Targeting the g 4c 2 repeat-containing RNA with a high-fidelity crispr-cas13 system improves abnormalities associated with ALS / ftd

Abstract

Methods and compositions for treating neurodegenerative disorders comprising crRNA molecules targeting exon1a and / or intron 1 of C9ORF72 and a Cas13 protein or a polynucleotide encoding a Cas13 are provided.

Description

[0001] TITLE: Targeting the G4C2 Repeat-Containing RNA With a High-Fidelity CRISPR- Cas13 System Improves Abnormalities Associated With ALS / FTD

[0002] GOVERNMENT SUPPORT

[0003] This invention was made with government support under 1 R01 NS123556-01 A1 , 1 U01 NS122102-01 A1, and 1 R01 GM141296-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] PRIORITY

[0005] This application claims the benefit of U.S. Ser. No. 63 / 605,767, filed on December 4, 2023, which is incorporated by reference in its entirety.

[0006] SUMMARY

[0007] Provided herein are methods for treating a neurodegenerative disorder comprising administering to the subject one or more crRNA molecules targeting exonl a and / or intron 1 of C9ORF72 and a Cas13 protein or a polynucleotide encoding a Cas13. The neurodegenerative disorder can comprise amyotrophic lateral sclerosis (ALS) or frontotemporal dementia. The amount of G4C2 repeat containing mRNA can be decreased and the amount of C9-L full length C9ORF72 mRNA cannot be affected or can be only minimally affected. The amount of full length C9ORF72 mRNA can be reduced by less than 30, 20, or 10%. The amount of G4C2 repeat containing mRNA can be decreased by 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. The amount of CBLN1 and / or CBLN2 mRNA can be reduced by about 20, 30, 40% or more in, for example, neurons. Transcriptional deficits due to the neurodegenerative disorder can be at least partially reversed. The one or more crRNA molecules can have greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3.

[0008] An aspect provides methods of decreasing a quantity of G4C2 repeat containing mRNA and / or cleaving G4C2 repeat containing mRNA in a subject or a cell. The method can comprise administering to the subject or cell: a fusion protein comprising a Cas13 protein and one or more nuclear localization signals or a polynucleotide encoding a fusion protein comprising a Cas13 protein and one or more nuclear localization signals; and one or more crRNA molecules targeting exonl a and / or intron 1 of C9ORF72 having greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3. A quantity of the G4C2 repeat containing mRNA is decreased and / or the G4C2 repeat containing mRNA is cleaved. The amount of G4C2 repeat containing mRNA can be decreased while the amount of C9-L full length C9ORF72 mRNA is not affected or is minimally affected. The amount of G4C2 repeat containing mRNA can be decreased by 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. The amount of C9-L full length C9ORF72 mRNA can be reduced by less than 30, 20, or 10%. The amount of CBLN1 and / or CBLN1 mRNA can be reduced by about 20, 30, 40% or more in, for example, neurons. The one or more crRNA molecules can have greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3.

[0009] An aspect provides a method of making a system for preventing or slowing the progression of one or more symptoms of a neurodegenerative disorder comprising combining one or more crRNA polynucleotides that target exon 1a and / or intron 1 of a C9ORF72 gene and a Cas13 protein or a polynucleotide encoding a Cas13 protein. The system can be delivered to a patient or cell. The Cas13 protein can be a Cas13d protein. The Cas13 protein can be from Ruminococcus flavefaciens. The Cas13 protein can be RfxCas13d-N2V7 or RfxCas13d-N2V8. The Cas13 protein can be PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a,

[0010] PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b,

[0011] PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dUrCas13d. The Cas13 protein can further comprise one or more a localization signals or one or more export signals. The Cas13 protein can further comprise one or more nuclear localization signals (NLS). The one or more NLSs can be selected from SEQ ID NO: 17-41. The Cas 13 protein can additionally comprise one or more epitope tags. The epitope tag can be FLAG (DYKDDDDK; SEQ ID NO:4), HA (YPYDVPDYAC; SEQ ID NO:5), myc (EQKLISEEDLC; SEQ ID NO:6), V5 (GKPIPNPLLGLDST; SEQ ID NOT), E-tag (GAPVPYPDPLEPR; SEQ ID NO:8), VSV-g (YTDIEMNRLGK; SEQ ID N0:9), 6xHis (HHHHHHH; SEQ ID NO:10), HSV (QPELAPEDPEDC; SEQ ID N0:11 ), or combinations thereof. The Cas13 protein can further comprise one or more fluorescent proteins. The one or more fluorescent proteins can be selected from: blue / UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalamal , Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, and mTFPI ), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, rnUKG, and mWasabi), yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, mKOK, mK02, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near-IR fluorescent proteins (for example, TagRFP657, IFP1.4, and iRFP), long stokes-shift proteins (for example, mKeima Red, LSS-mKate1 , and LSS-mKate2), photoactivatable fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP), photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGRI (green), KikGRI (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), fluorescein, rhodamine, photoswitchable fluorescent proteins (for example, Dronpa), or combinations thereof.

[0012] An aspect provides a CRISPR RNA (crRNA) comprising greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3. The crRNA can be less than 30 nucleotides in length. The crRNA can target exon 1a and / or intron 1 of a C9ORF72 gene.

[0013] An aspect provides a composition comprising one or more crRNA polynucleotides comprising SEQ ID NO:1 , 2, or 3 and a Cas13 protein or a polynucleotide encoding a Cas13 protein. In an aspect the one or more crRNA polynucleotides can have greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3. The composition can comprise two or more crRNA polynucleotides, wherein one or more crRNA polynucleotides target a sense RNA strand expressed from a C9ORF72 gene and one or more crRNA polynucleotides target an antisense RNA strand expressed from a C9ORF72 gene. In an aspect, the two or more crRNA polynucleotides can target exon 1a or intron 1 of the C9ORF72 gene. The Cas13 protein can be a Cas13d protein. The Cas13 protein can be from Ruminococcus flavefaciens. The Cas13 protein can be RfxCas13d-N2V7 or RfxCas13d-N2V8. The Cas13 protein can be PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d,

[0014] FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c,

[0015] HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a

[0016] LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b

[0017] PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b

[0018] Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a,

[0019] RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H,

[0020] PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dllrCas13d. The Cas13 protein can further comprise one or more localization signals or one or more export signals. The Cas13 protein can further comprise one or more nuclear localization signals (NLS). The one or more NLSs can be selected from SEQ ID NO: 17-41. The Cas 13 protein can additionally comprises one or more epitope tags. The epitope tag can be FLAG (DYKDDDDK; SEQ ID NO:4), HA (YPYDVPDYAC; SEQ ID NO:5), myc (EQKLISEEDLC; SEQ ID NO:6), V5 (GKPIPNPLLGLDST; SEQ ID NO:7), E-tag (GAPVPYPDPLEPR; SEQ ID NO:8), VSV-g (YTDIEMNRLGK; SEQ ID NO:9), 6xHis (HHHHHHH; SEQ ID NQ:10), HSV (QPELAPEDPEDC; SEQ ID NO:11 ), or combinations thereof. The Cas13 protein can further comprise one or more fluorescent proteins. The one or more fluorescent proteins can be selected from: blue / UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalamal , Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, and mTFPI ), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, and mWasabi), yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, mKOK, mK02, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near-IR fluorescent proteins (for example, TagRFP657, IFP1.4, and iRFP), long stokes-shift proteins (for example, mKeima Red, LSS-mKate1 , and LSS-mKate2), photoactivatable fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP), photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGRI (green), KikGRI (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), fluorescein, rhodamine, photoswitchable fluorescent proteins (for example, Dronpa), or combinations thereof.

[0021] An aspect provides a vector comprising a polynucleotide encoding a Cas13 protein and one or more crRNAs polynucleotides as set forth in SEQ ID NO:1 , 2, or 3.

[0022] One or more nuclear localization sequences (NLS) can be located 5’, 3’, or both 5’ and 3’ to the polynucleotide encoding the Cas13 protein. The vector can be a plasmid or a viral vector. The vector can be a retroviral vector, lentiviral vector, adenoviral vector, adeno- associated virus (AAV) vector, a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), a synthetic vector, a vector encapsulated within a lipid nanoparticle, or a vector complexed with a polymer. The vector can be an adeno- associated virus (AAV) vector, a naturally occurring AAV vector, or an engineered AAV vector. The vector can be AAV-1 , AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 , AAV-12, AAV-13, AAV-anc80, AAV rh.74, AAVrh.10, AAVPHP.B, or AAVPHP.eB. The vector can comprise three major viral proteins: VP1 , VP2 and VP3. The vector can comprise one or more AVV ITR sequences. The one or more ITR sequences can be selected from AAV1 ITR sequences, AAV2 ITR sequences, AAV4 ITR sequences, AAV5 ITR sequences, AAV6 ITR sequences, AAV7 ITR sequences, AAV8 ITR sequences, AAV9 ITR sequences, AAV 10 ITR sequences, AAV 11 ITR sequences, AAV12 ITR sequences, AAV13 ITR sequences, AAVrh74 ITR sequences, AAVrh.10 ITR sequences or any combination thereof. The vector can lack rep and cap genes. The polynucleotide encoding a Cas13 protein and the one or more crRNAs polynucleotides can be operably linked to one or more promoters. The one or more promoters can be a chicken fB-actin hybrid (CBh), U6, U7, tRNA, H1 , minimal CMV, synapsin, or T7 promoter.

[0023] An aspect provides a pharmaceutical composition comprising: (i) one or more crRNA polynucleotides comprising greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3; (ii) a Cas13 protein or a polynucleotide encoding a Cas13 protein; or the vector of any of claims 17-28; and (iii) a pharmaceutical excipient.

[0024] An aspect provides use of the pharmaceutical composition to treat a neurological disease. An aspect provides methods of decreasing an amount of one or more dipeptide repeat proteins (DRPs) in a subject or a cell. The methods can comprising administering to the subject or cell: a fusion protein comprising a Cas13 protein and one or more nuclear localization signals or a polynucleotide encoding a fusion protein comprising a Cas13 protein and one or more nuclear localization signals; and one or more crRNA molecules targeting exonla and / or intron 1 of C9ORF72 having greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3, wherein the amount of one or more DRPs is reduced in the subject or cell. The amount of the one or more DRPs can be decreased by 5, 10, 20, 30, 40, 50% or more as compared to an untreated cell or subject. The one or more DRPs can be glycine-alanine (GA) DRP, glycine-arginine (GR) DRP, proline-alanine (PA) DRP, proline-arginine (PR) DRP and / or glycine-proline (GP) DRP.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 RfxCas13d can be programmed to target C9ORF72. (a) Schematic of (top) the C9ORF72 gene and (bottom) the three main mRNA transcript variants expressed from it. V1 produces the short protein isoform (C9-S), while V2 and V3 produce the long protein isoform (C9-L). (b, c) Schematic of the dual-reporter system used to evaluate crRNAs. The platform consists of a (b) Renilla luciferase-encoding plasmid, pSV40-RLuc, whose 3’ untranslated region (UTR) carries a fragment of the C9ORF72 gene with 20 copies of the hexanucleotide repeat and 250- and 98-base pairs (bps) of the flanking upstream and downstream gene sequences, respectively, and (c) a firefly luciferase-encoding plasmid, pHSV-TK-FLuc, which is used a proxy for collateral cleavage, (d) Normalized Renilla and firefly luciferase expression in HEK293T cells after transfection with the pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d and one of the 15 candidate crRNAs. All values were normalized to cells transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d with a non-targeted (NTG) crRNA ( = 3). (e, f) Relative all-V and V3 mRNA in (e) HEK293T and (f) SH-SY5Y cells transfected with RfxCas13d and crRNAs 13, 7, and 1 or a NTG crRNA or one of two ASOs (n = 3). All values from HEK293T and SH- SY5Y cells were normalized to untreated cells. Values indicate means and error bars indicate SD. **P < 0.01 , ***P < 0.001 , ****P < 0.0001 ; one-tailed unpaired t-test comparing each crRNA treated to the NTG crRNA. All data points are biologically independent samples.

[0027] FIG. 2 RfxCas13d can target the G4C2 repeat-containing RNA and reduce the formation of RNA foci in the brain, (a) Cartoon illustrating the injection scheme, (b) Overview of the experimental plan to analyze C9ORF72 mRNA in EGFP-KASH+nuclei isolated by fluorescence-activated cell sorting (FACS), (c) Representative immunofluorescent staining of the hippocampus (HPC) and motor cortex (MC) in C9- BACexp mice two-months after injection with 2 x 1010VGs each of AAV-PHP.eB- RfxCas13d-crRNA and AAV-PHP.eB-EGFP-KASH. Scale bar, 20 pm. (d, e) Relative all- V and V3 mRNA in (d) the HPC and (e) MC of EGFP-KASH+nuclei from C9-BACexp injected with AAV-PHP.eB-RfxCas13d-crRNA-13, -7, -1 , or -NTG with AAV-PHP.eB- EGFP-KASH (n > 7). (f) Representative RNA FISH for the G4C2 repeat RNA (magenta) from the HPC of C9-BACexp mice injected with 2 x 1010GCs each of PHP.eB-RfxCasI 3d- crRNA and PHP.eB-EGFP-KASH. Scale bar, 5 pm. (g, h) Quantification of (g) the number of RNA foci per EGFP-KASH+cell in the HPC from injected C9-BACexp mice and (h) the percentage of EGFP-KASH+cells with 0, 1-2, 3-4, or >5 foci (n > 4). (g, h) 58-455 cells were counted per animal in the HPC for a total of 398, 845, and 467 cells for AAV- PHP.eB-RfxCas13d-crRNA-7, crRNA-13, and -NTG respectively (n > 4). RNA foci measurements were conducted by a blinded investigator. Values indicate means and error bars indicate SD. *P < 0.05, **P < 0.01 , ***P < 0.001 ; one-tailed unpaired t-test comparing each crRNA to the NTG crRNA. All data points are biologically independent samples.

[0028] Fig 3. High-fidelity RfxCas13d has improved specificity and can mediate targeting in cells derived from an ALS patient, (a) RfxCas13d domain organization with RfxCas13d-N2V7 and RfxCas13d-N2V8 mutations, (b, c) Relative all-V and V3 mRNA in (b) HEK293T and (c) SH-SY5Y cells transfected with RfxCas13d, RfxCas13d- N2V7 and RfxCas13d-N2V8 with crRNA-13. All values normalized to untreated cells (n = 3). (d) Volcano plot of the RNA-seq analysis comparing HEK293T cells transfected with (left) RfxCas13d or (right) RfxCasI 3d-N2V8 with crRNA-13 to each variant with a NTG crRNA (n = 3). Lines denote a >1 .25-fold change (FC) and an FDR-adjusted P < 0.01 . (e) Number of differentially expressed genes (DEGs) [>1.25-FC, FDR-adjusted P < 0.01 ] from (d). (f) Gene ontology (GO) and biological process (BP) term analysis for the DEGs in (d). Line denotes FDR-adjusted P < 0.05. (g) Venn diagram of overlapping DEGs. (h) Immunostaining of C9-ALS neurospheres (n = 3). Scale bar, 75 pm. (i) Brightfield and fluorescent images of C9-ALS neurospheres 14 days after treatment with PHP.eB-EGFP- KASH. Experiment was conducted once, (j, k) Relative (j) V3 to all-V mRNA ratio and (k) CBLN1 mRNA in C9-ALS or wild-type neurospheres treated with PHP.eB-RfxCasI 3d- N2V8-crRNA-13 or -NTG (n > 3). (j, k) Values normalized to the NTG crRNA. (k) All values normalized to the NTG in the C9-ALS cells. (I) Number of DEGs [>1.2-FC, FDR- adjusted P < 0.01] in C9-ALS cells treated with PHP.eB-RfxCas13d-N2V8-crRNA-13 or - NTG. Values indicate means and error bars indicate SD. (b, c) All-V and V3 mRNA for each RfxCas13d variant were compared to the same from untreated cells using a two- tailed unpaired t-test, with the exact P values shown, (d-f) FDR-adjusted P values were determined by a Global FDR correction across pairwise comparisons, (j, k) crRNA-13 and NTG were compared using a one-tailed unpaired t-test, with the exact P values shown. (I). FDR-adjusted P values were determined by a Global FDR correction across pairwise comparisons. All data points are biologically independent samples.

[0029] FIG. 4 High-fidelity RfxCas13d can target the G4C2 repeat -containing RNA and reverse deficits in C9-BACexp mice, (a) Cartoon illustrating the injection scheme, (b) Representative immunofluorescent staining of the hippocampus (HPC) and motor cortex (MC) in C9-BACexp mice two-months after injection with 2 x 1010GCs each of AAV-PHP.eB-RfxCas13d-N2V8-crRNA and AAV-PHP.eB-EGFP-KASH. Scale bar, 30 pm. (c, d) Relative all-V and V3 mRNA in EGFP-KASH+nuclei from (c) the HPC and (d) MC of C9-BACexp injected with AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13 or -NTG with AAV-PHP.eB-EGFP-KASH (n > 6). (e) Representative FISH for the G4C2 repeat RNA (Magenta) in EGFP-KASH+cells from the HPC of C9-BACexp mice injected with AAV- PHP.eB-RfxCas13d-N2V8-crRNA-13 or -NTG and AAV-PHP.eB-EGFP-KASH. Scale bar, 5 pm. (f) Quantification of the number of RNA foci per EGFP-KASH+cell in the (left) HPC and (right) MC of injected C9-BACexp mice (n > 6). (g) Soluble poly(GP) in the HPC of injected C9-BACexp mice (n > 6) (h, i) Volcano plot of the RNA-seq analysis comparing EGFP-KASH+cell nuclei from the MC of C9-BACexp mice injected with (h) AAV-PHP.eB- RfxCas13d-N2V8-NTG or (i) -crRNA-13 with AAV-PHP.eB-EGFP-KASH to wild-type littermates injected with the same formulation (n = 3-7). Lines denote a >1.2-fold change (FC) and an FDR-adjusted P < 0.05. (j, k) FC of the (j) down-regulated or (k) up-regulated DEGs from (h) for C9-BACexp mice injected with AAV-PHP.eB-RfxCas13d-N2V8- crRNA-13 relative to wild-type littermates. Values indicate means and error bars indicate SD. *P < 0.05, **P < 0.01 , ****P < 0.0001 ; (c, d, f, g) one-tailed unpaired t-test; (j, k) two- tailed unpaired t-test. (c, d, f, g). All tests compared vlaues for crRNA-13 to the NTG crRNA. All data points are biologically independent samples.

