WO2019136216A1

WO2019136216A1 - Therapeutic crispr/cas9 compositions and methods of use

Info

Publication number: WO2019136216A1
Application number: PCT/US2019/012300
Authority: WO
Inventors: Leonela AMOASII; Eric N. Olson; Yi-Li Min
Original assignee: The Board Of Regents Of The University Of Texas System
Priority date: 2018-01-05
Filing date: 2019-01-04
Publication date: 2019-07-11
Also published as: EP3735462A1

Abstract

CRISPR/Cas9-mediated genome editing holds clinical potential for treating genetic diseases, such as Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. Here, compositions and methods for the treatment of DMD are provided. In some embodiments, a composition providing therapeutically-effective ratios of a vector encoding a guide RNA of the disclosure and a vector encoding a Cas9 protein or nuclease domain thereof of the disclosure are provided for use in the treatment of DMD.

Description

THERAPEUTIC CRISPR/CAS9 COMPOSITIONS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims priority to U.S. Provisional Application Serial No. 62/724,341, filed August 29, 2018, U.S. Provisional Application Serial No. 62/672,255, filed May 16, 2018, U.S. Provisional Application Serial No. 62/639,871, filed March 7, 2018, and U.S. Provisional Application Serial No. 62/614,121, filed January 5, 2018, each of which is incorporated by reference herein in its entirety for all purposes. FEDERAL FUNDING SUPPORT CLAUSE

[02] This invention was made with government support under grant no. U54 HD 087351 awarded by National Institutes of Health. The government has certain rights in the invention.

INCORPORATION OF THE SEQUENCE LISTING

[03] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 4, 2019, is named UTFDP3354WO.txt and is 1,659,828 bytes in size.

FIELD OF THE DISCLOSURE

[04] The present disclosure relates to the fields of molecular biology, medicine and genetics. More particularly, the disclosure relates to compositions and uses thereof for genome editing to correct mutations in vivo using an exon-skipping and/or refraining approach.

BACKGROUND

[05] 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 expression of dystrophin, causing muscle membrane fragility and progressive muscle wasting.

SUMMARY

[06] Despite intense efforts to find cures through a variety of approaches, including myoblast transfer, viral delivery, and oligonucleotide-mediated exon skipping, there remains no cure for any type of muscular dystrophy. The disclosure provides compositions comprising a DMD guide RNA-Cas9 complex for disrupting a dystrophin splice acceptor site and inducing skipping and/or refraining of an exon of a DMD gene, therefore modifying a DMD gene in a cell or a subject. Compositions and method of the disclosure may be used to treat muscular dystrophy.

[07] In some embodiments, a composition comprising a first vector comprising a sequence encoding a DMD guide RNA and a second vector comprising a sequence encoding a Cas9 protein or a nuclease domain thereof, wherein the ratio of the first vector to the second vector is at least 1.5: 1, may be used for the treatment of muscular dystrophy. In some embodiments, the first vector and/or the second vector is a viral vector. In some embodiments, the first vector and/or the second vector is an adeno-associated viral (AAV) vector.

[08] The disclosure provides a composition comprising (i) a first vector comprising a nucleic acid comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD guide RNA, and (ii) a second vector comprising a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a sequence encoding a muscle-specific promoter, wherein the muscle-specific promoter drives expression of the sequence encoding a Cas9 or a nuclease domain thereof; wherein a ratio of the first vector to the second vector in the composition is between about 30: 1 to about 1 : 1 or about 1 : 1 to about 1 :30. In some embodiments, the ratio of the first vector to the second vector is about 1 : 1. In some embodiments, the ratio of the first vector to the second vector is about 1.7: 1. In some embodiments, the ratio of the first vector to the second vector is about 2: 1. In some embodiments, the ratio of the first vector to the second vector is about 2.5: 1. In some embodiments, the ratio of the first vector to the second vector is about 3: 1. In some embodiments, the ratio of the first vector to the second vector is about 4: 1. In some embodiments, the ratio of the first vector to the second vector is about 5: 1. In some embodiments, the ratio of the first vector to the second vector is about 6: 1. In some embodiments, the ratio of the first vector to the second vector is about 7: 1. In some embodiments, the ratio of the first vector to the second vector is about 8: 1. In some embodiments, the ratio of the first vector to the second vector is about 9: 1. In some embodiments, the ratio of the first vector to the second vector is about 10: 1. In some embodiments, the ratio of the first vector to the second vector is about 1 :2. In some embodiments, the ratio of the first vector to the second vector is about 1 :3. In some embodiments, the ratio of the first vector to the second vector is about 1 :4. In some embodiments, the ratio of the first vector to the second vector is about 1 :5. In some embodiments, the ratio of the first vector to the second vector is about 1 :6. In some embodiments, the ratio of the first vector to the second vector is about 1 :7. In some embodiments, the ratio of the first vector to the second vector is about 1 :8. In some embodiments, the ratio of the first vector to the second vector is about 1 :9. In some embodiments, the ratio of the first vector to the second vector is about 1 : 10. In some embodiments, the ratio of the first vector to the second vector is about 1 : 15. In some embodiments, the ratio of the first vector to the second vector is about 1 :20. In some embodiments, the ratio of the first vector to the second vector is about 1 :25. In some embodiments, the ratio of the first vector to the second vector is about 1 :30.

[09] In some embodiments, the first vector comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence; and at least one sequence encoding an additional promoter, wherein the at least one additional promoter drives expression of the at least one sequence encoding an additional DMD guide RNA. In some embodiments, the first vector or the second vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the first vector and the second vector comprise a sequence isolated or derived from an adeno-associated virus (AAV).

[010] In some embodiments, the sequence encoding the muscle-specific promoter comprises or consists of a sequence encoding a muscle-specific creatine kinase 8 (CK8) promoter. In some embodiments, the sequence encoding the muscle-specific promoter comprises or consists of a sequence encoding a CK8e promoter.

[Oil] In some embodiments of the compositions of the disclosure, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 60-371, SEQ ID NO: 383-694, SEQ ID NO: 682-697, SEQ ID NO: 715-717, SEQ ID NO: 790-862, SEQ ID NO: 1036-1051, SEQ ID NO: 1066-1380, SEQ ID NO: 1392-1467, or SEQ ID NO: 1484-1491. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039. In some embodiments, the sequence encoding the first DMD guide RNA and the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039. In some embodiments, the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 60-371, SEQ ID NO: 383- 694, SEQ ID NO: 682-697, SEQ ID NO: 715-717, SEQ ID NO: 790-862, SEQ ID NO: 1036- 1051, SEQ ID NO: 1066-1380, SEQ ID NO: 1392-1467, or SEQ ID NO: 1484-1491. In some embodiments, the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714. In some embodiments, the sequence encoding the first DMD guide RNA and the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714. In some embodiments, the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714.

[012] In some embodiments, the composition comprises between 5 x 10¹¹ viral genomes (vg)/kilogram (kg) and 1 x 10¹⁵ vg/kg, inclusive of the endpoints, of the first vector. In some embodiments, including those wherein the composition is administered locally or by an intramuscular delivery route, the composition comprises between 5 x 10¹¹ viral genomes (vg)/kilogram (kg) and 1 x 10¹⁵ vg/kg, inclusive of the endpoints, of the first vector. In some embodiments, including those wherein the composition is administered systemically or by an intravenous delivery route, the composition comprises between 5 x 10¹² viral genomes (vg)/kilogram (kg) and 1 x 10¹⁵ vg/kg, inclusive of the endpoints, of the first vector.

[013] In some embodiments, the composition comprises at least 5 x 10¹¹ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10¹² viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10¹² viral genomes (vg)/kilogram (kg) of the first vector.

In some embodiments, the composition comprises at least 1 x 10¹³ viral genomes

(vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10¹³ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10¹⁴ viral genomes

(vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10¹⁵ viral genomes (vg)/kilogram (kg) of the first vector.

[014] In some embodiments, the composition comprises at least 4 x 10¹² viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10¹² viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 6 x 10¹² viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10¹³ viral genomes

(vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 2 x 10¹³ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 3 x 10¹³ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 5 x 10¹³ viral genomes

(vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 1 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 2 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 3 x 10¹⁴ viral genomes

(vg)/kilogram (kg) of the first vector. In some embodiments, the composition comprises at least 4 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector. In some embodiments, the ratio of the first vector to the second vector is 1 : 1 and the composition comprises at least 6 x 10¹² vg/kg of the first vector and at least 6 x 10¹² vg/kg of the second AAV vector. In some embodiments, the ratio of the first vector to the second vector is 1 :1 and the composition comprises at least 2.6 x 10¹³ vg/kg of the first vector and at least 2.6 x 10¹³ vg/kg of the second AAV vector. In some embodiments, the ratio of the first vector to the second vector is 1 : 1 and the composition comprises at least 1 x 10¹⁴ vg/kg of the first vector and at least 1 x 10¹⁴ vg/kg of the second AAV vector. In some embodiments, the ratio of the first vector to the second vector is 2: 1 and the composition comprises at least 2 x 10¹⁴ vg/kg of the first vector and at least 1 x 10¹⁴ vg/kg of the second AAV vector. In some embodiments, the composition further comprises a pharmaceutically-acceptable carrier.

[015] In some embodiments, the composition comprises between 5 x 10¹¹ viral genomes (vg)/kilogram (kg) and 1 x 10¹⁵ vg/kg, inclusive of the endpoints, of the first vector. In some embodiments, the composition comprises between 5 x 10¹¹ viral genomes (vg)/kilogram (kg) and 1 x 10¹⁵ vg/kg, inclusive of the endpoints, of the second vector. [016] Also provided is a method of treating muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising (i) a first vector comprising a nucleic acid comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD guide RNA, and (ii) a second vector comprising a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a sequence encoding a muscle-specific promoter, wherein the muscle- specific promoter drives expression of the sequence encoding a Cas9 or a nuclease domain thereof; wherein a ratio of the first vector to the second vector in the composition is between about 10: 1 and about 1 :1 or about 1: 1 and about 1 : 10. In some embodiments, the

composition is administered locally. In some embodiments, the composition is administered directly to a muscle tissue. In some embodiments, the composition is administered by an intramuscular infusion or injection. In some embodiments, the muscle tissue comprises a tibialis anterior tissue, a quadriceps tissue, a soleus tissue, a diaphragm tissue, or a heart tissue. In some embodiments, the composition is administered by intra-cardiac injection. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered by an intravenous infusion or injection. In some embodiments, the subject is a neonate, an infant, a child, a young adult, or an adult. In some embodiments, the subject has muscular dystrophy. In some embodiments, the subject is a genetic carrier for muscular dystrophy. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the adult is at least 18 years old. In some embodiments, including those wherein the targeted muscle cell type is a cardiac muscle cell, the adult is at least 25 years old. In some embodiments, the adult is at least 20 kg. In some embodiments, the subject is a child. In some embodiments, the child is less than 18 years of age. In some embodiments, the child is 20 kg or less. In some embodiments, the subject is an infant. In some embodiments, the subject is less than 2 years old.

[017] In some embodiments of the method of treating of the disclosure, upon administering the therapeutically effective amount of the composition, the subject produces a minimal immune response to the composition. In some embodiments, the minimal immune response to the composition is reduced or eliminated ytreatment with an anti-inflammatory agent or an immune suppressive agent.

[018] In some embodiments of the method of treating of the disclosure, the composition does not induce breaks in a predicted alternative targeting site. In some embodiments, the predicted alternative targeting site comprises a coding sequence of the human genome and wherein the coding sequence comprises at least 2 mismatches with respect to the first genomic target sequence or the second genomic target sequence. In some embodiments, the predicted alternative targeting site comprises a coding sequence of the human genome and wherein the coding sequence comprises at least 3, 4, 5, 6, 7,8, 9, or 10 mismatches with respect to the first genomic target sequence or the second genomic target sequence. In some embodiments, the predicted alternative targeting site is identified using an algorithm (for example, those publicly available at Based on CRISPR design tools (crispr.mit.edu/ and benchling.com/). In some embodiments, confirmation that the composition does not induce breaks at predicted alternative targeting sites comprises DNA amplification, isolation of genomic PCR amplification products and sequencing of the isolated of genomic PCR amplification products spanning the predicted alternative targeting sites.

[019] In some embodiments of the method of treating of the disclosure, the administration of the therapeutically effective amount of the composition is provided as a single dose or provided within a single medical procedure.

[020] In some embodiments of the method of treating of the disclosure, the administration of the therapeutically effective amount of the composition is provided as multiple doses or provided over multiple medical procedures.

[021] As used herein in the specification,“a” or“an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word“comprising”, the words“a” or“an” may mean one or more than one.

[022] The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” As used herein “another” may mean at least a second or more.

[023] Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, for the method being employed to determine the value, or that exists among the study subjects. Such an inherent variation may be a variation of ±10% of the stated value. [024] Throughout this application, nucleotide sequences are listed in the 5’ to 3’ direction, and amino acid sequences are listed in the N-terminal to C-terminal direction, unless indicated otherwise.

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

BRIEF DESCRIPTION OF THE DRAWINGS

[026] 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.

[027] FIG. 1A-F.“Humanized’’-DEc50 mouse model. (FIG. 1A) Strategy showing CRISPR/Cas9-mediated genome editing approach to generate a humanized mouse model. (FIG. IB) RT-PCR analysis to validate deletion of exon 50 (DEc50). (FIG. 1C) Sequence of RT-PCR product to validate exon 50 deletion and generation of an out-of-frame sequence (nucleic acid sequence = SEQ ID NO: 1; amino acid sequence = SEQ ID NO: 2). (FIG. ID) Histochemistry of cardiac and skeletal muscle by hematoxylin and eosin (H&E) staining, and immunohistochemistry using dystrophin antibody. Scale bar: 50 mm. (FIG. IE) Western blot analysis of dystrophin and vinculin expression in tibialis anterior and heart tissues. (FIG. IF) Levels of serum CK, a marker of muscle dystrophy that reflects muscle damage and membrane leakage were measured in wildtype (WT), DEc50 and mdx mice.

[028] FIG. 2A-B. Exon 51 skipping. (FIG. 2A) RT-PCR of RNA from DEc50 mice 3 weeks after intramuscular injection indicates deletion of exon 51 (termed DEc50-51, lower band). (FIG. 2B) Sequence of the RT-PCR products of DEc50-51 band confirmed that exon 49 spliced directly to exon 52, excluding exon 51 (nucleic acid sequence = SEQ ID NO: 3; amino acid sequence = SEQ ID NO: 4).

[029] FIG. 3A-G. Correction of dystrophin expression using triplicate gRNA strategy 3 weeks after intra-muscular injection. (FIG. 3A) Strategy showing CRISPR/Cas9-mediated genome editing approach to correct the reading frame in DEc50 mouse model. (FIG. 3B) sgRNA targeting the splice acceptor site (sgRNA-ex5l-SA) sequence and schematic illustration of sgRNA binding position. Fig. 3B discloses SEQ ID NOS: 954-957, respectively, in order of appearance. (FIG. 3C) Schematic illustration of AAV injection plasmids and strategy. Double guide strategy using rAAV9-sgRNA plasmid for sgRNA- ex5l-SA and sgRNA-ex5l-SD. Triplicate using rAAV9-sgRNA plasmid containing 3 copies of sgRNA-ex5l-SA. Muscle creatine kinase 8 (CK8) promoter is used to express SpCas9.

U6, Hl and 7SK promoter for RNA polymerase III is used to express sgRNA. (FIG. 3D) Dystrophin immunohistochemistry staining of tibialis anterior muscle. (FIG. 3E)

Quantification of dystrophin positive fibers normalized by area. (FIG. 3F) Western blot analysis of dystrophin (DMD) and vincubn (VCL) expression 3 weeks after intramuscular injection. (FIG. 3G) Quantification of dystrophin expression after normalization to vinculin. Data are represented as mean ± SEM. **P<0.005. Scale bar: 50 mm

[030] FIG. 4A-B. Histological improvement of injected muscle after 3 weeks. (FIG. 4A)

Histochemistry of tibialis anterior muscle by hematoxylin and eosin (H&E) staining. (FIG. 4B) Quantification of fiber size and percentage of frequency. Data are represented as mean ± SEM. Scale bar: 50 mm.

[031] FIG. 5. Screen of sgRNA in human 293 cells and mouse 10T cells. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) on a 2% agarose gel. M denotes size marker lane bp indicates the length of the marker bands.

[032] FIG. 6. Dystrophin immunohistochemistry staining of entire tibialis anterior muscle. Immunohistochemistry analysis demonstrates that dystrophin levels were restored in mice treated using the double guide constructs and the triple guide constructs. Mice treated using the triple guide constructs had higher levels of dystrophin expression.

