Reference to Prior Application

This application claims priority to and benefit of US Provisional Application 62/340,501 , filed on May 23, 2016. This application is hereby incorporated by reference in its entirety.

Incorporation by Reference of Sequence Listing

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety

Statement of Government Support

This invention was made with government support under GM 103757, CA154697 and DE022957 awarded by National institutes of Health, The government has certain rights in the invention,

introduction

Gene therapy approaches for treatment of genetic diseases, such as hemophilia treatment through correction of Factor IX deficiency, face at least two significant challenges: stable, long-term expression, and reduction of hepatotoxicity. Long term correction of factor ΪΧ deficiency, via hepatic transduction with an adeiio-associated vector (AAV), represents a watershed event in the history of the gene therapy field. In spite of this success, there presentl exist barriers to the fullest implementation of hemophilia gene therapy. In the first regard, the limited packaging capacity of AAV for larger gene constructs practically confounds the use of this vector, in the second regard, vector-related toxicities have been most noteworthy for the context of in vivo

transduction of the liver.

Adenovirus (Ad) has been employed principally for short term transient gene expression applications. However, methods have been developed to accomplish long term coiTective gene expression via Ad. On this basis, integration capacities have been configured into Ad in an "in trans" manner allowing long term gene expression

(transposons, homology sequences, etc.) (11-14),

Most recently, the advent of the CR1SP Cas9 has provided a means to achieve effective gene editing in vivo via adenoviral vector-mediated transduction (15-18). A series of recent studies have validated that such targeted integration gene editing can he accomplished via vector-mediated in vivo delivery (19-22). It is noteworthy that all of these studies have been based upon liver transduction and none have yet sought to utilize other cellular sources in vivo. Additionally, Guan et al (EMBO Mo Med. 20.16 Mar 10. pi i; e201506039. doi: 10.15252/emmm.201.506039), used adenovirus to deliver Cas9 components and Factor IX corrections in mice, with liver targeting. They not only observed liver toxicity, but also failed to observe an .increase in clotting ability.

US patents 9,233,153, 9,629,906 and 9,617,560, all to GeriVec, Inc. describe the use of Gorilla ade ovirus vectors, but these patents do not describe the use of

CRISPR Cas9 or the correction of Factor IX in vivo.

Summary

The present inventor has developed vectors and methods for delivery of

C ISPR Cas9 components to cells i vivo. These vectors and methods can be used to deliver CRISPR/Cas9 components to targeted cells, and can also be used to effect transfer of therapeutic genes to a target cell. Delivery of CR1SPR Cas9 components to the relevant target ceils can he used to modify genomes for therapeutic purposes. This delivery can be efficient, selective and coordinated.

The present inventor has combined targeted adenovirus with CRISPR/Cas9 to accomplish in vivo editing at specific organ sites. The process allows editing of specific gene loci for gene therapy and other applications. The present inventor, therefore, has designed a CRISPR Cas9 deli ver system that can be targeted to pulmonary cells, and can thereby bypass the liver and can also reduce toxicity. In some configurations, the vectors and methods can be used to transfer to a recipient a therapeutic gene, such as Factor IX, for treatment of hemophilia, while avoiding liver toxicity, in some

configurations, the targeted cells can be pulmonary cells, and the targeted organ can be the lung.

The present teachings include integration-competent Ad which can provide gene expression longevity mandated for treatment of various genetic diseases, such as, for example hemophilia gene therapy.

in some embodiments, the present teachings can include, without limitation, an adenovirus vector comprising: a pulmonary targeting coding sequence, a Cas9 coding sequence, and a guideRNA coding sequence. In various configurations, the pulmonary targeting coding sequence can be a. myeloid binding protein (MBP) targeting iigand coding sequence. In some configurations, the MBP targeting iigand coding sequence can be a fiber-fibritin-MBP targeting ligand coding sequence. In various configurations, the pulmonary targeting sequence can comprise a vascular specific promoter and an integrin targeting peptide incorporated into a viral knob of an adenovirus. In various

configurations, the Cas9 coding sequence can be a codon-optimized Cas9 coding sequence. In some configurations, the adenovirus vector can be a gorilla adenoviral vector. In some configurations, the guideRNA can comprise a Pol III promoter. In various configurations, the guideRNA can encode one or more sequences targeted to a ROSA26 locus.

In various embodiments, the present teachings can include a two-vector system comprising a first vector in accordance with the present teachings and a second vector comprising left and right homology arms that are homologous to the guideRNA. In some configurations, the second vector can further comprise a promoter. In some

configurations, the promoter of the second vector can be a constitutive promoter. In some configurations, the constitutive promoter can be an EFla promoter that can include an ratron. In various configurations, the second vector ca further comprise a. multiple cloning site between the left and right homology arms configured such that when a sequence is inserted into the multiple cloning site, the second vector cannot be bound by Cas9. In various configurations, the second vector can farther comprise a gene of interest. In some configurations, a gene of interest can be Inserted into the multiple cloning site. In various configurations, the second vector can further comprise a Factor IX gene sequence. In various configurations, the pulmonary targeting coding sequence can be an MBP targeting ligand coding sequence. In various configurations, the guideRNA and the homology arms can comprise sequences targeted to the ROSA26 locus, In various configurations, the second vector can further comprise a Facto IX gene disrupting the sequence of the ROSA26 encoding homology arms. In various

configurations, the pulmonary targeting coding sequence can be a fiber-fibritm-MBP targeting ligand coding sequence, the Cas coding sequence can be a codon optimized coding sequence, the adenovirus vector can be a gorilla adenovirus vector, the guideRN A can be under the control of the Pol ΠΙ promoter. In some configurations, the guideRN A and homology arms can encode one or more sequences targeted to the ROSA26 locus and the second vector can further comprise a Factor IX gene which can disrupt the sequence of the ROSA26 encoding homology arms.

