DISEASES

RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of U.S. Provisional Application No. 63/442,271 filed January 31, 2023 and U.S. Provisional Application No. 63/332,097 filed April 18, 2022. The contents of each of these applications are hereby incorporated by reference in their entireties.

FIELD OF THE DISCLOSURE

[0002] The disclosure is directed to molecular biology, gene therapy, and compositions and methods for modifying expression and activity of RNA molecules.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (LOCN_021_001WO_SeqList_ST26.xml; Size: 326,544 bytes; and Date of Creation: April 18, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

[0004] There are long-felt but unmet needs in the art for providing effective gene therapies, particularly gene therapies which target the underlying pathogenic RNA causing a disease.

[0005] Over 20 unstable microsatellite repeat expansion (MRE) have been identified as the cause of neurological disease in humans. (Rohilla and Gagnon, Acta Neuropathologica Communications, (2017) 5:63.) Pathogenic RNAs expressed from these repetitive MRE tracts in microsatellite repeat expansion causes a range of debilitating and often devastating diseases and disorders. These repeat RNAs, their location within the genes, the ranges of normal and disease-causing repeat length and the clinical outcomes differ. Unstable repeats can be located in the coding or non-coding region of a gene. Available treatments address symptoms of these MRE diseases but do not target their underlying etiology.

[0006] A GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of C9ORF72 gene is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9ALS/FTD). Bidirectional transcription at the C9ORF72 repeat locus produces both sense G4C2 and antisense C4G2 containing transcripts. Studies to date have elucidated multiple pathogenic mechanisms including RNA gain-of-function of both sense and antisense HREs transcripts leading to formation of nuclear RNA foci and aggregating dipeptide repeat proteins generated by repeat-associated non- AUG (RAN) translation of both sense and antisense HREs, and a loss-of-function due to haploinsufficiency of the C9ORF72 protein. However, it remains unclear how much C9ORF72 protein loss contributes to neuronal death. As such, there remains a need to develop effective therapies for the knockdown of multiple pathogenic HRE sense, antisense, and/or flanking transcripts for the treatment of C9ORF72 diseases such as C9/ALS and FTD.

SUMMARY

[0007] The disclosure provides a composition comprising a nucleic acid sequence encoding an RNA-binding polypeptide capable of binding a first target RNA sequence and a second target RNA sequence, wherein the first target RNA comprises a toxic hexanucleotide repeat RNA sequence.

[0008] In some aspects, the toxic hexanucleotide repeat RNA sequence is GGGGCC (sense) or CCCCGG (antisense). In some aspects, the second target RNA sequence is GGGGCC, CCCCGG, or a flanking sequence thereof.

[0009] In some aspects, the RNA-binding polypeptide is a guided RNA-binding polypeptide, and the composition comprises one or more guide RNAs (gRNAs), wherein the one or more gRNAs comprise a direct repeat (DR) sequence capable of binding to the guided RNA-binding polypeptide and a spacer sequence capable of binding to the first or second target RNA sequence.

[0010] In some aspects, the spacer sequence is a sequence that comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to at least one of SEQ ID NO: 13 - SEQ ID NO: 131 or SEQ ID NO: 158 - SEQ ID NO: 169.

[0011] In some aspects, the DR sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to at least one of SEQ ID NO: 157, or SEQ ID NO: 170, SEQ ID NO: 171. [0012] In some aspects, the guided RNA-binding polypeptide is a Cas protein. In some aspects, the Cas protein is Casl3d. In some aspects, the Casl3d protein is encoded by a nucleic acid sequence that comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 147.

[0013] In some aspects, the nucleic acid sequence comprises one or more promoters. In some aspects, the one or more promoters are selected from a human U6 promoter, a mouse U6 promoter, a 7SK promoter, a tRAN valine promoter, an EFS promoter, or a CMV promoter.

[0014] The disclosure provides a vector comprising the composition of any one of the preceding claims. In some aspects, the vector is selected from the group consisting of: adeno- associated virus (AAV), retrovirus, lentivirus, adenovirus, nanoparticle, micelle, liposome, lipoplex, polymersome, polyplex, and dendrimer. In some aspects, the vector is an AAV vector.

[0015] The disclosure provides an AAV vector comprising a nucleic acid sequence encoding an RNA-binding polypeptide capable of binding a first target RNA sequence and a second target RNA sequence, wherein the first target RNA sequence comprises a toxic hexanucleotide repeat RNA sequence, wherein the AAV vector comprises: a first AAV ITR sequence; a first promoter sequence; a first gRNA comprising a spacer sequence capable of binding to the first target RNA sequence; a second gRNA comprising a spacer sequence capable of binding to the second target RNA sequence; a second promoter sequence; a polynucleotide sequence encoding for at least one repeat RNA-binding polypeptide; and a second AAV ITR sequence.

[0016] In some aspects, the RNA-binding polypeptide is a Cas protein. In some aspects, the first promoter sequence is a PolIII promoter sequence. In some aspects, the first promoter sequence is a U6 promoter sequence. In some aspects, the U6 promoter sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 156 or SEQ ID NO: 172 - SEQ ID NO: 174.

[0017] In some aspects, the spacer sequence of the first gRNA comprises comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 13 - SEQ ID NO: 131 or SEQ ID NO: 158 - SEQ ID NO: 169.

[0018] In some aspects, the spacer sequence of the second gRNA comprises comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 13 - SEQ ID NO: 131 or SEQ ID NO: 158 - SEQ ID NO: 169. [0019] In some aspects, the second promoter sequence is a constitutive promoter or a tissue-specific promoter. In some aspects, the second promoter sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 155 or SEQ ID NO: 186.

[0020] In some aspects, the Casl3d protein comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 146.

[0021] In some aspects, the Casl3d protein is encoded by a nucleic acid sequences that comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 147.

[0022] In some aspects, the first ITR sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 149 or SEQ ID NO: 150. In some aspects, the second ITR sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 149 or SEQ ID NO: 150.

[0023] In some aspects, the AAV vector comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 202 - SEQ ID NO: 218.

[0024] The disclosure provides a cell comprising the vector of any one of the preceding claims.

[0025] The disclosure provides a method of treating frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS) in a mammal having a toxic hexanucleotide repeat comprising administering a composition or AAV vector according to any one of the preceding claims to a toxic target repeat expansion RNA sequence comprising C4G2 and/or G4C2 in tissues of the mammal whereby the level of expression of the toxic target RNA is reduced.

[0026] In some aspects, the composition or AAV vector is administered to the subject intravenously, intrathecally, intracerebrally, intraventricularly, or subpially. In some aspects, the composition or AAV vector is administered to the subject intravenously. [0027] In some aspects, the reduced level of expression of the toxic target RNA thereby ameliorates symptoms of ALS or FTD in the mammal. In some aspects, the level of expression of the toxic target RNA is reduced compared to the level of expression of untreated toxic target repeat RNA. In some aspects, the level of reduction is between 1-fold and 20-fold.

[0028] In some aspects, compositions of the disclosure or vectors of the disclosure further comprise a third target RNA, wherein the third target RNA is selected from the group consisting of GGGGCC, CCCCGG, flanking sequences adjacent to GGGGCC, and flanking sequences adjacent to CCCCGG.

[0029] In some aspects, the Casl3d protei is a Casl3d variant engineered to improve on- target knockdown of G4C2 sense and C4G2 antisense flanking region.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which are included to provide a further understanding of the present disclosure, are incorporated in and constitute a part of this specification, illustrate aspects of the present disclosure and, together with the detailed description, serve to explain the principles of the present disclosure. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0031] FIGS. 1A-D show a series of schematics depicting exemplary CRISPR/Casl3d targeting systems and RNA expression reporter constructs used herein for the targeting of sense and anti-sense strands of C9ORF72 hexanucleotide repeat-containing RNA. FIG. 1A depicts an exemplary CRISPR/Casl3d targeting system with a single guide target of hexanucleotide repeat sense strand G4C2. FIG. IB depicts an exemplary CRISPR/Casl3d targeting system with a dual array target of sense strand G4C2 and flanking sequence adjacent of anti-sense C4G2. FIG. 1C depicts an exemplary G4C2 sense strand RNA expression reporter construct. FIG. ID depicts an exemplary anti-sense C4G2 flanking sequence RNA expression reporter construct.

[0032] FIGS. 2A-D show a series of bar graphs depicting knockdown of expression of RNA containing sense strand G4C2 and anti-sense C4G2 flanking regions in mammalian cells using the exemplary CRISPR/Casl3d targeting system in vitro. Mammalian cells were transfected with a first exemplary expression reporter construct expressing sense or anti-sense target RNA and a first exemplary CRISPR/Casl3d targeting system. Expression of target G4C2 RNA was determined using qPCR and normalized to GAPDH and cells treated with the non-targeting (NT) guide. Expression of the flanking antisense RNA was determined by luminescence of Firefly Luciferase normalized to Renilla luciferase signal (transfection control) and to NT guide. FIG. 2A shows a bar graph depicting expression of sense strand G4C2 RNA in cells transfected with the NT Guide (P000MJ Single Lambda Guide) or Single Guide (P000II Single G4C2gl) vector. The x-axis depicts the vector. The y-axis depicts normalized G4C2 expression. FIG. 2B shows a bar graph depicting Casl3d expression in cells transfected with the NT Guide (P000MJ Single Lambda Guide), Single Guide (P000II Single G4C2gl), Dual Array #1 (P02928C9flankg20_G4C2gl) or Dual Array #2 (P02929C9flankgl9_G4C2gl) vector. The x-axis depicts the vector. The y-axis depicts normalized Casl3d expression. FIG. 2C shows a bar graph depicting expression of anti-sense C4G2 flanking sequence in cells transfected with the Luciferase antisense reporter, NT Guide (P000MJ Single Lambda Guide), Dual Array #1 (P02928C9flankg20_G4C2gl) or Dual Array #2

(P02929C9flankgl9_G4C2gl). The x-axis depicts the vector. The y-axis depicts normalized C4G2 flanking expression. FIG. 2D shows a bar graph depicting expression of sense strand G4C2 in cells transfected with the NT Guide (P000MJ Single Lambda Guide), Single Guide (P000II Single G4C2gl), Dual Array #1 (P02928C9flankg20_G4C2gl) or Dual Array #2 (P02929C9flankgl9_G4C2gl) vector. The x-axis depicts the vector. The y-axis depicts G4C2 normalized expression.

[0033] FIGS. 3A-B show the elimination (knockdown) of sense strand G4C2 expression foci in host cells transfected with exemplary CRISPR/Casl3d targeting vectors. FIG. 3A shows a series of microscopy images of cells transfected with non-targeting (P000MJ Single Lambda Guide), Single Guide-G4C2 (P000II Single G4C2gl), Dual Array #1 (P02928C9flankg20_G4C2gl), or Dual Array #2 (P02929C9flankgl9_G4C2gl) vector. Expression of sense strand G4C2 was determined using RNA-FISH analysis. Cells were visualized by DAPI staining. FIG. 3B shows a bar graph depicting the average G4C2 expression foci per cell in the exemplary images depicted in FIG. 3A.

[0034] FIGS. 4A-B show an exemplary model of CRISPR/Casl3d based RNA targeting knockdown of sense hexanucleotide repeat expansions in vivo. FIG. 4A shows a table depicting mouse strain, dosage of vehicle or AAV9-Casl3d-G4C2 vector (targeting the sense G4C2 repeats) administered and sample collection timepoint of the exemplary mouse model. FIG. 4B shows a schematic of the exemplary subpial injection, the tissue sites of collection (cervical, thoracic, lumbar) and corresponding samples collected (RNA, protein) of the exemplary in vivo Cast 3d based RNA targeting knockdown model. [0035] FIGS. 5A-D show a series of bar graphs depicting exemplary Casl3d based RNA targeting knockdown of sense G4C2 RNA hexanucleotide repeat expansions in vivo using the exemplary mouse model depicted in FIG. 4B. FIG. 5A shows a bar graph depicting Cast 3d RNA expression in tissue following subpial injection of vector or AAV9-Casl3d-G4C2. The x-axis depicts the tissue. The y-axis depicts Casl3 expression. Casl3 expression was determined using ddPCR and normalized to Atp5b. FIG. 5B shows a bar graph depicting Cast 3 protein levels in tissue following subpial injection of vector or AAV9-Casl3d-G4C2. The x- axis depicts the tissue. The y-axis depicts Casl3 protein signal determined using Meso Scale Discovery (MSD).

[0036] FIG. 5C shows a bar graph depicting G4C2 sense strand (pathological variant) RNA expression relative to total C9ORF72 gene expression in mouse tissue following subpial injection of vector or AAV9-Casl3d-G4C2. The x-axis depicts the tissue. The y-axis depicts relative G4C2 RNA expression. FIG. 5D shows a bar graph depicting expression of C9 total RNA relative to Atp5b reference gene expression in mouse tissue following subpial injection of vector or AAV9-Casl3d-G4C2. The x-axis depicts the tissue. The y-axis depicts relative C9 total RNA expression.

