WO2021168216A1

WO2021168216A1 - Crispr/cas9 correction of mutations in dystrophin exons 43, 45 and 52

Info

Publication number: WO2021168216A1
Application number: PCT/US2021/018735
Authority: WO
Inventors: Yi Li MIN
Original assignee: The Board Of Regents Of The University Of Texas System
Priority date: 2020-02-21
Filing date: 2021-02-19
Publication date: 2021-08-26

Abstract

Duchenne muscular dystrophy (DMD), which affects 1 in 5,000 male births, is one of the most common genetic disorders of children. This disease is caused by an absence or deficiency of dystrophin protein in striated muscle. Here, three DMD mouse models are provided that can be used to test a variety of DMD exon skipping and refraining strategies. Compositions and methods for restoring the reading frame of exon 43, exon 45, and exon 52 deletion via CRISPR-mediated exon skipping and refraining are also provided. The compositions and method provided herein can be used to permanently correct genetic mutations.

Description

DESCRIPTION CRISPR/CAS9 CORRECTION OF MUTATIONS IN DYSTROPHIN EXONS 43, 45 AND 52 PRIORITY CLAIM [0001] The present application claims benefit of priority to U.S. Provisional Application Serial No. 62/979,790, filed February 21, 2020, the entire contents of which are hereby incorporated by reference. FIELD [0002] The present disclosure relates to the fields of molecular biology, medicine and genetics. More particularly, the disclosure relates to the use of genome editing to correct mutations causing genetic diseases such as Duchenne muscular dystrophy (DMD). SEQUENCE LISTING [0003] The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing was created on February 1, 2021, is named UTFDP3757WO_SEQLIST.txt and is ~116 kilobytes in size. FEDERAL FUNDING SUPPORT CLAUSE [0004] This invention was made with government support under grant nos. HL130253 and AR-067294 awarded by National Institutes of Health. The government has certain rights in the invention. BACKGROUND [0005] Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Duchenne muscular dystrophy (DMD) is one of the most severe forms of MD that affects approximately 1 in 5000 boys and is characterized by progressive muscle weakness and premature death. Cardiomyopathy and heart failure are common, incurable and lethal features of DMD. The disease is caused by mutations in the gene encoding dystrophin (DMD), a large intracellular protein that links the dystroglycan complex at the cell surface with the underlying cytoskeleton, thereby maintaining integrity of the muscle cell membrane during contraction. Mutations in the dystrophin gene result in loss of dystrophin function causing muscle membrane fragility and progressive muscle wasting. [0006] There remains a need in the art for treatments and cures for DMD. SUMMARY [0007] Provided herein is a nucleic acid comprising: a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence; wherein the spacer sequence comprises the sequence of SEQ ID NO: 60. In some embodiments, the scaffold sequence comprises the sequence of any one of SEQ ID NOs: 138-144. In some embodiments, the nucleic acid comprises one, two, three, four, or five copies of the sequence encoding the sgRNA. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA. In some embodiments, the nucleic acid comprises a promoter, wherein the promoter drives expression of the sgRNA. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, wherein the nucleic acid comprises a first promoter and expression of the first copy of the sgRNA is driven by the first promoter, wherein the nucleic acid comprises a second promoter and expression of the second copy of the sgRNA is driven by the second promoter, and wherein the nucleic acid comprises a third promoter and expression of the third copy of the sgRNA is driven by the third promoter. In some embodiments, the nucleic acid further comprises a sequence encoding a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes Cas9. In some embodiments, the Cas9 nuclease is a modified Cas9 nuclease. [0008] Also provided herein is a vector comprising a nucleic acid of the disclosure. In some embodiments, the vector is a plasmid. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the serotype of the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the serotype of the AAV vector is AAV9. In some embodiments, the AAV vector is replication-defective or conditionally replication defective. [0009] Also provided herein is a non-viral vector comprising a nucleic acid of the disclosure, wherein the non-viral vector comprises calcium phosphate, a liposome, a nanoparticle, and/or a lipid emulsion. [0010] Also provided herein is an AAV expression cassette comprising a first inverted terminal repeat (ITR), a first promoter, a nucleic acid of the disclosure, and a second ITR. In some embodiments, the AAV expression cassette further comprises a polyadenosine (polyA) sequence. In some embodiments, one or both of the first ITR and the second ITR are isolated or derived from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the expression cassette comprises three copies of the sequence encoding the sgRNA, wherein the nucleic acid comprises a first promoter and expression of the first copy of the sgRNA is driven by the first promoter, wherein the nucleic acid comprises a second promoter and expression of the second copy of the sgRNA is driven by the second promoter, and wherein the nucleic acid comprises a third promoter and expression of the third copy of the sgRNA is driven by the third promoter. [0011] Also provided herein is an AAV vector comprising a nucleic acid or an AAV expression cassette of the disclosure. In some embodiments, the AAV vector has the serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the serotype of the AAV vector is AAV9. In some embodiments, the AAV vector is replication-defective or conditionally replication defective. [0012] Also provided herein is a composition comprising a nucleic acid of the disclosure. In some embodiments, the composition further comprises a nucleic acid encoding a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes Cas9. In some embodiments, the Cas9 nuclease is a modified Cas9 nuclease. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. [0013] Also provided herein is a composition comprising an AAV expression cassette or an AAV vector of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. [0014] Also provided herein is a cell comprising a nucleic acid, an AAV expression cassette, an AAV vector, or a composition of the disclosure. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. [0015] Also provided herein is a composition comprising a cell of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. [0016] Also provided herein is a method of correcting a gene defect in a cell, the method comprising contacting the cell with a nucleic acid, a vector, a non-viral vector, an AAV vector, or a composition of the disclosure. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. [0017] Also provided herein is a method of treating a subject suffering from Duchenne muscular dystrophy, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a vector, a non-viral vector, an AAV vector, or a composition of the disclosure. [0018] Also provided herein is a composition comprising a first vector, wherein the first vector comprises a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 60, and a second vector, wherein the second vector encodes a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes Cas9. In some embodiments, the Cas9 nuclease is a modified Cas9 nuclease. In some embodiments, the second vector is a plasmid. In some embodiments, the second vector is an expression vector. In some embodiments, the second vector is a viral vector such as a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the second vector is a non-viral vector, wherein the non-viral vector comprises calcium phosphate, a liposome, a nanoparticle, and/or a lipid emulsion. [0019] Also provided herein is a method of treating a subject suffering from Duchenne muscular dystrophy, the method comprising administering to the subject a first vector, wherein the first vector comprises a nucleic acid comprising a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence and a second vector, wherein the second vector encodes a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes Cas9. In some embodiments, the Cas9 nuclease is a modified Cas9 nuclease. In some embodiments, the second vector is a plasmid. In some embodiments, the second vector is an expression vector. In some embodiments, the second vector is a viral vector. In some embodiments, the viral vector is a lenti viral vector, a retroviral vector, an adenoviral vector, or an adeno-associated vims (AAV) vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the second vector is a non- viral vector, wherein the non-viral vector comprises calcium phosphate, a liposome, a nanoparticle, and/or a lipid emulsion. In some embodiments, the administering induces a frameshift mutation in a target nucleic acid sequence in a cell of the patient. In some embodiments, the frameshift mutation comprises a deletion of at least one nucleotide, wherein the number of nucleotides deleted is not a multiple of 3. In some embodiments, the frameshift mutation comprises a deletion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 or 20 nucleotides. In some embodiments, the frameshift mutation comprises an insertion of at least one nucleotide, wherein the number of nucleotides inserted is not a multiple of 3. In some embodiments, the frameshift mutation comprises an insertion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 or 20 nucleotides. In some embodiments, the frameshift mutation comprises an insertion of 1 nucleotide. In some embodiments, the first vector and the second vector are administered simultaneously. In some embodiments, the first vector and the second vector are administered sequentially. In some embodiments, the first vector and the second vector are administered locally. In some embodiments, the first vector and the second vector are administered systemically. In some embodiments, the first vector and the second vector are administered by an oral, rectal, transmucosal, topical, transdermal, inhalation, intravenous, subcutaneous, intradermal, intramuscular, intra- articular, intrathecal, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular route of administration. In some embodiments, the subject is greater than or equal to 18 years old. In some embodiments, the subject is less than 18 years old. In some embodiments, the subject is less than 2 years old. In some embodiments, the subject is a human. In some embodiments, the ratio of the first vector to the second vector is 1:1 to 1:100. In some embodiments, the ratio of the second vector to the first vector is 1:1 to 1:100.

[0020] Also provided herein is a combination therapy comprising (a) a first composition, wherein the first composition comprises a first vector comprising a nucleic acid comprising a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence, and (b) a second composition wherein the second composition comprises a second vector comprising a nucleic acid that encodes a Cas9 nuclease. In some embodiments, at least one of the first and the second composition comprises a pharmaceutically acceptable carrier. In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes Cas9. In some embodiments, the Cas9 nuclease is a modified Cas9 nuclease.

[0021] Also provided herein is a nucleic acid encoding a single guide RNA (sgRNA) comprising a sequence of SEQ ID NO: 145, and compositions and kits comprising the same. [0022] Also provided herein is a method of correcting a dystrophin gene defect in exon 43, 45, or 52 of the DMD gene in a subject. As used herein, the term “correcting” or “correction” refers to restoring, at least partially, the function of dystrophin gene. The correction may be complete correction or partial correction. The correction may be a direct correction, wherein the underlying mutation within the dystrophin gene is reverted to the wild-type sequence. The correction may be an indirect correction, wherein the underlying mutation within the dystrophin gene remains, but a compensatory change in one or more positions other than the mutation site results in at least partial restoration of the normal function of dystrophin gene. In some embodiments, the method of correcting a dystrophin gene defect comprises contacting a cell in the subject with a nucleic acid encoding a Cpfl or Cas9 and a nucleic acid encoding a single guide RNA (sgRNA) comprising a sequence of SEQ ID NO: 145, resulting in selective skipping of a DMD exon. In some embodiments, the cell is a muscle cell, or a satellite cell. In some embodiments, Cas9, Cpfl and/or DMD guide RNA are provided to the cell through expression from one or more expression vectors coding therefor. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adeno- associated viral vector. In some embodiments, the expression vector is a non- viral vector. In some embodiments, a sequence encoding the Cas9 or a sequence encoding the Cpfl is provided to the cell as naked plasmid DNA or chemically-modified mRNA. In some embodiments, the method further comprises contacting the cell with a single- stranded DMD oligonucleotide to effect homology directed repair. In some embodiments, Cpfl or Cas9, DMD guide RNA and/or single-stranded DMD oligonucleotide, or expression vectors coding therefor, are provided to the cell in one or more nanoparticles. In some embodiments, the Cpfl or Cas9, DMD guide RNA and/or single-stranded DMD oligonucleotide are delivered directly to a muscle tissue. In some embodiments, the muscle tissue is tibialis anterior, quadricep, soleus, diaphragm or heart. In some embodiments, the Cpfl or Cas9, DMD guide RNA and/or single- stranded DMD oligonucleotide are delivered systemically. In some embodiments, the subject exhibits normal dystrophin-positive myofibers and/or mosaic dystrophin-positive myofibers containing centralized nuclei. In some embodiments, the subject exhibits a decreased serum CK level as compared to a serum CK level prior to contacting. In some embodiments, the subject exhibits improved grip strength as compared to a serum CK level prior to contacting. In some embodiments, the correction is permanent skipping of the DMD exon. In some embodiments, the correction is permanent skipping of more than one DMD exon. In some embodiments, the correction is permanent reframing of a DMD exon. In some embodiments, the contacting step comprising contacting the Cpfl or Cas9 and/or DMD guide RNA are delivered to a human iPSC in vitro to generate an edited iPSC and administering the edited iPSC to the subject. In some embodiments, the edited iPSC is administered directly to a muscle tissue. In some embodiments, the muscle tissue is tibialis anterior, quadricep, soleus, diaphragm or heart. In some embodiments, the edited iPSC is administered systemically. [0023] Also provided herein is a composition, a vector, or a non- viral vector of the disclosure for use as a medicament.

[0024] Also provided herein is a composition, vector, or non- viral vector of the disclosure for use in the treatment of Duchenne muscular dystrophy (DMD).