In various embodiments, the present teachings can include an adenovirus vector comprising a chimeric polypeptide comprising a de-knobbed adenovirus fiber, a T4 bacteriophage fibritin trimerizing foldon domain and a pulmonary targeting ligand. In some configurations, the de-knobbed adenovirus fiber can be a de-knobbed Ad5 fiber. In various configurations, the pulmonary targeting ligand can be a myeloid binding protein (MBP). in various configurations, the adenovirus can be a gorilla adenovirus. In. various configurations, an adenovirus vector in accordance with the present teachings can further comprise a UNA sequence encoding a chimeric polypeptide comprising a de-knobbed A.d5 fiber, a T4 bacteriophage fibritin irimerizing foldon domai and a pulmonar targeting ligand, In various configurations, an adenovirus vector in accordance with the present teachings can further comprise a nucleic acid sequence encoding CRISPR components, in various configurations, an adenovirus vector in accordance with the present teachings can further comprise a DNA sequence encoding a Cas9. In various configurations, the DNA sequence encoding a Cas9 can be a mammalian codon- optimized sequence encoding a Cas9, In various configurations, an adenovirus vector in accordance with the present teachings can further comprise a promoter operably linked to the DNA sequence encoding a Cas9. In some configurations, the promoter operably linked to the DNA sequence encoding a Cas9 can be a CMV promoter, in various configurations, an adenovirus vector in accordance with the present teachings can further comprise a DNA sequence encoding a guideRNA (gRNA). In various configurations, an adenovirus vector in accordance with the present teachings can further comprise a promoter operably linked to the DN A sequence encoding a gRNA. in some

configurations, the promoter operably linked to the D A sequence encoding a gR A can be a U6 promoter. In various configurations, an adenovirus vector in accordance with the present teachings can further comprise a DNA sequence targeted to an intromc region of a ROSA26 locus. In various configurations, an adenovirus vec tor in accordance wit the present teachings can further comprise a DNA sequence targeted to the second intromc region of a ROSA26 locus.

In. various embodiments, an adeno virus vector of the present teachings can comprise a plurality of integrin targeting peptides incorporated into an adenoviral fiber knob. In various configurations, an adenovirus vector in accordance with the present teachings can further comprise a T4 bacteriophage fibritin trimerizing foldon domain. In various configurations, an adenovirus vector in accordance with die present teachings can further comprise a vascular-specific promoter. In various configurations, the vascular-specific promoter can be a ROB04 enhancer/promoter.

In various embodiments, an adenovirus vector of the present teachings can comprise: a chimeric AD5-T4 phage fibritin shaft, a trimerization domain displaying a pulmonary targeting coding sequence, and a ROB04 enhancer/promoter operatively linked to a transgene of interest. In some configurations, the transgene of interest can be a CRISPR gene, in various configurations, the transgene of interest can be a Cas9 gene, in some configurations, the Cas9 gene can comprise a mammalian codon-optimi ed Cas9 coding sequence. In various configurations, the transgene of interest can be a guideR A (gRNA). in various configurations, the guideRNA can comprise a Pol ΠΪ promoter, in various configurations, the guideRNA can encode one or more sequences targeted to a ROSA26 loc us. In various configurations, the transgene of interest can encode a hemophilia factor. In various configurations, the transgene of interest can encode hemophili factor VIII or hemophilia factor IX. In various configurations, the adenovirus vector can be a gorilla adenoviral vector.

In various embodiments, the present teachings can include a two vector system comprising a first vector in accordance with the present teachings and a second vector comprising left and right homology arms that are homologous to a guideRNA. In some configurations, the second vector can farther comprise a promoter. In variou

configurations, the promoter of the second vector can be a constitutive promoter. In various configurations, the constitutive promoter can be an EF1 a promoter that includes an nitron. In various configurations, the second vector can further comprise a multiple cloning site between the left and right homology arms configured such that when a sequence is inserted into the multiple cloning site, the second vector cannot be bound by Cas9. in various configurations, the second vector can further comprise a gene of interest. In various configurations, a gene of interest can be inserted into the multiple cloning site. In some configurations, the gene of interest can be a Factor IX gene. In various configurations, the pulmonary targeting coding sequence can be an MBP targeting ligan coding sequence. In various configurations, the guideRNA and the homology arms can comprise sequences targeted to the ROSA26 locus. In various configurations, the second vector of a two vector system in accordance with the presen teachings can further comprise a Factor IX gene which disrupts the sequence of the ROSA26 encoding homology arras. In sorae configurations, the pulmonary targeting coding sequence can be a fiber- fihritra-MBP targeting ligand coding sequence, the adenovirus vector can be a gorilla adenoviral vector, the guideRNA can be under the control of the Pol 01 promoter, the guideRNA and homology arms can encode one or more sequences targeted to a ROSA26 locus, and the second vector can further comprise a Factor IX gene disrupting the sequence of the OSA26 encoding homology arms.

In various embodiments, a method of gene therapy can comprise adtninistering to a subject an adenovirus comprising a two vector system in accordance with the present teachings. In some configurations, the adenovirus can be a gorilla adenovirus.

In various embodiments, the present teachings can include an adenovirus vector comprising a pulmonary targeting ligand, a vascular-specific promoter and integrin targeting peptides incorporated into a viral knob.

Brief Description of the Drawings

FIG. 1. illustrates adenoviral retargeting via fiber replacement of the native Ad5 fiber protein. Normally Ad binds to the Coxsackse-adenovirus-receptor (CAR) via the knob domain of the fiber protein.

FIG. 2 A illustrates MBP peptide targeting of adenovirus to lung.

FIG. 2B illustrates targeted gene transfer localized to alveolar capillaries.