[0030] FIG. 5 panels a-b show targeting C9ORF72 with RfxCas13d. (a) Cartoon showing the locations of the crRNA binding sites for RfxCas13d within exon 1a and intron 1a of the C9ORF72 gene. Cas13 Design Resource was used to predict active crRNAs. Wessels, H. H. et al. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat. Biotechnol. 38, 722-727 (2020); Guo, X. et al. Cell Genom. 1 , doi: 10.1016 / j.xgen.2021.100001 (2021 ) Top strand is SEQ ID NO:56; Bottom strand is SEQ ID NO:57. (b) crRNA sequences used in this study. crRNA 1 is SEQ ID N0:1 , crRNA 2 is SEQ ID NO:44, crRNA 3 is SEQ ID NO:45, crRNA 4 is SEQ ID NO:46, crRNA 5 is SEQ ID NO:47, crRNA 6 is SEQ ID NO 48, crRNA 7 is SEQ ID N0:2, crRNA 8 is SEQ ID NO:49, crRNA 9 is SEQ ID NQ:50, crRNA 10 is SEQ ID NO:51 , crRNA 11 is SEQ ID NO:52, crRNA 12 is SEQ ID NO:53, crRNA 13 is SEQ ID NO:3, crRNA 14 is SEQ ID NO:54, crRNA 15 is SEQ ID NO:55.

[0031] FIG. 6 shows crRNA targeting scores. Targeting scores, defined as the ratio of Renilla (i.e. , target) to firefly (i.e., collateral) luciferase expression, were determined for each crRNA. A low targeting score indicates decreased collateral effects. Renilla and firefly luciferase expression were measured in HEK293T cells transfected with pSV40- RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d and one of the 15 candidate crRNAs (n = 3). Renilla and firefly luciferase values for each crRNA were normalized to cells transfected with pSV40-RLuc, pHSV-TK-FLuc, and an expression vector encoding RfxCas13d with a non-targeted (NTG) crRNA. Bars indicate means and error bars indicate SD. ***P < 0.001 , ****P < 0.0001 ; two-tailed unpaired t-test. All data points are biologically independent samples.

[0032] FIG. 7 shows quantification of EGFP-KASH expression in NeuN+cells in C9- BACexp mice. Percentage of NeuN+cells positive for EGFP-KASH in the motor cortex (MC) and hippocampus (HPC) of C9-BACexp mice injected with 2 x 1010VGs each of AAV-PHP.eB-EGFP-KASH and AAV-PHP.eB-RfxCas13d-crRNA. 61 -143 and 80-142 cells per animal were counted in the MC and HPC, respectively, for a total of 314 and 563 cells in the MC and HPC, respectively (n > 3). Bars indicate means and error bars indicate SD. All data points are biologically independent samples.

[0033] FIG. 8 panels a-b show quantification of the percentage of EGFP-KASH+cells positive for RfxCas13d in C9-BACexp mice, (a) Representative immunofluorescent staining of (top) the hippocampus (HPC) and (bottom) motor cortex (MC) of C9-BACexp mice injected with 2 x 1010VGs each of AAV-PHP.eB-EGFP-KASH and AAV-PHP.eB- RfxCas13d-crRNA. Scale bar; 20 pm (HPC); 50 pm (MC). (b) Percentage of EGFP- KASH* cells positive for RfxCas13d via its HA epitope within the injection sites in the MC and HPC of C9-BACexp mice. 79-132 and 84-212 cells were counted per animal in the MC and HPC, respectively, for a total of 316 and 715 cells from the MC and HPC, respectively (n > 3). Bars indicate means and error bars indicate SD. All data points are biologically independent samples.

[0034] FIG. 9 shows AAV-PHP.eB-EGFP-KASH co-injected with AAV-PHP.eB- RfxCas 13d -crRNA minimally transduced microglia and astrocytes in C9-BACexp mice. Representative immunofluorescent staining of (left) the hippocampus (HPC) and (right) motor cortex (MC) of C9-BACexp mice injected with 2 x 1010VGs each of AAV- PHP.eB-EGFP-KASH and AAV-PHP.eB-RfxCas13d-crRNA. Scale bar; 30 pm. Microglia marker: Iba1 , ionized calcium binding adaptor molecule 1. Astrocyte marker: GFAP, glial fibrillary acidic protein.

[0035] FIG. 10 panels a-b show RfxCas13d targeting did not affect the abundance of the C9-L protein in HEK293T cells, (a) Representative western blot of the C9-L protein in cell lysate from HEK293T cells transfected with an expression vector encoding RfxCas13d, RfxCas13d-N2V7, or RfxCasI 3d-N2V8 with either crRNA-13 or a nontargeted (NTG) crRNA. (b) Quantitation of the western blot. C9-L protein in each lane was normalized to [3-actin protein then to cells transfected with the NTG crRNA (n = 3). Bars represent means and error bars indicate SD. Two-tailed unpaired t-test. All data points are biologically independent samples.

[0036] FIG. 11 shows quantification of EGFP-KASH expression in NeuN+cells in C9- BACexp mice co-injected with AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13. Percentage of NeuN+cells positive for EGFP-KASH in the motor cortex (MC) and hippocampus (HPC) of C9-BACexp mice injected with 2 x 1010VGs each of AAV-PHP.eB- EGFP-KASH and AAV-PHP.eB-RfxCas13d-N2V8. 109-176 and 86-140 cells were counted per animal in the MC and HPC, respectively, for a total of 557 and 550 cells in the MC and HPC, respectively (n > 4). Bars indicate the means and error bars indicate SD. All data points are biologically independent samples.

[0037] FIG. 12 panels a-b show quantification of the percentage of EGFP-KASH* cells positive for RfxCasI 3d-N2V8 in C9-BACexp mice, (a) Representative immunofluorescent staining of (top) the hippocampus (HPC) and (bottom) motor cortex (MC) of C9-BACexp mice injected with 2 x 1010VGs each of AAV-PHP.eB-EGFP-KASH and AAV-PHP.eB-RfxCas13d-N2V8-crRNA. Scale bar; 15 pm (HPC); 50 pm (MC). (b) Percentage of EGFP-KASH* cells positive for RfxCasI 3d-N2V8 via its HA epitope near the injection sites in the MC and HPC of C9-BACexp mice. 95-114 and 81 -102 cells were counted per animal in the MC and HPC, for a total of 421 and 454 cells from the MC and HPC, respectively (n > 4). Bars indicate the means and error bars indicate SD. All data points are biologically independent samples.

[0038] FIG. 13 shows AAV-PHP.eB-EGFP-KASH co-injected with AAV-PHP.eB- RfxCas13d-N2V8-crRNA minimally transduced microglia and astrocytes in C9- BACexp mice. Representative immunofluorescent staining of (left) the motor cortex (MC) and (right) hippocampus (HPC) of C9BAC-exp mice injected with 2 x 1010VGs each of AAV-PHP.eB-EGFP-KASH and AAV-PHP.eB-RfxCas13d-N2V8-crRNA. Scale bar; 30 pm. Microglia marker: Iba1 , ionized calcium binding adaptor molecule 1. Astrocyte marker: GFAP, glial fibrillary acidic protein.

[0039] FIG. 14 panels a-b show the distribution of RNA foci in EGFP-KASH+cells from C9-BAC-exp mice, a, b Percentage of EGFP-KASIT cells with 0, 1-2, 3-4, or >5 foci positive for the G4C2 repeat-containing RNA in the (a) hippocampus (HPC) and (b) of mice injected with 2 x 1010GCs of AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13 or -NTG and AAV-PHP.eB-EGFP-KASH. RNA foci were determined by fluorescence in situ hybridization (FISH) using a previously described probe for the G4C2 repeat RNA (Lagier- Tourenne, et al. Proc. Natl. Acad. Sci. U. S. A. 110, E4530-4539, doi:10.1073 / pnas.1318835110 (2013)). 108-237 and 83-334 cells were counted per animal for the MC and HPC, respectively. In total, 1 ,139 and 919 cells were counted for AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13 and -NTG, respectively, for the MC, while a total of 1 ,432 and 1 ,016 cells were counted for AAV-PHP.eB-RfxCas13d-N2V8-crRNA- 13 and -NTG, respectively, for the HPC (n > 6). All measurements were conducted by a blinded investigator. Values indicate means.

[0040] FIG. 15 shows RfxCas13d-N2V8 reduced the poly(GP) protein by ~8% in bulk cortical tissue from C9-BACexp mice. Soluble poly(GP) from the motor cortex (MC) of C9-BACexp mice injected with PHP.eB-RfxCas13d-N2V8-crRNA-13 or -NTG, as measured by an MSD immunoassay platform (n = 6). Values indicate means and error bars indicate SD. n.s., P > 0.05; one-tailed unpaired t-test. All data points are biologically independent samples.

[0041] Fig. 16 shows a term analysis for the normalized DEGs in RfxCas13d-N2V8- treated C9-ALS neurospheres. Gene ontology (GO) term analysis for the reverted DEGs in Fig. 3I.

[0042] DETAILED DESCRIPTION

[0043] Introduction

[0044] Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, paralytic and invariably fatal disorder characterized by the selective loss of motor neurons in the brain and spinal cord1, and one that is thought to lie on a continuum of conditions that includes frontotemporal dementia (FTD)2-4, a syndrome defined by progressive cognitive impairment due to the degeneration of the frontal and temporal lobes of the brain56.

[0045] An abnormal expansion of a GGGGCC (G4C2) hexanucleotide repeat in the first intron of the chromosome 9 open-reading frame 72 (C9ORF72) gene is the most common genetic cause of both ALS and FTD7 8. To date, three non-exclusive mechanisms have been proposed to explain the pathogenicity of the hexanucleotide repeat expansion9-24. These include a loss-of-function of the C9ORF72 protein due to impaired transcription of the mutant allele9-11, an effect referred to as haploinsufficiency, and / or a toxic gain-of- function from the bidirectional transcription of sense12and antisense13repeat-containing transcripts that accumulate in foci within the nucleus of cells14, potentially with key RNA- binding proteins15-18. These repeat-containing transcripts can further serve as templates for the synthesis of one of five dipeptide repeat (DPR) proteins19-22that are believed to exert toxic effects23 24and are produced through a non-canonical translation mechanism19.

[0046] The hexanucleotide repeat-containing RNAs drive the pathogenesis of C9ORF72- linked ALS and FTD, hereafter reffered to as C9-ALS / FTD. Therefore, strategies for silencing their expression may treat the disorder, as lowering the repeat-containing RNAs can affect the accumulation of RNA foci and the production of DPR proteins. Cas1325-27, a class 2 type VI CRISPR effector protein that, when complexed with a CRISPR RNA (crRNA) molecule carrying complementarity to a target transcript, can cleave it via its intrinsic ribonuclease (RNase) activity. To date, four distinct Cas13 subtypes have been identified and used for gene silencing in eukaryotic cells25-28. Among these is the Cas13d nuclease from Ruminococcus flavefaciens XPD300225, known as RfxCas13d or CasRx, a CRISPR effector protein that is compact enough to fit within a single adeno-associated virus (AAV) vector to enable its in vivo delivery to the central nervous system29-32and whose modification with nuclear localization signal (NLS) sequence(s) can enable it to target transcripts in the nucleus25.

[0047] In an aspect, Cas13 systems, such as RfxCas13d are used to silence the hexanucleotide repeat-containing RNA to influence in vivo pathological hallmarks of C9- ALS / FTD.

[0048] Using a dual-luciferase reporter screen designed to assess both target engagement and collateral effects, Cas13d systems are demonstrated to facilitate the efficient targeting of the G4C2 repeat-containing RNA. Following in vivo delivery to C9- BACexp mice, which harbor the human C9ORF72 gene with a disease-associated expansion of the hexanucleotide repeat, Cas13d-based platforms can selectively curb the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 mRNA levels, an outcome that reduced the formation of RNA foci positive for the G4C2 repeat-containing RNA. Furthermore, high-fidelity forms of Cas13d have improved transcriptome-wide specificity compared to the native enzyme, and can also be used to target the G4C2 repeat-containing transcript, both in induced motor neuron-like cells from a patient with ALS and C9-BACexp mice, such that transcriptional deficits are reverted.

[0049] Polynucleotides

[0050] Polynucleotides can be single-stranded or double-stranded. In some aspects, a polynucleotide is DNA. In particular aspects, a polynucleotide is cDNA. In some aspects, a polynucleotide is RNA (e.g., mRNA, tRNA, rRNA). In some aspects, a polynucleotide is included within a nucleic acid construct. In some modalities, a construct is a replicable vector. In some aspects, a vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector and a non-episomal mammal vector.

[0051] In some aspects, a polynucleotide is operationally linked to one or more regulatory nucleotide sequences in an expression construct.

[0052] Unless otherwise indicated, the term polynucleotide, nucleic acid molecule, or gene includes reference to the specified sequence, as well as the complementary sequence thereof, and the corresponding RNA molecule. Polynucleotides can be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. As used herein, a polynucleotide can include both naturally occurring and non- naturally occurring nucleotides.

[0053] Polynucleotides can be obtained from nucleic acid molecules present in, for example, a mammalian cell. Polynucleotides can also be synthesized in the laboratory, for example, using an automatic synthesizer. Polynucleotides can be isolated. An isolated polynucleotide can be a naturally occurring polynucleotide that is not immediately contiguous with one or both of the 5' and 3’ flanking genom ic sequences that it is naturally associated with. An isolated polynucleotide can be, for example, a recombinant DNA molecule of any length, provided that the nucleic acid molecules naturally found immediately flanking the recombinant DNA molecule in a naturally occurring genome is removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules. “Isolated polynucleotides” can be (i) amplified in vitro, for example via polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, for example, by cleavage and separation by gel electrophoresis, (iv) synthesized, for example, by chemical synthesis, or (vi) extracted from a sample.

[0054] Cas13 and Cas7-11

[0055] A Cas13 or Cas13d polypeptide can be any Cas13 or Cas13d polypeptide (also called “Cas13” herein). Cas13 or Cas13d proteins can be directed to a specific RNA via an engineered CRISPR RNA (crRNA) guide molecule that encodes a programmable spacer sequence that mediates target engagement via RNA-RNA base complementarity. To date, several different Cas13 subtypes have been identified. Among these is the Cas13d nuclease from Ruminococcus fla vefaciens XPD3002 (RfxCas13d), a class II, type VI CRISPR effector protein that possesses favorable targeting capabilities to other Cas13 orthologs, has high programmability, as it does not require a protospacer flanking sequence to bind RNA, and can fit within a single adeno-associated virus (AAV) vector particle alongside a crRNA expression cassette to enable its in vivo gene transfer. Cas13 polypeptides are RNA-targeting programmable nucleases of the Type VI CRISPR-Cas systems. Type VI CRISPR-Cas systems are RNA-targeting immune systems derived from prokaryotes. The Cas13 family comprises at least four subtypes, including Cas13a (formerly C2c2), Cas13b, Cas13c and Cas13d. Type Vl-A and Vl-B systems have crRNA- dependent target cleavage activity and a non-specific, collateral RNase activity that is stimulated by target recognition and cleavage. Both of these activities are mediated by the two HEPN domains contained in type VI effectors Cas13a and Cas13b.

[0056] Any suitable Cas13 can be used. Plasmids encoding Cas13 are available from Addgene (Watertown MA, Addgene.org). For example, Cas13 can be encoded by Addgene plasmid number 176303, 176304, 176305, 176306, 176307, 82381 , 89898, 89906, 131012, 164862, 91905, 118963, 89901 , 141320, 164857, 164858, 164859, 165078, 91902, 91925, 155366, 91924, or any other suitable plasmid.

[0057] In an aspect, a dCas13 (a catalytically dead Cas13) can be used in the methods described herein. Any suitable dCas13 can be used. Plasmids encoding dCas13 are available from Addgene. For example, dCas13 can be encoded by Addgene plasmid number 119858, 154938, 154939, 155366, 155367, 100817, 157854, or any other suitable plasmid.

[0058] Cas13a / b / c exhibit high efficiency and specificity for RNA knockdown applications in mammalian cells. Instead of a preferred PAM sequence, Cas13a requires a 3' protospacer flanking sequence (PFS) of H, while Cas13b requires both a 3' PFS of NAN or NNA and a 5' PFS of D for effective RNA cleavage. Type IV-D CRISPR effectors (Cas13d), can be employed for RNA knockdown in mammalian cells. Target RNA cleavage by CRISPR / Cas13d is PFS-independent.

[0059] In some aspects a Cas13 polypeptide is a Cas13d protein. Cas13d polypeptides belong to the subtype Vl-D system, which is a variant of type VI CRISPR-Cas systems. Cas13d systems have robust target cleavage, indiscriminate RNase activities, and the ability to process pre-crRNA. Cas13d has a small size and can be packaged into viral vectors. Cas13 polypeptides can be guided by crRNAs, which provide target specificity. Cas13 polypeptides can complex with crRNA molecules through interactions with a short hairpin sequence within them. The crRNA molecules encode a spacer sequence which guides Cas13 to its target sequence, thereby conferring targeting specificity. In addition to targeted RNase activity, Cas13 systems have indiscriminate cleavage activity after recognition and cleavage of the target transcript, leading to non-specific cleavage of any nearby single stranded RNA transcripts regardless of complementarity to the spacer. Cas13 can process its own pre-crRNAs, allowing individual short single crRNAs to be customized to target RNA.