[0025] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0026] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0028] FIGS. 1A-1J: Exon 52, exon 43 or exon 45-deleted DMD iPSC-derived cardiomyocytes express dystrophin after CRISPR/Cas9 mediated genome editing. (FIG. 1A) Diagram for exon 53, exon 44, and exon 46 targeting strategy and potential products after editing. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. (FIG. IB) Indel analysis of sgRNAs targeting exon 53 in D52 DMD iPSCs. The diagram below the graph indicates the location of the sgRNAs in exon 53. Stop indicates stop codon generated by exon 52 deletion. (FIG.1C) Indel analysis of sgRNAs targeting exon 44 in Δ43 DMD iPSCs. The diagram below the graph indicates the location of the sgRNAs in exon 44. Stop indicates stop codon generated by exon 43 deletion. (FIG. 1D) Indel analysis of sgRNAs targeting exon 44 and exon 46 in Δ45 DMD iPSCs. The diagram below the graph indicates the location of the sgRNAs in exon 44 and exon 46. Stop in exon 44 indicates stop codon generated by 3n-1 INDELs and stop in exon 46 indicates stop codon generated by exon 45 deletion. (FIG.1E) Western blot analysis shows restoration of dystrophin protein expression in hE53g4 edited Δ52 iPSC-CMs. Vinculin is loading control. WT, iPSC- CMs from a healthy control. The second lane is unedited Δ52 iPSC-CMs. (FIG. 1F) Western blot analysis shows restoration of dystrophin protein expression in hE44g4 edited Δ43 iPSC- CMs. Vinculin is loading control. WT, iPSC-CMs from a healthy control. The second lane is unedited Δ43 iPSC-CMs. The third lane is hE44g1 edited Δ43 iPSC-CMs serving as negative control. (FIG.1G) Western blot analysis shows restoration of dystrophin protein expression in hE44g4-edited Δ45 iPSC-CMs. Vinculin is loading control. WT, iPSC-CMs from a healthy control. RF, exon reframed clones with -1 nt deletion. SK, exon skipped clones. The second lane is unedited Δ45 iPSC-CMs. (FIG. 1H) Immunostaining shows restoration of dystrophin expression in hE53g4-edited Δ52 iPSC-CMs. Dystrophin is shown in red. Cardiac troponin I is shown in green. Nuclei are marked by DAPI stain in blue. Scale bar is 50 µm. (FIG. 1I) Immunostaining shows restoration of dystrophin expression in hE44g4-edited Δ43 iPSC-CMs. Dystrophin is shown in red. Cardiac troponin I is shown in green. Nuclei are marked by DAPI stain in blue. Scale bar is 50 µm. (FIG. 1J) Immunostaining shows restoration of dystrophin expression in hE44g4 edited Δ45 iPSC-CMs. Dystrophin is shown in red. Cardiac troponin I is shown in green. Nuclei are marked by DAPI stain in blue. Scale bar is 50 µm. [0029] FIGS.2A-2D. Generation and characterization of mice with a DMD exon 52, 43, and 45 deletion. (FIG.2A) CRISPR/Cas9 editing strategy used for generation of exon 52, 43, and 45 deleted mice. (FIG. 2B) Serum creatine kinase (CK), a marker of muscle damage and membrane leakage, was measured in WT (C57BL/6 and C57BL/10), mdx, Δ52, Δ43, and Δ45 DMD mice. (FIG. 2C) Dystrophin staining of TA, diaphragm and heart of WT and Δ52, Δ43, and Δ45 DMD mice. Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue. Scale bar is 50 µm. (FIG. 2D) H&E staining of TA, diaphragm and heart. Note extensive inflammatory infiltrate and centralized myonuclei in Δ52, Δ43, and Δ45 DMD mice. Scale bar is 100 µm. [0030] FIGS. 3A-H. Correction of Dmd exon 52, exon 43 and exon 45 deletion in mice by intramuscular AAV9 delivery of gene editing components. (FIG. 3A) In vitro sgRNA screening in N2a mouse cell. Indel analysis of sgRNA targeting exon 53. mCTRL1 and mCTRL2 are positive controls. (FIG.3B) In vitro sgRNA screening in N2a mouse cell. Indel analysis of sgRNA targeting exon 44. mCTRL1 and mCTRL2 are positive controls. (FIG.3C, 3E, 3G) Total indel analysis of RT-PCR product in TA muscles from WT and Δ52, Δ43 and Δ45 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. ssAAV-Cas95x10¹⁰ vg/leg and scAAV-sgRNA (mE53g2, mE53g8 or mE44g7) 5x10¹⁰ vg/leg. (FIG.3D, 3F, 3H) Pie chart showing percentage of events detected at exon 53 or exon 44 after ssAAV-Cas9 and scAAV-sgRNA treatment using RT-PCR sequence analysis of TOPO-TA generated clones. RT-PCR products were divided into four groups: Not edited (NE), exon 53 or exon 44–skipped (SK), exon 53 or exon 44–reframed (RF), and out of frame (OF) are indicated. (n=3). [0031] FIGS. 4A-F. Intramuscular AAV9 delivery of gene editing components to Δ52, Δ43 and Δ45 mice rescues dystrophin expression. (FIG. 4A) Western blot analysis shows restoration of dystrophin expression in TA muscle and heart of Δ52 mice after AAV-Cas9 and AAV-mE53g2 or AAV-mE53g8 treatment. ssAAV-Cas95x10¹⁰ vg/leg and scAAV-mE53g2 or mE53g8 5x10¹⁰ vg/leg. Vinculin is a loading control. (FIG. 4B) Immunostaining shows restoration of dystrophin in TA muscle of Δ52 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. ssAAV-Cas95x10¹⁰ vg/leg and scAAV-mE53g2 or mE53g85x10¹⁰ vg/leg. Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue. (n=3). Scale bar is 100 µm. (FIG. 4C) Western blot analysis shows restoration of dystrophin expression in TA muscle and heart of Δ43 mice. Vinculin is loading control. ssAAV-Cas9 5x10¹⁰ vg/leg and scAAV-mE44g7 5x10¹⁰ vg/leg. (FIG. 4D) Immunostaining shows restoration of dystrophin in TA muscle of Δ43 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. ssAAV-Cas9 5x10¹⁰ vg/leg and scAAV-mE44g7 5x10¹⁰ vg/leg. Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue. (n=3). Scale bar is 100 µm. (FIG.4E) Western blot analysis shows restoration of dystrophin expression in TA muscle and heart of Δ45 mice. Vinculin is a loading control. ssAAV-Cas9 5x10¹⁰ vg/leg and scAAV-mE44g7 5x10¹⁰ vg/leg. (FIG. 4F) Immunostaining shows restoration of dystrophin in TA muscle of Δ45 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. ssAAV-Cas9 5x10¹⁰ vg/leg and scAAV-mE44g7 5x10¹⁰ vg/leg. Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue. (n=3). Scale bar is 100 µm. [0032] FIG.5. Table summarizing CRISPR gene editing strategies for exon 52, 43, and 45 deleted DMD.

{00879481} [0033] FIGS. 6A-6C. Editing strategy of human exon 53 sgRNAs that target the splice acceptor or donor sites for exon 52 deletion. (FIG. 6A) Diagram for exon 53 targeting strategy and potential products after editing. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. (FIG. 6B) human sgRNAs targeting exon 53. Category blue indicates sgRNAs targeting the 5’ end for exon skipping or 3n-1 reframing. Category light blue indicates sgRNAs for internal targeting. Category yellow indicates sgRNAs targeting the 3’ end for exon skipping. (FIG. 6C) In vitro sgRNA screening in 293T human cell. Indel analysis of sgRNA targeting exon 53. hCTRL1 and hCTRL2 are positive controls. Blue, light blue, and yellow bars corresponding to the categories indicated in (FIG.6B). [0034] FIGS. 7A-7C. Editing strategy of human exon 44 sgRNAs that target the splice acceptor or donor sites for exon 43 deletion. (FIG. 7A) Diagram for exon 44 targeting strategy and potential products after editing for exon 43 deletion. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. (FIG. 7B) human sgRNAs targeting exon 44. Category green indicates sgRNAs targeting the 5’ end for exon skipping and 3n-1 reframing. Category yellow indicates sgRNAs targeting the 3’ end for exon skipping. (FIG. 7C) In vitro sgRNA screening in 293T human cell. Indel analysis of sgRNA targeting exon 44. hCTRL1 and hCTRL2 are positive controls. Green and yellow bars corresponding to the categories indicated in (FIG.7B). [0035] FIGS. 8A-8C. Editing strategy of human exon 44 sgRNAs that target the splice acceptor or donor sites for exon 45 deletion. (FIG. 8A) Diagram for exon 44 targeting strategy and potential products after editing for exon 45 deletion. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. (FIG. 8B) Human sgRNAs targeting exon 44. Category green indicates sgRNAs targeting the 5’ end for exon skipping. Category yellow indicates sgRNAs targeting the 3’ end for exon skipping and 3n-1 reframing. (FIG.8C) In vitro sgRNA screening in 293T human cell. Indel analysis of sgRNA targeting exon 44. hCTRL1 and hCTRL2 are positive controls. Green and yellow bars corresponding to the categories indicated in (FIG.8B). [0036] FIGS. 9A-9C. Editing strategy of human exon 46 sgRNAs that target the splice acceptor or donor sites for exon 45 deletion. (FIG. 9A) Diagram for exon 46 targeting strategy and potential products after editing. Shapes of intron-exon junctions indicate complementarity that maintains the open reading frame upon splicing. (FIG. 9B) human sgRNAs targeting exon 46. Category purple indicates sgRNAs targeting the 5’ end for exon skipping and 3n-1 reframing. Category light blue indicates sgRNAs for internal targeting. Category yellow indicates sgRNAs targeting the 3’ end for exon skipping. (FIG. 9C) In vitro sgRNA screening in 293T human cell. Indel analysis of sgRNA targeting exon 46. hCTRL1 and hCTRL2 are positive controls. Purple, light blue, and yellow bars corresponding to the categories indicated in (FIG.9B). [0037] FIGS.10A-10G. Editing strategy of mouse exon 53, 44 and 46 sgRNAs for exon 52, 43 and 45 deletions. (FIG.10A) Mouse sgRNAs targeting exon 53. Category blue indicates sgRNAs targeting 5’ end for exon skipping or 3n-1 reframing. Category light blue indicates sgRNAs for internal targeting. Category yellow indicates sgRNAs targeting the 3’ end for exon skipping. (FIG. 10B) In vitro sgRNA screening in N2a mouse cell. Indel analysis of sgRNA targeting exon 53. mCTRL1 and mCTRL2 are positive controls. (FIG. 10C) Mouse sgRNAs targeting exon 44 for exon 43 deletion. Category green indicates sgRNAs targeting the 5’ end for exon skipping and 3n-1 reframing. Category yellow indicates sgRNAs targeting 3’ end for exon skipping. (FIG. 10D) Mouse sgRNAs targeting exon 44 for exon 45 deletion. Category green indicates sgRNAs targeting the 5’ end for exon skipping. Category yellow indicates sgRNAs targeting the 3’ end for exon skipping and 3n-1 reframing. (FIG.10E) In vitro sgRNA screening in N2a mouse cell. Indel analysis of sgRNA targeting exon 44. mCTRL1 and mCTRL2 are positive controls. (FIG.10F) mouse sgRNAs targeting exon 46. Category purple indicates sgRNAs targeting the 5’ end for exon skipping and 3n-1 reframing. Category light blue indicates sgRNAs for internal targeting. Category yellow indicates sgRNAs targeting the 3’ end for exon skipping. (FIG. 10G) In vitro sgRNA screening in N2a mouse cell. Indel analysis of sgRNA targeting exon 46. mCTRL1 and mCTRL2 are positive controls. [0038] FIGS. 11A-11F. Analysis of TA muscles from WT and Δ52, Δ43 or Δ45 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. (FIG. 11A, 11C, 11E) Total indel analysis of TA muscles from WT and Δ52, Δ43 or Δ45 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. (FIG. 11B, 11D, 11F) RT-PCR analysis of TA muscles from WT and Δ52, Δ43 or Δ45 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. Lower dystrophin bands indicate skipping of exon 53 (212 bp) or exon 44 (148 bp) in ΔEx52 (167 bp band), ΔEx43 (460 bp band) or ΔEx45 mice (318 bp band). [0039] FIGS. 12A-12C. Intramuscular AAV9 delivery of gene editing components rescues dystrophin expression in Δ52 mice. (FIG. 12A) Dystrophin immunostaining of TA muscle in corrected Δ52 DMD mice after 3 weeks of ssAAV-Cas9 and scAAV-mE53g2 or mE53g8 intramuscular injection (5 × 10¹⁰ vg/leg of ssAAV9-Cas9 and 5 × 10¹⁰ vg/leg of scAAV-mE53g2 or mE53g8). Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue.10X tile scan of the entire TA muscle. Scale bar is 500 µm. (FIG.12B) H&E staining of TA in WT, Δ52, and corrected Δ52 mice. (n=3). Scale bar is 100 µm. (FIG. 12C) Whole muscle scanning of TA of corrected Δ52 DMD mice. H&E staining of WT, Δ52 DMD and corrected Δ52 DMD 3 weeks after ssAAV-Cas9 and scAAV-mE53g2 or mE53g8 intramuscular injection (5 × 10¹⁰ vg/leg of ssAAV9-Cas9 and 5 × 10¹⁰ vg/leg of scAAV- mE53g2 or mE53g8). Tile scan (4X) of the entire muscle. (n=3). Scale bar is 500 µm. [0040] FIGS. 13A-13C. Intramuscular AAV9 delivery of gene editing components restore dystrophin expression in Δ43 mice. (FIG. 13A) Dystrophin immunostaining of TA muscle in corrected Δ43 DMD mice after 3 weeks of ssAAV-Cas9 and scAAV-mE44g7 intramuscular injection (5 × 10¹⁰ vg/leg of ssAAV9-Cas9 and 5 × 10¹⁰ vg/leg of scAAV- mE44g7). Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue. 10X tile scan of the entire TA muscle. Scale bar is 500 µm. (FIG. 13B) H&E staining of TA in WT, Δ43, and corrected Δ43 mice. (n=3). Scale bar is 100 µm. (FIG.13C) Whole muscle scanning of TA of corrected Δ43 DMD mice. H&E staining of WT, Δ43 DMD and corrected Δ43 DMD 3 weeks after ssAAV-Cas9 and scAAV-mE44g7 intramuscular injection (5 × 10¹⁰ vg of ssAAV9-Cas9 and 5 × 10¹⁰ vg/leg of scAAV-mE44g7). Scale bar is 500 µm. [0041] FIGS. 14A-14C. Intramuscular AAV9 delivery of gene editing components rescue dystrophin expression in Δ45 mice. (FIG. 14A) Dystrophin immunostaining of TA muscle in corrected Δ45 DMD mice after 3 weeks of ssAAV-Cas9 and scAAV-mE44g7 intramuscular injection (5 × 10¹⁰ vg/leg of ssAAV9-Cas9 and 5 × 10¹⁰ vg/leg of scAAV- mE44g7). Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue. 10X tile scan of the entire TA muscle. Scale bar is 500 µm. (FIG. 14B) H&E staining of TA in WT, Δ45, and corrected Δ45 mice. (n=3). Scale bar is 100 µm. (FIG.14C) Whole muscle scanning of TA of corrected Δ45 DMD mice. H&E staining of WT, Δ45 DMD and corrected Δ45 DMD 3 weeks after ssAAV-Cas9 and scAAV-mE44g7 intramuscular injection (5 × 10¹⁰ vg/leg of ssAAV9-Cas9 and 5 × 10¹⁰ vg/leg of scAAV-mE44g7). Scale bar is 500 µm. DETAILED DESCRIPTION [0042] Duchenne muscular dystrophy (DMD) is one of the most common genetic disorders of children. This disease is caused by an absence of dystrophin protein in striated muscle. Deletions of exons 43, 45 and 52 represent mutational “hot spots” in the dystrophin gene. Provided herein are three DMD mouse models harboring deletions of each of these exons. These mice were used to optimize CRISPR/Cas9 gene editing as a means of restoring dystrophin expression by exon skipping and refraining. Gene correction was also validated in cardiomyocytes obtained from induced pluripotent stem cells (iPSCs) with each deletion. The instant disclosure illustrates the variations in gene editing outcomes with different single guide RNAs and highlight the importance of guide RNA design and testing as a prelude for gene editing as a possible therapeutic strategy for DMD. These new strains of mice with common human DMD deletions represent an important platform for future studies of dystrophin gene correction therapies.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the detailed description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0044] All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are each incorporated by reference herein in their entirety.

[0045] The singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0046] Furthermore, the terms “about” and “approximately” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose, time, temperature, and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0047] Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0048] Reference to a vector or other DNA sequences as “recombinant” merely acknowledges the operable linkage of DNA sequences which are not typically operably linked as isolated from or found in nature.

[0049] The terms “gRNA” and “sgRNA” are used interchangeably herein, and refer to a short synthetic RNA composed of a “spacer” (or “targeting”) sequence and a “scaffold” sequence. In some embodiments, the gRNA may further comprise a poly-A tail.

[0050] In accordance with some embodiments of the disclosure, a “frameshift mutation” (or “frame-shift mutation” or “frameshift”) is caused by a deletion or insertion in a DNA sequence that shifts the reading frame of the DNA sequence.