FIG. 3 illustrates serum Al AT levels achieved via targeted and iintargeted adenoviral vectors.

FIG. 4 illustrates four GAd46 vectors containing the MBP targeting peptide.

FIG. 5 illustrates a ribbon diagram of the Gorilla Ad Fiber Domain.

FIG. 6 illustrates incorporatio of RGD-4C or flag peptide within the HI -loop of the gorilla Ad fiber knob domain.

FIG. 7 illustrates a polyacrylamide gel electrophoresis analysis of modified fiber (ROD or Flag peptide) incorporation into gorilla Ad capsid.

FIG. 8 illustrates a Western blot against anti-flag mAb, M2.

FIG. 9 illustrates a Western blot against anti-fiber tail mAb, 4D2.

FIG. 10 illustrates analysis of RGD-4C peptide incorporation - Ad gene transfer efficiency,

FIG. 1 1 illustrates analysis of RGD-4C peptide incorporation ~ Gorilla Ad gene transfer efficiency.

FIG. 12 illustrates analysis of RGD-4C peptide incorporation - Gorilla Ad gene transfer efficiency.

FIG. 13 illustrates analysis of RGD-4C peptide incorporation - Gorilla Ad gene transfer efficiency.

FIG. 1 illustrates that Ad.MBP preferentially targets lung in vivo by charting biodistributkm of Ad.MBP.Luc Expression in C57BL/6J mice.

FIG. 15 illustrates preferential targeting of Ad.MBP to lung in vivo. FIG. 16 .illustrates non-Gorilla adenovirus vectors Ad.CMVCas9, Ad.U6gRNA,

Ad.CMVCas~9-UgRNA, AdS.EFIeGFP (Rosa26 donor), Ad5-amFDC (Rosa26 donor) and AdS.EFlfxhAlAT (Rosa 26 donor).

FIG. 1.7 illustrates that adenoviral vector-mediated deliver of CRI$PR/Cas9 results in non-homologous end joining in-vitro through INDEL formation at ROSA26 target locus using Ad5.CMV-Cas9-gR A transduced into BNL-1NG cells.

FIG. I S illustrates adenoviral vector-mediated delivery of CRiSPR/Cas9 results in nonhomologous end joining in vivo through INDEL formation i an Ad5Cas9gENA injecied CB571J mouse.

FIG. 1 illustrates a FIX Western blot of supernatants of A549 cells with ADEFIamF9

(Rosa26 donor) at 1000 and 5000 MOI, at 72 hours post injection.

FIG. 20 illustrates A549 cells infected with AdS.EFiaGFP donor (10000 vp/celi).

FIG. 21 illustrates amount of OFF expressing cells 50 days after infection for various cell lines.

FIG. 22 illustrates targeted integration of hAl AT into ROSA26 locus permits extended gene expression via co-delivery CR3SPR/Cas9 and Donor Vector in vivo via ELISA, FIG. 23 illustrates mouse livers show extended GFP expression from both integrated genomic and episomal DMA at week 1 (Left column) and week 6 (right column).

Detailed Description

Whereas viral vectors have been employed for in vivo delivery of CRISPR/Cas9, these applications have been limited by the native ropism of the viral vecto used. The present teachings include modification of adenovirus to alter targeting to selected host cells, combined with CRISPR Cas9 delivery for editing of targeted cells.

The packaging capacity of Ad allows vector incorporation of al l CRISPR Cas9 elements ensuring coordinated tiuictionahty. The ability to modify Ad iropism can facilitate in vivo targeting. Targeted Ad thus embodies a set of attributes which can be useful for targeted in vivo editing.

The present inventor has developed an approac based upon gene delivery to the blood vessels of the lung. This strategy provides an alternative to liver sourcing of corrective hemophilia factors and can allow the application of gene therapy for diseases such as, for example, hemophilia, while avoiding liver toxicity. In some configurations, in vivo transduction of the pulmonar endothelium via targeted Ad can demonstrate that, pulmonary endothelium can serve as an effective platform to reconstitute deficient semm proteins. In some configurations, an Adenovirus can be modified to incorporate a range of gene constructs relevant to gene therapy for the full spectrum of hemophilia disorders.

In various embodiments, Ad technology can be fully commensurate with the CRISPR/Cas 9 system, and can allow in vivo genetic modifications for stable gene expression.

Various embodiments include utilizing the CRISPR Cas9 system i conjunction with pulmonary endothelial-targeted Ad, to achieve stable incorporation of corrective genes within the pulmonary endothelium, such as, for example, genes that can correct hemophilia. In. some configurations, the strategy can provide for stable genetic correction o hemophilia in a manner that can circumvent potential vector-associated toxicities for both factor IX and factor XIII hemophilia gene therapy. This approach thereby facilitates the application of corrective gene therapy for hemophilia.

In various configurations, an adenovirus vector of the present teachings can be used to effect stable expression from a pulmonary vascular source of introduced genes such as deficient hemophilia factors, while circumventing vector-mediated

hepaiotoxieiiies. I n various configurations, an adeno virus vector of the present teachings can be used for gene therapy for deficiency disorders for serum proteins such as, without limitation, factor VIO or factor IX.

In some embodiments, an adeno virus of the present teachings can be an adenovirus of a non-human primate such as a gorilla (Gad). In some configurations, adenovirus from a non-human primate can be used to avoid reactions of a human subject against human serotype 5-based Ad, including reactions due to preformed antibodies against human serotype 5-based Ad.