[033] FIG. 7A-B. Strategy for CRISPR/Cas9-mediated genome editing in AEx50 mice. (FIG. 7A) Strategy showing CRISPR/Cas9-mediated genome editing approach to correct the reading frame in AEx50 mouse model. (FIG. 7B) sgRNA targeting the splice acceptor site (sgRNA-ex5l-SA2) sequence (SEQ ID NO: 708) and schematic illustration of sgRNA binding position. Fig. 7B discloses SEQ ID NOS: 958-959, respectively, in order of appearance.

[034] FIG. 8A-F. sgRNA genomic analysis in mouse and human cells. (FIG. 8A) Cas9 was expressed in the presence or absence of mouse sgRNA-sgRNA-5l-SA2 in 10T1/2 cells and gene editing was monitored by T7E1 assay in fluorescence-based cell sorted (FACS) (+) and non-sorted cells (-). GFP was used as a control. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 77lbp PCR band. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay. M denotes size marker lane bp indicates the length of the marker bands.

(FIG. 8B) Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in 10T1/2 cells. Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and deletions (highlighted in red) (SEQ ID NOS: 960-966, respectively, in order of appearance). The line indicates the predicted exon splicing enhancers (ESEs) sequence located at the site of sgRNA. Black arrow indicates the cleavage site. (FIG. 8C) Mouse Exon 51 sequence (SEQ ID NO: 967) with the predicted exon splicing enhancers (ESEs) located at the site of sgRNA is indicated in red. Human Exon 51 sequence (SEQ ID NO: 968) with the predicted exon splicing enhancers (ESEs) located at the site of sgRNA is indicated in red. (FIG. 8D) Mouse and human ESE sites of exon 51 predicted using ESEfmder3. (FIG. 8E) Cas9 was expressed in the presence or absence of mouse sgRNA-sgRNA-5l-SA2 in 293 T human cells and gene editing was monitored by T7E1 assay in fluorescence-based cell sorted (FACS) (+) and non- sorted cells (-). GFP was used as a control. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 77lbp PCR band. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay. M denotes size marker lane bp indicates the length of the marker bands. (FIG. 8F) Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing deletions and insertions (SEQ ID NOS: 969-978, respectively, in order of appearance). Black arrowhead indicates the cleavage site.

[035] FIG. 9A-B. Schematic illustration of AAV injection plasmids and strategy. (FIG.

9 A) Muscle creatine kinase 8 (CK8) promoter to express SpCas9. (FIG. 9B) Triplicate using rAAV9-sgRNA plasmid containing 3 copies of sgRNA-ex5l-SA2. U6, Hl and 7SK promoter for RNA polymerase III were used to drive expression of sgRNA.

[036] FIG. 10A-B. In vivo Dmd gene editing. (FIG. 10A) Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel of TA (tibialis anterior) muscle samples from WT and AEx50 mice 3 weeks after intramuscular injection with AAV9-sgRNA-SA2 and AAV9-Cas9 expression vectors. Controls were injected with only AAV9-Cas9 not AAV9-sgRNA-SA2. Black arrowhead in the upper panel indicates the 77lbp PCR band. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay. M denotes size marker lane bp indicates the length of the marker bands. N=4. (FIG. 10B) Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in AEx50 mice injected with AAV9-sgRNA-5l-SA2 and AAV9-Cas9. Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and deletions (highlighted in red) (SEQ ID NOS: 979-1014, respectively, in order of appearance). Black arrowheads indicate the cleavage site. n=3.

[037] FIG. 11A-D. RT-PCR analysis of correction of reading frame. (FIG. 11A) RT- PCR of RNA from tibialis anterior muscles of wildtype (WT) and AEx50 mice 3 weeks after intramuscular injection of the sgRNA-5l-SA2 and Cas9 expression vectors. Lower dystrophin bands indicate deletion of exon 51. Primer positions in exons 48 and 53 are indicated (Fw, Rv). (FIG. 11B) Percentage of events detected at exon 51 after AAV9- sgRNA-5l-SA2 treatment using RT-PCR sequence analysis of TOPO-TA generated clones. For each of 4 different samples, 40 clones were generated and sequenced. RT-PCR products were divided into 4 groups: not-edited (NE), exon51 -skipped (SK), refrained (RF) and out of frame (OF). (FIG. 11C) Sequence of the RT-PCR products of the DEc50-51 mouse dystrophin lower band confirmed that exon 49 spliced directly to exon 52, excluding exon 51. Sequence of RT-PCR products of DEc50 refrained (AEx50-RF). Fig. 11C discloses SEQ ID NOS: 1015-1022, respectively, in order of appearance. (FIG. 11D) Deep sequencing analysis of RT-PCR products from the upper band containing DEc50 not-edited (NE) and \Ex50-RF. Sequence of RT-PCR products revealing insertions (highlighted in green) and deletions (highlighted in red). n=4. Data are represented as mean ± SEM. Fig. 11D discloses SEQ ID NOS: 1023-1026, respectively, in order appearance.

[038] FIG. 12A-D. Intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51-SA2 corrects dystrophin expression. (FIG. 12A) Tibialis anterior muscles of DEc50 mice were injected with AAV9 vector encoding sgRNA and Cas9 and analyzed 3 weeks later by immunostaining for dystrophin. Wild type control (WT-CTL) mice and DEc50 mice control ( \Ex50-CTL) were injected with AAV9-Cas9 alone without sgRNAs. Percentages of dystrophin-positive myofibers in \Ex50-CTL mice and in treated DEc50 mice (DEc50- AAV9-sgRNA-5l-SA2 and AAV9-Cas9) compared to WT-CTL are indicated in each panel. (FIG. 12B) Hematoxylin and eosin (H&E) staining of tibialis anterior muscles. (FIG. 12C) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in tibialis anterior muscles 3 weeks after intramuscular injection. (FIG. 12D) Quantification of dystrophin expression from blots after normalization to vinculin. Asterisk indicates non-specific immunoreactive bands. n=5 for AAV9-sgRNA-5l-SA2. Scale bar: 50mm.

[039] FIG. 13. Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mouse model. Dystrophin immunohistochemistry of entire tibialis anterior muscle. CTL mice were injected with AAV9-Cas9 alone without AAV9-sgRNA-5l-SA2. n=5.

[040] FIG. 14. Histological improvement of injected muscle after 3 weeks.

Histochemistry of tibialis anterior muscle by hematoxylin and eosin (H&E) staining.

[041] FIG. 15A-B. Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 combined with different AAV9s expressing single copy or triple copy of sgRNA in AEx50 mouse model. (FIG. 15A) The U6, Hl and 7SK promoters for RNA polymerase III were each individually used to express sgRNA in a single copy (AAV9-U6- sgRNA-5l-SA2; AAV9-Hl-sgRNA-5l-SA2; AAV9-7SK-sgRNA-5l-SA2) or triple copy. (FIG. 15B) Dystrophin immunohistochemistry of entire tibialis anterior muscle. Control (CTL) mice were injected with AAV9-Cas9 alone without AAV9-sgRNA-5l-SA2.

[042] FIG. 16A-B. Rescue of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mice. (FIG. 16A) Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 4 weeks after systemic injection of AAV9-sgRNA-5l. (FIG. 16B) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in TA, triceps, diaphragm muscles and heart. n=5 for each group. Scale bar: 50mm.

[043] FIG. 17A-B. Rescue of dystrophin expression 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mice. (FIG. 17A) Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 8 weeks after systemic injection of AAV9-sgRNA-5l. (FIG. 17B) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in TA, triceps, diaphragm muscles and heart. n=5 for each group. Scale bar: 50mm.

[044] FIG. 18A-B. Functional improvement 4 weeks after systemic delivery of AAV9- Cas9 and AAV9-sgRNA-51-SA2 in AEx50 mice. (FIG. 18A) Wild type (WT) mice, control AEx50 mice and AEx50 mice treated with AAV9-sgRNA-5l-SA2 (AEx50-AAV9-sgRNA- 51-SA2) were subjected to grip strength testing to measure muscle performance (grams of force). (FIG. 18B) Serum creatine kinase (CK) was measured in WT, AEx50 and AEx50- AAV9-sgRNA-5l-SA2 mice. n=5. Asterisk indicates non-specific immunoreactive bands. Data are represented as mean ± SEM.

[045] FIG. 19. Correction of dystrophin expression 6 weeks after intra-muscular injection in AEx50-MD Dog. Dystrophin immunohistochemistry staining of cranial tibialis muscle of a wild type dog (Nathan), a AEx50-MD Dog untreated, and two AEx50-MD Dogs (Newton (#1A) and Norman (#lB)) contralateral uninjected and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA-5l) SEQ ID NO: 863 injected cranial tibialis muscle. Scale bar: 50mih.

[046] FIG. 20A-B. Correction of dystrophin expression 6 weeks after intra-muscular injection in AEx50-MD Dog. (FIG. 20A) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in cranial tibialis muscles 6 weeks after intramuscular injection in 2 dogs (Newton (#lA) and Norman (#lB)). (FIG. 20B) Quantification of dystrophin expression from blots after normalization to vinculin.

[047] FIG. 21. Histological improvement of injected muscle after 6 weeks in AEx50-MD Dog. Histochemistry by hematoxylin and eosin (H&E) staining of cranial tibialis muscle from a wild type dog, AEx50 dog untreated, AEx50 contralateral uninjected and AEx50 dogs injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-Dog- #lA-AAV9s (Newton) and AEx50-Dog-#lB-AAV9s (Norman)). Scale bar: 50mm.

[048] FIG. 22A-B. Rescue of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) in AEx50 mice. (FIG. 22A) Dystrophin immunostaining of tibialis anterior (TA), triceps, diaphragm and cardiac muscles 4 weeks after systemic injection of different doses of AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA-5l. (Dose 1 = 2.6xl0¹³vg/kg of each AAV9; Dose 2 = 6xl0¹²vg/kg of each AAV9). (FIG. 22B) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in triceps, gastrocnemius/plantaris (G/P) diaphragm (Dia) muscles and heart.

[049] FIG. 23A-C. Correction of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) in 1 month old AEx50 mice. (FIG. 23A) Dystrophin immunostaining of tibialis anterior, triceps and cardiac muscles 4 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA-5l at a dose of 2.6xl0¹³vg/kg of each AAV9. (FIG. 23B)

Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in tibialis anterior muscle and heart. (FIG. 23C) Wild type (WT) mice, control AEx50 mice and AEx50 mice treated with AAV9-Cas9 and AAV9-sgRNA-5l (AEx50-AAV9) were subjected to grip strength testing to measure muscle performance (grams of force).

[050] FIG. 24A-C. Correction of dystrophin expression 8 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) in 1 month old AEx50 mice. (FIG. 24A) Dystrophin immunostaining of tibialis anterior, triceps, gastrocnemius, quadriceps, diaphragm and cardiac muscles 8 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51 with 2.6 x 10¹³ vg/kg of each AAV9. (FIG. 24B) Western blot analysis of dystrophin (DMD) and vinculin (VCL) expression in various tissues. (FIG. 24C) Wildtype (WT) mice, control AEx50 mice and AEx50 mice treated with AAV9- Cas9 and AAV9-sgRNA-5l (AEx50-AAV9) were subjected to grip strength testing to measure muscle performance (grams of force).

[051] FIG. 25A-B. In vivo investigation of correction of dystrophin expression using different AAV9-Cas9 and AAV9-sgRNA-51-SA2 (referred as AAV9-sgRNA-51) ratios by intravenous injection of AAV9s. (FIG. 25A) AEx50-KI-luciferase mice were injected with an AAV9 encoding Cas9 and an AAV9 encoding a sgRNA at a 1 : 1 ratio (1xl0¹⁴vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l) and at a 1 :2 ratio (1xl0¹⁴vg/kg of AAV9-Cas9 and 2xl0¹⁴vg/kg AAV9-sgRNA-5l). AEx50-KI-luciferase mice were analyzed weekly by bioluminescence. (FIG. 25B) Bioluminescence imaging of Dmd KI-luciferase reporter and AEx50-KI-luciferase reporter mice injected with an AAV9 encoding Cas9 and an AAV9 encoding an sgRNA at a 1:2 ratio (1xl0¹⁴vg/kg of AAV9-Cas9 and 2xl0¹⁴vg/kg of AAV9- sgRNA-5l) and at a 1: 1 ratio (1xl0¹⁴vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l), 2 weeks after injection.

[052] FIG. 26A-G.“Humanized”-AEx44 mouse model. (FIG. 26A) Outline of the CRISPR/Cas9 strategy used for generation of the AEx44 mice. (FIG. 26B) Outline of the CRISPR/Cas9 strategy to deplete exon 44. T7E1 assay using 10T1/2 mouse cells transfected with spCas9 with different sgRNAs targeting 5’ end (In44-l, In44-2 or In44-3) and 3’ end (In44-4, In44-5, In44-6) of exon 44 shows different cleavage efficiency at the Dmd exon 44. Red arrowheads show cleavage products of genome editing. (FIG. 26C) PCR genotyping of 10 Fl pups shows efficient exon 44 depletion by CRISPR/Cas9-mediated genome editing.

The lower band (red arrowheads) shows exon 44 deletion. (FIG. 26D) Serum creatine kinase (CK), a marker of muscle dystrophy that reflects muscle damage and membrane leakage was measured in wild type (WT; C57BL/6 and C57 BL/10), AEx44, and mdx mice. Data are represented as means ± SEM. Unpaired Student’s t-test was performed *P<0.05 (n=6). (FIG. 26E) Western blot analysis shows loss of dystrophin expression in heart, TA muscle, and gastrocnemius/plantaris (G/P) muscle of AEx44 mice. Vinculin was used as a loading control. (FIG. 26F) Dystrophin staining of TA, diaphragm and cardiac muscle. (FIG. 26G)

Hematoxylin and eosin (H&E) staining of TA, diaphragm and cardiac muscle. [053] FIG. 27A-E. Characterization of DEc44 mice. (FIG. 27A) Outline of the

CRISPR/Cas9 strategy used for generation of the DEc44 mice. (FIG. 27B) Activity of serum creatine kinase (CK), a marker of muscle dystrophy that reflects muscle damage and membrane leakage was measured in wild type (WT) and DEc44 mice. Maximal tetanic force (FIG. 27C), specific force (FIG. 27D), and forelimb grip strength (FIG. 27E) were reduced in DEc44 mice compared to wild type (WT) mice, indicating decreased muscle function. Data are represented as means ± SEM. **R<0.001. (n=6).

[054] FIG. 28A-G. Correction of Dmd exon 44 deletion in mice by intramuscular AAV9 delivery of gene editing components. (A) RT-PCR analysis of TA muscles from WT and DEc44 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. Lower dystrophin bands (179 bp) indicate skipping of exon 45. (B) Pie chart showing percentage of events detected at exon 45 after AAV-Cas9 and AAV-G6 treatment using RT- PCR sequence analysis of TOPO-TA (topoisomerase-based thymidine to adenosine) generated clones. RT-PCR products were divided into four groups: Not edited (NE), exon 45-skipped (SK), refrained (RF), and out of frame (OF) are indicated. Data are represented as mean ± SEM. (n=3) (C) Sequences of RT-PCR products of WT, DEc44 and corrected DEc44 mice. In-frame sequences are shown in blue, including WT and exon 45-skipped sequences. Refrained sequence is shown in green, and out of frame sequence is shown in red. Figure discloses SEQ ID NOS 2305-2312, respectively, in order of appearance. (D) Western blot analysis shows restoration of dystrophin expression in TA muscle and heart of DEc44 mice. Vinculin is loading control. (E) Quantification of the Western blot analysis in TA muscle. Relative dystrophin intensity was calibrated with vinculin internal control. Data are represented as mean ± SEM. Unpaired Student’s t-test was performed. *P<0.05. (n=3) (F) Immunostaining shows restoration of dystrophin in TA muscle of DEc44 mice 3 weeks after intramuscular injection of gene editing components carried by AAV9. Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. Scale bar is 100 mm. (n=3). (G) H&E staining of TA and heart in WT, DEc44, and corrected DEc44 mice. Inset box indicates area of magnification shown below. Scale bar is 50 mm. (n=3).