[0051] In accordance with some embodiments of the disclosure, “exon skipping” (or “exonskipping”) refers to a strategy which causes sections (e.g. mutated sections) of a gene to be “skipped” during RNA splicing, allowing the expression of a partially or fully functional protein. [0052] Unless the context indicates otherwise, it is specifically intended that the various features described herein can be used in any combination. I. Gene Editing Systems, e.g., CRISPR Systems [0053] Provided herein are gene editing systems which produce an insertion, deletion, or replacement of DNA at a specific site in the genome of an organism or cell. In some embodiments, the genome editing systems introduce a loss of function mutation or a gain of function mutation. In some embodiments, the genome editing systems of the disclosure are capable of modulating splicing or causing a frameshift in a target DNA sequence. In some embodiments, the genome editing systems correct DNA mutations in vitro and/or in vivo. [0054] The genome editing systems of the disclosure may comprise at least one nuclease (or catalytic domain thereof) and at least one gRNA, or nucleic acids encoding the at least one nuclease (or catalytic domain thereof) and the at least one gRNA. A sequence encoding the at least one nuclease and a sequence encoding the at least one gRNA may be delivered using the same vector (e.g., an AAV vector), or using different vectors (e.g., a first AAV vector for delivering the sequence encoding the nuclease, and a second AAV vector for delivering the sequence encoding the at least one gRNA). [0055] In some embodiments, the nuclease is a Type II, Type V-A, Type V-B, Type V-C, Type V-U, Type VI-B nuclease. In some embodiments, the nuclease is a transcription activator-like effector nuclease (TALEN), a meganuclease, or a zinc-finger nuclease. In some embodiments, the nuclease is a Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. For example, in some embodiments, the nuclease is a Cas9 nuclease or a Cpf1 nuclease. [0056] In some embodiments, the nuclease is a modified form or variant of a Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. In some embodiments, the nuclease is a modified form or variant of a TAL nuclease, a meganuclease, or a zinc-finger nuclease. A “modified” or “variant” nuclease is one that is, for example, truncated, fused to another protein (such as another nuclease), catalytically inactivated, etc. In some embodiments, the nuclease may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a naturally occurring Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B like, Cas13a (C2c2), Cas13b, Cas14 nuclease, or a TALEN, meganuclease, or zinc-finger nuclease. [0057] In embodiments, the nuclease is a Cas9 nuclease derived from S. pyogenes (SpCas9). An exemplary SpCas9 sequence is provided in SEQ ID NO: 1. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1, shown below:

[0058] In embodiments, the nuclease is a Cas9 derived from S. aureus (SaCas9). An exemplary SaCas9 sequence is provided in SEQ ID NO: 2. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2, shown below:

[0059] In embodiments, the nuclease is a Cpfl enzyme from Acidaminococcus (species

BV3L6, UniProt Accession No. U2UMQ6). For example, the Cpfl enzyme may have the sequence set forth below (SEQ ID NO: 3), or a sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto:

[0060] In some embodiments, the nuclease is a Cpfl enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3). An exemplary Lachnospiraceae Cpfl sequence is provided in SEQ ID NO: 4. In some embodiments, the nuclease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ

[0061] In some embodiments, a sequence encoding the nuclease is codon optimized for expression in mammalian cells. In some embodiments, the sequence encoding the nuclease is codon optimized for expression in human cells or mouse cells.

[0062] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence.

Spacer

[0063] A spacer sequence is a short nucleic acid sequence used to target a nuclease (e.g., a Cas9 nuclease) to a specific nucleotide region of interest (e.g., a genomic DNA sequence to be cleaved).

[0064] In some embodiments, the spacer may be about 17-24 base pairs in length, such as about 20 base pairs in length. In some embodiments, the spacer may be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 base pairs in length. In some embodiments, the spacer may be at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 base pairs in length. In some embodiments, the spacer may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In some embodiments, the spacer sequence has between about 40% to about 80% GC content.

[0065] In some embodiments, the spacer targets a site that immediately precedes a 5’ protospacer adjacent motif (PAM). The PAM sequence may be selected based on the desired nuclease. For example, the PAM sequence may be any one of the PAM sequences shown in Table 1 below, wherein N refers to any nucleic acid, R refers to A or G, Y refers to C or T, W refers to A or T, and V refers to A or C or G.

Table 1: Exemplary Nucleases and PAM sequences

[0066] In some embodiments, a spacer may target a sequence of a mammalian gene, such as a human gene. In some embodiments, the spacer may target a mutant gene. In some embodiments, the spacer may target a coding sequence. In some embodiments, the spacer targets the dystrophin ( DMD ) gene. An exemplary wild-type dystrophin sequence includes the human DNA sequence (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189), the sequence of which is reproduced below:

[0067] In some embodiments, the spacer sequence targets a sequence of the DMD gene. In some embodiments, the spacer targets an exon of the DMD gene. In some embodiments, the spacer targets exon 43, exon 44, exon 46, exon 50 or exon 53 of the DMD gene. [0068] In some embodiments, the spacer may have a sequence of any one of SEQ ID NOs:

13-137 (shown in Table 2 below). In some embodiments, a spacer may have a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of any one of SEQ ID NOs: 13-137. In some embodiments, a spacer may have a sequence of any one of the spacers shown in Table 2, or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

Table 2: DNA Sequences encoding gRNA spacer sequences

Scaffold

[0069] The scaffold sequence is the sequence within the gRNA that is responsible for nuclease (e.g., Cas9) binding. The scaffold sequence does not include the spacer/targeting sequence.

[0070] In some embodiments, the scaffold may be about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides in length. In some embodiments, the scaffold may be about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, or about 125 nucleotides in length. In some embodiments, the scaffold may be at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, or at least 125 nucleotides in length. In some embodiments, the scaffold may be 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 nucleotides in length. [0071] In some embodiments, the scaffold may comprise a sequence of any one of SEQ ID NOs: 138-144 (shown in Table 3 below), or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. Table 3: Exemplary scaffold sequences

[0072] In some embodiments, a gRNA (spacer + scaffold) comprises a scaffold and a spacer as shown in Table 4 below, wherein “X” indicates that the particular combination is contemplated by the instant disclosure. Table 4: Exemplary sgRNA (spacer + scaffold) sequences

[0073] In some embodiments, the sgRNA has a sequence (spacer + scaffold) of SEQ ID NO: 145 (shown in Table 5, below), or a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

Table 5: Exemplary sgRNA (spacer + scaffold) sequence

[0074] In some embodiments, a nucleic acid comprises one copy of the sequence encoding the sgRNA. In some embodiments, a nucleic acid comprises two, three, four, or five copies of the sequence encoding the sgRNA. [0075] In some embodiments, a nucleic acid comprises a sequence encoding a promoter, wherein the promoter drives expression of the sgRNA. In some embodiments, the nucleic acid comprises two copies of the sequence encoding a sgRNA, wherein expression of the first copy of the sgRNA is driven by a first promoter, and expression of the second copy of the sgRNA is driven by a second promoter. [0076] In some embodiments, the nucleic acid comprises three copies of the sequence encoding a sgRNA, wherein expression of the first copy of the sgRNA is driven by a first promoter, expression of the second copy of the sgRNA is driven by a second promoter, and expression of the third copy of the sgRNA is driven by a third promoter.

[0077] In some embodiments, the nucleic acid comprises four copies of the sequence encoding a sgRNA, wherein expression of the first copy of the sgRNA is driven by a first promoter, expression of the second copy of the sgRNA is driven by a second promoter, expression of the third copy of the sgRNA is driven by a third promoter, and expression of the fourth copy of the sgRNA is driven by a fourth promoter.

[0078] In some embodiments, the nucleic acid comprises five copies of the sequence encoding a sgRNA, wherein expression of the first copy of the sgRNA is driven by a first promoter, expression of the second copy of the sgRNA is driven by a second promoter, expression of the third copy of the sgRNA is driven by a third promoter, expression of the fourth copy of the sgRNA is driven by a fourth promoter, and expression of the fifth copy of the sgRNA is driven by a fifth promoter. [0079] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 13. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0080] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 14. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0081] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 15. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0082] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 16. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0083] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 17. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0084] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 18. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0085] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 19. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0086] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 20. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0087] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 21. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0088] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 22. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0089] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 23. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0090] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 24. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0091] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 25. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0092] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 26. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0093] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 27. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0094] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 28. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0095] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 29. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0096] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 30. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0097] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 31. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0098] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 32. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0099] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 33. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0100] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 34. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0101] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 35. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0102] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 36. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0103] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 37. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0104] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 38. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0105] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 39. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0106] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 40. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0107] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 41. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0108] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 42. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0109] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 43. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0110] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 44. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0111] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 45. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0112] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 46. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0113] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 47. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0114] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 48. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0115] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 49. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0116] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 50. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0117] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 51. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0118] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 52. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0119] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 53. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0120] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 54. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0121] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 55. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0122] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 56. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0123] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 57. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0124] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 58. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0125] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 59. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0126] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 60. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0127] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 61. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0128] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 62. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0129] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 63. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0130] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 64. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0131] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 65. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0132] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 66. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0133] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 67. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0134] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 68. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0135] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 69. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0136] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 70. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0137] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 71. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0138] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 72. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0139] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 73. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0140] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 74. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0141] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 75. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon.

[0142] In some embodiments, the disclosure provides a nucleic acid comprising a sequence encoding an sgRNA comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 76. In some embodiments, the scaffold comprises the sequence of any one of SEQ ID NOs: 138-144, such as SEQ ID NO: 138. In some embodiments, the nucleic acid comprises three copies of the sequence encoding the sgRNA, each driven by a different promoter. In some embodiments, the nucleic acid is comprised in a vector, e.g., an AAV vector such as an AAV9 vector. In some embodiments, a composition comprises a first nucleic acid according to this paragraph and a second nucleic acid encoding a Cas9 nuclease, such as a S. pyogenes Cas9. The first and second nucleic acids may be comprised in a single vector or in separate vectors. In some embodiments, the vector is an AAV vector, such as an AAV9 vector. In some embodiments, the disclosure provides a method of ameliorating a dystrophin gene defect in a human cell comprising contacting the cell with a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, the disclosure provides a method of treating a subject with DMD, comprising administering to the subject a therapeutically effective amount of a nucleic acid, vector, or composition as provided in this paragraph. In some embodiments, a method according to this paragraph results in skipping or refraining of a dystrophin gene exon. [0143] In some embodiments, a nucleic acid sequence comprising a sequence encoding a sgRNA further comprises a sequence encoding a nuclease. The nuclease may be, for example, a Type II, Type V-A, Type V-B, Type V-C, Type V-U, or Type VI-B nuclease. Exemplary nucleases include, but are not limited to a TALEN, a meganuclease, a zinc-finger nuclease, or a Cas9, Casl2a, Casl2b, Casl2c, Tnp-B like, Casl3a (C2c2), Casl3b, or Casl4 nuclease. [0144] In some embodiments, the nuclease is a Cas9 nuclease. In some embodiments, the Cas9 nuclease is a Streptococcus pyogenes or Streptococcus aureus Cas9. In some embodiments, the nuclease is a modified Cas9 nuclease. In some embodiments, the nuclease is a modified Streptococcus pyogenes Cas9 or a modified Streptococcus aureus Cas9.

[0145] CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a virus. CRISPRs are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with Cas genes that code for proteins related to CRISPRs. The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence these exogenous genetic elements like RNAi in eukaryotic organisms.

[0146] CRISPR repeats can range in size from 24 to 48 base pairs. They usually show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but are not truly palindromic. Repeats are separated by spacers of similar length.

[0147] CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. More than forty different Cas protein families have been described. Of these protein families, Casl appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat- associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer during microbial evolution.

[0148] Exogenous DNA is processed by proteins encoded by Cas genes into small elements (~30 base pairs in length), which are then inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constitutively expressed and are processed by Cas proteins to small RNAs composed of individual exogenously-derived sequence elements with a flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Evidence suggests functional diversity among CRISPR subtypes. The Cse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Casl and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

[0149] Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. One or both sites may be inactivated while preserving Cas9’s ability to locate its target DNA. tracrRNA (/._<?., a scaffold sequence) and spacer RNA may be combined into a "single-guide RNA" molecule that, mixed with Cas9, could find and cut the correct DNA targets. Such synthetic guide RNAs can be used for gene editing.

[0150] Cas9 proteins are highly enriched in pathogenic and commensal bacteria. CRISPR/Cas-mediated gene regulation may contribute to the regulation of endogenous bacterial genes, particularly during bacterial interaction with eukaryotic hosts. For example, Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein that is critical for F. novicida to dampen host response and promote virulence. Delivery of Cas9 DNA sequences also is contemplated.

[0151] Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpfl is a DNA-editing technology analogous to the CRISPR/Cas9 system. Cpfl is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in Prevotella and Francisella bacteria. It prevents genetic damage from viruses. Cpfl genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpfl is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. CRISPR/Cpfl has multiple applications, including treatment of genetic illnesses and degenerative conditions. [0152] Cpfl appears in many bacterial species. The Two Cpfl enzymes from Acidaminococcus and Lachnospiraceae display efficient genome-editing activity in human cells. [0153] A smaller version of Cas9 from the bacterium Staphylococcus aureus is a potential alternative to Cpf1. [0154] The systems CRISPR/Cas are separated into three classes. Class 1 uses several Cas proteins together with the CRISPR RNAs (crRNA) to build a functional endonuclease. Class 2 CRISPR systems use a single Cas protein with a crRNA. Cpf1 has been recently identified as a Class II, Type V CRISPR/Cas systems containing a 1,300 amino acid protein. [0155] The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. [0156] Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cas1, Cas2 and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the abundance of Cpf1-family proteins in many bacterial species. [0157] Functional Cpf1 doesn’t need the tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half as many nucleotides as Cas9). [0158] The Cpf1-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double-stranded break of 4 or 5 nucleotides overhang. [0159] The CRISPR/Cpf1 system consist of a Cpf1 enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA. CRISPR/Cpf1 systems activity has three stages: • Adaptation, during which Cas1 and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array; • Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein; and • Interference, in which the Cpf1 is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence. II. Nucleic Acid Delivery

[0160] As discussed above, in certain embodiments, expression cassettes are employed to express a transcription factor product, either for subsequent purification and delivery to a cell/subject, or for use directly in a genetic-based delivery approach. Expression requires that appropriate signals be provided in the vectors, and include various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.

A. Regulatory Elements

[0161] Throughout this application, the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, /._<?., is under the control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, responsible for initiating the specific transcription of a gene. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

[0162] The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0163] At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0164] In some embodiments, the sgRNA, Cas9 or Cpfl constructs of the disclosure are expressed by a muscle-cell specific promoter. This muscle-cell specific promoter may be constitutively active or may be an inducible promoter.

[0165] Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0166] In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma vims long terminal repeat, rat insulin promoter and glyceraldehyde- 3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product. [0167] Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole is able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter has one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. [0168] Below is a list of promoters/enhancers and inducible promoters/enhancers that could be used to drive expression of a nucleic acid encoding a gene of interest in an expression construct. Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

[0169] The promoter and/or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ alpha and/or DQ beta, b-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, b-Actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, oc-fetoprotein, t-globin, b-globin, c-fos, c-HA-ra.v, insulin, neural cell adhesion molecule (NCAM), α₁ -antitrypain, H2B (TH2B) histone, mouse and/or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), Duchenne muscular dystrophy, SV40, polyoma, retroviruses, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and gibbon ape leukemia vims.

[0170] In some embodiments, inducible elements may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor vims), b-interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, a-2- macroglobulin, vimentin, MHC class I gene H-2Kb, HSP70, proliferin, tumor necrosis factor, and/or thyroid stimulating hormone a gene. In some embodiments, the inducer is phorbol ester (TFA), heavy metals, glucocorticoids, poly(rI)x, poly(rc), E1A, phorbol ester (TPA), interferon, Newcastle Disease Virus, A23187, IL-6, semm, interferon, SV40 large T antigen, PMA, and/or thyroid hormone. Any of the inducible elements described herein may be used with any of the inducers described herein.

[0171] Of particular interest are muscle specific promoters. These include the myosin light chain-2 promoter, the a-actin promoter, the troponin 1 promoter; the Na⁺/Ca²⁺ exchanger promoter, the dystrophin promoter, the a7 integrin promoter, the brain natriuretic peptide promoter and the aB-crystallin/small heat shock protein promoter, a-myosin heavy chain promoter and the ANF promoter.

[0172] In some embodiments, the muscle specific promoter is the CK8 promoter. The CK8 promoter has the following sequence (SEQ ID NO: 146):

[0173] In some embodiments, the muscle-cell cell specific promoter is a variant of the CK8 promoter, called CK8e. The CK8e promoter has the following sequence (SEQ ID NO. 147):

[0174] Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. Any polyadenylation sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

B. Self-cleaving peptides

[0175] In some embodiments, the nucleic acids and/or expression constructs disclosed herein may encode a self-cleaving pepetide.