Methods

The methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et ah, Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Spector, D. L. et ah. Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; Nagy, A., Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), Cold Spring Harbor, MY, 2003 and Harlow, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laborator Press, Cold Spring Harbor, NY, 1999. Methods of administration of pharmaceuticals and dosage regimes, can be determined according to standard principles of pharmacology well known skilled artisans, using methods provided by standard reference texts such as Remington: the Science and Practice of Pharmacy ( Alfonso R. Gennaro ed. 1 th ed. 1995); Hardman, J.G., et at, Goodman & Gilman's The

Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R.C., et al, Handbook of Pharmaceutical Excipients, Fourth Eklition, Pharmaceutical Press, 2003. As used i the present description and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the contex indicates otherwise.

Examples

The present teachings include descriptions provided in the examples that are not itttettded to limit the scope of any aspect or claim. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The following non- limiting examples are provided to further illustrate the present teachings. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

Example 1

This example illustrates tropism modification of adenovirus via genetic capsid modification.

The present inventor altered tropism of Ad via genetic incorporation of binding ligand within specific domains of the adenoviral fiber protein (1), The present inventor has replaced the nati e fiber protein with a chimera of fiber plus the T4 Pol protein fibritin (2, 3). To accomplish Ad vector retargeting, the fiber protein is replaced by a chimera consisting of the tai l domain of the native fiber fused to the T4 Pol protei n fibritin, and an MBP pepiide binding to myeloid cells. Utilizing this method the inventor incorporated into the Ad capsid the pepiide iigand myeloid binding protein ''MBP" (FIG. 1). Studies have validated the specificity of the AdMBP to accomplish efficient and selective gene delivery to cells of myeloid lineage (4). We have used this system to test our hypothesis with respect to pulmonary vascular transductio via a "hitchhiking" mechanism (5). This system is an excellent mechanism for Cas Cr sper Ad vector virus delivery.

Example 2

This example illustrates targeted gene transfer to pulmonary vascular endothelium via a tropism-modified adenoviral vector.

In these experiments, mice were given via tail vein conventional untargeted h AdS vector encoding reporter (AdS.Luc) or tropism modified Ad with capsid

incorporated MBF (Ad.MBP.Luc). As can be seen in FIG. 2A and FIG. 2B,

Ad.MBP.Luc exhibited a statistically significant targeting effect with enhancement of gene delivery to the pulmonary endothelium and reduction of gene transfer to the liver, in comparison to AdS.Luc, Ad.MBP.Luc and Ad.Lue were injected in C57BL6 mice via tail vein. For FIG. 2A tissues were collected 24 hours later and assayed for lueiierase expression in lysate from various organs normalized to nig of total protein. For FIG. 2B, mice were injected (Lv.) with 2,5 x I0 ⁱ⁰ viral particles (VP) of MBP-targete Ad encoding the green protein reporter, Ad.MBP.GFP, 24 hours prior to sacrifice. Panels have been split into color channels in order to render the figure in black and white.

Panels show staining for the GPP transgene (FIG. 2B, bottom panel), von Willebrand factor (FIG. 2B, top panel), and nuclei (DAP!, FIG. 3B, middle panel) in (a) uninj cted conti'ols, mid mice injected with (b) control AdS.GFP, or (c, d) Ad.MBP.GFP, Scale bar, 40 μηι. Images are representative of n - 2-3 mice per group. Of note, the increase in the targeting ratio was more than five orders of magnitude compared to non-targeted adenovirus. The addition of MBP specifically targets Ad to lung tissue.

Example 3

Others ha ve highlighted the potential of extrahepatic sources of coagulation factors for gene therapy (6). This example illustrates that pulmonary endothelium can serve as a cellular source of a secretory protein to augment serum levels.

In these experiments, the present inventor utilized alpha 1 -antitrypsin (A I AT) which has biosynthesis and biodistribution analogies t serum coagulation factors. For these studies, mice were administered intravenously untargeted, or MBP-targeted Ad encoding human A1AT. At intervals post-challenge, serum A1.AT was analyzed (7). As can be seen in FIG. 3, the targeted Ad could augment serum Al AT in a manner

comparable to the control Ad. C57BL 6J mice were injected via tail vein with either 10 ^K viral particles of Ad5.CMV.AlAT or JO ¹⁰ viral particles of Ad5.MBP.CMV.Al AT. The mice were then sacrificed 8, 24 or 72 hours later, and their semm was collected to determine A1AT. n = 5 mice in each group at each time point. No A l AT was detectable in the serum at 8 hours. Both groups of mice had detectable serum concentrations at 24 and 72 hours, but there was no significant difference in serum Al AT concentration. These studies illustrate that pulmonary vasculature "sourcing" of a secretory protein can allow significant augmentation of serum levels.

The inventor has thus established herein; 1) modification of the tropism of adenoviral vectors via fiber replacement with the capsid incorporation of targeting peptides; 2) that Ad modified to accomplish myeloid cell binding achieves effective targeting to the pulmonary endothelium and wntargeting of the liver in vivo in murine models; and 3) that a pulmonary endothelial-sourced serum protein can directly augment serum levels.

In some embodiments, the present teachings include gorilla Ad with similar tropism modifications. Such vectors can circumvent preformed immunity to huAdS and can allow treatment of patients irrespective of anii-AdS immunity. Pulmonary

endothelium can thus be used as a cellular source for a serum protein such as factor Vili/factor IX. The present teachings include integration-competent Ad which can provide gene expression longevity mandated for treatment of various genetic diseases, such as, for example hemophilia gene therapy.

Example 4

This example illustrates the construction of a gorilla adenoviral vector (GAd) targeted to the pulmonary endothelium, and demonstration of augmentation of Factor IX (FIX ) levels in FIX hemophilia deficient mice.

In these experiments, G Ad targeted to the pulmonary endothelium are

constructed. The capacity of such targeted vectors to accomplish corrective levels of FIX in FIX hemophilia mice can be demonstrated.

