo logy- directed repair) by homologous recombination (HR), microhomology-mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase enzyme-mediated

modification, mtegrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinant enzyme-mediated modification. In certain embodiments, the modification to the genome by transgene insertion can occur via CRISPR-Cas9, TALEN, ZFNs, Cas-CLO VER, and cpfl .In certain embodiments of the methods of the disclosure, a cell with an in vivo or ex vivo genomic modification can be a germlme ceil or a somatic cell. In certain embodiments the modified cell can be a human, non-human, mammalian, rat, mouse, or dog ceil.

SUBSTITUTE SHEET (RULE 26) [0567] In certain embodiments, the modified cell can be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cell can be a stem cell. In certain embodiments, the modified cell can be differentiated, undifferentiated, or immortalized. In certain embodiments, the modified undifferentiated cell can be an induced pluri potent stem cell.

[0568] In certain embodiments, the modified cell can be a T cell, a hematopoietic stem cell, a natural killer cell, a macrophage, a dendritic cell, a monocyte, a megakaryocyte, or an osteoclast. In certain embodiments, the modified cell can be modified while the cell is quiescent, in an activated state, resting, interphase, in prophase, in metaphase, in anaphase, or in telophase. . In certain embodiments, the modified cell can be fresh, cryopreserved, bulk, sorted into sub populations, from whole blood, from leukapheresis, or from an immortalized cell line.

[Q569] Exemplary transposon/transposase systems of the disclosure include, but are not limited to, piggyBac and piggyBac-like transposons and transposases, Sleeping Beauty transposons and transposases, He!raiser transposons and transposases and Tol2 transposons and transposases.

[0570] The piggyBac transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and moves the contents between the ITRs into TTAA chromosomal sites. The piggyBac transposon system has no payload limit for the genes of interest that can be included between the ITRs. In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac™ or a Super piggyBac™ (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a Super piggyBac™ (SPB)

transposase, the sequence encoding the transposase is an mRNA sequence.

[0571] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac™ (PB) transposase enzyme. The piggyBac (PB) transposase enzyme may comprise or consist of an ammo acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFI DE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG

121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEI SLKRR ESMTGATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL I RCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYI PNKPSK YGIKILMMCD

301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ

SUBSTITUTE SHEET (RULE 26) 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC

421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NI5NILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV I CREHNIDMC QSCF (SEQ ID NO : 14487 ) .

[0578] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac™ (PB) transposase enzyme that comprises or consists of an ammo acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RI LTLPQRTI RGKNKHCmST S KSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEI I SEIVKW TNAEI SLKRR ESMTGATFRD TNEDEI AFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL I RCLRMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYI PNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLS SC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV I CREHNIDMC QSCF (SEQ ID NO : 14487) .

[0579] In certain embodiments, the transposase enzyme is a piggyBac™ (PB) transposase enzyme that comprises or consists of an amino acid sequence having an amnio acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac™ (PB) transposase enzyme that comprises or consists of an ammo acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac™ (PB) transposase enzyme that comprises or consists of an amino acid sequence having an ammo acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the ammo acid substitution at position 30 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 is a substitution of a serine (S) for a glycine (G). In certain embodiments, the amino acid substitution at position 282 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the

SUBSTITUTE SHEET (RULE 26) amino acid substitution at position 538 of the sequence of SEQ ID NO: 14487 is a substitution of a lysine (K) for an asparagine (N).

[0580] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggy Bac™ (SPB) transposase enzyme. In certain embodiments, the Super piggyBac™ (SPB) transposase enzymes of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 14487 wherein the ammo acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBac™ (SPB) transposase enzyme may comprise or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG

61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHC ST SKSTRRSRVS ALNIVR3QRG

121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF

181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL I RCLRMDF!KS IRFTLEENDV

241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD

301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ

361 EPYKLTIVGT VRSNKREI PE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC

421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN

481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV

541 PGTSDOSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV I CREHNI DMC QSCF (SEQ ID NO:

14484 }

[0581] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™ or Super piggyBac™ transposase enzyme may further comprise an ammo acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™ or Super piggyBac™ transposase enzyme may further comprise an ammo acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298,

SUBSTITUTE SHEET (RULE 26) 31 1 , 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an asparagine (N) for a serine (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the ammo acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the ammo acid substitution at position 82 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for an isoleucine (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 119 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain

embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a phenylalanine (F) In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for an alanine (A). In certain embodiments, the amino acid substitution at position 200 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a phenylalanine (F).In certain embodiments, the ammo acid substitution at position 207 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a valine (V). In certain

SUBSTITUTE SHEET (RULE 26) embodiments, the ammo acid substitution at position 209 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the ammo acid substitution at position 226 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a methionine (M). in certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a leucine (L). In certain embodiments, the amino acid substitution at position 240 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a proline (P). In certain embodiments, the amino acid substitution at position 258 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 31 1 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a proline (P). In certain embodiments, the ammo acid substitution at position 31 1 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine for a proline (P). In certain embodiments, the amino acid substitution at position 315 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for an arginine (R).In certain embodiments, the ammo acid substitution at position 319 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID

SUBSTITUTE SHEET (RULE 26) NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a tyrosine (Y). in certain embodiments, the amino acid substitution at position 328 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a tyrosine (Y). in certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a cysteine (C). In certain embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for the aspartic acid (D). In certain embodiments, the ammo acid substitution at position 436 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a valine (V) In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the amino acid substitution at position 470 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucme (I) for a methionine (M). In certain embodiments, the ammo acid substitution at position 552 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the ammo acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A) In certain embodiments, the ammo acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a glutamine (Q).

[0582] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™ transposase enzyme may comprise or the Super piggy Bac™ transposase enzyme may further comprise an ammo acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO:

SUBSTITUTE SHEET (RULE 26) 14484. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggy Bac™ transposase enzyme may comprise or the Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO:

14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™ transposase enzyme may comprise or the Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the ammo acid substituti on at position 194 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the ammo acid substitution at position 450 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a serine (S). In certain embodiments, the ammo acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487. In certain

embodiments, including those embodiments wherein the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO:

14487, the piggyBac™ transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, and a substitution of an

SUBSTITUTE SHEET (RULE 26) alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487. In certain embodiments, the piggyBac™ transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 14487.

[0583] The sleeping beauty transposon is transposed into the target genome by the Sleeping Beauty transposase that recognizes ITRs, and moves the contents between the ITRs into TA chromosomal sites. In various embodiments, SB transposon-mediated gene transfer, or gene transfer using any of a number of similar transposons, may be used in the compositions and methods of the disclosure.

[0584] In certain embodiments, and, in particular, those embodiments wherein the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase or a hyperactive Sleeping Beauty transposase (SBI0QX).

[0585] In certain embodiments of the methods of the disclosure, the Sleeping Beauty transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MGKSKEISQD LRKKIVDLHK SGSSLGAISK RLKVPRSSVQ TIVRKYKHHG TTQPSYRSGR

61 RRVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGRSARKK

121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN

181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMRKENYVD I LKQHLKTSV RKLKLGRKWV

241 FQ DNDPKHT SKWAKWLKD NKVKVLEWFS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL

301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 14485} ,

[0586] In certain embodiments of the methods of the disclosure, the hyperactive Sleeping Beauty (SB100X) transposase enzyme comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage m between identical to:

1 MGKSKEISQD LRKRIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR

61 RRVLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSARKK

121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN

181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMDAVQYVD I LKQHLKTSV RKLKLGRKWV

241 FQHDNDPKHT SKWAKWLKD NKVKVLEWFS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL

301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY (SEQ ID NO: 14665} .

SUBSTITUTE SHEET (RULE 26) [0587] The Helraiser transposon is transposed by the Helitron transposase. Helitron

transposases mobilize the Helraiser transposon, an ancient element from the bat genome that was active about 30 to 36 million years ago. An exemplary Helraiser transposon of the disclosure mcludes Helibatl, which comprises a nucleic acid sequence comprising:

1 TCCTATA AA TAAAAGAGAA ACATGCAAAT TGACCATCCC TCCGCTACGC TCAAGCCACG

61 CCCACCAGCC AATCAGAAGT GACTATGCAA ATTAACCCAA CAAAGATGGC AGTTAAATTT

121 GCATACGGAG GTGTCAAGCG CCCCAGGAGG CAACGGCGGC CGCGGGCTCC CAGGACCTTC

181 GCTGGCCCCG GGAGGCGAGG CCGGCCGCGC CTAGCCACAC CCGCGGGCTC CCGGGACCTT

241 CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG CGGAGAGCGG GAGGTTTGGA GGACTTGGCA

301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG CGAGAGAGAG GGTGGCTTGG AGGGCGTGGC

361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA CTGACAGCAG GGAGGAGGAA

421 GTCCCACCCC CACAGAATCA GCCAGAATCA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG

481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC AGACCGTGAC AGTAGAGAGG TGGGACTATG

541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG AAAGATGCCG GCGTTATCGA

601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAAA GAAGGCGGCG CCTGCAACAG

661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA ACAGCGGCGT

721 CATCGACAGA AAATGTCTAA AGACCAACGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA

781 CGACAGAA A TGTCTAGAGA ACAGTCATCA ACAAGTACTA CCAATACCGG TAGGAACTGC

841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG AACATAGTTG TGGTGGAATG

901 ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG ATGAAAAACC ATCCGATGGG

961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCCAA ATGATATACA TTTTCCAGAT

1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGTAA AAATTTCATG

1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT CCATGGGTGC AAATAT GCA

1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG GACAAGTTTA TCACCGTAGT

1201 GGAACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT

1261 ACAGCCGAAG CTACAAGTAA AAGATTAGCA ATGCCAGAAA ACCAGGGCTG CTCAGAAAGA

1321 CTCATGATCA ACATCAACAA CCTCATGCAT GAAATAAATG AATTAACAAA A CGTACAAG

1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA TCTGAAGCAG CAGCAAAAGG TATTGCTCCC

1441 ACAGAAGTAA CAATGGCGAT TAAATACGAT CGTAACAGTG ACCCAGGTAG ATATAATTCT

1501 CCCCGTGTAA CCGAGGTTGC TGTCATATTC AGAAACGAAG ATGGAGAACC TCCTTTTGAA

1561 AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CAAATGCCAC TAAAATGAAA

1621 CAAATCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT ATCCTATTCT TTTTCCACAT

1681 GGTGAAAAAG GCTGGGGAAC AGATATTGCA TTAAGACTCA GAGACAACAG TGTAATCGAC

1741 AATAATACTA GACAAAATGT AAGGACACGA GTCACACAAA TGCAGTATTA TGGATTTCAT

1801 CTCTCTGTGC GGGACACGTT CAATCCTATT TTAAATGCAG GAAAATTAAC TCAACAGTTT

1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAA ATTTCATCAA AGCAAACCAA

1921 TCTAAGTTGA GAGTTGAAAA ATATAGTGGT TTGATGGATT ATCTCAAATC TAGATC GAA

SUBSTITUTE SHEET (RULE 26) 1981 AATGACAATG T G C C G A T T G G T AAAAT GAT A AT AC T T C CAT CAT C T T T T G A G G GT AG T C C C 2041 AGAAAT AT G C AG C AG C GAT A T C AG GAT G C T AT G G CAAT T G TAACGAAGTA TGGCAAGCCC 2101 GATT TAT TCA T AAC CAT GAC AT G C AAC C C C AAAT G G G C AG AT AT T ACAAA CAATTTACAA 2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC TTGGTAGCCA GAGTTTTTAA TATTAAGCTG 2221 AATGCTCTTT TAAAT GAT AT ATGTAAATTC CAT T T AT TTG GCAAAGTAAT A G C T AA AT T 2281 CAT GT CAT T G AATTTCAGAA AC ' , GAC T G C C T C A C G C T C AC AT AT TAT T GATATTAGAT 2341 AGTGAGTCCA AATTACGTTC AGAAGAT GAC AT T GAC C GT A TAG T T AAG G C AGAAAT T CCA 2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA AT T G T AAAAT CAAATAT GGT AC AT G GAC C A 2461 TGTGGAATAC AAAATCCAAA TAG CCATGT AT G G AAAAT G GAAAATGTTC AAAG G GAT AT 2521 CCAAAAGAAT TTCAAAATGC GAC CAT T G GA A AT AT T GAT G GAT AT C C C AA AT ACAAAC GA

2581 AGATCTGGTA G C A C C A T G T C TATTGGAAAT A/A/AG T T G T C G AT A AC AC T T G GAT T GT C C C T 2641 TATAACCCGT ATTTGTGCCT TAAAT AT AAC T G T C AT AT AA ATGTTGAAGT CTGTGCATCA 2701 ATTAAAAGTG TCAAATATTT ATT TAAAT AC AT CT ATAAAG GGCACGATTG TG CAAATAT T 2761 CAAATTTCTG AAAAAAATAT TAT CAAT CAT GACGAAGTAC AGGACTTCAT TGACTCCAGG 2821 TATGTGAGCG C T C C T GAG G C TGTTTGGAGA CTTTTTGCAA TGCGAATGCA T GAC CAAT CT 2881 CAT GCAAT CA CAAGAT TAG C TAT T CAT T T G C C AAAT GAT C AGAATTTGTA T T T T CAT AC C 2941 GAT GATT TTG CTGAAGTTTT AG AT AG G G C T AAAAGGCATA ACT CGACTTT GATGGCTTGG 3001 TTCTTATTGA ATAGAGAAGA TTCTGATGCA CGTAATTATT ATTATT GGGA GATTCCACAG 3061 CATTATGTGT TTAATAATT C TTTGTGGACA AAACGCCGAA AGGGTGGGAA T AAAGT AT T A 3121 GGTAGACTGT TCACTGTGAG C T T T A G A G A A C CAGAAC GAT AT T AC C T T AG ACTTTTGCTT 3181 CTGCATGTAA A A G G T G C GAT AAGTTTTGAG G AT C T G C G A A C T G TAG / A/ Z T G T AAC T TAT 3241 GATACATTTC ATGAAGCTGC T AAAC AC C G A G GAT TAT T AC T T GAT GAC AC TAT C T G G AA 3301 GAT AC GAT TG AC GAT GCAAT CATCCTTAAT AT G C C C AAAC AACTACGGCA ACTTTTTGCA

3361 TATATATGTG TGTTTGGATG TCCTTCTGCT GCAGACAAAT TATGGGATGA GAAT AAAT C T

3421 CAT T T TAT T G AAGATTTCTG TTGGAAATTA CACCGAAGAG AAG GT G C CT G TGTGAACTGT 3481 GAAATGCATG C C C T T AAC G A AATTCAGGAG GTATTCACAT T G CAT G GAAT GAAATGTTCA 3541 CAT T T C AAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA ATACATGTGA TCAATTGTAC 3601 GAGCAACAAC AG G C AG AG G T TTTGATAAAT TCTCTGAATG ATGAACAGTT GGCAGCCTTT

3661 C AG AC TATAA CTTCAGCCAT CGAAGATCAA ACTGTACACC C C AAAT G C T T TTTCTTGGAT 3721 GGTCCAGGTG GTAGTGGAAA A/AC AT AT C T G TATAAAGTTT T AAC AC AT T A TAT TAG AG G T 3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT ACAGGAATTG C T G C AAT T T AC T T C T T G G T 3841 GGAAGAACCT TTCATTCCCA AT AT AAAT T A C CAAT TC CAT TAAAT GAAAC TTCAATTTCT 3901 AGACTCGATA TAAAGAGTGA AGTTGCTAAA AC CAT T AAAA AG G C C C AAC T TCTCATTATT 3961 GATGAATGCA CCATGGCATC CAGT CAT GCT AT AAAC G C CA TAGATAGATT ACTAAGAGAA

4021 ATTATGAATT TGAATGTTGC AT T T G GT G G G AAAG T T C T C C TTCTCGGAGG G GAT T T T C G A 4081 CAATGTCTCA GT AT T GT AC C AC AT G C TAT G C GAT C G G C C A TAG TAG AAAC GAGTTTAAAG 4141 TACTGTAATG TTTGGGGATG T T T CAGAAAG TTGTCTCTTA AAA CAAATAT GAGATCAGAG 4201 GATTCTGCTT ATAGTGAATG GT T AGTAAAA C T T G GAG AT G G C AAAC T T G A T A G C AG T T T T

SUBSTITUTE SHEET (RULE 26) 4261 CATTTAGGAA TGGATATTAT TGAAATCCCC CATGAAATGA TTTGTAACGG ATCTATTATT

4321 GAAGCTACCT TTGGAAATAG TATATCTATA GA AATATTA AAAATATATC TAAACGTGCA

4381 ATTCTTTGTC CAAAAAATGA GCATGTTCAA AAATTAAATG AAGAAATTTT GGATATACTT

4441 GATGGAGATT TTCACACATA TTTGAGTGAT GATTCCATTG ATTCAACAGA TGATGCTGAA

4501 AAGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC CTTCGGGAAT GCCGTGTCAT

4561 AAATTAAAAT TGAAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA TAGTAAATGG

4621 GGTCTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC GACCTAACAT TATCGAAGCT

4681 GAAGTATTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA TTCCAAGAAT TGATTTGTCC

4741 CCATCTGACA C!GGCCTCCC ATTTAAATTA ATTCGAAGAC AGTTTCCCGT GATGCCAGCA

4801 TTTGCGATGA CTATTAATAA ATCACAAGGA CAAACTCTAG ACAGAGTAGG AATATTCCTA

4861 CCTGAACCCG TTTTCGCACA TGGTCAGTTA TATGTTGCTT TCTCTCGAGT TCGAAGAGCA

4921 TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCAC AG GGAAATTAGT CAAGCACTCT

4981 GAAAGTGTTT TTACTCTTAA TGTGGTATAC AGGGAGATAT TAGAATAAGT TTAATCACTT

5041 TATCAGTCAT TGTTTGCATC AATGTTGTTT TTATATCATG TTTTTGTTGT TTTTATATCA

5101 TGTCTTTGTT GTTGTTATAT CATGTTGTTA TTGTTTATTT ATTAATAAAT TTATGTATTA

5161 TTTTCATATA CATTTTACTC ATTTCCTTTC ATCTCTCACA CTTCTATTAT AGAGAAAGGG

5221 CAAATAGCAA TATTAAAATA TTTCCTCTAA TTAATTCCCT TTCAATGTGC ACGAATTTCG

5281 TGCACCGGGC CACTAG (SEQ ID NO: 14667) .

Unlike other transposases, the Helitron transposase does not contain an RNase-H like catalytic domain, but instead comprises a RepHel motif made up of a replication initiator domain (Rep) and a DNA helicase domain. The Rep domain is a nuclease domain of the HUH superfamily of nucleases.

An exemplary' Helitron transposase of the disclosure comprises an amino acid sequence comprising:

1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR

61 RHRQKM3 KDQ RAFEVERRRW RRQNMSREQ3 STSTTNTGRN CLLSKNGVHE DAILEHSCGG

121 MTVRCEFCLS LNFSDEKPSD GKFTRCCS KG KVCPNDIHFP DYPAYLKRLM TNEDSDSKNF

181 MENIFSINSS FAFASMGANI ASPSGYGPYC FRIHGQVYHR TGTLHPSDGV SRKFAQLYIL

241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKS Y KMLHEVEKEA QS EAAAKGIA

301 PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FRNEDGEPPF ERDLLIHCKP DPNNPNATKM

361 KQI SILFPTL DAMTYPILFP HGEKGWGTDI ALRLRDNSVI DNNTRQNVRT RVTQMQYYGF

421 HLSVRDTFNP I LNAGKLTQQ FIVDSYSKME ANRINFIKAN QSKLRVEKYS GLMDYLKSRS

481 ENDNVPI GKM IILPSSFEGS PRNMQQRYQD AMAIVTKYGK PDLFITMTCN PKKADITNNL

541 QRWQKVENRP DLVARVFNI K LNALLNDI CK FHLFGKVIAK IHVIEFQKPG LPHAHILLIL

601 DSESKLRSED DIDRIVKAEI PDEDQCFRLF OIVKSNMVHG PCGIQNPNSP CMENGKCSKG

661 YPKEFQNATI GNIDGYPKYK RRSGSTMSIG NKWDNTWIV PYNPYLCLKY NCHINVEVCA

SUBSTITUTE SHEET (RULE 26) 721 SIKSVKYLFK YI YKGHDCAN IQISEKNIIN HDEVQDFIDS RYVSAPEAVW RLFAMRMHDQ

781 SHAITRLAIH LPNDQNLYFH TDDFAEVLDR AKRHNSTLMA WFLLNREDSD ARNYYYWEIP 841 QHYVFNNSLW TKRRKGGNKV LGRLFTVS FR EPERYYLRLL LLHVKGAI S F EDLRTVGGVT

901 YDT FHEAAKH RGLLLDDTIW KDTIDDAIIL NMPKQLRQLF AYI CVFGCPS AADKLWDENK 961 SHFIEDFCWK LHRREGACVN CEMHALNEIQ EVFTLHGMKC SHFKLPDYPL LMNANTCDQL 1021 YEQQQAEVLI NSLNDEQLAA FQTITSAIED QTVHPKCFFL DGPGGSGKTY LYKVLTHYIR 1081 GRGGTVLPTA STGIAANLLL GGRTFHSQYK LPIPLNETSI SRLDIRSEVA KTIKKAOLLI 1141 IDECTMASSH AINAIDRLLR EIMNLNVAFG GKVLLLGGDF RQCLSIVPHA MRSAIVQTSL 1201 KYCNVWGCFR KLSLKTNMRS EDSAYSEWLV KLGDGKLDSS FHLGMDIIEI PHEMICNGSI 1261 IEATFGNSIS IDNIKNISKR AILCPKNEHV QKLNEEI LDI LDGDFHTYLS DDSIDSTDDA 1321 EKENFPI EFL NSITPSGMPC HKLKLKVGAI IMLLRNLNSK WGLCNGTREI I KRLRPN11 E 1381 AEVLTGSAEG EVVLIPRIDL SPSDTGLPFK LIRRQFPVMP AFAMTINKSQ GQTLDRVGIF 1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVKWN TSSQGKLVKH SESVFTLNW YREILE (SEQ ID NO:

1450

[0590] In Helitron transpositions, a hairpin close to the 3’ end of the transposon functions as a terminator. However, this hairpin can be bypassed by the transposase, resulting in the transduction of flanking sequences. In addition, Helraiser transposition generates covalently closed circular intermediates. Furthermore, Helitron transpositions can lack target site duplications. In the Helraiser sequence, the transposase is flanked by 5’ and 3’ terminal sequences termed LTS and RTS. These sequences terminate with a conserved 5’-TC/CTAG-3’ motif. A 19 bp palindromic sequence with the potential to form the hairpin termination structure is located 11 nucleotides upstream of the RTS and consists of the sequence

GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 14500).

[0591] Tol2 transposons may be isolated or derived from the genome of the medaka fish, and may be similar to transposons of the hAT family. Exemplary Tol2 transposons of the disclosure are encoded by a sequence comprising about 4.7 kilobases and contain a gene encoding the To!2 transposase, which contains four exons. An exemplary Tol2 transposase of the disclosure comprises an amino acid sequence comprising the following:

1 MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSFKMKCVLC LPLNKEI SAF

61 KSSPSNLFKH IERMHPNYLK NYS KLTAQKR KIGTSTHASS SKQLKVDSVF PVKHVSPVTV

121 NKAILRYIIQ GLHPFSTVDL PSFKELISTL QPGISVITRP TLRSKIAEAA LIMKQKVTAA

181 MSEVEWIATT TDCWTARRKS FIGVTAHWIN PGSLERHSAA LACKRLMGSH TFEVLASAMN

241 DIHSEYEIRD KWCTTTDSG SNFMKAFRVF GVENNDIETE ARRCESDDTD SEGCGEGSDG

301 VEFQDASRVL DQDDGFEFQL PKHQKCACHL LNLVSSVDAQ KALSNEHYKK LYRSVFGKCQ

SUBSTITUTE SHEET (RULE 26) 361 ALWNKS SRSA LAAEAVESES RLQLLRPNQT RWNSTFMAVD RILQICKEAG EGALRNICTS

421 LEVPMFNPAE MLFLTEWANT MRPVAKVLDI LQAETNTQLG LLPSVHQLS LKLQRLHHSL

481 RYCDPLVDAL QQGIQTRFKH MFEDPEIIAA AILLPKFRTS WTNDETI IKR GMDYIRVHLE

541 PLDHKKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV SDTRESLLTF FAICSLSIKT

601 NTPLPASAAC EP.LFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE (SEQ ID NO: 14502).

[0592] An exemplary Tol2 transposon of the disclosure, including inverted repeats, subterminal sequences and the Tol2 transposase, is encoded by a nucleic acid sequence comprising the following:

1 CAGAGGTGTA AAGTACTTGA GTAATTTTAG TTGATTACTG TAGTTAAGTA TTATTTTTGG 61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA CTTAATTAGA 121 TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC 181 TTATTTTTTG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACCAAACC AATTGAATTG 241 CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT

301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA 361 TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG 421 GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA

481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTTCTT TAAGTGGTGT

541 AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC

601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT 661 TTTGTTTTAC TGATAGTTTT TTTTTGGGTG TGCATGTTTT 721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT

781 GACAATATAA GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT 841 CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATGA TTATCATTCC GTGCTCTCAT 901 TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT 961 TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA

1021 TTAAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT AATCTTTGTT AACGTTAGTT 1081 AATAGAAATA CAGATGTTCA TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT 1141 AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGCA GTTCATTATT 1201 AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA

1261 ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC 1321 AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA.

1381 TCAACAAGTA TTTAACATTA TAAAGTGTGC AATTGGCTGC AAATGTCAGT TTTATTAAAG 1441 GGTTAGTTCA CCCAAAAATG AAAATAATGT CATTAATGAC TCGCCCTCAT GTCGTTCCAA.

1501 GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATATTTTAGA TTTAGTCCGA 1561 GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGGT 1621 AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAAGCA 1681 TCGAATACAT TTTGGTCCAA AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC

SUBSTITUTE SHEET (RULE 26) 1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG AC T T C GC AGT GACGCTACAA T G C T GAAT AA 1801 AGTCGTAGGT TTTGTTATTT TTGGACCAAA AT G TAT T T T C GAT G C T T CAA ATAATT CTAC 1861 CTAACCCACT GAT GT C ACAT GGACTACTTT GA G T T T T T A TTACCTTTCT GGACATGGAC 1921 AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAGC TCTCGGACTA A C T AAAAT 1981 ATCTTAAACT GTGTTCCGAA GAT G AA C G GA GGTGTTACGG GCTTGGAACG ACATGAGGGT 2041 GAG T CAT T AA T G A CAT C T T T TCATTTTTGG G T G A A C T A A C CCTTTAATGC TGTAATCAGA 2101 GAG T G TAT G T GTAATTGTTA CATTTATTGC ATACAATATA AATAT TATT TGTTGTTTTT 2161 ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA T T G AC AG C AC AGAAGAGAAA 2221 LJA-L CGGGACC T C C AC C CAT G CTTCCAGCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC 2281 AGT CAAACAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA T CAT T C AAG G 2341 AC T T CAT C C T T T C A G C AC T G T T G AT C T G C C AT CAT T T AAA GAG C T GAT T A GTACACTGCA 2401 GCCTGGCATT T C T GT CAT T A CAAGGCCTAC TTTACGCTCC AAGATAGCTG AAGCTGCTCT 2461 GAT CAT G AAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAAT G GAT T G CAACCACAAC 2521 GGATTGTTGG AC T GCAC GT A GAAAG T CAT T CATTGGTGTA ACTGCTCACT GGATCAACCC 2581 TGGAAGTCTT GAAAGACAT T CCGCTGCACT T G C C T G CAAA AGATTAATGG G C T C T CAT AC 2641 TTTTGAGGTA C T G G C GAG T G C CAT GAAT GA T AT C C AC T CA GAGTATGAAA TACGTGACAA 2701 GGTTGTTTGC ACAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAG T T T T T G G 2761 TGTGGAAAAC AATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGT GAT G ACACTGATTC 2821 TGAAGGCTGT GGTGAGGGAA GTGATGGTGT G GAAT T C C AA GAT G C C T C AC GAGTCCTGGA 2881 CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACAT CAA AAGTGTGCCT GTCACTTACT

2941 T AAC CT AGT C T C AA G C G T T G AT c c c CAAAA A G C T C T C T C A AATGAACACT ACAAGAAACT 3001 CTACAGATCT GTCTTTGGCA AATGCCAAGC T T T T G G AT AAAAGCAGCC GATCGGCTCT 3061 AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT TTAAGGCCAA ACCAAACGCG 3121 GTGGAATTCA ACTTTTATGG CTGTTGACAG AAT CTTCAA ATTTGCAAAG AAGCAGGAGA 3181 A G G C G C A C T T C G GAAT AT AT G C A C C T C T C T T GAG GT T C CA AT GTAAGT GT TTTTCCCCTC 3241 TAT C GAT GT A AACAAAT GT G GGTTGTTTTT GTTTAATACT C T T T GAT TAT G C T GAT T T C T 3301 CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA GTGGGCCAAC ACAATGCGTC

3361 CAGTTGCAAA AGTACTCGAC ATCTTGCAAG C G GAAAC GAA TACACAGCT G GGGTGGCTGC 3421 TGCCTAGTGT C C AT CA GT T A AG C T T GAAAC TTCAGCGACT CCACCATTCT C T C AG G TAG T 3481 GT G AC C C AC T T G T G GAT G C C CTACAACAAG GAAT AAAC ACGATTCAAG C A TAT G T T T G 3541 AAGATCCTGA GAT CAT AG C A G C T G C CAT C C T T C T C C C T AA ATTTCGGACC T C T T G Gr AC AA 3601 AT GAT GAAAC CAT CAT AAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC 3661 TAATCTGGGC AACCTTTGAG C CAT AC CAAA ATTATTCTTT TAT T T AT T T A TTTTTGCACT 3721 TTTTAGGAAT GT TAT AT C C C ATCTTTGGCT G T GAT C T CAA TAT GAAT ATT GATGTAAAGT 3781 ATTCTTGCAG CAGGTT GTAG T T AT C C C T C A GTGTTTCTTG AAAC CAAACT C A TAT G TAT C 3841 AT AT GT G GT T TGGAAATGCA G T TAG AT T T T AT G C T AAAAT AAGGGATTTG CAT GAT T T T A 3901 GATGTAGATG AC T GCAC GT A AATGTAGTTA ATGACAAAAT CC AT AAAAT T TGTTCCCAGT 3961 CAGAAGCCCC TCAACCAAAC TTTTCTTTGT G T C T G C T C AC TGTGCTTGTA G G CAT G GAC T

SUBSTITUTE SHEET (RULE 26) 4021 ACAT CAGAGT GCATCTGGAG CCTTT GGACC ACAAGAAGGA AT T G G C C AAC AG T T CAT C T G

4081 ATGATGAAGA TTTTTTCGCT TCTTTGAAAC C GA_C AAC ACA TGAAGCCAGC AAAG AG T T G G

4141 AT G GAT AT C T G G C C T G T G T T TCAGACACCA G G G A G T C T C T GCTCACGTTT C C T G C T AT T T

4201 GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCATC GGCTGCCTGT GAGAGGCTTT

4261 TCAGCACTGC AGGATTGCTT T T C AG C C C C A AAAG AG C TAG GCTTGACACT AACAATTTTG

4321 AG A AT C A G C T TCTACTGAAG TTAAATCTGA GGTTTTACAA CTTTGAGTAG C GT GT AC T G G

4381 CAT TAG AT T G TCTGTCTTAT AGTTT GATAA T AAATACAA ACAGTT CTAA AG C AG GATAA

4441 AACCTTGTAT GCATTTCATT TAATGTTTTT T GAG AT T AAA AGCTTAAACA AGAATCTCTA

4501 GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA AGTACAATTT TAATGGAGTA

4561 CTTTTTTACT TTTACT CAAG TAAGATTCTA G C CAGAT ACT TTTACTTTTA AT T GAG T AAA

4621 AT T T T C C C T A AG AC T GT A CTTTCACTTG AGTAAAATTT T T GAG TAG T T TTTACACCTC

4681 TG (SEQ ID NO: 14668).

[0593] Exemplary transposon/transposase systems of the disclosure include, but are not limited to, piggyBac and piggyBac-iike transposons and transposases.

[0594] PiggyBac and piggyBac-iike transposases recognizes transposon-specific inverted term inal repeat sequences (ITRs) on the ends of the transposon, and m oves the contents between the ITRs into TTAA or TTAT chromosomal sites. The piggyBac or piggyBac-iike transposon system has no payload limit for the genes of interest that can be included between the ITRs.

[0595] In certain embodiments, and, in particular, those embodiments wherein the transposon is a piggyBac transposon, the transposase is a piggyBac™, Super piggyBac™ (SPB) transposase. In certain embodiments, and, in particular, those embodiments wherein the transposase is a piggyBac™, Super piggyBac™ (SPB), the sequence encoding the transposase is an mRNA sequence.

[0596] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-iike transposase enzyme.

[0597] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or a piggyBac-iike transposase enzyme. The piggyBac (PB) or piggyBac- like transposase enzyme may comprise or consist of an ammo acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG

61 S El LDEQNVI EQPGSSLASN R.I LTLPQRT I EGKNKHCWST SKSTRRSRVS ALNIVRSQRG

121 PTFMCRNIYD PLLCFKLFFT DE1ISEIVKW TNAEI SLKRR ESMTGATFRD TNEDEI YAFF

SUBSTITUTE SHEET (RULE 26) 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL I RCLRMDDKS IRPTLRENDV

241 FTPVRKIKDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYI PNKPSK YGIKILMMCD

301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RKITCDNWFT SIPLAKNLLQ

361 EPYKLTIVGT VRSNKREI PE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC

421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN

481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TS SFMRKRLE APTLKRYLRD NISNILPNEV

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID NO : 14487),

[0598] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme that comprises or consists of an amino acid sequence having an amino acid substitution at one or more of positions 30, 165, 282, or 538 of the sequence:

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG

61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG

121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF

181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL I RCLRMDDKS IRPTLRENDV

241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD

301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDN FT SIPLAKNLLQ

361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC

421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN

481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV

541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF (SEQ ID

NO : 14487).

[0599] In certain embodiments, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme that comprises or consists of an ammo acid sequence having an amino acid substitution at two or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac or piggyBac-hke transposase enzyme that comprises or consists of an ammo acid sequence having an amino acid substitution at three or more of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme that comprises or consists of an amino acid sequence having an ammo acid substitution at each of the following positions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 14487. In certain embodiments, the amino acid substituti on at positi on 30 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for an isoleucine (I). In certain

SUBSTITUTE SHEET (RULE 26) embodiments, the amino acid substitution at position 165 of the sequence of SEQ ID NO: 14487 is a substitution of a serrne (S) for a glycine (G). In certain embodiments, the ammo acid substitution at position 282 of the sequence of SEQ ID NO: 14487 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the ammo acid substitution at position 538 of the sequence of SEQ ID NO: 14487 is a substitution of a lysine (K) for an asparagine (N).

[0600] In certain embodiments of the methods of the disclosure, the transposase enzyme is a Super piggyBac™ (SPB) or piggyBac-iike transposase enzyme. In certain embodiments, the Super piggyBac™ (SPB) or piggyBac-iike transposase enzyme of the disclosure may comprise or consist of the amino acid sequence of the sequence of SEQ ID NO: 14487 wherein the amino acid substitution at position 30 is a substitution of a valine (V) for an isoleucine (I), the ammo acid substitution at position 165 is a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 is a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 is a substitution of a lysine (K) for an asparagine (N). In certain embodiments, the Super piggyBac™ (SPB) or piggyBac-iike transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG

61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG

121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEI SLKRR ESMTSATFRD TNEDEIYAFF

181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDREDFL I RCLRMDDKS IRPTLRENDV

241 FTPVRKIKDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD

301 SGTKYMINGM PYLGRGTQTK GVPLGEYYVK ELSKPVHGSC RKITCDNWFT SIPLAKNLLQ

361 EPYKLTIVGT VRSNKREI PE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC

421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN

481 S IF11YSHNVS SKGEKVQSRK KFMRNLYMSL TS SFMRKRLE APTLKRYLRD NISNILPKEV

541 PGTSDOSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV I CREHNI DMC QSCF (SEQ ID NO:

14484 } .

[0601] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™, Super piggyBac™ or piggyBac-iike transposase enzyme may further comprise an amino acid substituti on at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241 , 243, 258, 296, 298, 311, 315, 319, 327,

SUBSTITUTE SHEET (RULE 26) 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™, Super piggyBac™ or piggyBac-like transposase enzyme may further comprise an ammo acid substitution at one or more of positions 46, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and 570. In certain embodiments, the amino acid substitution at position 3 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an asparagine (N) for a serme (S). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an alanine (A). In certain embodiments, the amino acid substitution at position 46 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 82 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for an isoleucme (I). In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the amino acid substitution at position 1 19 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for an arginine (R). In certain embodiments, the ammo acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) a cysteine (C). In certain embodiments, the amino acid substitution at position 125 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a eysteme (C). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 177 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (II) for a tyrosine (Y). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a pheny lalanine (F). In certain embodiments, the amino acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a pheny lalanine (F). In certain embodiments, the ammo acid substitution at position 180 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valme (V) for a phenylalanine (F). In certain embodiments, the amino acid substitution at position 185 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a

SUBSTITUTE SHEET (RULE 26) methionine (M). In certain embodiments, the amino acid substitution at position 187 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for an alanine (A) in certain embodiments, the ammo acid substitution at position 200 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a phenylalanine (F). In certain embodiments, the ammo acid substitution at position 207 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a valine (V). In certain embodiments, the ammo acid substitution at position 209 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a valine (V). In certain embodiments, the amino acid substitution at position 226 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a methionine (M). In certain embodiments, the amino acid substitution at position 235 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a leucine (L). In certain

embodiments, the amino acid substitution at position 240 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 241 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a phenylalanine (F) In certain embodiments, the amino acid substitution at position 243 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a proline (P) In certain embodiments, the ammo acid substitution at position 258 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tryptophan (W) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a leucine (L). In certain embodiments, the amino acid substitution at position 296 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the ammo acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for a methionine (M). In certain embodiments, the ammo acid substitution at position 298 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine ( V) for a methionme (M). In certain embodiments, the amino acid substitution at position 31 1 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoieucine (I) for a proline (P). In certain embodiments, the amino acid substitution at position

SUBSTITUTE SHEET (RULE 26) 31 1 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine for a proline (P).

In certain embodiments, the ammo acid substitution at position 315 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for an arginine (R).In certain embodiments, the amino acid substitution at position 319 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a threonine (T). In certain embodiments, the amino acid substitution at position 327 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a tyrosine (Y). In certain embodiments, the ammo acid substitution at position 328 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a tyrosine (Y). In certain embodiments, the ammo acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a cysteine (C). In certain

embodiments, the amino acid substitution at position 340 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (I.) for a cysteine (C). In certain embodiments, the amino acid substitution at position 421 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a histidine (H) for the aspartic acid (D) In certain embodiments, the amino acid substitution at position 436 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a valine (V) In certain embodiments, the amino acid substitution at position 456 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a tyrosine (Y) for a methionine (M). In certain embodiments, the ammo acid substitution at position 470 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a phenylalanine (F) for a leucine (L). In certain embodiments, the amino acid substitution at position 485 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a serine (S). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a leucine (L) for a methionine (M). In certain embodiments, the amino acid substitution at position 503 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an isoleucine (I) for a methionine (M). In certain embodiments, the ammo acid substitution at position 552 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a lysine (K) for a valine (V). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a threonine (T) for an alanine (A). In certain embodiments, the amino acid substitution at position 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a glutamine (Q). In certain embodiments, the amino acid substitution at position

SUBSTITUTE SHEET (RULE 26) 591 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an arginine (R) for a glutamine (Q).

[0602] In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™ or piggyBac-like transposase enzyme or may comprise or the Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at one or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments of the methods of the disclosure, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™ or piggyBac-like transposase enzyme may comprise or the Super piggyBac™ transposase enzyme may further comprise an ammo acid substitution at two, three, four, five, six or more of positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, including those embodiments wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and/or 538, the piggyBac™ or piggyBac-like transposase enzyme may comprise or the Super piggyBac™ transposase enzyme may further comprise an amino acid substitution at positions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the amino acid substitution at position 103 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a proline (P) for a serine (S). In certain embodiments, the ammo acid substitution at position 194 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a valine (V) for a methionine (M). In certain embodiments, the amino acid substitution at position 372 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for an arginine (R). In certain embodiments, the amino acid substitution at position 375 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an alanine (A) for a lysine (K). In certain embodiments, the amino acid substitution at position 450 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of an asparagine (N) for an aspartic acid (D). In certain embodiments, the amino acid substitution at position 509 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a glycine (G) for a serine (S). In certain embodiments, the amino acid substitution at position 570 of SEQ ID NO: 14487 or SEQ ID NO: 14484 is a substitution of a serine (S) for an asparagine (N). In certain embodiments, the piggyBac™ or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine

SUBSTITUTE SHEET (RULE 26) (M) at position 194 of SEQ ID NO: 14487. In certain embodiments, including those embodiments wherein the piggyBac™ or piggy Bac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, the piggyBac™ or piggyBac-like transposase enzyme may further comprise an amino acid substitution at positions 372, 375 and 450 of the sequence of SEQ ID NO: 14487 or SEQ ID NO: 14484. In certain embodiments, the piggyBac™ or piggyBac-hke transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, and a substitution of an alanine (A) for a lysme (K) at position 375 of SEQ ID NO: 14487. In certain embodiments, the piggyBac™ or piggyBac-like transposase enzyme may comprise a substitution of a valine (V) for a methionine (M) at position 194 of SEQ ID NO: 14487, a substitution of an alanine (A) for an arginine (R) at position 372 of SEQ ID NO: 14487, a substitution of an alanine (A) for a lysine (K) at position 375 of SEQ ID NO: 14487 and a substitution of an asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO: 14487.

[0603] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia ni (GenBank

Accession No. AAA87375; SEQ ID NO: 14672), Argyrogramma agnata (GenBank Accession No. GU477713; SEQ ID NO: 14534, SEQ ID NO: 14673), Anopheles gambiae (GenBank Accession No. XP__312615 (SEQ ID NO: 14674); GenBank Accession No. XP__320414 (SEQ ID NO: 14772); GenBank Accession No. XP_310729 (SEQ ID NO: 14676), Aphis gossypii (GenBank Accession No. GU329918; SEQ ID NO: 14677, SEQ ID NO: 14678), Acyrihosiphon pisum (GenBank Accession No. XP_00l 948139; SEQ ID NO: 14679), A gratis ipsilon (GenBank Accession No. GU477714; SEQ ID NO: 14537, SEQ ID NO: 14680), Bornhyx mori (GenBank Accession No. BAD11135; SEQ ID NO: 14505), Chilo suppressalis (GenBank Accession No. JX294476; SEQ ID NO: 14681, SEQ ID NO: 14682), Drosophila melanogaster (GenBank Accession No. AAL39784; SEQ ID NO: 14683), Helicoverpa armigera (GenBank Accession No. ABS 18391 ; SEQ ID NO: 14525), Heliothis virescens (GenBank Accession No. ABD76335; SEQ ID NO: 14684), Macdunnoughia crassisigna (GenBank Accession No. EU287451; SEQ ID NO: 14685, SEQ ID NO: 14686), Pectinophora gossypieila (GenBank Accession No.

GU270322; SEQ ID NO: 14530, SEQ ID NO: 14735), Tnholmm castaneum (GenBank

Accession No. XP 001814566; SEQ ID NO: 14688), Ctenoplusia agnata (also called

SUBSTITUTE SHEET (RULE 26) Argyrogramma agnata), Messour houvieri. Megachile rotundata , Bombus impatiens, Mamestra bmssicae, Mayetiola destructor or Apis meUifera.

[0604] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Trichoplusia m (AAA87375).

[0605] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from an insect. In certain embodiments, the insect is Bombyx mori (BAD11135).

[0606] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from a crustacean. In certain embodiments, the crustacean is Daphnia pulicaria

(AAM76342, SEQ ID NO: 14689).

[0607] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from a vertebrate. In certain embodiments, the vertebrate is Xenopus tropicalis

(GenBank Accession No. BAE82026; SEQ ID NO: 14518), Homo sapiens (GenBank Accession No. NP_689808; SEQ ID NO: 14690), Mus musculus (GenBank Accession No. NP_74l958; SEQ ID NO: 14691), Macaca fascicularis (GenBank Accession No. AB 179012; SEQ ID NO: 14692, SEQ ID NO: 14693), Rattus norvegicus (GenBank Accession No. XPJ220453; SEQ ID NO: 14694) or Myotis lucifugus

[0608] In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from a urochordate. In certain embodiments, the urochordate is Ciona intestinalis (GenBank Accession No. XP_002123602; SEQ ID NO: 14695).

[0609] In certain embodiments, the piggyBac or piggyBac-like transposase inserts a transposon at the sequence 5’-TTAT-3’ within a chromosomal site (a TTAT target sequence).

[0610] In certain embodiments, the piggyBac or piggyBac-like transposase inserts a transposon at the sequence 5’-TTAA-3’ within a chromosomal site (a TTAA target sequence).