[0176] In some embodiments of self-cleaving peptides of the disclosure, the self-cleaving peptide is a 2A peptide. In some embodiments, a 2A-like self-cleaving domain from the insect virus Thosea asigna (TaV 2A peptide) (SEQ ID NO: 148, EGRGSLLTCGDVEENPGP) is used. These 2A-like domains have been shown to function across eukaryotes and cause cleavage of amino acids to occur co-translationally within the 2A-like peptide domain. Therefore, inclusion of TaV 2A peptide allows the expression of multiple proteins from a single mRNA transcript. Importantly, the domain of TaV when tested in eukaryotic systems has shown greater than 99% cleavage activity. Other acceptable 2A-like peptides include, but are not limited to, equine rhinitis A virus (ERAV) 2A peptide (SEQ ID NO: 149; QCTN Y ALLKL AGD VESNPGP) , porcine teschovirus- 1 (PTV1) 2A peptide (SEQ ID NO: 150; ATNFSLLKQAGDVEENPGP) and foot and mouth disease vims (FMDV) 2A peptide (SEQ ID NO: 151; PVKQLLNFDLLKLAGDVESNPGP) or modified versions thereof.

[0177] In some embodiments, the 2A peptide is used to express a reporter and a Cas9 or a Cpfl simultaneously. The reporter may be, for example, GFP.

[0178] Other self-cleaving peptides that may be used include, but are not limited to nuclear inclusion protein a (Nia) protease, a PI protease, a 3C protease, an L protease, a 3C-like protease, or modified versions thereof.

C. Delivery of Expression Vectors

[0179] In some embodiments, the gene editing compositions described herein are administered to a cell or to a subject using a non-viral vector or a viral vector. In some embodiments, the gene editing compositions described herein are administered to a cell or to a subject using a recombinant vector (e.g., a recombinant viral or a recombinant non-viral vector). In some embodiments, a recombinant vector comprises a nucleic acid of the disclosure, i.e., a nucleic acid comprising a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence targets an exon sequence of the DMD gene, such as a sequence of exon 43, 44, 46, 50, or 53. In some embodiments, the recombinant vector is a plasmid. In some embodiments, the recombinant vector is an expression vector.

[0180] Exemplary non-viral vectors for use with the compositions and methods described herein comprise nanoparticles (e.g., polymeric nanoparticles), liposomes (e.g., cationic liposomes), naked DNA, cationic lipid-DNA complexes, lipid emulsions, calcium phosphate, polymer complexes, or combinations thereof.

[0181] Exemplary viral vectors for use with the compositions and methods described herein include vectors based on adeno-associated vims (AAV), adenovirus, lentivirus, retrovirus, or a hybrid virus. In some embodiments, the viral vectors of the instant disclosure are replication defective, or at least conditionally replication defective.

[0182] The AAV genome may be from any naturally derived serotype or isolate or clade of AAV. Thus, the AAV genome may be the full genome of a naturally occurring AAV virus. As is known to the skilled person, AAV viruses occurring in nature may be classified according to various biological systems.

[0183] Commonly, AAV viruses are referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which owing to its profile of expression of capsid surface antigens has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, a vims having a particular AAV serotype does not efficiently cross-react with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11, and also recombinant serotypes, such as Rec2 and Rec3, recently identified from primate brain. The sequences of AAV genomes or of elements of AAV genomes including ITR sequences, rep or cap genes for use methods and compositions described herein may be derived from the following accession numbers for AAV whole genome sequences: Adeno- associated virus 1 NC_002077, AF063497; Adeno-associated virus 2 NC_001401; Adeno- associated virus 3 NC_001729; Adeno-associated vims 3B NC_001863; Adeno-associated virus 4 NC_001829; Adeno-associated virus 5 Y18065, AF085716; Adeno-associated virus 6 NC_001862; Avian AAV ATCC VR-865 AY186198, AY629583, NC_004828; Avian AAV strain DA-1 NC_006263, AY629583; Bovine AAV NC_005889, AY388617.

[0184] AAV vimses may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAV viruses, and typically to a phylogenetic group of AAV vimses which can be traced back to a common ancestor, and includes all descendants thereof. Additionally, AAV viruses may be referred to in terms of a specific isolate, /._<?., a genetic isolate of a specific AAV virus found in nature. The term genetic isolate describes a population of AAV viruses which has undergone limited genetic mixing with other naturally occurring AAV vimses, thereby defining a recognizably distinct population at a genetic level.

[0185] In some embodiments, the gene editing compositions of the instant disclosure are delivered to a cell or to a patient using one or more AAV vectors. An AAV vector typically comprises an AAV expression cassette encapsidated by an AAV capsid protein. The serotype of the AAV vector may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the AAV vector may be replication-defective or conditionally replication defective. [0186] In some embodiments, the AAV vector is selected from any of the AAV vectors disclosed in Table 1 of WO 2019/028306, which is incorporated by reference herein in its entirety. In some embodiments, the AAV vector is selected from one of the serotypes listed in Table 6, with the referenced patent application being incorporated by reference as well. Table 6: AAV Serotypes

[0187] The single-stranded DNA genome of wild-type AAV is about 4.7 kilobases (kb). In practice, AAV genomes of up to about 5.0 kb appear to be completely packaged, /._<?., be full- length, into AAV vims particles. With two AAV inverted terminal repeats (ITRs) of about 145 bases, the DNA packaging capacity of an AAV vector is such that a maximum of about 4.4 kb of protein.

[0188] There are a number of other ways in which expression vectors may introduced into cells. In certain embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor- mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells

Adenoviruses

[0189] “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

[0190] The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.

[0191] Adenovirus is particularly suitable for use as a gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5 ’-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.

[0192] In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild- type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of vims from an individual plaque and examine its genomic structure.

[0193] Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins. Since the E3 region is dispensable from the adenovirus genome, the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions. In nature, adenovirus can package approximately 105% of the wild-type genome, providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector- borne cytotoxicity. Also, the replication deficiency of the El -deleted vims is incomplete. [0194] Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovims. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.

[0195] The adenovirus may be of any of the 42 different known serotypes or subgroups A- F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use as described herein. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector. [0196] As stated above, the typical vector according to the present disclosure is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El- coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, or in the E4 region where a helper cell line or helper vims complements the E4 defect.

[0197] Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g. , 10⁹-10¹² plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.

Retroviruses

[0198] The retroviruses are a group of single- stranded RNA viruses characterized by an ability to convert their RNA to double- stranded DNA in infected cells by a process of reverse- transcription. The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5’ and 3’ ends of the viral genome.

[0199] In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells.

[0200] A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.

[0201] A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies are coupled via the biotin components by using streptavidin. Using antibodies against major histocompatibility complex class I and class II antigens, a variety of human cells that bear those surface antigens may be infected with an ecotropic virus in vitro.

Other viral vectors

[0202] Other viral vectors may be employed as expression constructs. For example, vectors derived from viruses such as vaccinia vims and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

Non-viral methods

[0203] Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.

[0204] Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

[0205] In yet another embodiment, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Polyomavirus DNA has been successfully injected in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Direct intraperitoneal injection of calcium phosphate- precipitated plasmids, resulting in expression of the transfected genes, may also be used. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

[0206] In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

[0207] Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the instant disclosure.

[0208] In a further embodiment, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes.

[0209] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. A reagent known as Lipofectamine 2000™ is widely used and commercially available. [0210] In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase. [0211] Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific. [0212] Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin. A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, may be used as a gene delivery vehicle. In some embodiments, epidermal growth factor (EGF) may be used to deliver genes. AAV Expression Cassettes and Recombinant AAV Vectors [0213] The wild-type AAV genome comprises two open reading frames, Rep and Cap, flanked by two inverted terminal repeats (ITRs). Typically, when producing a recombinant AAV, the sequence between the two ITRs is replaced with one or more sequence of interest (e.g., a transgene), and the Rep and Cap sequences are provided in trans. The recombinant AAV genome construct, comprising two ITRs flanking a sequence of interest (such as a transgene), is referred to herein as an AAV expression cassette. The disclosure provides AAV expression cassettes for production of AAV viral vectors. [0214] In some embodiments, an AAV expression cassette comprises a nucleic acid of the disclosure, i.e., a nucleic acid comprising a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence wherein the spacer sequence targets an exon sequence of the DMD gene, such as a sequence of exon 43, 44, 45, 50, or 53 of the DMD gene. [0215] In some embodiments, an AAV expression cassette comprises a first ITR, a transgene sequence, and a second ITR. In some embodiments, an AAV expression cassette comprises a first ITR, an expression control sequence (such as a promoter or enhancer), a transgene sequence, and a second ITR. In some embodiments, an AAV expression cassette comprises a first ITR, an expression control sequence (such as a promoter or enhancer), a transgene sequence, a staffer sequence, and a second ITR. The transgene may comprise all or part of a nucleic acid of the disclosure. For example, the transgene may comprise a gRNA sequence (/._<?., spacer + scaffold sequences), wherein the gRNA targets an exon sequence of the DMD gene, such as a sequence of exon 43, 44, 45, 50, or 53 of the DMD gene.

[0216] In some embodiments, an AAV expression cassette comprises a first ITR, a gRNA sequence, and a second ITR. In some embodiments, an AAV expression cassette comprises a first ITR, an expression control sequence (such as a promoter or enhancer), a gRNA sequence, and a second ITR. In some embodiments, an AAV expression cassette comprises a first ITR, an expression control sequence (such as a promoter or enhancer), a gRNA sequence, a staffer sequence, and a second ITR.

[0217] In some embodiments, the transgene comprises more than one guide RNA sequence, such as two, three, four, five, six, seven, or eight gRNA sequences. In some embodiments, the transgene comprises three, four or five gRNA sequences. In some embodiments, each gRNA sequence is operably linked to an expression control sequence (such as a promoter or enhancer). [0218] In some embodiments, an AAV expression cassette comprises a first ITR, a first expression control sequence (such as a promoter or enhancer), a first gRNA sequence, a second expression control sequence (such as a promoter or enhancer), a second gRNA sequence, and a second ITR.

[0219] In some embodiments, an AAV expression cassette comprises a first ITR, a first expression control sequence (such as a promoter or enhancer), a first gRNA sequence, a second expression control sequence (such as a promoter or enhancer), a second gRNA sequence, a third expression control sequence (such as a promoter or enhancer), a third gRNA sequence, and a second ITR.

[0220] In some embodiments, an AAV expression cassette comprises a first ITR, a first expression control sequence (such as a promoter or enhancer), a first gRNA sequence, a second expression control sequence (such as a promoter or enhancer), a second gRNA sequence, a third expression control sequence (such as a promoter or enhancer), a third gRNA sequence, a fourth expression control sequence (such as a promoter or enhancer), a fourth gRNA sequence, and a second ITR. [0221] In some embodiments, an AAV expression cassette comprises a first ITR, a first expression control sequence (such as a promoter or enhancer), a first gRNA sequence, a second expression control sequence (such as a promoter or enhancer), a second gRNA sequence, a third expression control sequence (such as a promoter or enhancer), a third gRNA sequence, a fourth expression control sequence (such as a promoter or enhancer), a fourth gRNA sequence, a fifth expression control sequence (such as a promoter or enhancer), a fifth gRNA sequence, and a second ITR.

[0222] In some embodiments, all of the gRNA sequences are the same. In some embodiments, two or more of the gRNA sequences are different. In some embodiments, all of the gRNA sequences are different. In some embodiments, the AAV expression cassette further comprises a staffer sequence. In some embodiments, the AAV expression cassette further comprises a polyadenosine (poly A) sequence.

[0223] In some embodiments, an AAV expression cassette comprises sequences encoding a first ITR, a first promoter, a first gRNA comprising a first spacer sequence, a second promoter, a second gRNA comprising a second spacer sequence, a third promoter, a third gRNA comprising a third spacer sequence; and a second ITR. At least one of the first, second, and third spacer sequences may target a sequence of the DMD gene (e.g., exon 43, exon 44, exon 46, exon 50 or exon 53 of the DMD gene). In some embodiments, the first, second, and third spacer sequences are each individually selected from any one of the gRNA spacer sequences in Table 2, or a sequence at least 95% identical thereto. In some embodiments, at least two of the first, second, and third spacer sequences are different. In some embodiments, the first, second, and third spacer sequences are the same. In some embodiments, the first, second, and/or third spacer sequences have a sequence that is at least 95% identical or 100% identical to the sequence of any one of SEQ ID NOs: 13-137.

[0224] In some embodiments, an AAV expression cassette comprises a first gRNA comprising a first spacer sequence, a second gRNA comprising a second spacer sequence, a third gRNA comprising a third spacer sequence, and a fourth gRNA comprising a fourth spacer sequence. In some embodiments, two, three, or four of the gRNAs are the same. In some embodiments, two, three, or four of the gRNAs are different. In some embodiments, an AAV expression cassette comprises a first promoter, a first gRNA comprising a first spacer sequence, a second promoter, a second gRNA comprising a second spacer sequence, a third promoter, a third gRNA comprising a third spacer sequence, a fourth promoter, and a fourth gRNA comprising a fourth spacer sequence. In some embodiments, an AAV expression cassette comprises a first ITR, a first promoter, a first gRNA comprising a first spacer sequence, a second promoter, a second gRNA comprising a second spacer sequence, a third promoter, a third gRNA comprising a third spacer sequence, a fourth promoter, a fourth gRNA comprising a fourth spacer sequence, and a second ITR. In some embodiments, the expression cassette further comprises a staffer sequence.

[0225] In some embodiments, an AAV expression cassette comprises a first gRNA comprising a first spacer sequence, a second gRNA comprising a second spacer sequence, a third gRNA comprising a third spacer sequence, a fourth gRNA comprising a fourth spacer sequence, and a fifth gRNA comprising a fifth spacer sequence. In some embodiments, two, three, four, or five of the gRNAs are the same. In some embodiments, two, three, four or five of the gRNAs are different. In some embodiments, an AAV expression cassette comprises a first promoter, a first gRNA comprising a first spacer sequence, a second promoter, a second gRNA comprising a second spacer sequence, a third promoter, a third gRNA comprising a third spacer sequence, a fourth promoter, a fourth gRNA comprising a fourth spacer sequence, a fifth promoter, and a fifth gRNA comprising a fifth spacer sequence. In some embodiments, an AAV expression cassette comprises a first ITR, a first promoter, a first gRNA comprising a first spacer sequence, a second promoter, a second gRNA comprising a second spacer sequence, a third promoter, a third gRNA comprising a third spacer sequence, a fourth promoter, a fourth gRNA comprising a fourth spacer sequence, a fifth promoter, a fifth gRNA comprising a fifth spacer sequence, and a second ITR. In some embodiments, the expression cassette further comprises a staffer sequence.

[0226] In some embodiments, an AAV expression cassette comprises sequences encoding a first inverted terminal repeat (ITR), a first promoter, a first gRNA comprising a first spacer sequence (e.g., a sequence at least 95% or 100% identical to any one of SEQ ID NOs: 13-137) and a scaffold sequence (e.g. , a scaffold sequence at least 95% or 100% identical to any of SEQ ID NO: 138-144); and a second ITR.