[055] FIG. 29A-E. Systemic AAV9 delivery of gene editing components to D44 mice rescues dystrophin expression. Different AAV9-Cas9 and AAV9-exon45-sgRNA-G6 ratios were injected into D44 mice: 1.7: 1 (8.5xl0¹³vg/kg of AAV9-exon45-sgRNA to 5xl0¹³vg/kg of AAV9-Cas9); 2: 1 (1xl0¹⁴vg/kg of AAV9-exon45-sgRNA to 5xl0¹³vg/kg of AAV9-Cas9); 2.5: 1 (l.25xl0¹³vg/kg of AAV9-exon45-sgRNA-G6 to 5xl0¹³vg/kg of AAV9-Cas9), 5: 1 (2xl0¹⁴vg/kg of AAV9-exon45-sgRNA-G6 to 5xl0¹³vg/kg of AAV9-Cas9) and 10: 1 (5xl0¹⁴vg/kg of AAV9-exon45-sgRNA-G6 to 5xl0¹³vg/kg of AAV9-Cas9). (FIG. 29A) Western blot analysis shows restoration of dystrophin expression in TA, diaphragm, triceps and cardiac muscles of D44 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9- Cas9/exon45-sgRNA4. Vinculin was used as a loading control. (FIG. 29B) Immunostaining shows restoration of dystrophin in TA, diaphragm, triceps and cardiac muscles of D44 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9-Cas9/exon45-sgRNA4. Dystrophin stains in red. Nucleus marks by DAPI stains in blue. (FIG. 29C) Reduction of serum creatine kinase activity in D44 mice 4 weeks after systemic delivery of AAV9-Cas9 or AAV9- Cas9/exon45-sgRNA4. (FIG. 29D) Maximal tetanic force of EDL muscles in WT (blue), AEx44 DMD (red), and corrected AEx44 DMD (green) mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-sgRNA at 1 :5 and 1: 10 ratios. PO.05. (n=6) (FIG. 29E) Specific force (mN/mm²) of EDL muscles in WT (blue), AEx44 DMD (red), and corrected AEx44 DMD (green) mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-sgRNA at 1 :5 and 1: 10 ratios. Data are represented as mean ± SEM. One-way ANOVA was performed followed by Newman-Keuls post hoc test. **P<0.00l. (n=6).

[056]

[057] FIG. 30. Immunostaining of dystrophin following intravenous delivery of AAV9- encoded gene editing components in AEx50-MD Dogs. Dystrophin immunohistochemistry staining of cranial tibialis, semitendinosus, biceps, triceps, diaphragm, heart and tongue muscles of wild type dog, untreated AEx50 dog, and AEx50 dogs injected systemically with AAV9-Cas9 and AAV9-sgRNA at 2xl0¹³vg/kg (total virus 4xl0¹³ vg/kg, referred as AEx50- Dog #2A-AAV9s) and 1x10¹⁴ vg/kg (total virus 2xl0¹⁴ vg/kg, referred as AEx50-Dog #2B- AAV9s) for each virus.

[058] FIG. 31A-D. Western blot of dystrophin following intravenous delivery of AAV9- encoded gene editing components. (FIG. 31A) Western blot analysis of dystrophin (DMD) and vinculin (VCL) of cranial tibialis, triceps, biceps muscles of wild type, untreated AEx50, and AEx50 injected with AAV9-Cas9 and AAV9-sgRNA at 2xl0¹³ vg/kg for each virus (referred as AEx50-Dog #2A-AAV9s). (FIG. 31B) Quantification of dystrophin expression from blots after normalization to vinculin. (FIG. 31C) Western blot analysis of dystrophin (DMD) and vinculin (VCL) of cranial tibialis, triceps, biceps, diaphragm, heart, tongue muscles of wild type, untreated AEx50, and AEx50 injected with AAV9-Cas9 and AAV9- sgRNA at 1x10¹⁴ vg/kg of each virus (referred as AEx50-Dog #2B-AAV9s). (FIG. 31D) Quantification of dystrophin expression from blots after normalization to vinculin. [059] FIG. 32. Muscle histology following intravenous delivery of AAV9-encoded gene editing. Histochemistry by hematoxylin and eosin (H&E) staining of cranial tibialis, diaphragm and biceps muscles of wild type dog untreated, AEx50-MD Dogs untreated and AAV9-Cas9-sgRNA injected with AAV9-Cas9/AAV9-sgRNA at 2xl0¹³vg/kg (Dog #2A) and 1xl0¹⁴vg/kg (Dog #2B) for each virus. Scale bar: 50mm.

[060] FIG. 33. Blood analysis 8 weeks after intra-venous injection. Creatine kinase (CK) activities in untreated wild type dog, untreated DEc50 dog, AEx50-Dog-#2A injected with 2xl0¹³vg/kg and AEx50-Dog-#2B injected with1xl0¹⁴vg/kg of each of AAV9-Cas9 and AAV9-sgRNA.

[061] FIG. 34A-F. AEx50-Dmd-Luc mouse model. (FIG. 34A) Strategy for creation of dystrophin reporter mice. Dystrophin ( Dmd) gene with exons is indicated in blue. Using CRISPR/Cas9 mutagenesis, the inventors inserted a DNA cassette encoding the Luciferase reporter with the protease 2A cleavage site at the 3’ end of the dystrophin coding region. (FIG. 34B) Bioluminescence imaging of wild-type (WT) and Dmd knock-in luciferase reporter (referred as WT-Dmd-Luc) mice. (FIG. 34C) Strategy for creation of AEx50-Dmd- Luc reporter mice. Dystrophin ( Dmd) gene with exons is indicated in blue. Using

CRISPR/Cas9 mutagenesis, the inventors deleted the exon 50 of Dmd gene. (FIG. 34D) Genotyping results of AEx50-Dmd-Luc reporter mice. Schematic of genotyping strategy and forward (Fw) and reverse (Rv) primers. (FIG. 34E) Bioluminescence imaging of wild-type (WT), WT-Dmd-Luc and AEx50-Dmd-Luc reporter mice. (FIG. 34F) Western blot analysis of dystrophin (DMD), Luciferase and vinculin (VCL) expression in skeletal muscle and heart tissues.

[062] FIG. 35A-E. Correction of dystrophin expression by intra-muscular injection of AAV9-encoded gene editing components. (FIG. 35A) The left tibialis anterior muscle of AEx50-Dmd-Luc mice were injected with AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA) SEQ. NO: 708. AEx50-Dmd-Luc mice were analyzed weekly by bioluminescence. Control mice were injected with saline. (FIG. 35B) Bioluminescence imaging of wild-type (WT), WT-Dmd-Luc and AEx50-Dmd-Luc mice injected with AAV9- Cas9 and AAV9-sgRNA 1 week and 4 weeks after injection. (FIG. 35C) Dystrophin immunohistochemistry of entire tibialis anterior muscle of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9- sgRNA, 4 weeks after injection. (FIG. 35D) Dystrophin immunohistochemistry of tibialis anterior muscle of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA, 4 weeks after injection. (FIG. 35E) Bioluminescent imaging (BLI) measurements of left hindlimb of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9- sgRNA. Data are represented as mean ± SEM. n=3. (**P<0.005, ***P<0.0005). Scale bar: 50mm.

[063] FIG. 36A-D. Correction of dystrophin expression by systemic delivery of AAV9- encoded gene editing components. (FIG. 36A) AEx50-Dmd-Luc mice were injected intraperitoneally with AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA) SEQ. NO: 708 and analyzed by bioluminescence. Control mice were injected with saline. (FIG. 36B) Bioluminescence imaging of WT-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA. (FIG. 36C) Bioluminescent imaging (BLI) measurements of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA. (FIG. 36D) Dystrophin

immunohistochemistry of diaphragm, heart, tibialis anterior and triceps muscles 10 weeks after systemic injection with AAV9-Cas9 and AAV9-sgRNA. Data are represented as mean ± SEM. n=4. (***P<0.0005). Scale bar: 50mm.

[064] FIG. 37A-B. Western blot of dystrophin and luciferase following systemic delivery of AAV9-encoded gene editing components. (FIG. 37A) Western blot analysis of dystrophin (DMD), Luciferase (Luc), Cas9 and vinculin (VCL) in diaphragm, heart, triceps muscles and tibialis anterior of WT-Dmd-Luc mice, control AEx50-Dmd-Luc mice and AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA-5l-SA2 (referred as AAV9-sgRNA) SEQ. NO: 708. (FIG. 37B) Quantification of dystrophin and Luciferase expression from blots after normalization to vinculin. Data are represented as mean ± SEM. n=4.

[065] FIG. 38. AEx50-Dmd-Luciferase mouse model analysis. Genomic sequence of targeted locus of WT-Dmd-Luc (top line) (SEQ ID NO: 2313) and AEx50-Dmd-Luciferase founder (bottom line) (SEQ ID NO: 2314) with a 215 base pair deletion that eliminated exon 50 (indicated in color green). sgRNA-#l and #2 are indicated in blue.

[066] FIG. 39. AEx50-Dmd-Luciferase mouse model muscle histological analysis.

Hematoxylin and eosin (H&E) staining of tibialis anterior, quadriceps and diaphragm muscles of 6 weeks old WT-Dmd-Luc and AEx50-Dmd-Luciferase mice. n=5. Scale bar: 50mm.

[067] FIG. 40. In vivo Dmd gene editing after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51. Percentage of indels detected at exon 51 after AAV9-Cas9 and AAV9- sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of RT-PCR products from diaphragm, heart, tibialis anterior and triceps muscle samples of untreated WT- Dmd-Luc, untreated AEx50-Dmd-Luciferase mice and AEx50-Dmd-Luciferase mice injected with AAV9-Cas9 and AAV9-sgRNA-5l. n=4.

[068] FIG. 41A-D. Single cut CRISPR editing of canine exon 50 in vivo and in vitro. (FIG. 41A) Scheme showing the CRISPR/Cas9-mediated genome editing approach to correct the reading frame in AEx50 dogs by refraining and skipping of exon 51. Gray exons are out of frame. (FIG. 4 IB) Illustration of sgRNA binding position and sequence for sgRNA-ex5l. PAM sequence for sgRNA is indicated in red. Black arrow indicates the cleavage site. Figure discloses SEQ ID NOS 2315-2316, respectively, in order of appearance. (FIG. 41C)

Sequence of the RT-PCR products of the AEx50-5l lower band confirmed that exon 49 spliced directly to exon 52, excluding exon 51. Sequence of RT-PCR products of AEx50 reframed (AEx50-RF). Figure discloses SEQ ID NOS 2317-2324, respectively, in order of appearance. (FIG. 41D) Cranial tibialis muscles of AEx50 dogs were injected with AAV9s encoding sgRNA-5l and Cas9 as schematized in FIG. 9A-B and analyzed 6 weeks later. Dystrophin immunohistochemistry staining of cranial tibialis muscle of wild type dog untreated, AEx50 dog untreated, AEx50 dogs contralateral (uninjected) muscle and AEx50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-#lA AAV9s and AEx50-#lB-AAV9s). Scale bar: 50mm.

[069] FIG. 42A-D. Rescue of dystrophin expression in human DMD iPSC-derived cardiomyocytes. (FIG. 42A) Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in iPS cells. Sequence of representative indels aligned with sgRNA sequence (indicated in blue) revealing insertions (highlighted in green) and deletions (highlighted in red). Black arrowhead indicates the cleavage site. Figure discloses SEQ ID NOS 2325-2336, respectively, in order of appearance. (FIG. 42B)

Immunocytochemistry of dystrophin expression (red) shows DMD iCMs lacking dystrophin expression. Following successful gene editing, the corrected DMD iCMs express dystrophin. Immunofluorescence (green) detects cardiac marker troponin-I. Nuclei are labeled by Hoechst dye (blue). (FIG. 42C) Western blot analysis of dystrophin (DMD) and vinculin (VCL) of WT untreated and GFP treated, iCM DMD carrying deletion from exon 48 to exon 50 untreated and GFP treated, iCM DMD carrying deletion from exon 48 to exon 50 from mixed clone treated with high and low concentration of Cas9 and human sgRNA-5l SEQ. NO: 714 (Cas9+hsgR). (FIG. 42D) Quantification of dystrophin expression from blots after normalization to vinculin. Scale bar: 50mm. [070] FIG. 43A-B. Validation of sgRNA-51 in dog and human 293T cells. (FIG. 43A)

Conservation of mouse (SEQ ID NO: 2337), dog (SEQ ID NO: 2338) and human (SEQ ID NO: 2339) exon 51 sequence. Single guide RNA sequence is indicated in blue. (FIG. 43B) Cas9 was expressed in the presence or absence of dog or human sgRNA-5l in dog primary muscle cells, Madin-Darby Canine Kidney (MDCK) dog cells and human 293T cells. Gene editing was monitored by T7E1 assay in presence (+) of dog sgRNA-5l (D-sgRNA-5l) or human sgRNA-5l (H-sgRNA-5l) and absence of sgRNA (-). Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 574bp PCR band amplified from human genomic samples. Grey arrowhead indicates the undigested 748bp PCR band amplified from dog genomic samples. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay from PCR bands amplified from human genomic DNA (H). Red arrowheads in the lower panel indicate the cut bands by T7E1 assay from PCR bands amplified from dog genomic DNA (D). M denotes size marker lane bp indicates the length of the marker bands.

[071] FIG. 44A-C. In vivo Dmd gene editing after intramuscular injection. (FIG. 44A) Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in DEc50 dogs injected with AAV9-sgRNA-5l and AAV9-Cas9. Sequences of representative indels are aligned with the sgRNA sequence (indicated in blue), revealing insertions

(highlighted in green) and deletions (highlighted in red). Figure discloses SEQ ID NOS 2340- 2359, respectively, in order of appearance. (FIG. 44B) RT-PCR of RNA from cranial tibialis muscles of wild type dog untreated, DEc50 dog untreated, DEc50 dogs contralateral uninjected and DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-# 1 A-AAV9s and AEx50-#lB-AAV9s) 6 weeks after intramuscular injection. Lower bands indicate deletion of exon 51. Primer positions in exons 48 and 53 are indicated (Fw, Rv). (FIG. 44C) Deep sequencing analysis of RT-PCR products containing AEx50 nonedited (NE) and AEx50-RF (reframed) sequences. Sequence of RT-PCR products revealing insertions (highlighted in green) and deletions (highlighted in red). Figure discloses SEQ ID NOS 2360-2364, respectively, in order of appearance.

[072] FIG. 45. List of potential off-target sites in the dog genome for sgRNA-51.

Mismatches in the target sequence are highlighted in red. Three potential genome-wide off- target sites (OT1 to OT3) were predicted in the coding regions of mitochondrial pyruvate carrier 2 (MCP2), microphthalmia-associated transcription factor (MITF) and prolyl 4- hydroxylase transmembrane (P4HTM). Figure discloses SEQ ID NOS 2360-2364, respectively, in order of appearance.

[073] FIG. 46. Deep sequencing analyses of off-target sites for sgRNA-51. Genomic deep sequencing analysis of PCR amplicons generated across the exonic off-target sites in cranial tibialis muscles. Mismatches in the target sequence are highlighted in red. Muscle samples from untreated wild type dog, untreated DEc50 dog and contralateral uninjected DEc50 dogs were used for analysis to determine the background of the sequencing analysis. Figure discloses SEQ ID NOS 2372-2425, respectively, in order of appearance.

[074] FIG. 47A-B. Intramuscular delivery of AAV9-Cas8 and AAV9-sgRNA-51 in AEx50 dogs reduces expression and numbers of develommental myosin (dMHC)-positive fibers. (FIG. 47A) Perlecan and develommental myosin (dMHC) immunohistochemistry of cranial tibialis muscles 6 weeks after intramuscular injection of AAV9s. (FIG. 47B) Western blot analysis of dMHC and vinculin (VCL) in cranial tibialis muscles 6 weeks after intramuscular injection. Scale bar: 50mm.

[075] FIG. 48. Intramuscular delivery of AAV9-Cas8 and AAV9-sgRNA-51 in DEc50 dogs restores dystroglycan complex protein expression b-dystroglycan

immunohistochemistry of cranial tibialis muscles 6 weeks after intramuscular injection of AAV9s. Scale bar: 50mm.

[076] FIG. 49. Lack of immune infiltration in muscles after intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51. CD4 and CD8 immunohistochemistry (green color) of wild type dog untreated, DEx50 dog untreated and DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-# 1 A-AAV9s and AEx50-#lb-AAV9s) 6 weeks after intramuscular injection. Scale bar: 50mm.

[077] FIG. 50. Blood analysis 6 weeks after intramuscular injection of AAV9-Cas9 and AAV9-sgRNA-51. Hematology analysis in wild type dog and DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l (referred as AEx50-#lA-AAV9s and AEx50-#lb- AAV9s). White blood cells (WBC), red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelet count (PLT) were evaluated.

[078] FIG. 51A-B. AAV9-Cas9 expression after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in dogs. (FIG. 51A) qRT-PCR analysis of Cas9 expression of cranial tibialis, heart left ventricle, heart right ventricle, heart septum, biceps, diaphragm, triceps, temporalis and masseter of untreated wild type dog, untreated AEx50 dog, AEx50-dog-#2A injected with 2xl0¹³vg/kg of AAV9-Cas9 and AAV9-sgRNA-5l. (FIG. 51B) qRT-PCR analysis of Cas9 expression of cranial tibialis, heart left ventricle, heart right ventricle, heart septum, biceps, diaphragm, triceps, temporalis and masseter of untreated wild type dog, untreated AEx50 dog, AEx50-dog-#2B injected with 1xl0¹⁴vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l. All data was normalized to canine 18S ribosomal RNA and represents. Fold difference relative to untreated animals (2 ^AAtT).