[0611] In certain embodiments, the target sequence of the piggyBac or piggyBac-like transposon comprises or consists of 5’-CTAA-3’, 5’ -XT AG-3’, 5’-ATAA-3’, 5 ^: ’-TCAA-3’, 5’AGTT-3’, 5’- ATT.L-3 5’-GITA-3’, 5’-TTGA-3\ 5’-TTTA-3’, 5’-TTAC-3’, 5’ -ACTA-3’, 5’-AGGG-3 \ 5’- CTAG-3’, 5 ^’ -TGAA-3 ^’ , 5 ^’ -AGGT-3\ 5 ^’ -ATCA-3\ 5’-CTCC-3\ f i’-T AAA-3’, 5’-TCTC-3’, 5’TGAA-3’, 5’-AAAT-3’, 5 ^’ -AATC-3’, 5’-ACAA-3\ 5 ^’ -ACAT-3 \ 5’-ACTC-3 ^’ , 5’-AGTG-3’, 5’ -AT AG-3’, 5’-CAAA-3’, 5’-CACA-3\ 5’-CATA-3’, 5’-CCAG- G, 5’-CCCA-3’, 5’-CGTA-3’ 5’-GTCC-3 ^’ , 5’-TAAG-3’, 5’-TCTA-3’, 5’-TGAG-3’, 5’-TGIT-3 ^’ , 5 ^’ -TTCA-3 ^’ 5’-TTCT-3’ and 5’-TTTT-3\

SUBSTITUTE SHEET (RULE 26) [0612] in certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Bombyx mori. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an ammo acid sequence at least

5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MDI ERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVPLPSE

121 NIVTEQAQVK NIAREASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS

181 FYMQETTLCE LKALIALLYL AGLI KSNRQS LKDLWRTDGT GVDI FRTTMS LQRFQFLQNN

241 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ CCQNAYS PSE FLTIDEMLLS FRGRCLFRVY

301 I PNKPAKYGI KI LALVDAKN FDWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR

361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL

421 VSYAPKKNKV WVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELSANYNVSR

481 NSKRWPMTLF YGVLNMAAIN ACIIYRANKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI

.541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KHSCNACAKP ICMEHAKFLC

601 ENCAELDSSL (SEQ ID NO 14504} ,

[0613] The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MDI ERQEERI RAMI,EEELSD YSDES S SEDE TDHCSEHEVN YDTEEERI DS VDVPSNSRQE

61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE

121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS

181 FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRTDGT GVDI FRTTMS LQRFQFLQNN

241 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ CCQNAYS PSE FLTIDEMLLS FRGRCLFRVY

301 I PNKPAKYGI KI LALVDAKN FYWNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR

361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL

421 VSYAPKKNKV WVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR

481 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI

541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC

601 ENCAELDS S L (SEQ ID NO 14505) .

In certain embodiments, the piggyBac or piggyBac-like transposase is fused to a nuclear localization signal. In certain embodiments, the ammo acid sequence of the piggyBac or piggyBac-like transposase fused to a nuclear localization signal is encoded by a polynucleotide sequence comprising:

i atggcaccca aaaagaaacg taaagtgatg gacattgaaa gacaggaaga aagaa tcagg

61 gcgatgctcg aagaagaact gagcgactac tccgacgaat cqtcatcaga ggatgaaacc 121 gaccactgta gcgagcatga ggttaactac gacaccgagg aggagagaat cgactctgtg 181 gatgtgccct ccaactcacg ccaagaagag gccaatgcaa ttatcgcaaa cqaatcggac 241 agcgatccag acgatgatct gccactgtcc ctcgtgcgcc agcgggccag cgcttcgaga 301 caagtgtcag gtccattcta cacttcgaag gacggcacta aq tgg ta ca a gaattgccag 361 cgacctaacg tcagactccg ctccgagaat atcgtgaccg aacaggctca ggtcaagaat 421 atcgcccgcg acgcctcgac tgagtacgag tgttggaata tcttcgtgac ttcqqacatg 481 c.tgcaagaaa ttctqacgca caccaac.agc tcgattaqqc atcgccagac c.aagactgca 541 gcggagaact catcggccqa aacctccttc tatatgcaag aqactactct gtgcgaactg

SUBSTITUTE SHEET (RULE 26) 601 aaggcgctga ttqcactqct q taettggee ggcctcatca aatcaaatag gcagagcctc

661 aaagatctct ggagaacgga tggaactgga gtggatatct tteggaegae tatgagettg

721 cagcqq t tcc agtttctgca aaacaata tc aqattegaeg acaagtccac ccqqqacqaa

781 aggaaacaga ctqacaacat ggctgcgttc cggtcaatat tcgatcagtt tgtgcagtgc

841 tgccaaaacq cttataqccc a teggaatte ctgaccatcg aeqaaa get tetet ccttc 901 cgggggcgct gcctgttccg agtgtacatc ccgaacaagc eggetaaata cggaatcaaa

961 atcctggccc tggtggacgc caagaatt tc tacgtcgtga atetegaagt gtacq caq ga 1021 aagcaaccgt cgqgaccgta cgctgtttcg aaccgcccgt ttgaagt cgt egageggett 1081 attcagccgg tggccaga tc cca ccgcaat gttacct teg acaa t tggtt caccggctac 1141 gagctgatgc ttcaccttct gaacqaqtac cggctcacta gcgtggggac tgteaggaag 1201 aacaaq egg c agatcccaga atccttca tc eg ca ccgacc gc cageetaa c teg tccq tg 1261 ttcggatttc aaaaggatat ca cgcttgtc tcgtacgccc ccaagaaaaa caaggtcgtg 1321 gtcgtgatga gcaccatgca tcacgacaac ageategaeg agtcaaccgg agaaaagcaa 1381 aagcccgaga tgatcacctt ctacaattca a cta a g g ccg gegtegaegt cgtqqatqaa 1441 ctgtgcgcga actataacgt gtcccggaac tetaageggt ggcctatgac tetet tctac 1501 ggagtgctga atatgqccqc aatcaacgcg tgcatcatct a:ceg caeca a caagaacgtg 1561 accatcaagc gcaccgagt t catcagatcg ctgggtttga gcatgatcta cgagcacctc 1621 cattcacgga a caagaag a gaatatccct acttacctga ggeagegtat egagaageag 1681 ttgggagaac caagcccgcg ccacgtgaac gtgccggggc gctacgtgcg gtgccaagat 1741 tgcccgtaca aaaaggaccg agatcgtgta acgcgtgcgc caaacctatc 1801 tgcatggagc a Lgccaa t c tctgtgtgaa aattgtgctg aa ctcga11 c ctccctg (SEQ

ID NO: 1 629 } ,

[0615] in certain embodiments, the piggyBac or piggyBac-like transposase is hyperactive. A hyperactive piggyBac or piggyBac-like transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase enzyme is isolated or derived from Bombyx mori. In certain embodiments, the piggyBac or piggyBac-like transposase is a hyperactive variant of SEQ ID NO: 14505. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to:

1 MDI ERQEERI RAMI.EEELS D YSDES S SEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE 61 EANAI IA ES DSDPDDDLPL S LVRQRASAS RQMSGFHYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY EOWNI FVT SD MLQEILTHTN SSIRWRQTKT AAENS SASTS 181 FYMQETTLCE LKALIGLLYI AGLIKSNEQS LKDLWRTDGT GVDI FRTTMS LQRFQFLQNN 241 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 I PNKPAKYGI KILALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKRWPMTLF YGVLNMAAIN ACI I YRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEPSPRHV NVPGRYVRCQ DCP YKKDRKT KRSCNACAKP I CMEHAKFLC 601 ENCAELDSHL (SEQ ID NO: 14576) .

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14576. In certain embodiments, the hyperactive piggyBac or piggyBac- like transposase comprises a sequence of:

1 MDI ERQEERI RAMIEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAI IANES DSDPDDDLPL S LVRQRASAS R.QVSGPFYTS KDGTKWYKNC QRPNVRLRSE

121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENS SAETS

181 FYMQETTLCE LKALIGLLYI AGLI KSNRQS LKDLWRTDGT GVDI FRTTMS LQRFQFLLNN

SUBSTITUTE SHEET (RULE 26) 241 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY

301 I PNKPAKYGI KI LALVDAKN FYVHNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR

361 NVT FDNWFTG YEVMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNS3 VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR

481 NSKRWPMTLF YGVLNMAAIN AC11 YRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEP S PRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAHLDS (SEQ ID NO: 14630) .

[0617] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

1 MDI ERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSASTS 181 FYMQETTLCE LKALI GLLYI AGLIKSNRQS LKDLWRTDGT GVDI FRTTMS LQRFQFLLNN 241 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 I PNKPAKYGI KI LALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVT FDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMS MHHD NSIDESTGEK QKPEMITFYN S KAGVDWD ELCANYNVSR 481 NSKR PMTLF YGVLNMAAIN ACI I YRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIAM QLGEP S PRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 EN CAELDS S L (SEQ ID NO : 14631) .

certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

1 MDI ERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE

121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSAETS

181 FYMQETTLCE LKALI GLLYI AGLIKSNRQS LKDLWRTDGT GVDI FRTTMS LQRFQFLLNN

241 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY

301 I PNKPAKYGI KI LALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR

361 NVT FDNWFTG YELMLHLLNE YRLTSVGTVR KNKTQI PENF IRTDRQPNSS VFGFQKDITL

421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELQANYNVSR

481 N5KR PMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI

541 PTYLRQRIEK QLGEP S PRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC

601 ENCAELDSSL (SEQ ID NO 14632) .

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

1 MDI ERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAIIANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRWRQTKT AAENSSAETS 181 FYMQETTLCE LKALI GLLYI AGLIKSNRQS LKDLWRTDGT GVDI FRTTMS LQRFQFLQNN 241 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 I PNKPAKYGI KI LALVDAKN FYVKNLEVYA GKQPSGPYAV SNRPFEWER LIQPVARSHR 361 NVT FDNWFTG YELMLHLLNE YRLTSVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 421 VSYAPKKNKV VWMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDWD ELCANYNVSR 481 NSKR PMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 541 PTYLRQRIEK QLGEP S PRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID NO 14633) .

[0620] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

SUBSTITUTE SHEET (RULE 26) i MDIERQEERI RAMLEEELSD YSDES S SEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAI IANES DSDPDDDLPL S LVRQRASAS PQVSGPFYTS KDGTKWYKNC QRPNVPLPSE

121 NIVTEQAQVK NIARDASTEY EC NI FVTSD MLQEI LTHTN S S I RHRQTKT AAENS SAETS

181 FYMQETTLCE LKALIALLYL AGLI KSNRQS LKDLWRTDGT GVDI FRTTMS LQRFQFLQNN

24 1 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ CCQNAYS PSE FLTI DEMLLS FRGRCLFRVY

301 I PNKPAKYGI KI LALVDAKN DYWNLEVYA GKQPSGPYAV SNRPFEWER L10PVARSHR

361 NVTFDNWFTG YELMLHLLNE YRLT SVGTVR KNKRQI PES F I RTDRQPNS S VFGFQKDI TL

421 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMITFYN STKAGVDVVD ELCANYNVSR

481 NSKRWPMTLF YGVLNMAAIN AC11 YRTNKN VTI KRTEFI R SLGLSMIYEH LHSRNKKKNI

541 PTYLRORIEK QLGEPSSRHV NVKGRYVRCQ DCPYKKDRKT KRSCNACAKP I CMEHAKFLC

601 ENCAELDSSL (SEQ ID NO 14634) .

[0621] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase is more active than the transposase of SEQ ID NO: 14505. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or any percentage in between identical to SEQ ID NO: 14505.

[0622] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution at a position selected from 92, 93, 96, 97, 165, 178, 189, 196, 200, 201, 21 1 , 215, 235, 238, 246, 253, 258, 261 , 263, 271, 303, 321, 324, 330, 373, 389, 399, 402, 403, 404, 448, 473, 484, 507,5 23, 527, 528, 543, 549, 550, 557,6 01, 605, 607, 609, 610 or a combination thereof (relative to SEQ ID NO: 14505). In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92A, V93L, V93M, P96G, F97H, F97C, H165E, H165W, E178S, E l 781 1. C189P, A196G,

L200I, A201 Q, L2I IA, W215Y, G219S, Q235Y, Q235G, Q238L, K246I, K253V, M258V,

F261 L, S263K, C271 S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M, N44IR, G448W, E449A, V469T, C473Q, R484K T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601V,

E605H, E605W, D607H, S609FI, L6101 or any combination thereof. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an amino acid substitution of Q92A, V93L, V93M, P96G, F97H, F97C, H I 6517 H165W, E178S, E178H, C189P, A196G,

L200I, A201Q, L21 1 A, W215Y, G219S, Q235Y, Q235G, Q238L, K246I, K253V, M258V, F261L, S263K, C271S, N303R, F321W, F321D, V324K, V324H, A330V, L373C, L373V, V389L, S399N, R402K, T403L, D404Q, D404S, D404M, N441R, G448W, E449A, V469T, C473Q, R484K T507C, G523A, I527M, Y528K Y543I, E549A, K550M, P557S, E601 V, 1:6051 1. E605W, 1)6071 1. S609H and 1.6101.

SUBSTITUTE SHEET (RULE 26) [0623] in certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid composes a substitution of E4X, A12X,

M 13X.1.14.X. E15X, D20X, E24X, S25X, S26X, S27X, D32X, H33X, E36X, E44X, E45X, E46X, I48X, D49X, R58X, A62X, N63X, A64X, I65X, I66X, N68X, E69X, D71X, S72X, D76X, P79X, R84X, Q85X, A87X, S88X, Q92X, V93X, S94X, G95X, P96X, F97X, Y98X, T99X, G145C, S149X, D150X, L152X, E154X, T157X, N160X, S161X, S162X, H165X, R166X, T168X, K169X, T170X, A171X, EG73C, S175X, S176X, EG78C, T179X, M183X, Q184X, T186X, T187X, L188X, C189X, L194X, I195X, A196X, L198X, L200X, A201X, L203X, I204X, K205X, A206X, N207X. Q209X, S210X, 1.21 I X. K212X, D213X, 1.2 I 4X. W215X, R2I6X, T217X, G219X, V222X, D223X, I224X, T227X, M229X, Q235X, L237X, Q238X, N239X, N240X, P302X, N303X, P305X, A306X, K307X, Y308X, 13 1 OX. K31 IX, I312X, L313X, A3 M X. L315X, V316X,D317X, A318X, K319X, N320X, F321X, Y322X, V323X, V324X, L326X, E327X, V328X, A330X, Q333X, P334X, S335X, G336X, P337X, A339X, V340X, S341X, N342X, R343X, P344X, F345X, E346X, Y347X. E349X, I352X, Q353X, V355X, A356X, R357X, N361X, D365X, W367X, T369X, G370X, L373X, M374X, L375X, H376X, N379X, E380X, R382X, V386X, V389X, N392X, R394X, Q395X, S399X, F400X, 140 I X, R402XT403X, D404X, R405X, Q406X, P407X, N408X, S409X, S410X,

V41 I X, F412X, F414X, Q415X, 1418X, T419X, L420X, N428XV432X, M434X, D440X, N441X, S442X, I443X, D444X, E445X, G448X, E449X, Q451X, K452X, M455X, I456X, T457X, F458X, S461 X, A464X, V466X, Q468X, V469X, E471 X, L472X, C473X, A474X, K483X, W485X, T488X, L489X, Y491X, G492X, V493X, M496X, I499X, C502X, I503X, T507X, K509X, N510X, V51 I X, T512X, I513X, R515X, E517X, S521X, G523X, L524X, S525X, 1527X, Y528X, E529X, H532X, S533X, N535X, K536X, K537X, N539X, I540X, T542X, Y543X, Q546X, E549X, K550X, Q551X, G553X, E554X, P555X, S556X, P557X, R558X, H559X, V560X, N561X, V562X, P563X, G564X, R565X, Y566X, V567X, Q570X, D571X, P573X, Y574X, K576X, K581X, S583X, A586X, A588X, E594X, F598X, L599X, E601X, N602X, C603X, A604X, E605X, L606X, D607X, S608X, S609X or L610X (relative to SEQ ID NO: 14505). A list of hyperactive ammo acid substitutions can be found in US patent No. 10,041 ,077, the contents of which are incorporated herein by reference in their entirety.

SUBSTITUTE SHEET (RULE 26) [0624] in certain embodiments, the piggyBac or piggyBac-like transposase is integration deficient in certain embodiments, an integration deficient piggyBac or piggyBac- like transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild type transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is an integration deficient variant of SEQ ID NO: 14505.

[0625] In certain embodiments, the excision competent, integration deficient piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of R9X, AI2X, M13X, D20X, Y21K, D23X, E24X, S25X, S26X, S27X, E28X, E30X, D32X, H33X, E36X, H37X, A39X, Y41X, D42X, T43X, E44X, E45X, E46X, R47X, D49X, S50X, S55X, A62X, N63X, A64X, I66X, A67X, N68X, E69X, D70X, D71X, S72X, D73X, P74X, D75X, D76X, D77X,I78X, S8IX,V83X, R84X, Q85X, A87X, S88X, A89X,S90X,R91X, Q92X, V93X. S94X, G95X, P96X, F97X, Y98X, T99X, W012X. G103X, Y107X, K108X, L117X, I122X, Q128X, 13 I 2X. D135X, S137X, E139X, Y140X, I145X, S149X, D150X, Q153X, E i 5 -IX. T157X, S161X, S162X, R164X, H165X, R166X, Q167X, T168X, K169X, T170X, Al 71 X, A172X, E173X, R174X, S175X, S176X, A177X, E178X, T179X,

S I 80X.Y 1 82X. Q184X, E185X, T187X, L188X, C189X, L194X, I195X, A196X, L198X, L200X, A201X, L203X, I204X, K205X, N207X, Q209X, 1.21 1 X D213X, L214X, W215X, R216X, T217X, G219X, T220X, V222X, D223X, 1224X. T227X, T228X, F234X, Q235X, L237X, Q238X, N239X, N24QX, N303X, K304X, I310X, I312X, L313X, A314X, L315X,

V316X,D317X, A318X, K319X, N320X, F321X, Y322X, V323X, V324X, N325X, L326X, E327X, V328X, A330X, G331 X, K332X, Q333X, S335X, P337X, P344X, F345X, E349X, H359X, N361X, V362X, D365X, F368X, Y371X, E372X, L373X, H376X, E380X, R382X, R382X, V386X, G387X, T388X, V389X, K391X, N392X, R394X, Q395X, E398X, S399X, F400X, 1401 X, R402XT403X, D404X, R405X, Q406X, P407X, N408X, S409X, S410X, Q415X,K416X, A424X, K426X, N428X, V430X, V432X, V433X, M434X, D436X, D440X, N441X, S442X, I443X, D444X, E445X, S446X, T447X, G448X, E449X, K450X, Q451X, E454X, M455X, I456X, T457X, F458X, S461X, A464X, V466X, Q468X, V469X, C473X, A474X, N475X, N477X, K483X, R484X, P486X, T488X, L489X, G492X, V493X, M496X, I499X, I503X, Y505X, T507X, N510X, V511X, T512X, L513X, K514X, T516X, E517X,

SUBSTITUTE SHEET (RULE 26) S521X, G523X, L524X, S525X, I527X, Y528X, [.531 X. H532X, S533X, N535X, I540X,

T542X, Y543X, R545.X. Q546X, E549X, L552X, G553X, E554X, P555X, S556X, P557X,

R558X, H559X, V560X, N561X, V562X, P563X, G564X, V567X, Q570X, D571X, P573X,

Y574X, K575X, K576X, N585X, A586X, M593X, K596X, E601X, N602X, A604X, E605X,

L606X, D607X, S608X, S609X or L610X (relative to SEQ ID NO: 14505). A list of integration deficient amino acid substitutions can be found in US patent No. 10,041,077, the contents of which are incorporated by reference m their entirety.

[0626] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence of:

i MDI ERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAI IANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 18 1 FYMQETTLCE LKALIALLYL AGLIKSNRQS LKDLWRKDGT GVDI FRTTMS LQRFQFLLNN 24 1 IRFDDI STRD ERKQTDNMAA FRSI FDQFVQ CCQNAYS PSE FLTIDEMLLS FRGRCLFRVY 301 I PNKPAKYGI KI LALVDAKN FYWNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLT SVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 42 1 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMIT FYN STKAGVDWD ELCANYNVSR 4 8 1 NSKKWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMMYEH LHSRNKKKNI 54 1 PTYLRQRIEK QLGEPVPRHV NVPGRYVRCQ DCPYKKDRKT KRSCNACAKP I CMEHAKFLC 601 ENCAELDSSL (SEQ ID NO 14 606) .

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence of:

1 MDI ERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAI IANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE 121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS 18 1 FYMQETTLCE LKALIGLLYL AGLI KSNRQS LKDLWRTDGT GVDI FRTTMS LQRFYFLQNN 24 1 IRFDDKSTLD ERKQTDNMAA FRSI FDQFVQ SCQNAYSPSE FLTIDEMLLS FRGRCLFRVY 301 I PNKPAKYGI KI LALVDAKN FYWNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR 361 NVTFDNWFTG YELMLHLLNE YRLT SVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL 42 1 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMIT FYN STKAGVDWD ELCANYNVSR 4 8 1 NSKRWPMTLF YGVLNMAAIN ACIIYRTNKN VTIKRTEFIR SLGLSMIYEH LHSRNKKKNI 54 1 PTYLRQRIEK QLGEPSPRHV NYPGRYVRCQ DCPYKKDRKT KRSCNACAKP I CMEHAKFLC 601 VNCAELDSSL (SEQ ID NO 14 607) .

In certain embodiments, the piggyBac or piggyBac-like transposase that is is integration deficient comprises a sequence of:

1 MDI ERQEERI RAMLEEELSD YSDESSSEDE TDHCSEHEVN YDTEEERIDS VDVPSNSRQE

61 EANAI IANES DSDPDDDLPL SLVRQRASAS RQVSGPFYTS KDGTKWYKNC QRPNVRLRSE

121 NIVTEQAQVK NIARDASTEY ECWNIFVTSD MLQEILTHTN SSIRHRQTKT AAENSSAETS

18 1 FYMQETTLCE LKALIALLYL AGLI KSNRQS LKDLWRKDGT GVDI FRTTMS LQRFQFLLNN

24 1 IRFDDKSTRD ERKQTDNMAA FRSI FDQFVQ CCQNAYS PSE FLTIDEMLLS FRGRCLFRVY

301 I PNKPAKYGI KI LALVDAKN DYWNLEVYA GKQPSGPYAV SNRPFEVVER LIQPVARSHR

361 NVTFDNWFTG YECMLHLLNE YRLT SVGTVR KNKRQIPESF IRTDRQPNSS VFGFQKDITL

42 1 VSYAPKKNKV VVVMSTMHHD NSIDESTGEK QKPEMIT FYN STKAGVDWD ELCANYNVSR

SUBSTITUTE SHEET (RULE 26) 481 NSKKWPMTLF YGVLNMAAIN AC11 YRTNKN VTIKRTEFIR SLGLSMIKEH LHSRNKKKNI 541 PTYLRORIEK QLGEPSPRHV NVFGRYVRCO DCPYKKDRKT KRSCNACAKP ICMEHAKFLC 601 ENCAELDSSL (SEQ ID O: 14608) ,

In certain embodiments, the integration deficient transposase comprises a sequence that is at least 90% identical to SEQ ID NO: 14608

[Q627] In certain embodiments, the piggyBae or piggyBac-like transposon is isolated or derived from Bomhyx mori. In certain embodiments, the piggyBae or piggyBac-like transposon comprises a sequence of:

1 ttatcccggc gagc tgagg cagggtat ct ca accctgg taaaatttta aagttgtgta

61 ttttataaaa ttttcgtctg acaacactag cgcgc cagt agctggaggc agqaq cq tgc

121 gggaggggat agtggcgtga tcgcagtgtg gcacgggaca ccggcgagat attcgtgtgc

181 aaacctgttt cgggtatgtt a accctgcc tcattgttga cg " a " " t t t L LLatgtaa t t

241 tttccgatta tt atttcaa ctgttttatt ggtattt tta tgttatccat tgttcttttt

301 ttatgattta ctgtatcggt tgtctttcgt tcc agtt gagttttttt t ta L La " "

361 cagtttttga tcaaa (SEQ ID NO: 14506) .

In certain embodiments, the piggyBae or piggyBac-like transposon comprises a sequence of:

1 tcatattttt agtttaaaaa aataatta a fcqfctttataa tgaaaagaat ctcatfcafcct

61 ttcagtatta ggttgattta tattccaaag aataatattt ttgttaaatt gttgattttt

121 gtaaacctct aaatgtttg tgctaaaatt actgtgttta agaaaaagat taataaataa

181 taataattt c at ttaaaa aett ettt ca ttgaatgcca ttaaataaac cattattt t a

241 caaaataaga tcaacataat tgagtaaata ataataagaa caatattata gtacaacaaa

301 atatgggtat gtcataccct gccacattct tgatgtaact ttttttcacc tcatgctcgc

361 cgggttat (SEQ ID NO: 14507) .

In certain embodiments, the piggyBae or piggy Bac-like transposon comprises a sequence of:

1 ttatcccggc gagcatgagg cagggtat ct cataccctgg taaaatttta aagttgtgta

61 ttttataaaa ttttcgtctq acaacactag cgcgctcagt agctqqaggc aggagcgtgc

121 gggaqqggat agtggcgtga tcgcagtgtq gcacgggaca ccggcgagat attcgtgtgc

181 aaacctgttt cgggtatgtt ataccctgcc teat (SEQ ID NO: 14508) ,

In certain embodiments, the piggyBae™ (PB) or piggyBac-like transposon comprises a sequence of:

1 taaataataa taaCttcata attaaaaact tctttcattg aatgccatta aataaaccat 61 tattttacaa aataagatca acataattga gtaaataata ataagaacaa tattatagta 121 caacaaaata tgggtatgtc ataccctgcc acattcttga tgtaactttt tttcacctca 181 tgctcgcccig gttat (SEQ ID NO: 14509) .

[0628] In certain embodiments, the piggyBae or piggyBac-like transposon comprises a 5’ sequence corresponding to SEQ ID NO: 14506 and a 3’ sequence corresponding to SEQ ID NO: 14507. In certain embodiments, one piggyBae or piggyBac-like transposon end is at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical or any percentage in between identical to SEQ ID NO: 14506 and the other piggyBae or piggyBac-like transposon end is at

SUBSTITUTE SHEET (RULE 26) least 85%, at least 90%, at least 95%, at least 98%, at least 99% or any percentage in between identical to SEQ ID NO: 14507. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14506 and SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14508 and SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the 5’ and 3’ transposon ends share a 16 bp repeat sequence at their ends of CCCGGCGAGCATGAGG (SEQ ID NO: 14510) immediately adjacent to the 5'-TTAT-3 target insertion site, which is inverted in the orientation m the two ends. In certain embodiments, 5’ transposon end begins with a sequence comprising 5'-TTATCCCGGCGAGCATGAGG-3 (SEQ ID NO: 14511), and the 3’ transposon ends with a sequence comprising the reverse complement of this sequence: 5'- CCTCATGCTCGCCGGGTTAT-3' (SEQ ID NO: 14512)

[0629] In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides of SEQ ID NO: 14506 or SEQ ID NO: 14508. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides of SEQ ID NO: 14507 or SEQ ID NO: 14509. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14506 or SEQ ID NO: 14508. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end with at least 90% identity to SEQ ID NO: 14507 or SEQ ID NO: 14509

[0630] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaacccggc gagcatgagg cagggtat ct cataccctgg taaaatttta aagttgtgt a 61 ttttataaaa ttttcgtctg acaacactag cgcgctcagt agctggaggc aggagcgtgc

121 qqqaggggat agtggcgtga tcgcagtgtq gcacgggaca ccggcgagat attcqtgtgc

181 aaacctgttt cgggtatgtt ataccctgcc tcattgttga cgtatttttt ttatgtaatt

241 tttccgatta ttaatttcaa ctgttttatt ggtatttbba tgttatccat tgttcttttt

301 ttabgattta ctgtatcggt tgtctttcgt tcctttagtt gagttttttt ttattatttt

361 cagtttttga tcaaa ( SEQ ID NO: 14515 ) .

0631] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

i tCcito t C agtttaaaaa aataattata c| ata a tgaaaagaat ctcattatct

61 ttcagtatta ggttgattta ua tt c.c.a a ag aataatattt L Lg 0 a a a 01- gttgattttt

121 gtaaacctct aaatgtttg t tgctaaaatt actgtgttta agaaaaagat taataaataa

181 taataatttc a taattaaaa acttctttca 0 g a a 0 c.c.a ttaaataatt cattatttta

241 caaaataaga tcaacataat tgagtaaata ataataagaa caatattata gtacaacaaa

301 atatgggtat gtcatacccfc tttttttttt tttttttttt btttttcggg tagagggccg

SUBSTITUTE SHEET (RULE 26) 361 aacctcctac gagqtccccg cgcaaaaggg gcgcgcgggg t tg tgaga c tcaacgatct 421 gcatggtgtt gtgagcagac cgcgggccca aggattttag agcccaccca ctaaacgact 481 cctctqcact cttacacccg acgtccga tc ccctccgagg tcagaacccg ga tqaqq ag 541 gggggetacc gcggtcaaca ctacaaccag acqgcgcggc tcaccccaag ga.cgcccagc 601 cgacggagcc ttcgaqqcqa a tcgaaggct ctgaaacgtc qqccq ctcg gtacggcagc 661 ccgtcgggcc gcccagacgg tgccgctggt gtcccggaat accccgctgg accagaacca 721 gcc tqccgg g tcgggacgcg atacaccg tc gaccggtcgc tctaatcact ccacqqcagc 781 gcgctagagt gctggta (SEQ ID NO: 14516; .

[0632] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of CCCGGCGAGCATGAGG (SEQ ID NO: 14510). In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of SEQ ID NO: 14510. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of TTATCCCGGCGAGCATGAGG (SEQ ID NO: 14511). In certain embodiments, the piggyBac or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14511. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of CCTCATGCTCGCCGGGTTAT (SEQ ID NO: 14512). In certain embodiments, the piggyBac or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14512. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising at least 16 contiguous nucleotides from SEQ ID NO: 14511 and one end comprising at least 16 contiguous nucleotides from SEQ ID NO: 14512. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14511 and SEQ ID NO:

14512. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of TTAACCCGGCGAGCATGAGG (SEQ ID NO: 14513). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of

CCT CAT GCT CGCCGGGTT A A (SEQ ID NO: 14514).

[0633] In certain embodiments, the piggyBac or piggyBac-like transposon may have ends comprising SEQ ID NO: 14506 and SEQ ID NO: 14507, or a variant of either or both of these having at least 90% sequence identity to SEQ ID NO: 14506 or SEQ ID NO: 14507, and the piggyBac or piggyBac-like transposase has the sequence of SEQ ID NO: 14504 or SEQ ID NO: 14505, or a sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,

60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage m between identity to SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a heterologous polynucleotide inserted between a pair of inverted repeats, where the transposon is capable of transposition by a piggyBac or piggyBac-like transposase

SUBSTITUTE SHEET (RULE 26) having at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identity to SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the transposon comprises two transposon ends, each of which comprises SEQ ID NO: 14510 in inverted orientations in the two transposon ends. In certain embodiments, each inverted terminal repeat (ITR) is at least 90% identical to SEQ ID NO: 14510.

[0634] In certain embodiments, the piggyBac or piggyBae-iike transposon is capable of insertion by a piggyBac or piggyBac-like transposase at the sequence 5'-TTAT-3 within a target nucleic acid. In certain embodiments, one end of the piggyBac or piggyBac-like transposon comprises at least 16 contiguous nucleotides from SEQ ID NO: 14506 and the other transposon end comprises at least 16 contiguous nucleotides from SEQ ID NO: 14507. In certain embodiments, one end of the piggyBac or piggyBac-like transposon comprises at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14506 and the other transposon end comprises at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14507.

[0635] In certain embodiments, the piggyBac or piggyBac-like transposon comprises transposon ends (each end comprising an ITR) corresponding to SEQ ID NO: 14506 and SEQ ID NO: 14507, and has a target sequence corresponding to 5'-TTAT3'. In certain embodiments, the piggyBac or piggyBac-like transposon also comprises a sequence encoding a transposase (e.g. SEQ ID NO: 14505). In certain embodiments, the piggyBac or piggyBac-like transposon comprises one transposon end corresponding to SEQ ID NO: 14506 and a second transposon end corresponding to SEQ ID NO: 14516 SEQ ID NO: 14516 is very similar to SEQ ID NO: 14507, but has a large insertion shortly before the ITR. Although the ITR sequences for the two transposon ends are identical (they are both identical to SEQ ID NO: 14510), they have different target sequences: the second transposon has a target sequence corresponding to 5'-TTAA-3', providing evidence that no change in ITR sequence is necessary to modify the target sequence specificity. The piggyBac or piggyBac-like transposase (SEQ ID NO: 14504), which is associated with the 5'-TTAA-3’ target she differs from the 5'-TTAT-3'-associated transposase (SEQ ID NO: 14505) by only 4 ammo acid changes (D322Y, S473C, A507T, H582R). In certain embodiments, the piggyBac or piggyBac-like transposase (SEQ ID NO: 14504), which is associated with the 5'-TTAA-3’ target site is less active than the 5'-TTAT-3'-associated

SUBSTITUTE SHEET (RULE 26) piggyBac or piggyBac-like transposase (SEQ ID NO: 14505) on the transposon with 5'-TTAT-3' ends. In certain embodiments, piggyBac or piggyBac-like transposons with 5'-TTAA-3’ target sites can be converted to piggyBac or piggyBac-like transposases with 5'-TTAT-3 target sites by replacing 5'-TTAA-3’ target sites with 5'-TTAT-3'. Such transposons can be used either with a piggyBac or piggyBac-like transposase such as SEQ ID NO: 14504 which recognizes the 5 ^! - TTAT-3’ target sequence, or with a variant of a transposase originally associated with the 5'- TTAA-3' transposon. In certain embodiments, the high similarity between the 5'-TTAA-3' and 5'-TTAT-3' piggyBac or piggyBac-like transposases demonstrates that very few changes to the ammo acid sequence of a piggyBac or piggyBac-like transposase alter target sequence specificity'. In certain embodiments, modification of any piggyBac or piggyBac-like transposon- transposase gene transfer system, in which 5'-TTAA-3’ target sequences are replaced with 5'-

TTAT-3'-target sequences, the ITRs remain the same, and the transposase is the original piggyBac or piggyBac-like transposase or a variant thereof resulting from using a low-level mutagenesis to introduce mutations into the transposase. In certain embodiments, piggyBac or piggyBae-!ike transposon transposase transfer systems can be formed by the modification of a 5'- TTAT~3'~active piggyBac or piggyBac-like transposon-transposase gene transfer systems in which 5'-TTAT-3’ target sequences are replaced with 5'-TTAA-3'-target sequences, the ITRs remain the same, and the piggyBac or piggyBac-like transposase is the original transposase or a variant thereof.

[0636] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Bombyx rnori. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 cccqqcqaqc atgaggcagg gtatctcata ccctggtaaa attttaaagt tqtqtatttt

61 ataaaatttt cgtctgacaa cactagcgcg ctcagtagct ggaggcagga gcgtgcggga

121 ggggatagtg gcqtqatcqc agtgtggcac gggacaccqq cqaqatattc gtgtgcaaac

181 ctgtttcggg tatgttatac cctgcctcat tgttgacgta t ( SEQ ID NO: 14577) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tttaagaaaa agattaataa ataataataa tttcataatt aaaaacttct ttcattgaat

61 qccattaaat aaaccattat tttacaaaat aagatcaaca taattgagta aataataata

121 agaacaatat tatagtacaa caaaatatgg gtatgtcata ccctgccaca ttcttgatgt

181 aacttttttt cacctcatgc tcgccggg (SEQ ID NO: 14578) .

In certain embodiments, the transposon comprises at least 16 contiguous bases from SEQ ID

NO: 14577 and at least 16 contiguous bases from SEQ ID NO: 14578, and inverted terminal repeats that are at least 87% identical to CCCGGCGAGCATGAGG (SEQ ID NO: 14510).

SUBSTITUTE SHEET (RULE 26) In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 cccqqcqaqc atgaggcagg gtatctcata ccctggtaaa attttaaagt tqtqtatttt

61 ataaaatttt cgtctgacaa cact agc.geg ctcagtagct ggaggcagga gcgtgcggga

121 ggggatagtg gcqtqa cqc agtgtggcac gggacaccqq cqaqa attc gtgtgcaaac

181 ctgtttcggg tatgttatac cctgcctcat tgttgacgta ttttttttat gtaattttt c

2 Ί 1 cqa 11a a a 111ca actgt 111a 11qq ta 11a tg 11 at cca11g 11 c 1111 a t

301 gatttactgt atcggttgtc ttt cgttcct ttagttgagt ttttttttat tattttcagt

361 ttttgatcaa a (SEQ ID NO: 14595) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tc.atattttt agtttaaaaa aataattata tgttttataa tgaaaagaat c.t cattat ct

61 ttcagtatta ggttqa a tattccaaag aataatattt q aaatt gttgattttt

121 qtaaacct ct aaatgtttgt tgctaaaatt actgtgttta agaaaaagat taataaataa

181 taataatttc ataattaaaa aettetttea ttqaatgcca ttaaataaac cattatttta

241 caaaataaga tcaacataat t gagtaaata ataataagaa caatattata gtacaacaaa

301 atatgggtat gtcataccc qccacattct tgatgtaact tttcacc tcatgctcgc

361 eggg (SEQ ID NO: 14596) .

[0637] In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596, and is transposed by the piggyBac or piggyBac-like transposase of SEQ ID NO: 14505. In certain embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID: 14596 are not flanked by a 5’-TTAA-3’ sequence. In certain embodiments, the ITRs of SEQ ID NO: 14595 and SEQ ID: 14596 are flanked by a 5’-TTAT-3’ sequence.

[0638] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 cccggcgagc atqaqqcaqq gtatctcata ccctggtaaa attttaaagt tgtgtatttt 61 ataaaatttt cgtctgacaa cactagcgcg ctcagtagct ggaggcagga gcgtgcggga

121 ggggatagtg gcqtqatcqc agtgtggcac gggacaccqq cqaqatattc gtgtgcaaac

181 ctgtttcggg tatgttatac cctgcctcat tgttgacgta ttttttttat gtaatttttc

241 egattattaa tttc actgt tttattggta tttttatgtt atccattgtt ctttttttat

301 g (SEQ ID NO: 14597) .

In certain embodiments, the piggyBac or piggyBac-Iike transposon comprises a sequence of:

1 caqqqta tct ca taccctgg taaaattt ta: aaq 11q tgta 1111ataaaa tt t tcq tctq

61 acaacactag cgcgctcagt agctggaggc aggagcgtgc gggaggggat agtggcgtga

121 tcgcagtgtg gcacqqqaca ccggcgagat attcgtgtqc aaacctgttt cgggtatgtt

181 ataccctgcc tcattgttga cgtatttttt ttatgtaatt tttccgatta ttaatttcaa

241 ctgttttatt ggtattttta tgttatccat tgttcttttt ttatg ( SEQ ID NO:

14598) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 caqqq atet ca taccctgg taaaattt a aaq ttgtgta ttttataaaa tt t eq etg 61 a ca aca ctag cgcgctcagt agct ggaggc aggagcgtgc gggaggggat agtggcgtga 121 tcgcagtgtg gcacqqqaca ccggcgagat attcgtgtqc aaacctgttt cgggt tgtt 181 ataccctgcc t cattgttga cgtat (SEQ ID NO: 14599) .

SUBSTITUTE SHEET (RULE 26) In certain embodiments, the 5’ end of the piggyBac or piggy Bac-like transposon comprises a sequence of SEQ ID NO: 14577, SEQ ID NO: 14595, or SEQ ID NOs: 14597-14599. In certain embodiments, the 5’ end of the piggyBac or piggyBac-like transposon is preceded by a 5’ target sequence.

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 bcababbbbb aqtttaaaaa aataattata bqbbbbabaa tgaaaagaat cbcabbabcb 61 ttcagtatta ggttqattta tattccaaag aataatattt ttgttaaatt gttgattttt

121 gtaaacctct aaatg gt tgctaaaatt actgtgttta aqaaaaagat taataaataa

181 taataatttc at ttaaaa acttctttca ttgaatgcca ttaaataaac cattatttta

241 caaaataaga tcaacataat tgagtaaata ataataagaa caatattata gtacaaca.aa

301 atatgggtat gtcataccct gccacattct tgatgtaact ttttttcacc. tcatgctcgc

361 cggg (SEQ ID NO: 14600) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tttaagaaaa agattaataa ataataataa tttcataatt aaaaacttct ttcattgaat

61 gccattaaat aaaccattat tttacaaaat aagatcaaca baabbqagba aataataata

121 aqaacaatat tatagtacaa caaaatatgg gtatgtcata ccctgccaca ttcttgatgt

181 aacttttttt ca (SEQ ID NO: 14601) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 cccggcgagc at gaggcagg gtatctcata ccctggtaaa attttaaagt tgtgtatttt

61 ataaaabbbb cgtctgacaa cactagcgcg ctcagtagct ggaggcagga gcgtgcggga

121 ggggatagtg gcqtqatcgc agtgtggcac gggacaccgg cqagatattc gtgtgcaaac

181 ctgtttcggg tatgttatac cctgcctcat tgttgacgta ttttttttat gtaatttttc

241 cgattattaa tttcaactgt tttattggta tttttatgtt atccattgtt ctttttttat

301 gatttactgt atcggttgtc tttcgttcct ttagttgagt ttttttttat tattttcagt

361 ttttgatcaa a (SEQ ID NO: 14602),

[0639] In certain embodiments, the 3’ end of the piggyBac or piggyBac-like transposon comprises a sequence of SEQ ID NO: 14578, SEQ ID NO: 14596, or SEQ ID NOs: 14600- 14601. In certain embodiments, the 3’ end of the piggyBac or piggyBac-like transposon is followed by a 3’ target sequence. In certain embodiments, the transposon is transposed by the transposase of SEQ ID NO: 14505. In certain embodiments, the 5’ and 3’ ends of the piggyBac or piggyBac-like transposon share a 16 bp repeat sequence of SEQ ID NO: 14510 in inverted orientation and immediately adjacent to the target sequence. In certain embodiments, the 5’ transposon end begins with SEQ ID NO: 14510, and the 3’ transposon end ends with the reverse complement of SEQ ID NO: 14510, 5’- CCTCATGCTCGCCGGG-3’ (SEQ ID NO: 14603). In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR with at least 93%, at least 87%, or at least 81% or any percentage in between identity to SEQ ID NO: 14510 or SEQ ID NO: 14603. In certain embodiments, the piggyBac or piggyBac-like transposon

SUBSTITUTE SHEET (RULE 26) comprises a target sequence followed by a 5’ transposon end comprising a sequence selected from SEQ ID NOs: 14577, 14595 or 14597 and a 3’ transposon end comprising SEQ ID NO: 14578 or 14596 followed by a target sequence in certain embodiments, the piggyBac or piggyBac like transposon comprises one end that comprises a sequence that is at least 90%, at least 95% or at least 99% or any percentage in between identical to SEQ ID NO: 14577 and one end that comprises a sequence that is at least 90%, at least 95% or at least 99% or any percentage in between identical to SEQ ID NO: 14578. In certain embodiments, one transposon end comprises at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14577 and one transposon end comprises at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14578.

[0640] In certain embodiments, the piggyBac or piggyBac-like transposon comprises two transposon ends wherein each transposon ends comprises a sequence that is at least 81% identical, at least 87% identical or at least 93% identical or any percentage in between identical to SEQ ID NO: 14510 in inverted orientation in the two transposon ends. One end may further comprise at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14599, and the other end may further comprise at least 14, at least 16, at least 18 or at least 20 contiguous bases from SEQ ID NO: 14601. The piggyBac or piggyBac-like transposon may be transposed by the transposase of SEQ ID NO: 14505, and the transposase may optionally be fused to a nuclear localization signal

[0641] In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14596 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac- like transposon comprises SEQ ID NO: 14597 and SEQ ID NO: 14596 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14595 and SEQ ID NO: 14578 and the piggyBac or piggyBac-like transposase composes SEQ ID NO: 14504 or SEQ ID NO: 14505. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14602 and SEQ ID NO: 14600 and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14504 or SEQ ID NO: 14505.

[0642] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 5’ end comprising 1, 2, 3, 4, 5, 6, or 7 sequences selected from ATGAGGCAGGGTAT (SEQ ID NO:

SUBSTITUTE SHEET (RULE 26) 14614), ATACCCTGCCTCAT (SEQ ID NO: 14615), GGC AGGGT AT (SEQ ID NO: 14616), ATACCCTGCC (SEQ ID NO: 14617), TAAAATTTTA (SEQ ID NO: 14618),

ATTTTATAAAAT (SEQ ID NO: 14619), TCATACCCTG (SEQ ID NO: 14620) and

TAAATAATAATAA (SEQ ID NO: 14621). In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 3’ end comprising 1 , 2 or 3 sequences selected from SEQ ID NO: 14617, SEQ ID NO: 14620 and SEQ ID NO: 14621.

[0643] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-iike transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Xenopus tropica!is. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,

90%, 95%, 99% or any percentage in between identical to:

1 MAKRFYSAEE AAAHCMASSS EXFS GSDS EY VPPASES DS S TEESWCS S ST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDT S NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLI D 241 SLSERFAAVY TPCQNICIDE S LLLFKGRLO FRQYIPS KRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPBLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKN ELLAI KF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGI Y LI QMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (3EQ ID NO:

14517 } .

[0644] In some embodiments, the piggyBac or piggyBac-like transposase is a hyperactive variant of SEQ ID NO: 14517. In certain embodiments, the piggyBac or piggyBac-like transposase is an integration defective variant of SEQ ID NO: 14517. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an ammo acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,

90%, 95%, 99% or any percentage in between identical to:

i MAKRFYSAEE AAAHCMAPS S EEFSGSDSEY VPPASES DS S TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDT S NFEPINFFQL

121 FMTEAILQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN

181 SLESYWNTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQPDHD RLHKLRPLID

241 SLSERFAAVY TPCQNICIDE SLLLFKGRLR FRQYI PS KRA RYGIKFYKLC ESSSGYTSYF

301 LIYEGKDSKL DP PGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT

361 PACGTINR R KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE

421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT SAWYKKVGIY LIQMALRNSY

481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMLP SDNVARLIGK HFIDTLPPTP

541 GKQRPQKGCK VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO:

14518 } .

SUBSTITUTE SHEET (RULE 26) [0645] in certain embodiments, the piggyBac or piggyBac-like transposase is isolated or derived from Xenopus tropiealis. In certain embodiments, the piggyBac or piggyBac-like transposase is a hyperactive piggyBac or piggyBac-like transposase. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence at least 90% identical to:

1 MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDS S TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDT S NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRG ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFANVY TPCONI Cl BE SLMLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSTGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRP PKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPD SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF El YHTQLHY (SEQ ID NO: 14572 ) .