[0227] In some embodiments, an AAV expression cassette comprises sequences encoding a first inverted terminal repeat (ITR), a first promoter, a first gRNA comprising a first spacer sequence (e.g., a sequence at least 95% or 100% identical to any one of SEQ ID NOs: 13-137) and a scaffold sequence (e.g. , a scaffold sequence at least 95% or 100% identical to any of SEQ ID NOs: 138-144), a second promoter, a second gRNA comprising a second spacer sequence (e.g., a sequence at least 95% or 100% identical to any one of SEQ ID NOs: 13-137) and a scaffold sequence (e.g., a scaffold sequence at least 95% or 100% identical to any of SEQ ID NOs: 138-144); and a second ITR. [0228] In some embodiments, an AAV expression cassette comprises sequences encoding a first inverted terminal repeat (ITR), a first promoter, a first gRNA comprising a first spacer sequence (e.g., a sequence at least 95% or 100% identical to any one of SEQ ID NOs: 13-137) and a scaffold sequence (e.g. , a scaffold sequence at least 95% or 100% identical to any of SEQ ID NOs: 138-144), a second promoter, a second gRNA comprising a second spacer sequence (e.g., a sequence at least 95% or 100% identical to any one of SEQ ID NOs: 13-137) and a scaffold sequence (e.g., a scaffold sequence at least 95% or 100% identical to any of SEQ ID NOs: 138-144), a third promoter, a third gRNA comprising a third spacer sequence (e.g., a sequence at least 95% or 100% identical to any one of SEQ ID NOs: 13-137) and a scaffold sequence (e.g., a scaffold sequence at least 95% or 100% identical to any of SEQ ID NOs: 138- 144); and a second ITR.

[0229] In some embodiments, an AAV expression cassette comprises a first inverted terminal repeat (ITR), a first promoter, a nucleic acid comprising a gRNA targeting a sequence of the DMD gene, such as a sequence of Exon 43, 44, 45, 50, or 53 of the DMD gene, and a second ITR. In some embodiments, the AAV expression cassette further comprises a polyadenosine (poly A) sequence.

[0230] In some embodiments, one or both of the first ITR and the second ITR are isolated or derived from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11 , AAV 12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV. In some embodiments, the expression cassette comprises multiple copies of the gRNA, such as 2, 3, 4, or 5 copies of the gRNA.

[0231] In some embodiments, an AAV expression cassette comprises a sequence to make the AAV vector less immunogenic (e.g., a “cloaking” sequence). In some embodiments, the sequence is isolated or derived from a telomere sequence. In some embodiments, the nucleotide sequence binds to a toll-like receptor, such as TLR9.

[0232] In some embodiments, an AAV expression cassette comprises sequences encoding a first ITR, a first promoter, a first gRNA comprising a first spacer sequence, a second promoter, a second gRNA comprising the first spacer sequence, a third promoter, a third gRNA comprising the first spacer sequence, and a second ITR.

[0233] In some embodiments, an AAV expression cassette comprises sequences encoding a first ITR, a first promoter, a first gRNA comprising a first spacer sequence, a second promoter, a second gRNA comprising the first spacer sequence, a third promoter, a third gRNA comprising the first spacer sequence, (optionally) a first staffer sequence, and a second ITR. The first spacer sequence may target the DMD gene, for example it may target exon 43, exon 44, exon 46, exon 50 or exon 53 of the DMD gene. In some embodiments, the first spacer sequence is selected from any one of the gRNA sequences in Table 2, or a sequence at least 95% identical thereto.

[0234] The AAV expression cassettes described herein may be incorporated into an AAV vector. In some embodiments, an AAV vector comprises an AAV expression cassette encapsidated by an AAV capsid protein.

[0235] In some embodiments, the AAV vector is based on one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV. In some embodiments, the AAV vector is based on a modified AAV, comprising one or more non-naturally occurring sequences. In some embodiments, the AAV vector is based on a chimeric AAV. The AAV vector may be replication-defective or conditionally replication defective.

III. Pharmaceutical Compositions and Delivery Methods

[0236] Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0237] One will generally desire to employ appropriate salts and buffers to render drugs, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the disclosure may comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.

[0238] The active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into muscle tissue. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra. [0239] The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

[0240] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0241] Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0242] The compositions generally may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like. [0243] Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered, and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards. IV. DMD Subject Characteristics and Clinical Presentation

[0244] Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy, affecting around 1 in 5000 boys, which results in muscle degeneration and premature death. The disorder is caused by a mutation in the gene dystrophin, located on the human X chromosome, which codes for the protein dystrophin. Dystrophin is an important component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. While both sexes can carry the mutation, females are rarely affected with the skeletal muscle form of the disease.

[0245] Mutations vary in nature and frequency. Large genetic deletions are found in about 60-70% of cases, large duplications are found in about 10% of cases, and point mutants or other small changes account for about 15-30% of cases. An examination of some 7000 mutations catalogued a total of 5,682 large mutations (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions, while 199 (14%) affected the splice sites. Point mutations totaled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).

A. Symptoms

[0246] Symptoms usually appear in boys between the ages of 2 and 3 and may be visible in early infancy. Even though symptoms do not appear until early infancy, laboratory testing can identify children who carry the active mutation at birth. Progressive proximal muscle weakness of the legs and pelvis associated with loss of muscle mass is observed first. Eventually this weakness spreads to the arms, neck, and other areas. Early signs may include pseudohypertrophy (enlargement of calf and deltoid muscles), low endurance, and difficulties in standing unaided or inability to ascend staircases. As the condition progresses, muscle tissue experiences wasting and is eventually replaced by fat and fibrotic tissue (fibrosis). By age 10, braces may be required to aid in walking but most patients are wheelchair dependent by age 12. Later symptoms may include abnormal bone development that lead to skeletal deformities, including curvature of the spine. Due to progressive deterioration of muscle, loss of movement occurs, eventually leading to paralysis. Intellectual impairment may or may not be present but if present, does not progressively worsen as the child ages. The average life expectancy for males afflicted with DMD is around 25.

[0247] The main symptom of Duchenne muscular dystrophy, a progressive neuromuscular disorder, is muscle weakness associated with muscle wasting with the voluntary muscles being first affected, especially those of the hips, pelvic area, thighs, shoulders, and calves. Muscle weakness also occurs later, in the arms, neck, and other areas. Calves are often enlarged. Symptoms usually appear before age 6 and may appear in early infancy. Other physical symptoms are:

• Awkward manner of walking, stepping, or running - (patients tend to walk on their forefeet, because of an increased calf muscle tone. Also, toe walking is a compensatory adaptation to knee extensor weakness.)

• Frequent falls

• Fatigue

• Difficulty with motor skills (running, hopping, jumping)

• Lumbar hyperlordosis, possibly leading to shortening of the hip-flexor muscles. This has an effect on overall posture and a manner of walking, stepping, or running.

• Muscle contractures of Achilles tendon and hamstrings impair functionality because the muscle fibers shorten and fibrose in connective tissue

• Progressive difficulty walking

• Muscle fiber deformities

• Pseudohypertrophy (enlarging) of tongue and calf muscles. The muscle tissue is eventually replaced by fat and connective tissue, hence the term pseudohypertrophy.

• Higher risk of neurobehavioral disorders (e.g., ADHD), learning disorders (dyslexia), and non-progressive weaknesses in specific cognitive skills (in particular short-term verbal memory), which are believed to be the result of absent or dysfunctional dystrophin in the brain.

• Eventual loss of ability to walk (usually by the age of 12)

• Skeletal deformities (including scoliosis in some cases)

• Trouble getting up from lying or sitting position

[0248] The condition can often be observed clinically from the moment the patient takes his first steps, and the ability to walk usually completely disintegrates between the time the boy is 9 to 12 years of age. Most men affected with DMD become essentially “paralyzed from the neck down” by the age of 21. Muscle wasting begins in the legs and pelvis, then progresses to the muscles of the shoulders and neck, followed by loss of arm muscles and respiratory muscles. Calf muscle enlargement (pseudohypertrophy) is quite obvious. Cardiomyopathy particularly (dilated cardiomyopathy) is common, but the development of congestive heart failure or arrhythmia (irregular heartbeat) is only occasional.

[0249] A positive Gowers' sign reflects the more severe impairment of the lower extremities muscles. The child helps himself to get up with upper extremities: first by rising to stand on his arms and knees, and then "walking" his hands up his legs to stand upright. Affected children usually tire more easily and have less overall strength than their peers. Creatine kinase (CPK- MM) levels in the bloodstream are extremely high. An electromyography (EMG) shows that weakness is caused by destruction of muscle tissue rather than by damage to nerves. Genetic testing can reveal genetic errors in the Xp21 gene. A muscle biopsy (immunohistochemistry or immunoblotting) or genetic test (blood test) confirms the absence of dystrophin, although improvements in genetic testing often make this unnecessary.

[0250] Additional symptoms may include:

• Abnormal heart muscle (cardiomyopathy)

• Congestive heart failure or irregular heart rhythm (arrhythmia)

• Deformities of the chest and back (scoliosis)

• Enlarged muscles of the calves, buttocks, and shoulders (around age 4 or 5). These muscles are eventually replaced by fat and connective tissue (pseudohypertrophy).

• Loss of muscle mass (atrophy)

• Muscle contractures in the heels, legs

• Muscle deformities

• Respiratory disorders, including pneumonia and swallowing with food or fluid passing into the lungs (in late stages of the disease)

B. Causes

[0251] Duchenne muscular dystrophy (DMD) is caused by a mutation of the dystrophin gene at locus Xp21, located on the short arm of the X chromosome. Dystrophin is responsible for connecting the cytoskeleton of each muscle fiber to the underlying basal lamina (extracellular matrix), through a protein complex containing many subunits. The absence of dystrophin permits excess calcium to penetrate the sarcolemma (the cell membrane). Alterations in calcium and signaling pathways cause water to enter into the mitochondria, which then burst. [0252] In skeletal muscle dystrophy, mitochondrial dysfunction gives rise to an amplification of stress-induced cytosolic calcium signals and an amplification of stress-induced reactive- oxygen species (ROS) production. In a complex cascading process that involves several pathways and is not clearly understood, increased oxidative stress within the cell damages the sarcolemma and eventually results in the death of the cell. Muscle fibers undergo necrosis and are ultimately replaced with adipose and connective tissue.

[0253] DMD is inherited in an X-linked recessive pattern. Females will typically be carriers for the disease while males will be affected. Typically, a female carrier will be unaware they carry a mutation until they have an affected son. The son of a carrier mother has a 50% chance of inheriting the defective gene from his mother. The daughter of a carrier mother has a 50% chance of being a carrier and a 50% chance of having two normal copies of the gene. In all cases, an unaffected father will either pass a normal Y to his son or a normal X to his daughter. Female carriers of an X-linked recessive condition, such as DMD, can show symptoms depending on their pattern of X-inactivation.

[0254] Duchenne muscular dystrophy has an incidence of 1 in 5,000 male infants. Mutations within the dystrophin gene can either be inherited or occur spontaneously during germline transmission.

C. Diagnosis

[0255] Genetic counseling is advised for people with a family history of the disorder. Duchenne muscular dystrophy can be detected with about 95% accuracy by genetic studies. [0256] DNA test. The muscle-specific isoform of the dystrophin gene is composed of 79 exons, and DNA testing and analysis can usually identify the specific type of mutation of the exon or exons that are affected. DNA testing confirms the diagnosis in most cases.

[0257] Muscle biopsy. If DNA testing fails to find the mutation, a muscle biopsy test may be performed. A small sample of muscle tissue is extracted (usually with a scalpel instead of a needle) and a dye is applied that reveals the presence of dystrophin. Complete absence of the protein indicates the condition.

[0258] Over the past several years DNA tests have been developed that detect more of the many mutations that cause the condition, and muscle biopsy is not required as often to confirm the presence of Duchenne's. [0259] Prenatal tests. DMD is carried by an X-linked recessive gene. Males have only one X chromosome, so one copy of the mutated gene will cause DMD. Fathers cannot pass X- linked traits on to their sons, so the mutation is transmitted by the mother.

[0260] If the mother is a carrier, and therefore one of her two X chromosomes has a DMD mutation, there is a 50% chance that a female child will inherit that mutation as one of her two X chromosomes, and be a carrier. There is a 50% chance that a male child will inherit that mutation as his one X chromosome, and therefore have DMD.

[0261] Prenatal tests can tell whether their unborn child has the most common mutations. There are many mutations responsible for DMD, and some have not been identified, so genetic testing only works when family members with DMD have a mutation that has been identified. [0262] Prior to invasive testing, determination of the fetal sex is important; while males are sometimes affected by this X-linked disease, female DMD is extremely rare. This can be achieved by ultrasound scan at 16 weeks or more recently by free fetal DNA testing. Chorion villus sampling (CVS) can be done at 11-14 weeks, and has a 1% risk of miscarriage. Amniocentesis can be done after 15 weeks, and has a 0.5% risk of miscarriage. Fetal blood sampling can be done at about 18 weeks. Another option in the case of unclear genetic test results is fetal muscle biopsy.

D. Treatment

[0263] There is no current cure for DMD, and an ongoing medical need has been recognized by regulatory authorities. Phase l-2a trials with exon skipping treatment for certain mutations have halted decline and produced clinical improvements in walking. Sarepta’s drug Exondys 51 ( eteplirsen ) has recently received FDA approval. However, treatment is generally aimed at controlling the onset of symptoms to maximize the quality of life, and include the following:

• Corticosteroids such as prednisolone and deflazacort increase energy and strength and defer severity of some symptoms.

• Randomized control trials have shown that beta-2-agonists increase muscle strength but do not modify disease progression. Follow-up time for most RCTs on beta2- agonists is only around 12 months and hence results cannot be extrapolated beyond that time frame.

• Mild, non-jarring physical activity such as swimming is encouraged. Inactivity (such as bed rest) can worsen the muscle disease.

• Physical therapy is helpful to maintain muscle strength, flexibility, and function. • Orthopedic appliances (such as braces and wheelchairs) may improve mobility and the ability for self-care. Form-fitting removable leg braces that hold the ankle in place during sleep can defer the onset of contractures.

• Appropriate respiratory support as the disease progresses is important.

[0264] Comprehensive multi-disciplinary care standards/guidelines for DMD have been developed by the Centers for Disease Control and Prevention (CDC), and are available at treat- nmd.eu/dmd/care/diagnosis-management-DMD.

1. Physical Therapy

[0265] Physical therapists are concerned with enabling patients to reach their maximum physical potential. Their aim is to:

• minimize the development of contractures and deformity by developing a program of stretches and exercises where appropriate

• anticipate and minimize other secondary complications of a physical nature by recommending bracing and durable medical equipment

• monitor respiratory function and advise on techniques to assist with breathing exercises and methods of clearing secretions

2. Respiration Assistance

[0266] Modem "volume ventilators/respirators," which deliver an adjustable volume (amount) of air to the person with each breath, are valuable in the treatment of people with muscular dystrophy related respiratory problems. The ventilator may require an invasive endotracheal or tracheotomy tube through which air is directly delivered, but, for some people non- invasive delivery through a face mask or mouthpiece is sufficient. Positive airway pressure machines, particularly bi-level ones, are sometimes used in this latter way. The respiratory equipment may easily fit on a ventilator tray on the bottom or back of a power wheelchair with an external battery for portability.