[0037] FIGS. 6A-D show a series of bar graphs depicting knockdown of expression of RNA containing sense G4C2 and antisense C4G2 repeats, as well as decrease in dipeptide repeats (DPRs) poly-GP aggregates in C9-ALS derived fibroblasts after treatment with Casl3d multi-targeting (MT) construct. C9-ALS derived fibroblasts were transduced with lentivirus expressing Casl3d and selected with puromycin 48 h after transduction. After selection cells were transduced with the lentivirus for guide expression targeting sense and antisense transcripts and harvested 72 h after the second transduction. Expression of target RNA was determined using ddPCR and normalized to GAPDH reference gene and cells treated with the non-targeting (NT) guide condition. Bar graph depicting expression of sense strand G4C2 RNA (FIG. 6A) and antisense RNA (FIG. 6B) in C9-ALS fibroblasts transduced with Cast 3d (Seq212) and with the NT guide or multi -targeting guide array (MT). The x-axis depicts the treatment. The y-axis depicts normalized sense or antisense expression. FIGs. 6C-D shows bar graphs depicting Cast 3d protein expression (FIG. 6C) or poly-GP DPR (FIG. 6D) by MSD in C9-ALS fibroblasts transduced with Casl3d and NT guide or multi -targeting (MT) guide array. The y-axis depicts firnol of Casl3d per total mg of protein (FIG. 6C) or raw signal for poly-GP (FIG. 6D). The x-axis depicts the treatment.

[0038] FIGS. 7A-F show a series of bar graphs depicting RNA knockdown of sense G4C2 and antisense C4G2 RNAs, as well as decrease in dipeptide repeats (DPRs) poly-GP aggregates in C9-ALS mouse model C9 B AC-500 (containing 500 G4C2 repeats) after 6 weeks subpial injection of AAV9 Casl3d multi-targeting (MT) (A02562). (FIG. 7A) shows a table depicting mouse strain, dosage of vehicle or AAV9-Casl3d MT vector administered and sample collection timepoint of the exemplary mouse model. (FIG. 7B) shows a schematic of the exemplary subpial injection, the tissue sites of collection (cervical, thoracic, lumbar) and corresponding samples collected (RNA, protein) of the exemplary in vivo Casl3d-MT based RNA targeting knockdown model. Decrease on sense RNA (V3 pathological isoform) (FIG. 7C) and antisense RNA (FIG. 7D) is shown by ddPCR as copies of the respective transcript per 1000 copies of the Atp5b reference gene. RNA was extracted from the lumbar region, close to the site of injection. The x-axis depicts the treatment. The y-axis depicts relative sense RNA expression (FIG. 7C) or antisense (FIG. 7D). Casl3d protein expression in tissue (thoracic region) following subpial injection of vector (Vehicle) or AAV9-Casl3d-MT (A02562) is shown in (FIG. 7E) and poly-GP DPRs in (FIG. 7F). The x-axis depicts the tissue (T represents Thoracic T9-12) and treatment, and the y-axis depicts Casl3 expression (fmol per mg of total protein) (FIG. 7E) or raw signal for poly-GP (FIG. 7F) using Meso Scale Discovery (MSD) assay.

[0039] FIGS. 8A-E show a series of bar graphs depicting RNA knockdown of G4C2 sense and antisense C4G2 repeats, as well as decrease in dipeptide repeats (DPRs) poly-GP aggregates in C9-ALS mouse model C9 BAC-500 (containing 500 G4C2 repeats) after 6 weeks intrastriatal injection of AAV9 Casl3d multi -targeting (MT) (A02562). Mice were injected unilaterally using AAV9 Casl3d MT with the non-injected side used as a negative control tissue, and two age groups were used, 17 weeks and 24 weeks-old mice, for evaluation. FIG. 8A shows a table depicting mouse strain, dosage of AAV9-Casl3d MT vector administered, mouse age at the treatment, and sample collection timepoint of the exemplary mouse model. Decrease on sense RNA (pathological isoform) (FIG. 8B) and antisense RNA (FIG. 8C) is shown by ddPCR as copies of the respective transcript per 1000 copies of the Atp5b reference gene. The x-axis depicts the groups and treatment. The y-axis depicts relative G4C2 sense RNA expression (FIG. 8B) or antisense RNA (FIG. 8C).

Casl3d expression in tissue following intrastriatal injection of AAV9-Casl3d-MT (A02562) is shown in (FIG. 8D) and poly-GP DPRs in (FIG. 8E) relative to untreated tissue (contralateral control). The x-axis depicts the groups and treatment, and the y-axis depicts Cast 3 expression (fmol per mg of total protein; in FIG. 8D) or relative poly-GP DPRs normalized to control untreated groups (FIG. 8E) using Meso Scale Discovery (MSD) assay. [0040] FIGS. 9A-B shows a schematic of the reporter construct containing the sense flanking region adjacent to the G4C2 repeats added downstream to firefly luciferase and a bar graph depicting knockdown of the sense flanking RNA in mammalian cells using the Cast 3d targeting system in vitro. FIG. 9A shows the reporter construct expressing the sense flanking region adjacent to the G4C2 repeats added downstream to firefly luciferase (FLUC) for screening purposes. FIG. 9B is bar graph depicting expression of sense flanking strand RNA in cells transfected with Casl3d and NT control or targeting guides. Mammalian cells were transfected with the luciferase reporter containing the sense flanking region, Cast 3d and the NT Guide (P000MJ Single Lambda Guide; negative control), firefly luciferase Guide (positive control), and guides targeting the sense flanking region. The x-axis depicts the guides used with Casl3d. The y-axis depicts expression of target RNA determined by firefly luciferase luminescence normalized to renilla luciferase (RLUC) transfection control and NT (nontargeting condition).

[0041] FIGS. 10A-E show optimized Casl3d variants that have been engineered to improved on-target knockdown of both G4C2 sense and C4G2 antisense flanking region. FIG. 10A is a schematic of reporter plasmid used to express G4C2 repeats driven by the CMV promoter. FIG. 10B is a bar graph showing quantitation of G4C2 RNA levels (y-axis) normalized to reference gene GAPDH and NT guide control condition after multiple treatments (x-axis). Wildtype (wt) Casl3d was co-transfected with non-targeting (NT) and G4C2- targeting guide (red and green, respectively). Engineered Casl3d variants were co-transfected with G4C2-targeting guide depicted in blue bars along x-axis. FIG. 10C is a schematic of C4G2 flanking sequence reporter plasmid cloned downstream of firefly luciferase ORF (FLUC). Renilla luciferase (RLUC) used as transfection control. (DZE) Bar graphs of two independent experiments testing C4G2 antisense flank knockdown in luciferase assay. Antisense C4G2 flanking level on y-axis, Seq212 variants on x-axis. FIG. 10D shows Seq212 point mutation variants, FIG. 10E shows a separate experiment testing RNA binding domains (RBD) tethered to Seq212. Dotted lines denote wildtype Seq212 knockdown levels.

DETAILED DESCRIPTION

[0042] The disclosure provides RNA-targeting gene therapy compositions and methods for treating HRE causing diseases and/or disorders such as ALS and FD.

[0043] A GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (c9ALS/FTD). Bidirectional transcription at the C9ORF72 repeat locus produces both sense G4C2 and antisense C4G2 containing transcripts. Studies to date have elucidated multiple pathogenic mechanisms including RNA gain-of-function of both sense and antisense HREs transcripts leading to formation of nuclear RNA foci and aggregating dipeptide repeat proteins generated by repeat-associated non- AUG (RAN) translation of both sense and antisense HREs, and a loss-of-function due to haploinsufficiency of the C9ORF72 protein. However, it remains unclear how much C9ORF72 protein loss contributes to neuronal death.

[0044] ALS is the most common motor neuron disease. The disease pathology involves the degeneration of upper and lower motor neurons, causing muscle weakness, spasticity, and paralysis leading to respiratory failure. Patients afflicted with ALS typically have about 700- 5000 repeats whereas a healthy patient has less than 30 repeats. See Ling, S.C. et al., Neuron (2013); Dejesus-Hernandez, M. et al., Neuron (2011); Renton et al., Neuron (2011); van Bittlerswijk et al. Curr Opin Neurol (2013). Potential pathogenic mechanisms include loss-of- function of the C9orf72 protein, RNA toxicity of the sense and antisense hexanucleotide repeats, and DPR (dipeptide proteins) toxicity caused by repeat associated non-ATG (RAN) translation which leads to expression of DPR. Braems, et al. Acta Neuropath (2020).

[0045] To explore therapeutic targeting of both sense and antisense HREs, novel CRISPR/Casl3d based RNA targeting systems with 2 guide RNAs (or more) targeting the sense and antisense repeat-containing-transcripts that can be packaged in a single AAV genome were engineered. Notably, qPCR and RNA-FISH analyses of cells transfected with G4C2 and C4G2 reporters showed efficient elimination of HREs. Furthermore, a G4C2 targeting guide RNA with two different orthologues of CRISPR/Casl3d into AAV9 with high yields was successfully packaged. These AAV9-packaged G4C2 targeting CRISPR/Casl3d compositions showed no overt safety concerns in wildtype mice at 8 weeks post-subpial delivery. Importantly, these constructs reduced the G4C2-HRE containing isoforms of C9ORF72 in both cultured neonatal cortical neurons and in the spinal cord of transgenic C9ORF72 mice containing 500- G4C2 repeats following subpial delivery, while largely preserving total C9ORF72 transcript (transcripts from both alleles and including normal non-expanded isoforms) levels. Similarly, a Casl3d-based multi-targeting (MT) guide targeting both sense and antisense was constructed and packaged (AAV9 Casl3d MT). RNA knockdown of G4C2 sense and C4G2 antisense transcripts, as well as decrease in dipeptide repeats (DPRs) poly- GP aggregates were shown in in vivo. In summary, a novel approach that can effectively target both sense and antisense HREs and/or flanking sequences thereof in C9ALS/FTD with a single product is disclosed herein.

RNA-Guided RNA-Binding Systems

[0046] In some embodiments, the disclosure provides RNA-guided RNA-binding systems. In some aspects, the RNA-guided RNA-binding system is an RNase Cas-based RNA-guided RNA-binding polypeptide. In some embodiments, a nucleic acid sequence encodes an RNA- guided RNA-binding polypeptide which is an RNase Cas protein (or a deactivated RNase Cas protein). In one embodiment, the nucleic acid sequence further comprises a gRNA sequence comprising a spacer sequence which binds to a toxic target repeat RNA and a direct repeat (DR) sequence which binds to the RNase Cas protein.

[0047] In one embodiment, a Casl3d system is catalytically active, in which case, the Casl3d nucleoprotein complex cleaves and destroys toxic RNA repeats. In another embodiment, a Casl3d system is catalytically inactive, in which case, the Casl3d nucleoprotein complex binds and blocks (but does not cleave) the RNA repeats. In yet another embodiment, a Cas 13d comprises a catalytically inactive Casl3d fused to an endonuclease which is capable of cleaving the toxic RNA repeats. In such an embodiment, the endonuclease is an active RNase. Exemplary endonucleases with RNase activity can be found herein, and these include, for example, a domain from a ZC3H12A zinc-finger (also referred herein as E17) or a PIN endonuclease.

[0048] In one embodiment, the RNase Cas protein is a Casl3 protein. In another embodiment, the Casl3 protein is a Casl3d protein. In another embodiment, the Casl3d protein is a deactivated RNase Casl3d protein (dCasl3d). In another embodiment, the dCasl3d protein is a fusion protein comprising 1) dCasl3d and 2) a polypeptide encoding a protein or fragment thereof having nuclease activity. In another embodiment, the dCasl3d protein is a fusion protein comprising 1) dCasl3d and 2) a nuclease domain of ZC3H12A, a zinc-finger endonuclease, (referred to as El 7 herein). In some embodiments, the Cas configuration comprises a signal sequence(s) such as NLS(s) and/or NES(s).

[0049] In some aspects, an SV-40 NLS can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 148. In some aspects, an SV-40 NLS can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 153. [0050] In some aspects, a cMyc NLS can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 179. In some aspects, a cMyc NLS can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 188.

[0051] In some aspects, a focal adhesion kinase (FAK) NES can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 178. In some aspects, an FAK NES can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 187.

[0052] In some embodiments, the dCasl3d is linked to E17 via a linker sequence. In one embodiment, the linker sequence is VDTANGS. In some embodiments, the nucleic acid sequence encoding the Cast 3d or dCasl3d fusion proteins are operably linked to at least one promoter sequence. In some embodiments, the promoter sequence comprises an enhancer and/or an intron. In some embodiments, the promoter sequence is a constitutive promoter such as, EIFla or its truncated form, the EFS promoter sequence, or the full length or truncated (tCAG) promoter sequence or the EFS/UBB promoter sequence. In other cases the promoter is a tissue specific promoter such as the neuron specific synapsin promoter sequence. In some embodiments, the nucleic acid sequence comprises a first promoter sequence that controls expression of a Cast 3d protein or Cast 3d fusion protein and a second promoter sequence that controls expression of the at least one guide RNA sequence.

[0053] In some embodiments of the compositions of the disclosure, the sequence encoding the RNA-guided RNA binding protein comprises a sequence isolated or derived from a protein with no DNA nuclease activity.

[0054] In some embodiments of the compositions of the disclosure, the sequence encoding the RNA-guided RNA binding protein disclosed herein comprises a sequence isolated or derived from a CRISPR Cas protein. In some embodiments, the CRISPR Cas protein is not a Type II CRISPR Cas protein. In some embodiments, the CRISPR Cas protein is not a Cas9 protein.