0646] In certain embodiments, piggyBac or piggyBac-like transposase is a hyperactive piggyBac or piggyBac-iike transposase. A hyperactive piggy Bac or piggyBac-Jike transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In certain embodiments, a hyperactive piggyBac or piggyBac-like transposase is more active than the transposase of SEQ ID NO: 14517. In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

i MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRG ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFANVY TPCQNICIDE SLMLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSTGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRP PKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPD SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF ElYHTQLHY (SEQ ID NO:

14572).

In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

i MAKRFYSAEE AAAHCMAS S S EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDT S NFEPINFFQL 121 FMTEAILQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTS YF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRP PKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP

SUBSTITUTE SHEET (RULE 26) 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14624 ) .

[0648] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

1 MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TFESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAI LQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLKIPVFSAT MSRNRYQLLL RFLHFNNNAT AVPPDQFGHD RLHKLPPLID 241 SLS ERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC E3SSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRP PKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGI Y LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY SEQ ID NO: 14625) .

0649] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

1 MAKRFYSAEE AAAHCMASSS EQTSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVD S NFEPINFFQL 121 FMTEAI LQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SIESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQFGHD RLHKLPPLID 241 SLSERFANVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRKPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGI Y LIQMALR SY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

1 MAKRFYSAEE AAAHCSASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAI LQDM VLYTNVYAEQ YLTQNPLTRG ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQFGHD RLHKLP.PLID 241 SLSERFANVY TPCQNICIDE SLMLFKGELQ FRQYIPSKRA RYGIKFYKLC ESSTGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRP PKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGI Y LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO:

14628 ) .

[0651] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises a sequence of:

i MAKRFYSAEE AAAHCMASSS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAI LQDM VLYTNVYAEQ YLTQNPLTRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFGAT MSRNRYQLLL RFLHFNNNAT AVPPDQFGHD RLHKLP.PLID 241 SLSERFANVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLNT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE

SUBSTITUTE SHEET (RULE 26) 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RHWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ ILPALLFGGV EEQTVPEMPP SDNVARLIGK HF1DTLPPTP 541 GKQRPQKGCK VCRKRGIRRD TRYYCPKCPR NPGLCRKPCF EIYHTQLHY (SEQ ID NO: 14664 } .

[0652] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises an ammo acid substitution at a position selected from ammo acid 6, 7, 16, 19, 20, 21, 22, 23, 24, 26, 28, 31, 34, 67, 73, 76, 77, 88, 91, 141, 145, 146, 148, 150, 157, 162, 179, 182,

189, 192, 193, 196, 198, 200, 210, 212, 218, 248, 263, 270, 294, 297, 308, 310, 333, 336, 354, 357, 358, 359, 377, 423, 426, 428, 438, 447, 450, 462, 469, 472, 498, 502, 517, 520, 523, 533, 534, 576, 577, 582, 583 or 587 (relative to SEQ ID NO: 14517). In certain embodiments, the hyperactive piggyBac or piggyBac-hke transposase comprises an amino acid substitution of Y6C, SVG, M16S, S19G, S20Q, S20G, S20D, E21D, E22Q, F23T, F23P, S24Y, S26V, S28Q, V31K, A34E, L67A, 073! E A76V, D77N, P88A, N91D, Y141Q, Y141A, N145E, N145V,

P146T, PI 46V, PI46K, PI48T, P148H, Y150G, Y150S, Y150C, H157Y, A162C, A179K,

LI 821, 1,182V, T189G, 1.1921 1. S193N, S193K, VI 961, S198G, T200W, L210H, F212N,

N218E, A248N, L263M, Q270L, S294T, T297M, S308R, L310R, L333M, Q336M, A354H, C357V, L358F, D359N, L377I, V 423H, P426K, K428R, S438A, T447G, T447A, L450V, A462H, A462Q, I469V, I472L, Q498M, L502V, E5171 , P520D, P520G, N523S, I533E, D534A, F576R, F576E, K577Ϊ, I582R, Y583F, L587Y or L587W, or any combination thereof including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of these mutations (relative to SEQ ID NO: 14517).

[0653] In certain embodiments, the hyperactive piggyBac or piggyBac-like transposase comprises one or more substitutions of an ammo acid that is not wild type, wherein the one or more substitutions a for wild type amino acid comprises a substitution of A2X, K3X, R4X, F5X, Y6X, S7X, A1 1X, A13X, C15X, M16X, A17X, S18X, S 19X, S20X, E21 X, E22X, F23X, S24X, G25X, 26X, D27X, S28X, E29X, E42X, E43X, S44X, C46X, S47X, S48X, S49X, T5QX, V51X, S52X, A53X, L54X, E55X, E56X, P57X, M58X, E59X, E62X, D63X, V64X, D65X, D66X, L67X, E68X, D69X, Q70X, E71X, A72X, G73X, D74X, R75X, A76X, D77X, A78X, A79X,

A 8 OX, G81X, G82X, E83X, P84X, A85X, W86X, G87X, P88X, P89X, C90X, N91X, F92X, P93X, E95X, I96X, P97X, P98X, F99X, T100X, T101X, P103X, G104X, V105X, K106X, V107X, D108X, T109X, Ni l IX, P114X, I1 15X, N1 16X, F117X, F118X, Q119X, M122X, T123X, E124X, A125X, I126X, L127X, Q128X, D129X, M130X, L132X, Y133X, V126X, Y127X, A138X, E139X, Q140X, Y141X, L142X, Q144X, N145X, P146X, L147X, P148X, Y150X, A151X, A155X, H157X, P158X , I161X, A162X, V168X, T171X, L172X, A173X,

SUBSTITUTE SHEET (RULE 26) M174X, I177X, A179X, L482X, D187X, T188X, T189X, T190X, L192X, S193X, I194X, P195X, V196X, S198X, A199X, T200X, S202X, L208X, L209X, L210X, R211X, F212X, F215X, N217X, N218X, A219X, T220X, A221X, V222X, P224X, D225X, Q226X, P227X, H229X, R231X, B233X, L235X, P237X, I239X, D240X, L242X, S243X, E244X, R244X, F246X, A247X, A248X, V249X, Y250X, T251X, P252X, C253X, Q254X, I256X, C257X,

I258X, D259X, E260X, S261X, L262X, L263X, L264X, F265X, K266X, G267X, R268X, L269X, Q270X, F271X, R272X, Q273X, Y274X, I275X, P276X, S277X, K278X, R279X, A280X, R281X, Y282X, G283X, I284X, K285X. F286X. Y287X. K288X, L289X, C290X,

E291X, S292X, S293XS294X, G295X, Y296X, T297X, S298X, Y299X, F300X, E304X,

L310X, P3I3X, G3I4X, P3I6X, P317X, D318X, L319X, T320X, V321X, K324X, E328X, 133 OX, S331X, P332X, L333X, L334X, G335X, Q336X, F338X, L340X, D343X, N344X, F345X, Y346X, S347X, L35IX, F352X, A354X, L355X, Y356X, C357X, L358X, D359X, T360X, R422X, Y423X, G424X, P426X, K428X, N429X, K430X, P431X, L432X, S434X, K435X, E436X, S438X, K439X, Y440X, G443X, R446X, T447X, L450X, Q451X, N455X, T460X, R46IX, A462X, K465X, V467X, G468X, I469X, Y470X, L471X, I472X, M474X, A475X, L476X, R477X, S479X, Y480X, V482XY483X, K484X, A485X, A486X, V487X, P488X, P490X, K491X, S493X, Y494X, Y495X, K496X, Y497T, Q498X, L499X, Q500X, 1501 X, L502X, P503X, A504X, L505X, L506X, F507X, G508X, G509X, V510X, E511X,

E512X, Q513X, T514X, V515X, E517X, M518X, P519X, P520X, S521X, D522X, N523X,

V524X, A525X, L527X, I528X, K53GX, H531 X, F532X, I533X, D534X, T535X, L536X, T539X, P540X,Q546X, K550X, R553X, K554X, R555X, G556X, Ϊ557C, R558X, R559X, D560X, T561X, Y564X, P566X, K567X, P569X, R570X, N571 X, L574X, C575X, F576X, K577X, P578X, F580X, E581 X, I582X, Y583X, T585X, Q586X, L587X, H588X or Y589X (relative to SEQ ID NO: 14517). A list of hyperactive amino acid substitutions can be found in US patent No. 10,041,077, the contents of which are incorporated by reference in their entirety.

[0654] in certain embodiments, the piggyBac or piggyBac-like transposase is integration deficient. In certain embodiments, an integration deficient piggyBac or piggyBac- like transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding naturally occurring transposase. In certain embodiments, the piggyBac or piggyBac-like transposase is an integration deficient

SUBSTITUTE SHEET (RULE 26) variant of SEQ ID NO: 14517. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase is deficient relative to SEQ ID NO: 14517.

[0655] In certain embodiments, the piggyBac or piggyBac-like transposase is active for excision but deficient m integration. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of

1 MAKRFYSAEE AAAHCMAS S S EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRVDAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL

121 FMTEAI LQDM VLYTNVYAEQ YLTQNPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN

181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL KFLHFNNEAT AVPPDQPGHD RLHKLRPLID

241 SLS ERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF

301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT

361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE

421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LI QMALRN S Y

481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP

541 GKQRPQKGCK. VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID

14605) ,

[0656] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

i MAKRFYSAEE AAAHCMAS SS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV

61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL

121 FMTEAI LQDM VLYTNVYAEQ YLTQVPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN

181 SLESYWDTTT VLNIPVFSAT MSRNRYQLLL RFLEFNNEAT AVPPDQPGHD RLHKLRPLID

241 SLSERFAAVY TPCQNICIDE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT

361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVI RE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LI QMALRN SY

481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHY (SEQ ID NO:

14604 } .

[0657] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

1 MAKRFYSAEE AAAHCMAS SS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGP PC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAI LQDM VLYTNVYAEQ YLTQNVLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNDAT AVPPDQPGHD RLHKLRPLID 241 SLTERFAAVY TPCQNI CI DE SLLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LIYEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRPPKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF EIYHTQLHYG RR (SEQ ID

NO : 14611) .

In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 1461 1 . In certain embodiments, the integration deficient

SUBSTITUTE SHEET (RULE 26) piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

1 MAIKRFYSAEE AAAHCMAS S S EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAP GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAI LQDM VLYTNVYAEQ YLTQVFLFRY ARAHAWHPTD IAEMKRFVGL TLAMGLIKAN 181 SLESYWDTTT VLSIPVFSAT MSRNRYQLLL RFLHFNNEAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE S LLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LI YEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACG INRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVI RE 421 QRVGRP PKN K PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RAWYKKVGIY LIQMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLIGK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF El YHTQLHYG RR (SEQ ID 14612} .

[0659] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14612. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises a sequence that is at least 90% identical to a sequence of:

1 MAKRFY SA.EE AAAHCMAS SS EEFSGSDSEY VPPASESDSS TEESWCSSST VSALEEPMEV 61 DEDVDDLEDQ EAGDRADAAA GGEPAWGPPC NFPPEIPPFT TVPGVKVDTS NFEPINFFQL 121 FMTEAI LQDM VLYTNVYAEQ YLTQVPLPRY ARAHAWHPTD IAEMKRFVGL TLAMGLI KAN 181 SLESYWDTTT VLNIPVFSAT MSRNRYQLLL RFLEFNNNAT AVPPDQPGHD RLHKLRPLID 241 SLSERFAAVY TPCQNICIDE S LLLFKGRLQ FRQYIPSKRA RYGIKFYKLC ESSSGYTSYF 301 LI YEGKDSKL DPPGCPPDLT VSGKIVWELI SPLLGQGFHL YVDNFYSSIP LFTALYCLDT 361 PACGTINRNR KGLPRALLDK KLNRGETYAL RKNELLAIKF FDKKNVFMLT SIHDESVIRE 421 QRVGRP PKNK PLCSKEYSKY MGGVDRTDQL QHYYNATRKT RA YKKVGI Y LI QMALRNSY 481 IVYKAAVPGP KLSYYKYQLQ I LPALLFGGV EEQTVPEMPP SDNVARLI GK HFIDTLPPTP 541 GKQRPQKGCK VCRKRGI RRD TRYYCPKCPR NPGLCFKPCF El YHTQLHYG RR (SEQ ID

4613) .

[Q66Q] In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14613. In certain embodiments, the integration deficient piggyBac or piggyBac-like transposase comprises an amino acid substitution wherein the Asn at position 218 is replaced by a Glu or an Asp (N218D or N218E) (relative to SEQ ID NO: 14517).

[0661] In certain embodiments, the excision competent, integration deficient piggyBac or piggyBac-like transposase comprises one or more substitutions of an amino acid that is not wild type, wherein the one or more substitutions a for wild type ammo acid comprises a substitution of A2X, K3X, R4X, F5X, Y6X, S7X, ASX, E9X, El OX, A11X, A12X, A13X, H14X, C15X, M16X, A17X, S1 8X, S 19X, S20X, E21X, E22X, F23X, S24X, G25X, 26X, D27X, S28X, E29X, V31X, P32X, P33X, A34X, S35X, E36X, S37X, D38X, S39X, S40X, T41X, E42X, E43X, S44X, W45X, C46X, S47X, S48X, S49X, T50X, V51X, S52X, A53X, L54X, E55X, E56X, P57X, M58X, E59X, V6QX, M122X, T123X, E124X, A125X, L127X, Q128X, D129X,

SUBSTITUTE SHEET (RULE 26) L132X, Y133X, V126X, Y127X, E139X, Q140X, Y141X, L142X, T143X, Q144X, N145X, P146X, L147X, P148X, R149X, Y150X, A151X, H154X, H157X, P158X, T159X, D160X, I161X, A162X, E163X, M164X, K165X, R166X, F167X, V168X, G169X, L170X, T171X, L172X, A173X, M174X, G175X, L176X, I177X, K178X, A179X, N180X, S181X, L182X, S184X, Y185X, D187X, T188X, T189X, T190X, V191X, L192X, S193X, I194X, P195X, V196X, F197X, S198X, A199X, T200X, M201X, S202X, R203X, N204X, R2Q5X, Y206X, Q207X, L208X, L209X, L210X, R21 IX, F212X, L213X, i 1241 X. F215X, N216X, N217X, N218X, A219X, T220X, A221X, \ 222 X. P223X, P224X, D225X, Q226X, P227X, G228X, H229X, D230X, R231X, H233X, K234X, L235X, R236X, L238X, I239X, D240X, L242X, S243X, E244X, R244X, F246X, L247C. A248X, V249X, Y250X, T251X, P252X, C253X, Q254X, N255X, I256X, C257X, I258X, D259X, E260X, S261X, L262X, L263X, L264X, F265X, K266X, G267X, R268X, L269X, Q270X, F271X, R272X, Q273X, Y274X, I275X, P276X, S277X, K278X, R279X, A280X, R281X, Y282X, G283X, I284X, K285X, F286X, Y287X, K288X, L289X, C290X, E291X, S292X, S293X, S294X, G295X, Y296X, T297X, S298X, Y299X, F300X, I302X, E304X, G305X,K306X, D307X, S308X, K309X, L310X, D3 I IX, P312X, P313X, G314X, C315X, P316X, P3I7X, D3I 8X, L319X, T320X, V321X, S322X, G323X, K324X, I325X, V326X, W327X, E328X, L329X, I330X, S331X, P332X, 1.333 X. L334X, G335X, Q336X, F338X, FI339X, L340X, V342X, N344X, F345X, Y346X, S347X, S348X, I349X, L351X, T353X, A354X, Y356X, C357X, L358X, D359X, T360X, P361X, A362X, C363X, G364X, I366X, N367X, R368X, D369X, K371 X, G372X, L373X, R375X, A376X, L377X, L378X, D379X, K380X, K381X, L382X, N383X, R384XG385X, T387X, Y388X, A389X, L390X, K392X, N393X, E394X, A397X, K399X, F400X, F401X, D402X, N405X, L406X, L409X, K422X. Y423X, G424X, E425X, P426X, K428X, N429X, K430X, P431X, L432X, S434X, K435X, E436X, S438X, K439X, Y440X, G442X, G443X, V444X, R446X, T447X, L450X, Q451X, H452X, X455X, T457X, R458X, T460X, R461X, A462X, Y464X, K465X, V467X, G468X, 1469X, L471X, 1472X, Q473X, M474X, L476X, R477X, N478X, S479X, Y480X, V482XY483X, K484X, A485X, A486X, V487X, P488X, G489X, P490X, K491X, L492X, S493X. Y494X, Y495X, K496X, Q498X, L499X, Q500X, I501X, L502X, P503X, A504X, L505X, L506X, F507X, G508X, G509X, V510X, E51 IX, E512X, Q513X, T514X, V515X, E517X, M518X, P519X, P520X, S521X, D522X, N523X, V524X, A525X, L527X, I528X. G529X, K530X, F532X I533X, D534X, T535X, L536X

SUBSTITUTE SHEET (RULE 26) P537X, P538X, T539X, P540X, G541X, F542X, Q543X, R544X. P545X, Q546X, K547X, G548X, C549X, K550X, V551X, C552X, R553X, K554X, R555X, G556X, I557X, R558X, R559X, D560X, T561X, R562X, Y563X, Y564X, C565X, P566X, K567X, C568X, P569X, R570X, N571X, P572X, G573X, L574X, C575X, F576X, K577X, P578X, C579X, F580X, E581X, I582X, Y583X, H584X, T585X, Q586X, L587X, H588X or Y589X (relative to SEQ ID

NO: 14517). A list of excision competent, integration deficient ammo acid substitutions can be found in US patent No. 10,041,077, the contents of which are incorporated by reference in their entirety.

[0662] In certain embodiments, the piggyBac or piggyBac-like transposase is fused to a nuclear localization signal. In certain embodiments, SEQ ID NO: 14517 or SEQ ID NO: 14518 is fused to a nuclear localization signal. In certain embodiments, the amino acid sequence of the piggyBac or piggyBac like transposase fused to a nuclear localization signal is encoded by a polynucleotide sequence comprising:

1 atggcaccca aaaagaaacg caaag tga tq gccaaaagat tttacagcgc egaagaagea 61 gcagcacatt gcatqqcatc g tca tccgaa gaattctcqq ggagcgattc cgaatatgtc 121 ccaccggcct eggaaagega ttcgagcact gaggagtegt ggtgttcctc ctcaactgtc 181 tcggctcttg aggageegat ggaagtgga t gag ga tgtgg acgacttgga ggaccaqqaa 241 gccggagaca ggqccgacgc tgccgcggga ggggagccqq cgtggggacc tccatgcaat 301 tttcctcccg aaatcccacc q ttcactact gtgccgggag tqaagg tega eacgtccaac 361 ttcgaaccga t caatttctt tcaactcttc atgactgaag cgatcctgca aqatatggtg 421 ctctacacta a tgtgtacgc cgagcagtac ctgactcaaa acccgctgcc teqe tacq eg 481 agagcgcatg cgtqqcaccc gaccgatatc gcggagatqa agcggttcgt gggactgacc 541 ctcgcaatgg gcctga tcaa g g ccaacagc ctcgagtcat ac tqqq a ta c cacgactgtg 601 cttagcattc cggtgttctc cgctaccatg tcccgtaacc gctaccaact cctgctgcgg 661 ttcctccact tcaacaacaa tgcgaccgct g tg cca cctg accagccagg acacqacaga 721 ctccacaagc tgcqqccatt gatcgactcg ctgagcgaqc gattcgccgc ggtgtacacc 781 ccttgccaaa acatttgca t cgacgagtcg cttctgctgt ttaaaggccg gcttcagttc 841 cgccagtaca tcccatcgaa gcgcgctcgc ta tggta tea aattctacaa actctgcgag 901 tcgtccagcg gctacacgtc atacttcttg atetaegagg ggaaggactc taagctggac 961 ccaccggggt gtccaccqqa tcttactgtc tccggaaaaa teg tg tggga actcatctca 1021 cctctcctcg gacaaggctt tcatctctac gtcgacaatt tctactcatc gatccctctg 1081 ttca:ccg ccc tctactgcct ggatactcca g cctg tggga ccattaacag aaaccggaag 1141 ggtctgccga gagcactgct ggataagaag ttgaacaggg gagagaetta egegetgaga 1201 aagaacgaac tcctcqcc t caaattcttc gacaagaaaa a tg tg 111a t gctcacctcc 1261 atccacgacg aatccgtca t ccgggagcag cgcgtgggca ggccgccgaa aaacaagccg 1321 ctgtgctcta aggaatactc caagtacatg gggggtgtcg accggaccga tcagctgcag 1381 cattactaca acqccactag aaagacccgg gcctggtaca agaaagt egg catctacctg 1441 atccaaatgg cactgaggaa t tcgtatatt gtctacaagg c tqccq t tec gggcccgaaa 1501 ctgtcatact acaagtacca gcttcaaatc ctgccggcgc tgctgttcgg tqgagtggaa 1561 gaacaqactg tgcccgagat gccgccatcc qacaacgtgg cccggttgat eggaaageae 1621 ttcattgata ccctqcctcc gacgcctgga aageagegge cacagaaggg atgcaaagtt 1681 tgccgcaagc geggaa tacg g cgcgatacc cgctactatt qcccqaagtg cccccgcaat 1741 cccggactgt gt ttcaagcc ctgttttgaa atctaccaca cccagttgca ttac (SEQ ID

NO : 14626) .

SUBSTITUTE SHEET (RULE 26) [0663] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Xenopus tropicalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaaccfcfcfct tactgccaat gacgcatgqq afcacgtcgtg gcagtaaaag ggcbbaaabg

61 ccaacgacgc gtcccatacg ttgttggcat tttaagtctt ctctctgcag cggcagcatg

121 tgccgccgct gcagagagbt bcbagcgatg acagcccctc bgggcaacga gccggggggg

181 ctgtc (SEQ ID NO: 14519; .

[0664] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tttqcabbbb bagacattta gaagccta a bcbbgbbaca gaattggaat tacacaaaaa

61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatatt g ttttcctggg

121 taaactaaaa gtccccbcga ggaaaggccc ctaaagtgaa acagbgcaaa acgttcaaaa

181 actgtctggc aatacaagtt ccactttqac caaaacggct ggcagtaaaa gggttaa (SEQ

ID NO: 14520} .

[0665] In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14519 and SEQ ID NO: 14520 In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaacccttt gcctgccaat cacgcatggg atacgt cgtg gcagtaaaag ggcttaaatg

61 ccaacgacgc gtcccatacg ttgttggcat tttaagtctt ctctctgcag cggcagcatg

121 tgccgccgct gcagagagtt bcbagcgatg acagcccctc tgggcaacga gccggggggg

181 ctgtc (SEQ ID NO: 14521) .

[0666] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tttqcatt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa 61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatattg ttttcctggg

121 ta actaaaa gtcccctcqa ggaaaggccc ctaaagtgaa acagbgcaaa acgttcaaaa

181 actgtctggc aatacaagtt ccactttggg acaaatcggc tggcagtgaa agggttaa (SEQ

ID NO: 14522} .

[0667] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaacctttt tactgccaat gacgcatgqq atacgtcgtg gcagtaaaag ggcttaaatg

61 cc acgacgc gtcccatacg ttgttggcat tttaattctt ctctctgcag cggcagcatg

121 tgccgccgct gcagagagtt tctagcgatg acagcccctc tgggcaacga gccggggggg

181 ctgtc (SEQ ID NO: 14523).

[0668] In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID

NO: 14520 and SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14522 and NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the

SUBSTITUTE SHEET (RULE 26) piggyBac or piggyBac-iike transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides from SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the piggyBac or piggyBac-like transposon comprises one end comprising at least 14, 16, 18, 20, 30 or 40 contiguous nucleotides from SEQ ID NO: 14520 or SEQ ID NO: 14522. In certain embodiments, the piggyBac or piggyBac-iike transposon comprises one end with at least 90% identity to SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523. In certain embodiments, the piggyBac or piggyBac-iike transposon comprises one end with at least 90% identity to SEQ ID NO: 14520 or SEQ ID NO: 14522. In one embodiment, one transposon end is at least 90% identical to SEQ ID NO: 14519 and the other transposon end is at least 90% identical to SEQ ID NO: 14520.

[0669] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of TTAACCTTTTTACTGCCA (SEQ ID NO: 14524). In certain embodiments, the piggyBac or piggyBac-iike transposon comprises a sequence of TT AACCCTTTGCCTGCC A (SEQ ID NO: 14526). In certain embodiments, the piggyBac or piggyBac-iike transposon comprises a sequence of TTAACCYTTTTACTGCCA (SEQ ID NO: 14527). In certain embodiments, the piggyBac or piggyBac-iike transposon comprises a sequence of

TGGCAGTAAAAGGGTTAA (SEQ ID NO: 14529). In certain embodiments, the piggyBac or piggyBac-iike transposon comprises a sequence of TGGCAGTGAAAGGGTTAA (SEQ ID NO: 14531). In certain embodiments, the piggyBac or piggyBac-iike transposon comprises a sequence of TTAACCYTTTKMCTGCCA (SEQ ID NO: 14533). In certain embodiments, one end of the piggyBac or piggyBac-iike transposon comprises a sequence selected from SEQ ID NO: 14524, SEQ ID NO: 14526 and SEQ ID NO: 14527. In certain embodiments, one end of the piggyBac™ (PB) or piggyBac-iike transposon comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531. In certain embodiments, each inverted terminal repeat of the piggyBac or piggyBac-iike transposon comprises a sequence of ITR sequence of

CCYTTTKMCTGCCA (SEQ ID NO: 14563). In certain embodiments, each end of the piggyBac™ (PB) or piggyBac-iike transposon comprises SEQ ID NO: 14563 in inverted orientations. In certain embodiments, one ITR of the piggy Bac or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14524, SEQ ID NO: 14526 and SEQ ID NO: 14527. In certain embodiments, one ITR of the piggyBac or piggyBac-like transposon comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531. In certain embodiments,

SUBSTITUTE SHEET (RULE 26) the piggyBac or piggyBac like transposon comprises SEQ ID NO: 14533 m inverted orientation in the two transposon ends.

[0670] In certain embodiments, The piggyBac or piggyBac-like transposon may have ends comprising SEQ ID NO: 14519 and SEQ ID NO: 14520 or a variant of either or both of these having at least 90% sequence identity to SEQ ID NO: 14519 or SEQ ID NO: 14520, and the piggyBac or piggyBac-like transposase has the sequence of SEQ ID NO: 14517 or a variant showing at least %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between sequence identity to SEQ ID NO: 14517 or SEQ ID NO: 14518. In certain embodiments, one piggyBac or piggyBac-like transposon end comprises at least 14 contiguous nucleotides from SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523, and the other transposon end comprises at least 14 contiguous nucleotides from SEQ ID NO: 14520 or SEQ ID NO: 14522. In certain

embodiments, one transposon end comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 contiguous nucleotides from SEQ ID NO: 14519, SEQ ID NO: 14521 or SEQ ID NO: 14523, and the other transposon end comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25 or at least 30 contiguous nucleotides from SEQ ID NO: 14520 or SEQ ID NO: 14522.

[0671] In certain embodiments, the piggyBac or piggyBac-like transposase recognizes a transposon end with a 5’ sequence corresponding to SEQ ID NO: 14519, and a 3’ sequence corresponding to SEQ ID NO: 14520 It will excise the transposon from one DNA molecule by cutting the DNA at the 5'-TTAA-3' sequence at the 5’ end of one transposon end to the 5'- TTAA-3' at the 3’ end of the second transposon end, including any heterologous DNA that is placed between them, and insert the excised sequence into a second DNA molecule. In certain embodiments, truncated and modified versions of the 5’ and 3’ transposon ends will also function as part of a transposon that can be transposed by the piggyBac or piggyBac-like transposase. For example, the 5’ transposon end can be replaced by a sequence corresponding to SEQ ID NO: 14521 or SEQ ID NO: 14523, the 3’ transposon end can be replaced by a shorter sequence corresponding to SEQ ID NO: 14522. In certain embodiments, the 5’ and 3’ transposon ends share an 18 bp almost perfectly repeated sequence at their ends (5'- TTAACCYTTTKMCTGCCA: SEQ ID NO: 14533) that includes the 5'-TTAA-3' insertion site, which sequence is inverted in the orientation in the two ends. That is in SEQ ID NO: 14519 and

SUBSTITUTE SHEET (RULE 26) SEQ ID NO: 14523 the 5’ transposon end begins with the sequence 5 ^! -

TT A A CC TTTTTA C TGC C A - 3 ^! (SEQ ID NO: 14524), or in SEQ ID NO: 14521 the 5’ transposon end begins with the sequence 5'-TTAACCCTTTGCCTGCCA-3' (SEQ ID NO:

14526); the 3’ transposon ends with approximately the reverse complement of this sequence: in SEQ ID NO: 14520 it ends 5’ TGGCAGTAAAAGGGTTAA-3' (SEQ ID NO: 14529), in SEQ ID NO: 14522 it ends 5'-TGGCAGTGAAAGGGTTAA-3' (SEQ ID NO: 14531.) One embodiment of the invention is a transposon that comprises a heterologous polynucleotide inserted between two transposon ends each comprising SEQ ID NO: 14533 m inverted orientations in the two transposon ends. In certain embodiments, one transposon end comprises a sequence selected from SEQ ID NOS: 14524, SEQ ID NO: 14526 and SEQ ID NO: 14527 In some embodiments, one transposon end comprises a sequence selected from SEQ ID NO: 14529 and SEQ ID NO: 14531.

[0672] In certain embodiments, the piggyBac™ (PB) or piggyBac-like transposon is isolated or derived from Xenopus tropicalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 ccctttgcct gccaatcacg catgggatac gtcgtggcag taaaagggct t aatgccaa 61 cgacgcgtcc catacgtt (SEQ ID NO: 14573}.

[0673] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 cctqqqtaaa ctaaaagtcc cctcgaqqaa aqqcccctaa agtgaaacag tqcaaaacqt 61 tcaaaaactg tctggcaata caagttccac tttgggacaa atcggctggc agtgaaaggg

(SEQ ID NO: 14574).

[0674] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at least 16 contiguous bases from SEQ ID NO: 14573 or SEQ ID NO: 14574, and inverted terminal repeat of CCYTTTBMCTGCCA (SEQ ID NO: 14575).

[0675] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 ccctttgcct gccaatcacg catgggatac gtcgtggcag taaaagggct taaatgccaa

61 cqacqcqtcc catacgttgt tggcatttta agtcttctct ctgcagcggc aqcatgtgcc

121 gccgctgcag agagtttcta gcgatgacag cccctctggg caacgagccg ggggggctgt

181 c (SEQ ID NO : 1 579) .

[0676] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 cctttttact qcc tg cg catgggatac gtcgtggcag taaaagggct taaatgccaa

SUBSTITUTE SHEET (RULE 26) 61 cgacgcgtcc catacgttgt tggcatttta attcttctct ctgcagcggc agcatgtgcc

121 gccgctgcag agagtttcta gcgatqacag cccctctggg caacgagccg ggggggctgt

181 c (SEQ ID NO: 14580) .

[0677] In certain embodiments, the piggyBac or piggyBac-Hke transposon comprises at a sequence of:

1 cctttttact gccaatgacg catgggatac gtcgtggcag taaaagggct taaatgccaa

61 cgacgcgtcc catacgttgt tggcatttta agtcttctct ctgcagcggc agcatgtgcc

121 gccgctgcag agagtttcta gcgatqacag cccctctggg caacgagccg ggggggctgt

181 c (SEQ ID NO: 14581) .

[0678] In certain embodiments, the piggyBac or piggyBac-Hke transposon comprises at a sequence of:

1 cctttttact gccaatgacg catgggatac gtcgtggcag taaaagggct taaatgccaa

61 cgacgcgtcc catacg gt tggcatttta agtcttctct ctgcagcggc agcatgtgcc

121 gccgctgcag agag (SEQ ID NO: 14582).

[0679] In certain embodiments, the piggyBac or piggyBac-!ike transposon comprises at a sequence of

1 cctttttact gccaatgacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta agtctt (SEQ ID NO: 14583).

[0680] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 ccctttgcct gccaatcacg catgggatac gtcgtggcag taaaagggct taaatgccaa 61 cgacgcgtcc catacgttgt tggcatttta agtctt (SEQ ID NO: 14584).

[0681] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 ttatcctttt tactgccaat gacgcatggg atacgtcgtg gcagtaaaag ggcttaaatg

61 ccaacgacgc gtcccatacg ttgttqqcat tttaagtctt ctctctgcag cgqcagcatg

121 tgccgccgct gcagagagtt tctagcgatg acagcccctc tgggcaacga gccggggggg

181 ctgtc (SEQ ID NO: 14585) .

[0682] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa

61 ttctaccata ttttgaaagc ttagqttqtt ctgaaaaaaa caatatattq ttttcctqgg

121 taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa acagtgcaaa acgttcaaaa

181 actgtctggc aatacaaqtt ccactttggg acaaatcqqc tqgcagtgaa aggg (SEQ ID

NO: 14586) .

[0683] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 5’ transposon end sequence selected from SEQ ID NO: 14573 and SEQ ID NQs: 14579-14585. In

SUBSTITUTE SHEET (RULE 26) certain embodiments, the 5’ transposon end sequence is preceded by a 5’ target sequence. In certain embodiments, the piggy Bac or piggyBac-like transposon comprises at a sequence of:

1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa

61 ttctaccata ttttgaaagc ttagqttqtt ctgaaaaaaa caatatattq ttttcctggg

121 taaactaaaa gtcccctcga gqaaaggccc cfcaaagtgaa acagtqcaaa acgttcaaaa

181 actgtctqqc aatacaagtt ccactttgac caaaacgqct ggcagtaaaa ggg ( SEQ ID

NO : 14587) .

[0684] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 ttgttctgaa aaaaacaata tattgttttc ctgggtaaac taaaagtccc ctcgaggaaa

61 ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt ctggcaatac aagttccact

121 ttgaccaaaa cggctggcag taaaaggg (SEQ ID NO: 14588).

[0685] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 tttgcatttt tagacattta gaagcctata tcttgttaca gaattggaat tacacaaaaa

61 ttctaccata ttttgaaagc ttaggttgtt ctgaaaaaaa caatatattg ttttcctggg

121 taaactaaaa gtcccctcga ggaaaggccc ctaaagtgaa acagtgcaaa acgttcaaaa

181 actgtctqqc aatacaagtt ccactttgac caaaacgqct ggcagtaaaa gggttat (SEQ

ID NO: 14589) .

[0686] In certain embodiments, the piggyBac or piggyBac-like transposon comprises at a sequence of:

1 ttgttctgaa aaaaacaata tattgttttc ctgggtaaac taaaagtccc ctcgaggaaa

61 ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt ctggcaatac aagttccact

121 ttgggacaaa tcggctggca gtgaaaggg (SEQ ID NO: 14590) .

[0687] In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 3’ transposon end sequence selected from SEQ ID NO: 14574 and SEQ ID NOs: 14587-14590. In certain embodiments, the 3’ transposon end sequence is followed by a 3’ target sequence. In certain embodiments, the 5’ and 3 transposon ends share a 14 repeated sequence inverted in orientation in the two ends (SEQ ID NO: 14575) adjacent to the target sequence. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a 5’ transposon end comprising a target sequence and a sequence that is selected from SEQ ID NOs: 14582-14584 and 14573, and a 3’ transposon end comprising a sequence selected from SEQ ID NOs: 14588-

14590 and 14574 followed by a 3’ target sequence.

[0688] In certain embodiments, the 5’ transposon end of the piggyBac or piggyBac-like transposon comprises

1 atcacqcatq qqat aegteg tggcagtaaa agggcttaaa tgccaacgac qcqtcccata

SUBSTITUTE SHEET (RULE 26) 61 cgtt

(SEQ ID NO: 14591), and an ITR In certain embodiments, the 5’ transposon end comprises

1 atgacgcatg ggatacqtcg ggcagtaaa agggcttaaa tqccaacg c gcgtcccata 61L cG11ot Lggc a L 111 q tc EL

(SEQ ID NO: 14592) and an ITR. In certain embodiments, the 3’ transposon end of the piggyBae or piggyBac-hke transposon comprises

1 cctggqtaaa ctaaaagtcc cctcgaggaa aggcccctaa agtgaaacag tgcaaaacgfc 61 tcaaaaactg tctgqcaata caagttccac tttgggacaa atcggc

(SEQ ID NO: 14593) and an ITR. In certain embodiments, the 3’ transposon end comprises

1 ttgttctgaa aaaaacaata tatt gttttc ctgggtaaac taaaagtccc c.tcgaggaaa

61 ggcccctaaa gtgaaacagt gcaaaacgtt caaaaactgt ctggcaatac aagttccact

12 1 1. 1. Q a c c a a a a c g g c

(SEQ ID NO: 14594) and an ITR.

[0689] In certain embodiments, one transposon end comprises a sequence that is at least 90%, at least 95%, at least 99% or any percentage in between identical to SEQ ID NO: 14573 and the other transposon end comprises a sequence that is at least 90%, at least 95%, at least 99% or any percentage in between identical to SEQ ID NO: 14574. In certain embodiments, one transposon end comprises at least 14, at least 16, at least 18, at least 20 or at least 25 contiguous nucleotides from SEQ ID NO: 14573 and one transposon end comprises at least 14, at least 16, at least 18, at least 20 or at least 25 contiguous nucleotides from SEQ ID NO: 14574. In certain embodiments, one transposon end comprises at least 14, at least 16, at least 18, at least 20 from SEQ ID NO: 14591, and the other end comprises at least 14, at least 16, at least 18, at least 20 from SEQ ID NO: 14593. In certain embodiments, each transposon end comprises SEQ ID NO: 14575 m inverted orientations.

[0690] In certain embodiments, the piggyBae or piggyBac-like transposon comprises a sequence selected from of SEQ ID NO: 14573, SEQ ID NO: 14579, SEQ ID NO: 14581 , SEQ ID NO: 14582, SEQ ID NO: 14583, and SEQ ID NO: 14588, and a sequence selected from SEQ ID NO: 14587, SEQ ID NO: 14588, SEQ ID NO: 14589 and SEQ ID NO: 14586 and the piggyBae or piggyBac-like transposase comprises SEQ ID NO: 14517 or SEQ ID NO: 14518.

[0691] In certain embodiments, the piggyBae or piggyBac-like transposon comprises ITRs of CCCTTTGCCTGCCA (SEQ ID NO: 14622) (5’ ITR) and TGGCAGTGAAAGGG (SEQ ID NO: 14623) (3’ ITR) adjacent to the target sequences.

SUBSTITUTE SHEET (RULE 26) [0692] in certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Helicoverpa armigera. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,

75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MAS RQRLNHD EΪATI LENDD DYSPLDSESE KEDCWEDDV WSDNEDAIVD FVEBTSAQED 61 PDNNIASRES PNLEVTSLTS HRIITLPQRS I PGKNNHWS TTKGRTTGRT SAINI IPTNR 121 GFTRMCRNIV DPLLCFQLFI TDEIIHEIVK WTNVEIIVKR QNLKDISASY RDTNTMEIWA 181 LVGILTLTAV MKDNHLSTDE LFDATFSGTR YVSV SRERF EFLIRCIRMD DKTLRPTLRS 241 DDAFLPVRKI WEIFINQCRQ NHVPGSNLTV DEQLLGFRGR CPFRMYIPNK PDKYGIKFPM 301 MCAAATKYMI DAIPYLGKST KTNGLPLGEF YVKDLTKTVH GTNRNITCDN WFTSI PLAKN 361 MLQAPYNLTI VGTIRSNKRE MPEEIKNSRS RPVGSSMFCF DGPLTLVSYK PKPSKMVFLL 421 SSCDENAVIN ESNGKPDMIL FYNQTKGGVD SFDQMCKSMS ANRKTNRWPM AVFYGMLNMA 481 FVNSYIIYCH NKINKQEKPI SRKEFMKKLS IQLTTPWMQE RLQAPTLKRT LRDNITNVLK 541 NWPASSENI SNEPEPKKRR YCGVCSYKKR RMTKAQCCKC KKAI CGEHNI DVCQDCI (SEQ

ID NO: 14525) .

[0693] in certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Helicoverpa armigera. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaaccctag aagcccaatc tacgtaaatt tgacgtatac cgcggcgaaa tatctctgtc 61 tctttcatgt ttaccgtcgg atcgccgcta acttctgaac caactcagta gccattggga

121 cctcgcagga cacagttgcg tcatctcggt aagtgccgcc attttgttgt actctctatt

181 acaacacacg t cac.qteaeg tcgttgcacg tcattttgac gtataattgg gctttgtgt a

2 1 ac 1. 1. 1. 1. gaa L tcaa EEL I. 6 6 6 1. 1.a t. g E. E_ E_ g a g a L L 111 g EELg 6 6aa t.c g E.a E. E. g

301 tcgttacatt tttcatataa taataatatt ttcaggttga gtacaaa (SEQ ID NO:

1457 o } . In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

i aga ctg111t tttctaagag a cttctaaaa tattattacg aq t; tga 11 ta attttatgaa

61 aacatttaaa actagttgat tttttttata attacataat tttaagaaaa agtgttagag

121 gc 00ga E- E. 1_.g 1_.tc 11. 1.000 c t-aaga E; E; tga ttaaa gtgccataat ag ba t taata

181 aagagtattt tttaacttaa aat gtatttt atttattaat taaaacttca attatgataa

241 ctcatgcaaa aatatagttc attaacagaa aaaaatagga aaactttgaa gttttgtttt 301 tacacgt cat 11 ttacgtat gattqqqctt tatagetagt taaatatgat tgggcttcta

361 gggttaa (SEQ ID O 14528} .

[0694] in certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Pectinophora gossypiella. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an ammo acid

SUBSTITUTE SHEET (RULE 26) sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70°/

75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

i MDLRKQDEKI RQWLEQDIEE DSKGESDNSS SETEDIVEME VHKNTSSESE VS SES DYEPV

61 CPSKRORTQI IESEESDNSE SIRPSRRQTS RVI DSDETDE DVMSSTPQNI PRNPNVIQPS

121 3RFLYGKNKH KWS SAAKPS S VRTSRRNIIH FI PGPKERAR EVSEPIDIFS LFISEDMLQQ

181 VVT FTNAEML IRKNKYKTET FTVS PTNLEE IRALLGLLFN AAAMKSNHLP TRMLFNTHRS

241 GTI FKACMSA ERLNFLIKCL RFDDKLTRNV RQRDDRFAPI RDLWQALISN FQKWYTPGSY 301 ITVDEQLVGF RGRCSFRMYI PNKPNKYGIK LVMAADVNSK YIVNAIPYLG KGTDPQNQPL

361 ATFFIKEITS TLHGTNRNIT MDNWFTSVPL ANELLMAPYN LTLVGTLRSN KREI PEKLKN 421 SKSRAIGTSM FCYDGDKTLV SYKAKSNKW FILSTIHDQP DINQETGKPE MIHFYNSTKG 481 AVDTVDQMCS SI STNRKTQR WPLCVFYNML NLSIINAYW YVYNNVRNNK KPMSRRDFVI 541 KLGDQLMEPW LRQRLQTVTL RRDIKVMIQD l L _* E,S S!)L£A PVPSVSNVRK IYYLCPSKAR 601 RMTKHRCIKC KQAICGPHNI DICSRCIE (: ;EQ ID NO: 4530) .

[0695] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Pectinophora gossypieila. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaaccctag ataactaaac attcgtccgc tcg a cg a cgc gctatgccgc gaaattgaag 61 tttacctatt attccgcgtc ccccgccccc gccgcttttt ctagcttcct gatttgcaaa 121 atagtgcatc gcgtgacacg ctcgaggtca cacgacaatt aggtcgaaag ttacaggaat 181 ttcgtcgtcc gctcgacgaa agtttagtaa ttacgtaagt ttggcaaagg taagtgaatg 241 aagtattttt ttataattat 11111aa 11c tttatagtga t acgtaagg 111 11 La a a 301 ttta tto.ccc DDatag11a L ttagccaatt gttataaatt ccttgttatt gctgaaaaat 361 ttgcctgttt tagtcaaaat L ta 11 a a c!.!. ttcgatcgtt ttttag (SEQ ID NO:

14532 ) . In certain embodiments, the piggy Bac or piggyBac-like transposon comprises a sequence of:

1 tttcactaag taattttgtt cctatttagt agataagtaa cacataatta ttgtgatatt 61 caaaacttaa gaggtttaat aaataataat aaaaaaaaaa tggtttttat ttcgtagtct 121 gctcgacgaa tgtttagtta ttacgtaacc gtgaatatag tttagtagt c tagggttaa (SEQ ID NO: 14571) .

[0696] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Ctenopiusia agnata. The piggyBac or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least

5%, 10%, 15%, 20%. 25%, 30%, 35%, 40%, 45%, 50%. 55%, 60%, 65%, 70%, 75%. 80%. 85%,

90%, 95%, 99% or any percentage in between identical to:

i MASRQHLYQD EIAAI LENED DYSPHDTDSE MEDCVTQDDV RSDVEDEMVD NIGNGTSPAS

61 RHEDPETPDP S S EASNLEVT LSSHRIIILP QRS I REKNNH IWSTTKGQSS GRTAAINIVR 121 TNRGPTRMCR NIVDPLLCFQ LFIKEEIVEE IVKWTNVEMV QKRVNLKDI S ASYRDTNEME 181 IWAIISMLTL SAVMKDNHLS TDELFNVSYG TRYVSVMSRE RFEFLLRLLR MGDKLLRPNL 241 RQEDAFTPVR KIWEI FINQC RLNYVPGTNL TVDEOLLGFR GRCPFRMYI P NKPDKYGIKF 301 PMVCDAATKY MVDAI PYLGK STKTQGLPLG EFYVKELTQT VHGTNRNVTC DNWFTSVPLA 361 KSLLNSPYNL TLVGTIRSNK REIPEEVKNS RSRQVGS SMF CFDGPLTLVS YKPKPSKMVF 421 LLSSCNEDAV VNQSNGKPDM ILFYNQTKGG VDSFDQMCSS MSTNRKTNRW PMAVFYGMLN

SUBSTITUTE SHEET (RULE 26) 481 MAFVNSYIIY CHNMLAKKEK PLSRKDFMKK LSTDLTTPSM QKRLEAPTLK RSLRDNITNV 541 LKIVPOAAI D TS FDEPEPKK RRYCGFCSYK KKRMTKTQCF KCKKPVCGEH NIDVCQDCI (SEQ ID NO: 14534} .