[0060] Cas13 polypeptides can be naturally occurring or non-naturally occurring. A Cas13 polypeptide can be a mutant (e.g., have one or more amino acid insertions, deletions, or substitutions) Cas13 polypeptide. A mutant Cas13 polypeptide can have altered biological activity as compared to a naturally occurring Cas13 polypeptide, such as altered nuclease activity without substantially diminished binding affinity to RNA. A mutant

[0061] Cas13 can have, for example, no nuclease activity. For example, a mutant Cas13 can be a ribonuclease that has the positively charged catalytic residues of the HEPN motifs inactivated, which eliminates programmable RNA cleavage without affecting guide RNA array processing or target RNA binding. A Cas13 polypeptide can be a Cas13d polypeptide. A Cas13d polypeptide can be from any suitable bacterial species, for example, Ruminococcus sp., Ruminoccocus flavefaciens, Ruminoccocus albus, and Eubacterium siraeum. In some aspects, the Cas13d polypeptide is derived from Ruminococcus flavefaciens strain XPD3002 (e.g., CasRx or RfxCas13d). In an aspect, a Cas13d polypeptide is a catalytically inactive version of CasRx (e.g. dCasRx). An exemplary sequence of CasRx (NLS-RfxCas13d-NLS) can be found at Plasmid #109049 (pXR001 : EF1a-CasRx-2A-EGFP, Addgene). In an aspect, a nucleotide sequence encoding a Cas13 polypeptide can be at least about 80% identical (e.g. at least about 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% identical) to the sequence of

[0062] RfxCas13d. In an aspect, the Cas13 protein is RfxCas13d-N2V7 or RfxCas13d-N2V8.

[0063] In an aspect, the Cas13 protein is PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d,

[0064] FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c,

[0065] HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a

[0066] LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b

[0067] PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b

[0068] Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a,

[0069] RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCas13b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dUrCas13d.

[0070] In an aspect, a Cas7-11 can be used, which has reduced or no risk of collateral trans cleavage of RNA. See Ozcan, Programmable RNA targeting with the single-protein CRISPR effector Cas7-11 . Nature 597, 720-725 (2021 ).

[0071] In some aspects, a polynucleotide can comprise a sequence encoding a Cas13 protein and one or more (1 , 2, 3, 4, 5, or more) localization signals. A localization signal tags a protein for transportation to a particular location in a cell. In an aspect a localization signal is a nuclear localization signal (NLS), which can be an amino acid sequence that tags a protein for import into the cell nucleus by nuclear transport. Localization signals can be operably linked to the sequence encoding a Cas13 protein. For example, the sequence encoding Cas13 can comprise two nuclear localization signals such that a Cas13 polypeptide is expressed that is fused to N- and C-terminal nuclear localization signals. An NLS can be, for example, SV40 large T antigen NLS, nucleoplasmin NLS, or any sequence in Table 1. Other NLSs are described in, for example, Konermann et al., Cell 173:665-676, 2018; Cokol et al., EMBO Rep. 1(5):411-415 (2000); Freitas & Cunha, Curr Genomics 10(8): 550-557 (2009).

[0072] A polynucleotide encoding a Cas13 or Cas13d polypeptide can be operably linked to a promoter such as ubiquitous promoters (e.g., ubiquitin promoter), tissue-specific promoters, inducible promoters, and constitutive promoters.

[0073] A polynucleotide encoding a Cas13 or Cas13d polypeptide can be operably linked to a sequence that encodes one or more reporter polynucleotides. Reporter polynucleotides include, for example, fluorescent reporters. crRNA Molecules and Cas13 Repeat Arrays crRNA molecules can comprise a Cas13-specific direct repeat (DR) region, which forms a hairpin structure when transcribed. A Cas13-specific direct repeat (DR) region can be specific for Cas13a, Cas13b, Cas13c, or Cas13d. The hairpin structure enables Cas13 to bind to the crRNA, effectively forming a Cas13-crRNA complex. crRNA molecules can also comprise a protospacer region, which has homology to a target nucleic acid molecule (e.g., a C9ORF72 G4C2 repeat-containing RNA).

[0074] Cas13 repeat array polynucleotides can encode one or more crRNAs and one or more Cas13-specific direct repeats,

[0075] A Cas13 repeat array polynucleotide can comprise one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, or more) crRNAs and one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, or more) Cas13-specific direct repeats. The crRNAs can have homology to the same or different target sequences in the same target RNA or can bind to different target RNAs. The crRNAs can be designed to have homology to any sequence in a target RNA. In instances where two or more crRNAs are included in a Cas13 repeat array polynucleotide, the crRNAs can have the same or different length. The crRNAs can comprise about 20 to 40 nucleotides (e.g., about 20, 25, 26, 27, 28, 29, 30, 35, or 40 nucleotides).

[0076] A Cas13d repeat array polynucleotide can comprise about 1 or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8 or more) Cas13-specific direct repeats. A direct repeat can be a repetitive sequence within a CRISPR locus that are interspersed by short spacers. A direct repeat sequence can have homology to a trans-activating CRISPR RNA. A direct repeat facilitates formation of a crRNA: tracrRNA duplex. The sequence and secondary structure of Cas13-specific direct repeats can be dependent on the specific Cas13. For instance, Cas13d from different species can have different direct repeat sequences and / or secondary structures. Exemplary direct repeat sequences for Cas13d can be found at e.g., Konnerman et al., Cell 1 / 3:665-6 / 6 (2018). Cas13 specific direct repeat sequences can be about 30 to about 40 nucleotides in length (e.g., about 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. A Cas13 specific direct repeat can form a hairpin structure that interacts with a Cas13 polypeptide to form a complex.

[0077] A Cas13d-specific direct repeat can be, for example, CAAGUAAACCCCUACCAACUGGUCGGGGUUUGAAAC (SEQ ID NO: 15) (can be used as a DNA sequence in a vector; the corresponding RNA sequence would be CAAGTAAACCCCTACCAACTGGTCGGGGTTTGAAAC SEQ ID NO: 107) or GAAACACCGAACCCCTACCAACTGGTCGGGGTTTG (SEQ ID NO: 16) (DNA to be expressed in cells from a transfected vector) or AACCCCTACCAACTGGTCGGGGTTTGAAAC (SEQ ID NO: 106) or at least 80% identical (e.g. at least 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% identical) to SEQ ID NO: 15, 16, 106, or 107) crRNAs can be arranged in tandem and interspersed by direct repeats. For example, crRNAs can be positioned between two direct repeats. Provided herein are nucleic acid molecules comprising Cas13-specific repeat arrays or crRNA molecules. Also provided herein are vectors comprising nucleic acid molecules comprising Cas13-specific repeat arrays or crRNA. Nucleic acid molecules encoding Cas13-specific repeat arrays or crRNA can be operably linked to one or more promoters. Any suitable promoter can be used including, for example, a polymerase III promoter, such as a polymerase-3 U6 (U6:3) promoter.

[0078] In some aspects a crRNA has homology to a C9ORF72 G4C2 repeat-containing RNA. The target nucleic acid sequence (the portion of the gene targeted by a crRNA and a Cas13 nuclease) of a G4C2 repeat can comprise about 20 nucleotides. The target nucleic acid molecule can comprise less than about 20 nucleotides. The target nucleic acid molecule can comprise more than 20 nucleotides. The target nucleic acid molecule can comprise at least about 5, 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30 or more nucleotides. The target nucleic acid molecule can comprise at most: 5, 10, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30 or more nucleotides.

[0079] A crRNA can be about 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or more nucleotides in length. The crRNA sequence that hybridizes to the target nucleic acid molecule can have a length of at least about 6 nucleotides (nt). The crRNA sequence can be at least about 6 nt, at least about 10 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt or at least about 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about 45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt, from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, from about 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about 19 nt to about 25 nt, from about

[0080] 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about

[0081] 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some examples, the crRNA can comprise 30 nucleotides. In some examples, the spacer sequence can comprise 28 nucleotides. In some examples, the spacer sequence can comprise 29 nucleotides. In some examples, the spacer sequence can comprise 31 nucleotides. In some examples, the spacer sequence can comprise 32 nucleotides. In some examples, the percent complementarity between the crRNA sequence and the target nucleic acid is at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some examples, the percent complementarity between the crRNA and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In some examples, the percent complementarity between the crRNA and the target nucleic acid can be 100% over the six contiguous 5'-most nucleotides of the target sequence of the complementary strand of the target nucleic acid molecule. The percent complementarity between the crRNA and the target nucleic acid molecule can be at least 60% over about 20 contiguous nucleotides. The length of the spacer sequence and the target nucleic acid can differ by 1 to 6 nucleotides, which may be thought of as a bulge or bulges.

[0082] A crRNA sequence can be designed or chosen using a computer program. The computer program can use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, frequency of genomic occurrence (e.g., of sequences that are identical or are similar but vary in one or more spots as a result of mismatch, insertion or deletion), methylation status, presence of SNPs, and the like.

[0083] A Cas13-specific repeat array or crRNA can be operably linked to nucleic acid molecules that encode one or more reporter genes, such as a fluorescent reporter gene. A Cas13 protein can be fused or linked to one or more fluorescent proteins. Examples of one or more fluorescent proteins include blue / UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalamal , Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, and mTFP1 ), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mllKG, and mWasabi), yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, mKOK, mK02, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near-IR fluorescent proteins (for example, TagRFP657, IFP1.4, and iRFP), long stokes-shift proteins (for example, mKeima Red, LSS-mKate1 , and LSS- mKate2), photoactivatable fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP), photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGRI (green), KikGRI (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), fluorescein, rhodamine, photoswitchable fluorescent proteins (for example, Dronpa), or combinations thereof.

[0084] A purified crRNA or Cas13-specific repeat array is a polynucleotide preparation that is substantially free of cellular material, other types of polynucleotides, chemical precursors, chemicals used in synthesis of the polynucleotide, or combinations thereof. A polynucleotide preparation that is substantially free of chemical precursors, chemicals used in synthesis, etc. of the polynucleotide has less than about 30%, 20%, 10%, 5%, 1 % or more of other polynucleotides, chemical precursors, and / or other chemicals used in synthesis. Therefore, a purified polynucleotide (e.g., a crRNA or Cas13-specific repeat array) is about 70%, 80%, 90%, 95%, 99% or more pure.

[0085] In an aspect a crRNA targets exon 1a and / or intron 1 of a C9ORF72 gene. In an aspect exonl a and / or intron 1 comprises:

[0086] TGCTGCCCGGTTGCTTCTCTTTTGGGGGCGGGGTCTAGCAAGAGCAGGTG TGGGTTTAGGAGGTGTGTGTTTTTGTTTTTCCCA (SEQ ID NO: 56)

[0087] In an aspect, the target is Exon 1 a:

[0088] TGCTGCCCGGTTGCTTCTCTTTTGGGGGCGGGGTCTAGCAAGAGCAGGTG TGGGTTTAGGAG (SEQ ID NO: 108)

[0089] In an aspect, the target is Intron 1 : GTGTGTGTTTTTGTTTTTCCCA (SEQ ID NO:109).

[0090] In an aspect a crRNA comprises greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3

[0091] SEQ ID NO:1 CAAAAGAGAAGCAACCGGGCAGC

[0092] SEQ ID NO:2 CACACCTGCTCTTGCTAGACCCC

[0093] SEQ ID NO:3 CCCCCAAAAGAGAAGCAACCGGG

[0094] In an aspect, a crRNA comprises greater than 80% sequence identity to any of crRNAI through crRNA15 as shown in Fig. 5b.

[0095] An aspect provides a composition comprising one or more crRNA polynucleotides targeting exon 1 a and / or intron 1 of C9ORF72 (e.g., crRNA comprising greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3 or other suitable crRNA) and a Cas13 protein or a polynucleotide encoding a Cas13 protein. Another aspect provides two or more crRNA polynucleotides, wherein one or more crRNA polynucleotides target a sense RNA expressed from a C9ORF72 gene and one or more crRNA polynucleotides target an antisense RNA expressed from a C9ORF72 gene.

[0096] Vectors

[0097] Vectors such as plasmid or viral vectors (e.g. AAV vectors) can comprise nucleic acid molecules encoding a Cas13 polypeptide (e.g. any Cas13 polypeptides described herein) and / or a nucleic acid molecule encoding a Cas13-specific repeat array or crRNA (e.g. any Cas13-specific repeat array or crRNA described herein). Any suitable vectors can be used. A vector can comprise, for example, any genetic element including, without limitation, naked DNA, a phage, transposon, cosmid, episome, plasmid, bacteria, or a virus, which expresses, or causes to be expressed, a desired nucleic acid construct (e.g., a crRNA and / or a Cas13 nuclease). Thus, in one aspect, the vector is a non-pathogenic virus. A vector can be, for example, a non-replicating virus. In one aspect, a viral vector can be a retroviral vector, such as a lentiviral vector. A viral vector can be, e.g., an adeno- associated viral vector (AAV).

[0098] AAV is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length, including two 145-nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555- 564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381 -6388 (2004); the AAV-10 genome is provided in Mol. Then, 13(1 ): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928, incorporated herein by reference in its entirety. U.S. Pat. No. 9,434,928 also provides the sequences of the capsid proteins and a self-complementary genome. In one aspect, an AAV genome is a self-complementary genome. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging, and host cell chromosome integration are contained within AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep / 8, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.

[0099] A cap gene can be expressed from the p40 promoter and encodes the three capsid proteins, VPI, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. More specifically, after the single mRNA from which each of the VP1 , VP2 and VP3 proteins are translated is transcribed, it can be spliced in two different manners: either a longer or shorter intron can be excised, resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. The longer intron is often preferred and thus the 2.3-kb- long mRNA can be called the major splice variant. This form lacks the first AUG codon, from which the synthesis of VP1 protein starts, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon that remains in the major splice variant is the initiation codon for the VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak (translation initiation) context. This contributes to a low level of synthesis of the VP2 protein, which is actually the VP3 protein with additional N terminal residues, as is VP1 , as described in Becerra et al., (December 1985). “Direct mapping of adeno- associated virus capsid proteins B and C: a possible ACG initiation codon”. Proceedings of the National Academy of Sciences of the United States of America. 82 (23): 7919-23, Cassinotti et al., (November 1988). “Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein 1”. Virology. 167 (1 ): 176-84, Muralidhar et al., (January 1994). “Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity” Journal of Virology. 68 (1 ): 170-6, and Trempe J P, Carter (1988). “Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein”. J. Virol. 62 (9): 3356-63, each of which is herein incorporated by reference. A single consensus polyA site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0100] Each VP1 protein contains a VP1 portion, a VP2 portion and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein that is unique to the VP1 protein. The VP2 portion is the amino acid sequence present within the VP1 protein that is also found in the N-terminal portion of the VP2 protein. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is the C-terminal portion of the VP1 protein that is shared with the VP1 and VP2 proteins.

[0101] The VP3 protein can be further divided into discrete variable surface regions l-IX (VR-I-IX). Each of the variable surface regions (VRs) can comprise or contain specific amino acid sequences that either alone or in combination with the specific amino acid sequences of each of the other VRs can confer unique infection phenotypes (e.g., decreased antigenicity, improved transduction and / or tissue-specific tropism relative to other AAV serotypes) to a particular serotype as described in DiMatta et al., “Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9” J. Virol., Vol. 86 (12): 6947-6958, 2012, the contents of which are incorporated herein by reference.

[0102] 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) can be replaced with foreign DNA to generate AAV vectors. The rep and cap proteins can be provided in trans. Another significant feature of AAV is that it is an extremely stable and hardy 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 can be lyophilized. Advantageously, AAV-infected cells are not resistant to superinfection.

[0103] AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh74, and AAVrh.10. Production of pseudotyped rAAV is disclosed in, for example, WO2001083692. 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).

[0104] A recombinant adeno-associated virus (rAAV) vector can comprise in 5' to 3' direction a first AAV inverted terminal repeat (ITR) sequence, a promoter sequence, a nucleic acid molecule encoding a Cas13 polypeptide, a polyA sequence, a crRNA sequence having homology to exon 1a and / or intron 1 of C9ORF72 and / or G4C2 repeat, and a second AAV ITR sequence.

[0105] In some aspects, a promoter as described herein can be used to drive expression of a crRNA sequence. Therefore, a promoter, such as a type III RNA polymerase III promoter (e.g., U6 promoter) can be present in a vector in association with crRNA sequences. For example, a promoter can be present 5' to a crRNA sequence or 5' to a series of crRNA sequences, where more than one crRNA sequence is used. In some aspects, where more than one crRNA sequences are present, a promoter can be present in association with each and every crRNA, e.g., 5' to each crRNA sequence.

[0106] Other elements can include, for example, one or more nuclear localization signals (NLSs), one or more promoters, and one or more tags.

[0107] An aspect provides one or more vectors comprising a polynucleotide encoding a Cas13 protein and one or more crRNA polynucleotides targeting exon 1a and / or intron 1 of C9ORF72 (e.g. crRNA having greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3 or other suitable crRNA). One or more nuclear localization sequences (NLS) can be located 5’, 3’, or both 5’ and 3’ to the polynucleotide encoding the Cas13 protein. The vector can be a plasmid or a viral vector. The vector can be a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated virus (AAV) vector, a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), a synthetic vector, a vector encapsulated within a lipid nanoparticle, or a vector complexed with a polymer. The vector can be an adeno-associated virus (AAV) vector, a naturally occurring AAV vector, or an engineered AAV vector. The vector can be AAV-1 , AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 , AAV-12, AAV-13, AAV-anc80, AAV rh.74, AAVrh.10, AAVPHP.B, or AAVPHP.eB. The vector can comprise three major viral proteins: VP1 , VP2 and VP3. The vector can comprise one or more AW ITR sequences.

[0108] The one or more ITR sequences can be selected from AAV1 ITR sequences, AAV2 ITR sequences, AAV4 ITR sequences, AAV5 ITR sequences, AAV6 ITR sequences, AAV7 ITR sequences, AAV8 ITR sequences, AAV9 ITR sequences, AAV 10 ITR sequences, AAV 11 ITR sequences, AAV12 ITR sequences, AAV13 ITR sequences, AAVrh74 ITR sequences, AAVrh.10 ITR sequences or any combination thereof. The vector can lack rep and cap genes. The polynucleotide encoding a Cas13 protein and one or more crRNA polynucleotides can be operably linked to one or more promoters. The promoter can be a chicken [3-actin hybrid (CBh), U6, U7, tRNA, H1 , minimal CMV, synapsin, or T7 promoter.