[079] FIG. 52A-D. In vivo DMD gene editing after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in dogs. (FIG. 52A) Percentage of indels detected at genomic locus of exon 51 after AAV9-Cas9 and AAV9-sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of PCR products amplifying the targeted region from cranial tibialis, heart left ventricle, heart right ventricle, heart septum, diaphragm, biceps and triceps samples of untreated wild type dog, untreated AEx50 dog, AEx50-Dog-#2A injected with 2xl0¹³vg/kg and AEx50-Dog-#2B injected with 1xl0¹⁴vg/kg of AAV9-Cas9 and AAV9- sgRNA. (FIG. 52B) RT-PCR of RNA from cranial tibialis, heart left ventricle, heart right ventricle, heart septum, diaphragm, biceps and triceps samples of untreated wild type dog, untreated AEx50 dog, AEx50-Dog-#2A injected with 2xl0¹³vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l (referred as AAV9s). Black arrowhead indicates the 822bp WT PCR. Red arrowhead indicates the 7l3bp AEx50 PCR. Lower bands indicate deletion of exon 51. (FIG. 52C) RT-PCR of RNA from cranial tibialis, heart left ventricle, heart right ventricle, heart septum, diaphragm, biceps and triceps samples of untreated wild type dog, untreated AEx50 dog, AEx50-Dog-#2B injected with 1xl0¹⁴vg/kg of AAV9-Cas9 and AAV9-sgRNA (referred as AAV9s). Black arrowhead indicates the 822bp WT PCR band. Red arrowhead indicates the 7l3bp AEx50 PCR band. Grey arrowhead indicates the deletion of exon 51 and 480bp AEx50-5l PCR band. (FIG. 52D) Percentage of indels detected at exon 51 after AAV9-Cas9 and AAV9-sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of RT-PCR products from cranial tibialis, heart left ventricle, heart right ventricle, heart septum, diaphragm, biceps and triceps samples of untreated wild type dog, untreated AEx50 dog, AEx50-Dog-#2A injected with 2xl0¹³vg/kg and AEx50-Dog-#2B injected with 1xl0¹⁴vg/kg of AAV9-Cas9 and AAV9-sgRNA.

[080] FIG. 53. Correction of dystroglycan complex protein expression after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in AEx50 dogs, b-dystroglycan

immunohistochemistry of cranial tibialis, biceps, triceps, diaphragm and tongue muscles 8 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-5l (referred as AAV9s). Scale bar: 50mm. [081] FIG. 54A-B. Decrease of regeneration markers after systemic delivery of AAV9- Cas9 and AAV9-sgRNA-51 in DEc50 dogs. (FIG. 54A) Perlecan and develomment myosin (dMHC) immunohistochemistry of triceps, diaphragm and semitendinosus muscles 8 weeks after systemic injection of AAV9-Cas9 and AAV9-sgRNA-5l (referred as AAV9s). (FIG. 54B) Western blot analysis of dMHC and vinculin (VCL) in cranial tibialis and diaphragm muscles of wild type, DEc50 injected with AAV9-Cas9 at 1x10¹⁴ vg/kg and untreated DEc50. Scale bar: 50mm.

[082] FIG. 55. ELISpot analysis before and after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51. T-cell reactivity to Cas9 measured using ELISpot analysis of peripheral blood mononuclear cells (PBMCs) of AEx50-Dog-#2A injected with 2xl0¹³vg/kg and AEx50-Dog-#2B injected with 1xl0¹⁴vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l. PBMCs were isolated from blood samples before injection (0), 1, 2, 4, 6 and 8 weeks after injection. PBMCs were stimulated with three different concentrations of Cas9 protein and phorbol l2-myristate 13- acetate (PMA)/ionomycin as a positive control. Medium alone served as the negative control. Spot forming units (SFU) were normalized to the number of responsive cells in the PBMCs used for the analyses. Each sample was measured in triplicate.

[083] FIG. 56A-B. Hematology and biochemistry analyses before and after systemic injection of AAV9-Cas9 and AAV9-sgRNA-51. Blood samples collected before injection (0), 1, 2, 4, 6 and 8 weeks after injection were used for the hematology and biochemistry analysis. (FIG. 56A) Hematology analyses in wild type dog untreated, DEc50 dog untreated, DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l. The numbers of white blood cells (WBC), red blood cells (RBC), Hemoglobin (HGB) concentration, Hematocrit (HCT), Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT) were examined in untreated wild type dog, DEc50 dog untreated, DEc50 dogs injected with AAV9-Cas9 and AAV9- sgRNA-5l. (FIG. 56B) Total protein, albumin, globulin, sodium, potassium, chloride, calcium, inorganic phosphate, urea, creatinine, cholesterol, total bilirubin, amylase, lipase, alanine transaminase (ALT) and alkaline phosphatase (ALP) were measured in serum samples from untreated wild type dog, untreated DEc50 dog, AEx50-Dog-#2A injected with 2xl0¹³vg/kg and AEx50-Dog-#2B injected with 1x10¹⁴ vg/kg of each of AAV9-Cas9 and AAV9-sgRNA.

[084] FIG. 57A-B. Validation of sgRNA-51 in human DMD iPS cells. (FIG. 57A) Cas9 was expressed in the presence or absence of human sgRNA-5l (hsgR) in human iPS cells and gene editing was monitored by T7E1 assay. High and low concentration of Cas9 and human sgRNA (hsgR) were tested. Undigested PCR products (upper panel) and T7E1 digestion (lower panel) are shown on a 2% agarose gel. Black arrowhead indicates the undigested 574bp PCR band. Green arrowheads in the lower panel indicate the cut bands by T7E1 assay. M denotes size marker lane bp indicates the length of the marker bands. (FIG. 57B)

Percentage of indels detected at exon 51 after Cas9 and sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of genomic PCR products.

[085] FIG. 58. Deep sequencing analysis of genomic DNA after systemic delivery of AAV9-Cas9 and AAV9 sgRNA-51 in dogs. Genomic deep sequencing analysis of PCR amplicons generated across the exon 51 target site in heart, biceps and triceps of DEx50 dogs systemically injected with AAV9-sgRNA-5l and AAV9-Cas9. Sequences of representative indels are aligned with the sgRNA sequence (indicated in blue), revealing insertions

(highlighted in green), deletions (highlighted in red), substitutions (underlined) and exonic splicing enhancer (ESE) site.

[086] FIG. 59A-B. Testes analyses after systemic delivery of AAV9-Cas9 and AAV9- sgRNA-51 in dogs. (FIG. 59A) Percentage of indels detected at genomic locus of exon 51 after AAV9-Cas9 and AAV9-sgRNA-5l treatment using tracking indels by decomposition (TIDE) analysis of PCR products amplifying the targeted region from testes, biceps and diaphragm samples of AEx50-Dog-#2B injected with 1xl0¹⁴vg/kg of each virus (total 2xl0¹⁴vg/kg). (FIG. 59B) Western blot analysis of Cas9 and vinculin (VCL) of testes of wild type, untreated AEx50, AEx50 injected with AAV9-Cas9 and AAV9-sgRNA at 2xl0¹³ vg/kg for each virus (total virus 4xl0¹³ vg/kg, referred as AEx50-Dog #2A-AAV9s), AEx50 injected with AAV9-Cas9 and AAV9-sgRNA at 1x10¹⁴ vg/kg for each virus (total virus 2xl0¹⁴ vg/kg, referred as AEx50-Dog #2B-AAV9s), biceps and diaphragm sample of AEx50 injected with AAV9-Cas9 and AAV9-sgRNA at 1x10¹⁴ vg/kg for each virus (total virus 2xl0¹⁴ vg/kg, referred as AEx50-Dog #2B-AAV9s).

[087] FIG. 60. Western blot titration of dystrophin following intravenous delivery of AAV9-encoded gene editing components. Western blot analysis of dystrophin (DMD) and vinculin (VCL) of cranial tibialis muscle of wild type, untreated AEx50, and AEx50 injected with AAV9-Cas9 and AAV9-sgRNA at 1x10¹⁴ vg/kg (total virus 2xl0¹⁴ vg/kg, referred as AEx50-Dog #2B-AAV9s).

[088] FIG. 61A-F. Exon 44-deleted DMD patient iPSC-derived cardiomyocytes express dystrophin after CRISPR/Cas9 mediated genome editing. (FIG. 61A) Schematic of the procedure for derivation and editing of DMD patient-derived iPSCs and iPSC-CMs. (FIG.

6 IB) Gene editing strategy for DMD exon 44 deletion. Deletion of exon 44 (black) results in splicing of exon 43 to 45, generating an out-of-frame stop mutation of dystrophin. Disruption of the splice junction of exon 43 or exon 45 results in splicing of exon 42 to 45 or exon 43 to 46, respectively, and restores the protein reading frame. The protein reading frame can also be restored by refraining exon 43 or 45 (green). (FIG. 61C) Sequence of sgRNAs targeting exon 43 splice acceptor and donor sites in the human DMD gene. The protospacer adjacent motif (PAM) (denoted as red nucleotides) of the sgRNAs is located near the exon 43 splice junctions. Exon sequence is bold upper case. Intron sequence is lower case. Arrowheads show sites of Cas9 DNA cutting with each sgRNA. Splice acceptor and donor sites are shaded in yellow. Figure discloses SEQ ID NOS 2486-2489, respectively, in order of appearance. (FIG. 61D) Sequence of sgRNAs targeting exon 45 splice acceptor site in the human DMD gene. The PAM (denoted as red nucleotides) of the sgRNAs is located near the exon 45 splice acceptor site. Human and mouse conserved sequence is shaded in light blue. Exon sequence is bold upper case. Intron sequence is lower case. Figure discloses SEQ ID NOS 2490-2491, respectively, in order of appearance. (FIG. 61E) Western blot analysis shows restoration of dystrophin expression in exon 43-edited (E43) and exon 45-edited (E45) AEx44 patient iPSCCMs with sgRNAs (G) 3, 4 and 6, as indicated. Vinculin is loading control. HC, iPSC-CMs from a healthy control. The second lane is unedited AEx44 patient iPSC-CMs. (FIG. 61F) Immunostaining shows restoration of dystrophin expression in exon 43-edited and exon 45-edited AEx44 patient iPSC-CMs. Dystrophin is shown in red. Cardiac troponin I is shown in green. Nuclei are marked by DAPI stain in blue.

[089] FIG. 62A-B. Analysis of mice with a DMD exon 44 deletion. (FIG. 62A) RT-PCR analysis of TA muscles to validate deletion of exon 44. RT-PCR primers were in exons 43 and 46, and the amplicon size is 503 base pairs (bp) for WT mice and 355 bp for AEx44 DMD mice. RT-PCR products are schematized on the right. (n=3) (FIG. 62B) Sequencing of RT-PCR products from AEx44 DMD mouse muscle confirmed deletion of exon 44 and generation of a premature stop codon in exon 45, indicated by red asterisk. Figure discloses SEQ ID NOS 2492-2943, respectively, in order of appearance.

[090] FIG. 63A-B. Rescue of dystrophin expression after systemic AAV9 delivery of gene editing components to AEx44 mice. (FIG. 63A) Maximal tetanic force of EDL muscles in WT (blue), AEx44 DMD (red), and corrected AEx44 DMD (green) mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-sgRNA at 1 :5 and 1: 10 ratios. (n=6) (FIG. 63B) Specific force (mN/mm²) of EDL muscles in WT (blue), AEx44 DMD (red), and corrected AEx44 DMD (green) mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-sgRNA at 1:5 and 1 : 10 ratios. (n=6). Data are represented as means ± SEM.

**PO.OOl. (n=6).

[091] FIG. 64A-E. Analysis of sgRNAs that target the splice acceptor or donor sites for exon 43 and 45. (FIG. 64A) Alignment of human and mouse DNA sequence at the intron-exon junction of exon 45. The conserved region is shaded in light blue. Exon sequence is in bold upper case and intron sequence is in lower case. (FIG. 64B) T7E1 assay using human 293 cells transfected with plasmids that express SpCas9 and exon 43 sgRNAl (Gl), sgRNA2 (G2), sgRNA3 (G3) or sgRNA4 (G4) shows cleavage of the DMD locus at the intron-exon junctions of exon 43. Red arrowheads denote cleavage products. PCR indicates the undigested PCR product. (FIG. 64C) T7E1 assay using mouse 10T1/2 and human 293 cells transfected with plasmids that express 5/;Cas9 and exon 45 sgRNA5 (G5), sgRNA6 (G6), sgRNA7 (G7) or sgRNA8 (G8) shows cleavage of the Dmd locus at the intron-exon junction of exon 45. Red arrowheads denote cleavage products. PCR indicates the undigested PCR product. (FIG. 64D) Sequences of the G6 edited 34 single clones. HC is sequence of the healthy human control. G6 sequence is shaded in blue. Insertions are shaded in green. Base modification and dystrophin gene status are listed on the right. Figure discloses SEQ ID NOS 2494-2528, respectively, in order of appearance. (FIG. 64E) Western blot analysis showing restoration of dystrophin expression in three exon 45-skipped single iPSC clones (clones #3, #11 and #13). Clone #3 and #11 were corrected through exon 45 skipping, and clone #9 was corrected through exon 45 reframing. HC, iPSC-derived CM from a healthy human control. NE, non-edited. Vinculin is loading control.

[092] FIG. 65A-B. Characterization of AEx44 mouse line. (FIG. 65A) sgRNA sequences are indicated in blue, protospacer adjacent motifs (PAMs) are indicated in red, and genotyping primers are highlighted in yellow. Exon 44 sequence is in bold upper case and intron sequence is in lower case. Figure discloses SEQ ID NO: 2529. (FIG. 65B) Picrosirius red staining of TA, diaphragm, and heart of WT and AEx44 mice. Scale bar is 50 mm.

[093] FIG. 66A-D. Intramuscular AAV9 delivery of gene editing components rescues dystrophin expression. (FIG. 66A) T7E1 assay shows cleavage of the Dmd locus at the intron-exon junction of exon 45 in mouse C2C12 cells with electroporation of G5 or G6 in PX458 or Trispr backbone. Red arrowheads show cleavage products of genome editing. PCR indicates the undigested PCR product. (FIG. 66B) T7E1 assay shows cleavage of the Dmd locus at the intron-exon junction of exon 45 in TA muscle of corrected AEx44 mice. Red arrowheads show cleavage products of genome editing. PCR indicates the undigested PCR product. On-target (FIG. 66C) cDNA and (FIG. 66D) genomic amplicon deep sequencing of DEc44 DMD, and corrected AF.\44 DMD mice after 3 weeks of AAV-Cas9 and AAV-G6 intramuscular injection (2.5 ^c 10¹⁰ vg of AAV9-Cas9 and 2.5 ^c 10¹⁰ vg of AAV-G6). Bold represents substitutions, red square is insertions,

is deletion. Vertical pink line indicates intron-exon junction in (FIG. 66C) and exon-exon junction in (FIG. 66D). Black arrowhead points to dotted vertical line representing the predicted cleavage site. Figures 66C-D disclose SEQ ID NOS 2530-2566, respectively, in order of appearance.

[094] FIG. 67A-C. Analysis of top ten potential off-target sites. (FIG. 67A) T7E1 analysis of the top 10 predicted off-target (OT) sites of sgRNA-G6 assayed in TA muscle 3 weeks following intramuscular injection of 2.5 ^c 10¹⁰ vg AAV9-Cas9 and 2.5 ^c 10¹⁰ vg AAV-G6. Red arrowheads denote on-target cleavage products. No off-target cleavage products were detected. PCR indicates the undigested PCR product. (FIG. 67B) Amplicon genomic deep sequencing analysis on the top 10 predicted off-target sites of G6. Muscle was analyzed 3 weeks following intramuscular injection of 2.5 ^c 10¹⁰ vg AAV9-Cas9 and 2.5 ^c 10¹⁰ vg AAV-G6. Mismatches in the target sequence are highlighted in red. Figure discloses SEQ ID NOS 2567-2577, respectively, in order of appearance. (FIG. 67C) Percentage of NHEJ in amplicon genomic deep sequencing analysis on the top 10 predicted off-target sites of G6. Blue indicates AAV-Cas9 only control, and red indicates AAV-Cas9/AAV-G6 injected TA muscle.

[095] FIG. 68. Correction of AEx44 mice by systemic delivery of AAV9 expressing gene editing components. Whole TA muscle scanning of AEx44 DMD and corrected AEx44 DMD 4 weeks after systemic injection of a 1 :5 ratio and 1: 10 ratio of AAV-Cas9 to AAV- G6. AAV-Cas9 was administered at 5 ^c 10¹³ vg/kg. Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. 10X tile scan of the entire TA muscle. Scale bar is 500 um.

[096] FIG. 69A-G. Western blot analysis of corrected AEx44 mice by systemic delivery of AAV9 expressing gene editing components. (FIG. 69A-F) Western blot analysis of dystrophin, Cas9, and GFP protein expression in TA, triceps, diaphragm, and heart of AEx44 mice 4 weeks after systemic delivery of AAVCas9 and AAV-G6 at the indicated ratios. AAV-Cas9 was administered at 5 ^c 10¹³ vg/kg. Vinculin is loading control. (n= 3) (FIG. 69G) Quantification of the Western blot analysis in TA, triceps, diaphragm, and heart.