[0697] In certain embodiments, the piggyBac or piggyBac-Hke transposon is isolated or derived from Ctenoplusia agnata. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaaccctag aagcccaatc taegteatte tgacgtgtat gtcgccgaaa atactctgtc 61 tctttctcct gcacgatcgg attgeegega aegetegatt caacccagt 2 ggegeegaga 121 tctattggag gactgcggcg 11 gatteggt aagtcccgcc attttgt cat agtaacagta 181 ttgcacgtca gettgaegta tatttgggct ttgtgttatt tttqtaaatt ttcaacgtta 241 gtttattatt gcatcttttt gttacattac tggtttattt gcatgtatta ct caaatat t 301 atttttattt tagegtagaa aataca ( SEQ ID NO: 14535) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 agactqtttt O01-1-gta 112 gca 22a 2a 2CL 22 2a 22 c2 a aagttgatt 2 aattetaaga 61 aaaaca t taa aataagtttc tttttgtaaa atttaattaa ttataagaaa aagtt taagt 121 tgatctcatt ttttataaaa atttgcaatg tttccaaagt 2a 22a 22g 2a aaagaataaa 181 taaaagtaaa ctgagtttta attgatgttt tat tat atc.a ttatactata tattaettaa 241 ataaaacaat a a ctgaatgt a 222 c 2aaaa qqaatca cta gaaaatatag tga tcaaaaa 301 tttacacgtc atttttgcgt atgattgggc tttataggtt ctaaaaatat gattgggcct 361 ctagggttaa (SEQ ID NO: 14536} .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCCTAGAAGCCCAATC (SEQ ID NO: 14564)

[0698] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Agrotis ipsilon. The piggyBac

(PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MESRQRLNQD EIATILENDD DYSPLDSDSE AEDRWEDDV WSDNEDAMI D YVEDTSRQED 61 PDNNIASQES ANLEVTSLTS HRI I SLPQRS ICGKNNHVWS TTKGRTTGRT SAINI IRTNR 121 GPTRMCRNIV DPLLCFQLFI TDEIIHEIVK WTNVEMIVKR QNLIDI SASY RDTNTMEMWA 181 LVGILTLTAV MKDNHLSTDE LFDATFSGTR YVSVMSRERF EFLIRCMRMD DKTLRPTLRS 241 DDAFI PVRKL WEIFINQCRL NYVPGGNLTV DEQLLGFRGR CPFRMYIPNK PDKYGIRFPM 301 MCDAATKYMI DAI PYLGK3T KTNGLPLGEF YVKELTKTVH GTNRNVTCDN WFTSI PLAKN 361 MLQAPYNLTI VGTIRSNKRE IPEEIKNSRS RPVGSSMFCF DGPLTLVSYK PKPSRMVFLL 421 SSCDENAVIN ESNGKPDMI L FYNQTKGGVD SFDQMCKSMS ANRKTNRWPM AVFYGMLNMA 481 FVNSYIIYCH NKINKQKKPI NRKEFMKNLS TDLTTPWMQE RLKAPTLKRT LRDNITNVLK 541 NWPPSPA N SEEPGPKKRS YCGFCSYKKR RMTKTQFYKC KKAICGEHNI DVCQDCV (SEQ

ID NO: 14537) .

In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Agrotis ipsilon. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

SUBSTITUTE SHEET (RULE 26) 1 ttaaccctag aaqcccaatc tacgtaaatt tgacgtatac cqcggcgaaa tatatctgtc 61 tctttcacgt ttaccgtcgg attcccqcta acttcggaac caactcagta qccattgaga 121 actcccaqqa ca cagttgcg tcatctcgq t aag tgccgcc attttgttgt aa bagacagg 181 ttgeaegtea ttttqacgta taattgggct ttgtgtaact tttgaaatt 111ataa111 241 ttattgatgt gattta 11 tg agttaatcgt a11q 111 cq t. taca t t t t t c atatgatatt 301 aatatttt ca gattgaatat aaa (SEQ ID NO: 14538} ,

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 dGdcto a L L- L L 11aa a a qq cooata a a g t attactattg cgCgaCCCaa tttt aaaa 61 atatttaaaa ccagttgatt tttttaataa ttacctaatt ttaagaaaaa atqttagaag 121 cttgatattt ttgttgattt ttttctaaga tttgattaaa aggccataat tgtattaata 181 aagagta111 11aac 11caa. a.111a1111a 111a 11aa 11 aaaacCCcaa ttatgataat 241 acatgcaaaa at atagttca t caacaqaaa aatataggaa aactctaata gttttatttt 301 tacacgtcat ttttacgtat gattgggctt Ca CageCagt caaatatgat tgqqcCCcCa 361 gggttaa {SEQ ID NO: 14539} ,

[0700] in certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Megachile rotundata. The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,

75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MNGKDSLGEF YLDDLSDCLD CRSASSTDDE SDSSNIAIRK RCPIPLIYSD SEDEDMNNNV

61 EDNNHFVKES NRYHYQIVEK YKITSKTKKW KDVTVTEMKK FLGLI I LMGQ VKKDVLYDYW

121 3TDPSIETPF FSKVMSRNRF LQIMQSWHFY NNNDISPNSH RLVKIQPVID YFKEKFNNVY

181 KSDQQLSLDE CLIPWRGRLS IKTYNPAKIT KYGILVRVLS EARTGYVSNF CVYAADGKKI

241 EETVLSVIGP YKNMWHHVYQ DNYYNSVNIA KI FLKNKLRV CGTIRKNRSL POILQTVKLS

301 RGQHQFLRNG HTLLEVWNNG KRNVNMI STI HSAQMAESRN RSRTSDCPIQ KPISIIDYNK

361 YMKGVDRADQ YLSYYSIFRK TKKWTKRWM FFINCALFNS FKVYTTLNGQ KITYKNFLHK | AALSLIEDCG TEEQGTDLPN SEPTTTRTTS RVDHPGRLEN FGKHKLVNIV TSGQCKKPLR

481 QCRVCASKKK LSRTGFACKY CNVPLHKGDC FERYHSLKKY (SEQ ID NO 14540} .

[0701] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Megachile rotundata. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaaataatg cccactctag a tga aettaa cactttaccq accggccgtc gattattega

61 cgtttgctcc ccagcgct Ca ccgaccggcc a Lcga L La 11 cgacgCCCgc ttcccagcgc

121 ttaccqaccg gtcatcgact tttgatcttt ccgttagatt tggttaggtc aqattgacaa

181 gtagcaagca CCtcgcattc tttattcaaa CaaCcggtgc ttttttctaa gctttagccc

241 ttagaa (SEQ ID NO: 14541} .

In certain embodiments, the the piggyBac or piggyBac-like transposon comprises a sequence of:

1 aca ettett ttttcaacaa a ta ttgttat atggattatt C C C Ca 111a tttatttatg

61 gtatatttta tg111a 111a cccatggtta ttatggtata ttccacgcaa ataataaact

121 qaaaacgaCt gtaatagatg aaataaatat tgttttaaca ctaatataat taaagtaaaa

181 gat CttaaCa aatttcgtta ccctacaata acacgaagcg tacaatttta ccagagttta

241 ttaa (SEQ ID NO: 14542} .

SUBSTITUTE SHEET (RULE 26) [0702] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Bombus impatiens. The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,

85%, 90%, 95%, 99% or any percentage in between identical to:

1 MNEKNGIGEF YLDDLSDCPD SYSRSNSGDE SDGSDTI IRK RGSVLPPRYS DSEDDEINNV

61 EDNANNVENN DDIWSTNDEA IILEPFEGSP GLKIMPS SAE SVTDNVNLFF GDDFFEHLVR

121 ESNRYHYQVM EKYKIPSKAK KWTDITVFEM KKFLGLIVLM GQIKKDVLYD YWSTDPSIET

181 PFFSQVMSRN RFVQIMQSWH FCNNDNIPHD SHRLAKIQPV IDYFRRKFND VYKPCQQLSL

241 DESIIPWRGR LSIKTYNPAK ITKYGILVRV LSEAVTGYVC NFDVYAADGK KLEDTAVIEP

301 YKNIWHQIYQ DNYYNSVKMA RILLKNKVRV CGTIRKNRGL PRSLKTIQLS RGQYEFRRNH

361 QILLEVWNNG RRNVNMISTI HSAQLMESRS KSKRSDVPIO KPNSIIDYNK YMKGVDRADQ

421 YLAYYSIFRK TKKWTKRWM FFINCALFNS FRVYTILNGK NITYKNFLHK VAVSWIEDGE

481 TNCTEQDDNL PNSEPTRRAP RLDHPGRLSN YGKHKLINIV TSGRSLKPOR QCRVCAVQKK

541 RSRTCFVCKF CNVPLHKGDC FERYHTLKKY (SEQ ID NO 14543) .

[Q7Q3] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Bomhus impatiens. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaatttttt aacattttac cgaccqatag ccgattaatc gggtttttgc cgctgacgct

61 taccqaccqa taacctatta atcggctttt fcgfccgtcgaa gcttaccaac ctataqccta

121 cctatagtta atcggttgcc atggcgataa acaatctttc tcattatat g agcagtaatt

181 tgttatttag tactaaqqta ccttgctcag ttgcgtcagt tqcq gc t tgtaagctcc

241 cacagtttta taccaattcg aaaaacttac cgttcgcg (SEQ ID NO: 14544).

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 actatttcac atttqaacta aaaaccgttg taatagataa aataaatata atttagtatt

61 aatattatgg aa c aaaga ttttattcaa tttaatt te ctatagtaac aaaaagcggc

121 caattttatc tgagcatacg aaaaqcacaq atactcccgc ccgacagtct aaaccqaaac

181 ag geeggeg ccagggagaa tctgcgcctg ageageeggt cggacgtgcg tttgctgttg

241 aaccgctagt ggtcaqtaaa ccagaaccag teagtaagee aqtaactgat cagttaacta

301 gattqtatag ttcaaattga aettaateta qtttttaagc gtttgaatgt tgtetaaett

361 cgttatatat tatattettt ttaa (SEQ ID NO: 14545) .

[0704] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Mameslra brassicae. The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MFSFVPNKEQ TRTVLIFCFH LKTTAAESHR PLVEAFGEQV PTVKTCERWF QRFKSGDFDV 61 DDKEHGKPPK RYEDAELQAL LDEDDAOTQK QLAEQLEVSQ QAVSNRLREG GKIQKVGRWV 121 PHELNERQRE RRKNTCEILL SRYKRKSFLH RIVTGEEKWI FFVNPKRKKS YVDPGQPATS

SUBSTITUTE SHEET (RULE 26) 181 TARPNRFGKK TRLCVWWDQS GVIYYELLKP GETVNT'ARYQ QQLINLNRAL QRKRPEYQKR

241 QHRVI FLHDN APSHTARAVR DTLETLNKEV LPHAAYS PDL APSDYHLFAS MGHALAEQRF

301 DSYESVEEWL DEWFAAKDDE FYWRGIHKLP ERWDNCVASD GKYFE (SEQ ID NO:

[0705] In certain embodiments, the piggyBae or piggyBac-like transposon is isolated or derived from Mamestra brassicae. In certain embodiments, the piggyBae or piggyBac-iike transposon comprises a sequence of:

1 ttattgggtt gcccaaaaag taattgcgga tttttcatat acctgtcttt taaacgtaca

61 tagggatega actcagtaaa act t tgac.ct tgtgaaataa caaacttgac tgtccaacca

121 ccatagtttg gegegaattg agegteataa ttgttttgac tttttgcagt caac ( SEQ ID

NO: 14547) .

In certain embodiments, the piggyBae or piggyBac-iike transposon comprises a sequence of:

1 atqatttttt ctttttaaac caattttaat taqttaattg atataaaaat ccqcaattac 61 tttttgggca acccaataa (SEQ ID NO: 14548) .

[0706] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBae or piggyBac-iike transposase enzyme. In certain embodiments, the piggyBae or piggyBac-iike transposase enzyme is isolated or derived from Mayetiola destructor. The piggyBae (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 1 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MENFENWRKR RHLREVLLGH FFAKKTAAES HRLLVEVYGE HALAKTQCFE KEQRFKSGDF 61 DTEDKERPGQ PKKFEDEELE ALLDEDCCQT QEELAKSLGV TQQAISKRLK AAGYIQKQGN 121 WVPHELKPRD VERRFCMSEM LLQRHKKKSF LSRIITGDEK WIHYDNSKRK KSYVKRGGRA 181 KSTPKSNLHG AKVMLCIWWD QRGVLYYELL EPGQTITGDL YRTQLI RLKO ALAEKRPEYA 241 KRHGAVIFHH DNARPHVALP VKNYLENSGW EVLPHPPYSP DLAPSDYHLF RSMQNDLAGK 301 RFTSEQGIRK WLDSFLAAKP AKFFEKGIHE LSERWEKVIA SDGQYFE (SEQ ID NO:

14549) .

[0707] In certain embodiments, the piggyBae or piggyBac-like transposon is isolated or derived from Mayetiola destructor. In certain embodiments, the piggyBae or piggyBac-like transposon comprises a sequence of:

1 taagacttcc aaaatttcca cccgaacttt accttccccg egeattatgt ctctcttttc

61 accctctgat ccctggtatt qttgtcgagc aegatttata ttgggtgtac aacttaaaaa

121 ccqgaattgg aegetagatg tccacactaa cgaatagtgt aaaagcacaa atttcatata

181 tacgtcafcfct tgaaggtaca tttgacagct afccaaaatca gtcaataaaa etattetate

241 tgtgtgcatc atattttttt attaact ( SEQ ID NO: 14550) .

In certain embodiments, the piggyBae or piggyBac-like transposon comprises a sequence of:

1 tgcattcatt cattttgtta tcgaaataaa gcattaattt tcactaaaaa attccggttt

61 ttaagttgta cacccaatat catccttagt gacaattttc aaatqqcttt cccattgagc

121 tgaaaccgtg getetagtaa gaaaaacgcc caacccgtca tcatatgcct tttttttctc

181 aacatccg (SEQ ID NO: 14551) .

SUBSTITUTE SHEET (RULE 26) [0708] in certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Apis mellifera. The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MENQKEHYRH ILLFYFRKGK NASQAHKKLC AVYGDEALKE RQCQNWFDKF RSGDFSLKDE 61 KRS GRPVEVD DDLIKAIIDS DRHSTTREIA EKLHVSHTCI ENHLKQLGYV QKLDTWVPHE 121 LKEKHLTQRI NSCDLLKKRN ENDPFLKRLI TGDEKWWYN NIKRKRSWSR PREPAQTT SK 181 AGI HRKKVLL SVWWDYKGIV YFELLPPNRT INSWYIEQL TKLNNAVEEK RPELTNRKGV 241 VFHHDNARPH TSLVTRQKLL ELGWDVLPHP PYSPDLAPSD YFLFRSLQNS LNGKNFNNDD 301 DIKSYLIQFF ANKNQKFYER GIMMLPERWQ KVIDQNGQHI TE (SEQ ID NO: 14552} . [Q7Q9] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Apis mellifera. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttgggttggc aactaagtaa ttgcggattt cactcataga tggcttcagt tgaattttta

61 ggtttgctgg cgtagtccaa atgtaaaaca cattttgtta tttgatagtt ggcaattcag

121 ctgtcaatca gtaaaaaaag ttttttgatc ggttgcgtaq ttttcgtttg gc.gtt cgttg

181 aaaa (SEQ ID NO: 14553} .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 agttatttag ttccatgaaa aaattgtctt tgattttcta aaaaaaatcc gcaattactt 61 agttgccaat ccaa (SEQ ID NO: 14554) .

[0710] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Messor bouvieri. The piggyBac (PB) or piggyBac-like transposase enzyme may comprise or consist of an amino acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MSSFVPENVH LRHALLFLFH QKKRAAESHR LLVETYGEHA PTIRTCETWF RQFKCGDFNV 61 QDKERPGRPK TFEDAELQEL LDEDSTQTQK QLAEKLNVSR VAICERLQAM GKIQKMGRWV 121 PHELNDRQME NRKIVSEMLL QRYERKSFLH RIVTGDEKWI YFENPKRKKS WLSPGEAGPS

181 TARPNRFGRK TMLCVWWDQI GWYYELLKP GETVNTDRYR QQMINLNCAL IEKRPQYAQR

241 HDKVI LQHDN APSHTAKPVK EMLKSLGWEV LSHPPYSPDL APSDYHLFAS MGHALAEQHF

301 ADFEEVKKWL DEWFSSKEKL FFWNGIHKLS ER TKCI ESN GQYFE (SEQ ID NO:

14555) .

[0711] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Messor bouvieri. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 aqt cagaaat ga cacctcga tcgacqacta atcgacgtct aatcgacgtc gattttatgt

SUBSTITUTE SHEET (RULE 26) 61 caacatgtta ccaqqtqtq t cggtaattcc tttccggttt ttccggcaga tgtcactagc 121 cataagtatg aaatgttatg atttqataca tatgt cattt tattctactg acattaacct 181 taaaactaca caagttacgt tccgccaaaa taacagcgtt atagatttat aa 666 q a 241 aa (SEQ ID NO: 14556) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ataaatttga actatccatt ctaagtaacg tgttttcttt aacgaaaaaa ccggaaaaga 61 attaccgaca ctcctqqtat gtcaacatgt tattttcqac attgaatcgc gtcgattcga 121 agtcgatcga ggtgtcattt ctgact ( SEQ ID NO: 14557).

[0712] In certain embodiments of the methods of the disclosure, the transposase enzyme is a piggyBac or piggyBac-like transposase enzyme. In certain embodiments, the piggyBac or piggyBac-like transposase enzyme is isolated or derived from Trichoplusia ni. The piggyBac

(PB) or piggyBac-like transposase enzyme may comprise or consist of an ammo acid sequence at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

1 MGS SLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGS SLASN RILTLPQRTI RGKNKHCKST SKSTRRSRVS ALNIVRSQRG 121 PTRMCRNIYD PLLCFKLFFT DEI I SEIVKW TNAEI SLKRR ESMTSATFRD TNEDEIYAFF 181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLEMDDKS IRPTLRENDV 241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD 301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ 361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC 421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN 481 SFI IYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV 541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV I CREHNI DMC QSCF {SEQ ID

NO : 14558) .

[0713] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Trichoplusia ni. In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ttaaccctag aaaqataqtc t:gcgtaaaat tga cgcatgc attcttgaaa tattgctctc 61 tctttctaaa tagcgcga t ccgtcgctgt gcatttagga catctcagtc gccgcttgga 121 qctcccgtga ggcgtgc11g t caatqcqqt aag tg tcact gattttgaac tataacgacc 181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg 241 ataattatat tg11a111ca tg11ctac11 acgtgataac t 0 L La 0 0a tatattttct 301 tgttatagat ate (SEQ ID NO: 14559) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tttgttactt tatagaagaa attttgagtt tttgtttttt tttaataaat aaataaacat 61 aaat a t tg tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat 121 atctattcaa attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt 181 ttacgcatga ttatctttaa cgtacgtcac aatatgatta tctttctagg gtt a {SEQ ID NO: 14560) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt

SUBSTITUTE SHEET (RULE 26) 61 tctaaatagc gcgaatccg t cgctgtgcat ttaggacatc tcagtcgccg cttggagctc

121 ccqtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata acgaccgcgt

181 gag tcaaaa t ga cgcatgat tatcttttac q tqactttta agatttaact ca tacqa taa

241 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt

301 atagatatc (SEQ ID NO: 14561} .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tttgttactt tataqaagaa attttgagtt tt gtt ttt tttaataaat aaataaacat

61 aaataaattg tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat

121 atctattcaa attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt

181 ttacgcatga ttatctttaa cgtacgtcac aatatgatta tctttctagg g ( SEQ ID NO:

14562 } .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tctaaatagc gcgaatccgt cgctgtgcat aggacatc tc g cgccg ct tggagctc

61 ccgtgaggcg tgcttgtcaa gcggtaagt g cactgatt ttgaactata acgaccgcgt

121 gagtcaaaat gacgcatqat tatcttttac gtgactttta agatttaact catacgataa

181 ttatattg a ttcatgtt ctacttacgt gataacttat ta atatata ttttcttg

241 atagatatc (SEQ ID NO: 14609) .

In certain embodiments, the piggyBac or piggyBac-like transposon comprises a sequence of:

1 tttgttactt tatagaagaa attttqaqtt tttgtttttt tttaataaat aaataaacat

61 aaataaattg tttgttgaat ttat ta t tag tatgtaagtg taaatataa t aaaacttaat

121 atctattcaa attaataaat aaacctcgat atacagaccq ataaaacaca tgcgtcaatt

181 ttacgcatga ttatctttaa cgtacgtcac aatatgatta tctttctagg g (SEQ ID NO:

14610} .

[0714] In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14561 and SEQ ID NO: 14562, and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14558. In certain embodiments, the piggyBac or piggyBac-like transposon comprises SEQ ID NO: 14609 and SEQ ID NO: 14610, and the piggyBac or piggyBac-like transposase comprises SEQ ID NO: 14558.

[0715] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Aphis gossypii. In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCTTCCAGCGGGCGCGC (SEQ ID NO: 14565).

[0716] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Chilo suppressalis. In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCCAGATTAGCCT (SEQ ID NO: 14566).

[0717] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Heliothis virescens. In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCCTTAATTACTCGCG (SEQ ID NO: 14567).

SUBSTITUTE SHEET (RULE 26) [0718] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Pectinophora gossypiella. In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCCTAGATAACTAAAC (SEQ ID NO: 14568).

[0719] In certain embodiments, the piggyBac or piggyBac-like transposon is isolated or derived from Anopheles stephensi. In certain embodiments, the piggyBac or piggyBac-like transposon comprises an ITR sequence of CCCTAGAAAGATA (SEQ ID NO: 14569).

[0720] DNA transposons in the hAT family are widespread in plants and animals. A number of active hAT transposon systems have been identified and found to be functional, including but not limited to, the Hermes transposon, Ac transposon, hobo transposon, and the Tol2 transposon. The hAT family is composed of two families that have been classified as the AC subfamily and the Buster subfamily, based on the primary sequence of their transposases. Members of the hAT family belong to Class II transposable elements. Class II mobile elements use a cut and paste mechanism of transposition hAT elements share similar transposases, short terminal inverted repeats, and an eight base-pairs duplication of genomic target.

[0721] Compositions and methods of the discl osure may comprise a TcBuster transposon and/or a TcBuster transposase.

[0722] Compositions and methods of the disclosure may comprise a TcBuster transposon and/or a hyperactive TcBuster transposase. A hyperactive TcBuster transposase demonstrates an increased excision and/or increased insertion frequency when compared to an excision and/or insertion frequency of a wild type TcBuster transposase. A hyperactive TcBuster transposase demonstrates an increased transposition frequency when compared to a transposition frequency of a wild type TcBuster transposase.

[0723] In some embodiments of the compositions and methods of the disclosure, a wild type

TcBuster transposase comprises or consists of the amino acid sequence of:

1 MMLNWLKSGK LESQSQEQSS CYLENSNCLP PTLDSTDIIG EENKAGTTSR KKRKYDEDYL 61 NFGFTWTGDK DEPNGLCVIC EQWNNS SLN FAKLKRHLDT KHPTLKGKSE YFKRKCNELN 121 QKKHTFERYV RDDNKNLLKA SYLVSLRIAK QGEAYTIAEK LI KPCTKDLT TCVFGEKFAS 181 KVDLVPLSDT TISRRIEDMS YFCEAVLVNR LENAKCGFTL QMDESTDVAG LAILLVFVRY 241 IHESSFEEDM LFCKALPTQT TGEEI FNLLN AYFEKHSIPW NLCYHICTDG AKAMVGVIKG 301 VIARIKKLVF DIKASHCCLH RHALAVKRI P NALHEVLNDA VKMINFIKSR PLNARVFALL 361 CDDLGSLHKN LLLHTEVRWL SRGKVLTRFW ELRDEIRIFF NEREFAGKLN DTSWLQNLAY 421 IADI FSYLNE VNLSLQGPNS TIFKVNSRIN SIKSKLKLWE ECITKNNTEC FANLNDFLET 481 SNTALDPNLK SNILEHLNGL KNTFLEYFPP TCNNISWVEN PFNECGNVDT LPIKEREQLI 541 DIRTDTTLKS SFVPDGIGPF IKLMDEFPE I SKRAVKELM PFVTTYLCEK S FSVYVATKT 601 KYRNRLDAED DMRLQLTTIH PDI DNLCNNK QAQKSH

(GenBank Accession No. ABF20545 and SEQ ID NO: 14735).

SUBSTITUTE SHEET (RULE 26) [0724] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a sequence having at least 20%, 25%, 30%, 35%, 40%,

45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage identity in between to a wild type TcBuster transposase comprising or consisting of the ammo acid sequence of:

1 MMLNWLKS GK LESQSQEQSS CYLENSNCLP PTLDSTDI I G EENKAGTTSR KKRKYDEDYL 61 NFGFTWTGDK DEPKGLCVIC EQWNNSSLN PAKLKRHLDT KHPTLKGKSE YFKRKCN ELN 121 QKKHT FERYV RDDNKNLLKA SYLVSLRIAK QGEAYT IAEK LIKPCTKDLT TCVFGEKFAS 181 KVDLVPLSDT TISRRIEDMS YFCEAVLVNR LENAKCGFTL QMDESTDVAG LAI LLVFVRY 241 IHESSFEEDM LFCKALPTQT TGEEI FNLLN AYFEKHSIPW NLCYHICTDG AKAMVGVI KG 301 VIARI KKLVP DIKASHCCLH RHALAVKRI P NALHEVLNDA VKMINFIKSR PLNARVFALL 361 CDDLGSLHKN LLLHTEVRWL SRGKVLTRFW ELRDEIRIFF NEREFAGKLN DTSWLQNLAY 421 IADIFSYLNE VNLSLQGPNS TIFKVNSRIN SIKSKLKLWE ECITKNNTEC FANLNDFLET 481 SNTALDPNLK SNILEHLNGL KNTFLEYFPP TCNNI SWVEN PFNECGNVDT LPIKEREQLI 541 DIRTDTTLKS SFVPDGIGPF WIKLMDEFPE I S KRAVKELM PFVTTYLCEK SFSVYVATKT 601 KYRNP.LDAED DMRLQLTTIH PDI DNLCNNK QAQKSH

(GenBank Accession No. ABF20545 and SEQ ID NO: 14735).

[0725] in some embodiments of the compositions and methods of the disclosure, a wild type

TcBuster transposase is encoded by a nucleic acid sequence comprising or consisting of:

1 atgatgttga attggctqaa aag tggaaag "ttgaaagtc aa Oc caq q a acagagttcc 61 tgctaccttg agaactctaa ctgcctqcca ccaacgctcg attctacaqa tattatcggt 121 gaagagaaca a agctggtac cacctctcqc aagaagcgga aatatgacga gqac ta ctg 181 aacttcggtt ttacatggac tggcgacaag gatgagccca acggactttg tgtgatttgc 241 gagcaggtag tcaacaa t tc ctcacttaac ccggccaaac tqaaacgcca tttggacaca 301 aagcat ccga egettaaagg caaqaqcgaa tacttcaaaa gaaaatgtaa cgagctcaat 361 caaaagaagc a tacttttga gcgatacq ta aq gga cgata acaagaacct cctgaaagct 421 tcttatctcg tcaqtttgag aat agctaaa cagggcqaqg catataccat agcggagaag 481 ttgatcaagc cttgcaccaa ggatctgaca acttgcgtat ttggagaaaa attcgcgagc 541 aaagttgatc tcgtccccct gtccqacacq actatttcaa ggcgaatcqa aqacatqagt 601 tacttctgtg aagccgtgct ggtgaacagg ttgaaaaatg ctaaatgtgg gtttacgctg 661 cagatggacg aqtcaacaqa tgttgccggt cttgcaatcc tgcttgtgtt tgttaggtac 721 atacatgaaa gctcttttga ggaggatatg ttgttctgca aagcacttcc cactcagacg 781 acaggggagg a gattttcaa tcttctcaat qcctatttcg aaaagcactc catccca tgg 841 aatctgtgtt accacatttg cacagacggt gccaaggcaa tggtaggagt tattaaagga 901 gtcatagcga gaataaaaaa actcgtccct gatataaaaq ctaqccactg ttgcctgcat 961 cgccacgctt tggctgtaaa gcgaataccg aatgcattgc acgaggtgct caatgacgct 1021 gtta:a:aa tga tcaacttcat caaqtctcqq ccgttgaatg cgcgcgtctt cqctttqctg 1081 tgtgacgatt tqqqgagcct gcataaaaat cttcttcttc ataccgaagt gaggtggctg 1141 tctagaggaa aggtgctgac ccgattttgg gaactgagaq a tgaaattag aattttcttc 1201 aacgaaaggg aatttgccgg gaaattqaac gacaccagtt ggttgcaaaa tttggcatat 1261 atagctgaca tattcagtta tctgaatqaa q t taa t cttt ccctgcaagg gccqaa Cage 1321 acaatcttca aggtaaatag ccgc ttaac agtattaaat caaagttgaa gttgtgggaa 1381 gagtgtataa cgaaaaa taa ca ctgagtgt tttgcgaacc tcaacga ttt tttggaaact 1441 tcaaacactg cgttggatcc aaacctqaag tctaatattt tggaacatct caacggtctt 1501 aagaacacct ttctggagta ttttccacct acgtgtaata atatctcctg qq tqqaqaa t

1561 c.cttt caatg aatqcggtaa cgt cgataca ctcccaataa aagagaggga acaattgatt

1621 ga catacgga ctqa taeqae a ttgaaatct tcattcgtgc c Oqa tggtat aggaccattc 1681 tggatcaaac tgatggacga atttccaqaa attagcaaac gagctgtcaa aqaqctcatg 1741 ccatttgtaa ccacttacct ctgtgagaaa tcattttccg tctatgtagc cacaaaaaca 1801 aa tatcgaa ataqacttqa tgctgaagac gatatgcqac tccaacttac tactatccat

SUBSTITUTE SHEET (RULE 26) 1861 ccaq cattg acaacctttg taacaacaaq caggctcaga aatcccacbg a

(GenBank Accession No. DQ481197 and SEQ ID NO: 14736).

[Q726] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a sequence having at least 20%, 25%, 30%, 35%, 40%,

45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage identity in between to a wild type TcBuster transposase encoded by a nucleic acid sequence comprising or consisting of:

1 atqatgttga attggctgaa aagtggaaaq cttgaaagtc aatcacagga acaqagttcc

61 bgcbacc 11q a gaa ctctaa ctgcctgcca ccaacgctcg attctacaga babba cggb 121 gaagagaa ca aaqctqqta c cacctctcgc aagaagcqqa aatatgacga ggactatctg 18 1 aacttcggtt ttacatggac tggcgacaag gatgagccca acggactttg tgtgatttgc 24 1 qaqcaggtag t caa caa11c ctcac11a:a:c ccg g ccaaa c tgaaa cgcca 111qga ca ca 30 1 aagca tccga cgcttaaagg caagagcgaa tacttcaaaa g aaatgt a cgagctcaat 361 caaaagaagc atac1111ga g eg ataegta agggacqata: a:caag a a cct cctgaaagct 42 1 tettateteg tcagtttgag aatagctaaa cagggcgagg catataccat agcggagaag 4 8 1 ttqatcaagc cttgcaccaa ggatctqaca aettgegtat 11ggagaaaa a11cg cg a g c 541 aaagttgatc tcgtccccct gtccgacacg actatttcaa ggegaatega agacatgagt 60 1 tacttctgtg aaqccqtqct ggtgaacagg ttgaaaaatq ctaaatgtgg gtttacgctg 661 cagatgga cg agtcaacaga tgttgccggt cttgcaatcc tgcttgtgtt tgttaggtac 72 1 abaca gaaa gctcttttga ggaggatatg ttgttctgca aagcacttcc cacbcagacg 7 8 1 acaggggagg aqattttcaa tcttctcaat gcctatttcg aaaagcactc catcccatgg 84 1 aatctgtg11 a cca ca 111g ca cagacggt gccaaggcaa tggtaggagt tattaaagga 90 1 qtcatagcga gaataaaaaa act cqtccct gait at aaaag ctagccactq ttgcctgcat 961 cgccacgctt tggctgt aa gcgaataccq aatgcattgc acgaggtget caa tga cg ct 1021 gttaaaatga t caacttcat caagtct egg ccgttgaatg egegegt ett cgctttgctg 1081 tgtgacgatt tggggagcct gcataaaaat cbtcttcttc ataccgaagt gaggtggctg 114 1 tetagaggaa aggtgctgac ccgattttgg gaact gagag atgaaattaq aattttcttc 1201 aacqaaaqgg aatttgccgg gaaattgaac gacaccagtt ggttgcaaaa tttggcatat 12 61 atagctgaca tatt cagtta t ct gaatgaa gttaat ettt ccctgcaagg geegaatage 132 1 acaatcttca aggtaaatag ccgcattaac agtattaaat caaagttgaa gttgtgggaa 138 1 qaqtq ta ta a egaaaaataa cactqaqtqt 111gcg aacc tcaacga111 111qgaa a ct 144 1 tcaaacactg cgttggatcc aaacctgaag tetaatattt tggaacatct caacggtctt 1501 aagaa cacct 11ctqqaqta ttttccacct acgtgtaata atatctccbg ggtggagaat 1561 cctttcaatg aatgcggtaa cgtcgataca ctcccaataa aagagaggga acaattgatt 1621 qa cata cg ga ctgatacgac a11qaaatct tcattcgtgc ctgatggtat aqqaccattc 168 1 tggatcaaac tgatggacga atttccagaa attagcaaac gagctgtcaa agagctcatg 174 1 ccattbgbaa ccacttacct ctgtgagaaa tcattttccq tetatgtage c c aaaaca 18 0 1 aaatatcgaa atagaettga bgcbgaagac gatatgegae tccaacttac tactatccat 1861 ccaqacattg acaacctt.bg taacaacaaq caggctcaga aatcccactq a

(GenBank Accession No. DQ481 197 and SEQ ID NO: 14736).

[0727] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a naturally occurring ammo acid sequence.

[0728] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase comprises or consists of a non-naturally occurring amino acid sequence.

SUBSTITUTE SHEET (RULE 26) [0729] in some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase is encoded by a sequence comprising or consisting of a naturally occurring nucleic acid sequence.

[0730] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase is encoded by a sequence comprising or consisting of a non-naturally occurring nucleic acid sequence.

[0731] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the wild type TcBuster Transposase comprises or consists of the amino acid sequence of SEQ ID NO: 14735. In some embodiments, the wild type TcBuster Transposase is encoded by a sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 14736. In some embodiments, the one or more sequence variations comprises one or more of a substitution, inversion, insertion, deletion, transposition, and frameshift. In some embodiments, the one or more sequence variations comprises a modified, synthetic, artificial or non-naturally occurring amino acid. In some embodiments, the one or more sequence variations comprises a modified, synthetic, artificial or non-naturally occurring nucleic acid.

[0732] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises an amino acid substitution in one or more of a DNA Binding and Oligomerization domain, an insertion domain and a Zn-BED domain.

[0733] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises an ammo acid substitution that increases a net charge a neutral pH when compared to a wild type TcBuster Transposase. In some embodiments, the wild type TcBuster Transposase comprises or consists of the amino acid sequence of SEQ ID NO: 14735. In some embodiments, the wild type TcBuster Transposase is encoded by a sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 14736. In some embodiments, the one or more sequence variations comprises an ammo acid substitution of the aspartic acid (D) at position 223 (D223), the aspartic

SUBSTITUTE SHEET (RULE 26) acid (D) at position 289 (D289) and the aspartic acid (£) at position 589 (E289) of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an ammo acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 223, 289 and/or 289 of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 70 amino acids of position 223, 289 and/or 289 of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an amino acid substitution within 80 amino acids of position 223, 289 and/or 289 of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an amino acid substitution of an aspartic acid (D) or a aspartic acid (E) to a neutral ammo acid, a lysine (L) or an arginine (R) (e.g. D223L,

D223R, D289L, D289R, E289L, E289R of SEQ ID NO: 14735)

[0734] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of Q82E, N85S,D99A, D132A, Q15 IS, Q151A, EI 53K, E153R, A154P, Y155H,

El 59 A, T171K, T171R, K I77E, D183K, D183R, D189A, T191E, S193K, S193R, Y201A, F202D, F202K, C203I, C203V, Q221 T, M222L, I223Q, E224G, S225W, D227A, R239EI, E243A, E247K, P257K, P257R, Q258T, E263A, E263K, E263R, E274K, E274R, S278K, N28I E, L282K, L282R, K292P, V297K, K299S, A303T, H322E, A332S, A358E, A358K, A358S, D376A, V377T, L380N, I398D, I398S, I398K, F400L, V431 L, S447E, N450K, N450R, I452F, E469K, K469K, P510D, P510N, E517R, R536S, V553S, P554T, P559D, P559S, P559K, K573E, E578L, K590T, Y595L, V596A, T598I, K599A, Q615A, T618K, T618K,

T61 8R, D622K and D622R of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an ammo acid substitution within 5, 10, 1 5, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of ammo acids in between of position

154, 1 55, 159, 171, 177, 183, 189, 191 , 193, 201, 202, 203, 221, 223, 224, 225, 227, 239, 243,

247, 257, 258, 263, 274, 278, 281, 282, 292, 297, 299, 303, 322, 332, 358, 376, 377, 380, 398,

400, 431. 447, 450, 452, 469, 510, 517. 536, 553, 554, 559, 573, 578, 590. 595, 596, 598, 599,

615, 618, and 622 of SEQ ID NO: 14735.

[0735] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type

SUBSTITUTE SHEET (RULE 26) TcBuster Transposase. In some embodiments, the one or more sequence variations composes one or more of E247K, V297K, A358K, S278K, E247R, E274R, V297R, A358R, S278R,

T171 R, D183R, S193R, P257K, E263R, L282K, T618K, D622R, E153K, N450K, ΊT71K, D183K, S193K, P257R, E263K, L282R, T618R, D622K, El 53R and N450R of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an ammo acid substitution withm 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of ammo acids in between of position 153, 171, 183, 193, 247, 257, 263, 274, 278, 282, 297, 358, 450, 618, 622 of SEQ ID NO: 14735.

[0736] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of U377T/Έ469K, V377T/E469K/R536S, A332S, V553S/P554T, E517R, K299S, Q615A/T618K, S278K, A303T, P510D, PSION, N28IS, N281E, K590T, Q258T, E247K, S447E, N85S, V297K, A358K, I452F, V377T/E469K/D189A, K573E/E578L,

1452F/V377T/E469K/D 189 A, A358K/V377T/E469K/D 189 A,

K573E/E578L/V377T/E469K/D189A, T171R, D183R, S193R, P257K, E263R, L282K, T618K, D622R, E153K, N450K, T171 K, D183K, S193K, P257R, E263K, L282R, T618R, D622K, E153R, N450R, E247K/E274K/V297K/A358K of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an ammo acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of position 85, 153, 171 , 189, 193, 247, 257, 258, 263, 274, 278, 281, 282, 297, 299, 303, 332, 358, 377, 450, 469, 447, 452, 469, 510, 517, 536, 553, 554, 573, 578, 590, 615, 618, 622 of SEQ ID NO: 14735.

[0737] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of V377T/E469K, V377T/E469K/R536S, V553S/P554T, Q615A/T618K, S278K, A303T, P510D, P510N, N281 S, N281E, K590T, Q258T, E247K, S447E, N85S, V297K,

A358K, I452F, V377T/E469K/D189A and K573E/E578L. In some embodiments, the one or more sequence variations comprises an ammo acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of amino acids in between of

SUBSTITUTE SHEET (RULE 26) position 85, 189, 247, 258, 278, 281, 297, 303, 358, 377, 447, 452, 469, 510, 536, 553, 554, 573, 578, 590, 615, 618 of SEQ ID NO: 14735.

[0738] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of Q151S, Q151A, A154P, Q615A, V553S, Y155H, Y201A, F202D, F202K,

C203I, C203V, F400L, I398D, I398S, I398K, V431L, P559D, P559S, P559K, M222L of SEQ ID NO: 14735. In some embodiments, the one or more sequence variations comprises an ammo acid substitution within 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,

100 or any number of amino acids in between of position 151, 154, 615, 553, 155, 201, 202, 203, 400, 398, 431, 559, 222 of SEQ ID NO: 14735.

[Q739] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 14735.

[0740] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of K573E and E578L, when numbered in accordance with SEQ ID NO: 14735 [0741] In some embodiments, the mutant TcBuster transposase comprises amino acid substitution I452K, when numbered in accordance with SEQ ID NO: 14735.

[0742] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of A358K, when numbered in accordance with SEQ ID NO: 14735.

[0743] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of V297K, when numbered in accordance with SEQ ID NO: 14735.

SUBSTITUTE SHEET (RULE 26) [0744] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of N85S, when numbered m accordance with SEQ ID NO: 1790147350.

[0745] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of I452F, V377T, E469K, and D189A, when numbered in accordance with SEQ ID NO: 14735.

[Q746] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of A358K, V377T, E469K, and D! 89 A, when numbered in accordance with SEQ ID NO: 14735.

[0747] In some embodiments of the compositions and methods of the disclosure, a mutant TcBuster Transposase comprises one or more sequence variations when compared to a wild type TcBuster Transposase. In some embodiments, the one or more sequence variations comprises one or more of V377T, E469K, D189A, K573E and E578L, when numbered in accordance with SEQ ID NO: 14735

[0748] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5’ inverted repeat comprising or consisting of the sequence of:

1 Cagtgttctt caacctttgc catccggcgg aaccctttqt cgagatattt ttttttatgg

61 aacccttcat ttagtaatac acccagatga gattttaggg acagctgcgt tgacttgtta

121 cgaa;caag gt gagcccgtgc tttggtcta:q cca a gggcat ggtaaaga ct ata:t tcg cgg

181 cgttgtgaca atttaccgaa caactccgcg gccgggaagc cgatctcggc ttgaacgaat

241 tg11aggtgg eggta:c11gg gtegatatca aagtgcatca: c 11c11cccg tatgcccaac

301 tttgtataga gagccactgc gggategtea ccgtaatctg ettgeaegta gatcacataa

361 gcaccaagcg cgttggcctc atgettgagg agattgatga gcgcggtggc aatgccctgc

421 ctccggtgct cgccggagac tgegagatea tagatata

(SEQ ID NO: 14737).

In some embodiments of the compositions and methods of the disclosure, a TcBuster

Transposase recognizes a 3’ inverted repeat comprising or consisting of the sequence of:

1 gatatcaagc ttatcgatac cgtcgacctc qagatttctg aaegatteta ggttaggatc

61 aaacaaaata caatttattt taaa.ac.tgta ag aactta cc get tg t ctaaaccaa

121 aaacaacaac aaaactacga ccacaagtac agttacata t tfcfcfcgaaaat taaggttaag

181 tgcagtgtaa gtc.aactatg egaatggata acatgtttca acatgaaact ccgattgacg

5UB5TITUTE SHEET (RULE 26) 241 catgtgca11 ctgaaqaq cg gcgcggccga cgtctctcga a t tga a gcaa tgactcgcgg

301 aaccccgaaa gcctttgggt ggaaccctag ggttccgcgg aacacaggtt gaagaacact

361 g

(SEQ ID NO: 14738).

[0749] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5’ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 14737 and a 3’ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 14738.

[0750] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5’ inverted repeat comprising or consisting of the sequence of:

1 Cctqcaqqag tgttcttcaa cctttgccat ccggcggaac cctttgtcga gatafcfcfcttt

61 tttatggaac cctt cattta gt aatacacc cagatgagat tttagggaca gctgcgttga

121 cttgttacga acaaqqtqaq cccgtgcttt ggtaataaaa actctaaata agatttaaat

181 ttgcatttat ttaaacaaac tttaaacaaa aagataaata ttccaaataa aataatatat

241 aaaataaaaa ataaaaatta atgacttttt tqcgcttgct tattattgca caaattatca

301 atatcgggat ggatcgttgt ttt tt

(SEQ ID NO: 14739)

[0751] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 3’ inverted repeat comprising or consisting of the sequence of:

1 Gagccaattc aqcatcatat ttctgaacga ttctaggtta ggatcaaaca aaatacaatt

61 tattttaaaa ctgtaagtta acttaccttt gcttgtctaa acctaaaaca acaacaaaac

121 tacgaccaca agtacagtta catatttttg aaaattaagg ttaagtgcag tgtaagtcaa

181 ctatgcqaat ggataacatg tttcaacatq aaactccgat tgacgcatgt gcattctgaa

241 gagcggcgcg gccgacgtct ctcgaattga agcaatqact cgcggaaccc cgaaagcctt

301 tgggtggaac cctagggttc cgcggaacac aggttgaaga acactg

(SEQ ID NO: 14740).

[0752] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a 5’ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 14739 and a 3’ inverted repeat comprising or consisting of the sequence of SEQ ID NO: 14740.

[0753] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95,% 97%, 99% or any percentage identify in between to one or more of SEQ ID NO: 14737, 14738, 14739 or 14740.

[Q754] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least

SUBSTITUTE SHEET (RULE 26) In some embodiments of the compositions and methods of the disclosure, a TcBuster

Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or any number of contiguous nucleotides in between having between 90 and 100% identity to SEQ ID NO: 14737, 14738, 14739 or 14740 or any portion thereof.

[0755] In some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes an inverted repeat comprising or consisting of a sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 99 or any number of discontinuous nucleotides in between having between 90 and 100% identity to SEQ ID NO: 14737, 14738, 14739 or 14740 or any portion thereof.

[0756] In some embodiments of the compositions and methods of the disclosure, a

TcBuster transposon comprises a 3’ inverted repeat and a 5’ inverted repeat in some embodiments of the compositions and methods of the disclosure, a TcBuster Transposase recognizes a TcBuster transposon comprising a 3’ inverted repeat and a S’ inverted repeat comprising or consisting of a sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,

55, 60, 65, 70, 75, 80 85, 90, 95, 97, 99 or any number of discontinuous nucleotides in between having between 90 and 100% identity to SEQ ID NO: 14737, 14738, 14739 or 14740 or any portion thereof.