Construction of tropism-modiiied gorilla adenoviral vector incorporating myeloid binding peptide (MBP). In some configurations, a t o-plasmid rescue method can be employed to construct a fiber-modified GAd incorporating a myeloid cell targeted heptapeptide into a de-knobbed huAdS fiber chimera with the trimerizing foldon domain of bacteriophage T4 fibritm (see FIG. 1). The recombinant GAd genome encoding chimeric fiber fused with MBP iigand (pGAd46FF~MBP) can be derived by homologous recombination in E, coli strain BJ5183 between a plasmid carrying the GAd46 genome lacking the fiber gene and a DNA fragment containing a fiber-fihrithv BP (FF-MBP) gene as previously described (2,4), E3 genes and the downstream fiber gene sequences can be deleted in the GAd46 genomic plasmid DNA and can be used for recombinatio with a shuttle plasraid carrying the chimeric fiber-fibritin-MBP gene flanked by surrounding GAd46 genome sequences . The FIX gene cDNA can be cloned from the plasmid pGEMmFlX (Sino Biological Inc., Beijing, China). This ORF can be Hgated into a shuttle plasmid carrying the CMV promoter connected to the S V40 pA via MCS and can be flanked by surrounding GAd46 genome sequences to generate pSh.uttleCMV.mFIX. The .recombinant GAd genome encoding CMV-FIX-pA cassette can be derived by homologous recombination in E. eoli strain BJ5183 between the plasraid carrying the GAd46FF-MBP genome but lacking the El gene, and the

pShuttleCMV.mFIX employing methods previously described (8) (See FIG. 4). Viral particles (vp) can be quantified by measuring absorbance of the dissociated virus at A260 nm using a conversion factor of 1.1x10 ^s " vp per absorbance unit (9), For validation of generic incorporation of binding peptides into fiber-fibritin purified GA.d46FF-MBP viral particles can be analyzed by PCR and Western blot. These analyses can show that FIX is expressed in the lung but not the liver.

Example 5

This example illustrates validation of myeloid binding specificity of tropism by modified GAd. For characterization of G Ad binding specificity, BMCs can be isolated and in vitro GAd binding and transduction can be evaluated as previously described (4, 5). Cells can be infected with GAd vectors at various multiplicities of infection and cellular-bound GAds can be detected as previously described (4). Additionally, the concentrations of FIX in the medi can be determined with the Factor IX Murine FE ISA Kit. Quantification Kit (Cloud-Clone Corp., Atlanta, GA). For blocking experiments, 1 x lO ^f> BMCs from FIX O mice can be pre-incubated with 25 _t ug of MB? peptide tor 30 min at 4°C before addition of virus.

Example 6

This example illustrates an analysis of biodistribution of tropism-modified GAd in murine models. In these experiments, FI KO mice can be injected via tail vein with PBS alone, 1 x10 ^U) vp of GAd46FF~MBP~inFIX, GAd46 expressing wild type fiber or GAd46FF vector expressing fiber-fibritin alone. The mice can be sacrificed 2, 24 or 72 hours later, and lungs, livers and spleens can be collected. The tissues ca be

homogenized in PBS, and genomic DMA can be extracted with the DMeasy Blood and Tissue Kits (Qiagen). The relative amount of viral DNA copies per ng of tissue genomic DNA can be determined by quantitative PCR (q-PCR). The mouse β-actin gene can be used as an internal standard for template loading of q-PCR. The q-PCR can show that FIX is expressed in the hmgs, but not the spleen or liver.

Example 7

Analysis of gene transfer targeting via tropism modified GAd in murine models. FIX KO mice can be injected via tail vein, as noted above. The mice can be sacrificed 2, 24 or 72 hours later, and blood can be collected and spun down allowing collection of the serum. The concentrations of itiFIX in the serum can be determined with a Factor ΪΧ Murine ELISA Kit Quantification Kit.

The inventor's strategy to source FIX from the pulmonary endothelium ca be based on in vivo gene delivery to this target cell.

Example 8

The inventor has also developed alternative targeting methods compatible with GAds. The inventor has developed a "combination targeting" method (.10) based upon a vascular-specific promoter in combination with integrra targeting peptides incorporated into the adenoviral fiber knob. This approach ca achieve a high ratio of .lung-to-liver targeting with transgene expression induced in vivo at the pulmonary endothelium. Example 9

This example illustrates the functional configuration of the CRISPR Cas9 nto pulmonary targeted GAd and demonstrate in vivo gene edi ting of target cells of the pulmonary endothelium .

The levels of targeted in vivo gene transfer can allow corrective gene editing that can be used for hemophilia gene therapy. Additionally, Ad's transient expression of CRISPR components can provide an additional level of safety whereby nuclease activity is restricted to a short temporal spa thereby reducing potential genotoxiciti.es. The present teachings include Ad-incorporated CRISPR Cas9 for in vivo gene editing withi pulmonary endothelial target cells. Feasibility studies can be demonstrated utilizing a GFP reporter. Studies herein can thus validate in vivo gene editing via genomic analysis as well as stabl e genetic analysis via monitoring of the incorporated reporter gene.

Example 10

A two-vector strategy for GAd~based targeted gene editing of ROSA26 safe harbo locus. A two- ector strategy can be used hi which one GAd vector encodes all necessary components of the Type II CRISPR7Cas9 gene editing system, targeted to the murine ROSA26 'safe harbor' locus, and a second vector encodes a constitutive ly expressed green fluorescent reporter gene with appropriate homology arras for integration. Non-limiting examples of these vectors are illustrated in FIG. 4, including a transient vector encoding mFIX, a vector encoding CRISPR components and a GFP reporter donor vector with Rosa26 homology arms for targeted genomic iniegration, and a Rosa26 donor vector encoding mFIX. This approach utilize three plasmids (provided by Genome Engineering Core (GEC) at Washington University in St, Louis). The first plasraid encodes mammalian codon-opfimized Cas9 and a nuclear leading sequence. This plasrnid is used to clone Cas9 gene into the MCS of our shuttle plasrnid (see example 3), resulting in pShuttle-CMV-Cas9. The pShutt!e-CMV-Cas9 is recombined into the El region of oar E1 _? E3 -deleted GAd plasrnid DMA upon co-transformation in Escherichia coti strain BJ518359. The plasrnid pGAd46 BP-CMVCas9 is digested to release the viral genome followed by transfeetion into HE 293 cells expressing El, allowing replication of GAd. Next, the replication defecti ve virus is amplified, purified via two ultracenlrimgations of CsCl gradients, and sequence validated using our standard techniques.