Relative dystrophin intensity was calibrated with vinculin internal control. Data are represented as mean ± SEM. One-way ANOVA was performed followed by Newman-Keuls post hoc test. *P<0.005, **R<0.001, ****R<0.0001 (n=3).

[097] FIG. 70. Histology of AEx44 mice after systemic delivery of AAV9 expressing gene editing components. H&E staining of TA, triceps, diaphragm and heart of AEx44 mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-G6 at the indicated ratios. AAV- Cas9 was administered at 5 ^c 10¹³ vg/kg.

[098] FIG. 71. Whole muscle scanning of TA, triceps, diaphragm and heart of corrected AEx44 DMD mice. H&E staining of WT, AEx44 DMD and corrected AEx44 DMD 4 weeks after systemic injection of a 1 :5 ratio and 1: 10 ratio of AAV-Cas9 to AAV-G6. AAV-Cas9 was administered at 5 ^c 10¹³ vg/kg. 4X tile scan of the entire muscle. Scale bar in TA, triceps, diaphragm is 500um, in heart is l.5mm.

[099] FIG. 72A-B. qPCR analysis of the skeletal and cardiac muscle groups comparing low and high doses of AAV-G6. (FIG. 72A) qPCR analysis of Cas9 mRNA expression in TA, triceps, diaphragm, and heart of AEx44 mice 4 weeks after systemic delivery of AAV- Cas9 and AAVG6 at the indicated ratios. AAV-Cas9 was administered at 5 ^c 10¹³ vg/kg. Normalized to 18S ribosomal RNA. Data are represented as means ± SEM. (n= 3) (FIG.

72B) qPCR analysis of GFP mRNA expression in TA, triceps, diaphragm, and heart of AEx44 mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-G6 at the indicated ratios. AAV-Cas9 was administered at 5 ^c 10¹³ vg/kg. Normalized to 18S ribosomal RNA. Data are represented as means ± SEM (n=3).

[0100] FIG. 73A-B. Histological analysis showing dystrophin restoration in EDL muscle of corrected AEx44 DMD mice. (FIG. 73A) Dystrophin immunostaining of EDL muscle in AEx44 DMD and corrected AEx44 DMD 4 weeks after systemic injection of a 1:5 ratio and 1: 10 ratio of AAV-Cas9 to AAV-G6. AAV-Cas9 was administered at 5 ^c 10¹³ vg/kg.

Dystrophin is shown in red. Nuclei are marked by DAPI stain in blue. (FIG. 73B) H&E staining of EDL muscle in AEx44 DMD and corrected AEx44 DMD 4 weeks after systemic injection of a 1:5 ratio and 1: 10 ratio of AAV-Cas9 to AAV-G6. AAV-Cas9 was

administered at 5 ^c 10¹³ vg/kg.

[0101] FIG. 74. Picrosirius red staining of TA, triceps, diaphragm and heart of corrected AEx44 DMD mice. Picrosirius red staining of TA, triceps, diaphragm and heart of AEx44 mice 4 weeks after systemic delivery of AAV-Cas9 and AAV-G6 at the indicated ratios. AAV-Cas9 was administered at 5 ^c 10¹³ vg/kg. Scale bar is 50 mm.

[0102] FIG. 75. Correction of dystrophin expression 4 weeks after systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51 in AEx50 mice using different AAV9-Cas9 and AAV9-sgRNA-51-SA2 ratios. Dystrophin immunostaining of diaphragm, triceps and quadriceps muscles 4 weeks after systemic injection of 5xl0¹³ vg/kg AAV9-Cas9 and 5xl0¹³ vg/kg AAV 9-sgRNA-51 (Ex-5l-SA2 SEQ. No: 708), 5x10¹³ vg/kg AAV9-Cas9 and 1x10¹⁴ vg/kg AAV9-sgRNA-5l and 5xl0¹³ vg/kg AAV9-Cas9 and 1x10¹⁴ vg/kg AAV9-sgRNA-5l.

DETAILED DESCRIPTION

[0103] DMD is a new mutation syndrome with more than 4,000 independent mutations that have been identified in humans. The majority of patient mutations include deletions that cluster in a hotspot, and thus a therapeutic approach for skipping and/or reframing certain exon applies to large group of patients. The rationale of the exon skipping and/or reframing approach is based on the genetic difference between DMD and Becker muscular dystrophy (BMD) patients. In DMD patients, the reading frame of dystrophin mRNA is disrupted resulting in prematurely truncated, non-functional dystrophin proteins. BMD patients have mutations in the DMD gene that maintain the reading frame allowing the production of internally deleted, but partially functional dystrophins leading to much milder disease symptoms compared to DMD patients.

[0104] The disclosure provides Clustered Regularly Interspaced Short Palindromic

Repeat/Cas9 (CRISPR/Cas9)-mediated genome editing compositions for correcting a dystrophin gene (DMD) mutation or for use in a method of correcting a dystrophin gene (DMD) mutation, a mutation which left untreated, results in the onset of DMD. The data presented herein show that in vivo AAV-mediated delivery of gene-editing components successfully remove the mutant genomic sequence by reframing and/or exon skipping in muscle cells of mice, dogs and humans. Using different modes of AAV9 delivery, dystrophin protein expression was restored in muscle cells of DMD mouse and dog models,

demonstrating that the therapy is safe and effective, within minimal off-targeting and immunogenetic effects.

[0105] Compositions and methods for treating DMD are provided herein. In some embodiments, an AAV construct is provided, wherein the AAV construct comprises a nucleic acid encoding three promoters that each drive expression of a DMD guide RNA. Using compositions and methods disclosed herein, a more robust and safe form of genome editing was achieved in a humanized mouse model for DMD with a deletion in exon 50, in a AEx50- MD Dog and in human IPS cells. These and other aspects of the disclosure are reproduced below.

[0106] As used herein,“refraining” is used to refer to a genome editing strategy in which small INDELs restore the protein reading frame. The term“skipping”or“exon skipping” is used to refer to a genom editing strategy wherein a splice donor or splice acceptor site is disrupted, which results in exon skipping and restoration of the protein reading frame.

CRISPR Systems

[0107] 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.

[0108] CRISPR repeats 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. Some CRISPR spacer sequences exactly match sequences from plasmids and phages, although some spacers match the prokaryote’s genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.

Guide RN A (gRNA)

[0109] As an RNA guided protein, Cas9 requires a short RNA to direct the recognition of DNA targets. Though Cas9 preferentially interrogates DNA sequences containing a PAM sequence NGG it can bind here without a protospacer target. However, the Cas9-gRNA complex requires a close match to the gRNA to create a double strand break. CRISPR sequences in bacteria are expressed in multiple RNAs and then processed to create guide strands for RNA. Because Eukaryotic systems lack some of the proteins required to process CRISPR RNAs the synthetic construct gRNA was created to combine the essential pieces of RNA for Cas9 targeting into a single RNA expressed with the RNA polymerase type III promoter U6. Synthetic gRNAs are slightly over 10Obp at the minimum length and contain a portion which is targets the 20 protospacer nucleotides immediately preceding the PAM sequence NGG; gRNAs do not contain a PAM sequence.

[0110] In some embodiments, the gRNA targets a site within a wildtype dystrophin gene. An exemplary wildtype dystrophin sequence includes the human sequence (see GenBank Accession NO. NC_000023.11), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5), the sequence of which is reproduced below:

[0111] In some embodiments, the gRNA targets a site within a mutant dystrophin gene. In some embodiments, the gRNA targets a dystrophin intron. In some embodiments, the gRNA targets a dystrophin exon. In some embodiments, the gRNA targets a site in a dystrophin exon that is expressed and is present in one or more of the dystrophin isoforms shown in

Table 1. In embodiments, the gRNA targets a dystrophin splice site. In some embodiments, the gRNA targets a splice donor site on the dystrophin gene. In embodiments, the gRNA targets a splice acceptor site on the dystrophin gene.

[0112] Table 1: Dystrophin isoforms

[0113] In embodiments, the guide RNA targets a mutant DMD exon. In some embodiments, the mutant exon is exon 23 or 51. In some embodiments, the guide RNA targets at least one of exons 1, 23, 41, 44, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 of the dystrophin gene. In embodiments, the guide RNA targets at least one of introns 44, 45, 50, 51, 52, 53, 54, or 55 of the dystrophin gene. In preferred embodiments, the guide RNAs are designed to induce skipping and/or reframing of exon 51 or exon 23. In embodiments, the gRNA is targeted to a splice acceptor site of exon 51 or exon 23.

[0114] Suitable gRNAs for use in various compositions and methods disclosed herein are provided as SEQ ID NOs: 383-705, 709-711, 715-717, 790-862, 864 (Tables 7, 9, 11, 13, and 15). In preferred embodiments, the gRNA is selected from any one of SEQ ID No: 790 to SEQ ID No: 862.

[0115] In some embodiments, gRNAs of the disclosure comprise a sequence that is complementary to a target sequence within a coding sequence or a non-coding sequence corresponding to the DMD gene, and, therefore, hybridize to the target sequence. In some embodiments, gRNAs for Cpfl comprise a single crRNA containing a direct repeat scaffold sequence followed by 24 nucleotides of guide sequence. In some embodiments, a“guide” sequence of the crRNA comprises a sequence of the gRNA that is complementary to a target sequence. In some embodiments, crRNA of the disclosure comprises a sequence of the gRNA that is not complementary to a target sequence.“Scaffold” sequences of the disclosure link the gRNA to the Cpfl polypeptide.“Scaffold” sequences of the disclosure are not equivalent to a tracrRNA sequence of a gRNA-Cas9 construct.

[0116] In some embodiments, a nucleic acid may comprise one or more sequences encoding a gRNA. In some embodiments, a nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 sequences encoding a gRNA. In some embodiments, all of the sequences encode the same gRNA. In some embodiments, all of the sequences encode different gRNAs. In some embodiments, at least 2 of the sequences encode the same gRNA, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encode the same gRNA.

Nucleases

Cas Nucleases

[0117] CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. As of 2013, more than forty different Cas protein families had 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, Apem, 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.

[0118] Exogenous DNA is apparently processed by proteins encoded by Cas genes into small elements (~30 base pairs in length), which are then somehow 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.

[0119] Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. The team demonstrated that they could disable one or both sites while preserving Cas9’s ability to locate its target DNA. Jinek et al. (2012) combined tracrRNA and spacer RNA into a "single-guide RNA" molecule that, mixed with Cas9, can find and cut the correct DNA targets and such synthetic guide RNAs are used for gene editing.

[0120] 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. Wang el al. (2013) showed that coinjection of Cas9 mRNA and sgRNAs into the germline (zygotes) generated mice with mutations. Delivery of Cas9 DNA sequences also is contemplated.

[0121] 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. Cpfl has been recently identified as a Class II, Type V CRISPR/Cas systems containing a 1,300 amino acid protein. See also U.S. Patent Publication 2014/0068797, which is incorporated by reference in its entirety.

[0122] In some embodiments, the compositions of the disclosure include a small version of a Cas9 from the bacterium Staphylococcus aureus (UniProt Accession No. J7RUA5). The small version of the Cas9 provides advantages over wildtype or full length Cas9. In some embodiments the Cas9 is a spCas9 (AddGene).

Cpfl Nucleases

[0123] Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 or CRISPR/Cpfl is a DNA-editing technology which shares some similarities with 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.

[0124] Cpfl appears in many bacterial species. The ultimate Cpfl endonuclease that was developed into a tool for genome editing was taken from one of the first 16 species known to harbor it. [0125] In embodiments, the Cpfl is a Cpfl enzyme from Acidaminococcus (species BV3L6,

UniProt Accession No. U2UMQ6; SEQ ID NO: 870), having the sequence set forth below:

[0126] In some embodiments, the Cpfl is a Cpfl enzyme from Lachnospiraceae (species ND2006, UniProt Accession No. A0A182DWE3; SEQ ID NO: 871), having the sequence set forth below:

[0127] In some embodiments, the Cpfl is codon optimized for expression in mammalian cells. In some embodiments, the Cpfl is codon optimized for expression in human cells or mouse cells. [0128] The Cpfl locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc fmger-like domain. The Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpfl does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have the alpha- helical recognition lobe of Cas9.

[0129] Cpfl CRISPR-Cas domain architecture shows that Cpfl is functionally unique, being classified as Class 2, type V CRISPR system. The Cpfl loci encode Casl, Cas2 and Cas4 proteins more similar to types I and III than from type II systems. Database searches suggest the abundance of Cpfl -family proteins in many bacterial species.

[0130] Functional Cpfl does not require a tracrRNA, therefore, only crRNA is required. This benefits genome editing because Cpfl is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half as many nucleotides as Cas9).

[0131] The Cpfl -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, Cpfl introduces a sticky-end-like DNA double- stranded break of 4 or 5 nucleotides overhang.

[0132] The CRISPR/Cpfl system consist of a Cpfl enzyme and a guide RNA that finds and positions the complex at the correct spot on the double helix to cleave target DNA.

CRISPR/Cpfl systems activity has three stages: 1) Adaptation, during which Casl and Cas2 proteins facilitate the adaptation of small fragments of DNA into the CRISPR array; 2) Formation of crRNAs: processing of pre-cr-RNAs producing of mature crRNAs to guide the Cas protein; and 3) Interference, in which the Cpfl is bound to a crRNA to form a binary complex to identify and cleave a target DNA sequence.

Cas9 versus Cpfl

[0133] Cas9 requires two RNA molecules to cut DNA while Cpfl needs one. The proteins also cut DNA at different places, offering researchers more options when selecting an editing site. Cas9 cuts both strands in a DNA molecule at the same position, leaving behind‘blunt’ ends. Cpfl leaves one strand longer than the other, creating 'sticky' ends that are easier to work with. Cpfl appears to be more able to insert new sequences at the cut site, compared to Cas9. Although the CRISPR/Cas9 system can efficiently disable genes, it is challenging to insert genes or generate a knock-in. Cpfl lacks tracrRNA, utilizes a T-rich PAM and cleaves DNA via a staggered DNA DSB. [0134] In summary, important differences between Cpfl and Cas9 systems are that Cpfl recognizes different PAMs, enabling new targeting possibilities, creates 4-5 nt long sticky ends, instead of blunt ends produced by Cas9, enhancing the efficiency of genetic insertions and specificity during NHEJ or HDR, and cuts target DNA further away from PAM, further away from the Cas9 cutting site, enabling new possibilities for cleaving the DNA.

[0135] Table 3: Differences between Cas9 and Cpfl

CRISPR -mediated gene editing

[0136] The first step in editing the DMD gene using CRISPR/Cpfl or CRISPR/Cas9 is to identify the genomic target sequence. The genomic target for the gRNAs of the disclosure can be any ~24 nucleotide DNA sequence, provided that the sequence is unique compared to the rest of the genome. In some embodiments, the genomic target sequence corresponds to a sequence within exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence is a 5’ or 3’ splice site of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. In some embodiments, the genomic target sequence corresponds to a sequence within an intron immediately upstream or downstream of exon 51, exon 45, exon 44, exon 53, exon 46, exon 52, exon 50, exon 43, exon 6, exon 7, exon 8, and/or exon 55 of the human dystrophin gene. Exemplary genomic target sequences can be found in Tables 6,

8, 10, 12, and 14.

[0137] The next step in editing the DMD gene is to identify all Protospacer Adjacent Motif (PAM) sequences within the genetic region to be targeted. The target sequence must be immediately upstream of a PAM. Once all possible PAM sequences and putative target sites have been identified, the next step is to choose which site is likely to result in the most efficient on-target cleavage. The gRNA targeting sequence needs to match the target sequence, and the gRNA targeting sequence must not match additional sites within the genome. In preferred embodiments, the gRNA targeting sequence has perfect homology to the target with no homology elsewhere in the genome. In some embodiments, a given gRNA targeting sequence will have additional sites throughout the genome where partial homology exists. These sites are called“off-targets” and should be considered when designing a gRNA. In general, off-target sites are not cleaved as efficiently when mismatches occur near the PAM sequence, so gRNAs with no homology or those with mismatches close to the PAM sequence will have the highest specificity. In addition to“off-target activity”, factors that maximize cleavage of the desired target sequence (“on-target activity”) must be considered. It is known to those of skill in the art that two gRNA targeting sequences, each having 100% homology to the target DNA may not result in equivalent cleavage efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides within the selected target sequence. Close examination of predicted on-target and off-target activity of each potential gRNA targeting sequence is necessary to design the best gRNA. Several gRNA design programs have been developed that are capable of locating potential PAM and target sequences and ranking the associated gRNAs based on their predicted on-target and off-target activity (e.g. CRISPRdirect, available at www.crispr.dbcls.jp).