[0109] Localization and Export Signals

[0110] In an aspect, a Cas13 protein can further comprise one or more localization signals (e.g., a nuclear localization signal) or one or more export signals. For example, a Cas13 protein can be present in a fusion protein along with one or more localization signals.

[0111] A nuclear localization signal (NLS) can drive a protein to the cell nucleus through the Nuclear Pore Complex and can be used to improve delivery efficiency. An NLS can generally comprise one or more short sequences of positively charged amino acids such as lysine or arginine. Examples of NLSs are shown in Table 1 .

[0112] Table 1 Nuclear Localization Signals ; Other non- VHSHKKKKIPTSPTFTTPKTLTLRRQPKYPRKSAPRRNKLDHY SEQ i classical i ID NO:37

[0113] Nucleic acid molecules encoding one or more NLSs can occur 5' to the nucleic acid molecule encoding the Cas13 polypeptide, 3' to the nucleic acid molecule encoding the Cas13 polypeptide, or both.

[0114] Promoters and Enhancers

[0115] A vector can comprise a promoter and / or an enhancer. A promoter or promoter sequence controls the initiation and rate of transcription of a coding sequence, such as a gene or a transgene. Promoters can be, for example, constitutive, inducible, repressible, or tissue-specific. Promoters can contain genetic elements for binding of regulatory proteins and molecules such as RNA polymerase and transcription factors. In some aspects, the promoter is a viral promoter, e.g., a CMV, HIV, adenovirus, or AAV promoter. Any suitable promoter can be used, such as a cytomegalovirus early enhancer / chicken P-actin (CAG) promoter, Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a p-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter (e.g., GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGA GAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGA CGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGG ACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT GTGGAAAGGAC (SEQ ID NO:42)), an H1 promoter, a ubiquitous chicken p-actin hybrid (CBh) promoter, EFla promoter, Ubc promoter, human p-actin promoter, TRE promoter, Ac5 promoter, polyhedrin promoter, CaMKIla promoter, Gall promoter, TEF1 promoter, GDS promoter, ADH1 promoter, Ubi promoter, a-1 -antitrypsin (hAAT) promoter, or small nuclear RNA (U 1 a or U 1 b) promoter.

[0116] In some aspects, the promoter is used together with at least one enhancer to increase the transcription efficiency. Non-limiting examples of enhancers include an interstitial retinoid-binding protein (IRBP) enhancer, an RSV enhancer or a CMV enhancer. An enhancer can increase the expression of a target sequence. A promoter / enhancer is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. An enhancer and / or promoter can be endogenous or exogenous (i.e., heterologous) An endogenous enhancer / promoter is naturally linked with a particular gene or nucleic acid sequence in the genome. An exogenous enhancer / promoter added or linked to a gene or nucleic acid sequence by genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. Examples of enhancer / promoters include a PDE promoter plus IRBP enhancer or a CMV enhancer plus U1a promoter. Enhancers can operate from a distance and irrespective of their orientation relative to the location of an endogenous or heterologous promoter. Therefore, an enhancer operating at a distance from a promoter is operably linked to that promoter irrespective of its location in the vector or its orientation relative to the location of the promoter.

[0117] Operably linked refers to the expression of a gene (i.e., a transgene) that is under the control of a promoter. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.

[0118] In an aspect, synthetic introns can be used to control gene expression by strategically inserting artificial intron sequences into a gene, which can significantly increase the level of protein produced by that gene due to intron-mediated enhancement. (IME). The presence of introns during RNA splicing process can boost mRNA stability and translation efficiency, leading to higher protein expression levels. Therefore, a synthetic intron can be used in place of or in addition to a promoter to control gene expression. Examples of useful synthetic introns include SV40 intron, CMV intron, hBG intron, and MTERFD3-derived intron.

[0119] Tags

[0120] A vector, such as a recombinant rAAV vector, can further comprise one or more tags, such as an epitope tag. For example, a human influenza hemagglutinin (HA) epitope tags can be present. An HA-tag can be encoded by TAC CCA TAC GAT GTT CCA GAT TAC GCT (SEQ ID NO: 12) or TAT CCA TAT GAT GTT CCA GAT TAT GCT (SEQ ID NO: 13). An HA epitope tag amino acid sequence can be YPYDVPDYA (SEQ ID NO: 14) or HA (YPYDVPDYAC; SEQ ID NO:5). Other suitable epitope tags can be used such as V5 epitope tag (GKPIPNPLLGLDST; SEQ ID NO:7), V5 E-tag (GAPVPYPDPLEPR; SEQ ID NO:8), VSV-g (YTDIEMNRLGK; SEQ ID NO:9), FLAG (DYKDDDDK; SEQ ID N0:4), tandem FLAG-tag, triple FLAG tag, tandem HA tag, triple HA tag (3*HA), sextuple Histidine tag (6xHIS) (HHHHHHH; SEQ ID NO:10), 6xHis HSV (QPELAPEDPEDC; SEQ ID NO: 11 ), biotin, c-MYC, Glutathione-S-transferase (GST) tag, Strep-tag, Strep-tag II, S-tag, natural histidine affinity tag (HAT), Calmodulin-binding peptide (CBP) tag, Streptavidin-binding peptide (SBP) tag, Chitin-binding domain, Maltose-binding protein (MBP), myc tag (EQKLISEEDLC; SEQ ID NO:6), or derivatives or combinations thereof.

[0121] PolyA

[0122] A vector, such as a recombinant rAAV vector, can comprise a polyadenylation (polyA) sequence. Any polyA sequence known in the art can be used. Non-limiting examples of polyA sequences include, but are not limited to, a bovine growth hormone (BGH) polyA sequence, a retinol dehydrogenase 1 (RDH1 ) polyA sequence, an SV40 polyA sequence, a SPA49 polyA sequence, a SNRP-TK65 polyA sequence, a sNRP polyA sequence, or a TK65 polyA sequence.

[0123] Bacterial Plasmids

[0124] In some aspects, rAAV vectors can be contained within a bacterial plasmid to allow for propagation of the rAAV vector in vitro. Therefore, provided herein are bacterial plasmids comprising any of the rAAV vectors described herein. A bacterial plasmid can further comprise an origin of replication sequence, an antibiotic resistance gene, a prokaryotic promoter, or a combination thereof.

[0125] Target RNA and Methods of Modifying a Target RNA in a Cell

[0126] Target RNA can be any G4C2 repeat containing mRNA molecules endogenous or exogenous to a eukaryotic cell. A Cas13-specific repeat array can include one or more crRNAs (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, or more) that can specifically hybridize with the same target RNA or at least two different target RNAs (e.g., 2, 3, 4, 5, 6, 7, 8, or more).

[0127] Methods are provided for modifying a target RNA in a cell. The methods can include introducing a nucleic acid molecule encoding a Cas13 polypeptide (e.g., any of the Cas13 polypeptides described herein) and a crRNA or Cas13-specific repeat array (e.g., any of the crRNA molecules or Cas13-specific repeat arrays described herein) into the cell. Nucleic acid molecules encoding a Cas13protein, crRNA, or Cas13-specific repeat arrays can be introduced into the cell in the same nucleic acid molecule or in different nucleic acid molecules.

[0128] A method can comprise contacting, via, e.g., transfection, the cell with (a) a nucleic acid molecule encoding a Cas13 polypeptide, and (b) a crRNA or a Cas13-specific repeat array comprising one or more crRNAs and one or more Cas13-specific direct repeats, wherein the one or more crRNAs can specifically hybridize with the target RNA. In some aspects a nucleic acid molecule encoding a Cas13 polypeptide can be introduced by a first vector and a Cas13-specific repeat array or a crRNA can be introduced by a second vector.

[0129] Pharmaceutical Compositions

[0130] In an aspect, pharmaceutical compositions comprising any of the isolated polynucleotides, vectors, rAAV vectors, rAAV viral vectors, and or crRNAs described herein are provided. An aspect provides a pharmaceutical composition comprising one or more crRNA polynucleotides targeting a G4C2 repeat. In an aspect, the one or more crRNA polynucleotides target exon 1 a and / or intron 1 of C9ORF72 (e.g., crRNA comprising greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3 or other suitable crRNA). A pharmaceutical composition can also be a Cas13 protein or a polynucleotide encoding a Cas13 protein, any of the vectors, rAAV vectors, rAAV viral vectors and / or crRNAs described herein; and a pharmaceutical excipient, or combinations thereof.

[0131] A pharmaceutical composition can be formulated by any suitable method, which include but are not limited to contacting the active ingredients (e.g., viral particles or recombinant vectors) with an excipient and / or additive or other accessory ingredient, dividing or packaging the product to a dose unit. Vectors such as viral particles can be formulated with desirable features, e.g., increased stability, increased cell transfection, sustained or delayed release, biodistributions or tropisms, modulated or enhanced translation of encoded protein in vivo, and the release profile of encoded protein in vivo.

[0132] Therefore, pharmaceutical compositions can comprise, for example, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transduced with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics, or combinations thereof. A pharmaceutical composition can be formulated as a nanoparticle such as a self-assembled nucleic acid nanoparticle.

[0133] A pharmaceutical composition can be prepared, packaged, and / or provided in bulk, as a single unit dose, and / or as a plurality of single unit doses. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Formulations can include one or more excipients and / or additives, each in an amount that together increases the stability of the viral vector, increases cell transfection or transduction by the viral vector, increases the expression of viral vector encoded protein, and / or alters the release profile of viral vector encoded proteins. In some aspects, a pharmaceutical composition can comprise an excipient and / or additives. Non limiting examples of excipients and / or additives include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, or combinations thereof.

[0134] A pharmaceutical composition can comprise a cryoprotectant, which is an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0135] A pharmaceutically acceptable carrier is any standard pharmaceutical carrier, such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. A pharmaceutical composition also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0136] Methods of Use and Methods of Treatment

[0137] Provided herein is the use of a composition or pharmaceutical composition for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, by administering or contacting the cell, tissue, organ, animal, or subject with a therapeutic effective amount of the composition or pharmaceutical composition. In one aspect, the subject is a mammal such as a human. A subject is not limited to a specific species and includes non-human animals subject to diagnosis or treatment or animal models, including, without limitation, simian, murine, rat, canine, or leporid species, as well as other livestock, sport animals, or pets.

[0138] An aspect provides methods of preventing or treating a neurological disorder or alleviating one or more symptoms of a neurological disorder by administering to a subject at least one therapeutically effective amount of any one of the pharmaceutical compositions disclosed herein. In some aspects the neurological disorder can be amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), progressive bulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, Kennedy's disease, and post-polio syndrome. Symptoms of these disorders can involve muscle weakness, muscle cramps and twitches, speech difficulties, uncontrollable emotions, fatigue, weight loss, difficulty using arms and legs, tremors, hand weakness, slowed movement, and swallowing problems. In some aspects, the disclosure provides methods of preventing or treating diseases or disorders caused by G4C2 repeat containing mRNA molecules.

[0139] A subject to be treated using the methods, compositions, pharmaceutical compositions, rAAV vectors or rAAV viral vectors of the present disclosure can have any of the diseases and / or symptoms described herein.

[0140] The methods of treatment and prevention disclosed herein can be combined with appropriate diagnostic techniques to identify and select patients for the therapy or prevention.

[0141] An aspect provides methods for treating a neurodegenerative disorder comprising administering to the subject one or more crRNA molecules targeting exonla and / or intronl of C9ORF72 (e.g. crRNA having greater than 80% sequence identity to SEQ ID NO: 1 , 2, or 3 or other suitable crRNA), and a Cas13 protein or a polynucleotide encoding a Cas13 protein. The neurodegenerative disorder can comprises amyotrophic lateral sclerosis (ALS), frontotemporal dementia, or other neurological disorder. The amount of G4C2 repeat containing mRNA can be decreased and the amount of C9-L full length C9ORF72 mRNA is not affected. The amount of C9-L full length C9ORF72 mRNA can be reduced by less than 30, 20, 10, 5, or 1 %. The amount of G4C2 repeat containing mRNA can be decreased by 50%, 60%, 70%, 80%, 90%, 95%, 99% or more in a cell, a tissue, or a patient.

[0142] In an aspect, the amount of CBLN1 mRNA, CBLN2 mRNA, or CBLN1 mRNA and CBLN2 mRNA is reduced by about 5, 10, 20, 30, 40% or more in neurons. In an aspect, the formation of RNA foci in the brain can be reduced by about 5, 10, 20, 30, 40% or more. In an aspect V3 mRNA of C9ORF72 can be reduced by about 5, 10, 20, 30, 40, 50% or more.

[0143] In an aspect, the amount of one or more di-peptide repeat (DPR) proteins can be reduced in a cell or patient. C9ORF72 expansion to pathogenesis is based on repeat- associated non-ATG (RAN) translation, a type of unconventional translation. RAN translation of C9ORF72 results in DPRs translated from both the sense and antisense strands of the expanded hexanucleotide repeats. From the six reading frames that undergo RAN translation, five distinct DPRs are generated: glycine-alanine (GA) and glycine-arginine (GR) DPRs from the sense RNA strand; proline-alanine (PA) and prolinearginine (PR) from the antisense strand; and glycine-proline (GP) translated from both the sense and antisense strands.

[0144] In an aspect, a method for decreasing an amount of one or more dipeptide repeat proteins (DRPs) in a subject or a cell is provided. The method can comprise administering to the subject or cell: a fusion protein comprising a Cas13 protein and one or more nuclear localization signals or a polynucleotide encoding a fusion protein comprising a Cas13 protein and one or more nuclear localization signals; and one or more crRNA molecules targeting exonla and / or intron 1 of C9ORF72 having greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3, wherein the amount of one or more DRPs is reduced in the subject or cell. The amount of the one or more DRPs is decreased by 10, 20, 30, 40, 50% or more as compared to an untreated cell or subject. The one or more DRPs can be glycine-alanine (GA) DRP, glycine-arginine (GR) DRP, proline-alanine (PA) DRP, proline-arginine (PR) DRP and / or glycine-proline (GP) DRP. In an aspect, the one or more DRPs are glycine-proline (GP) DRP.

[0145] In an aspect, transcriptional deficits due to the neurodegenerative disorder can be at least partially reversed. In particular, the number of up- and down-regulated differentially expressed genes in a diseased cell, tissue, or patient can be decreased after treatment as compared to an untreated cell, tissue or patient. In an aspect, the up- and down-regulated differentially expressed genes are attributed to the repeat expansion.

[0146] An aspect provides a method of decreasing a quantity of G4C2 repeat containing mRNA or cleaving G4C2 repeat containing mRNA in a subject or a cell. The method can comprise administering to the subject or cell: a fusion protein comprising a Cas13 protein and one or more nuclear localization signals or a polynucleotide encoding a fusion protein comprising a Cas13 protein and one or more nuclear localization signals; and one or more crRNAs targeting exonla and / or intron 1 of C9ORF72 (e.g. crRNAs having greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3 or other suitable crRNA). A quantity of G4C2 repeat containing mRNA can be decreased and / or the G4C2 repeat containing mRNA can be cleaved.

[0147] The amount of G4C2 repeat containing mRNA can be decreased and the amount of C9-L full length C9ORF72 mRNA is not affected. The amount of C9-L full length C9ORF72 can be the same as a control cell, tissue or patient not treated with Cas13 and crRNA. The amount of C9-L full length C9ORF72 mRNA can be reduced by less than 30, 20, 10, 5, or 1 % as compared to a control not receiving treatment. The amount of G4C2 repeat containing mRNA can be decreased by 50%, 60%, 70%, 80%, 90%, 95%, 99% or more in a cell, tissue, or a patient as compared to a control not receiving treatment. The amount of CBLN1 mRNA, CBLN2 mRNA, or CBLN1 mRNA and CBLN2 mRNA, can be reduced by about 10, 20, 30, 40% or more in a cell, tissue, or patient as compared to a control not receiving treatment. An aspect provides a method of making a system for preventing or slowing the progression of one or more symptoms of a neurodegenerative disorder comprising combining one or more crRNA polynucleotides that target exon 1a and / or intron 1 of a C9ORF72 gene and a Cas13 protein or a polynucleotide encoding a Cas13 protein, e.g. a Cas13d protein.

[0148] An aspect provides methods of reducing a level of G4C2 repeat containing mRNA molecule in a cell or patient by contacting the host cell or patient with any one of the polynucleotides, vectors, proteins, and / or rAAV viral vectors disclosed herein. In some aspects the amount of G4C2 repeat containing mRNA is reduced by about 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% or more as compared to untreated cells, tissues, or patients. In some aspects, the host cell is in vitro, in vivo, or ex vivo. In some aspects, the host cell is derived from a subject. In some aspects, the subject suffers from a disorder caused by G4C2 repeat containing mRNA molecules.

[0149] In some aspects, a subject can also be administered a prophylactic immunosuppressant treatment regimen in addition to being administered an rAAV vector. An immunosuppressant treatment regimen can comprise administering at least one immunosuppressive therapeutic. Non limiting examples of immunosuppressive therapeutics include, Sirolimus (rapamycin), acetaminophen, diphenhydramine, IV methylprednisolone, prednisone, or any combination thereof. An immunosuppressive therapeutic can be administered prior to the day of administration of the rAAV vector and / or rAAV viral vector, on the same day as the administration of the rAAV vector and / or rAAV viral vector, or any day following the administration of the rAAV vector and / or rAAV viral vector.

[0150] As used herein, “treating” or “treatment” of a disease in a subject refers to (1 ) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of the present technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. An effective amount means a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, an effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions. In some aspects, the effective amount of an rAAV viral vector is the amount sufficient to result in a decrease in the amount of G4C2 repeat containing mRNA molecules. A skilled artisan will be able to determine appropriate amounts depending on these and other factors.

[0151] In some aspects, the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target subject and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount can comprise one or more administrations of a composition (e.g., 1 , 2, 3, 4, 5, 10 or more) depending on the aspect.