[0055] In some embodiments of the compositions of the disclosure, the sequence encoding the RNA-guided RNA binding protein comprises a Type VI CRISPR Cas protein or portion thereof. In some embodiments, the Type VI CRISPR Cas protein comprises a Cas 13 protein or portion thereof. Exemplary Casl3 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, bacteria or archaea. Exemplary Cast 3 proteins of the disclosure may be isolated or derived from any species, including, but not limited to, Leptotrichia wadei, Listeria seeligeri serovar l/2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954), Lachnospiraceae bacterium, Clostridium aminophilum DSM 10710, Carnobacterium gallinarum DSM 4847, Paludibacter propionicigenes WB4, Listeria weihenstephanensis FSL R9-0317, Listeria weihenstephanensis FSL R9-0317, bacterium FSL M6-0635 (Listeria newyorkensis), Leptotrichia wadei F0279, Rhodobacter capsulatus SB 1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442 and Corynebacterium ulcerans. Exemplary Casl3 proteins of the disclosure may be DNA nuclease inactivated. Exemplary Casl3 proteins of the disclosure include, but are not limited to, Casl3a, Casl3b, Casl3c, Casl3d and orthologs thereof. Exemplary Casl3b proteins of the disclosure include, but are not limited to, subtypes 1 and 2 referred to herein as Csx27 and Csx28, respectively.

[0056] In some embodiments of the compositions of the disclosure, the sequence encoding the RNA binding protein comprises a sequence isolated or derived from a Casl3d protein. Casl3d is an effector of the type VI-D CRISPR-Cas systems. In some embodiments, the Casl3d protein is an RNA-guided RNA endonuclease enzyme that can cut or bind RNA. In some embodiments, the Cast 3d protein can include one or more higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains. In some embodiments, the Cast 3d protein can include either a wild-type or mutated HEPN domain. In some embodiments, the Cast 3d protein includes a mutated HEPN domain that cannot cut RNA but can process guide RNA. In some embodiments, the Cast 3d protein does not require a protospacer flanking sequence. Also see WO Publication No. W02019/040664 & US2019/0062724, which is incorporated herein by reference in its entirety, for further examples and sequences of Cast 3d protein, without limitation.

[0057] In some embodiments, Casl3d sequences of the disclosure include without limitation SEQ ID NOS: 1-296 of WO 2019/040664, so numbered herein and included herewith. Yan et al. (2018) Mol Cell. 70(2):327-339 (doi: 10.1016/j.molcel.2018.02.2018) and Konermann et al. (2018) Cell 173(3):665-676 (doi: 10.1016/j .cell/2018.02.033) have described Casl3d proteins and both of which are incorporated by reference herein in their entireties. Also see WO Publication Nos. WO2018/183403 (CasM, which is Casl3d) and W02019/006471 (Casl3d), which are incorporated herein by reference in their entirety.

[0058] In some aspects, a Casl3d seq212 protein can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 146. In some aspects, a Casl3d seq212 protein can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 147.

Casl3d Seq212 variants for enhanced C9 G4C2 sense and antisense knockdown

[0059] The disclosure provides Casl3d variants for enhanced C9 G4C2 sense and antisense knockdown. Exemplary Casl3d variants are referred to herein as Seq212. Exemplary Casl3d Seq212 variants can comprise, consist essentially of, or consist of a nucleotide sequence or amino acid sequences as set forth below.

[0060] In some aspects, Cas polypeptides are fused to at least one protein and/or enzyme. In some aspects, Cas polypeptides of the disclosure are fused to adenosine deaminase acting on RNA (ADAR) or a domain thereof. In some aspects, the ADAR is an AD ARI . In some aspects, the domain of ADAR can be the Z alpha (Za) or Z beta (Zb) domain.

[0061] Za and Zb ADAR binding domains can be tethered to Cas polypeptides, including Cast 3d Seq212, in order to enhance recruitment of target RNAs for cleavage. Without wishing to be bound by theory, positioning of the ADAR binding domains close to the active Cas 13d endonuclease site improves on -target RNA knockdown.

[0062] In some aspects, the Za ADAR binding domain can comprise, consist essentially of, or consist of the nucleotide sequence:

GGC T C TC AC AT GC T Gagtatctaccaagatcaggaacaaaggatcttaaagttcctggaagagcttggggaagg gaag gccaccacagcacatgatctgtctgggaaacttgggactccgaagaaagaaatcaatcga gttttatactccctggcaaagaagggca agctacagaaagaggcaggaacaccccctttgtggaaaatcgcggtctccactcaggctt ggaaccagcacagcgga (SEQ ID NO: 198).

[0063] In some aspects, the Za ADAR binding domain can comprise, consist essentially of, or consist of the amino acid sequence: GSHMLSIYQDQEQRILKFLEELGEGKATTAHDLSGKLGTPKKEINRVLYSLAKKGKL QKEAGTPPLWKIAVSTQAWNQHSG (SEQ ID NO: 199).

[0064] In some aspects, the Zb ADAR binding domain can comprise, consist essentially of, or consist of the nucleotide sequence:

[0065] GGCAGCCACATGGCCTCTttagacatggccgagatcaaggagaaaatctgcgactatctc ttca atgtgtctgactcctctgccctgaatttggctaaaaatattggccttaccaaggcccgag atataaatgctgtgctaattgacatggaaag gcagggggatgtctatagacaagggacaacccctcccatatggcatttgacagacaagaa gcgagagaggatgcaaatcaag (SEQ ID NO: 200).

[0066] In some aspects, the Zb ADAR binding domain can comprise, consist essentially of, or consist of the amino acid sequence:

GSHMASLDMAEIKEKICDYLFNVSDSSALNLAKNIGLTKARDINAVLIDMERQGDVY RQGTTPPIWHLTDKKRERMQIK (SEQ ID NO: 201).

[0067] In some aspects, a Casl3d seq212 protein can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 146. In some aspects, a Cast 3d seq212 protein can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 147.

[0068] In some aspects, a Casl3d variant having mutations D823A, D830A, T832A, K827A, T213A (P04493) can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1. In some aspects, a Casl3d variant having mutations D823A, D830A, T832A, K827A, T213A can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of anamino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 2.

[0069] In some aspects, a Casl3d variant having mutations D823A, D830A, T832A, K827A, Y215A (P04494) can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 3. In some aspects, a Casl3d variant having mutations D823A, D830A, T832A, K827A, Y215A can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 4.

[0070] In some aspects, a Cast 3d variant having mutations S808A, K825A, K826A, K827A (P04705) can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 5. In some aspects, a Cast 3d variant having mutations S808A, K825A, K826A, K827A can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 6.

[0071] In some aspects, a Casl3d variant having mutations A784S (P04603) can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 7. In some aspects, a Casl3d variant having mutations A784S can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 8.

[0072] In some aspects, a Cast 3d seq 212 sequencing having a Za ADAR domain tethered between L228 and K229 (P04617) can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 9. In some aspects, a Cast 3d seq 212 sequencing having a Za ADAR domain tethered between L228 and K229 can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 10.

[0073] In some aspects, a Cast 3d seq 212 sequencing having a Zb ADAR domain tethered between L228 and K229 (P04621) can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 11. In some aspects, a Cast 3d seq 212 sequencing having a Zb ADAR domain tethered between L228 and K229 can be encoded a nucleic acid sequence that can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 12.

Guide RNAs for RNA-Guided RNA-Binding Proteins

[0074] The terms guide RNA (gRNA) and single guide RNA (sgRNA) are used interchangeably throughout the disclosure.

[0075] Guide RNAs (gRNAs) of the disclosure may comprise of a “direct repeat” (DR) sequence and a spacer sequence. In some embodiments, a guide RNA is a single guide RNA (sgRNA) comprising a contiguous DR sequence and spacer sequence. In some embodiments, the spacer sequence and the DR sequence are not contiguous. In some embodiments, the gRNA comprises a DR sequence. DR sequences refer to the repetitive sequences in the CRISPR locus (naturally-occurring in a bacterial genome or plasmid) that are interspersed with the spacer sequences. It is well known that one would be able to infer the DR sequence of a corresponding (or cognate) Cas protein if the sequence of the associated CRISPR locus is known. In some embodiments, a guide RNA comprises a direct repeat (DR) sequence and a spacer sequence. In some embodiments, a sequence encoding a guide RNA or single guide RNA of the disclosure comprises or consists of a spacer sequence and a DR sequence, that are separated by a linker sequence. In some embodiments, the linker sequence may comprise or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any number of nucleotides (nt) in between. In some embodiments, the linker sequence may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any number of nucleotides in between. In some embodiments, the DR sequence is a Casl3d DR sequence.

[0076] In one embodiment, the gRNA that hybridizes with the one or more target RNA molecules in a Casl3d-mediated manner includes one or more direct repeat (DR) sequences, one or more spacer sequences, such as, e.g., one or more sequences comprising an array of DR- spacer-DR-spacer and so on. In one embodiment, a plurality of gRNAs are generated from a single array, wherein each gRNA can be different, for example target different RNAs or target multiple regions of a single RNA, or combinations thereof. In some embodiments, an isolated gRNA includes one or more direct repeat sequences, such as an unprocessed (e.g., about 36 nt) or processed DR (e.g., about 30 nt). In some embodiments, a gRNA can further include one or more spacer sequences specific for (e.g., is complementary to) the target RNA. In certain such embodiments, multiple polIII promoters can be used to drive multiple gRNAs, spacers and/or DRs. In one embodiment, a guide array comprises a DR (about 36nt)-spacer (about 30nt)-DR (about 36nt)-spacer (about 30nt).

[0077] Guide RNAs (gRNAs) of the disclosure may comprise non-naturally occurring nucleotides. In some embodiments, a guide RNA of the disclosure or a sequence encoding the guide RNA comprises or consists of modified or synthetic RNA nucleotides. Exemplary modified RNA nucleotides include, but are not limited to, pseudouridine ( ), dihydrouridine (D), inosine (I), and 7-m ethylguanosine (m7G), hypoxanthine, xanthine, xanthosine, 7- methylguanine, 5, 6-Dihydrouracil, 5-methylcytosine, 5-methylcytidine, 5- hydropxymethylcytosine, isoguanine, and isocytosine.

[0078] Guide RNAs (gRNAs) of the disclosure may bind modified RNA within a target sequence. Within a target sequence, guide RNAs (gRNAs) of the disclosure may bind modified or mutated (e.g., pathogenic) RNA. Exemplary epigenetically or post-transcriptionally modified RNA include, but are not limited to, 2’-O-Methylation (2’-0Me) (2’-O-methylation occurs on the oxygen of the free 2’-OH of the ribose moiety), N6-methyladenosine (m6A), and 5-methylcytosine (m5C).

[0079] In some embodiments of the compositions of the disclosure, a guide RNA of the disclosure comprises at least one sequence encoding a non-coding C/D box small nucleolar RNA (snoRNA) sequence. In some embodiments, the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the target sequence of the RNA molecule comprises at least one 2’-OMe. In some embodiments, the snoRNA sequence comprises at least one sequence that is complementary to the target RNA, wherein the at least one sequence that is complementary to the target RNA comprises a box C motif (RUGAUGA) and a box D motif (CUGA).

[0080] Spacer sequences of the disclosure bind to the target sequence of an RNA molecule. In some embodiments, spacer sequences of the disclosure bind to pathogenic target RNA.

[0081] In some embodiments of the compositions of the disclosure, the sequence comprising the gRNA further comprises a spacer sequence that specifically binds to the target RNA sequence. In some embodiments, the spacer sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 87%, 90%, 95%, 97%, 99% or any percentage in between of complementarity to the target RNA sequence. In some embodiments, the spacer sequence has 100% complementarity to the target RNA sequence. In some embodiments, the spacer sequence comprises or consists of 20 nucleotides. In some embodiments, the spacer sequence comprises or consists of 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucloetides, 36 nucleotides, 37 nucleotdes, 38 nucleotides, 39 nucleotides, or 40 nucleotides. In some embodiments, the spacer sequence comprises or consists of 26 nucleotides. In some embodiments, the spacer sequence is nonprocessed and comprises or consists of 30 nucleotides. In some embodiments the nonprocessed spacer sequence comprises or consists of 30-36 nucleotides.

[0082] DR sequences of the disclosure bind the Cas polypeptide of the disclosure. Upon binding of the spacer sequence of the gRNA to the target RNA sequence, the Cas protein bound to the DR sequence of the gRNA is positioned at the target RNA sequence. A DR sequence binds to the Cas protein via a nucleic acid - amino acid interaction, typically through hydrogen bonds and salt bridges. DR sequences disclosed herein have a sequence % identity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96, 97%, 98%, 99%, or any percentage identity in between. In some embodiments, a DR sequence disclosed herein has 100% sequence identity. In some embodiments, DR sequences of the disclosure comprise a secondary structure or a tertiary structure. Exemplary secondary structures include, but are not limited to, a helix, a stem loop, a bulge, a tetraloop and a pseudoknot. Exemplary tertiary structures include, but are not limited to, an A-form of a helix, a B-form of a helix, and a Z- form of a helix. Exemplary tertiary structures include, but are not limited to, a twisted or helicized stem loop. Exemplary tertiary structures include, but are not limited to, a twisted or helicized pseudoknot. In some embodiments, DR sequences of the disclosure comprise at least one secondary structure or at least one tertiary structure. In some embodiments, DR sequences of the disclosure comprise one or more secondary structure(s) or one or more tertiary structure(s).