[0757] In some embodiments of the compositions and methods of the disclosure, a piggyBat transposase of the disclosure is encoded by an ammo acid sequence comprising:

MSQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDS

ESDIEGGREEWSHVDNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEE SNR

YYNQNRNNFKLSKKSLKWKDITPQEMKKFLGLIVLMGQVRKDRRDDYWTTEPWTETP

YFGKTMTRDRFRQIWKAWHFNNNADIVNESDRLCKVRPVLDYFVPKFINIYKPHQQL SL

DECdVPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGEGKRLLETIQ TVV

SPYTDSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETK FIR

KNDILLQVWQSKKPVYUSSIHSAEMEESQNIDRTSKKKIVKPNALIDYNKHMKGVDR A

DQYLSYYSILRRTVKWTKRLAMYMINCALFNSYAVYKSVRQRKMGFKMFLKQTAIHW

LTDDIPEDMDIVPDLQPVPSTSGMRAKPPTSDPPCRLSMDMRKHTLQAIVGSGKKKN ILR

RCRVCSVHKLRSETRYMCKFCNIPLHKGACFEKYHTLKNY (SEQ ID NO: 14965).

SUBSTITUTE SHEET (RULE 26) [0758] In some embodiments of the compositions and methods of the disclosure, a piggy Bat transposase of the disclosure is encoded by an ammo acid sequence comprising:

atgtcgcagcattcagactatactcatgatgagttttgtgcagacaagttgtccaat tattcttgtgatagcgatcttgaaaatgcgagtacaagt gatgaagattctagtgatgatgaagtaatggtgcgtcccaggacattgaggcgacgaaga atttcgagctccagctctgactcagagtcaga tatagaaggcgggagagaagaatggtcgcatgttgataatccaccggtcttagaagattt tttagggcatcaaggattaaacacagatgctgt tataaataatatagaagatgccgtgaaattatttatcggagatgatttttttgaatttct tgtagaggagtcaaacaggtattataatcaaaatagga ataatttcaaactttcaaaaaaaagcctaaagtggaaagatataacccctcaagagatga agaagtttttagggttaattgttctcatgggacag gtgcgcaaagatagaagagatgactattggaccacggagccatggacggagacgccatat tttggtaaaacgatgacgagagacaggttc cgacagatatggaaagcttggcacttcaataataatgcggatatcgtaaatgaatcagat agactttgcaaagtgagaccagtactagattatt ttgtgcctaaatttataaatatttacaaacctcatcagcaattatcactagatgaaggga tcgtaccttggaggggaagattattctttagggtata taatgctggcaagatcgttaaatatggaatattggttcgtttgttgtgcgaaagtgatac aggatatatctgtaacatggaaatttattgcggcga aggaaagcgattattggaaacgatacaaacagtagtgtctccatacactgattcgtggta ccatatatatatggacaattattataatagcgtcg caaattgtgaagcacttatgaaaaacaaattcagaatatgtggaacaatccggaaaaatc gaggtatacctaaagattttcaaacaatttctttg aaaaaaggtgaaacaaaatttataaggaaaaatgatatattgttacaagtgtggcaatca aaaaagcctgtatacctgatttcttcgattcattct gcggagatggaagaaagtcagaatattgacagaacatcaaaaaagaaaattgtcaaaccg aatgcactcattgactacaataaacatatga aaggtgttgaccgggccgaccaatacctttcatattattcgatattgcggaggacggtca aatggacaaaaaggttggcaatgtatatgataa attgcgcattatttaattcttatgcagtttacaaatcagtgaggcaaagaaaaatgggtt ttaaaatgtttttgaaacaaacagctatccactggtt gacggatgatattccagaggacatggaca ttgttccagacc ttcaaccagtaccgtc tacttctggaatgcgggctaaaccacctacatctga tccaccatgcaggctatcgatggacatgagaaagcatacgttacaggcaattgtcggaag tggaaaaaagaaaaacattttgagaaggtgt cgcgtatgttccgttcataaattgcgcagtgagacacgctacatgtgcaaattttgcaat atacctctacataaaggggcgtgttttgaaaaatat catacgctaaaaaactattaa (SEQ ID NO: 14966).

[0759] In some embodiments of the compositions and methods of the disclosure, a piggyBat transposase of the disclosure is encoded by an ammo acid sequence comprising:

MPSLRKRKETNETDTLPEVFNDNLSDIPSEIEDADDCFDDSGDDSTDSTDSEIIRPVRKR K

VAVLSSDSDTDEATDNCWSE1DTPPRLQMFEGHAGVTTFPSQCDSVPSVTNLFFGDE LFE

MLCKELSNYHDQTAMKRKTPSRTLKWSPVTQKDIKKFLGLIILMGQTRKDSLKDYWS T

DPLICTPIFPQTMSRHRFEQIWTFWHFNDNAKMDSRSGRLFKIQPVLDYFLHKFRTI YKP

KQQLSLDEGMIPWRGRFKFRTYNPAKITKYGLLVRMVCESDTGYICSMEIYTAEGRK LQ

ETVLSVLGPYLGIWHHIYQDNYYNATSTAELLLQNKTRVCGTIRESRGLPPNLEMKT SR

MKKGDIIFSRKGDILLLAWKDKRVVRMISTIHDTSVSTTGKKNRKTGENIVKPTCIK EYN

AHMKGVDRADQFLSCCSILRKTMKWTKKWLYLINCGLFNSFRVYNVLNPQAKMKYK

SUBSTITUTE SHEET (RULE 26) QFLLSVARDWITDDNNEGSPEPETNLSSPSPGGARRAPRKDPPKRLSGDMKQHEPTCIPA SGKKKFPTRACRVCAAHGKRSESRYLCKFCLVPLHRGKCFTQYHTLKKY (SEQ ID NO:

14967).

[0760] In some embodiments of the compositions and methods of the disclosure, a piggyBat transposase of the disclosure is encoded by an amino acid sequence comprising:

ATGCCCTCTCTCAGAAAAAGGAAGGAAACCAACGAAACTGATACACTTCCGGAAGT

ATTTAACGATAATTTATCAGATATTCCTAGTGAGATCGAAGATGCGGATGACTGTTT

TGACGATTCCGGAGACGATTCTACTGATTCTACTGACAGTGAAATTATTAGACCTGT

AAGGAAGCGCAAGGTGGCGGTGCTTTCAAGTGATTCCGACACTGACGAAGCTACTG

ATAATTGTTGGTCTGAAATTGACACACCACCACGCTTACAAATGTTTGAAGGTCATG

CTGGGGTCACTACATTTCCGTCTCAGTGTGACTCTGTACCCTCTGTGACCAATCTCT T

TTTT GGT GAT GAATT GTTT GAGAT GTT GT GC A A AGAGCTGT CC A ACT AT C AC GAT C A

AACCGCAATGAAACGCAAAACACCATCTAGAACACTAAAGTGGTCTCCGGTTACAC

AGAAGGACATCAAGAAATTCCTTGGCCTAATTATTCTGATGGGTCAAACAAGAAAA

GATAGCTTGAAAGACTATTGGTCAACAGATCCTTTGATATGTACCCCTATATTTCCA

CAGACAATGAGTCGCCATAGATTTGAGCAAATATGGACATTCTGGCATTTCAATGAT

AACGCCAAAATGGACAGTCGCTCGGGGAGACTTTTCAAGATCCAACCTGTGCTGGA

TTATTTCCTGCATAAATTTCGAACAATATACAAACCAAAGCAACAGTTGTCTTTGGA

CGAGGGAATGATTCCATGGAGAGGACGTTTCAAATTTCGCACGTACAACCCAGCGA

AAATAACAAAATACGGTTTACTTGTTCGGATGGTGTGCGAGAGTGACACCGGCTAT

AT CT GC AGT ATGG AG A T A T A C ACTGCT G A AGG A A GG A A A TT GCA AGA A ACT GTT CT

TTCAGTCCTTGGACCCTATCTTGGCATATGGCACCATATTTACCAGGATAATTATTA C

AATGCTACATCTACTGCTGAATTGCTGCTACAGAACAAAACTAGAGTCTGTGGGACT

ATTAGGGAGAGTAGAGGTTTACCGCCAAATTTGGAAATGAAAACATCAAGAATGAA

GAAAGGTGACATAATATTTTCCAGAAAAGGCGATATTCTTCTCCTAGCATGGAAAG

ACAAGCGGGTTGTCCGAATGATATCAACGATCCATGACACTTCTGTCTCGACAACAG

GAAAAAAAAATAGAAAAACGGGAGAGAATATTGTAAAACCTACCTGCATCAAGGA

ATACAATGCCCACATGAAAGGCGTTGACCGTGCGGATCAATTCCTTTCGTGTTGTTC

CATTCTAAGGAAAACGATGAAATGGACAAAAAAAGTAGTGCTGTACCTTATAAACT

GTGGACTTTTCAATTCATTTAGAGTGTACAACGTCCTCAATCCACAAGCAAAAATGA

AGTATAAACAGTTTCTGCTATCGGTGGCGAGAGACTGGATAACGGATGACAATAAT

SUBSTITUTE SHEET (RULE 26) GAAGGCTCTCCGGAACCAGAGACAAATCTGTCCAGCCCTTCCCCTGGGGGTGCAAG

GAGAGCACCTCGTAAAGATCCACCCAAAAGGTTGTCAGGTGATATGAAGCAGCATG

AACCTACGTGTATTCCAGCGAGTGGAAAGAAAAAATTTCCTACGAGAGCCTGCAGA

GTTTGTGCCGCCCATGGAAAAAGGAGCGAATCTAGATACTTATGTAAATTTTGTTTG

GTCCCTCTTCATAGAGGAAAATGTTTTACGCAGTACCATACGTTAAAAAAGTACTAG

(SEQ ID NO: 14968).

[0761] In some embodiments of the compositions and methods of the disclosure, a piggyBat transposon of he disclosure is encoded by an amino acid sequence comprising:

CACTTGGATTGCGGGAAACGAGTTAAGTCGGCTCGCGTGAATTGCGCGTACTCCGC

GGGAGCCGTCTTAACTCGGTTCATATAGATTTGCGGTGGAGTGCGGGAAACGTGTA

AACTCGGGCCGATTGTAACTGCGTATTACCAAATATTTGTTTGTTTGCCGTTCACAA

AGATACCTACCTGCGTATGCGAACCGTTTCATACCCATTGGGGTAATAGGTATCATA

AAAATCCCTTGTAAGTAGAGAACACTTTATGGCGCTGGTCGAGTACAATACCGTTAG

GGTACCAATGTATTGTTTTCTCATTTTATAACCGATATCCCTGGATATCTCACCATG C

AAAGAGAATAGTTATTGAGAAATACTTCTTGTGTCCGGACGTCTTTAGTTTTGAGTT

TTGTGAATATTATTACGTGTTTAAATATTTAAATATATTGTGATAAAGACATTCATA T

AGAATTTTTTAACCAAATAAAATAAAATCGTTCCGCAAAGTGTTCGCATAATAATTA

TTTTTTT ATATCAGATATCT ATCGGAGTACCGCT ATTTTC AGTTAGT ATT ATTTTT ATT

TGCTTTAACCTAGAATGTCGCAGCATTCAGACTATAGTGATGATGAGTTTTGTGCAG

A C A AGTT GT CCA ATT ATT CTTGT G AT AGC GAT CTT GA A A AT GCGAGT A C A AGT GAT G

AAGATTCTAGTGATGATGAAGTAATGGTGCGTCCCAGGACATTGAGGCGACGAAGA

ATTTCGAGCTCCAGCTCTGACTCAGAGTCAGATATAGAAGGCGGGAGAGAAGAATG

GTCGCATGTTGATAATCCACCGGTCTTAGAAGATTTTTTAGGGCATCAAGGATTAAA

CACAGATGCTGTTATAAATAATATAGAAGATGCCGTGAAATTATTTATCGGAGATGA

_TTTTTTT GA _ATTTCTT G _TAGA GG _AGTC A _AACA GG _TATTAT A _ATCA A _AATA GGAATAA

TTTCAAACTTTCAAAAAAAAGCCTAAAGTGGAAAGATATAACCCCTCAAGAGATGA

AGAAGTTTTTAGGGTTAATTGTTCTCATGGGACAGGTGGGCAAAGATAGAAGAGAT

GACTATTGGACCACGGAGCCATGGACGGAGACGCCATATTTTGGTAAAACGATGAC

GAGAGACAGGTTCCGACAGATATGGAAAGCTTGGCACTTCAATAATAATGCGGATA

TCGTAAATGAATCAGATAGACTTTGCAAAGTGAGACCAGTACTAGATTATTTTGTGC

CTAAATTTATAAATATTTACAAACCTCATCAGCAATTATCACTAGATGAAGGGATCG

SUBSTITUTE SHEET (RULE 26) TACCTTGGAGGGGAAGATTATTCTTTAGGGTATATAATGCTGGCAAGATCGTTAAAT

ATGGAATATTGGTTCGTTTGTTGTGCGAAAGTGATACAGGATATATCTGTAACATGG

AAATTTATTGCGGCGAAGGAAAGCGATTATTGGAAACGATACAAACAGTAGTGTCT

CCATACACTGATTCGTGGTACCATATATATATGGACAATTATTATAATAGCGTCGCA

AATTGT GAAGC AC TT ATGAAAAAC AAATTC AGAAT ATGT GGAAC A ATC C GGAAAAA

TCGAGGTATACCTAAAGATTTTCAAACAATTTCTTTGAAAAAAGGTGAAACAAAATT

TATAAGGAAAAATGATATATTGTTACAAGTGTGGCAATCAAAAAAGCCTGTATACC

TGATTTCTTCGATTCATTCTGCGGAGATGGAAGAAAGTCAGAATATTGACAGAACAT

CAAAAAAGAAAATTGTCAAACCGAATGCACTCATTGACTACAATAAACATATGAAA

GGTGTTGACCGGGCCGACCAATACCTTTCATATTATTCGATATTGCGGAGGACGGTC

AAATGGACAAAAAGGTTGGCAATGTATATGATAAATTGCGCATTATTTAATTCTTAT

GCAGTTTACAAATCAGTGAGGCAAAGAAAAATGGGTTTTAAAATGTTTTTGAAACA

AACAGCTATCCACTGGTTGACGGATGATATTCCAGAGGACATGGACATTGTTCCAG

ACCTTCAACCAGTACCGTCTACTTCTGGAATGCGGGCTAAACCACCTACATCTGATC

CACCATGCAGGCTATCGATGGACATGAGAAAGCATACGTTACAGGCAATTGTCGGA

AGT GGAAAAA AGAAAAAC ATTTT GAGAAGGT GT CGCGT AT GTT CCGTT CAT AA ATT

GCGCAGTGAGACACGCTACATGTGCAAATTTTGCAATATACCTCTACATAAAGGGG

CGTGTTTTGAAAAATATCATACGCTAAAAAACTATTAATTTCTATTTGTTTTGATTT A

TT ATGAAA ATTTT ATTTT A AACTTTA AA A ATATGTGTATATTTTTAGGTA AT AAGTAT

TTTTTGGGTTTTTCGTATTTAGTTTATATATATCGAATTATTTATGTACTGAATAGA T

AAAAAAATGTCTGTGATTGAATAAATTTTCATTTTTTACACAAGAAACCGAAAATTT

CATTTCAATCGAACCCATACTTCAAAAGATATAGGCATTTTAAACTAACTCTGATTT

TGCGCGGGAAACCTAAATAATTGCCCGCGCCATCTTATATTTTGGCGGGAAATTCAC

CCGACACCGTAGTG (SEQ ID NO: 14963).

[0762] In some embodiments of the compositions and methods of the disclosure, a piggyBat transposon of he disclosure is encoded by an ammo acid sequence comprising:

CACATTGCGTACCGCTCACGAGTTTTCTCGTGTTTCGCGCGCCATCTGTTAAGGACC

GCTCACGAGTGTTCTCGTTTTTCACGCGCCATCTGTTATGGACCTTAGATGTCAACA C

ACTGTCTTGTCCACTGTGGGGCGCGGTTACAGTGTTTTGGCCAGGTTCAAGCCTCGG

ACTAATGAAAGGACAGGGTCCTCTCACTGCCACGTGCAAGTCCCAGCTGGAGGGCA

GGGCCCTCCCAGCACAATCATAGCCAACGGCTGTGGTTGTAAGCTTGAACCTATGGT

SUBSTITUTE SHEET (RULE 26) CCGAACACGTAGCCCCACGTGCCTTGTGATAGAGTTCGGGTGCATGTAGTTGAGTAG

GGTTGAGACTCACGAGAATGCTGTAAACAACGTGATCACGCCCCTACTTTGCCTCGT

GGCATCTGCTATAAAATAAAGACACGGCTTGTGGGCGCTGGCGTTGTCTCCTCTTCA

GGGAGCAGCGTCCCACCGAGACCCAGCTATTATTCTCTTGTCTGTCTTTCCTTAATC C

TTTCACCCCCCCACTCAGAGACACCCTTGGCCGTGCTGGCGCGGCACAGTCCACATC

GAATGAAGCGATCTCATTGGTGGAAACCGTGCAGGTCAATCTACGAAAAACTATAT

AATTGCACGAACCCATAAAGCATTGCAGTTACATTGTATTTTGGTCATTCGAATAGT

CTTCGTCTTCAAGTTCCTGGCGCTTTTAGAAATGCCCTCTCTCAGAAAAAGGAAGGA

AACCAACGAAACTGATACACTTCCGGAAGTATTTAACGATAATTTATCAGATATTCC

T AGT GAGAT CGA AGAT GCGGAT GACTGTTTT GAC GATT CCGG AGACGATT CT ACT GA

TTCTACTGACAGTGAAATTATTAGACCTGTAAGGAAGCGCAAGGTGGCGGTGCTTTC

AAGTGATTCCGACACTGACGAAGCTACTGATAATTGTTGGTCTGAAATTGACACACC

ACCACGCTTACAAATGTTTGAAGGTCATGCTGGGGTCACTACATTTCCGTCTCAGTG

TGACTCTGTACCCTCTGTGACCAATCTCTTTTTTGGTGATGAATTGTTTGAGATGTT G

TGCAAAGAGCTGTCCAACTATCACGATCAAACCGCAATGAAACGCAAAACACCATC

TAGAACACTAAAGTGGTCTCCGGTTACACAGAAGGACATCAAGAAATTCCTTGGCC

TAATTATTCTGATGGGTCAAACAAGAAAAGATAGCTTGAAAGACTATTGGTCAACA

GATCCTTTGATATGTACCCCTATATTTCCACAGACAATGAGTCGCCATAGATTTGAG

CAAATATGGACATTCTGGCATTTCAATGATAACGCCAAAATGGACAGTCGCTCGGG

GAGACTTTTCAAGATCCAACCTGTGCTGGATTATTTCCTGCATAAATTTCGAACAAT

ATACAAACCAAAGCAACAGTTGTCTTTGGACGAGGGAATGATTCCATGGAGAGGAC

GTTTCAAATTTCGCACGTACAACCCAGCGAAAATAACAAAATACGGTTTACTTGTTC

GGATGGTGTGCGAGAGTGACACCGGCTATATCTGCAGTATGGAGATATACACTGCT

GAAGGAAGGAAATTGCAAGAAACTGTTCTTTCAGTCCTTGGACCCTATCTTGGCATA

TGGCACCATATTTACCAGGATAATTATTACAATGCTACATCTACTGCTGAATTGCTG

CTACAGAACAAAACTAGAGTCTGTGGGACTATTAGGGAGAGTAGAGGTTTACCGCC

AAATTTGGAAATGAAAACATCAAGAATGAAGAAAGGTGACATAATATTTTCCAGAA

AAGGCGATATTCTTCTCCTAGCATGGAAAGACAAGCGGGTTGTCCGAATGATATCA

ACGATCCATGACACTTCTGTCTCGACAACAGGAAAAAAAAATAGAAAAACGGGAG

AGAATATTGTAAAACCTACCTGCATCAAGGAATACAATGCCCACATGAAAGGCGTT

GACCGTGCGGATCAATTCCTTTCGTGTTGTTCCATTCTAAGGAAAACGATGAAATGG

SUBSTITUTE SHEET (RULE 26) ACAAAAAAAGTAGTGCTGTACCTTATAAACTGTGGACTTTTCAATTCATTTAGAGTG

TACAACGTCCTCAATCCACAAGCAAAAATGAAGTATAAACAGTTTCTGCTATCGGTG

GCGAGAGACTGGATAACGGATGACAATAATGAAGGCTCTCCGGAACCAGAGACAA

ATCTGTCCAGCCCTTCCCCTGGGGGTGCAAGGAGAGCACCTCGTAAAGATCCACCC

AAAAGGTTGTCAGGTGATATGAAGCAGCATGAACCTACGTGTATTCCAGCGAGTGG

AAAGAAAAAATTTCCTACGAGAGCCTGCAGAGTTTGTGCCGCCCATGGAAAAAGGA

GCGAATCTAGATACTTATGTAAATTTTGTTTGGTCCCTCTTCATAGAGGAAAATGTT T

TACGCAGTACCATACGTTAAAAAAGTACTAGGAACTTTAATTGTTTAATTGTTTTTG T

AAATAAAAATGTTATAATTATTGAAAAACAACACCTAAAGTGCATTATGATCTGTAG

TTATGATGATTTAAATAACGTGCAGTTTGCCCAAAAACGTGTGGTCCCTGGCGTATG

TCTTAGAGATTTCTATGCGGTACGCAATGTG (SEQ ID NO: 14964).

[0763] Gene editing compositions of the disclosure may comprise a nuclease protein or a nuclease domain thereof. In some embodiments, the gene editing composition comprises a sequence encoding a nuclease protein or a sequence encoding a nuclease domain thereof. In some embodiments, the sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof comprises a DNA sequence, an RNA sequence, or a combination thereof. In some embodiments, the nuclease or the nuclease domain thereof comprises one or more of a CRISPR/Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease. In some embodiments, the nuclease or the nuclease domain thereof comprises one or more of a nuclease-inactivated Cas (dCas) protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease. In some embodiments, the nuclease or the nuclease domain thereof comprises a nuclease-inactivated Cas (dCas) protein and an endonuclease. In some embodiments, the nuclease or the nuclease domain thereof comprises a nuclease-inactivated Cas9 (dCas9) protein and an endonuclease, wherein the endonuclease comprises a Clo051 nuclease or a nuclease domain thereof. In some embodiments, the gene editing composition comprises a fusion protein. In some embodiments, the fusion protein comprises a nuclease-inactivated Cas9 (dCas9) protein and a Clo05l nuclease or a CloOSl nuclease domain. In some embodiments, the gene editing composition further comprises a guide sequence. In some embodiments, the guide sequence comprises an RNA sequence.

SUBSTITUTE SHEET (RULE 26) [0764] in some embodiments, the gene editing composition comprises a fusion protein. In some embodiments, the fusion protein comprises a nuclease-inactivated Cas9 (dCas9) protein and a Clo05l nuclease or a CloOSI nuclease domain. In some embodiments, the gene editing composition further comprises a guide sequence. In some embodiments, the guide sequence comprises an RNA sequence. In some embodiments, the fusion protein comprises or consists of the amino acid sequence:

MAPKKKRKVEGI KSNI SLLKDELRGQI SHI SHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDG IVYSTTLEDNFGI IVDTKAYSEGYSLPI SQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFI SGS FKGKF EEQLRRLSMTTGVNGSAVNWNLLLGAEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDK KYSIGLAI GTNSVGWA VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRI CYLQEI FSNEMAKVDDS

FFHRLEES FLVEEDKKHERHPI FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD

LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTP NFKSN FDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMI KRYDE HHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFI KPI LEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS GEQKKAIVDLLFKTNRK VTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ VSGQGDSLHEHIANLAGS PAIKKGI L0TVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRI EEGI K ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD DSIDNKVLTRSDKNRGK SDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI TKHVAQILDSRMNTKY DENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALI KKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKK TEVQTGGFSKES ILPKRN SDKLIARKKDWDPKKYGGFDS PTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSS E ^' EKNPI DFLEAKGYKEVKKDLI I KLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEI IEQI SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFD TTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGDGS PKKKRKVSS (SEQ ID NO: 14498} .In SOtne embodiments, the fusion protein is encoded by a nucleic acid comprising or consisting of the sequence:

1 atggcaccaa agaagaaaag aaaaqtqqag ggcat caagt caaacatcag cctgctgaaa 61 gacgaactgc ggggacagat tagtcaca tc aq tea egagt acctgtcact ga t tqa etg 121 gc.c.ttcgaca gcaaqcagaa tagactgttt gagatgaaag tgctggaact gctggtcaac 181 gagtatgget tcaagggcag acatctgggc gggtctagga aacctgacgg catcgtgtac 241 agtaccacac tggaagacaa cttcggaatc attgtegata ccaaggctta ttccgagggc 301 tactctctgc caattagtca ggcagatgag atggaaaggt aegtgegega aaactcaaat 361 agggaegagg aagtcaaccc caataagtgg tgggagaatt teagegagga agtgaagaaa 421 tactacttcg tctttatctc aggcagcttc aaagggaagt ttgaggaaca getgeggaga 481 ctgtccatga cta ccggggt gaacggatct gctgtcaacg tggtcaatct gctgctgggc 541 gcagaaaaga tcaggtccgg ggagatgaca attgaggaac tggaaegege catgt tcaac

SUBSTITUTE SHEET (RULE 26) 601 aattctgagt ttatcctqaa q ta tggagge gggggaagcg ataaqaaata ctccatcgga

661 ctqgccattg gcaccaattc cgtgggctgg gctgtcatc.a cagacgag a caaggtgcca

721 agcaagaag t tcaaggtcct ggggaacacc qa tcgccaca gtatcaagaa aaatctgatt

781 ggagccctgc tgttcgactc aggcgagact gc gaagcaa cccgactgaa gcggactgct

841 aggcgccgat atacccggag aaaaaatcgg atctgctacc tq caqqaaa t tttcagcaac

901 gagatggcca aggtggacga tagtttcttt caccgcctgg aggaatcatt cctggtggag

961 gaaqa taaq a aacacgagcg gcatccca c t t tggea aca ttgtggacga ag teqe t tat 1021 cacgagaagt accctactat ctat c.atctg aggaagaaac tggtggact c. caccgataag 1081 gcagacctgc gcctga tc ta tctggccctg gctcaca tga tcaaq t teeg ggggcatttt 1141 ctgatcgagg gagatctgaa ccctqacaat tctgatgtgg acaagctgtt catccagctg 1201 gtccagacat acaatcagct gtttgaggaa aacccaatta atgcct cagg cgtggacgca 1261 aaggccatcc tgaqcqccaq a ctg t cca a a tctaggcgcc tqqaaaacct gatcgctcag 1321 ctgccaggag agaagaaaaa cggcctgttt gggaatctga ttgcactgtc cctgggcctg 1381 acacccaact tcaagtctaa ttttgatctg geegaggaeg etaagetgea qctqtccaaa 1441 gacacttatg acgatgacct ggataacctg ctggctcaga teggegatea gtacgcagac 1501 ctgttcctgg ccqctaaqaa tctgagtgac gccatcctgc tqtcaqatat tctgcgcgtg 1561 aacacagaga ttactaaggc cccactgagt gcttcaatga tcaaaagata tgaegageae 1621 catcaggatc tgaccctgct gaagqctctq qtqaggcagc agctgcccga qaaatacaag 1681 gaaatcttct ttgatcagag caagaatgga tacgccggct atattgaegg cggggc cc 1741 caggaggagt tctacaaqt t ca tcaagccc at ctggaaa aqatqqacgg caccgaggaa 1801 ctqctggtga agetgaateg ggagqacctg ctgagaaaac agaggacatt tqataacgga 1861 agca tccctc a ccagattca tctgggcgaa c tq ca cgcca tcctgcgacg gcaqqaqqac 1921 ttctacccat ttctqaagga taacc.gc.gag aaaatcgaaa agatcctgac cttcagaatc 1981 ccctactatg tggggcctct q gca cgggga aa agtaga t t tq cc tgga t gacaagaaag 2041 tcagaggaaa ct atcacccc ctggaacttc gaggaagtgg tcgataaagq cqctagcgca 2101 cag tcc t tea ttgaaaggat gacaaatt 11 gacaagaacc tgccaaatga gaaqq tqctg 2161 cccaaacaca gcctqctgta egaatattte. acagtgtata aegagetgae taaagtgaag 2221 tacgtcaccg aagggatgcg caagcccgca ttcctgtccg qaqaqcagaa gaaagccatc 2281 qtqgacctgc tgtttaagac aaatcqqaaa gtgactgtca aacagctgaa qqaagactat 2341 ttcaaqaaaa ttgagtgttt cgattcag tq qaaa tea geg gegtegagga caqq 11 Caa c 2401 gcctccctgg ggacctacca cgatctgctg aagatcatca aqqataagga cttcctggac 2461 aacgaggaaa atgaggaca t cctggaggac attgtgctga cactgactct gtttgaggat 2521 cqcqaaatga tegaggaaeg actgaagact a tgcccatc tgttcgatga caaaqtqa tg 2581 aagcagctga aaagaaggcg ctacaccgga tggggacgcc tgagccgaaa actga tcaat 2641 gggattagag acaaqcaqaq cggaaaaact atcctggact ttctqaagtc cgatggcttc 2701 gccaacagga acttcatgca gctgattcac gatgactctc tgaccttcaa ggaggacatc

2761 caqaaagcac aggtgtctgg ccagqqqqac aqtctgcacg ageatatege aaacctgg cc 2821 ggcagccccg ccatcaagaa agggattc tg cagaccgtga aggtggtgga cgaactggtc 2881 aaggtcatgg gacgacacaa a cctgagaac atcgtgattg agatggcccg cgaa atcag 2941 acaactcaga agggccagaa aaacaqtcga gaacggatga agagaatega qqaaggeat c

3001 aagqaqctqg ggtcacagat cctgaaggag ca. tcctgtgg aaaacactca gc tgcagaa t 3061 gagaaactgt atctqtacta tctgc.agaat ggacgggata tgtacgtgga ccaggagctg 3121 gatattaaca gactgag tga t ta tgacgtg gatgcca teg tccctcagag cttcctgaag 3181 qatgactcca ttgacaacaa ggtgctqacc aggt ccgac.a agaaccgcgg caaatcagat 3241 aatq tq ccaa gegaggaagt ggtcaagaaa a. tqaaga act actggaggca gc tqc tqaa t 3301 gccaagctga tcacacagcg gaaatttgat aacctgacta aggeagaaag aggaggcctg 3361 tctgagctgg acaaggccgg cttcatcaag eggeage tgg tqqaqacaag acagatcact 3421 aaqcacq teg ctcagattct ggataqcaqa a tqaa ca caa agtacgatga aaacqacaag 3481 ctgatcaggg aggtgaaagt cattactc tg aaatccaagc tggtgtctga ctttagaaag 3541 gatttccagt tttataaaqt cagggagatc aacaactacc acca tqctca tgacgcatac 3601 ctgaacgcag tggtcgggac cgccctgatt aagaaatacc ccaagctgga gtccgagttc 3661 qtqtacqqag a ctataaagt gtacqatqtc cqqaagatga tcgccaaatc tqaqcaqqaa 3721 attggcaagg ccaccgctaa gtatttcttt tacagtaaca tcatgaattt ctttaagacc 3781 gaaatcacac tgqcaaatqq ggagatCaga aaaaggcctc tqa t tqaga c caacggggag 3841 acaggagaaa tcgtgtggga caagggaagg gattttgcta ccgtgcgcaa agtcctgt cc 3901 atqccccaag tgaatattgt caagaaaact gaagtgcaga ccgggggatt ctctaaqqag

5UB5TITUTE SHEET (RULE 26) 3961 agtattctgc ctaagcgaaa ctctgataaa ctgatcgccc qqaagaaaga ctgggacccc 4021 aagaagtatg gcgggttcga ctctccaaca gtggcttaca gtgtcctggt ggtcgcaaag 4081 gtggaaaagg ggaagtccaa gaaactgaag tctgtcaaag agctgctggg aa beaefca b 4141 atggaacgca gctccttcga gaagaatcct atcgattttc tggaagccaa gggctataaa 4201 gaggtgaaga aagacc bga t cattaagctg ccaaaatact cactg t ttga gctggaaaac 4261 ggacgaaagc gaatgctggc aagcgccgga gaactgcaga agggcaatga gctggccctg 4321 ccc tccaaa t a cgtgaactt cctgtatc tq qctagccact acgagaaact gaaqqqq tee 4381 cctgaggata acgaacagaa gcagctgttt gtggagcagc acaaacatta tctggacgag 4441 atcattgaac agatt tcaga q ttcages ag agagtgatcc tqqctqacgc aaatctggat 4501 aaagtcctga gcgc tacaa caagcaccga gacaaaccaa teegggagea ggccgaaaat 4561 atcattcatc tgttcaccct gacaaacctq ggcgcccctg cagccttcaa gtattttgac 4621 accacaatcg atcggaagag atacacttct accaaagagg tgctggatgc ta ccctgate 4681 caccagagta ttaccggcct gtatgagaca cgcatcgacc tg tcacagct gggaggegat 4741 gggagcccca agaaaaagcg gaaggtgtct agttaa (SEQ ID NO: 14499} .

[0765] In some embodiments, the gene editing composition comprises a fusion protein. In some embodiments, the fusion protein comprises a nuclease-inactivated Cas9 (dCas9) protein and a CloOSl nuclease or a Cio051 nuclease domain. In some embodiments, the gene editing composition further comprises a guide sequence. In some embodiments, the guide sequence comprises an RNA sequence. In some embodiments, the fusion protein comprises or consists of the amino acid sequence:

i MPKKKRKVEG IKSNI SLLKD ELRGQI SHI S HEYLSLIDLA FDSKQNRLFE MKVLELLVNE

61 YGFKGRHLGG SRKPDGIVYS TTLEDNFGI I VDTKAYSEGY SLPI SQADEM ERYVRENSNR

121 DEEVNPNKWW ENFSEEVKKY YFVFISGSFK GKFEEQLRRL SMTTGVNGSA VNWNLLLGA

181 EKIRSGEMTI EELERAMFNN SEFILKYGGG GSDKKYSIGL AI GTNSVGWA VITBEYKVPS

241 KKFKVLGNTD RHSIKKNLIG ALLFDSGETA EATRLKRTAR RRYTRRKNRI CYLQEIFSNE

301 MAKVDDSFFH RLEESFLVEE DKKHERHPIF GNIVDEVAYH EKYPTIYHLR KKLVDSTDKA 361 DLRLI YLALA HMIKFRGHFL IEGDLNPDNS DVDKLFIQLV QTYNQLFEEN PINASGVDAK 421 AILSARLSKS RRLENLIAQL PGEKKNGLFG NLIALSLGLT PNFKSNFDLA EDAKLQLSKD

481 TYDDDLDNLL AQIGDQYADL FLAAKNLSDA ILLSDILRVN TEITKAPLSA SMIKRYDEHH 541 QDLTLLKALV RQQLPEKYKE I FFDQSKNGY AGYI DGGASQ EEFYKFIKPI LEKMDGTEEL 601 LVKLNREDLL RKQRTFDNGS I PHQIHLGEL HAILRRQEDF YPFLKDNREK IEKILTFRIP 661 YYVGPLARGN SRFAWMTRKS EETITPWNFE EVVDKGASAQ SFIERMTNFD KNLPNEKVLP 721 KHSLLYEYFT VYNELTKVKY VTEGMRKPAF LSGEOKKAIV DLLFKTNRKV TVKQLKEDYF 781 KKI ECFDSVE ISGVEDRFNA SLGTYHDLLK IIKDKDFLDN EENEDI LEDI VLTLTLFEDP. 841 EMIEERLKTY AHLFDDKVMK QLKRRRYTGW GRLSRKLING IRDKQSGKTI LDFLKSDGFA 901 NRNFMQLIHD DSLTFKEDIQ KAQVSGQGDS LHEHIANLAG SPAIKKGILQ TVKVVDELVK 961 VMGRHKPENI VI EMARENQT TQKGQKNSRE RjyiKRIEEGIK ELGSQI LKEH PVENTQLQNE

1021 KLYLYYLQNG RDMYVDQELD INRLSDYDVD AIVPQSFLKD DSIDNKVLTR SDKNRGKSDN 1081 VPSEEWKKM KNYWRQLLNA KLITQRKFDN LTKAERGGLS ELDKAGFIKR QLVETRQITK 1141 HVAQILDSRM NTKYDENDKL I REVKVITLK SKLVSDFRKD FQFYKVREIN NYHHAHDAYL 1201 NAVVGTALI K KYPKLESEFV YGDYKVYDVR KMIAKSEQEI GKATAKYFFY SNIMNFFKTE

SUBSTITUTE SHEET (RULE 26) 1261 ITLANGEIRK RPLIETNGET GEIVWDKGRD FATVRKVLSM PQVNIVKKTE VQTGGFSKES

1321 ILPKRNSDKL IARKKDWDPK KYGGFDSPTV AYSVLWAKV EKGKSKKLKS VKELLGITIM 1381 ERSSFEKNPI DFLEAKGYKE VKKDLIIKLP KYSLFELENG RKRMLASAGE LQKGNELALP 1441 SKYVNFLYLA SHYEKLKGS P EDNEQKQLFV EQHKHYLDEI IEQISEFSKR VI LADANLDK 1501 VLSAYNKHRD KP I REQAENI IHLFTLTNLG APAAFKYFDT TIDRKRYTST KEVLDATLIH

1561 QSITGLYETR IDLSQLGGDG S PKKKRKV (SEQ ID NO: 14635} .

In some embodiments, the fusion protein is encoded by a nucleic acid comprising or consisting of the sequence:

1 atgcctaaga agaageggaa ggtggaaggc atcaaaagca acatctccct cctgaaag a c 61 gaactccggg ggcagattag c cacattagt cacgaatacc tctccctcat cgacctggct 121 11cgatagca agcagaacag getetttgag atgaaagtgc tggaactgct egte atgag 181 tacgggttca agggtcgaca cctcggcgga tctaggaaac cagacggcat cgtgtatagt 241 accacactgg aagacaactt tgggatcatt g tg g a ta cca aggcatactc tgagggttat 301 agtctgccca tttcacaggc egaegagatg gaacggtacg tgcgcgagaa ctcaaataga 361 gatgaggaag tcaaccctaa caagtggtgg gagaaettet ctgaggaagt gaagaaatac 421 tacttcgtct ttatcagcgg gtccttcaag ggtaaatttg aggaacagct caggagactg 481 agcatqacta ccggcgtgaa tggcagcgcc g tcaacgtgg tcaatctgct cc g eg ct 541 gaaaagattc ggageggaga ga gaccatc gaagagctgg agagggcaat gtttaataat 601 agcgagttta tcctgaaa ta cggtggcggt ggatccga ta aaaag tattc ta 11gg111a 661 gccatcggca ct aattccgt tggatgggct gtcataaccg atgaatacaa agtacctt ca 721 aagaaa C C ta aggtgttggg gaacacagac eg tea ttega ttaaaaagaa tc t a teggt 781 gccctcctat tcgatagtgg egaaaeggea gaggcgactc gcctgaaacg aaccgctcgg 841 agaaggtata cacgtcgcaa qaaccgaata tgttacttac aagaaa tttt tagcaatgag 901 atggccaaag ttgaegatte tttctttcac cgtttggaag agtccttcct tgtegaagag 961 gacaagaaac atgaaeggea ccccatcttt ggaaacatag tagatgaggt ggcatatcat 1021 gaaaagtacc caacgattta t ca cctca ga aaaaagctag ttgactcaa c tgataaagcg 1081 gacctgaggt taatctact ggctcttgcc catatgataa agttccgtgg gcactttctc 1141 a11gaggg tg atetaaat.ee ggacaactcg ga tg teg aca aactgttcat ccagt tag ta 1201 caaacctata atcagttgtt tgaagagaac cc ataaatg caagtggcgt ggatgegaag 1261 gctattctta gcgcccgcct ctctaaatcc egaeggetag aaaacctgat cgcacaatta 1321 cccggagaga agaaaaatgg gttgttcggt aaccttatag cgctctcact aggcctgaca 1381 ccaaatttta ag tegaaett egaettaget gaa g a tg cca aattgeaget tagtaaggac 1441 acgtacgatg aegatetega caatctactg gcacaaattg gagatcagta tgeggaetta 1501 11111ggctg ccaaaaacct tagegatgea atcctcctat ctgacatact gagagttaat 1561 actgagatta ccaaggcgcc gttatccgct tcaatgatca aaaggtacga tgaacatcac 1621 caagacttga ca cttctcaa ggccc tag c eg tcagcaac tgcctgagaa ata taaggaa 1681 atattctttg atcagtcgaa aaacgggtac gcaggttata ttgaeggegg agegagteaa

1741 gaggaa11ct acaag 11 ta t caaa cccata ttagagaaga tgga g gga c ggaagagttg 1801 cttgtaaaac caatcgcga agatctactg egaaageage ggactttcga caacggtagc

1861 a11 cca ca C c aaatccactt aggegaat g ca tg eta tac ttagaaggea ggagga 1111 1921 tatccgttcc tcaaagacaa t cgt gaaaag attgagaaaa tcctaacctt tcgcatacct 1981 tactatgtgg gacccctggc ccga gggaac tctcggttcg ca tggatga c aagaaagtcc 2041 gaagaaacga ttactccatg gaattttgag gaagttgtcg ataaaggtgc gtcagctcaa 2101 teg t tea teg agaggatgac caactttgac aagaa tttac cgaacgaaaa ag ta t tgcct 2161 aagcacagtt taetttaega gtatttcaca gtgtacaatg aactcacgaa agttaagtat 2221 gtcactgagg gcatgcgtaa acccgccttt etaageggag aacagaagaa agcaatagta 2281 gatctgttat tcaagaccaa ccgcaaagtg acagttaagc aattgaaaga ggactacttt 2341 aagaaaattg aatgettega ttctgtcgag atctccgggg tagaagateg atttaatgcg 2401 tcacttggta cgtatcatga cctcctaaag ataattaaag ataaggaett cctggataac 2461 gaagagaatg aagatatet agaagatata gtgttgactc ttaccctctt tgaagategg 2521 gaaatgattg aggaaagact aaaaacatac gctcacctgt tegaegataa ggttatgaaa

SUBSTITUTE SHEET (RULE 26) 2581 cagttaaaga ggegte eta ta cgggctgg ggacgattgt cgcggaaact tatcaacggg 2641 ataagagaca agcaaagtgg taaaactatt ctcgattttc taaagagcga cggcttcgcc 2701 aataggaact ttatgeaget gatccatgat qactctttaa ccttcaaaga ggata acaa 2761 aaggcacagg tttccggaca aggggactca ttgcacgaac atattgegaa tcttgctggt 2821 tcgccagcca tcaaaaaggg ca ta ctccag acagtcaaag tag egg a tga getagttaag 2881 gtcatgggac gt c caaacc ggaaaacatt gtaat egaga tggeaegega aaatcaaacg

2941 actcagaagg ggcaaaaaaa cagtcgagag egg a tga aga gaatagaaga ggg ta ttaaa 3001 ga ctgggca gccagatctt aaaggageat cctgtggaaa atacccaat t gcagaacgag 3061 aaactttacc tetat tacct acaaaatgga agggaca tg t a tg ttga tea ggaactggac 3121 ataaaccgtt tatetgatta egaegtegat gccattgtac cccaatcctt tttgaaggac 3181 gattcaatcg acaataaagt gcttacacgc teggataaga accgagggaa aag tgacaat 3241 gttccaagcg aggaagte t aaagaaaatg aagaactatt qqcggcagct cctaaatgcg 3301 aaactgataa cgcaaagaaa gttcgataac ttaactaaag ctgagagggg tggcttgtct

3361 gaacttgaca aggeeggatt tattaaacgt cag ctcgtgg aaacccgcca aatcacaaag 3421 catgttgcac agatactaga ttcccgaatg aatacgaaat aegaegagaa egataagetg 3481 attcgggaag tcaaagtaa t cactttaaag caaaattgg tgteggaett cag aggat 3541 tttcaa L LC L ataaagttag ggagataaat aactaccacc atgcgcacga egettatett 3601 aat ccg teg ta gggaccgc actcattaag aaa tacccga age agaaag tgagtttgtg 3661 tatggtgatt acaaagttta tgacgtccgt aagatgateg cgaaaagcga acaggagata 3721 ggcaaggcta cagccaaata cttcttttat tctaacatta tgaatttctt ta g cggaa 3781 atcactctgg caaacggaga gatacgcaaa cgacctttaa ttgaaaccaa tggggagaca 3841 ggtgaaa eg ta tgggataa gggccgggac t eg eg a egg tgagaaaagt tt tg tcca tg 3901 ccccaagtca acatagtaaa gaaaactgag gtgcagaccg gagggtttt c aaaggaateg

3961 attcttccaa aaaggaa tag tga taagctc ategetegta aaaaggactg ggacccgaaa 4021 aagtacggtg gettegatag ccctacagtt gcctatt ctg tcctagtagt ggcaaaagtt

4081 gagaagggaa aatccaagaa actgaagtca g tcaaagaat tattggggat aa ega t a tg 4141 gagegetegt cttttgaaaa gaacccc tc gacttccttg aggegaaagg ttacaaggaa 4201 gtaaaaaagg atetea taa taaactacca aagtatagtc tgtttgagtt agaaaatggc 4261 cgaaaacgga tgttggctag egeeggagag ett caaaagg ggaacgaact cgcactaccg 4321 tetaaa fcacg tg a a 1 cct gtatttagcq tccca ttacg agaagttgaa agq t tcacct 4381 gaagataacg aacagaagca actttttgtt gagcagcaca aacattatct egaegaaate 4441 atagagcaaa tttcggaat t cagtaagaga gtcatcctag ctgatgccaa tctggacaaa 4501 gtattaagcg catacaacaa gcacagggat aaa ccca tac gtgageagge ggaaaa ta tt 4561 atccatttgt ttactcttac caacctcggc gctccagccg cattcaagta ttttgacaca 4621 aegatagate gcaaacgata cacttctacc aaggaggtgc taga eg eg a c actg ttcac 4681 caatccatca egggattata tgaaactcgg atagatttgt cacagcttgg gggtgacgga 4741 tcccccaaga agaagagg a agtctga (SEQ ID NO: 14636} .