[0267] Ventilator treatment may start in the mid to late teens when the respiratory muscles can begin to collapse. If the vital capacity has dropped below 40% of normal, a volume ventilator/respirator may be used during sleeping hours, a time when the person is most likely to be under ventilating (“hypoventilating”). Hypoventilation during sleep is determined by a thorough history of sleep disorder with an oximetry study and a capillary blood gas (See Pulmonary Function Testing). A cough assist device can help with excess mucus in lungs by hyperinflation of the lungs with positive air pressure, then negative pressure to get the mucus up. If the vital capacity continues to decline to less than 30 percent of normal, a volume ventilator/respirator may also be needed during the day for more assistance. The person gradually will increase the amount of time using the ventilator/respirator during the day as needed. E. Prognosis [0268] Duchenne muscular dystrophy is a progressive disease which eventually affects all voluntary muscles and involves the heart and breathing muscles in later stages. The life expectancy is currently estimated to be around 25, but this varies from patient to patient. Recent advancements in medicine are extending the lives of those afflicted. The Muscular Dystrophy Campaign, which is a leading UK charity focusing on all muscle disease, states that “with high standards of medical care young men with Duchenne muscular dystrophy are often living well into their 30s.” [0269] In rare cases, persons with DMD have been seen to survive into the forties or early fifties, with the use of proper positioning in wheelchairs and beds, ventilator support (via tracheostomy or mouthpiece), airway clearance, and heart medications, if required. Early planning of the required supports for later-life care has shown greater longevity in people living with DMD. [0270] Curiously, in the mdx mouse model of Duchenne muscular dystrophy, the lack of dystrophin is associated with increased calcium levels and skeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) are protected and do not undergo myonecrosis. ILM have a calcium regulation system profile suggestive of a better ability to handle calcium changes in comparison to other muscles, and this may provide a mechanistic insight for their unique pathophysiological properties. The ILM may facilitate the development of novel strategies for the prevention and treatment of muscle wasting in a variety of clinical scenarios.

SEQUENCES Table 7: Sequence of Primers Used to Generate DMD Mouse Models, Genotyping and TIDE Analysis

V. EXAMPLES

[0271] In the examples below, gene editing strategies for DMD exons 53, 44 and 46 were systematically tested and optimized in vitro and were used to identify sgRNAs capable of restoring dystrophin expression through exon skipping or refraining in human iPS-derived cardiomyocytes bearing similar mutations, which represent -18% of human DMD mutations. Three new DMD mouse models were then generated, with the corresponding mutations, and demonstrated restoration of dystrophin expression in injected muscles through AAV delivery of Cas9 and optimized sgRNAs.

Example 1: Strategies to correct deletions of dystrophin exons 52, 43, and 45

[0272] Deletions of exon 52, exon 43, and exon 45 represent three mutational “hot spot” regions of dystrophin. In an effort to restore the open reading frame (ORF) of these DMD mutations, sgRNAs were designed to disrupt the splice acceptor sequences or exonic regions of exons 53, 44, and 46, respectively, which would be predicted to result in refraining of the next exon downstream of the deleted exon and restoration of the protein reading frame (FIG. 1A, FIG. 6A-6C, FIG. 7A-7C, FIG. 8A-8C, FIG. 9A-9C, FIG. 10A-10B, and Table 2). Correction of exon 53, 44, and 46 can potentially benefit 8%, 6% and 4% of DMD patients, respectively. Initially, each sgRNA was tested using TIDE analysis in human 293T cells or mouse N2A cells and optimal sgRNAs were then tested in human iPSCs bearing the corresponding exon deletion.

Example 2: Identification of optimal sgRNAs for correction of DMD exon 52 deletion

[0273] In the absence of exon 52, exon 53 is out-of-frame with preceding exons (FIG. 1A and FIG. 6A-6C). Skipping of exon 53 has the potential to restore the reading frame between exons 51 and 54. To target the splice acceptor site for exon 53 to allow exon skipping or to introduce 3n+l nucleotide INDELs in exon 53, allowing for reframing, 17 sgRNAs were designed at the 5’ end of human exon 53 (FIG. 6B (blue colored sgRNAs)). The cutting sites of the sgRNAs with refraining potential were designed to be located upstream of the premature stop codon in exon 53 that results from the deletion of single or multiple exons preceding exon 53 (FIG. 6B). Another 4 human sgRNAs that target sequences immediately after the premature stop codon were also designed to potentially introduce deletions that could abolish the stop codon and then reframe the dystrophin gene (FIG. 6B, (orange colored sgRNAs)). To target the splice donor site for exon skipping, 3 sgRNAs were designed each at the 3’ end of mouse or human exon 53 (FIG. 6B, (yellow colored sgRNAs)). The human sgRNAs were then screened in human 293T cells (FIG. 6C).

[0274] The Protospacer Adjacent Motif (PAM) is a short DNA sequence, usually 2-6 base pairs in length, that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9. The PAM is required for Cas9 to cut and is generally found 3-4 nucleotides downstream from the cut site. There is previous evidence showing that sgRNAs with NGG PAMs, in general, are more efficient than sgRNAs with NAG PAMs. Among the 24 human sgRNAs tested, sgRNAs with NGG PAM sequences showed higher efficiency of gene editing than sgRNAs with NAG PAM sequences, as determined by TIDE analysis (FIG. 6C and Table 2).

[0275] Exon 52-deleted human iPSCs (D52 DMD) were generated by reprogramming the peripheral blood mononuclear cells (PBMCs) from a DMD patient with exon 52 deletion. One human sgRNA for each strategy was selected for evaluation in D52 DMD iPSCs based on their editing efficiency and location (FIG. IB). It was found that hE53g4 and hE53gl0 both generated 20-25% of 3n+l INDELs and 15-20% of other INDELs, and hE53gl5 was less efficient than hE53g4 and hE53gl0, based on total INDELs generated (FIG. IB). The sgRNA hE53g4 target sequence is located at the 3’ boundary of exon 53, therefore only the larger deletions can enable exon skipping to restore the ORF (FIG. IB and FIG. 6B). hE53gl0 displayed the highest percentage of 3n+l INDELs, therefore using this sgRNA can potentially restore ORF by refraining exon 53 (FIG. IB and FIG. 6B).

[0276] D52 DMD iPSCs were subjected to editing with hE53g4 and the cells were differentiated into cardiomyocytes. iPSC cardiomyocytes edited with hE53g4 showed restoration of dystrophin expression as detected by both Western blot and immunostaining (FIG. IE and 1H).

Example 3: Identification of optimal sgRNAs for correction of DMD exon 43 and 45 deletions

[0277] Skipping or reframing of exon 44 can potentially restore dystrophin expression for exon 43 and exon 45 deletions of the dystrophin gene. However, deletion of exon 43 and exon 45 cannot be corrected by 3n+l reframing due to the reading frame of the exons. Instead, exon skipping and 3n-l reframing can potentially correct exon 43 and exon 45 deletions (FIG. 1A and FIG. 7A and FIG. 8A). To correct exon 43 deletion, 9 human sgRNAs located before the premature stop codon caused by exon 43 deletion were selected to target the 5’ end of the exon for exon 44 skipping or 3n-l reframing (FIG. 7B, (green colored sgRNAs)). Six human sgRNAs were selected to target the 3’ end for exon 44 skipping (FIG. 7B, (yellow colored sgRNA)). For correcting exon 45 deletion, 8 human sgRNAs were selected to target the 5’ end for exon 44 skipping (FIG. 8B, (green colored sgRNAs)). 14 human sgRNAs were selected to avoid generation of a stop codon and to target the 3’ end for exon 44 skipping or 3n-l reframing (FIG. 8B, (yellow colored sgRNAs)). Human exon 44 sgRNAs were then screened in 293T cells (FIG. 7C and 8C). Similar to the findings with exon 53, sgRNAs with NGG PAMs generally performed better than sgRNAs with NAG PAMs (FIG. 2D and 3C and Table 2). [0278] Exon 43 -deleted iPSCs (D43 DMD) were generated by removing exon 43 in a normal (WT) human iPSC line. Two human exon 44 sgRNAs (hE44g5 and hE44gl2) targeting the 5’ end of exon 44 (FIG. 1C and FIG. 7B, (green colored sgRNA)) and two human exon 44 sgRNAs (hE44g4 and hE44g8) targeting the 3’ end of exon 44 (FIG. 1C and FIG. 2C, (yellow colored sgRNA)) were selected for further testing in D43 DMD iPSCs. hE44g5 generated 30% of total editing efficiency, with 3n-l being the most dominant INDEL type, representing 22% of total editing efficiency (FIG. 1C). hE44g4, hE44g8 and hE44gl2 displayed similar total editing efficiency, but only hE44g4 had 12% of 3n-l INDEL efficiency (FIG. 1C). The major INDEL types generated by hE44g8 and hE44gl2 were not applicable to exon reframing but could potentially lead to exon skipping. hE44g4 edited D43 DMD iPSCs were selected for differentiation into cardiomyocytes based on their editing efficiency and higher percentage of 3n-l INDELs (FIG. IF). hE44gl edited D43 DMD iPSCs were differentiated into cardiomyocytes and served as a control (FIG. IF). Dystrophin protein expression was restored in hE44g4 edited iPSC derived cardiomyocytes, as shown by Western blot and immunostaining (FIG. IF and II).

[0279] Exon 45-deleted iPSCs (D45 DMD) were generated by removing exon 45 from a normal (WT) iPSC line. For correcting exon 45 deletion, hE44g4, hE44g8, and hE44gl 1 were selected for further analysis in D45 DMD iPSCs with respect to exon skipping and 3n-l nt refraining capability (FIG. ID, (green) and FIG. 8B, (yellow colored sgRNAs)). Among these three sgRNAs, hE44g4 introduced the highest percentage of 3n-l nt INDELs and other larger INDELs (FIG. ID), so single clones of hE44g4-edited iPSCs were differentiated into cardiomyocytes with either -lnt deletion at the 3’ end of exon 45 or large deletions that abolished the splice donor site of exon 45. Dystrophin expression was then assessed by Western blot analysis, and restoration of dystrophin protein expression was confirmed in DEc45 DMD derived cardiomyocytes that were edited by hE44g4 -lnt reframing and exon skipping (FIG. 1G). Immunostaining also showed that hE44g4 could restore dystrophin expression in D45 DMD iPSC-derived cardiomyocytes (FIG. 1J). [0280] To correct exon 45 deletion, an alternative strategy that targeted exon 46 was explored (FIG. 1 A and FIG. 9A). Eleven human sgRNAs located before the premature stop codon caused by exon 45 deletion were selected to target the 5 ’ end of the exon to allow for exon 46 skipping or 3n-l reframing (FIG. 9B, (purple colored sgRNAs)). Five human sgRNAs were selected to target the 3’ end of exon 44 to enable exon 44 skipping (FIG. 9B, (yellow colored sgRNAs)). Another 2 human sgRNAs that target sequences immediately after the premature stop codon were also designed to potentially abolish the stop codon and then reframe the dystrophin gene (FIG. 9B, (orange colored sgRNAs)). Human exon 46 sgRNAs were screened by TIDE analysis in 293T cells (FIG. 9C). Similar to the findings with exon 53 and exon 44, sgRNAs with NGG PAMs generally performed better than sgRNAs with NAG PAMs (FIG. 9C and Table 2).

[0281] Among the sgRNAs with the highest editing efficiency, hE46g2, hE46gl8 and hE46g8 were selected to be further tested in D45 DMD iPSC (FIG. ID, (purple)). sgRNA hE46g2 and hE46gl8 target the 5’ end of exon 46, and hE46g8 targets the 3’ end of exon 46 (FIG. 9B). Although hE46g8 showed 47% of total INDEL efficiency, this sgRNA is located at the 3’ end of exon 46 and the majority of the INDELS were 3n+l, which is not amenable for exon skipping of exon 46. Therefore, iPSC clones were not collected from exon 46 sgRNA edited iPSCs for differentiation.

[0282] Together, these findings demonstrated that hE53g4 can successfully restore dystrophin expression in D52 DMD iPSCs by 3n+l reframing or exon skipping. With different strategies, hE44g4 can successfully restore dystrophin expression in D43 DMD iPSC through exon skipping, but restoring dystrophin expression in D45 DMD iPSCs occurs through 3n-l refraining and exon skipping.

Example 4: Mice with deletions of dystrophin exons 52, 43, or 45 recapitulate DMD

[0283] To investigate CRISPR/Cas9-mediated exon skipping and reframing as a means of correcting common DMD mutations in vivo, mice with deletions of exon 52 (D52 DMD), exon 43 (D43 DMD), or exon 45 (D45 DMD) were generated using the CRISPR/Cas9 system directed by pairs of sgRNAs (FIG. 2A and Table 7). C57BL/6 zygotes were co-injected with in vitro transcribed Cas9 mRNA and sgRNAs, and then re-implanted into pseudo-pregnant females, yielding offspring that transmitted the mutant Dmd alleles through the germline. [0284] Deletion of Dmd exon 52, exon 43, or exon 45 was confirmed by RT-PCR analysis (FIG. 2A). Deletion of each exon placed the dystrophin gene out of frame, leading to the absence of dystrophin protein in skeletal muscle and heart (FIG. 2C). Mice lacking each exon showed pronounced dystrophic muscle at 1 -month of age (FIG. 2D). Serum analysis of the D52, D43, and D45 DMD mice showed elevated creatine kinase (CK) activity, a hallmark of muscle damage (FIG. 2B). Overall, the severity and progression of disease in these mice, as marked by absence of dystrophin protein expression, muscle histology, and serum CK (FIG. 2B-D) are comparable to other previously characterized DMD mouse models, such as mdx mice and D44 DMD mice that were described previously.

Example 5: Identification of optimal sgRNAs for targeting Dmd exon 53, 44 and 46

[0285] To test the CRISPR/Cas9-mediated exon skipping and refraining strategies for correcting common DMD mutations in vivo, sgRNAs were designed that target exon 53, exon 44 and exon 46 and conducted sgRNA screening in mouse N2a cells. For exon 53 targeting, 17 sgRNAs were designed at the 5’ end of mouse exon 53 for exon skipping and 3n+l refraining (FIG. 10A, (blue colored sgRNAs)), 3 mouse sgRNAs that target sequences immediately after the premature stop codon for INDELs that could potentially abolish the stop codon and then reframe the dystrophin gene (FIG. 10A, (orange colored sgRNAs)), and another 3 sgRNAs at the 3’ end of mouse exon 53 for exon skipping (FIG. 10A, (yellow colored sgRNA)).

[0286] Among the 23 mouse sgRNAs tested, sgRNAs with NGG PAM sequences showed higher total efficiency of gene editing than sgRNAs with NAG PAM sequences, as determined by TIDE analysis (FIG. 10B and Table 2). By screening corresponding sgRNAs that target exon 53 in mouse N2a cells, several sgRNAs were found that showed superior total INDEL efficiency by TIDE analysis and exon skipping potential (FIG. 10B). Mouse sgRNAs, mE53g2 and mE53g8, at the 5’ end of exon 53 were selected for testing exon skipping or exon reframing capability in the D52 DMD mouse model (FIG. 3A).

[0287] For exon 44 targeting, 5 mouse sgRNAs located before the premature stop codon caused by exon 43 deletion were designed to target the 5 ’ end of the exon for exon 44 skipping or 3n-l reframing (FIG. IOC, (green colored sgRNAs)). Six mouse sgRNAs were designed to target the 3’ end for exon 44 skipping (FIG. IOC, (yellow colored sgRNAs)). For correcting exon 45 deletion, the same 5 mouse sgRNAs (as indicated above) were used to target the 5’ end for exon 44 skipping (FIG. IOC, (green colored sgRNAs) and FIG. 10D, (green colored sgRNAs)). At the 3’ end, the design was expanded to 14 mouse sgRNAs so as to avoid generation of a stop codon and to target the 3’ end for exon 44 skipping or 3n-l reframing (FIG. 10D, (green colored sgRNAs)). Mouse exon 44 sgRNAs were screened in N2a cells (FIG. 10E). mE44g7 was selected for further in vivo analysis in D43 DMD or D45 DMD mouse models for its superior editing efficiency and conservation with human sgRNA hE44g4 (FIG. 3B).