[0083] In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof selectively binds to a tetraloop motif in an RNA molecule of the disclosure. In some embodiments, a target sequence of an RNA molecule comprises a tetraloop motif. In some embodiments, the tetraloop motif is a “GRNA” motif comprising or consisting of one or more of the sequences of GAAA, GUGA, GCAA or GAGA.

[0084] In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof that binds to a target sequence of an RNA molecule hybridizes to the target sequence of the RNA molecule. In some embodiments, a guide RNA or a portion thereof that binds to a first RNA binding protein or to a second RNA binding protein covalently binds to the first RNA binding protein or to the second RNA binding protein. In some embodiments, a guide RNA or a portion thereof that binds to a first RNA binding protein or to a second RNA binding protein non-covalently binds to the first RNA binding protein or to the second RNA binding protein.

[0085] In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof comprises or consists of between 10 and 100 nucleotides, inclusive of the endpoints. In some embodiments, a spacer sequence of the disclosure comprises or consists of between 10 and 30 nucleotides, inclusive of the endpoints. In some embodiments, a spacer sequence of the disclosure comprises or consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In some embodiments, the spacer sequence of the disclosure comprises or consists of 20 nucleotides. In some embodiments, the spacer sequence of the disclosure comprises or consists of 21 nucleotides. In some embodiments, the spacer sequence of the disclosure comprises or consists of 26 nucleotides.

[0086] Guide molecules generally exist in various states of processing. In one example, an unprocessed guide RNA is 36nt of DR followed by 30-32 nt of spacer. The guide RNA is processed (truncated/modified) by Cas 13d itself or other RNases into the shorter "mature" form. In some embodiments, an unprocessed guide sequence is about, or at least about 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 or more nucleotides (nt) in length. In some embodiments, an unprocessed guide sequence is 102 nt in length, e.g., DR 36 nt, spacer 30 nt and DR is 36 nt. In some embodiments, a processed guide sequence is about 44 to 70 nt (such as 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 nt). In some embodiments, an unprocessed spacer is about 28-40 nt long (such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nt) while the mature (processed) spacer can be about 10 to 30 nt, 10 to 25 nt, 14 to 25 nt, 20 to 22 nt, or 14-30 nt (such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nt). In some embodiments, an unprocessed DR is about 36 nt (such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41 nt), while the processed DR is about 30 nt (such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nt). In some embodiments, a DR sequence is truncated by 1-10 nucleotides (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, to 10 nucleotides at e.g., the 5’ end in order to be expressed as mature pre-processed guide RNAs.

[0087] In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof does not comprise a nuclear localization sequence (NLS).

[0088] In some embodiments of the compositions of the disclosure, a guide RNA or a portion thereof comprises a sequence complementary to a protospacer flanking sequence (PFS). In some embodiments, including those wherein a guide RNA or a portion thereof comprises a sequence complementary to a PFS, the first RNA binding protein may comprise a sequence isolated or derived from a Cas 13 protein. In some embodiments, including those wherein a guide RNA or a portion thereof comprises a sequence complementary to a PFS, the first RNA binding protein may comprise a sequence encoding a Cas 13 protein or an RNA-binding portion thereof. In some embodiments, the guide RNA or a portion thereof does not comprise a sequence complementary to a PFS.

[0089] In other embodiments, therapeutic targeting of multiple HRE targets, e.g., without limitation, sense and antisense HREs and flanking regions of the sense and/or antisense HREs are disclosed. Such multiple targets can be GGGGCC (G4C2), CCCCGG (C4G2), flanking sequences adjacent to GGGGCC (G4C2), or flanking sequences adjacent to CCCCGG (C4G2). [0090] In one embodiment, compositions and vectors are disclosed comprising two guide RNA molecules comprising spacer sequences binding distinct target RNA sequences. In one aspect, the first spacer sequence binds a G4C2 (sense) target RNA sequence and the second spacer sequence binds a sense flanking sequence. In one aspect, the first spacer sequence binds a C4G2 (antisense) target RNA sequence and the second spacer sequence binds an antisense flanking sequence.

[0091] In one such embodiment, two targets are selected from the group consisting of GGGGCC (G4C2) (sense), flanking sequences adjacent to GGGGCC (G4C2) (sense), and flanking sequences adjacent to CCCCGG (C4G2) (antisense). In one embodiments, two targets are selected from the group consisting of CCCCGG (C4G2) (antisense), flanking sequences adjacent to CCCCGG (C4G2) (antisense), and flanking sequences adjacent to GGGGCC (G4C2).

[0092] In one such embodiment, three targets that are selected from the group consisting of GGGGCC (G4C2), flanking sequences adjacent to GGGGCC (G4C2), and flanking sequences adjacent to CCCCGG (C4G2); or three targets selected from the group consisting of CCCCGG (C4G2), flanking sequences adjacent to CCCCGG (C4G2), and flanking sequences adjacent to GGGGCC (G4C2) are disclosed. In some embodiments, target sequences can comprise intronic or exonic sequences. In some embodiments, the intronic or exonic sequence can flank an HRE target sequence of the disclosure.

[0093] Exemplary G4C2 spacers for gRNA constructs disclosed herein are:

Table 1 : G4C2 spacers for gRNA constructs disclosed herein

[0094] Exemplary C4G2 spacers for gRNA constructs disclosed herein are:

Table 2: C4G2 spacers for gRNA constructs

[0095] Exemplary spacers for gRNAs comprising C9 C4G2 flanking regions are:

Table 3: Exemplary spacers for gRNAs comprising C9 C4G2 flanking regions

[0096] Exemplary guide array sequences targeting the sense intronic flanking region are:

Table 4: Exemplary guide array sequences targeting the sense intronic flanking region

[0097] The disclosure provides guide array nucleic acid sequences. In some aspects, a guide array comprises at least two guide RNA sequences. In some aspects, the at least two guide RNA sequences bind non-overlapping target RNA sequences. In some aspects, guide arrays of the disclosure comprise at least three guide RNA sequences. In some aspects, guide arrays of the disclosure comprise one gRNA that binds a sense strand and a second gRNA that binds an anti-sense strand. In some aspects, guide arrays of the disclosure comprise one gRNA that binds a sense strand and a second gRNA that also binds a sense strand. In some aspects, guide arrays of the disclosure comprise one gRNA that binds an anti-sense strand and a second gRNA that binds an anti-sense strand. Guide arrays of the disclosure can be delivered to a cell or subject as part of an AAV vector of the disclosure. In some aspects, following delivery to the subject or cell, the guide array is processed to yield individual gRNA sequences.

[0098] Guide arrays of the disclosure can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of SEQ ID NO: 132 - SEQ ID NO: 143. In some aspects, an AAV vector of the disclosure comprises a guide array that can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of SEQ ID NO: 132 - SEQ ID NO: 143.

[0099] Exemplary guide arrays (i.e. gRNA sequences) disclosed herein are:

Table 5: Exemplary guide arrays gRNA Target Sequences

[00100] The compositions of the disclosure bind and destroy a target sequence of an RNA molecule comprising a pathogenic repeat sequence. In one embodiment, the target RNA comprises a sequence motif corresponding to or complementary to a spacer sequence of the guide RNA corresponding to the RNA-guided RNA-binding protein. In some embodiments, one or more spacer sequences are used to target one or more target sequences. In some embodiments, multiple spacers are used to target multiple target RNAs. Such target RNAs can be different target sites within the same RNA molecule or can be different target sites within different RNA molecules. Spacer sequences can also target non-coding RNA. In some embodiments, multiple promoters, e.g., Pol III promoters can be used to drive multiple spacers in a gRNA for targeting multiple target RNAs. In one embodiment, the destruction or blocking of the target RNA(s) or target sequence motif(s) reduces expression of pathogenic HRE repeat RNA. In another embodiment, the destruction or blocking of the target repeat RNA(s) or target sequence motif(s) reduces expression of pathogenic HRE repeat RNA (C4G2 and/or G4C2, and/or flanking sequences thereof) thereby treating C9ORF72 disease such as ALS or FTD and/or ameliorating one or more symptoms associated with the diseases.

[00101] In some embodiments of the compositions and methods of the disclosure, the sequence motif of the target RNA is a signature of a disease or disorder.

[00102] A sequence motif of the disclosure may be isolated or derived from a sequence of foreign or exogenous sequence found in a genomic sequence, and therefore translated into an mRNA molecule of the disclosure or a sequence of foreign or exogenous sequence found in an RNA sequence of the disclosure.

[00103] A target sequence motif of the disclosure may comprise or consist of a repeated sequence. In some embodiments, the repeated sequence may be associated with a microsatellite instability (MSI). MSI at one or more loci results from impaired DNA mismatch repair mechanisms of a cell of the disclosure. A hypervariable sequence of DNA may be transcribed into an mRNA of the disclosure comprising a target sequence comprising or consisting of the hypervariable sequence.

[00104] A target sequence motif of the disclosure may comprise or consist of a biomarker. The biomarker may indicate a risk of developing a disease or disorder. The biomarker may indicate a healthy gene (and a low or no determinable risk of developing a disease or disorder). The biomarker may indicate an edited gene. Exemplary biomarkers include, but are not limited to, single nucleotide polymorphisms (SNPs), sequence variations or mutations, epigenetic marks, splice acceptor sites, exogenous sequences, heterologous sequences, and any combination thereof.

[00105] A target sequence motif of the disclosure may comprise or consist of a secondary, tertiary or quaternary structure. The secondary, tertiary or quaternary structure may be endogenous or naturally occurring. The secondary, tertiary or quaternary structure may be induced or non-naturally occurring. The secondary, tertiary or quaternary structure may be encoded by an endogenous, exogenous, or heterologous sequence.

[00106] In some embodiments of the compositions and methods of the disclosure, a target sequence of an RNA molecule comprises or consists of between 2 and 100 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of between 2 and 50 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of between 2 and 20 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of between 20-30 nucleotides or nucleic acid bases, inclusive of the endpoints. In some embodiments, the target sequence of an RNA molecule comprises or consists of about 26 nucleotides or nucleic acid bases, inclusive of the endpoints.

[00107] In some embodiments of the compositions and methods of the disclosure, a target sequence of an RNA molecule is continuous. In some embodiments, the target sequence of an RNA molecule is discontinuous. For example, the target sequence of an RNA molecule may comprise or consist of one or more nucleotides or nucleic acid bases that are not contiguous because one or more intermittent nucleotides are positioned in between the nucleotides of the target sequence.

[00108] In some embodiments of the compositions and methods of the disclosure, a target sequence of an RNA molecule is naturally occurring. In some embodiments, the target sequence of an RNA molecule is non-naturally occurring. Exemplary non-naturally occurring target sequences may comprise or consist of sequence variations or mutations, chimeric sequences, exogenous sequences, heterologous sequences, chimeric sequences, recombinant sequences, sequences comprising a modified or synthetic nucleotide or any combination thereof.

[00109] In some embodiments of the compositions and methods of the disclosure, a target sequence of an RNA molecule binds to a guide RNA of the disclosure. In some embodiments of the compositions and methods of the disclosure, one or more target sequences of an RNA molecule binds to one or more guide RNA spacer sequences of the disclosure.

[00110] In some embodiments of the compositions and methods of the disclosure, a target sequence of an RNA molecule binds to a first RNA binding protein of the disclosure.

[00111] In some embodiments of the compositions and methods of the disclosure, a target sequence of an RNA molecule binds to a second RNA binding protein of the disclosure.

[00112] Compositions of the disclosure comprise a gRNA comprising a spacer sequence that specifically binds to a target toxic RNA repeat sequence. In some embodiments, the spacer which binds the target RNA repeat sequence comprises or consists of about 20-30 nucleotides. In some embodiments, a gRNA comprises one or more spacer sequences.

AAV vectors [00113] An "AAV vector" as used herein refers to a vector comprising, consisting essentially of, or consisting of one or more nucleic acid molecules and one or more AAV inverted terminal repeat sequences (ITRs). In some aspects, the nucleic acid molecule encodes for a repeat targeting protein and/or composition of the disclosure. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that provides the functionality of rep and cap gene products, for example, by transfection of the host cell. In some aspects, AAV vectors contain a promoter, at least one nucleic acid that may encode at least one protein or RNA, and/or an enhancer and/or a terminator within the flanking ITRs that is packaged into the infectious AAV particle. The encapsidated nucleic acid portion may be referred to as the AAV vector genome. Plasmids containing AAV vectors may also contain elements for manufacturing purposes, e.g., antibiotic resistance genes, origin of replication sequences etc., but these are not encapsidated and thus do not form part of the AAV particle.

[00114] In some aspects, an AAV vector can comprise at least one nucleic acid molecule encoding a repeat targeting composition of the disclosure. In some aspects, an AAV vector can comprise at least one regulatory sequence. In some aspects, an AAV vector can comprise at least one AAV inverted terminal (ITR) sequence. In some aspects, an AAV vector can comprise a first ITR sequence and a second ITR sequence.