The second plasrnid encodes an efficient guideRN (gRNA), under the control, of the Pol III promoter U6, targeting the second intronic region of the ROSA26 locus and can be used for the generation of transgenic mice. To obtain a vector expressing both CRISPR components, the ^' U6.gR.NA expression cassette can be ligated into pShisttle- CMV-Cas9 to generate pShuttIe-CMV-Cas9-U6-g HA. The pShuttIe-CMV-Cas9-U6- g ^' RNA can be homoiogously recombined into the El region of our E1,E3 -deleted GAd plasrnid DNA containing the MBP targeting ligand, as above. The resulting plasrnid pGA.d46MBP~Cas9gRNA can he transfeeted into 293 cells for rescue _* amplified, purified, and sequence verified as described above (FIG. 4). The third plasrnid encodes donor homology arm (HA) sequences composing the left and right sequences (app. 800 bp ea.) surrounding the ROSA26gRNA target site, modified by a MCS between the HAs. This MCS destroys the gRNA target site on the donor plasrnid protecting it from cleavage by Cas9. This donor plasrnid can also be used as a ROSA26 donor. T he reporter eGFP can be inserted into pBApo (Ciontech Laboratories, Mountain View, CA) under the control of the constitutive mammalian EFla promoter which includes an nitron. This combination of promoter, transgene and polyA signal cassette can be Hg ted. into the ROSA26 donor piasmid and cloned into a GAd46 pShuttie. This shuttle vector can be recombined into a El deleted region to yield GAd46MBPE laGFP (Rosa26 Donor), rescued _* amplified, purified, and sequence verified as above (see FIG. 4). These vectors can be used to transform genes such as FIX into mammalian organisms.

Example i 1

Functional Validati on of Targeted Gene Editing by Genome Analysis.

To validate expression and fiinction of these vectors, titers can be detenrnned as described in Example 3. Gene products can be verified in vitro following infection with individual vectors at a MOI of 100 vp/ceil. The Cas9 can be detected via western blot, gRNA production can be verified with qRT-PCR with a probe specific to the A scaffold, and eGFP can be detected via florescence microscopy. Double stranded cleavage at the ROSA26 target sequence and integration can be validated in vitro using a marine BNL-1NG cell line, GAdCas9gRNA will be infected at MOIs of 1, 5, and 10 thousand viral particles/cell. 48 hrs post-infection (hpi), cells can be harvested, whole genomic DNA extracted and used for generation of a PCR library for deep sequencing analysis to detect mutations at the ROSA26 target site. The PCR primers consist of 5' and 3' oligos flanking the gRNA target sequence. To validate integration at the ROSA26 locus, BNL-IMG cells can be infected with Gad46Cas9gRNA and G Ad46EF I aeGFP (ROSA26 Donor) at a previously determined optimal cleavage MOL Cell cultures can be passaged and monitored for stable eGFP expression over a period longer (4÷ weeks) than is expected for transient unintegrated gene expression using fluorescent microscopy. Total integrated GFP expression in cells afte multiple passages can be quantified using ImageJ software for relative florescent. intensities compared to control infected and uninfected cell cultures. Following this period of extended culture, GFP cells can be sorted using flow cytometry and additional evidence of integration can be acquired, using PCR amplification of the R.OSA26 genomic region and southern blotting with the target site-specific probe to analyze migration differences due to insertions. These experiments can show that vectors of the present teachings are specifically targeted to lung tissue. Example 12

Functional Validation of Targeted Gene Editing with Reporter Gene Analysis.

Examination of in vivo efficacy of a ful ly targeted integration system can be performed,. In these experiments, extended gene expression levels and integration rates following co-infection of GAd46Cas9gRN A and GAd46EF1 aGFP in vivo can he analyzed using C57BL/J mice. Use of the GFP donor can allow optimization of an integration scheme without the added difficulty associated with handling hemophilia mice, which more readily die after injury (e.g. injection or blood draw) than wild type mice. For this analysis, wild-type CS7BL/6 mice grown to 8-10 weeks old can be co- injected with 100 μΐ. of PBS without virus or PBS containing equal amounts (!0 ^U 5xl0 ^kl , or 10 ^u total viral particles) of the CRISPR encoding vector and the donor vector Gad46MBP~EFl'¾GFF (w/ R26 homology arras) through tail veins. Initially, a ratio of 1 : 1 of each vector can be utilized but can subsequently be altered to optimize in vivo genomic integration. There can be 3 dose groups each containing 8 C57BL/6 mice and 4 uninfected, mice as a negative control. Additionally, 3 wild-type mice can be treated with GAd46MBPEF 1 *GFI* donor only (no nuclease) to serve as an experimental control for homology-directed repair (HDR) independent Integration, per dose group. These mice can be sacrificed 4, 8, and 16 wks post infection and whole genomic ON A can be extracted from lung cells for analysis of integration rates using LAM -PCR for an unbiased amplification of the target genomic locus (ROSA26) of both integrated and non-integrated alleles, as described elsewhere (23). These PCR products can be used for qPCR quantification of the integration rates at the ROSA26 locus using separate qPCR reactions of mm- integrated and integrated specific probes; B-actin can serve as an internal control to normalize the abundance of integrated vs non-integrated alleles.