[0152] As used herein, the term “administer” or “administration” is the delivery of a substance to a subject such as an animal or human. Administration can be effected in one dose, continuously, or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration can vary with the composition used for therapy, the purpose of the therapy, as well as the age, health, or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and other animals, treating veterinarian.

[0153] Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. It is noted that dosage can be impacted by the route of administration. Non-limiting examples of dosages can be as low as 108vector genomes to as much as 1017vector genomes per administration.

[0154] In some aspects of the methods described herein, the number of vector particles (e.g., rAAV viral vectors) administered to the subject ranges from about 108to about 1017. In some aspects, about 109to about 1012. In some aspects, about 1010to about 1012, about 101 1to about 1013, about 1011to about 1012, about 1011to about 1014, about 1012to about 1016, about 1013to about 1016, about 1014to about 1015, about 5x1 1to about 5*1012, or about 1012to about 1013viral particles are administered to the subject.

[0155] In some aspects the number of viral particles (e.g., rAAV viral vectors) administered to the subject can be at least about 1010, or at least about 101 1, or at least about 1012, or at least about 1013, or at least about 1014, or at least about 1015, or at least about 1016, or at least about 1017viral particles.

[0156] In some aspects, the amount of viral particles in a composition, pharmaceutical composition, or the amount of viral particles administered to a patient can calculated based on the percentage of viral particles that are predicted to contain viral genomes.

[0157] In some aspects, rAAV viral vectors can be introduced to the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intra-aurally, intra-ocularly, or peri-ocularly, orally, rectally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intracisternally, intranervally, intrapleurally, topically, intralymphatically, intracisternally, intra-arterial, intracardiac, subventricular, epidural, intracerebral, intracerebroventricular, sub-retinal, intravitreal, intraarticular, intraperitoneal, intrauterine, or any combination thereof. In some aspects, the rAAV vectors, rAAV viral vectors, compositions, or pharmaceutical compositions of this disclosure are parenterally administered by injection, infusion, or implantation. In some aspects, the vectors, e.g., viral particles, are delivered to a desired target tissue, e.g., to the brain, spinal cord, or CNS, as non-limiting examples. In some aspects, delivery of vectors such as viral particles is systemic. The intracisternal route of administration involves administration of a drug directly into the cerebrospinal fluid of the brain ventricles. It could be performed by direct injection into the cisterna magna or via a permanently positioned tube. In some aspects, vectors, such as rAAV viral vectors are administered intrathecally.

[0158] In some aspects, the vectors, such as rAAV viral vectors, show enhanced tropism for brain and cervical spine (e.g., AAV2, AAV9, AAV-Se1 / 2, BR1 N, AAVHSCs, AAV.CAP.B10. Other rAAV viral vectors can show enhanced tropism for other specific organs or tissues. In some aspects, vectors, such as rAAV viral vectors of the disclosure can cross the blood-brain-barrier (BBB).

[0159] Transgenic Organisms

[0160] Provided herein are transgenic organisms, which can include a non-human animal in where one or more of the cells of the organism includes a transgene. The organism can be a vertebrate or an invertebrate, such as an arthropod. A transgenic organism can have one or more recombinant nucleic acid molecules stably integrated into the genome of the organism, wherein the recombinant nucleic acid molecule encodes a Cas13 polypeptide (e.g. any of the Cas13 polypeptides described herein). In some aspects a transgenic organism can have two or more recombinant nucleic acid molecules stably integrated into the genome of the organism, comprising at least a first recombinant nucleic acid molecule that encodes a Cas13 polypeptide, and a second recombinant nucleic acid molecule that comprises a sequence that encodes a crRNA or a Cas13-specific repeat array.

[0161] A transgenic animal comprising a recombinant nucleic acid molecule encoding a Cas13 polypeptide can be identified based upon the presence of the nucleic acid sequence in its genome and / or expression of Cas13 in tissues or cells of the animal. A transgenic animal comprising a recombinant nucleic acid molecule encoding a crRNA can be identified based upon the presence of the nucleic acid sequence in its genome. A transgenic animal can be used to breed additional animals carrying the one or more transgenes. A transgenic animal can be heterozygous or homozygous for the one or more transgenes.

[0162] Methods for making transgenic animals include, for example, pronuclear microinjection retrovirus mediated gene transfer into germ lines, gene targeting into embryonic stem cells, electroporation of embryos, and in vitro transformation of somatic cells, such as cumulus or mammary cells, followed by nuclear transplantation.

[0163] Also provided herein are populations of cells isolated from a transgenic organism, as well as primary or cultured host cells, e.g., isolated host cells, engineered to include a nucleic acid molecule sequence that encodes one or more Cas13 proteins and / or crRNAs. The cells can be isolated from any of the transgenic animals described above. Also provided herein are methods of introducing transgenes described herein into a host cell (e.g., primary cells or cultured cells) by, for example, viral delivery.

[0164] The compositions and methods are more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. The terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.

[0165] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference as well as the singular reference unless the context clearly dictates otherwise. The term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105). All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The aspects illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of” can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claims. Thus, it should be understood that although the present methods and compositions have been specifically disclosed by aspects and optional features, modifications and variations of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.

[0166] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.

[0167] Whenever a range is given in the specification, for example, a temperature range, a time range, a composition, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements or steps that are included in the description herein can be excluded from the claimed compositions or methods.

[0168] In addition, where features or aspects of the compositions and methods are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[0169] The following are provided for exemplification purposes only and are not intended to limit the scope of the aspects described in broad terms above.

[0170] EXAMPLES

[0171] Example 1 : Programming RfxCas13d to target C9ORF72 The human C9ORF72 gene consists of 11 exons that are transcribed to three major transcript variants, V1 , V2 and V3, with the repeat expansion located in intron 1 between exons 1 a and 1 b (Fig. 1a) These three transcript variants produce two protein isoforms: a 222-amino acid short isoform, known as C9-S, which is translated from V1 , and a 481 -amino acid long isoform, C9-L, translated from V2 and V3 (Fig. 1a)10’33.

[0172] Both V1 and V3 encode the repeat expansion, while transcription of V2, the predominant variant that accounts for ~85-95% of the total C9ORF72 transcripts in the brain3435, is initiated downstream of the repeat (Fig. 1a)13. Because the partial loss of the C9ORF72 protein has been hypothesized as a potential source of the pathogenicity of the repeat expansion3637, we sought to use RfxCas13d to target either exon 1a or intron 1 to preferentially silence V1 and V3 (Fig. 1a), a strategy we expected would spare V2, a major source of the C9-L protein.

[0173] To facilitate the design of crRNAs to silence the repeat-containing RNA, we utilized the Cas13 Design Resource. We searched exon 1a and the intron 1 sequences flanking the hexanucleotide repeat before selecting the 15 highest ranking crRNAs for detailed testing (Fig. 5). We then created a dual-luciferase reporter screen to determine the ability of the crRNAs to target their respective sequences. This platform consists in part of a Renilla luciferase transgene whose 3’ untranslated region (UTR) is fused to a fragment of the C9ORF72 gene encoding 20 copies of the hexanucleotide repeat with 250- and 98- base pairs (bps) of the flanking upstream and downstream gene sequences, respectively (Fig. 1b). When complexed with a functional crRNA, RfxCas13d is expected to target the 3’ UTR of the Renilla luciferase transcript, which in turn is anticipated to decrease its expression.

[0174] In addition, because RfxCas13d has the potential to degrade non-specific RNAs via its frans-cleavage activity3839, we utilized firefly luciferase as a secondary reporter to measure collateral effects (Fig. 1c) which, by this approach, is expected to be decreased if frans-cleaved by RfxCas13d. As the extent of frans-cleavage has been observed to depend in some cases on the crRNA4041, we reasoned this strategy could aid in the identification of crRNA targeting sequences that minimize this effect.

[0175] To test the crRNAs, we transfected human embryonic kidney (HEK) 293T cells with each reporter plasmid and an expression vector encoding a RfxCas13d variant with two NLS sequences, one attached to the N-terminus and the other to the C-terminus, and one of the 15 candidate repeat-targeting crRNAs. From this screen, we found that all 15 crRNAs decreased Renilla luciferase by >80% at 48 hr post-transfection (P < 0.0001 ) and that 14 of the 15 crRNAs decreased it by at least 95% (P < 0.0001 ; Fig. 1d); however, we found that the majority of the crRNAs also triggered a reduction in the expression of firefly luciferase (Fig. 1 d), indicating they exerted collateral effects. This notwithstanding, we identified two crRNAs, crRNAs 13 and 1 , that decreased Renilla luciferase by ~95% (P < 0.0001 ) and had no significant effect on firefly luciferase compared to the controls (P > 0.05; Fig. 1d).

[0176] We next evaluated if RfxCas13d and three crRNAs, specifically crRNAs 13, 7 and 1 , could target C9ORF72 in HEK293T cells. Notably, among the six crRNAs found to fra s-cleavage firefly luciferase with efficiencies less than 50%, these three crRNAs possessed the most favorable targeting scores from the dual-reporter screen, a number we defined as the ratio of Renilla (i.e., target) to firefly (i.e. , collateral) luciferase (Fig. 6). To determine targeting, we used validated qPCR probes to measure V3, which served as the readout for the repeat-containing transcript, and the all-variant (all-V) pool, which consists predominately of V2.

[0177] Compared to cells transfected with RfxCas13d and a non-targeted crRNA, we measured by qPCR that each of the three repeat-targeting crRNAs decreased the relative abundance of V3 mRNA by ~90-95% (P < 0.0001 for all; Fig. 1e). Importantly, relative to the same controls, we measured that each of three crRNAs also had no significant effect on all-V (P < 0.05 for all; Fig. 1e), indicating that RfxCas13d preferentially targeted V3.

[0178] As an additional control, we tested in HEK293T cells two previously validated antisense oligonucleotides (ASOs) for C9ORF72: ASO-2, which binds intron 1 and selectively targets V1 and V342, and ASO-4, which binds exon 2 and thus targets the three main transcript variants42. As expected, qPCR revealed that only ASO-2 lowered V3 (P < 0.001 ) and that ASO-4 decreased both V3 and all-V (P < 0.001 for both; Fig. 1e), reinforcing the validity of our measurement methods.

[0179] We next evaluated if RfxCas13d could preferentially target V3 in a second human cell line, SH-SY5Y neuroblastoma cells. Compared to cells transfected with RfxCas13d and a non-targeting crRNA, we found by qPCR that crRNAs 13, 7 and 1 decreased V3 mRNA by 80-95% (P < 0.001 for all; Fig. 1f) and that these crRNAs either had no effect on all-V (crRNAs 13 and 1 ; P > 0.05) or a limited effect on it (crRNA-7; Fig. 1f). Consistent with our prior control measurements, ASO-2 was again found to only lower V3 in SH- SY5Y cells (P < 0.001 ), while ASO-4 decreased both V3 and all-V (P < 0.001 for both; Fig. 1f).

[0180] In sum, we find that a dual-luciferase screen that considers collateral effects can identity crRNAs for targeting C9ORF72, and that RfxCasI 3d can be used to preferentially silence V3, a transcript variant that carries the repeat expansion. Example 2 RfxCas13d targeting can decrease the G4C2 repeat -containing RNA and reduce RNA foci formation in a C9-ALS / FTD mouse model

[0181] We next sought to determine whether RfxCas13d could target the G4C2 repeat transcript in a mouse model of C9-ALS / FTD, specifically C9-BACexp mice43, which harbor a bacterial artificial chromosome (BAC) encoding the full-length human C9ORF72 gene with -100-1 ,000 copies of the hexanucleotide repeat. Similar to other mouse models of C9-ALS / FTD44, C9-BACexp mice do not manifest an overt motor phenotype, however, they do develop several hallmarks of the disorder, including the formation of RNA foci composed of the G4C2 repeat-containing RNA.

[0182] To deliver RfxCas13d, we used the adeno-associated virus (AAV) vector variant PHP.eB45which, when injected to the brain parenchyma, can transduce cells to a similar extent as AAV946but, in the experience of our laboratory, packages at higher titer. The hippocampus (HPC) and motor cortex (MC) of two-month-old C9-BACexp mice were thus injected with 2 x 101° genome copies (GCs) of an AAV-PHP.eB vector carrying a CBh- driven RfxCas13d variant with one of three repeat-targeting crRNA, crRNAs 13, 7 and 1 (i.e., AAV-PHP.eB-RfxCas13d-crRNA-13, -7 or -1 ), or a non-targeting crRNA (i.e., AAV- PHP.eB-RfxCas13d-NTG; Fig. 2a).

[0183] To facilitate the isolation of transduced nuclei for a more detailed analysis of RfxCas13d-mediated outcomes, we co-injected C9-BACexp mice with 2 x 1010GCs of a second AAV-PHP.eB vector encoding a CBh-driven EGFP variant fused to KASH (Klarsicht / ANC-1 / Syne-1 homology; AAV-PHP.eB-EGFP-KASH), a domain that promotes EGFP localization to the outer nuclear membrane47, which can enable the isolation of transduced nuclei by fluorescence-activated cell sorting (FACS)4849(Fig. 2a- b).

[0184] Based on an immunofluorescent analysis conducted at two-months post-injection, we observed strong EGFP-KASH expression at the sites of injection in C9-BACexp mice. Within the HPC and MC, we determined that -91 % and -53% of the cells positive for the pan-neuronal marker NeuN, respectively, were positive for EGFP-KASH (Fig. 2c and Fig. 7) and that -72% and -75% of the EGFP-KASH+cells in the HPC and MC, respectively, were positive for RfxCas13d by its hemagglutinin (HA) epitope (Fig. 8). Despite relying on the ubiquitous CBh promoter to drive its expression, EGFP-KASH was largely confined to NeuN+cells, as we observed limited expression in GFAP+astrocytes and Iba1+microglia in both the HPC and MC (Fig. 9).

[0185] We next used FACS to isolate EGFP-KASH+nuclei from the HPC and MC of injected C9-BACexp mice to determine the relative abundance of V3 and all-V by qPCR (Fig. 2b). Relative to EGFP-KASH+cells from mice injected with AAV-PHP.eB- RfxCas13d-NTG, we measured that each repeat-targeting crRNA effectively decreased V3 mRNA (Fig. 2d-e), with the most potent and consistent crRNA, the exon 1a-targeting crRNA-13, found to suppress V3 by ~48% in the HPC (P < 0.001 ; Fig. 2d) and ~52% in the MC (P < 0.001 ; Fig. 2e). Critically, all three repeat-targeting crRNAs were found to have no effect on all-V (P > 0.05 for each for the HPC and MC; Fig. 2d-e), indicating that RfxCas13d preferentially targeted V3 in vivo.

[0186] Using fluorescence in situ hybridization (FISH), we next determined whether targeting the repeat RNA with RfxCas13d affected RNA foci formation in C9-BACexp mice, specifically in the HPC where they develop in 40-60% of cells43. Given their improved targeting compared to crRNA-1 , we quantified RNA foci in EGFP-KASH+cells for mice only treated with RfxCasI 3d and crRNAs 13 and 7.

[0187] Compared to animals infused with control vector, C9-BACexp mice injected with AAV-PHP.eB-RfxCas13d-crRNA-13 and AAV-PHP.eB-RfxCas13d-crRNA-7 had a -37% and a -31 % decrease, respectively, in RNA foci positive for the G4C2 repeat RNA (P < 0.05 for crRNA 13; P = 0.06 for crRNA 7; Fig. 2f-g). Additionally, we measured a shift in the distribution of the average number of foci per cell for the treated animals, with C9- BACexp mice injected with AAV-PHP.eB-RfxCas13d-crRNA-13 and AAV-PHP.eB- RfxCas13d-crRNA-7 having an 11-13% increase in the in the percentage of EGFP- KASH+cells with no detectable foci and a two-fold decrease in the percentage of EGFP- KASH+ cells with 3-4 foci per cell compared to the controls (P < 0.05 for both crRNAs; Fig. 2h)

[0188] Thus, we find that RfxCasI 3d can be delivered to a C9-ALS / FTD rodent model by an AAV vector, where it could preferentially suppress the expression of the G4C2 repeatcontaining RNA, which we show could affect the formation of RNA foci in neurons. Example 3 High-fidelity RfxCasI 3d can target C9ORF72 with improved transcriptome-wide specificity

[0189] Because of their risk for inducing collateral effects in cells3839, high-fidelity forms of Cas13 that possess a reduced capacity to trans-cleavage non-target RNAs have been developed41. Given their potential for enhancing silencing, we determined the ability of these higher fidelity variants to target C9ORF72.

[0190] To this end, HEK293T cells were transfected with an expression vector encoding the native RfxCasI 3d or one of two high-fidelity variants, RfxCasI 3d-N2V7 and RfxCasI 3d-N2V8 (Fig. 3a) with either crRNA-13, the top-performing repeat-targeting crRNA from our first study, or a non-targeted crRNA. Based on qPCR, we found that both RfxCasI 3d-N2V7 and RfxCas13d-N2V8 decreased V3 mRNA in HEK293T cells by >90% compared to cells transfected with the same variant but with a non-targeted crRNA (P < 0.001 for both; Fig. 3b), an effect that was on par with our observations for the native RfxCas13d protein (Fig. 3b). Consistent with our earlier measurements, each high-fidelity variant was also found to have no effect on all-V compared to their respective controls (P > 0.05 for both; Fig. 3b). We then tested the targeting ability of RfxCas13d-N2V7 and - N2V8 in SH-SY5Y cells, where we found by qPCR that each high-fidelity variant decreased V3 mRNA by >90% (P < 0.001 for both; Fig. 3c) and exerted no significant effect on all-V (P > 0.05 for both; Fig. 3c), which altogether demonstrates the compatibility of the high-fidelity RfxCasI 3d variants with previously validated crRNAs.

[0191] To further interrogate the effect of targeting V3 by RfxCasI 3d, we used western blot to measure C9-L, the full-length C9ORF72 protein isoform, in HEK293T cells. Because our approach preferentially targets V3 and because C9-L is encoded by V2 (Fig. 1a), we expected that RfxCasI 3d would have no effect, or a minimal effect, on the relative abundance of C9-L. Consistent with this reasoning, no significant difference in the C9-L protein was measured in cells transfected with RfxCasI 3d, RfxCasI 3d-N2V7 or RfxCasI 3d-N2V8 with crRNA-13 relative to their controls (P > 0.05 for all; Fig. 10). C9- S, the short C9ORF72 protein isoform encoded by V3, could not be detected, and thus was not analyzed.