[00115] In some aspects, a first ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 149 or SEQ ID NO: 150. In some aspects, a second ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 149 or SEQ ID NO: 150.

[00116] In some aspects, an AAV vector can comprise at least one promoter sequence. In some aspects, an AAV vector can comprise at least one enhancer sequence. In some aspects, an AAV vector can comprise at least one polyA sequence. In some aspects, an AAV vector can comprise at least one linker sequence. In some aspects, an AAV vector of the disclosure can comprise at least one nuclear localization signal, or nuclear export signal and or both.

[00117] In some aspects, an AAV vector of the disclosure will comprise a WPRE (woodchuck hepatitis virus post-trancriptional regulatory element) or portion thereof. In some aspects, a WPRE sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 151 or SEQ ID NO: 191. [00118] In some aspects, an AAV vector of the disclosure can comprise a Cas protein, peptide, or fragment thereof.

[00119] In some aspects, an AAV vector of the disclosure can comprise a polyadenylation (poly A) sequence. In some aspects, a polyA sequence can comprise an SV- 40 polyA sequence

[00120] In some aspects, an SV-40 polyA sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 152. [00121] In some aspects, an AAV vector of the disclosure can comprise an endonuclease protein, peptide, or fragment thereof. In some aspects, an AAV vector of the disclosure can comprise a guide RNA, in some cases a repeat targeting guide RNA. In some aspects, AAV vectors of the disclosure can comprise a fusion protein comprising one or more elements of the disclosure, including, but not limited to, an RNA-targeting protein (such as a Cas, PUF, or PUMBY) and an endonuclease. Optionally, fusion proteins of the AAV vector can further comprise a linker amino acid sequence between the one or more elements of the disclosure.

[00122] In some aspects, a AAV vector can comprise a first AAV ITR sequence, a promoter sequence, a RNA-targeting composition nucleic acid molecule, a regulatory sequence and a second AAV ITR sequence. In some aspects, an AAV vector can comprise, in the 5’ to 3’ direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, and a second AAV ITR sequence.

[00123] In some embodiments of the compositions and methods of the disclosure, a vector comprises a guide RNA of the disclosure. In some embodiments, the vector comprises at least one guide RNA of the disclosure. In some embodiments, the vector comprises one or more guide RNA(s) of the disclosure. In some embodiments, the vector comprises two or more guide RNAs of the disclosure. In one embodiment, the vector comprises three guide RNAs. In one embodiment, the vector comprises four guide RNAs. In some embodiments, the vector comprises a PolIII promoter, one or multiple guides, a PolII promoter, the Cas protein, a regulatory element and a polyA sequence. In some embodiments, the vector further comprises a guided or non-guided RNA-binding protein of the disclosure. In some embodiments, the vector further comprises an RNA-binding fusion protein of the disclosure. In some embodiments, the fusion protein comprises a first RNA binding protein and a second RNA binding protein. In some embodiments, the RNA-guided RNA-binding systems comprising an RNA-binding protein and a gRNA are in a single vector. In a particular embodiment, the single vector comprises the RNA-guided RNA-binding systems which are Casl3d RNA-guided RNA-binding systems or catalytic deactivated Cast 3d (dCasl3d) RNA-guided RNA-binding systems. In one embodiment, the single vector comprises the Casl3d RNA-guided RNA- binding systems which are CasRx or dCasRx RNA-guided RNA-binding systems. In another embodiment, the single vector comprises the Casl3d RNA-guided RNA-binding systems which are Seq212 Cast 3d or Seq212 dCasl3d or Seq212 Cast 3d variants RNA-guided RNA- binding systems. In another embodiment, the single vector comprises a non-guided RNA- binding system comprising a PUF or PUMBY-based protein. In another embodiment, the single vector comprises a non-guided RNA-binding system comprising a PUF or PUMBY- based protein fused with a nuclease domain from ZC3H12A, such as E17 (SEQ ID NO: 358). In another embodiment, the single vector comprises a dCasl3d RNA-binding system fused with a nuclease domain from ZC3H12A, such as E17 (SEQ ID NO: 359).

[00124] In some aspects, the nuclease domain from ZC3H12A, E17 can comprise, consist essentially of, or consist of the amino acid sequence:

GGGTPKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGILLAVNWFL ERGHTDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFTPSRRVGGKRVVCYDD RFIVKLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGR HGPSLDNFLRKKPLTLE (SEQ ID NO: 144).

[00125] In some aspects, the nuclease domain from ZC3H12A, E17 can comprise, consist essentially of, or consist of the amino acid sequence:

SGPCGEKPVLEASPTMSLWEFEDSHSRQGTPRPGQELAAEEASALELQMKVDFFRKL GYSSTEIHSVLQKLGVQADTNTVLGELVKHGTATERERQTSPDPCPQLPLVPRGGGT PKAPNLEPPLPEEEKEGSDLRPVVIDGSNVAMSHGNKEVFSCRGILLAVNWFLERGH TDITVFVPSWRKEQPRPDVPITDQHILRELEKKKILVFTPSRRVGGKRVVCYDDRFIV KLAYESDGIVVSNDTYRDLQGERQEWKRFIEERLLMYSFVNDKFMPPDDPLGRHGP SLDNFLRKKPLTLEHRKQPCPYGRKCTYGIKCRFFHPERPSCPQRSVADELRANALLS PPRAPSKDKNGRRPSPSSQSSSLLTESEQCSLDGKKLGAQASPGSRQEGLTQTYAPSG RSLAPSGGSGSSFGPTDWLPQTLDSLPYVSQDCLDSGIGSLESQMSELWGVRGGGPG EPGPPRAP YTGYSP YGSELP ATAAF S AFGRAMGAGHF S VP AD YPP APP AFPPREYWS EPYPLPPPTSVLQEPPVQSPGAGRSPWGRAGSLAKEQASVYTKLCGVFPPHLVEAVM GRFPQLLDPQQLAAEILSYKSQHPSE (SEQ ID NO: 145).

[00126] In some embodiments of the compositions and methods of the disclosure, a first vector comprises a guide RNA of the disclosure and a second vector comprises an RNA- binding protein or RNA-binding fusion protein of the disclosure. In some embodiments, the first vector comprises at least one guide RNA of the disclosure. In some embodiments, the first vector comprises one or more guide RNA(s) of the disclosure. In some embodiments, the first vector comprises two or more guide RNA(s) of the disclosure. In some embodiments, the fusion protein comprises a first RNA binding protein and a second RNA binding protein. In some embodiments, the first vector and the second vector are identical vectors or vector serotypes. In some embodiments, the first vector and the second vector are not identical vectors or vector serotypes. In some embodiments of the compositions and methods of the disclosure, the RNA-binding systems capable of targeting toxic RNA repeats are in a single vector.

[00127] One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally -derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno- associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. In some embodiments, the vector is a lentivirus (such as an integration-deficient lentiviral vector) or adeno-associated viral (AAV) vector. Vectors are capable of autonomous replication in a host cell into which they are introduced such as e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors and other vectors such as, e.g., non-episomal mammalian vectors, are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[00128] In some embodiments, vectors such as e.g., expression vectors, are capable of directing the expression of genes to which they are operatively-linked. Common expression vectors are often in the form of plasmids. In some embodiments, recombinant expression vectors comprise a nucleic acid provided herein such as e.g., a guide RNA which can be expressed from a DNA sequence, and a nucleic acid encoding a Cas 13d protein, in a form suitable for expression of a protein in a host cell. Recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence such as e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell. Certain embodiments of a vector depend on factors such as the choice of the host cell to be transformed, and the level of expression desired. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein such as, e.g., CRISPR transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.

[00129] In some embodiments of the compositions and methods of the disclosure, a vector of the disclosure is a viral vector. In some embodiments, the viral vector comprises a sequence isolated or derived from a retrovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is replication incompetent. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self- complementary.

[00130] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169- 228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized after the publication dates of the reviews because it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1- 61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. [00131] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA to generate AAV vectors. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[00132] Recombinant AAV (rAAV) genomes of the invention comprise, consist essentially of, or consist of a nucleic acid molecule encoding a repeat targeting composition (such as a PUF, PUMB Y, or RNA-guided protein) and one or more AAV ITRs flanking the nucleic acid molecule. Production of pseudotyped rAAV is disclosed in, for example, W02001083692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, e.g., Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[00133] In some embodiments of the compositions and methods of the disclosure, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector comprises an inverted terminal repeat sequence or a capsid sequence that is isolated or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAVrhlO, AAV11, AAV12, AAVrhlO, or AAVrh74. In some embodiments, the AAV serotype is AAVrh74. In some embodiments, the AAV serotype is AAV9. In one embodiment, the AAV vector comprises a modified capsid. In one embodiment the AAV vector is an AAV2-Tyr mutant vector. In one embodiment the AAV vector comprises a capsid with a non-tyrosine amino acid at a position that corresponds to a surface-exposed tyrosine residue in position Tyr252, Tyr272, Tyr275, Tyr281, Tyr508, Tyr612, Tyr704, Tyr720, Tyr730 or Tyr673 of wild-type AAV2. See also WO 2008/124724 incorporated herein in its entirety. In some embodiments, the AAV vector comprises an engineered capsid. AAV vectors comprising engineered capsids include without limitation, AAV2.7m8, AAV9.7m8, AAV2 2tYF, and AAV8 Y733F). In some embodiments, the viral vector is replication incompetent. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV). [00134] In some embodiments of the compositions and methods of the disclosure, a vector of the disclosure is a non-viral vector. In some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer. In some embodiments, the vector is an expression vector or recombinant expression system. As used herein, the term “recombinant expression system” refers to a genetic construct for the expression of certain genetic material formed by recombination.

[00135] In some embodiments of the compositions and methods of the disclosure, an expression vector, viral vector or non-viral vector provided herein, includes without limitation, an expression control element. An “expression control element” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene. Exemplary expression control elements include but are not limited to promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, introns, and 5’ and 3’ UTR sequences (to provide RNA stability and/or improve transcription/ translation or promote RNA nuclear export). Expression control elements may be constitutive, inducible, repressible, or tissue-specific, for example. A “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. In some embodiments, expression control by a promoter is tissue-specific. In some embodiments, expression control by a promoter is constitutive or ubiquitous. Non-limiting exemplary promoters include a Pol III promoter such as, e.g., U6 and Hl promoters and/or a Pol II promoter e.g., SV40, CMV (optionally including the CMV enhancer), RSV (Rous Sarcoma Virus LTR promoter (optionally including RSV enhancer), CBA (hybrid CMV enhancer/ chicken B-actin), CAG (hybrid CMV enhancer fused to chicken B-actin), truncated CAG, Cbh (hybrid CBA), EF-la (human elongation factor alpha- 1) or EFS (short intron-less EF-1 alpha), PGK (phosphoglycerol kinase), CEF (chicken embryo fibroblasts), UBC (ubiquitinC), GUSB (lysosomal enzyme beta-glucuronidase), UCOE (ubiquitous chromatin opening element), hAAT (alpha- 1 antitrypsin), TBG (thyroxine binding globulin), Desmin (full-length (SEQ ID NO: 655)or truncated (SEQ ID NO: 656)), MCK (muscle creatine kinase), C5-12 (synthetic muscle promoter), CK8e (creatin kinase 8), NSE (neuron-specific enolase), Synapsin, Synapsin-1 (SYN-1), opsin, PDGF (platelet-derived growth factor), PDGF-A, MecP2 (methyl CpG-binding protein 2), CaMKII (Calcium/ Calmodulin-dependent protein kinase II), mGluR2 (metabotropic glutamate receptor 2), NFL (neurofilament light), NFH (neurofilament heavy), nP2, PPE (rat preproenkephalin), ENK (preproenkephalin), Preproenkephalin-neurofilament chimeric promoter, EAAT2 (glutamate transporter), GFAP (glial fibrillary acidic protein), MBP (myelin basic protein), human rhodopsin kinase promoter (hGRKl), B-actin promoter, dihydrofolate reductase promoter, MHCK7 (hybrid promoter of enhancer/ promoter regions of muscle creatine kinase and alpha myosin heavy-chain genes) and combinations thereof. An “enhancer” is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription. Non-limiting exemplary enhancers and posttranscriptional regulatory elements include the CMV enhancer, MCK enhancer, R-U5’ segment in LTR of HTLV-1, SV40 enhancer, the intron sequence between exons 2 and 3 of rabbit B-globin, and Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). In some embodiments an intron is used to enhance promoter activity such as a UBB intron. In some embodiments, the UBB intron is used with an EFS promoter.

[00136] In some aspects, a CMV enhancer/promoter sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 186.

[00137] In some aspects, an EFS promoter sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 155. [00138] In some embodiments of the compositions and methods of the disclosure, an expression vector, viral vector or non-viral vector provided herein, includes without limitation, vector elements such as an IRES or 2A peptide sites for configuration of “multicistronic” or “polycistronic” or “bicistronic” or tricistronic” constructs, i.e., having double or triple or multiple coding areas or exons, and as such will have the capability to express from mRNA two or more proteins from a single construct. Multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. The two strategies most widely used for constructing multicistronic configurations are through the use of an IRES or a 2A self-cleaving site. An “IRES” refers to an internal ribosome entry site or portion thereof of viral, prokaryotic, or eukaryotic origin which are used within polycistronic vector constructs. In some embodiments, an IRES is an RNA element that allows for translation initiation in a cap- independent manner. The term “self-cleaving peptides” or “sequences encoding self-cleaving peptides” or “2A self-cleaving site” refer to linking sequences which are used within vector constructs to incorporate sites to promote ribosomal skipping and thus to generate two polypeptides from a single promoter, such self-cleaving peptides include without limitation, T2A, and P2A peptides or other sequences encoding the self-cleaving peptides.