[0766] In some embodiments, the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphyloccocus pyogenes. In some embodiments, the dCas9 comprises a dCas9 with substitutions at positions 10 and 840 of the ammo acid sequence of the dCas9, which inactivate the catalytic site. In some embodiments, these substitutions are DI0A and H840A. In some embodiments, the“X” residue at position 1 of the dCas9 sequence is a methionine (M). In some embodiments, the amino acid sequence of the dCas9 comprises the sequence of:

1 XDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE

61 ATRLKRTARR RYTRRKNRIC YLQEI FSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG

121 NIVDEVAYHE KYPTI YHLRK KLVDSTDKAD LRLIYLALAH MI KFRGHFLI EGDLNPDNSD

181 VDKLEIQLVQ TYNQLFEENP INAS GVDAKA ILSARLSKSE RLENLIAQLP GEKKNGLFGN

241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI

SUBSTITUTE SHEET (RULE 26) 301 LLSDILRVNT EITKAPLSAS MI KRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA

361 GYI DGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGS I PHQIHLGELH 42 1 AILRRQEDFY PFLKDNREKI EKILTFRI PY YVGFLARGNS RFAWMTRKS E ETITPWNFEE 4 8 1 WDKGASAQS FI ERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 54 1 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DELKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 72 1 HEHIANLAGS PAIKKGILQT VKWDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER 7 8 1 MKRIEEGIKE LGSQI LKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDA 84 1 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEWKKMK NYWRQLLNAK LITQRKFDNL 901 TKAERGGLS E LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AWGTALIKK PKLESEFVY GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEI RKR PLIETNGETG EIWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1141 Y3VLWAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLI IKLPK 1201 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGS PE DNEQKQLFVE 12 61 QHKHYLDEI I EQI SEFSKRV I LADANLDKV LSAYNKHRDK PI REQAENI I HLFTLTNLGA

132 1 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD (i EQ ID NO:

0767] In some embodiments, the dCas9 of the disclosure comprises a dCas9 isolated or derived from Staphylococcus aureus. In some embodiments, the dCas9 comprises a dCas9 with substitutions at positions 10 and 580 of the amino acid sequence of the dCas9 which inactivate the catalytic site. In some embodiments, these substitutions are D10A and N580A. In some embodiments, the dCas9 is a small and inactive Cas9 (dSaCas9). In some embodiments, the ammo acid sequence of the dSaCas9 comprises the sequence of:

mkrnyilglA lglts vgygi idyetrdvid agvrlf kean ver egrrsk rga Ikr r r

61 rhriqrvkkl lfdynlltdh s e 1 s g 1 tipye arvkglsqkl seeefsaall hlakrrgvhn 121 vneveedtgn el s tkeql s r ns kaleekyv aelqlerlkk dgevrgsinr fktsdyvkea

18 1 kqllkvqkay hqldqs fidt yidlletrrt yyegpgegsp fgwkdlkewy emlmghctyf

24 1 peelrs vkya ynadlynaln dlnnlvitrd enekleyyek fqiienvfkq kkkptlkqia

30 1 keilvneedi kgyrvtstgk peftnlkvyh dikditarke llenaelldq iakiltiyqs

361 sediqeeltn Inseltqeei eqisnlkgyt gthnlslkai nlildelwht ndnqiaifnr

42 1 lklvpkkvdl sqqkeipttl vddfilspvv krsfiqslkv inaiikkygl pndiiielar

4 8 1 eknskdaqkm inemqkrnrq tnerieeiir ttgkenakyl iekiklhdmq egkclyslea

54 1 ipledllnnp fnyevdhllp rs vs fdns fn nkvlvkqeeA skkgnrtpfq yls s sds kis

60 1 yet fkkhiln lakgkgrisk tkkeylleer dinrf svqkd fi rnlvdtr yatrglmnl 1

SUBSTITUTE SHEET (RULE 26) 661 rsyfrvnnld VKVKsmggf ts fi rr wkf kkernKg Kh haedal i ian adf1 fkewkk

721 ldkakkvmen qmfeekqaes mpeieteqey keifitphqi khikdfkdyk yshrvdkkpn

781 relindtlys trkddkgntl ivnnlnglyd kdndklkkli nkspekllmy hhdpqtyqkl

841 klimeqygde knplykyyee tgnyltkysk kdngpvikki kyygnklnah Iditddypns

901 rnkvvklslk pyrfdvyldn gvykfvtvkn ldvikkenyy evnskcyeea kklkkisnqa

961 e fi as fynnd likingelyr vigvnndlln rievnn dit yreylenmnd krppri ikti

1021 asktqsikky stdilgnlye vkskkhpqii kkg (SEQ ID NO: 14638} .

[Q768] Therapeutic HSCs of the disclosure may be modified to express a therapeutic protein.

Therapeutic HSCs of the disclosure may be modified to express a secreted therapeutic protein, including secreted human proteins. In some embodiments, the therapeutic protein is not secreted, but rather functions intracellularly (e.g. hemoglobin for hemoglobinopathies). In some embodiments, the therapeutic protein is not secreted, but rather directs a modified HSC of the disclosure to a cell niche of a subject’s body (e.g. expression of CXCR4 increase homing of modified HSCs of the disclosure to the bone marrow of a subject).

[0769] In some embodiments, the therapeutic protein is a human hemoglobin. In some embodiments, the therapeutic protein comprises a human beta-globin. In some embodiments, the human beta-globin composes a substitution of Glutamine (Q) for Threonine at position 87 (T87Q). In some embodiments, the human beta globm is encoded by the amino acid sequence comprising:

MVHLTPEEKS AVTALWGKVN VDEVGGEALG RLLWYPWTQ RFFESFGDLS TPDAVMGNPK 60

VKAHGKKVLG AFSDGLAHLD NLKGTFA LS ELHCDKLHVD PENFRLLGNV LVCVLAHHFG 120

KEFTPPVQAA YQKWAGVAN ALAHKYH (SEQ ID NO: 14639}

147

In some embodiments, the human beta-globin is encoded by a nucleic acid sequence comprising: ctaggtattq aataagaaaa atgaagttaa ggtqgttgat ggt aacacta tgctaataac 60 tgcagagcca gaagcaccat aaqggacatg ataagggagc cagcaqacct ctgatctctt 120 cctgaatgct aatcttaaac atcctgagga agaatgggac ttccatttgg ggtgggccta 180 tgatagggta a taagacagt agtgaatatc aagctacaaa aagccccctt tcaaattctt 240 ctcagtccta acttttcata ctaagcccag tccttcca a gcagactgtq aaagagtgat 300 agttccggqa qactagcact gcagattccg ggtcactgtg agtgggggag gcagggaaqa 360 agggctcaca ggacagtcaa accatgcccc ctgtttttcc ttcttcaagt agacctctat 420 aagacaacag agacaactaa ggctgagtgg ccaggcgagg agaaaccatc tcgccgtaaa 480 acatggaagg aacacfcfccag gggaaaggtg gtatctctaa qcaagagaac tgagtggagt 540 caaqqctgag agatgcagga taaqcaaatq ggtagtgaaa agacattcat qaqqacagct 600

SUBSTITUTE SHEET (RULE 26) aaaacaataa gt tgtaaa atacaqcata gcaaa cttt aacctccaaa tcaagcctct 660 acttgaatcc ttttctgagg gatgaataag gcataggcat caggggctgt tgccaatgtg 720 cattagct gt ttgcagcctc accttctttc at ggagttta agatatagtg tattttccca 780 aggtttgaac tagetettea tttcttta g a a atg c actgacctcc cacattccct 840 ttttagtaaa a ta t tcagaa ataatttaaa tacatca t tq caa tgaaaat aaatg 111 900 tattaggcag aatccagatg ctcaaggccc cataat t cccccagt t t aq tagttgga 960 cttagggaac aaaggaacct ttaatagaaa ttggacagca agaaagegag ettagtgata 1020 ct_g i_gggcc agggeattag ccacaccagc caccactttc tgataggcag cctgcactgg 1080 tggggtgaat tctttgccaa agtgatgggc cagcacacag accagcacgt tgcccaggag 1140 ctgtgggagg aaqa taa gag gtatgaacat ga ttagcaaa agggcctagc ttggactcag 1200 taa tccag ccttatccca accataaaa t aa agcag a tggtagctgg attgtagctg 1260 ctattagcaa tatgaaacct cttacatcag ttacaattta tatgeagaaa tatt a atg 1320 cagaaatatt gctattgcct taacccagaa attatcactg ccacccttta gaatggtgca 1380 aagaggcatg atacattgta tcattat tgc cctgaaagaa agagattagg gaaagtatta 1440 gaaataagat aaacaaaaaa gtatattaaa agaagaaaq c a ttttttaaa attacaaatg 1500 c aaa t L a cc ctgatttggt caatatqtq t accctgttac ttctcccc11 ccta tga cat 1560 gaacttaacc atagaaaaga aggggaaaga aaacatcaag ggt cccatag actcaccctg 1620 aagtt ctcag ggtccacgtg cagcttgtca cagtgcagct cactcagctg ggcaaaggtg 1680 cccttgaggt tgtccaggtg agccaqq cca tcactaaagg caccgagcac tttcttgcca 1740 tgagecttea cc 11aqq g11 gcccataaca gcatcaqqaq tgga cagate cccaaaggac 1800 tcaaa g a a cc tctgggtcca aggqtaqacc a ccagcagcc taagggtggg aaaa ta g a cc 1860 aataggeaga gagagteagt gcctatcaga aacccaagag tcttctctgt ctccacatgc 1920 ccagtttcta ttggtctcct taaacctgtc ttgtaacctt gataccaacc tgcccagggc 1980 ct caeca cca acttcatcca egt tcacctt gccccacagg gcagtaacqq cagacttctc 2040 ctcaggagtc aqqtqcacca tggtgtctgt ttqaqqttqc tagtgaacac agttgtgtca 2100 gaagedSS tg taagcaatag atggctctgc cctgac . .gt atgcccagcc ctggct cc.tg 2160 ccctccctgc tcctgggagt agattggcca accctagggt gtggctccac agggtgaggt 2220 ctaagtgatg acagccgtac ctgtccttgg ctcttc tgge actggcttag gagttggact 2280 teaaa ccctc agccctccct ctaaqa ta ta tctcttggcc ccata cca tc ag ta caa att 2340 gctactaaaa acatcctcct ttgcaagtgt atttacctaq aatatgtcac attctgtctc aggeat ccat tttctttatg atgccgtttg aggtggagtt ttagtcaggt ggteagette 2460 tcccccrctt tgccatctgc cctgtaagca tcctgctggg gacccagata ggagteatea 2520 ct c aaggc tg agaacatctg ggcacacacc etaagee tea gcatgactca tcatgactca 2580 qca t tgctgt gcttgagcca gaaqq 11 tq c ttagaaggtt acacagaacc agaaggeggg 2640 ggtggggcac tqaccccga c aggggcctgg ccagaactqc teatgettgg actatggqaq 2700 gtcactaatg gagacacaca gaaatgtaac aggaactaag gaaaaactga agettattta 2760 atcagagatg aggatgctgg aagggataga gggagctgag cttgtaaaaa gtatagtaat 2820 cattcagcaa a tqq 111 q a agcacctgct ggatgc taaa ca eta ttttc agtgcttgaa 2880

SUBSTITUTE SHEET (RULE 26) tcataaataa gaataaaaca tqt tet ta t tcccca caag agtccaaqta aaaaataaca 2940 gttaattata atgtgctctg tcccccaggc tgqaqtgcag tggcacgatg tcagctcact 3000 gcaacct ccg cctcccgggg qactagtctc gagggctggt tagaaqqttc tactggagga 3060 gggtcccagc ccattgctaa attaaca tea ggctctgaga ctggcagtat atctctaaca 3120 gtggttgatg c ta tc t ctg gaacttgcct gctaca t tqa q a cca ctgac ccataca tag 3180 gaagcccata gctctgtcct qaactqttag gccactggtc cagagaqtqt gcatctcctt 3240 tgatcctcat aataacccta tgagatagac acaattatta ctcttacttt atagatgatq 3300 atCC G gaaaa cataggagtc aaggcacttg cccctagctg ggggtatagg ggagcagtcc 3360 ca tgtagtag tagaatgaaa aatgctgcta tgctgtgcct cccccacctt tcccatgtct 3420 gccctctact ca tqq teta t ctctcctggc tcctggqaq t ca tggactcc acccagcacc 3480 accaa cctga cctaaccacc tatetgagee tgccagccta taacccatct gggccctgat 3540 agctggtgqc cagccctgac cccaccccac cctccctgga a cct ctgata gacacatctq 3600 gcacaccagc tcgcaaagtc accgtgaggg tcttgtgttt gctgagtcaa aatt ccttga 3660 aatccaagtc cttagagact cctgctccca aatttacagt catagacttc ttcatggctg 3720 tctcctttat ccacaqaa tg a 11 cc111gc ttcattqccc ca tccatctg ateetee tea 3780 tcag tgca gc acagggccca tqaqcaq tag ctgcagagtc tcacataqqt ctgg cactgc 3840 ctctgacatg tccgacctta ggcaaatgct tgactcttct gccct c.gaga agettateqa 3900 tggctagtgc atgcaaatct gacactcagt gggcctggg t gaaggtgaga attttattgc 3960 tqaa tgagag cctctgggga cate t tq cca gtcaatgagt ctcagg t tea a 111 cct tct 4020 cagtcttgga g taacaqaag ctcatgcatt taataaacgg aaa ttttgta ttgaaatgag 4080 agccattgga aatcatttac tccaqactcc ta ett taaa aagagaaact gaggctcaga 4140 gaagggtgqq qaetttetea gtatgacatg gaaatqatca ggcttggatt caaagctcct 4200 gactttctgt ctag tgtatg tgcagtgagc cccttttcct ctaactgaaa gaaggaaaaa 4260 aaaatggaac ccaaaatatt ctaca tag 11 tccatgtcac agccagggct qqqcagtctc 4320 ctgttatttc ttttaaaata aatatatcat ttaaatqc t aaata gcaa accctgctcq 4380 ggaatgggag ggagagtctc tqqagtccac cccttctcgg ccctgqctct gcagatagtg 4440 ctatcaaagc cctgacagag ccctgcccat tgctgggcct tggagtgagt cagcctagta 4500 gagaggcagg gcaagccatc tcatagctgc tgagtgggag agagaaaagg gctcattgtc 4560 ta taaactca ggtcatggct a 11 c t ta t tc tcacactaag aaaaagaa tg aqatgtctac 4620 atataccctq cqtcccctct tgtgtactgg ggtccccaag agetetetaa aagtgatqqc 4680 aaagt cat tg egetagatge catcccatct at tataaacc tgcatttgtc tccacacacc 4740 agtcatggac aataaccctc ctcccaggtc cacgtgcttg tctttgt ta atactcaagt 4800 aa a a G cggaa aatgtattct ttcaatcttg ttctgttatt cctgtttcaa tggcttagta 4860 gaaaaagtac a tac11g 111 tccca taaa t tgacaataga caatttcaca tcaatgtcta 4920 tatgggtcgt tgtgtttgct gtgtttgcaa aaactcacaa taactttata ttgttactac 4980 tctaagaaag ttacaacatg qtqaatacaa gagaaageta ttacaagtcc agaaaat aaa 5040 agttatcatc ttgaggccat t 5061

(SEQ ID NO: 14640}

SUBSTITUTE SHEET (RULE 26) [0770] In some embodiments of the methods of the disclosure, at least one HSC of the plurality of therapeutic HSCs is genetically modified in some embodiments, each HSC of the plurality of therapeutic HSCs is genetically modified.

[0771] In some embodiments of the methods of the disclosure, the subject has a disease or disorder and the plurality of therapeutic HSCs improves a sign or symptom of the disease or disorder, optionally by providing (e.g. secreting) a therapeutic protein systemically or locally within the subject that acts upon the HSC or upon a second cell in the subject.

[0772] In some embodiments of the methods of the disclosure, the subject has an immune disease or disorder and wherein the plurality of therapeutic HSCs improves a sign or symptom of the immune disease or disorder. In some embodiments, at least one HSC of the plurality of therapeutic HSCs is genetically modified to improve a sign or symptom of the immune disease or disorder of the subject. In some embodiments, each HSC of the plurality of therapeutic HSCs is genetically modified to improve a sign or symptom of the immune disease or disorder of the subject. Exemplary immune disorders include, hut are not limited to, inflammation and autoimmune conditions.

[0773] In some embodiments of the methods of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood cell, an immune cell circulating in the blood, a bone marrow cell or a precursor cell thereof and the plurality of therapeutic HSCs improves a sign or symptom of the disease or disorder. In some embodiments, the disease or disorder is a clotting disorder. In some embodiments, at least one HSC of the plurality of therapeutic HSCs has been modified to secrete a protein that improves a sign or symptom of the clotting disorder. In some embodiments, a majority of HSCs of the plurality of therapeutic HSCs have been modified to secrete a protein that improves a sign or symptom of the clotting disorder. In some embodiments, each HSC of the plurality of therapeutic HSCs has been modified to secrete a protein that improves a sign or symptom of the clotting disorder. In some embodiments, the at least one HSC, the majority of HSCs or each HSC of the plurality of therapeutic HSCs are modified to secrete a protein that improves a sign or symptom of the clotting disorder. In some embodiments, the at least one HSC, the majority of HSCs or each HSC of the plurality of therapeutic HSCs are modified to secrete one or more clotting factors.

SUBSTITUTE SHEET (RULE 26) [0774] in some embodiments of the methods of the disclosure, the subject has a genetic or epigenetic marker for a glycogen storage disease or disorder and the plurality of therapeutic HSCs improves a sign or symptom of the glycogen storage disease or disorder. In some embodiments, the glycogen storage disease or disorder is glycogen storage disease (GSD) type 0, GSD type I, GSD type II, GSD type III, GSD type IV, GSD type V, GSD type VI, GSD type VII, GSD type IX, GSD type X, GSD type XI, GSD type XII or GSD type XIII In some embodiments, at least one HSC, a majority of HSCs or each HSC of the plurality of therapeutic HSCs are modified to secrete one or more of glycogen synthase, glucose-6-phosphatase, acid aipha-giucosidase, glycogen debranching enzyme, glycogen branching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter GLUT2, Aldolase A or b-enolase and wherein the plurality of therapeutic HSCs improves a sign or symptom of GSD type 0, GSD type I, GSD type II, GSD type III, GSD type IV, GSD type V, GSD type VI, GSD type VII, GSD type IX, GSD type X, GSD type XI, GSD type XII or GSD type XIII, respectively.

[0775] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject is human.

[0776] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has an immune system disease or disorder or the subject is at risk of developing an immune system disease or disorder.

[0777] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,

autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal & neuronal neuropathies,

Balo disease, Behcet’s disease, bullous pemphigoid, cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, chronic inflammatory demyelmating polyneuropathy (CIDP), chronic

SUBSTITUTE SHEET (RULE 26) recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’s disease, Cogans syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelmatmg neuropathies, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), discoid lupus, Dressier’ s syndrome, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4- related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (Type 1 diabetes), juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastie vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), Lupus (SLE, Lyme disease, chronic Meniere’s disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic’s), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH) Party Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I autoimmune polyglandular syndrome, type II autoimmune polyglandular syndrome, type III autoimmune polyglandular syndrome, polymyalgia rheumatica, polymyositis, postmyocardiai infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red ceil aplasia, Raynauds phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter’s syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome, subacute bacterial

SUBSTITUTE SHEET (RULE 26) endocarditis (SBE), susac’s syndrome, sympathetic ophthalmia, Takayasu’s arteritis, temporal arteritis/Giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesiculobullous dermatosis or vitiligo.

[0778] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject is immunocompromised.

[0779] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has an inflammatory disorder.

[0780] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has an immune system disease or disorder or the subject is at risk of developing an immune system disease or disorder. In some embodiments, the subject has a genetic or epigenetic marker for the immune system disease or disorder. In some embodiments, the immune system disease or disorder is induced a medical intervention.

[0781] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for the immune system disease or disorder.

[Q782] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood cell, an immune cell circulating in the blood, a bone marrow cell or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC).

[0783] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood cell, an immune cell circulating in the blood, a bone marrow cell or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC).

[0784] In some embodiments of the methods of treating a disease or disorder of the disclosure, the disease or disorder is cancer. In some embodiments, the cancer is a lymphoma, a leukemia, a myeloma or a malignant immunoproliferative disease. In some embodiments, the lymphoma is Hodgkin lymphoma, Non-Hodgkin lymphoma, anaplastic large cell lymphoma,

angioimmunoblastic T-cell lymphoma (AILT), hepatosplenic T-cell lymphoma, B-cell lymphoma, retieu!oendothe!iosis, reticulosis, microglioma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphatic tissue lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B cell lymphoma,

SUBSTITUTE SHEET (RULE 26) Waldenstrom's macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis or nodular lymphocyte predominant Hodgkin's lymphoma.

[0785] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood cell, an immune cell circulating in the blood, a bone marrow cell or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC). In some

embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a lymphoma, a leukemia, a myeloma or a malignant immunoproliferative disease. In some embodiments, the leukemia is plasma cell leukemia (PCL), acute erythraemia and erythroleukaemia, acute erythremie myelosis, acute erythroid leukemia, Heilmeyer-Schoner disease, acute

megakaryoblastic leukemia (AMKL), mast cell leukemia, panmyelosis, acute panmyelosis with myelofibrosis (APMF), lymphosarcoma cell leukemia, blastic phase chronic myelogenous leukemia, stem cell leukemia, accelerated phase chronic myelogenous leukemia, acute myeloid leukemia (AML), polycythemia vera, acute promyelocytic leukemia, acute basophilic leukemia, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, B-eell leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, chronic lymphocytic leukemia, hairy cell leukemia or chronic idiopathic myelofibrosis.

[0786] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood cell, an immune cell circulating m the blood, a bone marrow ceil or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC). In some

embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a lymphoma, a leukemia, a myeloma or a malignant immunoproliferative disease. In some embodiments, the myeloma is multiple myeloma, Kah!ers disease, myelomatosis, solitary myeloma, plasma ceil leukemia, extramedullary plasmacytoma, malignant plasma cell tumour or plasmacytoma.

[0787] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood

SUBSTITUTE SHEET (RULE 26) cell, an immune cell circulating in the blood, a bone marrow cell or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC). In some

embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a lymphoma, a leukemia, a myeloma or a malignant immunoproliferative disease. In some embodiments, the malignant immunoproliferative disease is alpha heavy chain disease or gamma heavy chain disease.

[0788] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests m a blood cell, an immune cell circulating m the blood, a bone marrow cell or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC). In some

embodiments, the disease or disorder is an anemia. In some embodiments, the anemia is a hemolytic anemia, an autoimmune hemolytic anemia, a congenital hemolytic anemia, an aplastic anemia, a b-thalassemia, a congenital erythroid aplasia, a congenital dyserythropoietic anemia, a glucose-6-phosphate dehydrogenase deficiency, a Fanconi anemia, a hereditary spherocytosis, a hereditary elliptocytosis, a hereditary pyropoikilocytosis, a hereditary' persistence of fetal hemoglobin, a hereditary stomatocytosis, a hexokinase deficiency, a hyperanaemia, a

hypochromic anemia, an ineffective erythropoiesis, a macrocytic anemia, a megaloblastic anemia, a myelophthisic anemia, a neuroacanthocytosis, a chorea-acanthocytosis, a paroxysmal nocturnal hemoglobinuria, a pyruvate kinase deficiency, a Rh deficiency syndrome, a sickle-cell disease, a sideroblastic anemia, a stornatocytic ovalocytosis, a thalassemia, a triosephosphate isomerase (TPI) deficiency or a warm autoimmune hemolytic anemia. In some embodiments, the disease or disorder is a hemoglobinopathy. In some embodiments, the hemoglobinopathy is a b- thalassemia or a sickle-cell disease.

[0789] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood cell, an immune cell circulating in the blood, a bone marrow cell or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC). In some

embodiments, the disease or disorder is a clotting disorder or a hemorrhagic condition. In some embodiments, the disease or disorder is a clotting disorder. In some embodiments, the clotting disorder is a defibrination syndrome, a protein C deficiency, a protein S deficiency, Factor V

SUBSTITUTE SHEET (RULE 26) Leiden, thrombocytosis, thrombosis, recurrent thrombosis, antiphospholipid syndrome, primary antiphospholipid syndrome or thrombotic thrombocytopenic purpura (TTP).

[0790] in some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a blood cell, an immune cell circulating m the blood, a bone marrow ceil or a precursor cell thereof. In some embodiments, the precursor cell is a hematopoietic stem cell (HSC). In some

embodiments, the disease or disorder is a clotting disorder or a hemorrhagic condition. In some embodiments, the disease or disorder is a hemorrhagic condition. In some embodiments, the hemorrhagic condition is thrombocytopenia, hemophilia, hemophilia A, hemophilia B, hemophilia C, Von Willebrand disease (vWD), hereditary Von Willebrand disease (vWD), vWD type I, vWD type 2, vWD type 3, Glanzmann's thrombasthenia or Wiskott-Aldrich syndrome (WAS)

[0791] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a secondary target cell that may be contacted by the composition comprising a plurality of modified HSCs In some embodiments, the secondary target cell is a stem cell or a progenitor cell. In some embodiments, the stem cell is a somatic stem cell. In some embodiments, the stem cell is a target HSC, a mesenchymal stem cell, an epidermal stem cell, an epithelial stem cell, a neural stem cell. In some embodiments, the secondary target cell is a differentiated cell. In some embodiments, the differentiated cell is a red blood cell, a white blood cell, a monocyte, a granulocyte, a platelet, or a dendritic cell.

[0792] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests in a secondary target cell that may be contacted by the composition comprising a plurality of modified HSCs. In some embodiments, the secondary target ceil is a stem cell or a progenitor cell. In some embodiments, the progenitor ceil is an osteoclast. In some embodiments, the at least one HSC of the composition comprising a plurality of modified HSCs is modified to secrete a ligand, peptide or protein that enhances an activity of an osteoclast. In some embodiments, the composition comprising a plurality of modified HSCs treats or prevents a disease or disorder associated with aberrant osteoclast function. In some embodiments, the subject has one or more genetic or epigenetic markers for the disease or disorder associated with aberrant osteoclast

SUBSTITUTE SHEET (RULE 26) function. In some embodiments, the disease or disorder associated with aberrant osteoclast function is Paget’s disease, hypophosphatasia or ostesopetrosis.

[0793] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has a genetic or epigenetic marker for a disease or disorder that manifests m a secondary target cell that may be contacted by the composition comprising a plurality of modified HSCs. In some embodiments, the secondary target ceil is a differentiated cell. In some embodiments, the differentiated cell is a red blood cell, a white blood cell, a monocyte, a granulocyte, a platelet, or a dendritic ceil. In some embodiments, the at least one HSC of the composition comprising a plurality' of modified HSCs is modified to secrete a ligand, peptide or protein that enhances an activity of a granulocyte. In some embodiments, the composition comprising a plurality of modified HSCs treats or prevents a disease or disorder associated with aberrant granulocyte function. In some embodiments, the subject has one or more genetic or epigenetic markers for the disease or disorder associated with aberrant granulocyte function. In some embodiments, the disease or disorder associated with aberrant granulocyte function is Chronic Granulomatous Disease.

[Q794] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has an immune system disease or disorder or the subject is at risk of developing an immune system disease or disorder. In some embodiments, the immune system disease or disorder is induced a medical intervention. In some embodiments, the subject is at risk of developing an immune system disease or disorder due to a past, present or future medical intervention.

[0795] In some embodiments of the methods of treating a disease or disorder of the disclosure, the subject has an immune system disease or disorder or the subject is at risk of developing an immune system disease or disorder. In some embodiments, the immune system disease or disorder was induced by an infection. In some embodiments, the subject is at risk of developing an immune system disease or disorder due to a past, present or potential infection. In some embodiments, the infection is viral, bacterial and/or microbial. In some embodiments, the infection is viral. In some embodiments, the infection is viral and the subject becomes immunocompromised as a result of the infection. In some embodiments, the subject was exposed to or infected with HIV. In some embodiments, the subject has developed AIDS. In some

SUBSTITUTE SHEET (RULE 26) embodiments, the infection is viral in some embodiments, the infection is viral and the subject develops cancer.

[0796] in some embodiments of the methods of treating a disease or disorder of the disclosure, the subject’s endogenous HSCs have been eliminated prior to administering a composition of the disclosure.

[0797] Exemplary therapeutic secreted proteins may be used as a monotherapy or in combination with another therapy in the treatment or prevention of any disease or disorder.

These secreted proteins may be used as a monotherapy or in combination with another therapy for enzyme replacement and/or administration of biologic therapeutics. A database of human secreted proteins can be found at

proteinatlas.org/search/protein_class:Predicted%20secrete d%20proteins, the contents of which are incorporated herein by reference. Exemplary human secreted proteins can be found, but are not limited to the human secreted proteins in Table 1.

[0798] Table 1. Human Therapeutic Proteins

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

[0799] Inducible proapoptotic polypeptides of the disclosure are superior to existing inducible polypeptides because the inducible proapoptotic polypeptides of the disclosure are far less immunogenic. While inducible proapoptotic polypeptides of the disclosure are recombinant polypeptides, and, therefore, non-naturally occurring, the sequences that are recombined to produce the inducible proapoptotic polypeptides of the disclosure do not comprise non-human sequences that the host human immune system could recognize as“non-self’ and, consequently, induce an immune response m the subject receiving an inducible proapoptotic polypeptide of the disclosure, a cell comprising the inducible proapoptotic polypeptide or a composition comprising the inducible proapoptotic polypeptide or the cell comprising the inducible proapoptotic polypeptide.

[0800] Transposons of the disclosure may comprise an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In some embodiments, the non-human sequence comprises a restriction site. In some embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible proapoptotic polypeptides of the disclosure may also be referred to as an“iC9 safety switch”. In some embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In some embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In some embodiments, transposons of the disclosure may comprise an inducible caspase polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide,

SUBSTITUTE SHEET (RULE 26) wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In some embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide. In some embodiments, the amino acid sequence of the ligand binding region that comprise a FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of valme (V) for phenylalanine (F) at position 36 (F36V).

[0801] In some embodiments, the FKBP12 polypeptide is encoded by an ammo acid sequence comprising

GVQVETISPGDGRTFPKRGQTCVVHYTGMI .EDGKKVDSSRDRNKPFKFMf.GKQFVlRG WEEGVAQMS VGQRAKLTI SPD Y AY GAT GHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 14641 ).

[0802] In some embodiments, the FKBP12 polypeptide is encoded by a nucleic acid sequence comprising

GGGGTCCAGGTCGAGACTATTTCACCAGGGGATGGGCGAACATTTCCAAAAAGGGG

CCAGACTTGCGTCGTGCATTACACCGGGATGCTGGAGGACGGGAAGAAAGTGGACA

GCTCCAGGGATCGCAACAAGCCCTTCAAGTTCATGCTGGGAAAGCAGGAAGTGATC GAGGATGGGAGGAAGGCGTGGCACAGATGTCAGTCGGCCAGCGGGCCAAACTGA

CCATTAGCCCTGACTACGCTTATGGAGCAACAGGCCACCCAGGGATCATTCCCCCTC ATGCCACCCTGGTCTTCGAT GTGGAACTGCTGAAGCTGGAG (SEQ ID NO; 14642). In some embodiments, the induction agent specific for the ligand binding region may comprise a FK506 binding protein 12 (FKBP12) polypeptide having a substitution of valine (V) for phenylalanine (F) at position 36 (F36V) comprises AP20187 and/or AP1903, both synthetic drugs.

[0803] In some embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the linker region is encoded by an amino acid comprising GGGGS (SEQ ID NO; 14643) or a nucleic acid sequence comprising GGAGGAGGAGGATCC (SEQ ID NO; 14644). In some embodiments, the nucleic acid sequence encoding the linker does not comprise a restriction site.

[0804] In some embodiments of the truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an

SUBSTITUTE SHEET (RULE 26) arginine (R) at position 87 of the sequence. Alternatively, or m addition, m some embodiments of the inducible proapoptotic polypeptides, inducible easpase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid sequence that does not comprise an alanine (A) at position 282 the sequence in some embodiments of the inducible proapoptotic polypeptides, inducible caspase polypeptides or truncated caspase 9 polypeptides of the disclosure, the truncated caspase 9 polypeptide is encoded by an amino acid comprising

GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVY GTDGCPVSYEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN P EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFE Q WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 14645) or a nucleic acid sequence comprising

TTTGGGGACGTGGGGGCCCTGGAGTCTCTGCGAGGAAATGCCGATCTGGCTTACATC CTGAGC AT GGAACC CT GCGGC C ACT GTCTGAT C ATT A AC A AT GT GA ACTT CT GC AGA GAAAGCGGACTGCGAACACGGACTGGCTCCAATATTGACTGTGAGAAGCTGCGGAG AAGGTTCTCTAGTCTGCACTTTATGGTCGAAGTGAAAGGGGATCTGACCGCCAAGA

AAATGGTGCTGGCCCTGCTGGAGCTGGCTCAGCAGGACCATGGAGCTCTGGATTGC

TGCGTGGTCGTGATCCTGTCCCACGGGTGCCAGGCTTCTCATCTGCAGTTCCCCGGA

GCAGTGTACGGAACAGACGGCTGTCCTGTCAGCGTGGAGAAGATCGTCAACATCTT

CAACGGCACTTCTTGCCCTAGTCTGGGGGGAAAGCCAAAACTGTTCTTTATCCAGGC

:TGTGGCGGGGAACAGAAAGATCACGGCTTCGAGGTGGCCAGCACCAGCCCTGAGG

ACGAATCACCAGGGAGCAACCCTGAACCAGATGCAACTCCATTCCAGGAGGGACTG

AGGACCTTTGACCAGCTGGATGCTATCTCAAGCCTGCCCACTCCTAGTGACATTTTC GTGTCTTACAGTACCTTCCCAGGCTTTGTCTCATGGCGCGATCCCAAGTCAGGGAGC TGGTACGTGGAGACACTGGACGACATCTTTGAACAGTGGGCCCATTCAGAGGACCT GCAGAGCCTGCTGCTGCGAGTGGCAAACGCTGTCTCTGTGAAGGGCATCTACAAAC AGATGCCCGGGTGCTTCAATTTTCTGAGAAAGAAACTGTTCTTTAAGACTTCC (SEQ ID NO: 14646).

[0805] In some embodiments of the inducible proapoptotic polypeptides, wherein the polypeptide comprises a truncated caspase 9 polypeptide, the inducible proapoptotic polypeptide

SUBSTITUTE SHEET (RULE 26) is encoded by an amino acid sequence comprising

GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKK\¾SSRDRNKPFKFMLGKQEVIRG

WEEGVAQMSVGGRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGFG D

VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFS SLHF

MVEVKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGC

PFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHS E DLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 14647) or the nucleic acid sequence comprising

ggggtccaggtcgagactatttcaccaggggatgggcgaacatttccaaaaaggggccag acttgcgtcgtgcattacaccgggatgctg gaggacgggaagaaagtggacagctccagggatcgcaacaagcccttcaagttcatgctg ggaaagcaggaagtgatccgaggatggg aggaaggcgtggcacagatgtcagtcggccagcgggccaaactgaccattagccctgact acgcttatggagcaacaggccacccagg gatcattccccctcatgccaccctggtcttcgatgtggaactgctgaagctggagggagg aggaggatccggatttggggacgtgggggc cctggagtctctgcgaggaaatgccgatctggcttacatcctgagcatggaaccctgcgg ccactgtctgatcattaacaatgtgaacttctg cagagaaagcggactgcgaacacggactggctccaatattgactgtgagaagctgcggag aaggttctctagtctgcactttatggtcgaa gtgaaaggggatctgaccgccaagaaaatggtgctggccctgctggagctggctcagcag gaccatggagctctggattgctgcgtggtc gtgatcctgtcccacgggtgccaggcttctcatctgcagttccccggagcagtgtacgga acagacggctgtcctgtcagcgtggagaaga tcgtcaacatcttcaacggcacttcttgccctagtctggggggaaagccaaaactgttct ttatccaggcctgtggcggggaacagaaagat cacggcttcgaggtggccagcaccagccctgaggacgaatcaccagggagcaaccctgaa ccagatgcaactccattccaggagggac tgaggacctttgaccagctggatgctatctcaagcctgcccactcctagtgacattttcg tgtcttacagtaccttcccaggctttgtctcatggc gcgatcccaagtcagggagctggtacgtggagacactggacgacatctttgaacagtggg cccattcagaggacctgcagagcctgctgc tgcgagtggcaaacgctgtctctgtgaagggcatctacaaacagatgcccgggtgcttca attttctgagaaagaaactgttctttaagacttc c (SEQ ID NO: 14670).

[0806] Transposons and other delivery vectors of the disclosure may comprise at least one self- cleaving peptide(s) located, for example, between one or more of a sequence encoding an inducible proapoptotic polypeptide of the disclosure, a sequence encoding a therapeutic protein of the disclosure and a selection gene of the disclosure.

[0807] Transposons and other delivery vectors of the disclosure may comprise at least two self cleaving peptide(s), a first self-cleavmg peptide located, for example, upstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure of the disclosure and a

SUBSTITUTE SHEET (RULE 26) second first self-cleaving peptide located, for example, downstream or immediately upstream of an inducible proapoptotic polypeptide of the disclosure of the disclosure.

[0808] The at least one self-cleaving peptide may comprise, for example, a T2A peptide, GSG- T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 14648) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising

EGRGSLLTCGDVEENPGP (SEQ ID NO: 14649). A GSG-T2A peptide may comprise an ammo acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 14650) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 14651) . A GSG-T2A peptide may comprise a nucleic acid sequence comprising

ggatctggagagggaaggggaagcctgctgaectgtggagacgtggaggaaaaceca ggacca (SEQ ID NO: 14666). An E2A peptide may comprise an amino acid sequence comprising

QCTNY ALLKLAGDVESNPGP (SEQ ID NO: 14652) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the ammo acid sequence comprising

QCTNY ALLKLAGDVESNPGP (SEQ ID NO: 14653) A GSG-E2A peptide may comprise an ammo acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 14654) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 14655) An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14656) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14657) . A GSG-F2A peptide may comprise an amino acid sequence comprising

GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14658) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising

GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14659) . A P2A peptide may comprise an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 14660) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the ammo acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 14661) . A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:

SUBSTITUTE SHEET (RULE 26) 14662) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GS GATNF SLLKQ AGD VEENPGP (SEQ ID NO: 14663).

[0809] Transposons and other delivery vectors of the disclosure may comprise a first and a second self-cleaving peptide, the first self-cleaving peptide located, for example, upstream of one or more of a sequence encoding a therapeutic protein of the disclosure the second self- cleaving peptide located, for example, downstream of a sequence encoding a therapeutic protein of the disclosure. The first and/or the second self-cleaving peptide may comprise, for example, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG- F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide may comprise an ammo acid sequence comprising EGRGSLLTCGD VEENPGP (SEQ ID NO: 14648) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 14649). A GSG-T2A peptide may comprise an amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 14650) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 14651) . A GSG-T2A peptide may comprise a nucleic acid sequence comprising

ggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaaccca ggacca (SEQ ID NO: 14669). An E2A peptide may comprise an amino acid sequence comprising

QCTNYALLKLAGDVESNPGP (SEQ ID NO: 14652) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising

QCTNYALLKLAGDVESNPGP (SEQ ID NO: 14653) A GSG-E2A peptide may comprise an ammo acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 14654) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 14655). An F2A peptide may comprise an amino acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14656) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the ammo acid sequence comprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14657) . A GSG-F2A peptide may comprise an amino acid sequence comprising

GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14658) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising

GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 14659) . A P2A peptide may comprise

SUBSTITUTE SHEET (RULE 26) an amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 14660) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 14661) . A GSG-P2A peptide may comprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 14662) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 14663). Transposons of the disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective cell culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.

[Q81Q] In some embodiments of the compositions and method of the disclosure, the selection agent kills dividing cells and the presence and/or expression of the selection gene in an HSC of the disclosure protects the modified HSC during cell division, thereby permitting only modified HSCs to proliferate in the presence of the selection agent. For example, the selection agent, methotrexate (MTX) kills divi ding cells and the presence and/or expressi on of the selection gene, DHFR, in an HSC of the disclosure protects the modified HSC during cell division, thereby permitting only modified HSCs to proliferate in the presence of MTX.

[0811] Allogeneic cells of the disclosure are engineered to prevent adverse reactions to engraftment following administration to a subject. Allogeneic cells may be any type of cell.

[0812] In some embodiments of the composition and methods of the disclosure, allogeneic cells are stem cells. In some embodiments, allogeneic cells are derived from stem cells.

Exemplary stem cells include, but are not limited to, embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs), multipotent stem cells, pluripotent stem cells, and hematopoetic stem cells (HSCs).

[0813] In some embodiments of the composition and methods of the disclosure, allogeneic cells are differentiated somatic cells.

[0814] In some embodiments of the composition and methods of the disclosure, allogeneic cells are immune cells. In some embodiments, allogeneic cells are T lymphocytes (T cells). In some embodiments, allogeneic cells are T ceils that do not express one or more components of a naturally-occurring T-cell Receptor (TCR). In some embodiments, allogeneic cells are T cells

SUBSTITUTE SHEET (RULE 26) that express a non-naturally occurring antigen receptor. Alternatively, or in addition, in some embodiments, allogeneic cells are T cells that express a non-naturally occurring Chimeric Stimulatory Receptor (CSR). In some embodiments, the non-naturally occurring CSR comprises or consists of a switch receptor. In some embodiments, the switch receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some

embodiments, the extracellular domain of the switch receptor binds to a TCR co-stimulatory molecule and transduces a signal to the intracellular space of the allogeneic cell that recapitulates TCR signaling or TCR co-stimulatory signaling.

[0815] Adoptive cell compositions that are“universally” safe for administration to any patient requires a significant reduction or elimination of alloreactivity.

[0816] Towards this end, allogeneic cells of the disclosure are modified to interrupt expression or function of a T-cell Receptor (TCR) and/or a class of Major Histocompatibility Complex (MHC). The TCR mediates graft vs host (GvH) reactions whereas the MHC mediates host vs graft (HvG) reactions. In preferred embodiments, any expression and/or function of the TCR is eliminated in allogeneic cells of the disclosure to prevent T-cell mediated GvH that could cause death to the subject. Thus, m particularly preferred embodiments, the disclosure provides a pure TCR-negative allogeneic T-cell composition (e.g. each cell of the composition expresses at a level so low as to either be undetectable or non-existent).

[0817] In preferred embodiments, expression and/or function of MHC class I (MHC-I, specifically, HLA-A, HLA-B, and HLA-C) is reduced or eliminated in allogeneic cells of the disclosure to prevent HvG and, consequently, to improve engraftment of allogeneic cells of the disclosure in a subject. Improved engraftment of the allogeneic cells of the disclosure results in longer persistence of the cells, and, therefore, a larger therapeutic window for the subject.

Specifically, in the allogeneic cells of the disclosure, expression and/or function of a structural element of MHC-I, Beta-2-Microglobulm (B2M), is reduced or eliminated in allogeneic cells of the disclosure.

[0818] The above strategies for generating an allogeneic cell of the disclosure induce further challenges. T Ceil Receptor (TCR) knockout (KO) in T ceils results m loss of expression of CD3-zeta (CD3z or CD3 , which is part of the TCR complex. The loss of (Z03z in TCR-KO T- ce!ls dramatically reduces the ability of optimally activating and expanding these cells using standard stimulation/activation reagents, including, but not limited to, agonist anti-CD3 niAb.

SUBSTITUTE SHEET (RULE 26) When the expression or function of any one component of the TCR complex is interrupted, all components of the complex are lost, including TCR-alpha (TCRa), TCR-beta ! TCR if). CD3- gamma (CD3y), CD3-epsilon (CD3E), CD3-delta (CD35), and CD3-zeta (CD3Q. Both CD3E and ί/B3z are required for T cell activation and expansion. Agonist anti-CD3 mAbs typically recognize CD3e and possibly another protein within the complex which, in turn, signals to €B3z.€B3z provides the primary stimulus for T cell activation (along with a secondary co- stimulatory signal) for optimal activation and expansion. Under normal conditions, full T-cell activation depends on the engagement of the TCR in conjunction with a second signal mediated by one or more co-stimulatory receptors (e.g. CB28, CD2, 4-1BBL, etc... ) that boost the immune response. However, when the TCR is not present, T cell expansion is severely reduced when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAh. In fact, T cell expansion is reduced to only 20-40% of the normal level of expansion when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAb

[0819] The disclosure provides a Chimeric Stimulatory Receptor (CSR) to deliver CD3z primary stimulation to allogeneic T cells in the absence of an endogenous TCR (and, consequently, an endogenous CDS z) when stimulated using standard activation/stimulation reagents, including agonist anti-CD3 mAb.

[0820] In the absence of an endogenous TCR, Chimeric Stimulatory Receptors (CSRs) of the disclosure provide a ί/B3z stimulus to enhance activation and expansion of allogeneic T cells. In other words, in the absence of an endogenous TCR, Chimeric Stimulatory Receptors (CSRs) of the disclosure rescue the allogeneic cell from an activation-based disadvantage when compared to non-all ogeneic T-cells that express an endogenous TCR. In some embodiments, CSRs of the disclosure comprise an agonist mAb epitope extracellularly and a ί/B3z stimulatory domain intracellularly and, functionally, convert an anti-CD28 or anti-CD2 binding event on the surface into a CB3z signaling event in an allogeneic T cell modified to express the CSR. In some embodiments, a CSR comprises a wild type CB28 or CD2 protein and a CB3z intracellular stimulation domain, to produce CD28z CSR and CD2z CSR, respectively. In preferred embodiments, CD28z CSR and/or CB2z CSR further express a non-natural!y occurring antigen receptor and/or a therapeutic protein. In preferred embodiments, the non-natura!ly occurring antigen receptor comprises a Chimeric Antigen Receptor.

SUBSTITUTE SHEET (RULE 26) [0821] The data provided herein demonstrate that modified allogeneic T cells of the disclosure comprising/expressing a CSR of the disclosure improve or rescue, the expansion of allogeneic T cells that no longer express endogenous TCR when compared to those cells that do not comprise/express a CSR of the disclosure.