[0192] We next used RNA-seq to compare the transcriptome-wide specificities of RfxCasI 3d with its high-fidelity counterparts. When programmed with crRNA-13, the native RfxCasI 3d protein was found to perturb the expression of 1214 genes in HEK293T cells (>1.25-fold change [FC]; false discovery rate [FDR]-adjusted P < 0.05), while RfxCasI 3d-N2V8 was observed to affect only 230 genes, a ~5.8-fold decrease (Fig. 3d- e). Interestingly, we found that RfxCasI 3d-N2V7 influenced a similar number of genes (1355) as the native protein (Fig. 3e), indicating it possessed decreased transcriptomewide specificity relative to RfxCasI 3d-N2V8 when paired with crRNA-13.

[0193] Through an over-representation analysis of gene ontology (GO) and biological process (BP) terms, we next compared the themes enriched for the genes affected by the native RfxCasI 3d protein and RfxCasI 3d-N2V8. For RfxCasI 3d, we observed enrichment (FDR-adjusted P < 0.05) for nine biological functions (Fig. 3f), including RNA processing, protein-DNA complex assembly, intrinsic apoptotic signaling and chromatin assembly and disassembly, several of which were previously linked to RfxCasI 3d and its collateral effects. However, no enrichment (FDR-adjusted P > 0.05) was observed for these or any other themes in cells transfected with RfxCasI 3d-N2V8 (Fig. 3f). As expected, given our approach spares V2, the predominant C9ORF72 transcript variant, we observed no enrichment forfunction(s) related to the C9ORF72 protein for any group. Interestingly, unlike for RfxCasI 3d and RfxCasI 3d-N2V7, the majority (68%) of the genes affected by RfxCasI 3d-N2V8 were up-regulated (Fig. 3e); however, no themes (FDR- adjusted P > 0.05) were identified for these differentially expressed genes (DEGs).

[0194] Last for this analysis, we analyzed the shared DEGs between the native RfxCasI 3d protein, RfxCasI 3d-N2V7 and RfxCasI 3d-N2V8. In total, only 21 DEGs were found to be shared among all three variants, though 94 DEGs were shared between RfxCasI 3d and RfxCasI 3d-N2V8 (Fig. 3g), with a subsequent GO and BP term analysis found to reveal an enrichment in functions related to the positive regulation of cellular component organization and epithelial cell differentiation. These DEGs thus could represent effects related from targeting V3 in HEK293T cells.

[0195] As our RNA-seq study revealed that RfxCasI 3d-N2V8 possessed improved transcriptome-wide specificity compared to its native form and RfxCasI 3d-N2V7, we next determined its ability to target V3 in a more physiologically relevant model: iPSC-derived motor neuron-like cells from a 74-year-old female ALS patient with >1 5 copies of the hexanucleotide repeat in the C9ORF72 gene.

[0196] Starting from neural progenitor cells, we conducted a two-week differentiation protocol that led to the formation of HB9+and ChAT+neurospheres (Fig. 3h), which we treated with AAV-PHP.eB vector encoding a CBh-driven RfxCasI 3d-N2V8 variant with either crRNA-13 (i.e., AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13) or a non-targeted crRNA (i.e., AAV-PHP.eB-RfxCas13d-N2V8-NTG), with the AAV applied to cells at a multiplicity of infection (MOI) of ~2 x 106. To visualize transduction, we separately treated HB9+and ChAT+neurospheres with an AAV-PHP.eB vector encoding a CBh-driven EGFP at the same MOI.

[0197] After determining qualitatively that EGFP was expressed in the neuron-like clusters at 14 days post-transduction (Fig. 3i), we used qPCR to measure V3 and all-V at the same time point in the RfxCasI 3d-treated cells, which were analyzed as a bulk population. To account for variation in C9ORF72 expression between wells, we determined the relative ratio of V3 to all-V for each sample, finding that C9-ALS neurospheres treated with the PHP.eB-RfxCas13d-N2V8-crRNA-13 vector had an ~40% decrease in this ratio compared to cells treated with the non-targeting crRNA (P < 0.05; Fig. 3j). This ratio was also decreased in wild-type neurospheres treated with PHP.eB- RfxCasI 3d-N2V8-crRNA-13, though the measured effect was not as robust as in the C9- ALS cells, as we observed a ~22% decrease in the V3 to all-V ratio within the wild-type neurospheres (P < 0.05; Fig. 3j). As crRNA-13 is not selective for the hexanucleotide repeat expansion and can target the wild-type V3 transcript, these data demonstrate targeting in second iPSC-based cell line, which helps to support the reproducibility of this approach within this cell culture model.

[0198] RNA-seq was next used to determine the transcriptome-wide specificity of RfxCas13d-N2V8 in the wild-type neurospheres at 14 days post-transduction. When paired with crRNA-13, RfxCas13d-N2V8 was found to perturb the expression of only two genes (BMP3 and COL6A5; >1.25-FC to non-targeted crRNA; FDR-adjusted P < 0.01 ) both of which were down-regulated. Interestingly, BMP3 and COL6A5 are both involved in cartilage signaling, a finding that could benefit from further study, though we note that such an analysis is outside the scope of the current work.

[0199] We next asked if RfxCas13d-N2V8 could reverse, or partially reverse, a disease-associated transcriptional alteration in the C9-ALS neurospheres. To answer this, we used qPCR to measure the expression of CBLN1 , a member of the cerebellin family of proteins that normally contributes to the formation and function of synapses. CBLN1 has been found to be up-regulated in neuron-like cells derived from C9-ALS patients, a finding we corroborated by qPCR, which showed a —14-fold increase in its expression in the C9-ALS iMNs versus the wild-type cells (P < 0.0001 ; Fig. 3k).

[0200] At 14 days post-transduction, we measured by qPCR that the C9-ALS neurospheres treated with RfxCas13d-N2V8 had a -35% decrease in CBLN1 mRNA relative to cells treated with the non-targeted crRNA (P < 0.01 ; Fig. 3k), with no change in its expression observed in the wild-type cells treated with PHP.eB-RfxCas13d-N2V8- crRNA-13 (P > 0.05; Fig. 3k).

[0201] Finally, RNA-seq was used to determine the transcriptome-wide effects of RfxCas13d-N2V8 in the C9-ALS neurospheres. At 14 days post-transduction, we found that the C9-ALS iMNs treated with PHP.eB-RfxCasI 3d-N2V8-crRNA-13 had -18% fewer DEGs than cells treated with the non-targeted crRNA (>1 ,25-FC to wild-type iMNs; FDR- adjusted P < 0.01 ; Fig. 3I). For the DEGs whose expression was normalized by RfxCas13d-N2V8-mediated targeting, a term analysis revealed enrichment for functions related to RNA metabolism, including RNA splicing and mRNA splicing, the regulation of RNA and mRNA splicing, and the regulation of mRNA processing (Fig. 16).

[0202] Altogether, our results demonstrate that a high-fidelity form of RfxCas13d can be used to target V3 and that the most effective of these variants, RfxCas13d-N2V8, possesses improved transcriptome-wide specificity compared to the native enzyme. We also find that this high-fidelity variant can reduce the relative abundance of the G4C2 repeat RNA in neuron-like cells derived from a C9-ALS patient.

[0203] Example 5 High-fidelity RfxCas13d can target the G4C2 repeat RNA and revert transcriptional deficits in vivo

[0204] Given its improved targeting capabilities compared to the native protein, we next evaluated if RfxCas13d-N2V8 could target the G4C2 repeat-containing RNA in vivo. Mirroring our earlier study, we injected the HPC and MC of two-month-old C9-BACexp mice with 2 x 1010GCs of AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13 or AAV-PHP.eB- RfxCas13d-N2V8-NTG. Further, to facilitate the isolation of transduced nuclei for a detailed analysis of RfxCas13d-N2V8-mediated outcomes, each site was co-injected with 2 x 1010GCs of AAV-PHP.eB-EGFP-KASH (Fig. 4a).

[0205] Consistent with our earlier study, we observed robust EGFP-KASH expression in the brain, finding at four-weeks post-injection that ~87% and ~49% of the cells positive for NeuN in the HPC and MC, respectively, were positive for EGFP-KASH (Fig. 4b and Fig. 11) and that -81% and -78% of the EGFP-KASH* cells in the HPC and MC, respectively, were positive for RfxCas13d-N2V8 (Fig. 4b and Fig. 12). As before, we observed limited delivery to glial cells, which included GFAP+astrocytes and Iba1* microglia (Fig. 13).

[0206] Following their isolation by FACS, we used qPCR to measure targeting in EGFP- KASH* nuclei. Compared to controls, C9-BACexp mice injected with AAV-PHP.eB- RfxCas13d-N2V8-crRNA-13 had a two-fold decrease in V3 mRNA, both in the HPC and the MC (Fig. 4c-d; P < 0.01 for the HPC and P < 0.001 for the MC) and showed no difference in all-V in either region (Fig. 4c-d; P > 0.1 for both the HPC and MC), indicating that RfxCas13d-N2V8 preferentially targeted V3 in vivo.

[0207] Using FISH, we next determined if RfxCas13d-N2V8 affected RNA foci formation in C9-BACexp mice. Within the HPC, we measured a -37% decrease in RNA foci positive for the G4C2 repeat in EGFP-KASH* cells (Fig. 4e-f), in addition to a shift in the distribution of the number of foci per cell (Fig. 14a). We further quantified RNA foci in the MC, finding that C9-BACexp mice treated with RfxCas13d-N2V8 had a -27% decrease in foci per cell (Fig. 4e-f; P < 0.05) alongside a corresponding shift in their distribution (Fig. 14b).

[0208] We next evaluated if RfxCas13d-N2V8 decreased poly(GP), a DPR protein translated from the G4C2 repeat RNA and which is produced in C9-BACexp mice43. Using a Meso Scale Discovery immunoassay platform1452'54, we measured an -18% decrease in soluble poly(GP) in bulk hippocampal tissue from mice injected with PHP.eB-RfxCas13d-N2V8-crRNA-13 (P < 0.05; Fig. 4g). We also measured an -8% decrease in soluble poly(GP) within bulk cortical tissue (Fig. 15), though this result did not meet the threshold for statistical significance for our study (P > 0.05). We attribute this weaker effect to the more limited transduction observed in the MC, where only ~49% of the NeuN+cells in the injected area were found to be positive for EGFP-KASH versus the HPC, where -87% of the NeuN+cells in the injected area were EGFP-KASH positive (Fig. 11), a nearly two-fold difference. This is particularly meaningful given that the bulk tissue used for these analyses consists of a mixture of transduced and nontransduced cells, with the presence of the latter expected to dampen the measured effect, as observed in the case of the MC. This is in contrast to: (i) our analysis of the V3 mRNA, which was conducted on RNA isolated from EGFP-KASH+cell populations enriched by FACS, and (ii) our quantification of the RNA foci, which was limited to EGFP-KASH* cells.

[0209] Finally, we evaluated if RfxCas13d-N2V8 could reverse the transcriptional abnormalities that manifest in C9-BACexp mice26. To determine this, we injected the MC of C9-BACexp mice and their wild-type C57BL / 6J littermates with 2 x 1010GCs of AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13 or AAV-PHP.eB-RfxCas13d-N2V8-NTG alongside 2 x 101° GCs of AAV-PHP.eB-EGFP-KASH, which we used to facilitate the isolation of transduced nuclei. We then conducted RNA-seq on the EGFP-KASH* nuclei isolated from each animal by FACS.

[0210] To uncover the gene alterations that could be attributed to the repeat expansion, a pairwise DEG analysis was conducted between C9-BACexp mice and their C57BL / 6J littermates, which were also injected with the same vector formulations. In total, we identified 839 genes whose expression was altered in C9-BACexp mice (>1 ,2-FC; FDR- adjusted P < 0.05; Fig. 4h), 488 of which were up-regulated, with an average FC from wild-type of 1.83 (Fig. 4j), and 348 of which were down-regulated, with an average FC from wild-type of 1.71 (Fig. 4k). Among the DEGs, enrichment (P < 0.05) was observed for terms related to autophagy, RNA splicing and RNA regulation, each of which has been linked to C9ORF72 and / or the repeat expansion.

[0211] To determine whether RfxCas13d-N2V8 targeting had an effect on the C9- ALS / FTD-associated DEGs, we next conducted a pairwise analysis between C9-BACexp and C57BL / 6J mice injected with AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13. Of the 831 genes whose expression was altered in C9-BACexp mice injected with AAV-PHP.eB- RfxCas13d-N2V8-NTG, we found that only 73 of them (-9%) were affected (that is, deviated significantly from wild-type; >1.2-FC; FDR-adjusted P < 0.05) in C9-BACexp mice treated with crRNA-13 (Fig. 4i). In particular, the same 488 and 348 up- and down- regulated DEGs that deviated from wild-type by a FC of 1 .83 and 1.71 , respectively, were found to deviate from wild-type by a FC of only 1.18 and 1.16 in C9-BACexp mice injected AAV-PHP.eB-RfxCas13d-N2V8-crRNA-13 (Fig. 4j-k; P < 0.0001 for both compared to NTG).

[0212] Most notably, we found that 401 (82%) and 253 (73%) of the originally identified up- and down-regulated DEGs, respectively, reverted back to wild-type by a FC difference of at least 50%, while 247 (51 %) and 144 (41 %) of the DEGs reverted back to wild-type by a FC difference of at least 75% (Fig. 4j-k). These results thus demonstrate that RfxCas13d can at least partially reverse the transcriptional deficits that manifest in C9- BACexp mice.

[0213] In conclusion, we find that RfxCas13d and a high-fidelity version of it can be used to curb the production of the G4C2 repeat-containing RNA, while preserving normal C9ORF72 mRNA levels, further finding that RfxCas13d targeting can improve abnormalities associated with the repeat expansion. These results illustrate the potential of CRISPR-Cas13 technology for C9ORF72-linked ALS / FTD.

[0214] Example 7 Discussion

[0215] An abnormal expansion of a G4C2 repeat in the first intron of the C9ORF72 gene is the most common genetic cause of ALS78, accounting for up to 40% of familial forms of the disorder and 5-10% of all sporadic cases of the disease in the United States, Europe and Australia55. To date, three non-exclusive mechanisms have been proposed to explain the pathogenicity of the repeat expansion9-24. These include haploinsufficiency or loss of C9ORF72 protein functon9and / or acquired toxicity from the effects of bidirectionally transcribed repeat-containing RNAs13 15-18and / or their non-canonical translation to one of five DPR proteins19-22.

[0216] Given the role that the repeat transcripts may play in C9-ALS / FTD, gene silencing has emerged as a promising strategy for the disorder. Accordingly, both ASOs42’50’52’56’57and miRNAs34’58have both been used to target the G4C2 repeat-containing RNA. These strategies, however, possess limitations that could limit their effectiveness for C9- ALS / FTD. For example, ASOs have a transient lifecycle, which can require a lifetime of administrations to sustain a therapeutic effect59 60. This can lead to periods of diminished activity and also impose a physical burden on patients. Conversely, while modalities for RNA interference can be expressed for an extended period of time from a viral vector, they rely on endogenous RNA processing pathways that are predominately located in the cytoplasm61 -63which, in the case of C9-ALS / FTD, could limit their effectiveness, as both the repeat transcripts and the RNA foci are located predominately in the nucleus42’6465 One emerging technology whose properties could overcome these limitations is CRISPR-Cas13, particularly RfxCas13d, a class II, type VI CRISPR effector protein that is highly programmable, capable of cleaving target RNAs via its intrinsic RNase activity, is compact enough to be encoded within a single AAV vector for a potential single-dose treatment25and can be modified to access the nucleus2566to more directly engage with the repeat RNAs. In this proof-of-concept study, we demonstrate that RfxCas13d can be used to blunt the expression of the G4C2 repeat-containing RNA, both in induced motor neuron-like cells from a patient with C9-ALS and in C9-BACexp mice, a transgenic mouse model that harbors the full-length human C9ORF72 gene with ~100-1 , 000 copies of the hexanucleotide repeat43. Specifically, following its in vivo delivery to C9-BACexp mice via AAV, we found that RfxCas13d could be used to reduce the accumulation of RNA foci and at least partially reverse its transcriptional deficits.

[0217] After binding its target transcript, RfxCas13d undergoes a conformational rearrangement that can unlock its intrinsic ability to frans-cleave RNAs. This can result in the degradation of non-specific transcripts38’39 67, which can limit the implementation of the technology for certain applications. We hypothesized that a reporter screen that offered a unique readout for a collaterally trans-cleaved substrate could aid in the identification of crRNAs that minimize this effect. For this reason, we used a dual-reporter screen consisting of: (i) a Renilla luciferase transgene whose 3’ UTR was fused to a fragment of the C9ORF72 gene and thus was used as the target transcript and (ii) a constitutively expressed firefly luciferase, whose was expression was expected to be decreased if frans-cleaved. Though most crRNAs screened by this method triggered collateral effects, we nonetheless still identified several that effectively targeted Renilla luciferase, without significantly affecting the expression of firefly luciferase, a finding that supports the utility of dual-reporter systems for identifying crRNAs for Cas13 proteins that trigger relatively minimal collateral effects in cells.

[0218] Given concern for collateral effects, we also implemented into our platform higher fidelity forms of RfxCas13d with reduced frans-cleavage activity41, specifically RfxCas13d-N2V8 and RfxCas13d-N2V7. In addition to maintaining its ability to effectively target the G4C2 repeat RNA, we found that the high-fidelity RfxCas13d variant RfxCas13d-N2V8 had improved transcriptome-wide specificity compared to its native form, affecting the expression of a relatively limited number of genes (230 for RfxCasI 3d- N2V8 vs. 1214 for RfxCasI 3d). Further, using a GO and BP term analysis, we compared the enriched themes for the DEGs affected by RfxCasI 3d and RfxCasI 3d-N2V8. While we observed enrichment for nine biological functions for the native RfxCasI 3d protein, including terms linked to its collateral effects41, we measured no significant enrichment for these or any other themes for RfxCas13d-N2V8. Thus, coupling a dual-reporter system for discovering crRNAs that weighs collateral effects with high-fidelity forms of the technology can be an effective means for establishing CRISPR-Cas13-based platforms.