[00139] In one embodiment, exemplary vector configurations are shown in Figures 1A-1B. Exemplary vector configurations comprise a promoter or regulatory sequence (promoter/enhancer combination) driving the expression of the nucleic acid encoding the RNA- targeting Casl3d-based system. In another embodiment, a vector configuration comprises a promoter driving expression of the RNA-guided Cas RNase RNA-binding protein, or dCas protein fusion in operable linkage with a second promoter driving expressing of a cognate gRNA. In another embodiment, the vector configuration comprises a linker and one or more tags.

[00140] In some embodiments of the compositions of the disclosure, vectors comprising guide RNA sequences of the disclosure comprises a promoter sequence to drive expression of the guide RNA. In some embodiments, a vector comprising a guide RNA sequence of the disclosure comprises a promoter sequence to drive expression of the guide RNA. In some embodiments, the promoter to drive expression of the guide RNA is a constitutive promoter. In some embodiments, the promoter sequence is an inducible promoter. In some embodiments, the promoter is a sequence is a tissue-specific and/or cell-type specific promoter. In some embodiments, the promoter is a hybrid or a recombinant promoter. In some embodiments, the promoter is a promoter capable of expressing the guide RNA in a mammalian cell. In some embodiments, the promoter is a promoter capable of expressing the guide RNA in a human cell. In some embodiments, the promoter is a promoter capable of expressing the guide RNA and restricting the guide RNA to the nucleus of the cell. In some embodiments, the promoter is a human RNA polymerase promoter or a sequence isolated or derived from a sequence encoding a human RNA polymerase promoter. In some embodiments, the promoter is a U6 promoter or a sequence isolated or derived from a sequence encoding a U6 promoter. In some embodiments, the U6 promoter is a human or mouse U6 promoter. In some embodiments, the promoter is a human or mouse tRNA promoter or a sequence isolated or derived from a sequence encoding a human or mouse tRNA promoter. In some embodiments, the promoter is a human or mouse valine tRNA promoter or a sequence isolated or derived from a sequence encoding a human or mouse valine tRNA promoter. [00141] In some embodiments of the compositions of the disclosure, a promoter to drive expression of the guide RNA further comprises a regulatory element. In some embodiments, a vector comprising a promoter sequence to drive expression of the guide RNA further comprises a regulatory element. In some embodiments, a regulatory element enhances expression of the guide RNA. Exemplary regulatory elements include, but are not limited to, an enhancer element, an intron, an exon, or a combination thereof.

[00142] In some embodiments of the compositions of the disclosure, a vector of the disclosure comprises one or more of a sequence encoding a guide RNA, a promoter sequence to drive expression of the guide RNA and a sequence encoding a regulatory element. In some embodiments of the compositions of the disclosure, the vector further comprises a sequence encoding a fusion protein of the disclosure.

[00143] In some embodiments, the vector is a viral vector. In some embodiments, the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector. In some embodiments, the vector is a retroviral vector, an adenoviral/retroviral chimera vector, a herpes simplex viral I or II vector, a parvoviral vector, a reticuloendotheliosis viral vector, a polioviral vector, a papillomaviral vector, a vaccinia viral vector, or any hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors. In some embodiments, the vector further comprises one or more expression control elements operably linked to the polynucleotide. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the AAV vector has low toxicity. In some embodiments, the AAV vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis. In some embodiments, the AAV vector can encode a range of total polynucleotides from 4.5 kb to 4.75 kb. In some embodiments, exemplary AAV vectors that may be used in any of the herein described compositions, systems, methods, and kits can include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV2-Tyr mutant vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rhlO vector, a modified AAV.rhlO vector, an AAVrh.74, an AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64Rl vector, and a modified AAV.rh64Rl vector, an AAV-Tyr mutant vector, and any combinations or equivalents thereof. In some embodiments, the lentiviral vector is an integrase-competent lentiviral vector (ICLV). In some embodiments, the lentiviral vector can refer to the transgene plasmid vector as well as the transgene plasmid vector in conjunction with related plasmids (e.g., a packaging plasmid, a rev expressing plasmid, an envelope plasmid) as well as a lentiviral-based particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Lentiviral vectors are well-known in the art (see, e.g., Trono D. (2002) Lentiviral vectors, New York: Spring-Verlag Berlin Heidelberg and Durand et al. (2011) Viruses 3(2): 132-159 doi: 10.3390/v3020132). In some embodiments, exemplary lentiviral vectors that may be used in any of the herein described compositions, systems, methods, and kits can include a human immunodeficiency virus (HIV) 1 vector, a modified human immunodeficiency virus (HIV) 1 vector, a human immunodeficiency virus (HIV) 2 vector, a modified human immunodeficiency virus (HIV) 2 vector, a sooty mangabey simian immunodeficiency virus (SIVSM) vector, a modified sooty mangabey simian immunodeficiency virus (SIVSM) vector, a African green monkey simian immunodeficiency virus (SIVAGM) vector, a modified African green monkey simian immunodeficiency virus (SIVAGM) vector, an equine infectious anemia virus (EIAV) vector, a modified equine infectious anemia virus (EIAV) vector, a feline immunodeficiency virus (FIV) vector, a modified feline immunodeficiency virus (FIV) vector, a Visna/maedi virus (VNV/VMV) vector, a modified Visna/maedi virus (VNV/VMV) vector, a caprine arthritisencephalitis virus (CAEV) vector, a modified caprine arthritis-encephalitis virus (CAEV) vector, a bovine immunodeficiency virus (BIV), or a modified bovine immunodeficiency virus (BIV).

[00144] The disclosure provides adeno-associated virus (AAV) vectors comprising RNA- binding polypeptides capable of binding toxic hexanucleotide repeat RNA sequences. In some aspects, AAV vetors of the disclosure compise Casl3d constructs capable of binding tergat RNA sequences disclosed herein.

[00145] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ : a first ITR sequence, a first promoter sequence, at least one gRNA sequence, a second promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence, and a second ITR sequence.

[00146] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’: a first ITR sequence, a first promoter sequence, a first gRNA sequence, a second gRNA sequence, a second promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence, and a second ITR sequence.

[00147] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’: a first ITR sequence, a first promoter sequence, a first gRNA sequence, a second gRNA sequence, a third gRNA sequence, a second promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence, and a second ITR sequence.

[00148] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ : a first ITR sequence, a human U6 promoter sequence, at least one gRNA sequence, an EFS promoter sequence, a cas polypeptide, a WPRE sequence, an SV-40 polyA sequence, and a second ITR sequence.

[00149] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ : a first ITR sequence, a human U6 promoter sequence, a first gRNA sequence, a second gRNA sequence, an EFS promoter sequence, a cas polypeptide, a WPRE sequence, an SV-40 polyA sequence, and a second ITR sequence.

[00150] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ : a first ITR sequence, a human U6 promoter sequence, a first gRNA sequence, a second gRNA sequence, an EFS promoter sequence, a cas polypeptide, a linker sequence, an SV-40 nuclear localization sequence, a WPRE sequence, an SV-40 polyA sequence, and a second ITR sequence.

[00151] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ : a first ITR sequence, a first promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence, a second promoter sequence, at least one gRNA sequence, and a second ITR sequence.

[00152] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ : a first ITR sequence, a first promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence, a second promoter sequence, a first gRNA sequence, a second gRNA sequence and a second ITR sequence.

[00153] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3 ’a first promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence, a second promoter sequence, a first gRNA sequence, and a second gRNA sequence.

[00154] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ : a first ITR sequence, an EFS promoter sequence, a cas polypeptide, a linker sequence, an SV-40 nuclear localization sequence, a WPRE sequence, an SV-40 polyA sequence, a human U6 promoter sequence, a first gRNA sequence, a second gRNA sequence, and a second ITR sequence.

[00155] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3 ’a first promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence, a second promoter sequence, a first gRNA sequence, a second gRNA sequence, and a third gRNA sequence. [00156] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’ a first gRNA sequence, a second gRNA sequence, and a third gRNA sequence, a first promoter sequence (reverse strand), a second promoter sequence, a cas polypeptide, a posttranscriptional regulatory element sequence, a polyA sequence.

[00157] In some aspects, an AAV vector of the disclosure comprises from 5’ to 3’: a first ITR sequence, an EFS promoter sequence, a cas polypeptide, a linker sequence, an SV-40 nuclear localization sequence, a WPRE sequence, an SV-40 polyA sequence, a first gRNA sequence, a second gRNA sequence, a human U6 promoter sequence (reverse strand), and a second ITR sequence.

[00158] Exemplary Unitary AAV Casl3d-based constructs disclosed herein:

A02557: pAV-hU6 C9 C4G2 flanking g29 G4C2gl EFS_Seq212-WPRE3

[00159] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00160] AAV vector A02557 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02557 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 202.

[00161] Nucleotide sequences of A02557 elements in order from 5’ to 3’ A02558: pAV-hU6 G4C2gl C9 C4G2 flanking g29 EFS_Seq212-WPRE3

[00162] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00163] AAV vector A02558 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02558 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 203.

[00164] Nucleotide sequences of A02558 elements in order from 5’ to 3’

A02559: pAV-hU6 C9 C4G2 flanking g60 G4C2gl EFS_Seq212-WPRE3

[00165] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00166] AAV vector A02559 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02559 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 204.

[00167] Nucleotide sequences of A02559 elements in order from 5’ to 3’

A02560: pAV-hU6 G4C2gl C9 C4G2 flanking g60_ EFS_Seq212-WPRE3

[00168] Amino acid sequences of plasmid elements in order N-terminal to C-terminal

[00169] AAV vector A02560 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02560 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 205.

[00170] Nucleotide sequences of A02560 elements in order from 5’ to 3’

A02561: pAV-hU6 C9 C4G2 flanking g36 G4C2gl EFS_Seq212-WPRE3

[00171] Amino acid sequences of plasmid elements in order N-terminal to C-terminal

[00172] AAV vector A02561 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02561 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 206.

[00173] Nucleotide sequences of A02561 elements in order from 5’ to 3’

A02562: pAV-hU6 C9 C4G2 flanking g20 G4C2gl EFS_Seq212-WPRE3

[00174] Amino acid sequences of plasmid elements in order N-terminal to C-terminal

[00175] AAV vector A02562 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02562 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 207.

[00176] Nucleotide sequences of A02562 elements in order from 5’ to 3’

A02563: pAV-hU6 C9 C4G2 flanking g48 G4C2gl EFS_Seq212-WPRE3

[00177] Amino acid sequences of plasmid elements in order N-terminal to C-terminal

[00178] AAV vector A02563 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02563 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 208.

[00179] Nucleotide sequences of A02563 elements in order from 5’ to 3’

A02564: pAV-hU6 C9 C4G2 flanking g59 G4C2gl EFS_Seq212-WPRE3 [00180] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00181] AAV vector A02564 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02564 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 209.

[00182] Nucleotide sequences of A02564 elements in order from 5’ to 3’

A02565: pAV-hU6 C9 C4G2 flanking gl9 G4C2gl EFS_Seq212-WPRE3 [00183] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00184] AAV vector A02565 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02565 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 210.

[00185] Nucleotide sequences of A02565 elements in order from 5’ to 3’

A02566: pAV-hU6 C9 C4G2 flanking g24 G4C2gl EFS_Seq212-WPRE3 [00186] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00187] AAV vector A02566 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02566 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 211.

[00188] Nucleotide sequences of A02566 elements in order from 5’ to 3’

A02567: pAV-hU6 C9 C4G2 flanking g!2 G4C2gl EFS_Seq212-WPRE3 [00189] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00190] AAV vector A02567 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02567 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 212.

[00191] Nucleotide sequences of A02567 elements in order from 5’ to 3’

A02568: pAV-hU6 C9 C4G2 flanking g34 G4C2gl EFS_Seq212-WPRE3 [00192] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00193] AAV vector A02568 can comprise from 5’ to 3’ the elements set forth below.

AAV vector A02568 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 213.

[00194] Nucleotide sequences of A02568 elements in order from 5’ to 3’

[00195] In some aspects, the disclosure provides constructs having PolIII promoter and guide expression cassette orientated upstream or downstream to the Cas and on the sense or antisense strands. In some aspects, these can comprise constructs: P03189, P03190, and P03191.

P03189: pcDNA3.1_EFS_Seq212-WPRE3 hU6_ C9 C4G2 flanking_g20_G4C2gl

[00196] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00197] AAV vector P03189 can comprise from 5’ to 3’ the elements set forth below.

AAV vector P03189 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 214.

[00198] Nucleotide sequences of plasmid elements in order from 5’ to 3’

P03190: pcDNA3.1_hU6_ C9 C4G2 flanking_g20_G4C2gl (antisense strand)EFS_Seq212-

WPRE3 SV40PA

[00199] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00200] AAV vector P03190 can comprise from 5’ to 3’ the elements set forth below.