[0822] A wildtype/iiatural human CD28 protein (NCBI: CD28_HUMAN; UniProt/Swiss-Prot: PI 0747.1) comprises or consists of the amino acid sequence of:

MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYS YNLFSREFRASLHKGLDSAVE VCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYV QTDI YFCKIEVMYPPPYLDNEKS NGT I IHVKGKHLCPS PLFPGPSKPFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYM NMT P RRP G P T RKH Y Q P YAP P RD FAA YR S (SEQ ID NO: 14741)

[0823] A nucleotide sequence encoding wildtype/iiatural CD28 protein (NCBI: CCDS2361.1) comprises or consists of the nucleotide sequence of:

AT G C T GAG G C T G C T C T T G G C T C T C AAC T TAT T C C C T T C AAT T C AAG T AAC AG G AAAC AAG AT T T T G G T GAAG GAG T C G C C CAT G C T T G TAG C G TAG GAC AAT G C G G T C AC C T TAG C T G C AAG TAT T C C TAG AAT C T C T T C T C AAG G GAG T T C C G G G CAT C C C T T C AC AAAG GAC T G GAT AG T G C T G T G GAA G T C T G T G T T G TAT AT G G G AA TAG T C C C AG C AG C T T GAG G T T TAG T C AAAAAC G G G G T T C AAC T G T GAT G G GAAAT T G G G C AAT G AAT C AG T GAC AT T C TAG C T C C AGAAT T T G TAT G T T AAC C AAAC AGAT AT T T AC T T C T G CAAAA T GAAG T T AT G T AT C C T C C T C C T T AC C T AGAC AAT GAGAAGAG C AAT G G AAC CAT TAT C CAT G T GAAAG G GAAAC AC C T T T G T C C AAG T C C C C T AT T T C C C G GAC C T T CTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAG TAAC AG T G G C C T T TAT TAT T T T C T G G G T GAG GAG T AAGAG GAG C AG G C T C C T G C AC AG T GAC T AC AT G AAC AT GAC T C C C C G C C G C C C C G G G C C C AC C C G C AAG CAT T AC C AG C C C TAT G C C C C AC C AC G C G AC T T C G C AG C C T AT C G C T C C T G A (SEQ ID NO: 14742)

[0824] An exemplar CSR CD28z protein of the disclosure comprises or consists of the ammo acid sequence of (CD28 Signal peptide, CD 28 Extracellular Domain, CD28 Transmembrane domain. CD28 Cytoplasmic Domain, CD3z Intracellular Domain):

MLRJYLLM LFPSI^TGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVE VCVVYGNYS QQLQVYSKTGFNCDGKL GNES VTFYL ONE YVNQTDI YFCKIE VMY PPPYLDNEKS NGT 11 HVKGKHL CPS PL FPGPS K PFWVLVWGGVLAC YS LLVTVAF 11FWVRSKRSRLLHSDYM NMTPERPGPTBKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLY E GRREEYOVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDT YDALHMQAL P PR (SEQ ID NO: 14743)

CD28 Signal peptide:

MLRLLLALNLFPS I QVTG (SEQ ID NO: 14744)

CD 28 Extracellular Domain :

NK I L VKQS PML VAY DNAVNL S C K Y S YNL F S RE ERAS LHKGL D S AVE VC VVY GN Y S QQL QV Y S KT

SUBSTITUTE SHEET (RULE 26) GFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLF PGPSKP (SEQ ID NO: 14745)

CD28 Transmembrane domain:

FWVLVWGGVLAC YS LLVTVAF11 FWV (SEQ ID NO: 14746)

CD 28 Cytoplasmic Domain.

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 14747)

CD3z Intracellular Domain:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14748)

[0825] An exemplar nucleotide sequence encoding a CSR CD28z protein of the disclosure is depicted in Fig. 16 and comprises or consists of the nucleotide sequence of (CD28 Signal peptide, CD28 Extracellular Domain, CD28 Transmembrane domain. CD28 Cytoplasmic Domain , CD3z Intracellular Domain):

ATGCTGAGACTGCTGCTGGCCCTGAATCTGTTCCCCAGCATCCAAGTGACCGGCAGCAAS ATCC

TGGTCAAGCAGAGCCCTATGCTGGTGGCCTACGACAACGCCGTGAACCTGAGCTGCA AGTACAG CTACAACCTGTTCAGCAGAGAGTTCCGGGCCAGCCTGCACAAAGGACTGGATTCTGCTGT GGAA GTGTGCGTGGTGTACGGCAACTACAGCCAGCAGCTGCAGGTCTACAGCAAGACCGGCTTC AACT GCGACGGCAAGCTGGGCAATGAGAGCGTGACCTTCTACCTGCAAAACCTGTACGTGAACC AGAC CGACATCTATTTCTGCAAGATCGAAGTGATGTACCCGCCTCCTTACCTGGACAACGAGAA GTCC AACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGA CCTA GCAAGCCTTTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGG TTAC AGTGGCCTTCATCATCTTTTGGGTC CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACA TG AACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCT AGAG ACTTCGCCGCCTACCGGTCCAGAGTGAAGTTCTCCAGATCCGCCGATGCTCCCGCCTATA AGCA GGGCGAGA,ACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGATGTGC TGGAG AAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAG GGCC TGTACAATGAACTGCAGAAAGACAAGATGGCCGAGGCCTAGAGCGAGATCGGAATGAAGG GCGA GCGCAGAAGAGGCAAGGGAGACGATGGAGTGTAGGAGGGCCTGAGCACCGCCACC AGGATACC TATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA (SEQ ID NO: 14749)

CD28 Signal peptide:

ATGCTGAGACTGCTGCTGGCCCTGAATCTGTTCCCCAGCATCCAAGTGAGCGGC (SEQ ID NO: 14750)

CD28 Extracellular Domain :

AAC AGATCCTGGTGAAGGAGAGCCCTATGCTGGTGGCCTACGAC ACGCCGTGAACCTGAGCT GCAAGTACAGCTACAACCTGTTCAGCAGAGAGTTCCGGGCCAGCCTGCACAAAGGAATGG ATTC TGCTGTGGAAGTGTGCGTGGTGTAGGGCAACTACAGCCAGCAGCTGCAGGTCTACAGCAA GACC GGCTTC ACTGCGACGGCAAGCTGGGCAATGAGAGCGTGACCTTCTACCTGCAAAACCTGTAGG

SUBSTITUTE SHEET (RULE 26) TGAAGGAGACCGACATCTATTTCTGCAAGATCGAAGTGATGTACCCGCCTCCTTAGCTGG ACAA CGAGAAGTCCAACGGGAGCATCATCGAGGTGAAGGGCAAGGAGCTGTGTCCTTCTCGAGT GTTC CCCGGACCTAGCAAGCCT (SEQ ID NO: 14751)

CD28 Transmembrane domain:

TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACAGTG GCCT TCATCATCTTTTGGGTC (SEQ ID NO: 14752)

CD28 Cytoplasmic Domain:

CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTAGATGAACATGACCCCTAGACGGCCC GGAC CAACCAGAAAGCACTACCAGCCTTAGGCTCCTCCTAGAGACTTCGCCGCCTAGCGGTCC

(SEQ ID NO: 14753)

CD3z Intracellular Domain:

AGAGTGAAGTTCTCCAGATCCGCCGATGCTCCCGCCTATAAGCAGGGCCAGAACCAGCTG TAGA ACGAGCTGAACCTGGGGAGAAGAGAAGAGTAGGATGTGCTGGACAAGCGGAGAGGCAGAG ATCC TGAGATGGGCGGGAAGCCGAGACGGAAGAATCCTCAAGAGGGCCTGTACAATGAAGTGCA GAAA GACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGGAAG GGAG ACGATGGACTGTAGCAGGGCCTGAGGAGCGCGAGGAAGGATACCTATGATGCCCTGCACA TGCA GGCCCTGCCTCCAAGA (SEQ ID NO: 14754)

[0826] A wildtype/natural human CD 2 protein (NCBI: CD2JHUMAN ; UniProt/Swiss-Prot: P06729.2) comprises or consists of the amino acid sequence of:

MSFPCKFVASFLLIFNVSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIKWE KTSD KKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDL KIQE RVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTA GNKV SKESSVEPVSCPEKGLDIYLI IGICGGGSLLMVFVALLVFYITKRKKQRSRRNTDEELETRAHRV ATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPS GTQV HQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 14755)

[0827] A nucleotide sequence encoding wildtype/natural CD2 protein (NCBI: CCDS889.1) comprises or consists of the nucleotide sequence of:

ATGAGCTTTCCATGTAAATTTGTAGCGAGCTTCCTTCTGATTTTCAATGTTTCTTCCAAA GGTG CAGTCTCCAAAGAGATTAGGAATGCCTTGGAAACCTGGGGTGCCTTGGGTGAGGACATGA AGTT GGACATTCCTAGTTTTCAAATGAGTGATGATATTGACGATATAAAATGGGAAAAAACTTC AGAG AAGAAAAAGATTGCACAATTCAGAAAAGAGAAAGAGACTTTCAAGGAAAAAGATACATAT AAGC TATTTAAAAATGGAACTCTGAAAATTAAGCATCTGAAGACCGATGATCAGGATATCTACA AGGT ATCAATATATGATACAAAAGGAAAAAATGTGTTGGAAAAAATATTTGATTTGAAGATTCA AGAG AGGGTCTCAAAACCAAAGATCTCCTGGACTTGTATGAAGACAACCCTGACCTGTGAGGTA ATGA ATGGAACTGACCCCGAATTAAAGCTGTATGAAGATGGGAAACATCTAAAAGTTTCTCAGA GGGT CATCAGAGACAAGTGGACCACCAGCCTGAGTGCAAAATTGAAGTGCAGAGGAGGGAACAA AGTC AGCAAGGAATCGAGTGTCGAGCCTGTCAGCTGTCCAGAGAAAGGTCTGGACATCTATCTC ATCA TTGGCATATGTGGAGGAGGCAGCCTCTTGATGGTCTTTGTGGCACTGCTCGTTTTCTATA TCAC CAA AGGAAAAAACAG GGAGTCGGAGAAATGATGAGGAGCTGGAGACAAGAGCCCACAGAGTA

SUBSTITUTE SHEET (RULE 26) G C TACT G AA G AAAG G G G C C G G AAG C C C C A C C AAAT T C C AG C 1 T C A AC C C C T C AG A AT C C A G C A A C T T C C C AAC AT C C T C C T C C AC C AC C T G G T CAT C G T T C C C AG G C AC C TAG T CAT C G T C C C C C G C C TCCTGGACACCGTGTTCAGCACCAGCCTCAGAAGAGGCCTCCTGCTCCGTCGGGCACACA AGTT C AC GAG GAG AAAG G C C C G C C C C T C C C C AGAC C T C G AG T T C AG C C AAAAC C T C C C CAT G G G G C AG C AG AAAAC T CAT T G T C C C C T T C C T C T AAT T AA (SEQ ID NO: 14757)

[0828] An exemplary CSR CD2z protein of the disclosure is depicted in Fig.17 and comprises or consists of the amino acid sequence of (CD2 Signal peptide, CD2 Extracellular Domain, CD2 Transmembrane domain. CD2 Cytoplasmic Domain, CD3z Intracellular Domain):

MSFPCKFVASFELIFNVSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIKWE KTSD KKK 1 AQ tRKEKE TFKEKD TY KL FKNG TL K IKHL K TDDQD 1 YKVS I YD TKGK NVL EKltDL KI QE

RVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFK CTAGNKV SKESS VEPVS CPEKGLDI YL 11 GI CGGGS LLMVFVALLVF Y I GKRKKQRSKRMDEELETRAHRV ATEERGBKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHBBPPPGHRVQHQBQKBPPAPS GTQV HQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLYNEUCLGKR EEYO VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STAT KDT YDALHMQAL P PR (SEQ ID NO: 14757)

CD2 Signal peptide: MSFPCKFVASFLLIFNVSSKGAVS (SEQ ID NO: 14758)

CD2 Extracellular Domain.

KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTY KLFK NGTLKIKHLKTDDQDI YKVSI YDTKGKNVLEKIFDLKIQERVSKPKI S TCI TTLTCEVNINGT DPE LNL YQDGKHLKL S QRVI THKWTT S L SAKFKCTAGNKVS KE S S VE PVS CPEKGLD (SEQ ID NO: 14759)

CD2 Transmembrane domain: IYLIIGICGGGSLLMVFVALLVFYIT (SEQ ID NO:

14760)

CD 2 Cytoplasmic Domain·.

KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPP PGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 14761)

CD3z Intracellular Domain:

RVKFSRSADAPAYKQGQNQLYNELNLGR.REEYDVLDKRR.GRDPEMGGKPRRKNPQEGL YNELQK

DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:

14748)

[0829] The present disclosure provides a non-naturally occurring CSR CD2 protein

comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14756. The

SUBSTITUTE SHEET (RULE 26) present disclosure provides a CD2 signal peptide comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14758. The present disclosure provides a CD2 extracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14759. The present disclosure provides a CD2 transmembrande domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14760. The present disclosure provides a CD2 cytoplasmic domain comprising, consisting essential of) or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14761. The present disclosure provides a CD3z intracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14748

[0830] An exemplary nucleotide sequence encoding a CSR CD2z protein of the disclosure comprises or consists of the amino acid sequence of (CD 2 Signal peptide, CD2 Extracellular

Domain, CD 2 Transmembrane domain CD2 Cytoplasmic Domain, CD3z Intracellular Domain):

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCT AAGGGCG

CCGTGTCCAAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATA TTAACCT

GGACATCCCCAGCTTCCAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAAC CAGCGAC AAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTAC AAGC TGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCAGGACA TCTATAAGGT GTCCATCTACGACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCA AGAG CGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTG ATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGC GCGT GA TCACCCACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAG TG TCTAAAGAGTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACTGGACATCTACCGG AACA T C G G CAT C T G T G G C G G C G GAAG C C T G C T GAT G G T G T T T G T G G C T C T G C T G G T G T T C TAG AT C AC

GAAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCA TAGAGTG

GCCACCGAGGAAAGAGGGAGAAAGCCCCACCAGATTCCAGCCAGCACAGCCCAGAAT CCTGCGA

5UB5TITUTE SHEET (RULE 26)

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAG GGCG CCGTGTCC (SEQ ID NO: 14763)

CD 2 Extracellular Domain :

AAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCTGGAC ATCC CCAGCTTCCAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGA AGAA GATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTAG AGCTGTTCAAG AACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACGAGGACATCTATAAGGTGTCC ATCT ACGACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCAAGAGCGGG TGTC CAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTGATGAACGG CACA GACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATC ACCC ACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTA AAGA GTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACTGGAC (SEQ ID NO: 14764)

CD2 Transmembrane domain:

ATCTACCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTG CTGG TGTTCTACATCACC (SEQ ID NO: 14765)

CD 2 Cytoplasmic Domain:

AAGCGGAAGAAGGAGCGGAGCAGACGGAACGAGGAGGAACTGGAAACACGGGCCCATAGA GTGG CCACCGAGGAAAGAGGCAGAAAGCCCCACGAGATTCGAGCGAGCACACCCCAGAATCCTG CCAC CTCTCAACACCCTCCACCTCCACCTGGAGACAGATCTCAGGCCCCATCTCACAGACCTCC ACCA CCTGGTCATCGGGTGGAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCACACAG GTGC ACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGCG CCGC TGAGAACAGCCTGTCTCCAAGCAGCAAC (SEQ ID NO: 14766)

CD3z Intracellular Domain:

AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTG TACA ACGAGCTGAATCTGGGGCGCAGAGAAGAGTACGATGTGCTGGACAAGCGGAGAGGCAGAG ATCC TGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCA GAAA GACAAGATGGCCGAGGCCTACAGCGAGATCGG ATGAAGGGCGAGCGCAGAAGAGGGAAGGGAC ACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACA TGCA GGCCCTGCCTCCAAGA (SEQ ID NO: 14767)

SUBSTITUTE SHEET (RULE 26) [0831] An exemplar mutant CSR CD2z-Dl 11H protein of the disclosure comprises or consists of the annno acid sequence of (CD2 Signal peptide, CD2 Extracellular domain with D 111H mutation within the CD 2 Extracellular domain CD2 Transmembrane domain CD2

Cytoplasmic domain , CD3z Intracellular domain):

MSFPCKFVASFIYLlFWsrSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIK WEKTSD

KKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQD1YKVS IYHTKGKNVLEK1FDLKIQE

RVSKPKISWTCINTTL TCEVMNGTDPELNL YQDGKHLKLSQRVI THKWTTSLSAKFKCTAGNKV

SKESSVEPVSCPEKGLD1YL11GICGGG S LLKVFVA LVFY l YKRKKQRSRRNDEELETRAHRV ATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPS GTQV

HQQKGPPLPRPRVQPKPPHGA ENSLSPSSWRVKFS 3NQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQG: STAT KDTYDALHMQALPPR (SEQ ID NO: 14 768)

CD2 Signal peptide: MS FPCKFVAS FLLI FNVS SKGAVS (SEQ ID NO: 14758)

CD2 Extracellular domain with D111H mutation within the CD2 Extracellular domain:

KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEK DTYKLFK NGTLKIKHLKTDDQDIYKVSIYHTKGKNVLEKIFDLKIQERVSKPKI SWTCINTTLTCEVMNGT DPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGL (SEQ ID NO: 14769)

CD2 Transmembrane domain:

IYLI IGICGGGSLLMVFVALLVFYIT (SEQ ID NO: 14760)

CD 2 Cytoplasmic domain',

KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPP

PGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLS PS SN ( SEQ ID NO :

14761)

CD3z Intracellular domain:

RVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DK AEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR ( SEQ ID NO:

147 8)

[0832] The present disclosure provides a non-natural!y occurring CSR CD2 protein

comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14768. The present disclosure provides a CD2 extracellular domain comprising, consisting essential of, or consisting of an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at

SUBSTITUTE SHEET (RULE 26) least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14769.

[0833] An exemplar nucleotide sequence encoding a mutant CSR CD2z-Dl 11H protein of the disclosure comprises or consists of the ammo acid sequence of (CD2 Signal peptide, CD2 Extracellular domain with D111H mutation within the CD 2 Extracellular domain , CD2

Transmembrane domain, CD2 Cytoplasmic domain, CD3z Intracellular domain):

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAG GGCG

CCGTG CCAAAGAGATCACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCCAGGATATTAACCT GGACA TCCCCAGCTTCCAGATGAGCGACGACATCGA TGACATCAAGTGGGAGAAAACCAGCGAC AAGAAGAAGATCGCCCAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTAC AAGC TGTTCAAGAACGGCACCCTGAAGATCAAGCACCTGAAAACCGACGACCAGGACATCTATA AGGT GTCCATCTACCACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTCAAGATCCA AGAG CGGGTGTCCAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCGAAGTG ATGA ACGGCACAGACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGC GCGT GATCACCCACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAA AGTG TCTAAAGAGTCCAGCGTCGAGCCCGTGTC TGCCCTGAAAAAGGACTGGACAECTACCTGATCA TCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTGTTTGTGGCTCTGCTGGTGTTCTACA TCAC

^ Q GQA QA GCAG QQ QC QA GQ C CSA QAGQA TGGA CA GQG C l^AQ Q^Q GCCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCT GCCA P PYPΆ Ά *Ά HGΉYHHΆ PH HHΆ 3 *Ά G*Ά Ά YP HΆ (2P ⁹ *G* 'Ά G*Ά HΆ ί Ά GΉYHHΆ HG*

ACCTGGTCATCGGGTGCAGCACCAGCCTCAGAAAAGACCTCCTGCTCCTAGCGGCAC ACAGGTG CACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAGTGCAGCCTAAACCTCCTCATGGC GCCG CTGAGAACAGCCTGTCTCCAAGCAGCAACAGAGTGAAGTTGAGCCGGAGCGCCGATGCTC CTGC CTATAAGGAGGGACAGAACGAGCTGTAGAACGAGCTGAATCTGGGGCGCAGAGAAGAGTA GGAT GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGCAAGCCCAGACGGAAGAAT CCTC AAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTAGAGCGAGATCG GAAT GAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGC CACC AAGGATACCTATGATGCCCTGCACATGGAGGCCCTGCCTCCAAGA (SEQ ID NO: 14770)

ATGAGCTTCCCTTGCAAGTTCGTGGCCAGCTTCCTGCTGATCTTCAACGTGTCCTCTAAG GGCG CCGTGTCC (SEQ ID NO: 14763)

CD2 Extracellular domain with DillH mutation within the CD2 Extracellular domain:

AAAGAGATGACAAACGCCCTGGAAACCTGGGGAGCCCTCGGCGAGGATATTAACCTG GACATCC CCAGCTTCGAGATGAGCGACGACATCGATGACATCAAGTGGGAGAAAACCAGCGACAAGA AGAA GATCGCCGAGTTCCGGAAAGAGAAAGAGACATTCAAAGAGAAGGACACCTAG AGCTGTTCAAG AACGGCACCCTGAAGATCAAGGACCTGAAAACCGACGACGAGGACATCTATAAGGTGTCC ATCT ACCACACCAAGGGCAAGAACGTGCTGGAAAAGATCTTCGACCTC AGATCCAAGAGCGGGTGTC CAAGCCTAAGATCAGCTGGACCTGCATCAACACCACACTGACCTGCG AGTGATGAACGGCACA GACCCCGAGCTGAACCTGTACCAGGATGGCAAACACCTGAAGCTGAGCCAGCGCGTGATC ACCC ACAAGTGGACAACAAGCCTGAGCGCCAAGTTCAAGTGCACCGCCGGAAACAAAGTGTCTA AAGA GTCCAGCGTCGAGCCCGTGTCTTGCCCTGAAAAAGGACTGGAC (SEQ ID NO: 14771)

SUBSTITUTE SHEET (RULE 26) CD2 Transmembrane domain:

ATC AGCTGATCATCGGCATCTGTGGCGGCGGAAGCCTGCTGATGGTG TTGTGGCTCTGCTGG TGT CTACATCACC (SEQ ID NO: 14765)

Cl) 2 Cytoplasmic domain :

AAGCGGAAGAAGCAGCGGAGCAGACGGAACGACGAGGAACTGGAAACACGGGCCCATAGA GTGG CCACCGAGGAAAGAGGCAGAAAGCCCCACCAGATTCCAGCCAGCACACCCCAGAATCCTG CCAC CTCTCAACACCCTCCACCTCCACCTGGACACAGATCTCAGGCCCCATCTCACAGACCTCC ACCA CCTGGTCATCGGGTGGAGCACCAGCCTCAGAAAAGACCTCCTGCTCC AGCGGCACACAGG GC ACCAGCAAAAAGGACCTCCACTGCCTCGGCCTAGAG GCAGCCTAAACCTCCTCATGGCGCCGC TGAGAACAGCCTGTCTCC.AAGCAGCAAC (SEQ ID NO: 14766)

CD3z Intracellular domain:

AGAGTGAAGTTCAGCCGCAGCGCCGATGCTCCTGCCTATAAGCAGGGACAGAACCAGCTG TAGA ACGAGCTGAATCTGGGGCGCAGAGAAGAGTAGGATGTGCTGGACAAGCGGAGAGGCAGAG ATCC TGAGATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCA GAAA GACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAG GGAC

ACGATGGACTGTATCAGGGCCTGAGCACCGCCACCAAGGATACC ATGATGCCCTGCACATGCA

GC O A ' V FPn T n wn ·

[Q834] Gene editing compositions of the disclosure, including but not limited to, RN A-guided fusion proteins comprising dCas9~Clo051, may be used to target and decrease or eliminate expression of an endogenous T-cell receptor of an allogeneic cell of the disclosure. In preferred embodiments, the gene editing compositions of the disclosure target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding an endogenous T-cell receptor of an allogeneic cell of the disclosure.

[0835] Nonlimiting examples of primers (including a T7 promoter, genome target sequence, and gRNA scaffold) for the generation of guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-a) are provided in Table 2.

[0836] Table 2. Target sequences underlined

SUBSTITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26)

[0837] Nonlimiting examples of primers for the generation of guide RNA (gRNA) templates for targeting and deleting TCR-beta (TCR-b) are provided in Table 3.

0838] Table 3. Target sequences underlined

5UB5TITUTE SHEET (RULE 26)

[0839] Nonlimiting examples of primers for the generation of guide RNA (gRNA) templates for targeting and deleting beta-2-microglobulin (b2M) are provided in Table 4

0840] Table 4. Target sequences imder!ined

5UB5TITUTE SHEET (RULE 26)

[0841] Gene editing compositions of the disclosure, including but not limited to, RNA-guided fusion proteins comprising dCas9-Clo051, may be used to target and decrease or eliminate expression of an endogenous MHO, MHCII, or MHC activator of an allogeneic cell of the disclosure. In preferred embodiments, the gene editing compositions of the disclosure target and delete a gene, a portion of a gene, or a regulatory element of a gene (such as a promoter) encoding one or more components of an endogenous MHCI, MHCII, or MHC activator of an allogeneic cell of the disclosure.

[0842] Nonlimiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are provided in Tables 5 and 6

[0843] Table 5.

5UB5TITUTE SHEET (RULE 26)

5UB5TITUTE SHEET (RULE 26) [0844] Table 6

[0845] MHO knockout (KQ) renders cells resistant to killing by T cells, but also makes them susceptible to natural killer (NK) cell-mediated cytotoxicity (“Missing-self hypothesis”). It is hypothesized that NK rejection would reduce the in vivo efficacy and/or persistence of these KO cells in a therapeutic setting, such as allogeneic (alio) CAR-T therapy. Retention of MHO on the surface of alio CAR-T cells would render them susceptible to killing by host T cells, as observed in the classic mixed lymphocyte reaction (MLR) experiment. It is estimated that up to 10% of a person’s T cells are specific to foreign MHC, which would mediate the rejection of foreign cells and tissues. A targeted KO of MHCI, specifically HLA-A, B and C, which can be achieved by targeted KO of B2M, results in a loss of additional HLA molecules including HI A-E. Loss of HLA-E, for example, renders the KO ceils more susceptible to NK cell-mediated cytotoxicity due to the“Missing-self Hypothesis”. NK-mediated cytotoxicity' against missing-self cells is a defense mechanism against pathogens that downregulate MHC on the surface of infected cells to evade detection and killing by cells of the adaptive immune system.

[0846] Two strategies are contemplated by the disclosure for engineering alio (MHCI-neg) T cells (including CAR-T cells) more resistant to NK cell-mediated cytotoxicity . In some embodiments, a sequence encoding a molecule (such as single-chain HLA-E) that reduces or prevents NK killing is introduced or delivered to an allogeneic cell. Alternatively, or in addition, gene editing methods of the disclosure retain certain endogenous HLA molecules (such as endogenous HLA-E). For example, the first approach involves piggy Bac (PB) delivery of a single-chain (sc)HLA-E molecule to B2M KO T cells.

[0847] The second approach uses a gene editing composition with guide RNAs selective for HLA-A, HLA-B and HLA-C, but not, for example, HLA-E or other molecules that are protective against natural-killer cell mediated cytotoxicity for MHCI KO cells.

[0848] Alternative or additional molecules to HLA-E that are protective against NK cell- mediated cytotoxicity include, but are not limited to, CD47, interferon alpha/beta receptor 1 (IFNAR1), human IFNAR1, interferon alpha/beta receptor 2 (IFNAR2), human IFNAR2, HLA- Gl , HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, human carcino embryonic antigen-related cell adhesion molecule 1 (CEACAM1), viral hemoagglutinins, CD48, LLT1 (also referred to as C-type lectin domain family 2 member (CLC2D)), ULBP2, ULBP3, and sMICA or a variant thereof. [0849] An exemplary CD47 protein of the disclosure composes or consists of the amino acid sequence of (Signal peptide. Extracellular, TM, Cytoplasmic)·.

MWPLVAALLLGSACCGSAOLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKW

KFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCE VT

ELTREGETIIELKYRW;

L VAGL V1TV1 VIVGAILFVPGE YSLKNA TGLGLIVTSTGl VFSTAiGL TSFVIAIL V

IQ VIA YILA WGLSLCMACIPMHGPUJSGLSILALAQLLGL VYMKFVASNOKTIQPPRKA V EEPLNAFKESKGMMNDE (SEQ ID NO: 14902).

[0850] An exemplary INFAR1 protein of the disclosure comprises or consists of the ammo acid sequence of (Signal peptide. Extracellular, TM, Cytoplasmic).

MMWLLGATTLVLVAVAPWLSAAAGGKNLKSPQKVEVDI IDONEILRWNRSDESVGNVT FSFDYQKTGMDKWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSF TPFRKAQIGPPEV LEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERI ENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPPPENIEVSVQ NQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQK GIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLPPVFNIRSLSDSFHIYIGAPKQSGNTP VIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSV FSOAVCEKTKPGmSKIWLIVGICIALFALPFVIYAAKVFLRCINYVFFPSLKPSSSIDE YES EQPLKNLL L S TSEE Q IEK CFI I ENI S TIA TVEE TNQ IDE DHKK YSSQ TS QOS GNYS N EDESESKTSEELQQDFV (SEQ ID NO: 14903) .

[0851] An exemplary INFAR2 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide. Extracellular, TM, Cytoplasmic)·.

MLLSQNAFI FRSLNLVLMVYISLVFGISYDSPDYTDESCTFKISLRNFRSILSWELKNHS IVPTHYTLLYTIMSKPEDLKWKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTTLF SCSHNFWLAIDMSFEPPEFEIVGFTNHINVMVKFPSI\FEEELQFDLSLVIEEQSEGIVK K HKPEIKGNMSGNFTYI IDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAE SAK IGGIITVFLIAL VL TSTIVTL KWIG YICLRNSL PKVLNFHNFL AWPFPNL PPLEAMD MVEVIYINRKKKVWDYNYDDESDSDTEAAPRTSGGGYTMHGLTVRPLGQASATSTESQLI DPESEEEPDLPEVDVELPTMPKDSPQQLELLSGPCERRKSPLQDPFPEEDYSSTEGSGGR ITFNVDLNSVFLRVLDDEDSDDLEAPLMLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPT FPS PSSEGL WSEDAPSDQSDTSESDVDLGDGY IMR (SEQ ID NO: 14904) .

[0852] An exemplar} HLA-G1 protein of the disclosure composes or consists of the amino acid sequence of (Alpha chain 1 , Alpha chain 2 , Alpha chain 3):

MWMAPRTL FLLLS GALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDS

ACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSflTLQ fv GCDLG

SDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLE GTCVEWL

iJRYLF GKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVE TRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLWL AA WT GAAVAAVLWRKKS S D (SEQ ID NO: 14905} . [0853] An exemplary HLA-G2 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1 , Alpha chain 2, Alpha chain 3):

MWMAPRTL FLLLS GALTLTET WAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDS ACPRME PRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEADPPKTHVTHHPVFD

YEATLRCWALGFYPAE I I LTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCH T GAAVAAVLWRKKS S D (SEQ ID NO:

[0854] An exemplary HLA-G3 protein of the disclosure comprises or consists of the ammo acid sequence of (Alpha chain 1 , Alpha chain 2 , Alpha chain 3):

MWMAPRTL FLLLS GALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDS ACPRME PRAFWVEQEGPE YWEEE TRNTKAHAQTDRMNLQTLRGY YNQSEAKQS S L PT I P IMG I V

AGLWLAAWTGAAVAAVLWRKKS SD (SEQ I D NO: 14907) .

[0855] An exemplar}' HLA-G4 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1 , Alpha chain 2, Alpha chain 3):

MWMAPRTL FLLLS GALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDS ACPRME PRAFWVEQEGPE YWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSMRLGRMIGCDGG

SDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLE GTCVEWL

HRYLENGKEMLQRAKQS S L PT I P IMG I VAGLWLAAWT GAAVAAVLWRKK S S D (SEQ ID NO:

14908) .

[0856] An exemplary HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1 , Alpha chain 2, Alpha chain 3. intron 4)

MWMAPRTL FLLLS GALTLTET WAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDS ACPRME PRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQ!V TGCDLG

SDGRLLRGYEOYAYDGKDYLALNEDLRSWTAADTAAQ1SKRKCEAANVAEQRRAYLE GTCVEWL HR YLENGKEML QRADPPKTHVTHHPVFDYEATLRCWALGFYPAE I I LTWQRDGEDQTQDVELVE TRPAGDGTFQKWAAV PSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRS-LSEDL

(SEQ ID NO: 14909) .

[0857] An exemplar}' HLA-G5 protein of the disclosure composes or consists of the amino acid sequence of (Alpha chain 1 , Alpha chain 2, Alpha chain 3. intron 4):

MWMAPRTL FLLLS GALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDS ACPRME PRAPWVEQEGPEYWEEETRNTKAHAQTPRMNLQTLRGYYNQSEADPPKTHVTHHPVFD

YEATLRCWALGFYPAE I ILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGEEQRYTCH VQHE GL PE P LMLRWSKEGDGGIMSVKESRSLSEDL (SEQ ID NO: 14910) . [0858] An exemplary HLA-G5 protein of the disclosure comprises or consists of the amino acid sequence of (Alpha chain 1 , Alpha chain 2, Alpha chain 3, intron 2) \

MWMAPRTLFLLLSGALTLTETWAGSHS RYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDS

ACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNL TLRGYYNQSEASE (SEQ ID NO: 14911) .

[0859] An exemplary CEACAM1 protein of the disclosure comprises or consists of the amino acid sequence of (Extracellular, TM, Cytoplasmic):

MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQ QLFGYSWYKGERVDGNRQ1VGYAIGTQQATPGPANSGRETIYPNASLL1Q VTQNDTGFY TLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYL WI NNQSLPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLNVTYGPDTP TISPSDTYYRPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCH ANNSVTGCNRTTVKTJ IVTELSPVVAKPQJKASKTTVTGDKDSWLTCSTNDTGISJRWF FKNQSLPSSERMKLSQGNTTLSINPVKREDAGTYWCEVFNPISKNQSDPIMLN YNALP QENGLSPGAJAGJVIGVVALVALJAVALACFIHFGKTGRASDQRDLTEHKPSVSNHTQDH SNDPPNKMNEVTYSTLNFEAQQPTQPTSASPSLTATEI IYSEVKKQ (SEQ ID NO: 14912) .

[Q86Q] An exemplar}' viral hemagglutinin protein of the disclosure comprises or consists of the amino acid sequence of (HAforInfluenzaAvirus(A/NewCaledonia/20/1999(HlNl); TM):

MKAKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEK TVTHSVNLLEDSHNGKLCL LKGIAPLQLGNCSVAGWILGNPECELLI SKESWSYIVETPNPENGTCYPGYFADYEELRE QLSSVSSFERFEIFPKESSWPNHTVTGVSASCSHNGKSSFYRNLLWLTGKNGLYPNLSKS YWNKEKEVLVLWGVHHPPNIGNQRALYHTENAYVSWSSHYSRRFTPEIAKRPKVRDQE GRINYYWTLLEPGDTI IFEANGNLIAPWYAFALSRGFGSGI ITSNAPMDECDAKCQTPQG AINSSLPFQNVHPVTIGECPKYVRSAKLRMVTGLRNIPSIQSRGLFGAIAGFIEGGWTGM VDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKWSVIEKMNTQFTAVGKEFNKLERRM ENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGC FEFYHKCNNECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSL VL L VS L GA I S F MC S N G S L Q C R I C I (SEQ ID NO: 14913) ,

[0861] An exemplar}' CD48 protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide. Chain, Pro peptide removed in mature form):

SRG D' ALELLLLPLS LL ;IQGHLVHMTWSGS VTLN1SESLP

ENYKQLTWFYTFDQKIVEWDSRKSKYFESKFKGRVRLDPQSGALYISKVQ KEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKIEKIEDMDDNCYL KLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSN SVSSKNGTVCLSPPCTLARSFGVEWIASWLVVTVPTILGLLLT (SEQ ID NO: 14914) .

[0862] An exemplar}' LLT1 protein of the disclosure comprises or consists of the amino acid sequence of (Cytoplasmic, TM, Extracellular): MHDSNlSrVEKDITPSELPANPGCLHSKEHSIKATLIWRLF LIMFLTIIV77G WlALSAIj

ANCHQEPSVCLQAACPESWIGFQRKCFYFSDDTKNWTSSQRFCDSQDADLAQVESFQELN

FI RYKGPSDHW GLS QGQP^WINGTEWTRQFPILGAGECAYLNDKGASSARHYTER

KWICSKSDIHV (SEQ ID NO: 14915) .

[0863] An exemplary ULBP2 protein of the disclosure comprises or consists of the ammo acid sequence of (also known as NKG2D ligand; Genbank ACCESSION No. AAQ89028):

1 maaaaatkil lclpllllls gwsragradp hslcyditvi pkfrpgprwc avqgqvdekt

61 flhydcgnkt vtpvsplgkk lnvttawkaq npvlrevvdi lteqlrdiql enytpkeplt

121 Iqarmsceqk aeghssgswq fsfdgqlfll fdsekrmwtt vhpgarkmke kwendkvvam

181 sfhyfsmgdc igwledflmg mdstlepsag aplamssgtt qlratattli lcclliilpc

241 fiipgi (SEQ ID NO: 14916) .

[0864] An exemplary ULBP3 protein of the disclosure comprises or consists of the ammo acid sequence of (also known as NKG2D ligand; Genbank ACCESSION No. NP 078794):

1 maaaaspail prlailpyll fdwsgtgrad ahslwynfti ihlprhgqqw cevqsqvdqk

61 nflsydcgsd kvlsmghlee qlyatdawgk qlemlrevgq rlrleladte ledftpsgpl

121 tlqvrmscec eadgyirgsw qfsfdgrkfl lfdsnnrkwt vvhagarrmk ekwekdsglt

181 tffkmvsmrd ckswlrdflm hrkkrlepta pptmapglaq pkaiattlsp wsfliilcfi

241 ipgi (SEQ ID NO: 14917) .

[0865] An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signal Peptide. Portion of Extracellular domain, JM and cytoplasmic domain ) (Genbank Accession No. Q29983):

1 mglgpvflll agifpfappg aaaephslry nltvlswdgs vqsgfltevh ldgqpflrcd

61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce

121 ihednstrss qhfyydgelf Isqnletke tmpqssraqt lantnvrnflk edamkt thy

181 hamhadclqe Irrylksgw Irrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr

241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv

301 1 vlqshwqtf hvsavaaaai fviiifyyrc ckkktsaaeg pelvslqyld qhpygtsdhr

361 datqlgfqpl msdlgstgst ega (SEQ ID NO: 14918).

[0866] An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Alpha- 1. Alpha-2, Alpha- 3):

1 mglgpvflll agifpfappg aaaephslry nltvlswdgs vqsgfltevh ldgqpflrcd

61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce

121 ihednstrss qhfyydgelf Isqnletkew tmpqssraqt lamnvrnflk edamktkthy

181 hamhadclqe Irrylksgw Irrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr

241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpypsqkv

301 1vlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvld qhpvgtsdhr

361 datqlgfqpl msdlgstgst ega (SEQ ID NO: 14919).

[0867] An exemplary sMICA protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide ; Alpha- 1. Alpha-2, Alpha- 3): MGGVLLTQB LLSLVLALLFBSM&SM ephslry nltylswdgs vqsgfltevh ldqqpflrcd

61 rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce

121 i ednstrss qhfyydgelf Isqnletkew tmpqssraqt 1 thy

181 hamhadclqe Irrylksgw Ixxtvppmvn vtrseasegn itvtcrasgf ypwnitlswr

241 qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh s thpvpsgkv

301 lviqshw (SEQ ID NO: 14920).

[0868] An exemplar ' sMlCA protein of the disclosure comprises or consists of the amino acid sequence of (Signal peptide).

MSGV iTGRTiLSIiVLALL PS ASMEPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDR

QKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKLDLRMTLAHIKLDQKEGLHSLQEI RVCEIHE DNSTRSSQHFYYNGELFLSQNLETKEWTMPQSSRAQTLTHYHAMHADCLQELRRYLKSGW LRR TVPPMVDVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGT YQTW VATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW (SEQ ID NO: 14921) .

[0869] An exemplary bGBE Tnrner (270G and 4 MS) protein of the disclosure comprises or consists of the ammo acid sequence of:

MSRSVALAVLALLSLSGLEAVMAPRTLI LGGGGSGGGGSGGGGS IQRTPKIQVYSRHRΆENGKS

NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDE YACRVNH

VTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRF ISVGYVD

DTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYN QSEAGSH

TLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDA SEAEHQR

AYLEDTC\7EWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEI TLTWQQDG

EGHTQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVTLRWKPASQ PTIPIV

GI IAGLVLLGSWSGAWAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL* (SEQ ID NO:

14922).

[0870] An exemplar}' bGBE Trimer (270G and 484S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

atgtctcgcagcgtggccctggccgtgctggccctgctgtccctgtctggcctggag gccgtga tggccccccggaccctgatcctgggaggaggaggcagcggcggaggaggctccggaggcg gcgg ctctatccagcgcacacctaagatccaggtgtattctcggcacccagccgagaacggcaa gagc aacttcctgaattgctacgtgagcggctttcacccttccgacatcgaggtggatctgctg aaga atggcgagagaatcgagaaggtggagcactccgacctgagcttctccaaggattggtctt ttta tctgctgtactataccgagtttacccctacagagaaggacgagtacgcctgtcgcgtgaa ccac gtgacactgtcccagccaaagatcgtgaagtgggaccgggatatgggc ggcggcggctctggcg gcggcggcagcggcggcggcggctccggaggaggcggctctggcagccactccctgaagt attt ccacacctctgtgagccggccaggcagaggagagccacggtteatetctgtgggctacgt ggac gatacacagttcgtgaggtttgacaatgatgccgccagcccaagaatggtgcctagggcc ccat ggatgga gca ggagggcagcgagta11gggacagggagacccggagcgccagagacacagcaca gattttccgggtgaacctgagaaccctgaggggctactataatcagtccgaggccggctc tcac acactccagtggatgcacggatgcgagctgggaccagatcjgccgcttcctgcggggcta cgagc agtttgcctatgacggcaaggattacctgaccctgaacgaggacctgagatcctggaccg ccgt ggatacagccgcccagatcagcgagcagaagtccaatgacgcatctga ggcagagcaccaga gg gcatatctggaggatacctgcgtggagtggctgcacaagtacctggagaagggcaaggag acac tgctgcacctggagccccctaagacccacgtgacacaccacccaatcagcgaccacgagg ccac cctgaggtgttgggcactgggcttctatcccgccgagatcaccctgacatggcagcagga cgga gagggacacacccaggatacagagctggtggagaccaggcccgccggcgatggcacattt caga agtgggccgccgtggtggtgcc11ccggagaggagcagagatacacctgtcacgtgcagc acga gggactgccagagccagtgaccctgaggtggaagcctgccagccagcccacaatccctat cgtg ggaatcatcgcaggcctggtgctgctgggctctgtggtgagcggagcagtggtggccgcc gtga tctggcggaagaagagcagcggaggcaagggaggctcctactccaaggcagagtggagcg actc cgcccagggctctgagagccactccctgtga (SEQ ID NO: 14923) .

[0871] An exemplar} bGBE Trimer (270M and 484S) protein of the disclosure comprises or consists of the ammo acid sequence of:

MSRSVALAVLALLSLSGLEAVMAPRTLI LGGGGSGGGGSGGGGS IQRTPKIQVYSRHRΆENGKS NFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC RVNH VTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISV GYVD DTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSE AGSH TLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEA EHQR AYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTW QQDG EGHTQDTELVETRPAGDGTFQKWAAVWPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTI PIV

GIIAGLVLLGSWSGAWAAVIWRKKSSGGKGGSY&KAEWSDSAQGSESHSL*(S EQIDNO:

14924).

[0872] An exemplary bGBE Tnrner (270R and 484S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

atgtctcgcagcgtggccctggccgtgctggccctgctgtccctgtctggcctggag gccgtga tggccccccggaccctgatcctgggaggaggaggcagcggcggaggaggctccggaggcg gcgg ctctatccagcgcacacctaagatccaggtgtattctcggcacccagccgagaacggcaa gagc aacttcctgaattgctacgtgagcggctttcacccttccgacatcgaggtggatctgctg aaga atggcgagagaatcgagaaggtggagcactccgacctgagcttctccaaggattggtctt ttta tctgctgtactataccgag111acccctacagagaaggacgagtacgcctgtcgcgtgaa ccac gtgacactgtcccagccaaagatcgtgaagtgggaccgggatatgggcggcggcggctct ggcg gcggcggcagcggcggcggcggctccggaggaggcggctctggcagccactccctgaagt attt ccacacctctgtgagccggccaggcagaggagagccacggtteatetctgtgggctacgt ggac gatacacag11cgtgagg111gacaatgatgccgccagcccaagaatggtgcctagggcc ccat ggatggagcaggagggcagcgagtattgggacagggagacccggagcgccagagacacag caca gattttccgggtgaacctgagaaccctgaggggctactataatcagtccgaggccggctc tcac acactccagtggatgcacggatgcgagctgggaccagategccgc11cctgcggggc tacgagc agtttgcctatgacggcaaggattacctgaccctgaacgaggacctgagatcctggaccg ccgt ggatacagccgcccagatcagcgagcagaagtccaatgacgcatctgaggcagagcacca gagg gcatatctggaggatacctgcgtggagtggctgcacaagtacctggagaagggcaaggag acac tgctgcacctggagccccctaagacccacgtgacacaccacccaatcagcgaccacgagg ccac cctgaggtg11gggcactgggc1 ctatcccgccgagatcaccctgacatggcagcaggacgga gagggacacacccaggatacagagctggtggagaccaggcccgccggcgatggcaca111 caga agtgggccgccgtggtggtgccttccggagaggagcagagatacacctgtcacgtgcagc acga g g g a c t g c c a g a g c c a g t g a c c c t g a g g t g g a a g c c t g c c a g c c ag c c c a c a a t c c c t a t c g t g ggaatcatcgcaggcctggtgctgctgggctctgtggtgagcggagcagtggtggccgcc gtga t c t g g c gg a ag a a g a g c a g c gg a gg c a a g g g a g g c t c c t a c t cc a a g g c a g a g t gg a g c g a c t c cgcccagggctctgagagccactccctgtga (SEQ ID NO: 14925) .