[0219] Using FISH, we determined that both RfxCas13d and RfxCas13d-N2V8, its high- fidelity counterpart, could reduce RNA foci positive for the G4C2 repeat RNA in C9- BACexp mice, demonstrating the ability for this technology to influence a hallmark of 09- ALS / FTD. Additionally, we tested whether RfxCas13d-N2V8 could reduce a DPR protein using specialized immunoassay platforms1452'54and found an ~18% reduction within the HPC of mice injected with PHP-eB-RfxCas13d-N2V8-crRNA-13. These findings are further corroborated with RNA-seq data showing RfxCas13d-N2V8 could at least partially revert a majority of the transcriptional abnormalities found to manifest in C9-BACexp mice.

[0220] For our proof-of-concept, AAV was delivered directly to the MC and HPC of C9- BACexp mice, where RfxCas13d was observed to be expressed primarily to neurons. While neurons are principally affected in C9-ALS / FTD, other cell types, including astrocytes and microglia, have been implicated in the pathogenesis of the disorder. Thus, additional studies are needed to determine the ideal AAV capsid and delivery route for C9-ALS / FTD to maximize transduction to all affected cell types. In addition, we note that our approach was used to target only the sense strand transcript; however, RfxCas13d is a multiplexable enzyme25that, in the future, can be configured to target both the sense and antisense repeat-containing RNAs, the latter of which is also thought to contribute to pathogenesis of C9-ALS / FTD15'18.

[0221] In conclusion, we demonstrate that RfxCas13d can be used to improve abnormalities associated with C9ORF72-linked ALS / FTD via its programming to preferentially degrade the G4C2 repeat-containing RNA transcribed from the C9ORF72 gene. Our results thus illustrate the potential of RNA-targeting CRISPR technologies for C9ORF72-linked ALS / FTD and adds to the growing number of examples that demonstrate the applicability of CRISPR-based approaches for ALS29’31 68'71.

[0222] Example 8 Methods

[0223] Plasmid construction

[0224] The plasmid pAAV-CBh-RfxCas13d-U6-crRNA vector was constructed as previously described29.

[0225] To construct pSV40-RLuc, the Renilla luciferase reporter plasmid, a 27-nucleotide spacer sequence was inserted between the Bbsl and Clal restriction sites of psiCHECK- 2 (Promega) to remove the firefly luciferase gene sequence and its promoter sequence. A fragment of the C9ORF72 gene encoding 20 copies of the G4C2 hexanucleotide repeat with 250- and 98-bps of the flanking upstream and downstream sequences from the C9ORF72 gene, respectively, was synthesized (Genscript) and then inserted between the Xhol and Notl restriction sites of the previously modified psiCHECK-2 plasmid, which were located in the 3’ UTR of the Renilla transgene.

[0226] To construct pHSV-TK-FLuc, the firefly luciferase reporter, a 32-nucleotide spacer sequence was inserted between the Bglll and Bbsl restriction sites of psiCHECK-2 to remove Renilla luciferase and its promoter sequence.

[0227] To construct pAAV-CBh-RfxCas13d-N2V7-U6-cRNA and pAAV-CBh-RfxCas13d- N2V8-U6-crRNA, the plasmids encoding the high-fidelity RfxCas13d variants, the native RfxCas13d gene sequence was PCR amplified from pAAV-CBh-RfxCas13d-U6-crRNA as two fragments using the primers: (1 ) Fusion-Ncol-Fwd-v2 and Fusion-N2V7-Rev or Fusion-N2V8-Rev; and (2) Fusion-N2V7-Fwd or Fusion-N2V8-Fwd with Fusion-Sacl- Reverse (Table 2). The resulting amplicons from the fusion PCR were then ligated into the Ncol and Sacl restriction sites of pAAV-CBh-RfxCas13d-U6-crRNA. crRNAs were cloned into respective plasmids as previously described29. Briefly, oligonucleotides encoding the crRNA sequences were synthesized (Integrated DNA Technologies) and incubated with T4 polynucleotide kinase (NEB) for 30 min at 37°C and subsequently heat inactivated at 95°C for 5 min. The duplexed oligonucleotides were then annealed by cooling to 4°C at a rate of -0.1 °C / s and subsequently ligated into the Bbsl restriction sites in pAAV-CBh-RfxCas13d-U6-cRNA, AAV-CBh-RfxCas13d-N2V7-U6- cRNA, and pAAV-CBh-RfxCas13d-N2V8-U6-cRNA.

[0228] Sanger sequencing (ACGT) was used to confirm the identity of all plasmids. All primer sequences are provided in Table 2.

[0229] Table 2. Primer sequences

[0230]

[0231] HEK293T and SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with 10% (v / v) fetal bovine serum (FBS; Gibco) and 1 % (v / v) antibiotic-antimycotic (Gibco) in a humidified 5% CO2 incubator at 37°C.

[0232] For the dual-luciferase screen, HEK293T cells were seeded onto a 96 well plate at a density of 2 x 104cells per well and transfected the following day with 100 ng of pAAV-CAG-RfxCas13d-U6-crRNA, 1 ng of pSV40-RLuc and 10 ng of pHSV-TK-FLuc using polyethylenimine (1 mg / mL; PEI).

[0233] At 72 hr post-transfection, HEK293T cells were lysed with Passive Lysis Buffer (Promega). Renilla and firefly luciferase luminescence was then measured using the Dual-Glo Luciferase Assay System (Promega) with a Synergy HTX Plate Reader (BioTek). Renilla and firefly luminescence values for each sample were normalized to values from cells transfected with RfxCas13d with a non-targeted crRNA.

[0234] For targeting V3 and all-V, HEK293T and SH-SY5Y cells were seeded onto a 24- well plate at a density of 2 x 105cells per well and transfected the following day with 1 pg of pAAV-CAG-RfxCas13d-U6-crRNA using Lipofectamine 3000 (ThermoFisher Scientific), according to the manufacturer’s instructions. qPCR

[0235] RNA from transfected cells or tissue was purified using the RNeasy Plus Mini Kit (Qiagen) and immediately converted to complementary DNA (cDNA) using an iScript cDNA Synthesis Kit (BioRad). qPCR was conducted on a 96-well plate using 30 ng of cDNA with TaqMan Fast Advanced Master Mix (ThermoFisher Scientific) and probes for human HPRT1 (Hs02800695_m1 ; ThermoFisher Scientific), mouse HPRT1 (MM01318743_M1 ; ThermoFisher Scientific), C9ORF72-all-V (Hs00376619_m1 ; ThermoFisher Scientific) and C9ORF72-V3 (Hs00948764_m1 ; ThermoFisher Scientific). Reaction volumes were 20 pL and probe concentrations were 150 nM and 250 nM for HPRT1 and C9ORF72-all-V and -V3, respectively. All qPCR reactions were conducted as recommended by the manufacturer’s instructions (ThermoFisher Scientific). Western blot

[0236] Cells were lysed by radioimmunoprecipitation assay (RIPA) buffer [0.2% IGEPAL CA-620, 0.02% SDS with Protease Inhibitor Cocktails (VWR Life Science, 97063-010)]. Protein concentration was then determined using the DC Protein Assay Kit (Bio-Rad). A total of 20 pg of protein per sample was electrophoresed by SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane in transfer buffer [20 mM Tris-HCI, 150 mM glycine, and 20% (v / v) methanol] for 30 min at 100 V using a Criterion Blotter (Bio-Rad). Membranes were blocked with 5% (v / v) blotting-grade blocker (Bio-Rad) in TBS (10 mM Tris-HCI and 150 mM NaCI, pH 7.5) with 0.05% Tween- 20 (TBS-T) for 1 hr and then incubated with primary antibody in blocking solution at 4 °C overnight. The following primary antibodies were used: Rabbit anti-[3-actin (1 :1000; Cell Signaling Technology, 4970S) and rabbit anti-C9ORF72 (1 :1000; Proteintech, 22637-1- AP).

[0237] After incubation with primary antibody, membranes were washed three times with TBS-T and incubated with goat anti-rabbit horseradish peroxidase conjugate (1 :4000; ThermoFisher Scientific, 65-6120) in blocking solution for 1 hr at room temperature (RT). Membranes were then washed three final times with TBS-T and treated with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Chemiluminescence was detected using a ChemiDoc XRS+ (Bio-Rad). Band intensities were quantified using Image Lab Software (Bio-Rad) and normalized to the reference protein in each sample. RNA sequencing Library construction was conducted by the Roy J. Carver Biotechnology Center (University of Illinois Urbana-Champaign, Urbana, IL) as previously described29. Briefly, RNAs were purified using the RNeasy Plus Mini Kit (Qiagen) and subsequently treated with DNase. RNAs were then converted into individually barcoded polyadenylated mRNA sequencing libraries using the Kapa Hyper Stranded mRNA library kit (Roche) and fused with unique dual indexes. Adaptor-ligated double-stranded cDNAs that were synthesized from the mRNA libraries were then PCR-amplified for eight cycles with KAPA HiFi DNA Polymerase (Roche), quantitated by PCR and subsequently pooled in equimolar concentration. The libraries were then sequenced by a NovaSeq 6000 (Illumina) using 2x150nt reads on an S1 lane. The FASTQ files that were generated from the sequencing were demultiplexed using the bcl2fastq v2.20 Conversion Software (Illumina). The quality of the demultiplexed FastQ files was then evaluated using FastQC (version 0.11.9).

[0238] Mouse RNAs were purified using the RNeasy Plus Mini Kit (Qiagen) and subsequently treated with DNase. Libraries were then prepared using the Universal RNA- Seq Kit (Tecan) with probes to deplete mouse rRNAs, with the quality of each sample assessed using a 5200 Fragment analyzer (Agilent). The final barcoded RNA-seq libraries were pooled in equimolar concentration and sequenced by a NovaSeq 6000 (Illumina) using 1x100nt reads on one S2 lane for 101 cycles. The FASTQ files that were generated from the sequencing were demultiplexed using the bcl2fastq v2.20 Conversion Software (Illumina). The quality of the demultiplexed FastQ files was then evaluated using FastQC (version 0.11.9).

[0239] Data analysis was conducted by the High-Performance Biological Computing Core (University of Illinois Urbana-Champaign, Urbana, IL). Salmons v1 .5.2 was used to quasimap reads to the transcriptome and to quantify the abundance of each transcript. T ranscriptomes were then indexed using the decoy-aware method with the entire genome file as a decoy. Gene-level counts were estimated on the basis of transcript-level counts using the “lengthScaled TPM” method from the tximport package72. Read counts were normalized using the trimmed mean of M-values (TMM) method from the edgeR package73, and differential gene expression was tested using the limma-trend method73.

[0240] Overrepresentation analyses on DEGs was c using NetworkAnalyst 3.074and Enrichr75, with term analyses conducted using the GO:BP databases76.

[0241] AAV packaging

[0242] AAV vectors were packaged as described77. Briefly, 2 x 107HEK293T cells were seeded onto 15-cm plates in DMEM supplemented with 10% (v / v) FBS and 1 % (v / v) antibiotic-antimycotic (ThermoFisher Scientific). At 16 hr after seeding, cells were transfected with 15 g of pAAV-CBh-RfxCas13d-U6-crRNA-13, -7, -1 and -NTG, pAAV- CBh-RfxCas13d-N2V8-U6-crRNA-13 and -NTG, pAAV-CBh-EGFP-KASH or pAAV-CBh- EGFP with 15 pg of pAAV-PHP.eB and 15 pg of pHelper using 135 pL of polyethylamine (1 pg / PL).

[0243] Cells were harvested by a cell scraper at five-days post-transfection and centrifuged at 4000g for 5 min at RT. Cells were then resuspended in lysis buffer (50 mM Tris-HCI and 150 mM NaCI, pH 8.0) and subsequently freeze-thawed three consecutive times using liquid nitrogen and a 37°C water bath. Afterwards, cells was incubated with benzonase (10 units per 1 mL of suspension; Sigma-Aldrich) for 30 min at a 37°C. The suspension was then centrifuged for 30 min at 18,500g at RT, with the ensuing supernatant layered on an iodixanol density gradient, as described. The iodixanol density gradient was centrifuged for 2 hr at 140,000g at 18°C and virus was extracted, washed three times with 15 mL of PBS with 0.001 % Tween-20 and concentrated to less than 250 pL using an Ultra-15 Centrifugal Filter Unit (Amicon). Viral titer was determined by qPCR using iTaq Universal SYBR Green Supermix (Bio-Rad).

[0244] Stereotaxic injections

[0245] All procedures were approved by the Illinois Institutional Animal Care and Use Committee (IACUC) at the University of Illinois Urbana-Champaign and conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

[0246] Two-month-old C9-BACexp mice [C57BL / 6J-Tg(C9ORF72_i3)112Lutzy / J; Jackson Laboratory, Stock #023099] were injected at stereotaxic coordinates anterior- posterior (AP) = 1.2 mm; medial-lateral (ML) = ±1.4 mm; and dorsal-ventral (DV) = 2 mm and 1.7 mm for the MC and stereotaxic coordinates AP = -1.9 mm; ML = ±1.3 mm; and DV = 1.8 mm and 1.3 mm for the HPC. 1 x 1010GCs of each vector was delivered per depth in 2.4 uL of saline solution for a total of 2 x 1010GCs per animal. Injections were performed using a drill and microinjection robot (NeuroStar).

[0247] Nuclei isolation

[0248] Neuronal nuclei were isolated from dissected brain as described78. Briefly, tissue from the MC and HPC were dissected and homogenized in 2 mL of Nuclei EZ Lysis Buffer (Sigma-Aldrich) using a KIMBLE Dounce Tissue Grinder (Sigma-Aldrich). After the addition of an additional 2 mL of Nuclei EZ Lysis Buffer, samples were incubated at RT for 5 min. Homogenized tissues were then centrifuged at 500g for 5 min. After removing the supernatant, nuclei were resuspended in 4 mL of Nuclei Suspension Buffer (PBS with 100 pg / mL of BSA) and centrifuged at 500g for an additional 5 min. The resulting pellet was then resuspended in 1 mL of Nuclei Suspension Buffer for FACS. Nuclei were strained through Round-Bottom Polystyrene Test Tubes with a 35 pm Cell Strainer Snap Cap (Falcon) and subjected to FACS using a BD FACSAria II Cell Sorter (Roy J. Carver Biotechnology Center Flow Cytometry Facility, University of Illinois Urbana-Champaign, Urbana, IL). Nuclei were collected in 350 pL of RNeasy Plus Kit Lysis Buffer (Qiagen) and at least 15,000 nuclei were collected for each sample.

[0249] Immunofluorescent analysis

[0250] Immunofluorescent analyses were performed as described68. Briefly, after their extraction, brains were fixed in 4% paraformaldehyde (PFA) overnight at 4°C. Fixed tissues were then sliced to 40 pM sagittal sections on a CM3050 S cryostat (Leica) and stored in cryoprotectant solution at -20°C. For staining, sections were washed three times in PBS for 15 min and incubated in blocking solution [PBS with 10% (v / v) donkey serum (Abeam) and 0.5% Triton X-100] for 2 hr at RT. Sections were then stained with primary antibodies in blocking solution for 72 hr at 4°C. After incubation, sections were washed three times with PBS and incubated for 2 hr with the secondary antibodies at RT. Sections were then washed three final times with PBS and mounted onto slides using VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories).

[0251] Sections were imaged using a Leica TCS SP8 confocal microscope (Beckman Institute Imaging Technology Microscopy Suit, University of Illinois Urbana-Champaign, Urbana, IL). Images were analyzed using Imaged imaging software by a blinded investigator.

[0252] Primary antibodies were rabbit anti-HA (1 :500; Cell Signaling Technology, 3724S) and Mouse anti-NeuN (1 :1000; Millipore Sigma, MAB377).

[0253] Secondary antibodies were donkey anti-mouse Alexa Fluor 647 (Jackson ImmunoResearch, 715-605-151 ) and donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152).

[0254] FISH

[0255] Tissue sections were incubated in blocking solution [DEPC PBS with 10% (v / v) donkey serum and 0.5% (v / v) Triton X-10] for 2 hr at RT. Sections were then washed three times with DEPC PBS and incubated in hybridization solution [2x DEPC saline- sodium citrate (SSC) with 50% (v / v) formamide, 10% (w / v) dextran sulfate, 50 mM sodium phosphate and 0.5% (v / v) Triton X-100] with 40 nM of the FISH probe for 2 hr in the dark at 37°C. Afterwards, sections were incubated in the dark at 66°C for 2 hr. Tissue sections were then washed once in 2x DEPC SSC and subsequently washed twice with 0.1x DEPC SSC at RT. Tissues were stained with DAPI, mounted onto slides with VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories) and stored at 37°C. Sections were then imaged using a Leica TCS SP8 microscope (Beckman Institute Imaging Technology Microscopy Suit, University of Illinois Urbana-Champaign, Urbana, IL). Images were analyzed using Imaged by a blinded investigator.

[0256] The sequence of the FISH probe used to detect the G4C2-containing RNA foci was: 5TYE563 / CCCCGGCCCCGGCCCC / 3TYE563. SEQ ID NO:43. The probe was previously described and validated42and custom-synthesized by Qiagen.

[0257] Differentiations

[0258] Neural progenitor cells (NPCs) derived from a 74-year-old female ALS patient with >145 copies of the hexanucleotide repeat expansion in the C9ORF72 gene were obtained from AXOL Bioscience (ax0073). Upon arrival, NPCs were immediately resuspended at a concentration of ~2.6 x 105cells per mL in Motor Neuron Maintenance Medium (AXOL Bioscience, ax0072) with 0.2 pM Compound E (Abeam, ab142164), 0.1 pM retinoic acid (Sigma-Aldrich, R2625) and 10 pM ROCK Inhibitor (Focus Biomolecules, 10-2301 ). The cells were then seeded on 24-well plate that was pre-coated with 0.5 mg / mL poly-D- Lysine (Sigma-Aldrich, P7405) and vitronectin (ThermoFisher, A14700) at a density of ~1.3 x 105cells per well.