[0288] The potential of targeting exon 46 for correcting exon 45 deletion was again explored. Five mouse sgRNAs located before the premature stop codon caused by exon 45 deletion were selected to target the 5’ end of the exon to allow for exon 46 skipping or 3n-l refraining (FIG. 10F, (purple colored sgRNAs)). Seven mouse sgRNAs were selected to target the 3’ end of exon 46 to enable exon 46 skipping (FIG. 10G, (yellow colored sgRNAs)). One mouse sgRNA that targets sequences immediately after the premature stop codon was also designed to potentially abolish the stop codon and then reframe the dystrophin gene (FIG. 10F, (orange colored sgRNAs)). Mouse exon 46 sgRNAs were screened by TIDE analysis in mouse N2a cells (FIG. 10G). Similar to the findings with exon 53 and exon 44, sgRNAs with NGG PAMs generally performed better than sgRNAs with NAG PAMs (FIG. 10G and Table 2). Due to the lack of human genomic DNA conservation, exon 46 sgRNAs were not pursued.

Example 6: Correction of DMD exon 43, 45, and 52 deletion in mice by intramuscular AAV 9 delivery of gene editing components

[0289] To test the editing efficiency of the sgRNAs in vivo, an SpCas9 expression cassette was packaged in single- stranded AAV9 (ssAAV9), and mE53g2, mE53g8 or mE44g7 sgRNA expression cassettes were packaged in three different self-complementary AAV9 (scAAV9) viruses. AAV9 is a single-stranded DNA vims that displays tropism to both skeletal muscle and heart and has been used in numerous clinical trials. To further achieve muscle-specific gene editing, the CK8e regulatory cassette that combines enhancer and promoter regions of the muscle CK gene was utilized to drive SpCas9 expression in skeletal muscle. For delivery of sgRNA, three RNA polymerase III promoters (U6, HI, and 7SK) were used to express three copies of each sgRNA.

[0290] To validate the efficacy of the single cut gene editing strategy in the D52, D43, and D45 mouse models, localized intramuscular (IM) injection of ssAAV9 encoding SpCas9 (ssAAV-Cas9) and scAAV9 encoding sgRNA (scAAV-mE53g2, scAAV-mE53g8 or scAAV- mE44g7) was performed in the tibialis anterior (TA) muscle of postnatal day 12 (P12) mice. As a control group, WT and DMD mice were injected with ssAAV-Cas9 without scAAV- sgRNA. In initial studies, 50 pi of AAV9 was injected per leg, containing equal doses of ssAAV-Cas9 (5xl0¹⁰vg/leg) and scAAV-sgRNAs (5xl0¹⁰vg/leg). Three weeks after IM injection, the TA muscles were collected for analysis.

[0291] Similar to the in vitro sgRNA efficiency observed in N2a cells, it was found that mE53g2 had better editing efficiency compared to mE53g8 in vivo (FIG. 11 A). In vivo gene editing by scAAV-mE53g2 and scAAV-mE53g8 was then compared by RT-PCR and a clear exon skipping band was observed at 167bp below the D52 DMD band at 379 bp (FIG. 1 IB). TIDE analysis of the RT-PCR product also revealed that mE53g2 generated a higher percentage of total indels than mE53g8 (FIG. 3C).

[0292] To further evaluate the mutations generated by gene editing, topoisomerase-based thymidine to adenosine (TOPO-TA) cloning was performed using the RT-PCR amplification products and sequenced the cDNA products. TOPO-TA cloning and sequencing of the mE53g2 (5xl0¹⁰ vg/leg scAAV) edited clones showed that 13.3% of the transcripts successfully skipped exon 53 and 4.7% of the transcripts were the product of 3n-l reframing (FIG. 3E), indicating a total of 18% of corrected transcripts in TA muscle of AEx52 DMD mice.

[0293] In mE44g7 treated D43 DMD muscle, similar total indel percentages were observed with IM injection of scAAV-mE44g7 (5xl0¹⁰vg/leg) (FIG. 11C). scAAV-mE44g7 treated D43 DMD TA-muscles were then analyzed by RT-PCR and a clear exon skipping band was observed at 460bp below the D43 DMD band at 608 bp (FIG. 11D). TIDE analysis of the RT- PCR product revealed that mE44g7 generated -15% of average total indels with the IM injection (FIG. 3E). TOPO-TA cloning and sequencing of the mE44g7 (5xl0¹⁰ vg/leg scAAV) edited clones showed that 18.9% of the transcripts successfully skipped exon 44 and none of the transcripts were the product of 3n-l refraining (FIG. 3F). These results demonstrated that the predicted strategy for mE44g7 in D43 DMD is accurate. mE44g7 is a sgRNA that targets at the 3’ end of exon 44, and for correction of exon 43 deletion, it can only induce exon skipping but not exon refraining in exon 44 (FIG. IOC).

[0294] Interestingly, using the same scAAV-mE44g7 to treat AEx45 DMD muscle, similar total indel percentages were observed with scAAV-mE44g7 (5xl0¹⁰ vg/leg) (FIG. 11C). scAAV-mE44g7 treated AEx45 DMD TA-muscles were then analyzed by RT-PCR and revealed a clear exon skipping band at 318bp below the AEx43 DMD band at 466bp (FIG. 1 IF). TIDE analysis of the RT-PCR product revealed that mE44g7 generated -27% of average total indels in muscles treated with all three dosages (FIG. 3G). TOPO-TA cloning and sequencing of the mE44g7 (5xl0¹⁰ vg/leg scAAV) edited clones showed that 8.0% of the transcripts successfully skipped exon 44 and 6.4% of the transcripts were the product of 3n-l reframing (FIG.3H). These findings validated that the strategy for mE44g7 in ∆45 DMD mice is accurate. As a sgRNA that targets the 3’ end of exon 44, mE44g7 can correct exon 45 deletion by inducing both exon skipping and exon reframing in exon 44 (FIG.10D). Example 7: Restoration of dystrophin expression and the dystrophic phenotype in exon 52, exon 43 or exon 45 deleted mice [0295] To evaluate dystrophin protein restoration after IM injection with ssAAV-Cas9 and scAAV-sgRNAs, Western blot analysis was performed on the TA muscles of scAAV-mE53g2 or scAAV-mE53g8 treated ∆Ex52 mice and scAAV-mE44g7 treated ∆43 and ∆45 mice. In scAAV-mE53g2 treated TA muscle, 47% (5x10¹⁰ vg/leg) of dystrophin protein restoration (FIG. 4A) was observed. Dystrophin restoration was significantly higher than in scAAV- mE53g8 treated TA muscle, which showed an average of 21% (5x10¹⁰ vg/leg) of dystrophin protein restoration (FIG. 4A). Immunostaining and whole muscle scanning also revealed that scAAV-mE53g2 treated muscles restored ~72% of dystrophin+ fibers, and only ~24% of dystrophin+ fibers in scAAV-mE53g8 treated muscle (FIG. 4B and FIG. 12A). Histological whole muscle scanning analysis and hematoxylin and eosin (H&E) staining showed that scAAV-mE53g2 rescued the dystrophic phenotype, eliminating necrotic cells and central nuclei, but scAAV-mE53g8 barely improved with the phenotype (FIG.12B and 12C). [0296] In scAAV-mE44g7 treated ∆43 DMD muscle, the restoration of dystrophin was about 23% of wild-type levels (FIG. 4C). Immunostaining and whole muscle scanning also revealed that scAAV-mE44g7 treated muscles restored ~36% of dystrophin+ fibers (FIG.4D and FIG. 13A). H&E staining and whole muscle scanning showed that scAAV-mE44g7 slightly improved the dystrophic phenotype (FIG.13B and 13C). [0297] Using the same scAAV-mE44g7, ∆45 DMD muscle was treated, and it was observed that dystrophin protein expression was restored to 32% (5x10¹⁰ vg/leg) of the WT level (FIG. 4E). Immunostaining and whole muscle scanning also revealed that scAAV-mE44g7 treated muscles show 60% of dystrophin+ fibers in the TA muscle (FIG. 4F and FIG. 14A). H&E staining and whole muscle scanning showed that scAAV-mE44g7 improved the dystrophic phenotype of the injected muscle (FIG.14B and 14C). Example 8: Materials and Methods

Study design

[0298] This study was designed with the primary aim to identify strategies to correct exon 52, exon 43 and exon 45 mutations in corresponding DMD mouse models and human DMD iPSCs. Secondary objectives were to investigate and compare the amount of exon skipping, expression of dystrophin protein, and histological phenotype in corrected DMD mice. PBMCs from healthy individuals and DMD patients were generated at the UT Southwestern Wellstone Myoediting Core. Male donors’ PBMCs were used in all experiments. PBMCs were collected based on the mutation of the patients; exclusion, randomization, or blinding approaches were not used to select the donors. Animal work described in this manuscript has been approved and conducted under the oversight of the UT Southwestern Institutional Animal Care and Use Committee. Animals were allocated to experimental groups based on genotype; exclusion, randomization, or blinding approaches were not used to assign the animals for the experiments. AAV injection and dissection experiments were conducted in a nonblinded fashion. Blinding approaches were used during histology validation and immunostaining analysis. For each experiment, sample size reflects the number of independent biological replicates and was provided in the brief description of the drawings.

Plasmids and cloning

[0299] The pSpCas9(BB)-2A-GFP (PX458) plasmid contained the human codon optimized SpCas9 gene with 2A-EGFP. Cloning of sgRNA was done using Bbs I sites. The sgRNAs in this study, listed in Table 2, were selected using prediction of crispr.mit.edu. sgRNA sequences were cloned into PX458, then tested in tissue culture using HEK 293 and N2a cells.

[0300] The AAV TRISPR-sgRNAs-CK8e-GFP plasmid contained three sgRNAs driven by the U6, HI or 7SK promoters. The expression cassette was synthesized (Genscript), digested with restriction enzymes and subcloned into the pSJG self-complementary AAV plasmid, a gift from S. Gray (UT Southwestern).

Human iPSCs maintenance and nucleofection

[0301] Human iPSCs were cultured in mTeSRl media (cat. 05850, Stemcell Technologies) and passaged approximately every 3-4 days (1:6-1:18 split ratio). One hour before nucleofection, iPSCs were treated with 10 mM ROCK inhibitor, Y-27632 (cat. S1049, Selleckchem), and dissociated into single cells using Accutase (cat. A6964, Innovative Cell Technologies Inc.)· iPSCs (8 x 10⁵) were mixed with 5 μg total of pSpCas9(BB)-2A-GFP (PX458) Addgene plasmid 48138 which contains gRNA as indicated, and then nucleofected using the P3 Primary Cell 4D-Nucleofector X kit (cat. V4XP-3024, Lonza) according to manufacturer’s protocol. After nucleofection, iPSCs were cultured in mTeSRl media supplemented with 10 μM ROCK inhibitor and 100 μg/ml Primocin (InvivoGen), and the next day, the media was switched to fresh mTeSRl. Two days after nucleofection, GFP(+) and GFP(-) cells were sorted by FACS and subjected to genotyping by PCR. Single clones derived from GFP(+) iPSCs were picked, expanded, genotyped, and sequenced.

Human iPSC-CMs differentiation

[0302] To differentiate the iPSCs into cardiomyocytes, cells were cultured in CDM3 media (supplemented with 4-6 μM of CHIR99021 (cat. S2924, Selleckchem) for 2 days (days 1-2), followed by CDM3 supplemented with 2 μM WNT-C59 (cat. S7037, Selleckchem) for 2 days (days 3-4). Starting from day 5, cells were cultured in BASAL media (RPMI-1640, cat. 11875- 093, Gibco, supplemented with B27-supplement, cat.17504044, Thermo Fisher Scientific) for 6 days (days 5-10). On day 10 after differentiation initiation, media was changed to SELECTIVE media (RPMI-1640, no glucose, cat. 11879-020, Gibco, supplemented with B27- supplement) for 10 days (days 11-20) and, last, by BASAL media for 2 to 6 days. Then, the cardiomyocytes were dissociated using TrypLE Express media (cat. 12605-028, Gibco) and re plated at 2 x 10⁶ cells per well in a six-well dish. Cardiomyocytes were used for experiments on days 30-40 after initiation of differentiation.

Mice

[0303] Mice were housed in a barrier facility with a 12-hour light/dark cycle and maintained on standard chow (2916 Teklad Global). AEx52, AEx43 and AEx45 DMD mice were generated in the C57/BL6N background using the CRISPR/Cas9 system. The sgRNAs for generating the mouse models and primers for genotyping, are listed in Table 7.

Genomic DNA isolation, PCR amplification and TIDE analysis of PCR products [0304] Genomic DNA of mouse N2a cells and human HEK 293T cells and human iPSCs was isolated using DirectPCR (cell) lysis reagent (VIAGEN) according to manufacturer's protocol. Genomic DNA of mouse muscle tissues was isolated using GeneJET genomic DNA purification kit (Qiagen DNeasy blood and tissue kit) according to manufacturer’s protocol. Genomic DNA was PCR-amplified using GoTaq DNA polymerase (Promega) or with primers. RT-PCR products were subcloned into pCRII-TOPO vector (Invitrogen) according to the manufacturer's protocol. Individual clones were picked, and the DNA was sequenced. Primer sequences are listed in Table 7. AAV vector production [0305] AAVs were prepared by Boston Children's Hospital Viral Core. AAV vectors were purified by discontinuous iodixanol gradients (Cosmo Bio, AXS-1114542-5), then concentrated with Millipore Amicon filter unit (UFC910008, 100KDa). AAV Titers were determined by quantitative real-time PCR assays. Briefly, 4 μl of the AAV vector was treated with DNase I (NEB M0303S) and 2M NaOH, followed by neutralization. The mixture was serially diluted, and Droplet Digital PCR (ddPCR) (Bio-Rad Laboratories) was performed according to the manufacturer’s protocol. AAV9 delivery to ΔEx52, ΔEx43, and ΔEx45 DMD mice [0306] Before intramuscular injection, the ΔEx52, ΔEx43, and ΔEx45 DMD mice were anesthetized. For AAV9 intramuscular injection, the TA muscle of P12 male ΔEx52, ΔEx43 and ΔEx45 DMD mice was injected using an ultrafine needle (31 gauge) with 50 μl of AAV9 preparations or with saline solution. Dystrophin Western blot analysis [0307] For Western blot of iPSC-derived cardiomyocytes, 2 x 10⁶ cardiomyocytes were harvested and lysed with lysis buffer (10% SDS, 62.5 mM Tris pH 6.8, 1 mM EDTA, and protease inhibitor). For Western blot of skeletal muscles, tissues were crushed into fine powder using a liquid nitrogen-frozen crushing apparatus. Cell or tissue lysates were passed through a 25G syringe and then a 27G syringe, 10 times each. Protein concentration was determined by BCA assay and 50 µg of total protein was loaded onto a 4-20% acrylamide gel. Gels were run at 100V 15min, and switched to 200V for 45 minutes followed by 1 hour 20 min transfer to a PVDF membrane at 100V at 4^oC. The blot was incubated with mouse anti-dystrophin antibody (MANDYS8, Sigma-Aldrich, D8168), at 4^oC overnight, then with goat anti-mouse HRP antibody (Bio-Rad Laboratories) at room temperature for 1 hour. The blot was developed using Western Blotting Luminol Reagent (Santa Cruz, sc-2048). The loading control was determined by blotting with mouse anti-vinculin antibody (Sigma-Aldrich, V9131). Histological analysis of muscles

[0308] Skeletal muscles from WT and D52, D43, and D45 DMD mice were individually dissected and cryo-embedded in a 1:2 volume mixture of Gum Tragacanth powder (Sigma- Aldrich) to Tissue Freezing Medium (TFM) (Triangle Bioscience). All embeds were snap frozen in isopentane heat extractant supercooled to -155°C. Resulting blocks were stored at - 80°C prior to sectioning. Eight-micron transverse sections of skeletal muscle, and frontal sections of heart were prepared on a Leica CM3050 cryostat and air-dried prior to staining on the same day. H&E staining was performed according to established staining protocols and dystrophin immunohistochemistry was performed using MANDYS8 monoclonal antibody (Sigma- Aldrich) with modifications to manufacturer’s instructions. In brief, cryostat sections were thawed and rehydrated/delipidated in 1% triton/phosphate-buffered-saline, pH 7.4 (PBS). Following delipidation, sections were washed free of Triton, incubated with mouse IgG blocking reagent (M.O.M. Kit, Vector Laboratories), washed, and sequentially equilibrated with MOM protein concentrate/PBS, and M ANDYS 8 diluted 1:1800 in MOM protein concentrate/PBS. Following overnight primary antibody incubation at 4°C, sections were washed, incubated with MOM biotinylated anti-mouse IgG, washed, and detection completed with incubation of Vector fluorescein-avidin DCS. Nuclei were counterstained with propidium iodide (Molecular Probes) prior to cover slipping with Vectashield.