Additionally, liver tissue sections can be generated for irnniuno.fi uorescent staining of eGFP positive cells to visualize expression resulting from integration. Injections and data analysis can be performed in a double blinded mariner to ensure an unbiased

experimentation and analysis of this strategy. Extraction RNA from lung tissue of treated and non-treated mice at the above time points ca be performed to quantify GFP gene expression using GFP specific RT-PCR probes, with B-aetin probes to normalize GFP mRNA quantity.

Additionally, injection of new bom mice can increase homology-direcied repair. Administration of a small molecule drug (Ser7) can inhibit non-homologous end joining (NHEJ) and increase HDR rate.

These experiments can demonstrate the ability of vectors of the present teachings to incorporate into the ROSA26 locus.

Example 13

This example illustrates the use of a composite GAd vector system, which includes pulmonary vascular targeting and CRISPR/Cas9. This system can be used tor gene therapy correctio of hemophilia in a murine model, and can achieve stable, long term correction of factor IX. deficient mice. Pulmonary vascular endothelium can serve as a cellular source for FIX to achieve correction levels of serum reconstitution . In vivo editing of pulmonary endothelial cells can be achieved vi vector-mediated delivery of components of the CRJSPR/Cas9 system. Based on these key feasibilities, gene therapy correction in a mammalian model of a genetic disease can be achie ved, in the aggrega te, vectors and methods of the present teachings ca be used for treatment of genetic diseases such as hemophilia in humans.

Example 14

Generatio of a ROSA26 targeted donor vector encoding murine FIX. A mFIX donor vector targeted for gene insertion at the murine ROSA26 locus can be produced in a similar manner to the eGFP donor vector in Example 2. Briefly, mFIX cDNA (Sino Biological Inc) can be inserted into pBApo (Clontech Laboratories) under the control of the constitutive mammalian EF l a promoter which includes an intron. This promoter, transgene, and polyA signal cassette can be ligated into the OSA26 donor plasmid and cloned into a GAd46 pShuttle. This shuttle vector is reeombined into E l deleted region to yield G Ad46MBPEF 1 omFIX (Rosa26 Donor), rescued, amplified, purified, and sequence verified as above (see FIG. 4). Transgene expression of this donor vector can be confirmed in human A549 ceils following transduction at 1000 MOl, followed by Western Blot detection of the secreted -mFIX in the culture supernatant. These

experiments can illustrate that FIX can be transformed into the OSA26 locus.

Example 15

Functional Validation of Targeted In Vivo Gene Editing by Genome Analysis, FIX O mice grown to 8-1 weeks old are co-injected with 100 [iL of PBS without virus or PBS containing equal amounts (10 ¹⁰ , 5x10 ^U \ or .10 ^H total viral particles) of the

C 1SPR encoding vector and the donor vector Gad46MBP-BF 1 amFIX (w/ R26

homology anus) through tail veins. 3 dose groups can each contain 8 C57BL/6 mice and 4 uninfected mice as a negative control. Additionally, 3 wild-type mice can be treated with GAd46MBPEFlamFIX donor only (no nuclease) as an experimental control for HDR independent integration, per dose group. These mice can be sacrificed 4, 8, and 16 wks post infection and whole genomic D A can. be extracted from lung cells to analyze integration rates using L AM-PCR for an unbiased amplification of the target genomic locus (ROSA26) of both in tegra ted and non integrated alleles, as described above and elsewhere (23), Lung tissue can be harvested for whole protein extract for Western Blot detection of mFIX production as well as generation of liver tissue sections for

immunoflourescent staining of mFI X positive cells to visualize expression resulting from integration. Further evidence can be obtained by extracting RMA from lung tissue of treated and non-treated mice at the above time points to quantify mFIX gene expression using mFIX-speciftc RT-PCR probes, with B-actin probes to normalize GFP mRNA quantity. These experiments can illustrate that FIX can be transformed into the ROSA26 locus.

Example 16

Validation of Phenotype Rescue in Targeted Gene Editing in hemophilic mice. Factor IX KO mice (8-10 weeks old) can be co-injected with 100 μΕ of PBS without virus or PBS containing equal amounts (lO' ^*1 , I0 ^itt , or 10 ¹ ' total viral particles) of the CRISPR encoding vector and the donor vector Gad46MBP-EFlamFIX (w/ R26 homology arms) through tail veins. Scheduled blood samples can be collected from the retro-orbital plexus and the plasma can be stored at -80 C for future ELISAs of raFlX levels and activated partial thromboplastin time (APTT) assays to measure FIX coagulation activity. Blood collection can occur at 24 hrs, 48 hrs, 1 wk, and every two weeks thereafter for up to 60 weeks (or until mf 1 X is no longer detectable) post- administration. Plasma can also be collected in parallel from untreated mice and wild- type C57BL/6 mice of the same age to serve as negative and positive controls, respectively. Additional experimental evidence can be obtained using an AAV vector encoding murine FIX. 3 dose groups each contain 15 KO mice and 4 mice (C57BL 6) as a positive control. 3 FIX KO mice can be treated with GAd46MBPEF l mFlX donor only (no nuclease) to serve as an experimental control for HDR independent integration, per dose group. The total for the experiment can be therefore 54 FIX KO mice and 12 C57B1/6 mice. 3 randomly selected mice of each hemophiliac background in each dose group can be subjected to a tail bleed assay at 8 weeks post administration for further validation of increased coagulation ability with non-treated and wild type mice used as controls. These experiments can show that vectors of the present teachings can be used to rescue the hemophilia phenotype.