AAV vector P03190 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 215.

[00201] Nucleotide sequences of P03190 elements in order from 5’ to 3’

P03191: pcDNA3.1_EFS_Seq212-WPRE3_SV40PA_hU6_ C9 C4G2 flanking_g20_G4C2gl (reverse strand)

[00202] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

Plasmid Element Amino Acid

[00203] AAV vector P03191 can comprise from 5’ to 3’ the elements set forth below.

AAV vector P03191 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 216.

[00204] Nucleotide sequences of P03191 elements in order from 5’ to 3’

Promoter sequences

[00205] HRE targeting compositions and AAV vectors of the disclosure can comprise promoter sequences which regulate expression of RNA-targeting and/or binding polypeptides of the disclosure, such as Casl3d polypeptides, and guide RNA sequences of the disclosure.

[00206] In some aspects, vectors of the disclosure can comprise two Pol III promoters. Exemplary PolIII promoters can comprise, consist essentially of, or consist of a nucleic acid as set forth below.

[00207] In some aspects, a human U6 promoter can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 156.

[00208] In some aspects, a human U6 promoter (reverse strand) can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:

172.

[00209] In some aspects, a human U6 promoter with a point mutation can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:

173.

[00210] In some aspects, a mouse U6 promoter can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 174.

[00211] In some aspects, an Hl promoter can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 175.

[00212] In some aspects, a 7SK promoter can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 176.

[00213] In some aspects, a valine tRNA promoter can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 177.

[00214] Exemplary constructs using two such promoters are:

[00215] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00216] AAV vector P03235 can comprise from 5’ to 3’ the elements set forth below.

AAV vector P03235 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 217.

[00217] Nucleotide sequences of P03235 elements in order from 5’ to 3’

P03236: pcDNA3.1_hU6_G4C2gl _hU6 C9 C4G2 flanking_ g20_EFS_Seq212-

WPRE3 SV40PA

[00218] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00219] AAV vector P03236 can comprise from 5’ to 3’ the elements set forth below.

AAV vector P03236 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 218.

[00220] Nucleotide sequences of P03236 elements in order from 5’ to 3’

Lentiviral Vectors

[00221] In some aspects, the disclosure provides lentiviral vectors comprising RNA repeat targeting compositions of the disclosure. L04473: Seq212 with C-terminal NES and C-terminal NLS x2 for nucleus/cytoplasm shuttling

[00222] Amino acid sequences of plasmid elements in order N-terminal to C-terminal

[00223] Lentiviral AAV vector L04473 can comprise from 5’ to 3’ the elements set forth below. AAV vector L04473 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 219.

[00224] Nucleotide sequences of L04473 elements in order from 5’ to 3’

L04474: Seq212 with N-terminal NLS x2 and C-terminal NES for nucleus/cytoplasm shuttling [00225] Amino acid sequences of plasmid elements in order N-terminal to C- terminal

[00226] Lentiviral AAV vector L04474 can comprise from 5’ to 3’ the elements set forth below. AAV vector L04474 can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 220.

[00227] Nucleotide sequences of plasmid elements in order from 5’ to 3’

Nucleic Acids

[00228] Provided herein are the nucleic acid sequences encoding RNA-binding G4C2 repeattargeting systems disclosed herein for use in gene transfer and expression techniques described herein. It should be understood, although not always explicitly stated that the sequences provided herein can be used to provide the expression product as well as substantially identical sequences that produce a protein that has the same biological properties. These “biologically equivalent” or “biologically active” or “equivalent” polypeptides are encoded by equivalent polynucleotides as described herein. They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions. Specific polypeptide sequences are provided as examples of particular embodiments. Modifications to the sequences to amino acids with alternate amino acids that have similar charge. Additionally, an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement or in reference to a polypeptide, a polypeptide encoded by a polynucleotide that hybridizes to the reference encoding polynucleotide under stringent conditions or its complementary strand. Alternatively, an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.

[00229] The nucleic acid sequences (e.g., polynucleotide sequences) disclosed herein may be codon-optimized which is a technique well known in the art. In some embodiments disclosed herein, exemplary Cas sequences are codon optimized for expression in human cells. Codon optimization refers to the fact that different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. It is also possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in a particular cell type. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms. Based on the genetic code, nucleic acid sequences coding for, e.g., a Cas protein, can be generated. In some embodiments, such a sequence is optimized for expression in a host or target cell, such as a host cell used to express the Cas protein or a cell in which the disclosed methods are practiced (such as in a mammalian cell, e.g., a human cell). Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a Cas protein (such as one encoding a protein having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type protein) that takes advantage of the codon usage preferences of that particular species. For example, the Cas proteins disclosed herein can be designed to have codons that are preferentially used by a particular organism of interest. In one example, a Cas nucleic acid sequence is optimized for expression in human cells, such as one having at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% sequence identity to its corresponding wild-type or originating nucleic acid sequence. In some embodiments, an isolated nucleic acid molecule encoding at least one Cas protein (which can be part of a vector) includes at least one Cas protein coding sequence that is codon optimized for expression in a eukaryotic cell, or at least one Cas protein coding sequence codon optimized for expression in a human cell. In one embodiment, such a codon optimized Cas coding sequence has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating sequence. In another embodiment, a eukaryotic cell codon optimized nucleic acid sequence encodes a Cas protein having at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating protein. In another embodiment, a variety of clones containing functionally equivalent nucleic acids may be routinely generated, such as nucleic acids which differ in sequence but which encode the same Cas protein sequence. Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (see, for example, Stryer, 1988, Biochemistry, 3 ^rd Edition, W.H. 5 Freeman and Co., NY).

[00230] It will be understood that RNA sequences disclosed herein, such a s guide RNA sequences or target RNA sequences, can be depicted as DNA sequences. In such cases, a uracil (U) may be denoted as a threonine (T). In some aspects, an RNA sequence will be expressed as a DNA sequence encoding said RNA sequence.

[00231] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi -stranded complex, a single self- hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[00232] Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0. lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, O. lx SSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.

[00233] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.

Cells

[00234] In some embodiments of the compositions and methods of the disclosure, a cell of the disclosure is a prokaryotic cell.

[00235] In some embodiments of the compositions and methods of the disclosure, a cell of the disclosure is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bovine, murine, feline, equine, porcine, canine, simian, or human cell. In some embodiments, the cell is a non-human mammalian cell such as a non-human primate cell. [00236] In some embodiments, a cell of the disclosure is a somatic cell. In some embodiments, a cell of the disclosure is a germline cell. In some embodiments, a germline cell of the disclosure is not a human cell.

[00237] In some embodiments of the compositions and methods of the disclosure, a cell of the disclosure is a stem cell. In some embodiments, a cell of the disclosure is an embryonic stem cell. In some embodiments, an embryonic stem cell of the disclosure is not a human cell. In some embodiments, a cell of the disclosure is a multipotent stem cell or a pluripotent stem cell. In some embodiments, a cell of the disclosure is an adult stem cell. In some embodiments, a cell of the disclosure is an induced pluripotent stem cell (iPSC). In some embodiments, a cell of the disclosure is a hematopoietic stem cell (HSC).

[00238] In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is a neuronal cell. In one embodiment, a cell or cells of a patient treated with compositions disclosed herein include, without limitation, central nervous system (neurons), peripheral nervous system (neurons), peripheral motor neurons, and/or sensory neurons. In one embodiment, a neuronal cell is a glial cell.

[00239] In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is a fibroblast or an epithelial cell. In some embodiments, an epithelial cell of the disclosure forms a squamous cell epithelium, a cuboidal cell epithelium, a columnar cell epithelium, a stratified cell epithelium, a pseudostratified columnar cell epithelium or a transitional cell epithelium. In some embodiments, an epithelial cell of the disclosure forms a gland including, but not limited to, a pineal gland, a thymus gland, a pituitary gland, a thyroid gland, an adrenal gland, an apocrine gland, a holocrine gland, a merocrine gland, a serous gland, a mucous gland and a sebaceous gland. In some embodiments, an epithelial cell of the disclosure contacts an outer surface of an organ including, but not limited to, a lung, a spleen, a stomach, a pancreas, a bladder, an intestine, a kidney, a gallbladder, a liver, a larynx or a pharynx. In some embodiments, an epithelial cell of the disclosure contacts an outer surface of a blood vessel or a vein.

[00240] In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is a primary cell.

[00241] In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is a cultured cell.

[00242] In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is in vivo, in vitro, ex vivo or in situ. [00243] In some embodiments of the compositions and methods of the disclosure, a somatic cell of the disclosure is autologous or allogeneic.

Methods of Use

[00244] The disclosure provides a method of modifying level of expression of an RNA molecule of the disclosure or a protein encoded by the RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or RNA-binding fusion protein (or a portion thereof) to the RNA molecule.

[00245] The disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA- binding protein or the fusion protein (or a portion thereof) to the RNA molecule.

[00246] The disclosure provides a method of modifying level of expression of an RNA molecule of the disclosure or a protein encoded by the RNA molecule comprising contacting the composition of the disclosure and a cell comprising the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or fusion protein (or a portion thereof) to the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In some embodiments, the composition of the disclosure comprises a vector comprising a guide RNA of the disclosure and an RNA-binding protein or fusion protein of the disclosure. In some embodiments, the vector is an AAV.

[00247] The disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and a cell comprising the RNA molecule under conditions suitable for binding of one or more of the guide RNA or the RNA-binding protein or fusion protein (or a portion thereof) to the RNA molecule. [00248] The disclosure provides a method of modifying the level of expression of an RNA molecule of the disclosure or a protein encoded by the RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for RNA nuclease activity wherein the RNA-binding protein or fusion protein induces a break in the RNA molecule.

[00249] The disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition of the disclosure and the RNA molecule under conditions suitable for RNA nuclease activity wherein the RNA-binding protein or fusion protein induces a break in the RNA molecule. [00250] The disclosure provides a method of modifying a level of expression of an RNA molecule of the disclosure or a protein encoded by the RNA molecule comprising contacting the composition of the disclosure and a cell comprising the RNA molecule under conditions suitable for RNA nuclease activity wherein the RNA-binding protein or fusion protein induces a break in the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In some embodiments, the composition comprises a vector comprising composition comprising a guide RNA of the disclosure and an RNA-binding fusion protein of the disclosure. In some embodiments, the vector is an AAV.

[00251] The disclosure provides a method of modifying an activity of a protein encoded by an RNA molecule comprising contacting the composition and a cell comprising the RNA molecule under conditions suitable for RNA nuclease activity wherein the RNA-binding protein or fusion protein induces a break in the RNA molecule. In some embodiments, the cell is in vivo, in vitro, ex vivo or in situ. In some embodiments, the composition comprises a vector comprising composition comprising a guide RNA or a single guide RNA of the disclosure and a nucleic acid sequence encoding an RNA-binding protein or fusion protein of the disclosure. In some embodiments, the vector is an AAV.

[00252] The disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of a composition of the disclosure. In one embodiment, the disclosure provides a method of treating toxic hexanucleotide repeat diseases. In another embodiment, the repeat disorder is ALS and FTD.

[00253] The disclosure provides a method of treating a repeat disease, wherein the composition modifies, reduces, destroys, knocks down, blocks or ablates a level of expression of a toxic repeat RNA (compared to the level of expression of a toxic repeat RNA treated with a non-targeting (NT) control or compared to no treatment). In another embodiment, the level of reduction is 1-fold or greater. In another embodiment, the level of reduction is 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold. In another embodiment, the level of reduction is 10-fold or greater. In another embodiment, the level of reduction is between 10- fold and 20-fold. In another embodiment, the level of reduction is 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold. In another embodiment, the gene therapy compositions disclosed herein when administered to a patient lead to 20%-100% destruction of the toxic G4C2 repeat RNA. In one embodiment, the % elimination of the toxic repeat RNA is any of 20-99%, 25%-99%, 50%-99%, 80%-99%, 90%-99%, 95%-99%. In one embodiment, the % elimination is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In another embodiment, % elimination is complete elimination or 100% elimination of the toxic repeat RNA.

[00254] In some embodiments of the methods of the disclosure, a subject of the disclosure is female. In some embodiments of the methods of the disclosure, a subject of the disclosure is male. In some embodiments, a subject of the disclosure has two XX or XY chromosomes. In some embodiments, a subject of the disclosure has two XX or XY chromosomes and a third chromosome, either an X or a Y.

[00255] In some embodiments of the methods of the disclosure, a subject of the disclosure is a neonate, an infant, a child, an adult, a senior adult, or an elderly adult. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or 31 days old. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months old. In some embodiments of the methods of the disclosure, a subject of the disclosure is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number of years or partial years in between of age.

[00256] n some embodiments of the methods of the disclosure, a subject of the disclosure is a mammal. In some embodiments, a subject of the disclosure is a non-human mammal.

[00257] In some embodiments of the methods of the disclosure, a subject of the disclosure is a human.

[00258] In some embodiments of the methods of the disclosure, a therapeutically effective amount comprises a single dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises at least one dose of a composition of the disclosure. In some embodiments, a therapeutically effective amount comprises a therapeutically effective amount comprises one or more dose(s) of a composition of the disclosure.