[0138] The next step is to synthesize and clone desired gRNAs. Targeting oligos can be synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector that is chosen. The gRNAs for Cpfl are notably simpler than the gRNAs for Cas9, and only consist of a single crRNA containing direct repeat scaffold sequence followed by ~24 nucleotides of guide sequence.

[0139] Each gRNA should then be validated in one or more target cell lines. For example, after the Cas9 or Cpfl and the gRNA are delivered to the cell, the genomic target region may be amplified using PCR and sequenced according to methods known to those of skill in the art.

[0140] In some embodiments, gene editing may be performed in vitro or ex vivo. In some embodiments, cells are contacted in vitro or ex vivo with a Cas9 or a Cpfl and a gRNA that targets a dystrophin splice site. In some embodiments, the cells are contacted with one or more nucleic acids encoding the Cas9 or Cpfl and the guide RNA. In some embodiments, the one or more nucleic acids are introduced into the cells using, for example, lipofection or electroporation. Gene editing may also be performed in zygotes. In embodiments, zygotes may be injected with one or more nucleic acids encoding Cas9 or Cpfl and a gRNA that targets a dystrophin splice site. The zygotes may subsequently be injected into a host. [0141] In some embodiments, the Cas9 or Cpfl is provided on a vector. In embodiments, the vector contains a Cas9 derived from S. pyogenes (SpCas9, SEQ ID NO: 872). In

embodiments, the vector contains a Cas9 derived from S. aureus (SaCas9, SEQ ID NO: 873). In embodiments, the vector contains a Cpfl sequence derived from a Lachnospiraceae bacterium. See, for example, Uniprot Accession No. A0A182DWE3; SEQ ID NO: 871. In embodiments, the vector contains a Cpfl sequence derived from an Acidaminococcus bacterium. See, for example, Uniprot Accession No. U2UMQ6; SEQ ID NO: 870. In some embodiments, the Cas9 or Cpfl sequence is codon optimized for expression in human cells or mouse cells. In some embodiments, the vector further contains a sequence encoding a fluorescent protein, such as GFP, which allows Cas 9 or Cpfl -expressing cells to be sorted using fluorescence activated cell sorting (FACS). In some embodiments, the vector is a viral vector such as an adeno-associated viral vector.

[0142] In some embodiments, the gRNA is provided on a vector. In some embodiments, the vector is a viral vector such as an adeno-associated viral vector. In embodiments, the Cas9 or Cpfl and the guide RNA are provided on the same vector. In embodiments, the Cas9 or Cpfl and the guide RNA are provided on different vectors.

[0143] In some embodiments, the cells are additionally contacted with a single-stranded DMD oligonucleotide to effect homology directed repair. In some embodiments, small INDELs restore the protein reading frame of dystrophin (“refraining” strategy). When the refraining strategy is used, the cells may be contacted with a single gRNA. In embodiments, a splice donor or splice acceptor site is disrupted, which results in exon skipping and restoration of the protein reading frame (“exon skipping” strategy). When the exon skipping strategy is used, the cells may be contacted with two or more gRNAs.

[0144] Efficiency of in vitro or ex vivo Cas9 or Cpfl -mediated DNA cleavage may be assessed using techniques known to those of skill in the art, such as the T7 El assay.

Restoration of DMD expression may be confirmed using techniques known to those of skill in the art, such as RT-PCR, western blotting, and immunocytochemistry.

[0145] In some embodiments, in vitro or ex vivo gene editing is performed in a muscle or satellite cell. In some embodiments, gene editing is performed in iPSC or iCM cells. In embodiments, the iPSC cells are differentiated after gene editing. For example, the iPSC cells may be differentiated into a muscle cell or a satellite cell after editing. In embodiments, the iPSC cells are differentiated into cardiac muscle cells, skeletal muscle cells, or smooth muscle cells. In embodiments, the iPSC cells are differentiated into cardiomyocytes. iPSC cells may be induced to differentiate according to methods known to those of skill in the art. [0146] In some embodiments, contacting the cell with the Cas9 or the Cpfl and the gRNA restores dystrophin expression. In embodiments, cells which have been edited in vitro or ex vivo, or cells derived therefrom, show levels of dystrophin protein that is comparable to wildtype cells. In embodiments, the edited cells, or cells derived therefrom, express dystrophin at a level that is 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between of wildtype dystrophin expression levels. In embodiments, the cells which have been edited in vitro or ex vivo, or cells derived therefrom, have a mitochondrial number that is comparable to that of wildtype cells. In embodiments the edited cells, or cells derived therefrom, have 50%, 60%, 70%, 80%, 90%, 95% or any percentage in between as many mitochondria as wildtype cells. In embodiments, the edited cells, or cells derived therefrom, show an increase in oxygen consumption rate (OCR) compared to non-edited cells at baseline.

Nucleic Acid Expression Vectors

[0147] 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. Provided herein are expression vectors which contain one or more nucleic acids encoding Cas9 or Cpfl and at least one DMD guide RNA that targets a dystrophin splice site. In some embodiments, a nucleic acid encoding Cas9 or Cpfl and a nucleic acid encoding at least one guide RNA are provided on the same vector. In further embodiments, a nucleic acid encoding Cas9 or Cpfl and a nucleic acid encoding least one guide RNA are provided on separate vectors.

[0148] 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.

[0149] 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, i.e., 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, required to initiate 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.

Regulatory Elements

[0150] 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.

[0151] 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.

RNA Polymerase and Pol III Promoters

[0152] In eukaryotes, RNA polymerase III (also called Pol III) transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. The genes transcribed by RNA Pol III fall in the category of“housekeeping” genes whose expression is required in all cell types and most environmental conditions. Therefore, the regulation of Pol III transcription is primarily tied to the regulation of cell growth and the cell cycle, thus requiring fewer regulatory proteins than RNA polymerase II. Under stress conditions however, the protein Mafl represses Pol III activity.

[0153] In the process of transcription (by any polymerase) there are three main stages: (i) initiation, requiring construction of the RNA polymerase complex on the gene's promoter; (ii) elongation, the synthesis of the RNA transcript; and (iii) termination, the finishing of RNA transcription and disassembly of the RNA polymerase complex.

[0154] Promoters under the control of RNA Pol III include those for ribosomal 5S rRNA, tRNA and few other small RNAs such as U6 spliceosomal RNA, RNase P and RNase MRP RNA, 7SL RNA (the RNA component of the signal recognition particles), Vault RNAs, Y RNA, SINEs (short interspersed repetitive elements), 7SK RNA, two microRNAs, several small nucleolar RNAs and several few regulatory antisense RNAs.

Additional Promoters and Elements

[0155] In some embodiments, the 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.

[0156] 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.

[0157] In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus 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.

[0158] 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 must be 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 must have 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.

[0159] Below is a list of promoters/enhancers and inducible promoters/enhancers that could be used in combination with the 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.

[0160] The promoter and/or enhancer may be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ b, 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, a-fetoprotein, t-globin, b-globin, c-fos, c-HA-ra.v, insulin, neural cell adhesion molecule (NCAM), ai-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 virus.

[0161] In some embodiments, inducible elements may be used. In some embodiments, the inducible element is, for example, MTII, MMTV (mouse mammary tumor virus), b- interferon, adenovirus 5 E2, collagenase, stromelysin, SV40, murine MX gene, GRP78 gene, a-2-macroglobulin, vimentin, MHC class I gene H-2k±>, 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 (TP A), interferon, Newcastle Disease Virus, A23187, IL-6, serum, 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.

[0162] 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.

[0163] In some embodiments, the muscle specific promoter is the CK8 promoter. The CK8 promoter has the following sequence (SEQ ID NO: 874):

[0164] 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. 875):

[0165] 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.

Self-cleaving peptides

[0166] 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: 876,

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: 877; QCTNYALLKLAGDVESNPGP), porcine teschovirus-l (PTV1) 2A peptide (SEQ ID NO: 878; ATNFSLLKQAGDVEENPGP) and foot and mouth disease virus (FMDV) 2A peptide (SEQ ID NO: 879; PVKQLLNFDLLKLAGDVESNPGP) or modified versions thereof.

[0167] 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.

[0168] Other self-cleaving peptides that may be used include, but are not limited to nuclear inclusion protein a (Nia) protease, a Pl protease, a 3C protease, an L protease, a 3C-like protease, or modified versions thereof.

Therapeutic Compositions

AAV-Cas9 vectors

[0169] In some embodiments, a Cas9 may be packaged into an AAV vector. In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10 or any combination thereof.

[0170] Exemplary AAV-Cas9 vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the Cas9 sequence. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV 10, AAV11 AAVrh74, AAVrh10 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 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, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,

139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some

embodiments, the ITRs comprise or consist of 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, 125, 126,

127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,

145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs have a length of 110 ± 10 base pairs. In some embodiments, the ITRs have a length of 120 ± 10 base pairs. In some embodiments, the ITRs have a length of 130 ± 10 base pairs. In some embodiments, the ITRs have a length of 140 ± 10 base pairs. In some embodiments, the ITRs have a length of 150 ± 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, the ITRs have a sequence selected from SEQ. ID. NO:

880, SEQ ID NO: 881, SEQ ID NO: 882, SEQ ID NO: 883 and SEQ ID. NO: 946.

[0171] In some embodiments, the AAV-Cas9 vector may contain one or more nuclear localization signals (NLS). In some embodiments, the AAV-Cas9 vector contains 1, 2, 3, 4, or 5 nuclear localization signals. Exemplary NLS include the c-myc NLS (SEQ ID NO:

884), the SV40 NLS (SEQ ID NO: 885), the hnRNPAI M9 NLS (SEQ ID NO: 886), the nucleoplasmin NLS (SEQ ID NO: 887), the sequence

RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 888) of the IBB domain from importin-alpha, the sequences VSRKRPRP (SEQ ID NO: 889) and PPKKARED (SEQ ID NO: 890) of the myoma T protein, the sequence PQPKKKPL (SEQ ID NO: 891) of human p53, the sequence SALIKKKKKMAP (SEQ ID NO: 892) of mouse c- abl IV, the sequences DRLRR (SEQ ID NO: 893) and KQKKRK (SEQ ID NO: 894) of the influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO: 895) of the Hepatitis virus delta antigen and the sequence REKKKFLKRR (SEQ ID NO: 896) of the mouse Mxl protein. Further acceptable nuclear localization signals

includebipartite nuclear localization sequences such as the sequence

KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 897) of the human poly(ADP-ribose) polymerase or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 898) of the steroid hormone receptors (human) glucocorticoid.

[0172] In some embodiments, the AAV-Cas9 vector may comprise additional elements to facilitate packaging of the vector and expression of the Cas9. In some embodiments, the AAV-Cas9 vector may comprise a poly A sequence. In some embodiments, the polyA sequence may be a mini-poly A sequence. In some embodiments, the AAV-Cas9 vector may comprise a transposable element. In some embodiments, the AAV-Cas9 vector may comprise a regulator element. In some embodiments, the regulator element is an activator or a repressor.

[0173] In some embodiments, the AAV-Cas9 may contain one or more promoters. In some embodiments, the one or more promoters drive expression of the Cas9. In some

embodiments, the one or more promoters are muscle-specific promoters. Exemplary muscle- specific promoters include 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, the aB-crystallin/small heat shock protein promoter, a-myosin heavy chain promoter, the ANF promoter, the CK8 promoter and the CK8e promoter.

[0174] In some embodiments, the AAV-Cas9 vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-Cas9 vector may be optimized for expression in human cells. In some embodiments, the AAV- Cas9 vector may be optimized for expression in a bacculovirus expression system.

[0175] In some embodiments, the AAV-Cas9 vector comprises a sequence selected from

SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, or SEQ ID NO: 902, as shown in Table 4.

Table 4: Exemplary gene editing constructs (from ITR to ITR for delivery via AAV vector)

[0176] In some embodiments of the gene editing constructs of the disclosure, including those embodiments encompassing SEQ ID NOs: 899-902, the construct comprises or consists of a promoter and a nuclease. In some embodiments, the construct comprises or consists of a CK8e promoter and a Cas9 nuclease. In some embodiments, the construct comprises or consists of a CK8e promoter and a Cas9 nuclease isolated or derived from Staphylococcus pyogenes (“SpCas9”). In some embodiments, the CK8e promoter comprises or consists of a nucleotide sequence of

embodiments, the construct comprising a promoter and a nuclease further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences from isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences each comprising or consisting of a nucleotide sequence of

GGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGC CCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA (SEQ ID NO:880). In SOIUe

embodiments, the construct comprising a promoter and a nuclease further comprises at least two ITR sequences, wherein the first ITR sequence comprises or consists of a nucleotide sequence of

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID

NO: 881) and the second ITR sequence comprises or consists of a nucleotide sequence of

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGG TCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID

NO: 882). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease and a second AAV2 ITR. In some embodiments, the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease and a second ITR, further comprises a poly A sequence. In some embodiments, the polyA sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of

TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGT GT GTTGGTTTTTTGATCAGGCGCG (SEQ ID NO:

903). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a minipoly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least one nuclear localization signal. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a nuclease, a poly A sequence and a second ITR, further comprises at least two nuclear localization signals. Exemplary nuclear localization signals of the disclosure comprise or consist of a nucleotide

Sequence Of AAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAA (SEQ ID NO: 887), or a nucleotide sequence of ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC

(SEQ ID NO: 885). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a poly A sequence and a second ITR, further comprises a stop codon. The stop codon may have a sequence of TAG (SEQ ID NO: 904), TAA (SEQ ID NO: 905), or TGA (SEQ ID NO: 906). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a stop codon, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon, a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon a minipoly A sequence and a second AAV2 ITR. In some embodiments, the construct comprising or consisting of, from 5’ to 3’ a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence and a second ITR, further comprises transposable element inverted repeats. Exemplary transposable element inverted repeats of the disclosure comprise or consist of a nucleotide sequence of

(SEQ ID NO: 908). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat having a sequence of SEQ ID NO: 907, a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon a minipoly A sequence, a second AAV2 ITR, and a second transposable element inverted repeat having a sequence of SEQ ID NO: 908. In some embodiments, the construct comprising or consisting of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, and a second transposable element inverted repeat, further comprises a regulatory sequence. Exemplary regulatory sequences of the disclosure comprise or consist of a nucleotide sequence of

(SEQ ID NO: 909). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a sequence encoding a promoter, a sequence encoding a first nuclear localization signal, a sequence encoding a nuclease, a sequence encoding a second nuclear localization signal, a stop codon, a poly A sequence, a second ITR, a regulatory sequence and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat having a sequence of SEQ ID NO: 907, a first AAV2 ITR, a sequence encoding a CK8e promoter, a nuclear localization signal having a sequence of SEQ ID NO: 885, a sequence encoding a SpCas9 nuclease, a nuclear localization signal having a sequence of SEQ ID NO: 887, a stop codon, a minipoly A sequence, a second AAV2 ITR, a regulatory sequence having a sequence of SEQ ID NO: 909, and a second transposable element inverted repeat having a sequence of SEQ ID NO: 908. In some embodiments, the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure have length from 1-1500 nucleotides, inclusive of all ranges therebetween. In some embodiments, the spacer sequences may be located either 5’ to or 3’ to an ITR, a promoter, a nuclear localization sequence, a nuclease, a stop codon, a polyA sequence, a transposable element inverted repeat, and/or a regulator element. In some embodiments, the construct may have a sequence comprising or consisting of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, or SEQ ID NO: 902.

AAV-sgRNA vectors

[0177] In some embodiments, at least a first sequence encoding a gRNA and a second sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, and a fourth sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, at least a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, a fourth sequence encoding a gRNA, and a fifth sequence encoding a gRNA may be packaged into an AAV vector. In some embodiments, a plurality of sequences encoding a gRNA are packaged into an AAV vector. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 sequences encoding a gRNA may be packaged into an AAV vector. In some embodiments, each sequence encoding a gRNA is different. In some embodiments, at least 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the sequences encoding a gRNA are the same. In some embodiments, all of the sequence encoding a gRNA are the same.

[0178] In some embodiments, the AAV vector is a wildtype AAV vector. In some embodiments, the AAV vector contains one or more mutations. In some embodiments, the AAV vector is isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, AAVrh10 or any combination thereof.

[0179] Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV 3, AAV4, AAV 5, AAV6, AAV7, AAV 8, AAV9, AAV 10, AAV11, AAVrh74, AAVrh10 or any combination thereof. In some embodiments, the ITRs are isolated or derived from an AAV vector of a first serotype and a sequence encoding a capsid protein of the AAV-sgRNA vector is isolated or derived from an AAV vector of a second serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, or AAVrh10. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, or AAVrh10. In some embodiments, the first serotype is AAV2 and the second serotype is AAV1, AAV2, AAV 3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, AAVrh74, or AAVrh10. In some embodiments, the first serotype is AAV2 and the second serotype is AAV9.