Example 17

This example illustrates Gorilla Ad Fiber Knob domain gene transfer efficiency. FIG. 5 illustrates the 3-din ensional structure of the Gorilla Ad fiber knob, including die location of the HI loops. FIG. 6 illustrates the incorpora tion of RGD-4C (GAd KnobHS- RGD) and FLAG (GAd Knob 01 -Flag) tags into the i ll loo of the gorilla adenovirus knob. FIG. 7 illustrates total protein staining of gel purified Gorilla Ad. FIG. 8 illustrates an anti-flag Western blot of flag tagged Gorilla adenovirus knobs derived from gel purified Gorilla Ad. FIG. 9 illustrates a Western blot detected with anti-fiber tail antibody, as before.

In these experiments, plasrnids were transformed into A549 (lung), OV -4 (ovary), RD, SKOV3 (ovary), RD (muscle) and SVEC4-10 cell (endothelial) lines. Expression was measured by Relative Light Units (RLU) indicating reporter luc ferase gene expression (FIG. 10-13). AD5L/G-RGD showed better integration than AD5L/G, with a maximum of 7.0 x ID ⁶ relative light units detected (FIG. 30). GC46L.HI-RGD showed better incorporation than either GC46.HI-Flag or untagged GC46L in most cell lines, although in S OV3 all constructs showed equal integration, and in SVEC4-10, very little GC46L.HI-RGD was detected, but GC46L.HI-Flag expressed well (FIG. 1 1). In general, GC46L JTl-RGD upper expressed higher than GC46L.HI-RGD, except in the O V-4 cell line where they were about equal (FIG. 12). GC46L.HI-RG.D integrated more readily relative to GC46L.HI-FLAG or GC46L (FIG.13).

Example 18

This example illustrates lung targeting of Ad.MBP in vivo. C57BL 6J mice were transformed with Ad.MBPLnc, and expression of MBP Luc was measured with lneiferase assay. FIG. 14 illustrates the biodistributioia of the luciferase construct in various tissues hi different concentrations. Targeting index, lung/liver is 94 at 10' vp/mouse, 645 at 10 ^s vp/mouse, 1218 at 10 vp/mouse, and 3953 at 10 ¹ ' ⁰ vp/mouse. FIG. 15 compares the biodistribution of expression of Ad .5 Luc to AdMBP.Luc in C57BL/6J mice. AdS.Luc expresses highly in the liver, which can cause liver toxicity. In contrast, AdMBP.Luc is expressed highly in the lungs and very lowly in the liver, allowing for high expression of constructs with low liver toxicity.

Example 1

This example illustrates Cas/CRiSPR targeting constructs using Adenovirus constructs described in FIG. 16. The first vector set consists of CRISP components. Control vector Ad.CMVCas9 expresses Cas from the El deleted region. Control: vector Ad.U6gRNA expresses a ROSA26 specific gRNA from the E3 deleted region. The experimental vector Ad,CMVCas~9~UgRN A expresses Cas9 from the El deleted region and a ROSA26 specific gRNA from the E3 deleted region. The second vector set contains donor cassettes for integration composed of transgenes (EGFP reporter or secretaire A3 AT) driven b the constituti ve EF3 a promoter/intron flanked by up- and downstream sequences (-.8Kb) surrounding the ROSA26 gRNA restriction she. These vectors are: AdS.EFleGFP, AdS-amFIX and Ad5.EFlahA.lAT (FIG. 16).

To test Rosa targeting, Ad5,CMV-Cas9-gRNA transduced into BNL-ING cells. Targeted Hlmmn deep sequencing of the ROSA26 from genomic DN A of BNL-ING murine liver cells shows insertions and deletions (mdel) formation resulting from NHEJ DNA repair of DSBs increases with increasing multiplicity of infection with AdS.C V- Cas9,lJ6-gRNA (FIG. 17). Furthermore, a C57B1/J mouse injected with Ad5Cas9gRNA showed 15% 1NDEL formation in li er, and near control levels the kidney and spleen (FIG. 18).

AdS.EFornFIX was transformed into A549 cells at 1000 and 5000 MOT A

Western blot against FIX antibody was performed, and increased FIX expression was observed with increased MOI (FIG. 19). A549 cells were infected with Ad.S.EFlaGFP at 10,000 vp celi and expression was observed at 1 dpi (FIG. 20). Further ceil transformations using the constructs indicated in FIG, 21 also illiistrated stability of Cas9 constructs 50 days after infection. These experiments illustrate that vectors of the present teachings can be used to stably transform genes into mammalian cells.

Example 20

This example ill ustrates the stability of expression using constructs of the present teachings. As shown in FIG. 22, non-integrative mouse groups were injected with either PBS (filled triangle), 7.SE10 VP of hAlAT donor vector (AdS.BF 1 a~h Al AT) and

2.5E10 sham vector (Ad5.CMV-EGFP _} open square, broken line) or 5E10 VP of each (open diamond, dotted line), integrative mouse groups were injected with 7.5E10 VP of hAl AT donor vector (AdS.EFla-hAlAT) and 2.5ΕΪ0 CRlSPR-contaming vector

(Ad5.CMV-Cas.U6-gRNA, open circle), 5E10 VP of each (open triangle), or 2.5EI0 VP ofhAIAT donor vector (Ad5.EFla-hAl AT) and 7.5E10 CRIS ^' PR-containing vector (Ad5.CMV-Cas.U6-gRNA, filled square). Plasma levels of hAl AT were determined weekly or bi-weekly via ELISA demonstrating that hAl AT level were more stable over time in mice receiving a CRISPR/Cas9 integration system compared to an equivalent episomai based expression system.

This effect without a targeting moiety is further illustrated in FIG. 23, which illustrates that Ad constructs can be expressed at week 1 (left column) and week 6 (right column) in mouse liver cells (amplified 4x), These experiments further illustrate the stabil ity of transformations performed using the presen t teachings.

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