[0259] For the differentiation, as specified by the manufactures instructions, a full volume medium exchange was conducted each day using Complete Motor Neuron Maintenance Medium with 0.2 pM Compound E (Abeam, ab142164), 0.5 pM retinoic acid (Sigma- Aldrich, R2625), 10 ng / mL recombinant human ciliary-derived neurotrophic factor (CNTF) (ax139888), 5 ng / mL recombinant human brain-derived neurotrophic factor (BDNF) (ax139800), 10 ng / mL recombinant human brain-derived neurotrophic factor (GDNF) (ax139855), and motor neuron maturation accelerator supplement (ax0179).

[0260] At day 14 post-seeding, cells were treated with AAV-PHP.eB vector encoding: (i) a RfxCas13d-N2V8 with either crRNA-13 or a non-targeted crRNA or (ii) EGFP in the presence of 1 % (v / v) antibiotic-antimycotic (Gibco). Vector was added to cells at an MOI of ~2 106.

[0261] Immunocytochemistry

[0262] Differentiated neuron-like cells were fixed in 200 pL of 4% (v / v) PFA for 15 min at RT and subsequently incubated with PBS with 0.1 % Triton X-100 for 10 min at RT, after which they washed three times with PBS. To block, cells were incubated in blocking solution [PBS with 10% (v / v) donkey serum (Abeam) and 0.1 % Tween 20] for 30 min at RT. The cells were then incubated with primary antibodies in blocking solution overnight at 4°C. After incubation, cells were washed three times with PBS and incubated with secondary antibodies in blocking solution for 1 hr at RT, after which they were washed three times with PBS.

[0263] Primary antibodies were mouse anti-HB9 (1 :200; Thermo Fisher Scientific, PA5- 23407), goat anti-ChAT (1 :50; EMD Millipore, AB144P), rabbit anti-lba1 (1 :500; Wako Pure Chemicals Industries, 019-19741 ), and chicken anti-GFAP (1 :1 ,000; Abeam, ab4674).

[0264] Secondary antibodies were donkey anti-goat Alexa Fluor 647 (Jackson ImmunoResearch, 705-605-147), donkey anti-mouse Cy3 (Jackson ImmunoResearch, 715-165-150) donkey anti-rabbit Alexa Fluor 647 (Jackson ImmunoResearch, 711-605- 152), Donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711 -165-152), and Donkey anti-chicken Alexa Fluor 647 (Jackson ImmunoResearch, 703-605-155).

[0265] Cells were imaged using a Zeiss Observer Z1 microscope (Beckman Institute Imaging Technology Microscopy Suit, University of Illinois Urbana-Champaign, Urbana, IL). Images were processed using ImageJ imaging software. poly(GP) immunoassay

[0266] Meso Scale Discovery (MSD) Gold 96-well Small Spot SA SECTOR plates (L45SA; MSD) were coated with 2 pg / mL of anti-GP antibody (24494-1 -AP; Proteintech) overnight at 4°C. The following day, plates were incubated with 150 pL of 2.5% (v / v) MSD Blocker A (R93BA; MSD) in PBS for 1 hr at RT with shaking (750 rpm) and then washed with PBS with 0.05% (v / v) Tween-20 (PBS-T). Plates were then incubated with 200 pg of bulk tissue lysate for 75 min at RT with shaking (750 rpm), at which point they were washed three times with 150 pL of PBS-T and incubated with 30 pL of anti-poly(GP) detection antibody (2 pg / mL; Proteintech, 24494-1 -AP) for 1 hr at RT with shaking (750 rpm). Plates were then washed three final times with 150 pL of PBS-T and incubated with 150 pL MSD Read Buffer B (R60AM; MSD). Signal was acquired using the MESO QuickPlex SQ 120MM plate reader (MSD).

[0267] Statistical analysis

[0268] Statistical analysis was performed using GraphPad Prism 8. Unless otherwise noted, all measurements were compared using a one-tailed unpaired t-test.

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Claims

CLAIMS:What is claimed is:1 . A method for treating a neurodegenerative disorder comprising administering to the subject one or more crRNA molecules targeting exonl a and / or intron 1 of C9ORF72 and a Cas13 protein or a polynucleotide encoding a Cas13.

2. The method of claim 1 , wherein the neurodegenerative disorder comprises amyotrophic lateral sclerosis (ALS) or frontotemporal dementia.

3. The method of any of claims 1-2, wherein the amount of G4C2 repeat containing mRNA is decreased and the amount of C9-L full length C9ORF72 mRNA is not affected.

4. The method of any of claims 1-3, wherein the amount of full length C9ORF72 mRNA is reduced by less than 30, 20, or 10%.

5. The method of any of claims 1-4, wherein the amount of G4C2 repeat containing mRNA is decreased by 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.

6. The method of any of claims 1-5, wherein the amount of CBLN1 mRNA is reduced by about 20, 30, 40% or more in neurons.

7. The method of any of claims 1-6, wherein transcriptional deficits due to the neurodegenerative disorder is at least partially reversed.

8. The method of any of claims 1-7, wherein the one or more crRNA molecules have greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3.

9. A method of decreasing a quantity of G4C2 repeat containing mRNA and / or cleaving G4C2 repeat containing mRNA in a subject or a cell, the method comprising administering to the subject or cell:a fusion protein comprising a Cas13 protein and one or more nuclear localization signals or a polynucleotide encoding a fusion protein comprising a Cas13 protein and one or more nuclear localization signals; and one or more crRNA molecules targeting exonl a and / or intron 1 of C9ORF72 having greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3, wherein a quantity of the G4C2 repeat containing mRNA is decreased and / or the G4C2 repeat containing mRNA is cleaved.

10. The method of claim 9, wherein the amount of G4C2 repeat containing mRNA is decreased and the amount of C9-L full length C9ORF72 mRNA is not affected.11 . The method of any of claims 9-10, wherein the amount of G4C2 repeat containing mRNA is decreased by 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.

12. The method of any of claims 9-11 , wherein the amount of CBLN1 mRNA is reduced by about 20, 30, 40% or more.

13. The method of any of claims 9-12, wherein the one or more crRNA molecules have greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3.

14. A method of making a system for preventing or slowing the progression of one or more symptoms of a neurodegenerative disorder comprising combining one or more crRNA polynucleotides that target exon 1a and / or intron 1 of a C9ORF72 gene and a Cas13 protein or a polynucleotide encoding a Cas13 protein.

15. The method of any of claims 1-14, wherein the Cas13 protein is a Cas13d protein.

16. The method of any of claims 1-15, wherein the Cas13 protein is from Ruminococcus flavefaciens.

17. The method of any of claims 1-16, wherein the Cas13 protein is RfxCas13d-N2V7 or RfxCas13d-N2V8.

18. The method of any of claims 1 -14, wherein the Cas13 protein is PspCasI 3b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a,BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCasI 3b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dllrCas13d.

19. The method of any of claims 1 -18, wherein the Cas13 protein further comprises one or more a localization signals or one or more export signals.

20. The method of any of claims 1 -19, wherein the Cas13 protein further comprises one or more nuclear localization signals (NLS).21 . The method of claim 20, wherein the one or more NLSs are selected from SEQ ID NO:17-41.

22. The method of any of claims 1 -21 , wherein the Cas 13 protein additionally comprises one or more epitope tags.

23. The method of claim 22, wherein the epitope tag is FLAG (DYKDDDDK; SEQ ID NO:4), HA (YPYDVPDYAC; SEQ ID NO:5), myc (EQKLISEEDLC; SEQ ID NO:6), V5 (GKPIPNPLLGLDST; SEQ ID NO:7), E-tag (GAPVPYPDPLEPR; SEQ ID NO:8), VSV-g (YTDIEMNRLGK; SEQ ID NO:9), 6xHis (HHHHHHH; SEQ ID NQ:10), HSV (QPELAPEDPEDC; SEQ ID NO:11 ), or combinations thereof.

24. The method of any of claims 1-23, wherein the Cas13 protein further comprises one or more fluorescent proteins.

25. The method of claim 24, wherein the one or more fluorescent proteins are selected from: blue / UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalamal , Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, and mTFP1), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, and mWasabi), yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, mKOK, mK02, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near- IR fluorescent proteins (for example, TagRFP657, IFP1.4, and iRFP), long stokes-shift proteins (for example, mKeima Red, LSS-mKate1 , and LSS-mKate2), photoactivatable fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP), photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGRI (green), KikGRI (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), fluorescein, rhodamine, photoswitchable fluorescent proteins (for example, Dronpa), or combinations thereof.

26. A CRISPR RNA (crRNA) comprising greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3.

27. The crRNA of claim 26, wherein the crRNA is less than 30 nucleotides in length.

28. The crRNA of claim 26, wherein the crRNA targets exon 1a and / or intron 1 of a C9ORF72 gene.

29. A composition comprising one or more crRNA polynucleotides comprising SEQ ID NO: 1 , 2, or 3 and a Cas13 protein or a polynucleotide encoding a Cas13 protein.

30. The composition of claim 29, comprising two or more crRNA polynucleotides, wherein one or more crRNA polynucleotides target a sense RNA strand expressed froma C9ORF72 gene and one or more crRNA polynucleotides target an antisense RNA strand expressed from a C9ORF72 gene.31 . The composition of claim 30, wherein the two or more crRNA polynucleotides target exon 1a or intron 1 of the C9ORF72 gene.

32. The composition of any of claims 30-31 , wherein the Cas13 protein is a Cas13d protein.

33. The composition of any of claims 30-32, wherein the Cas13 protein is from Rumi nococcus flavefaciens.

34. The composition of any of claims 30-33, wherein the Cas13 protein is RfxCas13d- N2V7 or RfxCas13d-N2V8.

35. The composition of any of claims 30-32, wherein the Cas13 protein is PspCas13b, PspCas13b Truncation, AdmCas13d, AspCas13b, AspCas13c, BmaCas13a, BzoCas13b, CamCas13a, CcaCas13b, Cga2Cas13a, CgaCas13a, EbaCas13a, EreCas13a, EsCas13d, FbrCas13b, FnbCas13c, FndCas13c, FnfCas13c, FnsCas13c, FpeCas13c, FulCas13c, HheCas13a, LbfCas13a, LbmCas13a, LbnCas13a, LbuCas13a, LseCas13a, LshCas13a, LspCas13a, Lwa2cas13a, LwaCas13a, LweCas13a, PauCas13b, PbuCas13b, PgiCas13b, PguCas13b, Pin2Cas13b, Pin3Cas13b, PinCas13b, Pprcas13a, PsaCas13b, PsmCas13b, RaCas13d, RanCas13b, RcdCas13a, RcrCas13a, RcsCas13a, RfxCas13d, UrCas13d, dPspCas13b, PspCas13b_A133H, PspCas13b_A1058H, dPspCasI 3b truncation, dAdmCas13d, dAspCas13b, dAspCas13c, dBmaCas13a, dBzoCas13b, dCamCas13a, dCcaCas13b, dCga2Cas13a, dCgaCas13a, dEbaCas13a, dEreCas13a, dEsCas13d, dFbrCas13b, dFnbCas13c, dFndCas13c, dFnfCas13c, dFnsCas13c, dFpeCas13c, dFulCas13c, dHheCas13a, dLbfCas13a, dLbmCas13a, dLbnCas13a, dLbuCas13a, dLseCas13a, dLshCas13a, dLspCas13a, dLwa2cas13a, dLwaCas13a, dLweCas13a, dPauCas13b, dPbuCas13b, dPgiCas13b, dPguCas13b, dPin2Cas13b, dPin3Cas13b, dPinCas13b, dPprCas13a, dPsaCas13b, dPsmCas13b, dRaCas13d, dRanCas13b, dRcdCas13a, dRcrCas13a, dRcsCas13a, dRfxCas13d, or dllrCas13d.

36. The composition of any of claims 30-35, wherein the Cas13 protein further comprises one or more localization signals or one or more export signals.

37. The composition of any of claims 30-36, wherein the Cas13 protein further comprises one or more nuclear localization signals (NLS).

38. The composition of claim 37, wherein the one or more NLSs are selected from SEQ ID NO:17-41.

39. The composition of any of claims 30-38, wherein the Cas 13 protein additionally comprises one or more epitope tags.

40. The composition of claim 39, wherein the epitope tag is FLAG (DYKDDDDK; SEQ ID NO:4), HA (YPYDVPDYAC; SEQ ID NO:5), myc (EQKLISEEDLC; SEQ ID NO:6), V5 (GKPIPNPLLGLDST; SEQ ID NOT), E-tag (GAPVPYPDPLEPR; SEQ ID NO:8), VSV-g (YTDIEMNRLGK; SEQ ID NO:9), 6xHis (HHHHHHH; SEQ ID NO:10), HSV (QPELAPEDPEDC; SEQ ID NO:11 ), or combinations thereof.41 . The composition of any of claims 30-40, wherein the Cas13 protein further comprises one or more fluorescent proteins.

42. The composition of claim 41 , wherein the one or more fluorescent proteins are selected from: blue / UV fluorescent proteins (for example, TagBFP, Azurite, EBFP2, mKalamal , Sirius, Sapphire, and T-Sapphire), cyan fluorescent proteins (for example, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, and mTFP1 ), green fluorescent proteins (for example, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, and mWasabi), yellow fluorescent proteins (for example, EYFP, Citrine, Venus, SYFP2, and TagYFP), orange fluorescent proteins (for example, Monomeric Kusabira-Orange, mKOK, mK02, mOrange, and mOrange2), red fluorescent proteins (for example, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, and mRuby), far-red fluorescent proteins (for example, mPlum, HcRed-Tandem, mKate2, mNeptune, and NirFP), near- IR fluorescent proteins (for example, TagRFP657, IFP1.4, and iRFP), long stokes-shift proteins (for example, mKeima Red, LSS-mKate1 , and LSS-mKate2), photoactivatable fluorescent proteins (for example, PA-GFP, PAmCherryl, and PATagRFP),photoconvertible fluorescent proteins (for example, Kaede (green), Kaede (red), KikGRI (green), KikGRI (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and PSmOrange), fluorescein, rhodamine, photoswitchable fluorescent proteins (for example, Dronpa), or combinations thereof.

43. A vector comprising a polynucleotide encoding a Cas13 protein and one or more crRNAs polynucleotides as set forth in SEQ ID NO:1 , 2, or 3.

44. The vector of claim 43, wherein one or more nuclear localization sequences (NLS) are located 5’, 3’, or both 5’ and 3’ to the polynucleotide encoding the Cas13 protein.

45. The vector of any of claims 43-44, wherein the vector is a plasmid or a viral vector.

46. The vector of any of claims 43-45, wherein the vector is a retroviral vector, lentiviral vector, adenoviral vector, adeno-associated virus (AAV) vector, a recombinant AAV (rAAV), a self-complementary recombinant AAV (scAAV), a synthetic vector, a vector encapsulated within a lipid nanoparticle, or a vector complexed with a polymer.

47. The vector of any of claims 43-46, wherein the vector is an adeno-associated virus (AAV) vector, a naturally occurring AAV vector, or an engineered AAV vector48. The vector of any of claims 43-47, wherein the vector is AAV-1 , AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 , AAV-12, AAV-13, AAV-anc80, AAV rh.74, AAVrh.10, AAVPHP.B, or AAVPHP.eB.

49. The vector any of claims 47-48, wherein the vector comprises three major viral proteins: VP1 , VP2 and VP3.

50. The vector of any of claims 47-49, wherein the vector comprises one or more AW ITR sequences.51 . The vector of claim 50, wherein the one or more ITR sequences are selected from AAV1 ITR sequences, AAV2 ITR sequences, AAV4 ITR sequences, AAV5 ITR sequences, AAV6 ITR sequences, AAV7 ITR sequences, AAV8 ITR sequences, AAV9 ITR sequences, AAV 10 ITR sequences, AAV 11 ITR sequences, AAV12 ITRsequences, AAV13 ITR sequences, AAVrh74 ITR sequences, AAVrh.10 ITR sequences or any combination thereof.

52. The vector of any of claims 46-51 , wherein the vector lacks rep and cap genes.

53. The vector of any of claims 43-52, wherein the polynucleotide encoding a Cas13 protein and the one or more crRNAs polynucleotides are operably linked to one or more promoters.

54. The vector of claim 53, wherein the one or more promoters are a chicken p-actin hybrid (CBh), U6, U7, tRNA, H1 , minimal CMV, synapsin, or T7 promoter.

55. A pharmaceutical composition comprising:(i) one or more crRNA polynucleotides comprising greater than 80% sequence identity to SEQ ID NO: 1 , 2, or 3;(ii) a Cas13 protein or a polynucleotide encoding a Cas13 protein; or the vector of any of claims 17-28; and(iii) a pharmaceutical excipient.

56. Use of the pharmaceutical composition of claim 55 to treat a neurological disease.

57. A method of decreasing an amount of one or more dipeptide repeat proteins (DRPs) in a subject or a cell, the method comprising administering to the subject or cell: a fusion protein comprising a Cas13 protein and one or more nuclear localization signals or a polynucleotide encoding a fusion protein comprising a Cas13 protein and one or more nuclear localization signals; and one or more crRNA molecules targeting exonl a and / or intron 1 of C9ORF72 having greater than 80% sequence identity to SEQ ID NO:1 , 2, or 3, wherein the amount of one or more DRPs is reduced in the subject or cell.

58. The method of claim 57, wherein the amount of the one or more DRPs is decreased by 5, 10, 20, 30, 40, 50% or more as compared to an untreated cell or subject.

59. The method of claim 57, wherein the one or more DRPs are glycine-alanine (GA)DRP, glycine-arginine (GR) DRP, proline-alanine (PA) DRP, proline-arginine (PR) DRP and / or glycine-proline (GP) DRP.

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