Statistics

[0309] All data are presented as mean ± S.E.M. Unpaired two-tailed Student’s t-tests were performed for comparison between the respective two groups (wild-type and DMD mice, wild- type and DMD-AAV9 treated mice, and DMD control and DMD-AAV9 treated mice). Data analyses were performed with statistical software (GraphPad Prism Software, San Diego, CA, USA). P values less than 0.05 were considered statistically significant.

[0310] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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Claims

What is claimed is:

1. A nucleic acid comprising: a sequence encoding a single guide RNA (sgRNA) comprising a spacer sequence and a scaffold sequence, wherein the spacer sequence comprises the sequence of SEQ ID NO: 60.

2. The nucleic acid of claim 1, wherein the scaffold sequence comprises the sequence of any one of SEQ ID NOs: 138-144.

3. The nucleic acid of claim 1 or 2, wherein the nucleic acid comprises one, two, three, four, or five copies of the sequence encoding the sgRNA.

4. The nucleic acid of claim 3, wherein the nucleic acid comprises three copies of the sequence encoding the sgRNA.

5. The nucleic acid of any one of claims 1-4, wherein the nucleic acid comprises a promoter, wherein the promoter drives expression of the sgRNA.

6. The nucleic acid of any one of claims 1-5, wherein the nucleic acid comprises three copies of the sequence encoding the sgRNA, wherein the nucleic acid comprises a first promoter and expression of the first copy of the sgRNA is driven by the first promoter, wherein the nucleic acid comprises a second promoter and expression of the second copy of the sgRNA is driven by the second promoter, and wherein the nucleic acid comprises a third promoter and expression of the third copy of the sgRNA is driven by the third promoter.

7. The nucleic acid of any one of claims 1-6, wherein the nucleic acid further comprises a sequence encoding a Cas9 nuclease.

8. The nucleic acid of claim 7, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9.

9. The nucleic acid of claim 7 or 8, wherein the Cas9 nuclease is a modified Cas9 nuclease.

10. A vector comprising the nucleic acid of any one of claims 1 to 9.

11. The vector of claim 10, wherein the vector is a plasmid.

12. The vector of claim 10 or 11, wherein the vector is an expression vector.

13. The vector of claim 10, wherein the vector is a viral vector.

14. The vector of claim 13, wherein the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated vims (AAV) vector.

15. The vector of claim 10, 13 or 14, wherein the viral vector is an adeno-associated virus (AAV) vector.

16. The vector of claim 15 , wherein the serotype of the AAV vector is selected from AAV 1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.

17. The vector of claim 15 or 16, wherein the AAV vector is replication-defective or conditionally replication defective.

18. The vector of any one of claims 15-17, wherein the serotype of the AAV vector is AAV9.

19. A non- viral vector comprising the nucleic acid of any one of claims 1 to 18, wherein the non-viral vector comprises calcium phosphate, a liposome, a nanoparticle, and/or a lipid emulsion.

20. An AAV expression cassette comprising: a first inverted terminal repeat (ITR); a first promoter; the nucleic acid of any one of claims 1 to 9; and a second ITR.

21. The AAV expression cassette of claim 20, wherein the AAV expression cassette further comprises a polyadenosine (poly A) sequence.

22. The AAV expression cassette of claim 20 or 21, wherein one or both of the first ITR and the second ITR are isolated or derived from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.

23. The AAV expression cassette of any one of claims 20-22, wherein the expression cassette comprises the nucleic acid of claim 6.

24. An AAV vector comprising the nucleic acid of any one of claims 1-9 or the AAV expression cassette of any one of claims 20-23.

25. The AAV vector of claim 24, wherein the AAV vector has the serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, or ovine AAV.

26. The AAV vector of claim 24 or 25, wherein the AAV vector is replication-defective or conditionally replication defective.

27. The AAV vector of any one of claims 24-26, wherein the serotype of the AAV vector is AAV9.

28. A composition comprising the nucleic acid of any one of claims 1 to 6.

29. The composition of claim 28, wherein the composition further comprises a nucleic acid encoding a Cas9 nuclease.

30. The composition of claim 29, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9.

31. The composition of claim 29 or 30, wherein the Cas9 nuclease is a modified Cas9 nuclease.

32. The composition of any one of claims 28-31, further comprising a pharmaceutically acceptable carrier.

33. A composition comprising the AAV expression cassette of any one of claims 20-23, or the AAV vector of any one of claims 24-27.

34. The composition of claim 33, further comprising a pharmaceutically acceptable carrier.

35. A cell comprising the nucleic acid of any one of claims 1-9, the AAV expression cassette of any one of claims 20-23, the AAV vector of any one of claims 24-27, or the composition of any one of claims 28-34.

36. The cell of claim 35, wherein the cell is a stem cell.

37. The cell of claim 35 or 36, wherein the cell is a mammalian cell.

38. The cell of any one of claims 35-37, wherein the cell is a human cell.

39. A composition comprising the cell of any one of claims 35-38.

40. The composition of claim 39, further comprising a pharmaceutically acceptable carrier.

41. A method of correcting a gene defect in a cell, the method comprising contacting the cell with: the nucleic acid of any one of claims 1-9; the vector of any one of claims 10-18; the non-viral vector of claim 19; the AAV vector of any one of claims 20-27 ; or the composition of any one of claims 28-34.

42. The method of claim 41, wherein the cell is a stem cell.

43. The method of claim 41 or 42, wherein the cell is a mammalian cell.

44. The method of any one of claims 41-43, wherein the cell is a human cell.

45. A method of treating a subject suffering from Duchenne muscular dystrophy, the method comprising administering to the subject a therapeutically effective amount of: the nucleic acid of any one of claims 1 to 9; the vector of any one of claims 10-18; the non-viral vector of claim 19; the AAV vector of any one of claims 20-27; or the composition of any one of claims 28-34.

46. A composition comprising: a first vector, wherein the first vector is the vector of any one of claims 10-18 or the non-viral vector of claim 19, and a second vector, wherein the second vector encodes a Cas9 nuclease.

47. The composition of claim 46, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9.

48. The composition of claim 46 or 47, wherein the Cas9 nuclease is a modified Cas9 nuclease.

49. The composition of any one of claims 46-48, wherein the second vector is a plasmid.

50. The composition of any one of claims 46-49, wherein the second vector is an expression vector.

51. The composition of any one of claims 46-48, wherein the second vector is a viral vector.

52. The composition of claim 51, wherein the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

53. The composition of claim 52, wherein the viral vector is an adeno-associated vims (AAV) vector.

54. The composition of claim 53, wherein the serotype of the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAVRh74, AAV2i8, AAVRhlO, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.

55. The composition of any one of claims 46-54, wherein the second vector is a non- viral vector, wherein the non- viral vector comprises calcium phosphate, a liposome, a nanoparticle, and/or a lipid emulsion.

56. A method of treating a subject suffering from Duchenne muscular dystrophy, the method comprising administering to the subject: a first vector, wherein the first vector is the vector of any one of claims 10-18 or the non- viral vector of claim 19, and a second vector, wherein the second vector encodes a Cas9 nuclease.

57. The method of claim 56, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9.

58. The method of claim 56 or 57, wherein the Cas9 nuclease is a modified Cas9 nuclease.

59. The method of any one of claims 56-58, wherein the second vector is a plasmid.

60. The method of any one of claims 56-69, wherein the second vector is an expression vector.

61. The method of any one of claims 56-58, wherein the second vector is a viral vector.

62. The method of claim 61, wherein the viral vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

63. The method of claim 62, wherein the viral vector is an adeno-associated virus (AAV) vector.

64. The method of claim 63, wherein the serotype of the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVRh74, AAV2i8, AAVRh10, AAV39, AAV43, AAVRh8, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.

65. The method of any one of claims 56-60, wherein the second vector is a non-viral vector, wherein the non-viral vector comprises calcium phosphate, a liposome, a nanoparticle, and/or a lipid emulsion.

66. The method of any one of claims 56-65, wherein the administering induces a frameshift mutation in a target nucleic acid sequence in a cell of the patient.

67. The method of claim 66, wherein the frameshift mutation comprises a deletion of at least one nucleotide, wherein the number of nucleotides deleted is not a multiple of 3.

68. The method of claim 67, wherein the frameshift mutation comprises a deletion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 or 20 nucleotides.

69. The method of claim 66, wherein the frameshift mutation comprises an insertion of at least one nucleotide, wherein the number of nucleotides inserted is not a multiple of 3.

70. The method of claim 69, wherein the frameshift mutation comprises an insertion of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 or 20 nucleotides.

71. The method of claim 70, wherein the frameshift mutation comprises an insertion of 1 nucleotide.

72. The method of any one of claims 56-71, wherein the first vector and the second vector are administered simultaneously.

73. The method of any one of claims 56-71, wherein the first vector and the second vector are administered sequentially.

74. The method of any one of claims 56-73, wherein the first vector and the second vector are administered locally.

75. The method of any one of claims 56-73, wherein the first vector and the second vector are administered systemically.

76. The method of any one of claims 56-73, wherein the first vector and the second vector are administered by an oral, rectal, transmucosal, topical, transdermal, inhalation, intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intrathecal, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular route of administration·

77. The method of any one of claims 56-76, wherein the subject is greater than or equal to 18 years old.

78. The method of any one of claims 56-76, wherein the subject is less than 18 years old.

79. The method of claim 78, wherein the subject is less than 2 years old.

80. The method of any one of claims 56-79, wherein the subject is a human.

81. The method of any one of claims 56-80, wherein the ratio of the first vector to the second vector is 1:1 to 1:100.

82. The method of any one of claims 56-80, wherein the ratio of the second vector to the first vector is 1:1 to 1:100.

83. A combination therapy comprising; a first composition comprising a first vector comprising the nucleic acid of any one of claims 1 to 9; and a second composition comprising a second vector comprising a nucleic acid that encodes a Cas9 nuclease.

84. The combination therapy of claim 83, wherein at least one of the first and the second composition comprises a pharmaceutically acceptable carrier.

85. The combination therapy of claim 83 or 84, wherein the Cas9 nuclease is a Streptococcus pyogenes Cas9.

86. The combination therapy of claim 85, wherein the Cas9 nuclease is a modified Cas9 nuclease.

87. The composition of any one of claims 28-34, 39-40, or 46-55, the vector of any one of claims 10-18, or the non- viral vector of claim 19 for use as a medicament.

88. The composition of any one of claims 28-34, 39-40, or 46-55, or the vector of any one of claims 10-18, or the non-viral vector of claim 19 for use in the treatment of Duchenne muscular dystrophy.

89. A nucleic acid encoding a single guide RNA (sgRNA) comprising a sequence of SEQ ID NO: 145.

90. A kit comprising the nucleic acid of claim 89.

91. A method of correcting a dystrophin gene defect in exon 41 of the DMD gene in a subject comprising contacting a cell in the subject with a nucleic acid encoding a Cpfl or Cas9 and the nucleic acid of claim 89, resulting in selective skipping of a DMD exon.

92. The method of claim 91, wherein the cell is a muscle cell, or a satellite cell.

93. The method of claim 91 or 92, wherein Cas9, Cpfl and/or DMD guide RNA are provided to the cell through expression from one or more expression vectors coding therefor.

94. The method of claim 93, wherein the expression vector is a viral vector.

95. The method of claim 94, wherein the viral vector is an adeno-associated viral vector.

96. The method of claim 93, wherein the expression vector is a non-viral vector.

97. The method of claim 91 , wherein a sequence encoding the Cas9 or a sequence encoding the Cpfl is provided to the cell as naked plasmid DNA or chemically-modified mRNA.

98. The method of any one of claims 91-97, further comprising contacting the cell with a single-stranded DMD oligonucleotide to effect homology directed repair.

99. The method of any one of claims 91-97, wherein Cpf1 or Cas9, DMD guide RNA and/or single-stranded DMD oligonucleotide, or expression vectors coding therefor, are provided to the cell in one or more nanoparticles.

100. The method of any one of claims 91-99, wherein the Cpf1 or Cas9, DMD guide RNA and/or single-stranded DMD oligonucleotide are delivered directly to a muscle tissue.

101. The method of claim 100, wherein the muscle tissue is tibialis anterior, quadricep, soleus, diaphragm or heart.

102. The method of any one of claims 91-99, wherein the Cpf1 or Cas9, DMD guide RNA and/or single-stranded DMD oligonucleotide are delivered systemically.

103. The method of any one of claims 91-102, wherein the subject exhibits normal dystrophin-positive myofibers and/or mosaic dystrophin-positive myofibers containing centralized nuclei.

104. The method of any one of claims 91-103, wherein the subject exhibits a decreased serum CK level as compared to a serum CK level prior to contacting.

105. The method of any one of claims 91-104, wherein the subject exhibits improved grip strength as compared to a serum CK level prior to contacting.

106. The method of any one of claims 91-105, wherein the correction is permanent skipping of the DMD exon.

107. The method of any one of claims 91-105, wherein the correction is permanent skipping of more than one DMD exon.

108. The method of any one of claims 91, 93-93 or 103-107, wherein the contacting step comprising contacting the Cpf1 or Cas9 and/or DMD guide RNA are delivered to a human iPSC in vitro to generate an edited iPSC and administering the edited iPSC to the subject.

109. The method of claim 108, wherein the edited iPSC is administered directly to a muscle tissue.

110. The method of claim 109, wherein the muscle tissue is tibialis anterior, quadricep, soleus, diaphragm or heart.

111. The method of claim 108, wherein the edited iPSC is administered systemically.