[00259] In some embodiments of the methods of the disclosure, a therapeutically effective amount eliminates a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount reduces a severity of a sign or symptom of the disease or disorder.

[00260] In some embodiments of the methods of the disclosure, a therapeutically effective amount eliminates the disease or disorder.

[00261] In some embodiments of the methods of the disclosure, a therapeutically effective amount prevents an onset of a disease or disorder. In some embodiments, a therapeutically effective amount delays the onset of a disease or disorder. In some embodiments, a therapeutically effective amount reduces the severity of a sign or symptom of the disease or disorder. In some embodiments, a therapeutically effective amount improves a prognosis for the subject.

[00262] In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject via intrathecal, intravenous (IV), or subpial administration. In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject via intracerebral administration. In some embodiments, the composition of the disclosure is administered to the subj ect by an intrastriatal route. In some embodiments, the composition of the disclosure is administered to the subject by a stereotaxic injection or an infusion. In some embodiments, the composition is administered to the brain. In some embodiments of the methods of the disclosure, a composition of the disclosure is administered to the subject locally.

[00263] In some embodiments, the compositions disclosed herein are formulated as pharmaceutical compositions. Briefly, pharmaceutical compositions for use as disclosed herein may comprise a protein(s) or a polynucleotide encoding the protein(s), optionally comprised in an AAV, which is optionally also immune orthogonal, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the disclosure may be formulated for routes of administration, such as e.g., oral, enteral, topical, transdermal, intranasal, and/or inhalation; and for routes of administration via injection or infusion such as, e.g., intravenous, intramuscular, subpial, intrathecal, intraparenchymal, intrathecal, intrastriatal, subcutaneous, intradermal, intraperitoneal, intratumoral, intravenous, intraocular, and/or parenteral administration. In certain embodiments, the compositions of the present disclosure are formulated for intracerebral or intrastriatal administration.

EXAMPLES

EXAMPLE 1: AAV9 MEDIATED DELIVERY OF RNA TARGETING SYSTEMS

ELIMINATES HEXANUCLEOTIDE REPEAT EXPANSIONS IN C9ORF72

ALS/FTD MODELS [00264] GGGGCC (G4C2) hexanucleotide repeat expansions (HRE) in the first intron of C9ORF72 gene are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Bidirectional transcription at the C9ORF72 repeat locus produces both sense G4C2 and antisense C4G2 containing transcripts. Studies to date have elucidated multiple pathogenic mechanisms including RNA-gain of function of both sense and antisense HREs, dipeptide repeats generated by RAN translation of both sense and antisense HREs, and haploinsufficiency of the C9ORF72 gene. However, it remains unclear how much C9ORF72 protein loss contributes to neuronal death. To explore therapeutic targeting of both sense and antisense HREs, we engineered novel CRISPR/Cas 13d based RNA targeting systems with 2 guide RNAs targeting G4C2 and C4G2 containing transcripts that can be packaged in a single AAV genome, as depicted in FIGS. 1A-B. FIGS. 2A-D show qPCR and FIGS. 3A-B show RNA-FISH analyses of cells transfected with G4C2 and C4G2 reporters showed efficient elimination of HREs. Initially, we packaged a G4C2 targeting guide RNA with two different orthologues of CRISPR/Casl3d in AAV9 with high yields. These AAV9-packaged G4C2 targeting CRISPR/Cas 13 ds showed no overt safety concerns in wildtype mice 8 weeks post subpial delivery. FIG. 4B shows a schematic depicting the subpial delivery model. Further, these constructs reduced the HRE containing isoforms of C9ORF72 in both cultured neonatal cortical neurons and in the spinal cord of BAC C9-500 poly-HRE mice, following subpial delivery, while largely preserving total C9ORF72 transcript (transcripts from both alleles and including normal non-expanded isoforms) levels. FIGS. 5C-D show the reduced levels of HRE containing isoforms of C9ORF72 in the spinal cord and the preserved total C9ORF72 transcript in BAC C9-500-poly-HRE mice, following subpial delivery of the exemplary CRISPR/Casl3d based RNA targeting system. In summary, we show a novel approach that can effectively target both sense and antisense HREs in C9ORF72 ALS/FTD with a single product.

EXAMPLE 2: In vitro-Cas!3d-multi-targeting expanded sense/anti-sense repeatcontaining RNA

[00265] Mammalian cells (COSM6, HEK293T) were transfected with CRISPR/Casl3d based RNA targeting vectors and reporters expressing the G4C2 (60 to 90 repeats) or C4G2 flanking region added downstream of firefly luciferase. Then, for quantification of G4C2 expression RNA was extracted from the cells and expression of G4C2 was analyzed by qRT-PCR normalizing to a reference gene and non-targeting condition (NT). For the antisense flanking sequence RNA knockdown was analyzed by Firefly Luciferase luminescence and normalized to Renilla Luciferase transfection control and NT condition. FIGS. 2A-D shows qRT-PCR and Luminescence analyses of cells transfected with G4C2 and C4G2 flanking reporters showed efficient elimination of sense and antisense HRE containing transcripts. Expression of G4C2 was also examined in transfected mammalian cells using RNA-FISH. FIGS. 3A-B show RNA- FISH analyses of cells transfected with G4C2 and showed efficient elimination of HRE. Methods

[00266] For transfection of mammalian cells, 6xl0 ⁴ COSM6 or 8xl0 ⁴ HEK293T cells were seeded per well in 24 well plates. The next day, cells were transfected with 50 ng of the pAV- FH-G4C2 RapidAmp product (~90 target repeats) plasmid and 500 ng of Cas expressing vector. On day 4, cells were harvested, RNA extracted, and qRT-PCR performed.

[00267] RNA was analyzed using qRT-PCR. Results are shown as G4C2 expression normalized to GAPDH and relative to non-targeting (NT) guide in untreated cells (n=2). The following primers were used for GAPDH, G4C2: (GAPDH F: CAGCCTCAAGATCATCAGCAA (SEQ ID NO: 194), R:TGTGGTCATGAGTCCTTCCA (SEQ ID NO: 195); G4C2 F2: GCGCTCTCGAGGATTATAAGG (SEQ ID NO: 196), R2: CGGTGGATCGGATAAACCTT (SEQ ID NO: 197))

[00268] For the C4G2 flanking analysis 2xl0 ⁴ HEK293T cells were seeded per well in 96 well plates. The next day, cells were transfected with 5 ng of the pFLUC-C4G2 flanking reporter plasmid, 50 ng of Cas expressing vector and 50 ng of sgRNA expressing vector. On day 4, cells were lysed, and luminescence determined for firefly and renilla control for analysis.

EXAMPLE 3: IN F/FD- VA9-CAS13D-G4C2 EXPRESSION AND EFFICACY OF

TARGETING SENSE-EXPANDED-REPEAT DNA

[00269] A G4C2 targeting guide RNA with two different orthologues of CRISPR/Casl3d was packaged in AAV9 with high yields. The exemplary AAV9-packaged G4C2 targeting CRISPR/Casl3d was administered to BAC C500 mice by bilateral subpial injection. FIG. 4B shows a schematic depicting the subpial delivery model, and the samples that were taken (RNA, protein) and the associated mouse tissue following subpial delivery of the vehicle or AAV9-packaged G4C2 targeting CRISPR/Casl3d targeting system. Administration of the AAV9-packaged G4C2 targeting CRISPR/Casl3d vectors showed no overt safety concerns in wildtype mice 8 weeks post subpial delivery. FIG. 4A shows the vector, mouse strain, and dosage of vector administered per animal. The vector was administered at a dose of 5E10 vg/animal (5E12 vg/ml, 5 pL/injection site). The indicated samples were collected ten weeks following administration of the treatment. RNA was extracted from the cervical and lumbar tissue and quantified by ddPCR. Protein was extracted from the cervical and thoracic tissue and quantified by MSD.

[00270] Administration of the AAV9-packaged G4C2 targeting CRISPR/Casl3d system reduced the sense HRE containing pathological isoforms of C9ORF72 in the spinal cord of BAC C9-500 poly-HRE mice, following subpial delivery, while largely preserving total C9ORF72 transcript (transcripts from both alleles and including normal non-expanded isoforms) levels. FIGS. 5C-D show the reduced levels of HRE containing isoforms of C9ORF72 in the spinal cord and the preserved total C9ORF72 transcript in BAC C9-500-poly- HRE mice, following subpial delivery of the exemplary CRISPR/Casl3d based RNA targeting system. FIGS. 5A-B show the relative RNA and protein levels of Cast 3d in mouse tissue following subpial injection of the AAV9-packaged G4C2 targeting CRISPR/Casl3d vector or administration of a vehicle. Casl3d expression was normalized to Atp5b reference gene expression.

Methods

[00271] Mice were administered either vehicle or AAV9-Casl3d-G4C2 vector by bilateral subpial injection targeting spinal cord. A Th 13 Laminectomy was used to expose LI spinal cord. The vector was administered at a dose of 5E10 vg/animal (5E12 vg/ml, 5 pL/injection site).

EXAMPLE 4: REDUCTION IN SENSE AND ANTISENSE RNA AND POLY-GP IN MULTI-TARGETING (MT) CAS13D TREATED C9-ALS FIBROBLASTS

[00272] Casl3d multi-targeting (MT) construct targeting sense strand G4C2 and antisense C4G2 flanking region was used to evaluate knockdown of both sense and antisense transcripts and decrease of DPRS in C9-ALS derived fibroblasts. Knockdown of the sense and antisense targets and a decrease in dipeptide repeats (DPRs) poly-GP aggregates in C9-ALS fibroblasts after treatment with Cast 3d MT construct were observed. See FIG. 6A-6D.

Methods

[00273] The fibroblasts were transduced with lentivirus expressing Cast 3d and selected with puromycin 48 hrs after transduction. After selection cells were transduced with the lentivirus for MT guide expression targeting sense and antisense transcripts and harvested 72 hrs after the second transduction.

EXAMPLE 5: IN VIVO SUBPIAL INJECTION OF AAV9 CAS13D MT (MULTI¬

TARGETING) (ALS05) [00274] RNA knockdown of G4C2 sense and C4G2 antisense transcripts, as well as decrease in dipeptide repeats (DPRs) poly-GP aggregates in C9-ALS mouse model C9 BAC-500 (containing 500 G4C2 repeats) were observed after 6 weeks subpial injection of AAV9 Casl3d MT (A02562). See FIG. 7A-7F.

Methods

[00275] Mice were injected subpially. RNA was extracted from the lumbar region, close to the site of injection and measured using ddPCR as copies of the respective transcript per 1000 copies of the Atp5b reference gene. Cast 3d MT expression in thoracic tissue region and poly- GP DPRs were measured using Meso Scale Discovery (MSD) assay.

EXAMPLE 6: IN VIVO INTRASTRIATAL INJECTION OF AAV9 CAS13D MT (MULTI-TARGETING) (ALS07)

[00276] RNA knockdown of G4C2 sense and antisense transcripts, as well as decrease in dipeptide repeats (DPRs) poly-GP aggregates in C9-ALS mouse model C9 BAC-500 (containing 500 G4C2 repeats) were observed after 6 weeks intrastriatal injection of AAV9 Casl3d MT (A02562). See FIG. 8A-8E.

Methods

[00277] Mice were injected unilaterally with the non-injected side used as a control, and two age groups were used 17 weeks and 24 weeks-old mice for evaluation. Decrease on sense and antisense RNAs (pathological isoforms) were shown by ddPCR as copies of the respective transcript per 1000 copies of the Atp5b reference gene. Casl3d expression in tissue following intrastriatal injection of AAV9-Casl3d-MT (A02562) and poly-GP DPRs relative to untreated tissue (contralateral control) were measured using the Meso Scale Discovery (MSD) assay.

EXAMPLE 7: KNOCKDOWN OF SENSE G4C2 FLANKING REGION WITH SEQ212 USING LUCIFERASE REPORTER ASSAY

[00278] Mammalian cells were transfected with the luciferase reporter containing the sense flanking region, Cast 3d, and the NT Guide (P000MJ Single Lambda Guide; negative control), firefly luciferase Guide (positive control), and guides targeting the sense flanking region. Knockdown of sense G4C2 flanking region in the mammalian cells was observed. See FIG.

9A-9B

EXAMPLE 8: SEQ212 VARIANTS FOR IMPROVED G4C2 AND ANTISENSE

FLANKING REGION KNOCKDOWN [00279] Casl3d variants were engineered to improve on-target knockdown of both G4C2 sense and C4G2 antisense flanking region. Two reporter plasmids were constructed to express G4C2 repeats driven by the CMV promoter (10A) and a Luciferase reporter containing the antisense flanking region (10C). See FIG. 10A-10E.

Methods

[00280] Wildtype (wt) Cast 3d was co-transfected with non-targeting (NT) and G4C2- targeting guide. Engineered Cast 3d variants were co-transfected with G4C2-targeting guide. C4G2 flanking sequence reporter plasmid was cloned downstream of firefly luciferase ORF (FLUC). Renilla luciferase (RLUC) was used as a transfection control. Two independent experiments tested C4G2 antisense flank knockdown in luciferase assay. A separate experiment tested RNA binding domains (RBD) tethered to Seq212.