[0873] An exemplary gBE Dimer (R and S) protein of the disclosure comprises or consists of the ammo acid sequence of:

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEV DLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDN DAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQI FRVNLRTLRGYYNQSEAGSHTLQWM HGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRA YLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQ QDGEGHTQDTELVETRPAGDGTFQKWAAWVPSGEEQRYTCHVQHEGLPEPVTLRWKPAS QPTIPIVGI IAGLVLLGSWSGAWAAVIWRKKSSGGKGGS YSKAEWSDSAQGSESHSL (SEQ ID NO: 14926) .

[0874] An exemplary gBE Dimer (R and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

AT GAG C AG AT C T G Ϊ G G CCCTGGCTGTTCTGGCTCTGCTGTCTCTGT C T G G C C T G GAAG C CAT C C AG C G GAC C C C T AAG AT C C AG G T G AG AG C AG AC AC C C C G C C G AGAAC G G C AAG AG C A C T T C C T GAAC T G C T AC G T G T C C G G C T T T C AC C C C AG C GAC AT GAG G T G GAC C T G C T GAAGAAC G G C GAG C G GAT C GAG AAG G G G AAC AC AG C GAT C T GAG C T T C AG C AAG GAC T G G T C C T T C AC C T G C T G T AC TAG AC C GAG T T C AC C C C TAG C GAGAAG GAC GAG TAG G C C T G C AGAG T GAAC C AC G T GAC AC T GAG C C AG C C T .AAG AT C G T GAAG T G G GAC AG AG AT AT G G G C G GAG G C G GAT C T G G T G G C G GAG G A A G T G G C G G C G GAG G A T C T G G C G G T G G T G G T T C T G G AT C T C AC AG C C T GAAG T AC T T T C AC AC C T C C G T G T C C AG AC C T G G C AGAG G C GAG C C T AGAT CA C AG C G T G G G C AC G G GAC GAC AC C C A GTTCGTCAGATTCGACAACGACGCCGCCTCTCCTCGGATGGTTCCTAGAGCACCCTGGAT GGAA C AAGAG G G GAG C GAG T AC T G G GAT C G C G AGAC AAG AAG C G C C AGAGAC .AC AG C C C AGAT C T T C C G C G T GAAC C T GAGAAG C C T G C G G G G C T AC T AC AAT C AG T C T GAG G C C G G C T C T C AC AC C C T G C A G T G GAT G CAT G GAT G T GAAC T G G G C C C C GAC AGACGGT T C C T GAG AG G C T AT GAG C AG T T C G C C TAG GAC G G C AAG GAC TAG C T GAC AC T GAAC GAG GAC C T GAGAAG C T G GAC C G C C G T G GAT AC AG C C G C T C AGAT C AG C GAG C AGAAG T C T AAC GAC G C C AG C GAG G C C GAAC AC C AGAG AG C C T AT C T G GAAGAT AC C T G C G T G GAAT G G C T G GAC AAG TAG C T G G AAAAG G G C AAAGAG AC AC T G C T G C AC C T G GAAC C T C C AAAGAC AC A G T G AC C C AC CAT C C T AT C AG C GAC C AC GAG G C C AC AC T G AGAT G T T G G G C C C T G G G C T T T TAG C C T G C C G AGA C AC AC T GAC AT G G C AG C AG GAT G G C GAG G G C C A C AC .AC AG GAT AC AG AG C T G G T G GAAAC AAGAC C T G C C G G C G AC G G C AC C T T C C AGAAAT G G G C T G C T G T G G T T G T G C C C AG C G G C GAG GAAC AGAGAT AC AC C T G T C AC G T G C AG C AC GAG G GAC T G C C T G AAC C T G T G AC T C T GAG AT G G AA G C C T G C C AG C GAG C C A A C A AT C C C C AT C G T G G GAAT C AT T G C C G G C C T G G T G C T G C T G G GAT C T G T G G T T T C T G G T G C T G T G G T G G C C G C C G T GAT T T G GAGA AAG AAG T C C T C T G G C G G C AAAG G C G G C T C C T AC TCTAAG G C C GAG T G GAG C GAT T C T G C C C AG G G C T C T GAAAG C C AC AG C C T G T AGAT AA (SEQ ID NO: 14927). [0875] An exemplary gBE Dimer (G and S) protein of the disclosure comprises or consists of the ammo acid sequence of:

DLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVG YVDDTQFVRFDN DAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQI FRVNLRTLRGYYNQSEAGSHTLQWM HGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRA YLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQ QDGEGHTQDTELVETRPAGDGTFQKWAAWVPSGEEQRYTCHVQHEGLPEPVTLRWKPAS

[0876] An exemplary gBE Dimer (G and S) protein of the disclosure comprises or consists of the ammo acid sequence of:

ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC ATCC AG C G GAC C C C T AAG AT C GAG G T G T AC AG C AG AC AC C C C G C C GAGAAC G G C AAGAG C AAC T T C C T GAACTGCTACGTGTCCGGCTTTCACCCCAGCGACATTGAGGTGGACCTGCTGAAGAACGG CGAG C G GAT C GAG AAG G T G G AAC AC AG C GAT C T GAG C T T GAG C AAG GAC T G G T C C T T C T AC C T G C T G T AC TAG AC C GAG T T C AC C C C TAG C G AGAAG GAG GAG TAG G C C T G GAG AG T GAAC C AC G T GA C AC T GAG C GAG C C T AAG AT C G T GAAG T G G GAC AG AGAT AT G G G C G GAG G C G GAT C T G G T G G C G GAG GA AGTGGCGGCGGAGGATCTGGCGGTGGTGGTTCTGGATCTCACAGCCTGAAGTACTTTCAC ACCT C C G T G T C C AGAC C T G G C AGAG G C GAG C C TAG AT T CAT C AG C G T G G G C T AC G T G GAG GAC AC C C A G T T C G T C AG AT T C GAC AAC GAC G C C G C C T C T C C T C G GAT G G T T C C TAG AG C AC C C T G GAT G G AA C AAGAG G G C AG C GAG T AC T G G GAT C G C G AGAC AAG AAG C G C C AGAGAC ACAGC C CAGAT C T T CC G C G T GAAC C T GAGAAC C C T G C G G G G C T AC T AC AAT C AG T C T GAG G C C G G C T C T C AC AC C C T G C A G T G GAT G C T G GAT G T GAAC T G G G C C C C GAC AGACAGT T C C T GAG AG G C T AT GAG C AG T T C G C C TACGACGGCAAGGACTACCTGACACTGAACGAGGACCTGAGAAGCTGGACCGCCGTGGAT ACAG C C G C T CAGA T C AG C GAG C AGAAG T C T AAC GAC G C C AG C GAG G C C GAAC AC C AGA G AG C C T AT C T G GAAGAT AC C T G C G T G GAAT G G C T G C AC AAG TAG C T G G AAAAG G G C AAAGAG AC AC T G C T G C AC C T G GAAC C T C C AAAGAC AC AT G T G AC C C AC CAT C C T AT C AG C GAC C AC GAG G C C AC AC T G AGAT G T T G G G C C C T G G G C T T T TAC C C T G C C GAGAT C AC AC T GAC AT G G C AG C AG GAT G G C GAG G G C C A C AC AC AG GA AC AG AG C T G G G GAAAC AAGAC C T G C C G G C GAC G G C AC C T T C C AGAAAT G G G C T G C T G T G G T T G T G C C C AG C G G C GAG GAAC AGAGAT AC AC C T G T C AC G T G C AG C AC GAG G GAC T G C C T GAAC C T G T GAC T C T GAGAT G GAAG C C T G C C AG C C AG C C AAC AAT C C C CAT C G T G G GAAT CAT TGCCGGCCTGGTGCTGCTGGGATCTGTGGTTTCTGGTGCTGTGGTGGCCGCCGTGATTTG GAGA AAG AAG T C C T C T G G C G G C AAAG G C G G C T C C AC TCTAAG G C C GAG T G GAG C G AT T C T G C C C AG G G C T C T GAA.AG C C AC AG C C T G T A GAT AA (SEQ ID NO: 14929).

[0877] An exemplary WT HLA-E Monomer (R and S) protein of the disclosure comprises or consists of the amino acid sequence of:

MSRSVALAVLALLSLSGLEAGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRF

DNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSH TLQ

WMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEA EHQ

RAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEI TLT WQQDGE GHT QDTE LVE TRPAGDGT FQKWAAWVP S GEE QRYT CHVQHE GL PE PVT LRWKP AS QPT I P IVGI IAGLVLLGSWS GAWAAVIWRKKS S GGKGGSYSKAEWS DSAQGSE SHS

L (SEQ ID NO: 14930).

[0878] An exemplary WT HLA-E Monomer (R and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

ATGAGCAGATCTGTGGCCCTGGCTGTTC TGGCTCT GCTGTCTCTGTCT GGACTGGAAGCCGGCA G C C AC AG C C T G AAG T AC T T T C A C AC C AG C G T G T C C AGAC C T G G C AGAG G C GAG C C T AGAT T C AT GAG C G T G G G C TAG G T G GAG G AC AC C GAG T T C G T C AGAT T C G AC AAC GAC G C C G C C T C T C C T C G G AT G G T T C C T AGAG GAG C C T G GAT G GAAC AAGAG G G GAG C GAG TAG T G G GAC AGAGAGAC AAG AA G C G C GAG AG AC AC AG C C C AGAT C T T GAG AG G AAC C T G C G G AC C C T G C G G G G C T AC T AC AAT C A G T C T GAAG C C G G C T C T C AC AC C C T G C AG T G GAT G GAG G GAT G T GAAC T G G G C C C C GAC AGAAGA T T C C T GAGAG G C AG GAG GAG T T C G C C AG GAC G G C AAG GAC T AC C T GAC AC T GAAC GAG GAC C T GAG AG C T G GAC C G C C G T G GAT AC AG C C G C T C AG AT C AG C GAG C AGAAG T C T AAC GAC G C C T C T GAG G C C GAAC AC C AG AGAG C C T AC C T G GAAGAT AC C T G C G T G GAAT G G C T G C AC AAG T AC C T G GAAAAG G G C AAAGAGA C AC T G C T G C AC C T G GAAC C T C C AAAG AC AC AC G T GAC C C AC CAT C C T A T C AG C GAC C AC GAG G C C AC AC T G AGAT G T T G G G C C C T G G G C T T T AC C C C G C C G AGAT C AC AC T GAC AT G G C AG C AG GAT G G C GAG G G C C AC AC AC AG GA AC AG AG C T G G T G GAAAC AAGAC C T G C C G G C GAC G G C AC C T T C C AG AΆAT G G G C T G C T G T G G T G G T T C C C AG C G G C GAG GAAC AGAGA T AC A C C T G T C AC G T G C AG C AC GAG G GAC T G C C T GAAC C T G T GAC AC T GAG G T G GAAG C C T G C C A G C C A G C C T AC AAT C C C CAT C G T G G GAAT CAT T G C C G G C C T G G T G C T G C T G G GAT C T G T G G T T T C T G G T G C AG T G G T G G C C G C C G T GAT C T G G C G GAAAAAAAG C T C AG G C G G C AAAG G C G G C T C C AC TCCA

[0879] An exemplary WT HLA-E Monomer (G and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

MS RSVALAVLALL S L S GLEAGS HS LKYFHT SVS RP GRGE PR F I SVGYVDDT QFVRF DNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQ WMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQI S EQKSNDASEAEHQ RAYLEDTCVEWLHKYLEKGKET LLHLE P PKTHVTHHP I S DHEATLRCWALGFYPAE I TLT W QQDGE GHT QDTE LVE TRPAGDGT FQKWAAVWP S GEE QR YT CHVQHE GL PE PVT LRWKP AS QPT I P IVG I IAGLVLLGS WS GAWAAVIWRKKS S GGKGGS Y SKAEWS DSAQG SE SHS

L (SEQ ID NO: 14932) .

[0880] An exemplary WT HLA-E Monomer (G and S) protein of the disclosure comprises or consists of the nucleic acid sequence of:

AT G AG C AGAT C T G T G G C C C T G G C T G T T C T G G C T C T G C T G T C T C T G T C T G GAC T G GAAG C C G G C A G C C AC AG C C T GAAG TAG T T T C AC AC C AG C G G T C C AGAC C T G G C AG AG G C GAG C C T AGAT T CAT C AG C G T G G G C T AC G T G GAC GAC AC C C AG T T C G T C AGAT T C GAC AAC GAC G C C G C C T C T C C T C G G AT G G T T C C T AGAG C AC C C T G GAT G GAAC AAGAG G G C AG C GAG TAG T G G GAC AGAG AGAC AAGAA G C G C C AG AG AC AC AG C C C AGAT C T T C AG AG T G AC C T G C G GAC CCTGCGGGG C T AC TAG AAT C A G T C T GAAG C C G G C T C T C AC AC C C T G C AG T G GAT G C AC G GAT G T GAAC T G G G C C C C GAC GGAAGA T T C C T GAG AG G C TAG GAG C AG T T C G C C T AC G AC G G C AAG GAC TAG C T G AC AC T G AAC GAG GAC C T GAGAAG C T G GAC C G C C G T G GAT AC AG C C G C T C AG AT C AG C GAG C AGAAG T C T AAC GAC G C C T C T GAG G C C GAAC AC C AG AGAG C C TAG C T G GAAGAT AC C T G C G T G GAAT G G C T G C AC AAG T AC C T G G AAAAG G G C AAAGAG AC AC T G C T G C AC C T G GAAC C T C C AAAGAC AC AC G T GAC C C AC CAT C C T A T C AG C GAC C AC GAG G C C AC AC T GAGAT G T T G G G C C C T G G G C T T T T AC C C C G C C G AGAT C A C AC T GAC AT G G GAG GAG GAT G G C GAG G G C C AC AC AC AG GAT AC AG AG C T G G T G GAAAC AAGAC C T G C C GGCGACGGCACCTTCCAGAAAT GGGCTGCTGTGGT GGTTCCCAGCGGCGAGGAACAGAGATACA C C T G T C AC G T G GAG C AC GAG G GAC T G C C T GAAC C T G T GAC AC T GAG G T G GAAG C C T G C C AG C C A GCCTACAAT CCCCATCGTGGGAATCATT GCCGGCCTGGTGCTGCTGGGATCTGT GGTTTCTGGT GCAGTGGTGGCCGCCGTGATCT GGCGGAAAAAAAGCTCAGGCGGCAAAGGCGGCTCCTAC rCCA AAG C C GAG T G GAG C GAT T C T G C C GAG G G C T C T GAAAG C C AC T C T C T G T AGAT AA (SEQ ID NO: 14933) .

[0881] In some embodiments of the methods of the disclosure, a modified HSC or modified

HSC descendent cell of the disclosure may be produced by introducing a transgene into an HSC or an HSC descendent cell of the disclosure. The introducing step may comprise delivery of a nucleic acid sequence and/or a genomic editing construct via a non -transposition delivery system.

[0882] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo , in vitro or in situ comprises one or more of topical delivery, adsorption, absorption, electroporation, spm-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery. In some

embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ by mechanical transfection comprises cell squeezing, cell bombardment, or gene gun techniques. In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection comprises liposomal delivery, delivery by micelles, and delivery by polymerosomes.

[0883] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ comprises a non-viral vector. In some embodiments, the non-viral vector comprises a nucleic acid. In some embodiments, the non-viral vector comprises plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises a transposon of the disclosure.

[0884] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent ceil ex vivo, in vivo, in vitro or in situ comprises a viral vector. In some embodiments, the viral vector is a non- integrating non-chromosomal vector. Exemplary non- integrating non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes viruses. In some embodiments, the viral vector is an integrating chromosomal vector. Integrating chromosomal vectors include, but are not limited to, adeno-associated vectors (AAV),

Lentiviruses, and gamma-retroviruses.

[0885] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, m vivo, in vitro or in situ comprises a combination of vectors. Exemplar}', non-limiting vector combinations include: viral and non-viral vectors, a plurality of non-viral vectors, or a plurality of viral vectors. Exemplary' but non-limiting vectors combinations include: a combination of a DNA-denved and an RNA- derived vector, a combination of an RNA and a reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

[0886] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ stably integrates a nucleic acid sequence, transiently integrates a nucleic acid sequence, produces site-specific integration a nucleic acid sequence, or produces a biased integration of a nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

[0887] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and/or a genomic editing construct into an HSC or HSC descendent cell ex vivo, in vivo, in vitro or in situ stably integrates a nucleic acid sequence. In some embodiments, the stable chromosomal integration can be a random integration, a site- specific integration, or a biased integration in some embodiments, the site-specific integration can be non-assisted or assisted. In some embodiments, the assisted site-specific integration is co delivered with a site-directed nuclease. In some embodiments, the site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In some embodiments, the transgene with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end joining. In some embodiments the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumi n gene, the adeno- associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

[0888] In some embodiments, the site-specific transgene integration occurs at a site that disrupts expression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at nitrons, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-specific integration include but are not limited to TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allo-rejection.

[0889] In some embodiments, the site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In some embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0890] In some embodiments of the methods of the disclosure, enzymes may be used to create strand breaks m the host genome to facilitate delivery or integration of the transgene. In some embodiments, enzymes create single-strand breaks. In some embodiments, enzymes create double-strand breaks. In some embodiments, examples of break-inducing enzymes include but are not limited to: transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVER™, and CPF1. In some embodiments, break-inducing enzymes can be delivered to the cell encoded m DNA, encoded in mRNA, as a protein, as a nucleoprotein complex with a guide RNA (gRNA).

[0891] In some embodiments of the methods of the disclosure, the site-specific transgene integration is controlled by a vector-mediated integration site bias. In some embodiments vector- mediated integration site bias is controlled by the chosen lentiviral vector. In some embodiments vector-mediated integration site bias is controlled by the chosen gamma-retroviral vector.

[0892] In some embodiments of the methods of the disclosure, the site-specific transgene integration site is a non-stable chromosomal insertion. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

[0893] In some embodiments of the methods of the disclosure, the genome modification is a non-stable integration of a transgene. In some embodiments, the non-stable integration can be a transient non-chromosomal integration, a semi-stable non chromosomal integration, a semi- persistent non-chromosoma! insertion, or a non-stable chromosomal insertion. In some embodiments, the transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic.

[0894] In some embodiments, the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

[0895] In some embodiments of the methods of the disclosure, the genome modification is a semi-stable or persistent non-chromosomal integration of a transgene. In some embodiments, a DNA vector encodes a Scaffold/matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.

[0896] In some embodiments of the methods of the disclosure, the genome modification is a non-stable chromosomal integration of a transgene. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

[0897] In some embodiments of the methods of the disclosure, the modification to the genome by transgene insertion can occur via host cell-directed double-strand breakage repair (homology- directed repair) by homologous recombination (HR), microhomology -mediated end joining (MMEJ), nonhomologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or recombinant enzyme-mediated modification. In some embodiments, the modification to the genome by transgene insertion can occur via CRISPR-Cas9, TALEN, ZFNs, Cas-CLOVER, and cpf 1 _·

Vectors

[0898] The disclosure provides a vector comprising a transposon of the disclosure. In some embodiments, the vector is a viral vector. The vector may be a recombinant vector.

[0899] Viral vectors of the disclosure may comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof. The viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplary' adeno-associated viruses and recombinant adeno-associated viruses of the disclosure comprise two or more inverted terminal repeat (ITR) sequences located in cis next to a sequence encoding an inducible proapoptotic polypeptide, a sequence encoding a therapeutic protein, and/or a sequence encoding a selection marker of the disclosure. Exemplary' adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to ail serotypes (e.g.

AAV! , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno- associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g. AAV2/5, AAV-DJ and A AV-DJ8).

Exemplary adeno-associated viruses and recombinant adeno-associated viruses of the disclosure include, but are not limited to, rAAV-LK03.

[0900] The disclosure provides a vector comprising a transposon of the disclosure. In some embodiments, the vector is a nanoparticle. Exemplary nanoparticle vectors of the disclosure include, but are not limited to, nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof ), ammo acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g. polymersomes), micelles, lipids (e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g. calcium phosphate or gold) or any combination thereof. A nanoparticle vector may be passively or actively transported across a cell membrane.

[0901] Vectors of the disclosure may comprise a selection gene. Transposons of the disclosure may comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. Selective ceil culture conditions may comprise a compound harmful to cell viability or survival and wherein the gene product confers resistance to the compound.

[0902] In some embodiments of the compositions and method of the disclosure, the selection agent kills dividing cells and the presence and/or expression of the selection gene m an HSC of the disclosure protects the modified HSC during cell division, thereby permitting only modified HSCs to proliferate in the presence of the selection agent. For example, the selection agent, methotrexate (MTX) kills dividing cells and the presence and/or expression of the selection gene, DHFR, in an HSC of the disclosure protects the modified HSC during cell division, thereby permitting only modified HSCs to proliferate in the presence of MTX.

[0903] Vectors of the disclosure may comprise an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence. In some embodiments, the non-human sequence comprises a restriction site. In some embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible proapoptotic polypeptides of the disclosure may also be referred to as an“iC9 safety switch”.

[0904] The disclosure provides a composition comprising a vector of the disclosure.

[09Q5] Nucleic acid molecules of the disclosure can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.

[0906] Isolated nucleic acid molecules of the disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally, with one or more introns, e.g., but not limited to, nucleic acid molecules comprising the coding sequence for a protein, an inducible proapoptotic polypeptide, a selection gene product, a transposase, a gene editing composition or effector domain thereof, an HD AC inhibitor and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the proteins of the disclosure as described herein and/or as known in the art. Of course, the genetic code is well known m the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific protein scaffolds of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.

[0907] As indicated herein, nucleic acid molecules of the disclosure include, but are not limited to, those encoding the ammo acid sequence of a protein of the disclosure, by itself; the coding sequence for the entire protein or a portion thereof; the coding sequence for a protein, fragment or portion, as w ^r ell as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one iniron together with additional, non-coding sequences, including but not limited to, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example, ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding a protein can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein scaffold comprising a protein scaffold fragment or portion.

[0908] The amino acids that make up protein scaffolds of the disclosure are often abbreviated. The ammo acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al, Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). A protein scaffold of the disclosure can include one or more ammo acid

substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein. Ammo acids in a protein scaffold of the disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces single alanine mutations at ever residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites that are critical for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et a!., Science 255:306-312 (1992)).

[0909] HSC compositions or any portion thereof may be genetically modified using, for example, a nucleofection strategy such as electroporation. The total number of cells to be nucleofected, the total volume of the nucleofection reaction, and the precise timing of the preparation of the sample may be optimized to yield cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies.

[0910] Nucleofection and/or electroporation may be accomplished using, for example, Lonza Amaxa, MaxCyte PulseAgile, Harvard Apparatus BTX, and/or Invitrogen Neon. Non-metal electrode systems, including, but not limited to, plastic polymer electrodes, may be preferred for nucleofection.

[0911] Prior to genetic modification by nucleofection, HSC compositions or any portion thereof may be resuspended in a nucleofection buffer. Nucl eofection buffers of the disclosure include commercially-available nucleofection buffers. Nucleofection buffers of the disclosure may be optimized to yield cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or

greater/faster expansion upon addition of expansion technologies. Nucleofection buffers of the disclosure may include, but are not limited to, PBS, BBSS, OptiMEM, BTXpress, Amaxa Nucleofector and any combination thereof. Nucleofection buffers of the disclosure may comprise one or more supplemental factors to yield cells that have greater viability, nucleofect with higher efficiency, exhibit greater viability post-nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Exemplary

supplemental factors include, but are not limited to, salts, minerals, metabolites or any combination thereof. Nucleofection buffers of the disclosure may include, an HD AC inhibitor composition of the disclosure.

[0912] Transposition reagents, including a transposon and a transposase, may be added to a nucleofection reaction of the disclosure prior to, simultaneously with, or after an addition of cells to a nucleofection buffer (optionally, contained within a nucleofection reaction vial or cuvette). Transposons of the disclosure may comprise plasmid DXA, linearized plasmid DNA, a PCR product, DOGGYBONE™ DNA, an mRNA template, a single or double-stranded DNA, a protein-nucleic acid combination or any combination thereof. Transposons of the disclosure may comprised one or more sequences that encode one or more TTAA site(s), one or more inverted terminal repeat(s) (ITRs), one or more long terminal repeat(s) (LTRs), one or more insulator(s), one or more promotor(s), one or more full-length or truncated gene(s), one or more poly A signal(s), one or more self-cleaving 2A peptide cleavage site(s), one or more internal ribosome entry site(s) (IRES), one or more enhancer(s), one or more regulator(s), one or more replication ongin(s), and any combination thereof.

[0913] Transposons of the disclosure may comprise one or more sequences that encode one or more full-length or truncated gene(s). Full-length and/or truncated gene(s) introduced by transposons of the disclosure may encode one or more of a signal peptide, a therapeutic protein, a drug resistance gene, or any combination thereof.

[0914] Transposons of the disclosure may be prepared in water, TAE, TBE, PBS, HBSS, media, a supplemental factor of the disclosure or any combination thereof.

[0915] Transposons of the disclosure may be designed to optimize clinical safety and/or improve manufacturability. As a non-limiting example, transposons of the disclosure may be designed to optimize clinical safety and/or improve manufacturability by eliminating unnecessary sequences or regions. Transposons of the disclosure may or may not be GMP grade.

[0916] Transposase enzymes of the disclosure may be encoded by one or more sequences of plasmid DNA, mRNA, protein, protein-nucleic acid combination or any combination thereof.

[0917] Transposase enzymes of the disclosure may be prepared in water, TAE, TBE, PBS, BBSS, media, a supplemental factor of the disclosure or any combination thereof. Transposase enzymes of the disclosure or the sequences/constructs encoding or delivering them may or may not be GMP grade.

[0918] Transposons and transposase enzymes of the disclosure may be delivered to a cell by any means.

[0919] Although compositions and methods of the disclosure include delivery of a transposon and/or transposase of the disclosure to a cell by plasmid DNA (pDNA), the use of a plasmid for delivery may allow the transposon and/or transposase to be integrated into the chromosomal DNA of the cell, which may lead to continued transposase expression. Accordingly, transposon and/or transposase enzymes of the disclosure may be delivered to a cell as either mRNA or protein to remove any possibility for chromosomal integration. [0920] Transposons and transposases of the disclosure may be pre-incubated alone or in combination with one another prior to the introduction of the transposon and/or transposase into a nucleofection reaction. The absolute amounts of each of the transposon and the transposase, as well as the relative amounts, e.g., a ratio of transposon to transposase may be optimized.

[0921] Following preparation of nucleofection reaction, optionally, in a vial or cuvette, the reaction may be loaded into a nucleofector apparatus and activated for delivery' of an electric pulse according to the manufacturer’s protocol. Electric pulse conditions used for delivery of a transposon and/or a transposase of the disclosure (or a sequence encoding a transposon and/or a transposase of the disclosure) to a ceil may be optimized for yielding cells with enhanced viability'·, higher nucleofection efficiency, greater viability post-nucleofection, desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. When using Amaxa nucleofector technology, each of the various nucl eofection programs for the Amaxa :2B or 4D nucleofector are contemplated.

[09:2:2] Following a nucleofection reaction of the disclosure, cells may be gently added to a cell medium. Post-nucleofection cell media of the disclosure may comprise an ' one or more commercially-available media. Post-nucleofection cell media of the disclosure may be optimized to yield cells with greater viability, higher nucleofection efficiency, exhibit greater viability post- nucleofection, display a more desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Post-nucleofection cell media of the disclosure may comprise an FID AC inhibitor composition of the disclosure.

[0923] Post-nucleofection cell media of the disclosure may be used at room temperature or pre warmed to, for example to between 32°C to 37°C, inclusive of the endpoints. Post-nucleofection cell media of the disclosure may be pre-warmed to any temperature that maintains or enhances cell viability and/or expression of a transposon or portion thereof of the disclosure.

[0924] Post-nucleofection cell media of the disclosure may be contained in tissue culture flasks or dishes, G-Rex flasks, Bioreactor or cell culture bags, or any other standard receptacle. Post- nucleofection cell cultures of the disclosure may be may be kept still, or, alternatively, they may ^¬ be perturbed (e.g. rocked, swirled, or shaken).

[0925] Post-nucleofection cell cultures may comprise genetically-modified cells. Post- nucleofection FISC cell cultures may comprise genetically-modified HSCs. Genetically modified cells of the disclosure may be either rested for a defined period of time or stimulated for expansion by, for example, the addition of an HD AC inhibitor composition. In some embodiments, genetically modified cells of the disclosure may be either rested for a defined period of time or immediately stimulated for expansion by, for example, the addition of an HD AC inhibitor composition. Genetically modified cells of the disclosure may be rested to allow them sufficient time to acclimate, time for transposition to occur, and/or time for positive or negative selection, resulting m cells with enhanced viability, higher nucleofection efficiency, greater viability post-nucleofection, desirable cell phenotype, and/or greater/faster expansion upon addition of expansion technologies. Genetically modified cells of the disclosure may be rested, for example, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more hours. In some embodiments, genetically modified cells of the disclosure may be rested, for example, for an overnight. In some aspects, an overnight is about 12 hours.

Genetically modified cells of the disclosure may be rested, for example, for I, 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14 or more days.

[0926] Genetically modified cells of the disclosure may be selected following a nucleofection reaction and prior to addition of an expander technology.

[0927] Genetically modified cells of the disclosure may be simultaneously selected and expanded following a nucleofection reaction.

[0928] As early as 24-hours post-nucleofection, expression of a selection marker of the disclosure may be detectable in modified HSCs upon successful nucleofection of a transposon of the disclosure. Due to epi -chromosomal expression of the transposon, expression of a selection marker alone may not distinguish modified HSCs (those cells in which the transposon has been successfully integrated) from unmodified HSCs (those cells in which the transposon was not successfully integrated). When epi -chromosomal expression of the transposon obscures the detection of modified cells by the selection marker, the nucleofected cells (both modified and unmodified cells) may be rested for a period of time (e.g. 2-14 days) to allow the cells to cease expression or lose all epi-chromosomal transposon expression. Following this extended resting period, only modified HSCs should remain positive for expression of selection marker. The length of this extended resting period may be optimized for each nucleofection reaction and selection process. When epi-chromosomal expression of the transposon obscures the detection of modified cells by the selection marker, selection may be performed without tins extended resting period, however, an additional selection step may be included at a later time point (e.g. either during or after the expansion stage).

[0929] The disclosure provides modified HSCs for administration to a subject m need thereof. Modified ceils of the disclosure may be formulated for storage at any temperature including room temperature and body temperature. Modified cells of the disclosure may be formu lated for cryopreservation and subsequent thawing. Modified cells of the disclosure may be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. Modified ceils of the disclosure may be formulated in a pharmaceutically acceptable carrier with an indicator of ceil viability. Modified cells of the disclosure may be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more reagents to inhibit further expansion and/or prevent cell death.

[0930] In some embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion.

[0931] In some embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises between IxlCr and lx10 ¹⁰ cells per kg of body weight of the subject per administration, or any range, value or fraction thereof.

[0932] In some embodiments of the disclosure, modified cells of the disclosure are delivered to a patient via injection or intravenous infusion. In some embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises a single or multiple doses. In some embodiments, a therapeutically effective dose of a composition of the disclosure or of compositions comprising modified cells of the disclosure comprises a single dose.

[0933] While particular embodiments of the disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the disclosure. The scope of the appended claims includes all such changes and modifications that are within the scope of this disclosure.

EXAMPLES

Example 1: Selection and Expansion of isolated hematopoietic stem cells (HSCs) using VP A or SR1/UM171/UNC0638 [0934] CD34+ HSCs were expanded for 7 days in culture in the presence or absence of valproic acid (VP A). CD34+ HSCs were treated with lmM valproic acid (VP A),

SR1+UM171+UNC0638 (SUU) or control for 7 days. Fig. 1 is a schematic diagram depicting the selection and expansion of isolated hematopoietic stem cells (HSCs) from mobilized peripheral blood. HSCs are isolated from mobilized peripheral blood and corrected for gene delivery. Transposed HSCs are selected and expanded prior to transplantation, allowing for higher levels of engraftment and/ reduced intensity of myeloablative conditioning regimens such as busulfan chemotherapy. Fig. 2 is a schematic diagram showing HSC expansion. As shown, a low percentage of transposed HSCs results after selection. One possible mechanism to increase the HSC population is to block differentiation while expanding self-renewal and proliferation properties of the HSCs.

[0935] Cells were subsequently separated into subsets based on the expression of cell surface markers and ranked by increasing primitiveness (Fig. 3). Fig. 3 is a bar graph depicting the fold expansion of different hematopoietic cell subsets over 7 days of cell culture. Cells on Day 7 were separated into subsets of hematopoietic cells based on the expression of cell surface markers and were rank ordered based on increasing primitiveness.

[0936] The fold expansion of Day 7 over Day 0 control cells was ploted on the y-axis (logarithmic scale). A 720-fold expansion of the most primitive subset of ceils (CD34+38- 90+45RA-49f+l 33 +) was observed with lmM VP A. To a lesser degree, treatment with

SR1 /UM171/UNC0638 also increased the fold expansion of the most primitive subset of cells. This shows that treatment of HSCs with VPA or SR1/UM171/UNC0638 causes an expansion of HSC population in cell culture.

[0937] Next, the effects of VPA and SR1/UM171/UNC0638 were tested on HSCs transposed with a DHFR mutein. CD34+ cells w¾re modified using piggyBac transposition to express the DHFR mutein which confers methotrexate (MTX) resistance (Fig. 4). The elongation factor-1 alpha (EFlo) is used as a constitutive promoter to drive the bi-cistromc cassette consisting of the enhanced green fluorescence (GFP) reporter and the dihydrofolate reductase resistance (DHFR) mutein genes. The SV40 polyA signal and the 250 bp cHS4 chromatin insulator are indicated. During transposition, the co-delivered PB transposase recognizes the transposon-specific inverted terminal repeat sequence (ITR) located on both ends of the transposon vector and efficiently moves the contents from the original sites in the delivered DNA plasmid and efficiently integrates them into TTAA chromosomal sites. Addition of MTX to the growth media results in the selection of only cells which express the DHFR gene. Fig. 5A shows a schematic timeline of the experimental set up. Cells were nucleofected on Day 0. Ceils were selected with MTX and expanded using VP A or SR1/XJM171/UNC0638 on Day 4. Cells w ^? ere collected on Day 11, analyzed by flo ' cytometry and separated into subsets based on the expression of cell surface markers and ranked by increasing primitiveness. Treatment with VP A or

SR1/LM171/UNC0638 caused an increased expansion of MTX selected cells in comparison to the bulk population of cells (Figs. 6 and 7). Fig. 6 is a senes of flow' cytometry scatterplots showing the GFP expression profiles of hematopoietic cell subsets on Day 11 of cell culture. In each plot the y-axis is a measure of forward scater (FSC-A) and is plotted from 0 - 250K in increments of 50K. In each plot, the x-axis is a measure of GFP and is ploted in logarithmic scale. mPB CD34+ cells were treated with VPA or SR1/UM171/UNC0638, in the presence or absence of MTX. Cells on Day 11 were separated into subsets of hematopoietic cells based on the expression of cell surface markers and were rank ordered from left to right based on increasing primitiveness. Treatment of cells with VPA or SR1/XJM171/UNC0638 alone caused a mild increase in frequency of GFP+ cells with increased primitiveness (CD34+38-900+45RA- 49f+ cells). For both treatment conditions, the addition of MTX treatment resulted in significant purity of GFP+ cells with increased primitiveness. Fig. 7 is a series of bar graphs showing the quantification of the flow cytometry results from Fig. 6 The x-axis represents the hematopoietic cell subsets rank ordered based on increasing primitiveness and the y-axis represents the frequency of GFP+ cells (%). This shows effective enrichment of PB transposed CD 34+ cells among all subsets by MTX selection

[0938] This is especially evident with the most primitive subset of CD34+38-90+45RA-49f+ HSCs. In this most primitive subset, there was a 37.7-fold and 1.83-fold increase in the absolute number of cells in comparison to Day 0 control cells, following treatment with VPA or

SR1/UM171/TJNC0638, respectively (Fig. 8B). This shows effective enrichment and expansion of PB transposed CD34+ cells by MTX selection.

Together, this data demonstrates the ability to simultaneously select DHFR transposed HSCs using methotrexate and expand the cell population of selected cells using VPA or

SR1/UM171/UNC0638. [0939] Alternatively, selection can also be performed with methotrexate, pralatrexate, pyrimeihannne, dapsone, raltitrexed, trimetrexate, metoprine, iclaprim, aminopterin, lometrexol, nolatrexed, brodimoprim, trimethoprim, pemetrexed, proguanil, piritrexim, eycloguanii, or any combination thereof.

Example 2: Expression of Therapeutic Proteins in HSCs

[Q94Q] Therapeutic HSCs may be modified to express a therapeutic protein. Human hemoglobin tetramers consist of two alpha globin chains and two beta globin chains. Mutations in beta globin causes sickle cell anemia. Absence or reduced expression of beta globin causes beta-thalassemia. Therapeutic HSCs can be modified to express non-secreted proteins that function mtraceliularly, such as a beta globin, for these hemoglobinopathies. A schematic diagram of a construct expressing human therapeutic beta-globin, iC9 and DHFR (PB-HBB- PGK-iC9-T2A-DHFR construct) is shown in Fig. 9. The PB-HBB-PGK-iC9-T2A-DHFR construct is encoded by a nucleic acid sequence comprising SEQ ID NO: 14974.

[0941] Fig. 10A shows a schematic diagram of a construct for erythroid-speeifie expression of the human therapeutic beta-globin and the constitutive expression of GFP and DHFR (PB-HBB- PGK-GFP-T2A-DHFR construct) (SEQ ID NO: 14936). Fig. 10B is a schematic timel ine of the piggyBac transposition of a therapeutic vector containing T87Q beta-globin, followed by expansion with a combination of SRI , UM171 and UNC0638 treatment and selection with methotrexate. Fig. I OC shows flow cytometry scatterplots depicting the frequency of GFP+ cells at day 4, before selection and expansion, and at Day 1 1 , after 7 days of selection and expansion.

Example 3; Expression of Gamma Globin Inducers (GGIs) in HSCs

[0942] Up-regulation of fetal hemoglobin (HbF) is one method for treating sickle cell disease and beta-thalassemia. During erythropoiesis, there is a switch from g to b globin expression dunng the fetal to adult cell transition. One method of increasing the population of fetal hemoglobin in these hemoglobinopathies is to increase the expression of gamma globin inducers (GGIs). For example, in sickle cell disease, the delivery of a GGIs can promote anti-sickling properties.

[0943] Therapeutic HSCs may be modified to express targeted shRNA. B-cell

lymphoma/leukemia 1 1 A (BCL1 1 A) is a gene that encodes a regulatory C2H2 type zinc-finger protein that can bind to DNA. BCL11 A regulates hemoglobin switching during development. Disruption of BCLl 1 A results in the reversion to fetal (gamma) hemoglobin expression. As such, a therapeutic strategy to increase gamma globin production in diseases such as beta thalassemia and sickle cell anemia is to inhibit BCLl 1 A. Therapeutic HSCs can be modified to express BCLl 1 A targeted shRNA for treatment of these hemoglobinopathies. A schematic diagram of a construct expressing BCLl 1 A shRNA and DHFR (nano-PB-V5-JctR-Ank-miRE- sh49-BCLl 1 A-EFla-GFP-T2A-DHFR construct) is shown in Fig. 11 A. The nucleic acid sequence encoding the nano-PB-V5-JctR-Ank-miRE-sh49-BCLl 1 A-EFla-GFP-T2A-DHFR construct and its gene features is comprised of SEQ ID NO: 14975.

[0944] Therapeutic HSCs may be modified to express proteins that mediate gamma hemoglobin expression. Insulin-like growth-factor 2 mRNA-binding protein 1 (IGF2BPI) plays a role in reactivating fetal hemoglobin (HbF) expression in adult cells and IGF2BPI

overexpression causes a reversal from the adult cell phenotype towards a fetal-like globin phenotype. One therapeutic strategy to increase fetal globin production in diseases such as beta thalassemia and sickle cell anemia is to increase the expression of IGF2BP1. Therapeutic HSCs can be modified to express IGF2BP1 for treatment of these hemoglobinopathies. A schematic diagram of a construct expressing IGF2BPI and DHFR (nano-PB~V5-JetR-SPTAI-IGF2BPI- EF1 a-GFP-T2A-DHFR construct) is shown in Fig. 1 IB. The nucleic acid sequence encoding the nano-FB-V5-.JctR-SPTA1 -iGF2BPl-EFla-GFP-T2A-DHFR construct and its gene comprises SEQ ID NO: 14977). Fig. 11C is a schematic timeline of the piggyBac transposition of each therapeutic vector, followed by expansion with a combination of SRI, UM171 and UNC0638 (SUU) and selection with methotrexate. Fig. 11D shows flow cytometry scatterplots depicting the frequency of GFP+ cells at day 4, before selection and expansion, and at Day 1 1 , after 7 days of selection and expansion. This protocol yielded a high selection purity of CD34+ cells expressing a therapeutic vector.

[0945] Selection can also be performed with pralatrexate, pyrimethamine, dapsone, raltitrexed, trimetrexate, metoprine, iclaprim, ammopterin, !ometrexol, nolatrexed, brodimoprim, trimethoprim, pemetrexed, proguanil, piritrexim, or cycloguanil, or any combination thereof.

or SR1/UM171/UN C0638

[0946] Human mobilized peripheral blood CD34+ ceils were treated with transposition, transposition boosting, selection, and multiple expansion reagents, followed by characterization via both in vitro and m vivo assays. The schematic timeline of piggyBac transposition by nucleofection, transposition boosting treatment with VP A and UNC0638, methotrexate selection and expansion treatment with either cytokines alone, VP A, VPA/UM171/UNC0638 (VUU), or SR1/UM171/UNC0638 (SUU) is depicted in Fig. 12A. This scheme was used to generate ceils used m the assays shown in Figs. 12B-12E. At Day 11 after expansion and selection the absolute number of transposed (GFP+) ceils yielded was quantitated (Fig. 12B).Fig. 12C is a bar graph showing the absolute numbers of GFP+ and GFP- cells within the CD34+/38-/90+/45RA-/49f+ hematopoietic stem cell subset as determined using flow ^r cytometry. The fold expansion of HSCs in the expansion conditions over a Day 0 control are noted. Treatment with VP A,

VPA/UM171/UNC0638, or SR1/UM171/UNC0638 resulted in increased numbers of modified phenotypic HSCs compared to cytokine control or Day 0. Fig. 12D is a box plot showing the output of a long-term culture initiating cell (LTC-IC) assay. The absolute number of cobblestone area forming cells (CAFCs) in each condition as assessed 4 weeks post plating the assay is shown. Data is represented as mean with a 95% confidence interval. There is no significant difference between in the absolute number of CAFCs in VTA, VUU or SUU expansion conditions as compared to a Day 0 control. There are significantly less Week 4 CAFCs present in the cytokine only control as compared to the Day 0 control.

[0947] Fig. 12E shows the engraftment of uncultured mobilized peripheral blood CD34 cells compared to cytokine, VP A, VUU and SUU expansion conditions as assessed in the bone marrow of NSG miee 20 weeks post transplant. There was no significant difference in engraftment observed when compared uncultured cells to VP A expansion or SUU expansion conditions.

[0948] Selection can also be performed with pralatrexate, pyrimethamine, dapsone, raltitrexed, trimetrexate, metoprine, iclaprim, aminopterin, !ometrexol, nolatrexed, brodimoprim, trimethoprim, pemetrexed, proguanil, piritrexim, or cye!oguanil, or any combination thereof. Example 5; Pralatrexate as a selection agent

[0949] Pralatrexlate (PTX) was used as a selection agent in human mobilized peripheral blood CD34+ cells modified with a thereutic piggyBac transposon. Fig. 13 A shows a schematic diagram of a construct for erythroid-specific expression of a BCLl 1 A targeted shRNA and the constitutive expression of GFP and DHFR (PB-Ank-BCLl 1 ashRNA-MND.GFP-T2A-DHFR construct). Fig. 13B is a schematic diagram of the culture conditions tested. VU is treated with the recovery treatment, SUU expansion and PTX selection. VU + SRI additionally includes culture with SRI from cell thaw through Day 11. VU + SRI + UM171 additionally includes culture with SR1/UM171/UNC0638 from cell thaw through Day 11. Fig. 13C is a bar graph depicting the absolute number of transposed (GFP+) phenotypic HSCs as determined using the CD34+/CD90+/CD45RA- subset. The VU + SRI + UM171 culture condition yields

approximately a 3-fold increase in phenotypic HSCs compared to the VU culture condition. Example 6: Selection with methoexatem pralatrexate, or pralatrexate + dipyridamole

[0950] Multiple selection agents were evaluated in human mobilized peripheral blood CD34+ cells with piggyBac transposition modified with piggyBac transposons. Fig. 14A is a schematic timeline of the piggyBac transposition by nucleofection, expansion treatment

(SR1/UM171/UNC0638), and selection treatment via either methotrexate, pralatrexate or pralatrexate + dipyridamole. Fig. 14B is a representative flow cytometry scatterplots of the GFP expression at Day 4, before selection, and Day 11, after selection. Selection efficiency is increased with pralatrexate compared to methotrexate, and further increased with the addition of dipyridamole.

[0951] Selection can also be performed with pralatrexate, pyrimethamine, dapsone, raltitrexed, trimetrexate, metoprine, iclaprim, aminopterin, lometrexol, nolatrexed, brodimoprim, trimethoprim, pemetrexed, proguanil, piritrexim, or cycloguaml, or any combination thereof. Example 7: Transposition of Interleukin 2 Receptor subunit gamma in CD34+ cells

[0952] Fig. 15A-15B is a schematic diagram and scatterplot depicting the delivery of a therapeutic piggyBac transposon to human mobilized peripheral blood CD34+ cells. Fig. 15 A is a schematic diagram of a construct for constitutive expression of Interleukin 2 Receptor subunit gamma, GFP and DF!FR (PB-EFl a-IL2RG-T2A-GFP-T2A-DHFR). Fig. 1 5B is a flow cytometry scatterplot depicting the frequency of GFP+ cells 7 days after electroporation (4.97%).