[0180] Exemplary AAV-sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the gRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV 3, AAV4, AAV 5, AAV6, AAV7, AAV 8, AAV9, AAV 10, AAV11, AAVrh74, AAVrh10 or any combination thereof. In some embodiments, a first ITR is isolated or derived from an AAV vector of a first serotype, a second ITR is isolated or derived from an AAV vector of a second serotype and a sequence encoding a capsid protein of the AAV- sgRNA vector is isolated or derived from an AAV vector of a third serotype. In some embodiments, the first serotype and the second serotype are the same. In some embodiments, the first serotype and the second serotype are not the same. In some embodiments, the first serotype, the second serotype, and the third serotype are the same. In some embodiments, the first serotype, the second serotype, and the third serotype are not the same. In some embodiments, the first serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, or AAVrh10. In some embodiments, the second serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, or AAVrh10. In some embodiments, the third serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV 7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, or AAVrh10. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV1, AAV2, AAV 3, AAV4, AAV5, AAV6, AAV7, AAV 8, AAV9, AAV10, AAV11, AAVrh74, or AAVrh10. In some embodiments, the first serotype is AAV2, the second serotype is AAV4 and the third serotype is AAV9. Exemplary AAV- sgRNA vectors contain two ITR (inverted terminal repeat) sequences which flank a central sequence region comprising the sgRNA sequences. In some embodiments, the ITRs are isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAVrh74, or AAVrh10 or any combination thereof. In some embodiments, the ITRs comprise or consist of full-length and/or wildtype sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of truncated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of elongated sequences for an AAV serotype. In some embodiments, the ITRs comprise or consist of sequences comprising a sequence variation compared to a wildtype sequence for the same AAV serotype. In some embodiments, the sequence variation comprises one or more of a substitution, deletion, insertion, inversion, or transposition. In some embodiments, the ITRs comprise or consist of at least 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, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

142, 143, 144, 145, 146, 147, 148, 149 or 150 base pairs. In some embodiments, the ITRs comprise or consist of 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, 125, 126, 127, 128, 129, 130, 131,

132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or

150 base pairs. In some embodiments, the ITRs have a length of 110 ± 10 base pairs. In some embodiments, the ITRs have a length of 120 ± 10 base pairs. In some embodiments, the ITRs have a length of 130 ± 10 base pairs. In some embodiments, the ITRs have a length of 140 ± 10 base pairs. In some embodiments, the ITRs have a length of 150 ± 10 base pairs. In some embodiments, the ITRs have a length of 115, 145, or 141 base pairs. In some embodiments, the ITRs have a sequence selected from SEQ ID NO: 880, SEQ ID NO: 881, SEQ ID NO: 882, or SEQ ID NO: 883.

[0181] In some embodiments, the AAV-sgRNA vector may comprise additional elements to facilitate packaging of the vector and expression of the sgRNA. In some embodiments, the AAV-sgRNA vector may comprise a transposable element. In some embodiments, the AAV- sgRNA vector may comprise a regulatory element. In some embodiments, the regulatory element comprises an activator or a repressor. In some embodiments, the AAV-sgRNA sequence may comprise a non-functional or“stuffer” sequence. Exemplary stuffer sequences of the disclosure may have some (a non-zero percentage of) identity or homology to a genomic sequence of a mammal (including a human). Alternatively, exemplary stuffer sequences of the disclosure may have no identify or homology to a genomic sequence of a mammal (including a human). Exemplary stuffer sequences of the disclosure may comprise or consist of naturally occurring non-coding sequences or sequences that are neither transcribed nor translated following administration of the AAV vector to a subject.

[0182] In some embodiments, the AAV-sgRNA vector may be optimized for production in yeast, bacteria, insect cells, or mammalian cells. In some embodiments, the AAV-sgRNA vector may be optimized for expression in human cells. In some embodiments, the AAV- Cas9 vector may be optimized for expression in a bacculovirus expression system.

[0183] In some embodiments, the AAV-sgRNA vector comprises at least one promoter. In some embodiments, the AAV-sgRNA vector comprises at least two promoters. In some embodiments, the AAV-sgRNA vector comprises at least three promoters. In some embodiments, the AAV-sgRNA vector comprises at least four promoters. In some embodiments, the AAV-sgRNA vector comprises at least five promoters. Exemplary promoters include, for example, immunoglobulin light chain, immunoglobulin heavy chain, T-cell receptor, HLA DQ a and/or DQ b, 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, a -fetoprotein, t- globin, b-globin, c-fos, c-HA-ra.v insulin, neural cell adhesion molecule (NCAM), ai- 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 virus. Further exemplary promoters include the U6 promoter, the Hl promoter, and the 7SK promoter.

[0184] In some embodiments, the sequence encoding the gRNA comprises a sequence selected from SEQ ID Nos: 383-705, 709-711, 715-717, 790-862, and 864.

[0185] In some embodiments, the AAV vector comprises a first sequence encoding a gRNA and a second sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA and a second promoter drives expression of the second sequence encoding a gRNA. In some embodiments, the first and second promoters are the same. In some embodiments, the first and second promoters are different. In some embodiments, the first and second promoters are selected from the Hl promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA and the second sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA and the second sequence encoding a gRNA are not identical.

[0186] In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, and a third sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, and a third promoter drives expression of a third sequence encoding a gRNA. In some embodiments, at least two of the first, second, and third promoters are the same. In some embodiments, each of the first, second, and third promoters are different. In some embodiments, the first, second, and third promoters are selected from the Hl promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first promoter is the U6 promoter. In some embodiments, the second promoter is the Hl promoter. In some embodiments, the third promoter is the 7SK promoter. In some embodiments, the first promoter is the U6 promoter, the second promoter is the Hl promoter, and the third promoter is the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, and the third sequence encoding a gRNA are not identical.

[0187] In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, and a fourth sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, a third promoter drives expression of the third sequence encoding a gRNA, and a fourth promoter drives expression of the fourth sequence encoding a gRNA. In some embodiments, at least two of the first, second, third, and fourth promoters are the same. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third and fourth promoters are selected from the Hl promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, and the fourth sequence encoding a gRNA are not identical.

[0188] In some embodiments, the AAV vector comprises a first sequence encoding a gRNA, a second sequence encoding a gRNA, a third sequence encoding a gRNA, a fourth sequence encoding a gRNA, and a fifth sequence encoding a gRNA, a first promoter drives expression of the first sequence encoding a gRNA, a second promoter drives expression of the second sequence encoding a gRNA, a third promoter drives expression of the third sequence encoding a gRNA, a fourth promoter drives expression of the fourth sequence encoding a gRNA, and a fifth promoter drives expression of the fifth sequence encoding a gRNA. In some embodiments, at least two of the first, second, third, fourth, and fifth promoters are the same. In some embodiments, each of the first, second, third, fourth, and fifth promoters are different. In some embodiments, each of the first, second, third, and fourth promoters are different. In some embodiments, each of the first, second, third, fourth and fifth promoters are selected from the Hl promoter, the U6 promoter, and the 7SK promoter. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are identical. In some embodiments, the first sequence encoding a gRNA, the second sequence encoding a gRNA, the third sequence encoding a gRNA, the fourth sequence encoding a gRNA, and the fifth sequence encoding a gRNA are not identical.

[0189] In some embodiments, the AAV-sgRNA vector comprises a sequence selected from SEQ ID NO: 910, SEQ ID NO: 911, SEQ ID NO: 912, or SEQ ID NO: 913. In some embodiments, the AAV-sgRNA vector comprises a sequence selected from SEQ ID NO: 914, SEQ ID NO: 915, SEQ ID NO: 916, or SEQ ID NO: 917. In some embodiments, the AAV- sgRNA vector comprises a sequence selected from SEQ ID NO: 918, SEQ ID NO: 919, SEQ ID NO: 920, or SEQ ID NO: 921. Exemplary AAV-sgRNA vectors are provided in Table 5. Table 5. Exemplary AAV-sgRNA vectors.

[0190] In some embodiments of the gene editing constructs of the disclosure, including those embodiments encompassing SEQ ID NOs: 910 to 921, the construct comprises or consists of a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA.

Exemplary sequences encoding gRNAs of the disclosure are SEQ ID NO: 383-705, 709-711, 715-717, 790-862, 864. In some embodiments, the sequence encoding the gRNA is

CACTAGAGTAACAGTCTGAC (SEQ ID NO: 708). In some embodiments, the sequence encoding the gRNA is C AC C AGAGT AAC AGT CT GAG (SEQ ID NO: 714). In some embodiments, the sequence encoding the gRNA is C ACC AGAGT AAC AGTCTGAC (SEQ ID NO: 863). In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO.: 708, a second promoter, a second sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO: 708. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, a second sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO: 714. In some embodiments, the construct comprises, from 5’ to 3’, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, a second sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, and a third sequence encoding a gRNA of SEQ ID NO: 863. Exemplary promoters of the disclosure include the U6 promoter having a sequence of

CGAGTCCAACACCCGTGGGAATCCCATGGGCACCATGGCCCCTCGCTCCAAAAATGCTTTCGCGTCGCGCAGACA

TATATAGCTTGTGCGCCGCCTGGGTA (SEQ ID NO: 924). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 922), the Hl promoter (SEQ ID NO: 923), and the 7SK promoter (SEQ ID NO: 924). In some embodiments, the first, second, and third promoter are each individually selected from the U6 promoter (SEQ ID NO: 922), and the Hl promoter (SEQ ID NO: 923). In some

embodiments, the construct comprises, from 5’ to 3’, a U6 promoter, a first sequence encoding a gRNA, a Hl promoter, a second sequence encoding a gRNA, a 7SK promoter, and a third sequence encoding a gRNA. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, a second sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 708. In some embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, a second sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 714. In some

embodiments, the construct comprises, from 5’ to 3’, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 863, the Hl promoter, a second sequence encoding a gRNA of SEQ ID NO: 863, the 7SK promoter, and a third sequence encoding a gRNA of SEQ ID NO: 863. In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two inverted terminal repeat (ITR) sequences. In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences isolated or derived from an AAV of serotype 2 (AAV2). In some embodiments, the construct comprising a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, and a third sequence encoding a gRNA further comprises at least two ITR sequences, wherein the first ITR sequence is isolated or derived from an AAV of serotype 4 (AAV4) and the second ITR sequence is isolated or derived from an AAV of serotype 2 (AAV2). Exemplary ITR sequences are

NO: 882). In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6 promoter, a first sequence encoding a gRNA, a Hl promoter, and a second sequence encoding a gRNA, a 7SK promoter, a third sequence encoding a gRNA, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, and the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6, the sequence encoding a gRNA of SEQ ID NO: 708, a Hl promoter, and the sequence encoding a gRNA of SEQ ID NO: 708, a 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a U6, the sequence encoding a gRNA of SEQ ID NO: 714, a Hl promoter, and the sequence encoding a gRNA of SEQ ID NO: 714, a 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, and a second ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, and a second ITR, further comprises a poly A sequence. In some embodiments, the polyA sequence comprises or consists of a minipolyA sequence. Exemplary minipolyA sequences of the disclosure comprise or consist of a nucleotide sequence of

TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGT GT GTTGGTTTTTTGATCAGGCGCG (SEQ ID NO:

903). In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding s gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO.: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a minipolyA sequence, and a second ITR.

In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprising, from 5’ to 3’ a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR further comprises transposable element inverted repeats. Exemplary transposable element inverted repeats of the disclosure comprise or consist of a nucleotide sequence of

(SEQ ID NO: 908). In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’, a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, and a second transposable element inverted repeat. In some embodiments, the construct comprising a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a second transposable element inverted repeat, further comprises a regulatory sequence.

Exemplary regulatory sequences of the disclosure comprise or consist of a nucleotide sequence of

TGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGA

AGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGC GGAGTTGTTCGGTAAATTGTCACAACGCCG (SEQ ID NO: 909). In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, a sequence encoding a gRNA of SEQ ID NO: 714, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprising a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, and a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a minipolyA sequence, and a second ITR, further comprises a stuffer sequence. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO. 714, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, and a second ITR. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, and a second ITR.

In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, a first sequence encoding a gRNA, a second promoter, a second sequence encoding a gRNA, a third promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some

embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a first sequence encoding a gRNA, the Hl promoter, a second sequence encoding a gRNA, the 7SK promoter, a third sequence encoding a gRNA, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, a first promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a second promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a third promoter, the sequence encoding a gRNA of SEQ ID NO: 863, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the Hl promoter, the sequence encoding a gRNA of SEQ ID NO: 708, the 7SK promoter, the sequence encoding a gRNA of SEQ ID NO: 708, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some

embodiments, the construct comprises or consists of, from 5’ to 3’ a first transposable element inverted repeat, a first ITR, the U6 promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the Hl promoter, a sequence encoding a gRNA of SEQ ID NO: 714, the 7SK promoter, a sequence encoding a gRNA of SEQ ID NO: 714, a stuffer sequence, a minipolyA sequence, a second ITR, a regulatory sequence, and a second transposable element inverted repeat. In some embodiments, the construct may further comprise one or more spacer sequences. Exemplary spacer sequences of the disclosure have length from 1-1500 nucleotides, inclusive of all ranges therebetween. In some embodiments, the spacer sequences may be located at a position that is 5’ to or 3’ to an ITR, a promoter, a sequence encoding a gRNA, a polyA sequence, a transposable element inverted repeat, a stuffer sequence, and/or a regulator element.

Exemplary Therapeutic Nucleic Acids and Vectors

[0191] In some embodiments, a nucleic acid comprises a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor site.

[0192] In some embodiments, the sequence encoding the first promoter and the sequence encoding the second promoter are identical. In some embodiments, the sequence encoding the first promoter and the sequence encoding the second promoter not identical. In some embodiments, the sequence encoding the first promoter and the sequence encoding the second promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity.

[0193] In some embodiments, the first genomic target sequence and the second genomic target sequence are identical. In some embodiments, the first genomic target sequence and the second genomic target sequence are not identical. In some embodiments, the first genomic target sequence and the second genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, the first genomic target sequence and the second genomic target sequence are complementary.

[0194] In some embodiments, the nucleic acid further comprises a sequence encoding a third DMD guide RNA targeting a third genomic target sequence, and a sequence encoding a third promoter wherein the third promoter drives expression of the sequence encoding the third DMD guide RNA, and wherein the third genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the sequences encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter are identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter are not identical. In some embodiments, at least two of the sequences encoding the first promoter, the sequence encoding the second promoter, and the sequence encoding the third promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, and the third genomic target sequence are complementary.

[0195] In some embodiments, the nucleic acid further comprises a sequence encoding a fourth DMD guide RNA targeting a fourth genomic target sequence, and a sequence encoding a fourth promoter, wherein the fourth promoter drives expression of the fourth sequence encoding a DMD guide RNA, wherein the fourth genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter are identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter are not identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, and the sequence encoding the fourth promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, and the fourth genomic target sequence are complementary. [0196] In some embodiments, the nucleic acid further comprises a sequence encoding a fifth DMD guide RNA targeting a fifth genomic target sequence, and a sequence encoding a fifth promoter, wherein the fifth promoter drives expression of the sequence encoding the fifth DMD guide RNA, wherein the fifth genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter are identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter are not identical. In some embodiments, at least two of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are not identical. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence share at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity. In some embodiments, at least two of the first genomic target sequence, the second genomic target sequence, the third genomic target sequence, the fourth genomic target sequence, and the fifth genomic target sequence are complementary.

[0197] In some embodiments, the nucleic acid further comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence, and at least one additional promoter, wherein the additional promoter drives expression of the sequence encoding the additional DMD guide RNA, wherein the additional genomic target sequence comprises a dystrophin splice acceptor site. In some embodiments, the dystrophin splice acceptor site comprises the 5’ splice acceptor site of exon 51. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a constitutive promoter. In some embodiments, the first promoter or the second promoter comprises a constitutive promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a sequence encoding a constitutive promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises a constitutive promoter. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding an inducible promoter. In some embodiments, at least one of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter comprises an inducible promoter. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a cell-type specific promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a cell-type specific promoter. In some embodiments, the cell type specific promoter comprises a muscle-specific promoter. In some embodiments, the sequence encoding the first promoter or the sequence encoding the second promoter comprises a sequence encoding a U6 promoter, an Hl promoter, or a 7SK promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a U6 promoter, an Hl promoter, or a 7SK promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a U6 promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises an Hl promoter. In some embodiments, at least one of the sequence encoding the first promoter, the sequence encoding the second promoter, the sequence encoding the third promoter, the sequence encoding the fourth promoter, and the sequence encoding the fifth promoter comprises a 7SK promoter.

[0198] In some embodiments, the sequence encoding the first DMD guide RNA, the sequence encoding the second DMD guide RNA, and sequence encoding the third DMD guide RNA are identical, and the 5’ splice acceptor site comprises a 5’ splice acceptor site of exon 51. In some embodiments, the sequence encoding the first promoter comprises a sequence encoding a U6 promoter, the sequence encoding the second promoter comprises a sequence encoding an Hl promoter, and the sequence encoding the third promoter comprises a 7SK promoter. In some embodiments, the nucleic acid comprises a DNA sequence. In some embodiments, the nucleic acid comprises an RNA sequence.

[0199] In some embodiments, the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR). In some embodiments, the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno- associated virus (AAV). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 2 (AAV2). In some

embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 4 (AAV4). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 145 nucleotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 115 nucleotides. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 141 nucleotides. In some embodiments, the nucleic acid further comprises a polyadenosine (poly A) sequence. In some embodiments, the poly A sequence is a mini poly A sequence. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of any one of SEQ ID NOs: 60-382, 706-708 and 712-789. In some embodiments, the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises the sequence of SEQ ID NO: 714.

[0200] Also provided is a vector comprising a nucleic acid comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor site. In some embodiments, the vector further comprises a sequence encoding an inverted terminal repeat of a transposable element. In some embodiments, the transposable element is a transposon. In some embodiments, the transposon is a Tn7 transposon. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is replication-defective or conditionally replication defective. In some embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6),

7 (AAV7), 8 (AAV8), 9 (AAV 9), 10 (AAV 10), 11 (AAV 11), AAVrh74, AAVrh10 or any combination thereof. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV 9). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells. In some embodiments, the vector comprises the sequence of SEQ ID NO: 914, SEQ ID NO: 915, SEQ ID NO: 916, or SEQ ID NO: 917.

[0201] Also provided is a nucleic acid comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter. In some embodiments, the sequence encoding the muscle-specific promoter comprises a sequence encoding a CK8 promoter. In some embodiments, the sequence encoding the muscle-specific promoter comprises a sequence encoding a CK8e promoter. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding an S. pyogenes Cas9 or a nuclease domain thereof. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is isolated or derived from a sequence encoding S. aureus Cas9 or a nuclease domain thereof. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a mammal. In some embodiments, the sequence encoding the Cas9 or the nuclease domain thereof is codon optimized for expression in a human.

[0202] In some embodiments, the nucleic acid comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof further comprises a polyA sequence. In some embodiments, the polyA sequence is a mini polyA sequence. In some embodiments, the nucleic acid further comprises one or more sequences encoding an inverted terminal repeat (ITR). In some embodiments, the nucleic acid further comprises a sequence encoding a 5’ inverted terminal repeat (ITR) and a sequence encoding a 3’ ITR. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno- associated virus (AAV) of serotype 2 (AAV2). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV2. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an adeno-associated virus (AAV) of serotype 4 (AAV4). In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) and the sequence encoding a 3’ ITR comprises a sequence isolated or derived from an AAV4. In some embodiments, the sequence encoding the 5’ inverted terminal repeat (ITR) or the sequence encoding a 3’ ITR comprises or consists of 145 nucleotides, 115 nucleotides, or 141 nucleotides. In some embodiments, the nucleic acid further comprises a nuclear localization signal. In some embodiments, the nucleic acid is optimized for expression in mammalian cells. In some embodiments, the nucleic acid is optimized for expression in human cells.

[0203] Also provided is a vector comprising a nucleic acid comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, the vector further comprises a sequence encoding an inverted terminal repeat (ITR) of a transposable element. In some embodiments, the transposable element is a transposon. In some embodiments, the transposon is a Tn7 transposon. In some embodiments, the vector further comprises a sequence encoding a 5’ ITR of a T7 transposon and a sequence encoding a 3’ ITR of a T7 transposon. In some embodiments, the vector is a non- viral vector. In some embodiments, the non-viral vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is replication-defective or conditionally replication defective. In some embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV 6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11), AAVrh74, AAVrh10 or any combination thereof. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 9 (AAV9). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 2 (AAV2). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV2 and a sequence isolated or derived from an AAV9. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype 4 (AAV4). In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV4 and a sequence isolated or derived from an AAV9. In some embodiments, wherein the vector is optimized for expression in mammalian cells. In some embodiments, the vector is optimized for expression in human cells. In some embodiments, the vector comprises the nucleic acid sequence of SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, or SEQ ID NO: 902.

Pharmaceutical Compositions and Delivery Methods

[0204] Also provided herein are compositions comprising one or more vectors and/or nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0205] For clinical applications, pharmaceutical compositions are prepared in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0206] Appropriate salts and buffers are used to render drugs, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the present disclosure 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. Any conventional media or agent that is not incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions may be used. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.

[0207] In some embodiments, 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] In some embodiments, the compositions of the present disclosure are 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.

[0212] 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" l5th 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 Biologies standards.

[0213] In some embodiments, a first vector and a second vector are administered to a patient. In some embodiments, the first vector comprises a nucleic acid comprising a first sequence encoding a first DMD guide RNA targeting a first genomic target sequence; a sequence encoding a second DMD guide RNA targeting a second genomic target sequence; a sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA; and a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD guide RNA. In some embodiments, the second vector comprise a nucleic acid comprising a sequence encoding a Cas9 or a nuclease domain thereof; a sequence encoding a muscle- specific promoter, wherein the muscle-specific promoter drives expression of the sequence encoding a Cas9 or a nuclease domain thereof.

Therapeutically-Effecitve Ratios

[0214] In some embodiments, a first vector and a second vector are administered to a patient in a therapeutically effective ratio. As used herein, the term“ratio” may refer to a ratio of the amount of vector in a composition (concentration), amount delivered to a patient (dosage), amount available to a therapeutic site (bioavailability), amount expressed by a target cell (copy number), amount of modifications made (efficacy), amount of DNA, or number of coding sequences (e.g., sequences encoding a gRNA or a Cas9).

[0215] In some embodiments, the ratio of the first vector and the second vector is between

1 : 1 and 1 :30. In other embodiments, the ratio of the first vector and the second vector is between 30: 1 and 1 :1. In some embodiments, the ratio of the first vector and the second vector is any one of the ratios shown in the“Ratio Table” below:

[0216] In some embodiments, the ratio of the amount of the first vector and amount of the second vector is between 1 : 1 and 1:30. In other embodiments, the ratio of the amount of the first vector and amount of the second vector is between 30: 1 and 1: 1. In some embodiments, the ratio of the amount of first vector and the amount of the second vector is any one of the ratios shown in the“Ratio Table.”

[0217] In some embodiments, the first vector is an AAV-Cas9 vector of the disclosure and the second vector is an AAV-sgRNA vector of the disclosure. In some embodiments, the ratio of the AAV-Cas9 vector to the AAV-sgRNA vector is any one of the ratios shown in the “Ratio Table.”

[0218] In some embodiments, the ratio of the first vector to the second vector is greater than 10: 1. For example, the ratio of the first vector to the second vector may be about 11 : 1, about 12: 1, about 13: 1, about 14: 1, about 15:1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 25: 1, about 30: 1, about 35: 1, about 40: 1, about 50: 1, about 75: 1, or about 100: 1. In some embodiments, the ratio of an AAV-sgRNA vector to an AAV-Cas9 vector is greater than 10: 1; for example, the ratio may be about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17:1, about 18: 1, about 19: 1, about 20: 1, about 25: 1, about 30: 1, about 35: 1, about 40:1, about 50: 1, about 75: 1, or about 100: 1.

[0219] In some embodiments, between 4 x 10¹² viral genomes (vg)/kilogram (kg) and 3 x 10¹³ vg/kg, inclusive of the endpoints, of the first and/or the second vector are administered to the patient. In some embodiments, between 4 x 10¹² viral genomes (vg)/kilogram (kg) and 3 x 10¹³ vg/kg, inclusive of the endpoints, of the first and/or the second vector are administered to the patient. In some embodiments, at least 5 x 10¹² viral genomes

(vg)/kilogram (kg), 6 x 10¹² viral genomes (vg)/kilogram (kg), 1 x 10¹³ viral genomes (vg)/kilogram (kg), 2 x 10¹³ viral genomes (vg)/kilogram (kg), 3 x 10¹³ viral genomes (vg)/kilogram (kg), 5 x 10¹³ viral genomes (vg)/kilogram (kg), 1 x 10¹⁴ viral genomes (vg)/kilogram (kg), 2 x 10¹⁴ viral genomes (vg)/kilogram (kg), 3 x 10¹⁴ viral genomes (vg)/kilogram (kg), or 4 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first and/or the second vector are administered to the patient.

[0220] In some embodiments, the Cas9 or Cpfl and gRNAs described herein may be delivered to the patient using adoptive cell transfer (ACT). In adoptive cell transfer, one or more expression constructs are provided ex vivo to cells which have originated from the patient (autologous) or from one or more individual(s) other than the patient (allogeneic). The cells are subsequently introduced or reintroduced into the patient. Thus, in some embodiments, one or more nucleic acids encoding Cas9 or Cpfl and a guide RNA that targets a dystrophin splice site are provided to a cell ex vivo before the cell is introduced or reintroduced to a patient.

[0221] In some embodiments, a composition comprises (i) a first nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor and (ii) a second nucleic acid sequence comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0222] In some embodiments, a composition comprises (i) a first vector comprising a nucleic acid sequence comprising a sequence encoding a first DMD guide RNA targeting a first genomic target sequence, a sequence encoding a second DMD guide RNA targeting a second genomic target sequence, a sequence encoding a first promoter wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and a sequence encoding a second promoter wherein the first promoter drives expression of the sequence encoding the second DMD guide RNA, wherein the first genomic target sequence and the second genomic target sequence each comprise a dystrophin splice acceptor and (ii) a second vector comprising a nucleic acid sequence comprising a sequence encoding a promoter and a sequence encoding a Cas9 or a nuclease domain thereof, wherein the sequence encoding the promoter comprises a sequence encoding a muscle-specific promoter such as the CK8 or CK8e promoter. In some embodiments, at least one of the first vector and the second vectors are AAVs. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

Cells and Cell Compositions

[0223] Also provided is a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell. Also provided is a composition comprising a cell comprising one or more nucleic acids of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0224] Also provided is a cell comprising a composition comprising one or more vectors of the disclosure. In some embodiments, the cell is a human cell. In some embodiments, the cell is a muscle cell or satellite cell. In some embodiments, the cell is an induced pluripotent stem (iPS) cell. Therapeutic Methods and Uses

[0225] Also provided is a method for correcting a dystrophin defect, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping of a DMD exon and/or reframing. In some embodiments, the at least one DMD guide RNA- Cas9 complex disrupts a dystrophin splice site and induces a refraining of a dystrophin reading frame. In some embodiments, the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and produces an insertion which restores the dystrophin protein reading frame. In some embodiments, the insertion comprises an insertion of a single adenosine.

[0226] Also provided is a method for inducing selective skipping and/or refraining of a DMD exon, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or refraining of a DMD exon.

[0227] Also provided is a method for inducing a reframing event in the dystrophin reading frame, the method comprising contacting a cell with one or more compositions of the disclosure under conditions suitable for expression of the first DMD guide RNA, the second DMD guide RNA and the Cas9 protein or a nuclease domain thereof, wherein at least one of first DMD guide RNA or the second DMD guide RNA forms a complex with the Cas9 protein or the nuclease domain thereof to form at least one DMD guide RNA-Cas9 complex, wherein the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or refraining of a DMD exon. In some embodiments, the at least one DMD guide RNA-Cas9 complex disrupts a dystrophin splice site and induces selective skipping and/or reframing of exon 51 of a human DMD gene.

[0228] Also provided is a method of treating muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more compositions of the disclosure. In some embodiments, the composition is administered locally. In some embodiments, the composition is administered directly to a muscle tissue.

In some embodiments, the composition is administered by an intramuscular infusion or injection. In some embodiments, the muscle tissue comprises a tibialis anterior tissue, a quadriceps tissue, a soleus tissue, a diaphragm tissue, or a heart tissue. In some

embodiments, the composition is administered by an intra-cardiac injection. In some embodiments, the composition is administered systemically. In some embodiments, the composition is administered by an intravenous infusion or injection. In some embodiments, following administration of the composition, the subject exhibits normal dystrophin-positive myofibers, and mosaic dystrophin-positive myofibers containing centralized nuclei, or a combination thereof. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of normal dystrophin positive myofibers when compared to an absence or a level of abundance of normal dystrophin-positive myofibers prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits an emergence or an increase in a level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei when compared to an absence or an level of abundance of mosaic dystrophin-positive myofibers containing centralized nuclei prior to administration of the composition. In some embodiments, following administration of the composition, the subject exhibits a decreased serum CK level when compared to a serum CK level prior to administration of the composition. In some embodiment, following administration of the composition, the subject exhibits improved grip strength when compared to a grip strength prior to administration of the composition. In some embodiments, the subject is a neonate, an infant, a child, a young adult, or an adult. In some embodiments, the subject has muscular dystrophy. In some embodiments, the subject is a genetic carrier for muscular dystrophy. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject appears to be asymptomatic and a genetic diagnosis reveals a mutation in one or both copies of a DMD gene that impairs function of the DMD gene product. In some embodiments, the subject presents an early sign or symptom of muscular dystrophy. In some embodiments, the early sign or symptom of muscular dystrophy comprises loss of muscle mass or proximal muscle weakness. In some embodiments, the loss of muscle mass or proximal muscle weakness occurs in one or both leg(s) and/or a pelvis, followed by one or more upper body muscle(s). In some embodiments, the early sign or symptom of muscular dystrophy further comprises pseudohypertrophy, low endurance, difficulty standing, difficulty walking, difficulty ascending a staircase or a combination thereof. In some embodiments, the subject presents a progressive sign or symptom of muscular dystrophy. In some embodiments, the progressive sign or symptom of muscular dystrophy comprises muscle tissue wasting, replacement of muscle tissue with fat, or replacement of muscle tissue with fibrotic tissue. In some embodiments, the subject presents a later sign or symptom of muscular dystrophy. In some embodiments, the later sign or symptom of muscular dystrophy comprises abnormal bone develomment, curvature of the spine, loss of movement, and paralysis. In some embodiments, the subject presents a neurological sign or symptom of muscular dystrophy. In some embodiments, the

neurological sign or symptom of muscular dystrophy comprises intellectual impairment and paralysis. In some embodiments, administration of the composition occurs prior to the subject presenting one or more progressive, later or neurological signs or symptoms of muscular dystrophy. In some embodiments, the subject is less than 10 years old, less than 5 years old, or less than 2 years old.

[0229] Also provided is the use of a therapeutically-effective amount of one or more compositions of the disclosure for treating muscular dystrophy in a subject in need thereof.

[0230] Tables 6-25 provide exemplary primer and genomic targeting sequences for use in connection with the compositions and methods disclosed herein.

Delivery Vectors

[0231] There are a number of ways in which expression vectors may be 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. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals.

[0232] One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. “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.

[0233] 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.

[0234] Adenovirus is particularly suitable for use as a gene transfer vector because of its mid sized 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 (El A 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 mRNAs for translation. 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 virus from an individual plaque and examine its genomic structure.

[0235] 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-home cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.

[0236] 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 adenovirus. 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.

[0237] Improved methods for culturing 293 cells and propagating adenovirus are known in the art. In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rmm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h. [0238] The adenoviruses of the disclosure are replication defective, or at least conditionally replication defective. 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 in the present disclosure.

[0239] 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 virus complements the E4 defect.

[0240] 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.

[0241] Adenovirus vectors have been used in eukaryotic gene expression and vaccine develomment. Animal studies suggested that recombinant adenovirus could be used for gene therapy. Studies in administering recombinant adenovirus to different tissues include trachea instillation, muscle injection, peripheral intravenous injections and stereotactic inoculation into the brain.

[0242] 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. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome.

[0243] 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.

[0244] 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 sialogly coprotein receptors.

[0245] A different approach to targeting of recombinant retroviruses may be used, in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor are used. The antibodies are coupled via the biotin components by using

streptavidin. Using antibodies against major histocompatibility complex class I and class II antigens, it has been demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux el al, 1989).

[0246] There are certain limitations to the use of retrovirus vectors in all aspects of the present disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes. Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (see, for example, Markowitz et al, 1988; Hersdorffer el al, 1990). [0247] Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus adeno-associated virus (AAV) and herpesviruses may be employed. They offer several attractive features for various mammalian cells.

[0248] In embodiments, the AAV vector is replication-defective or conditionally replication defective. In embodiments, the AAV vector is a recombinant AAV vector. In some embodiments, the AAV vector comprises a sequence isolated or derived from an AAV vector of serotype AAV1, AAV2, AAV3, AAV4, AAV 5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11 or any combination thereof.

[0249] In some embodiments, a single viral vector is used to deliver a nucleic acid encoding a Cas9 or a Cpfl and at least one gRNA to a cell. In some embodiments, Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector.

[0250] In some embodiments, a single viral vector is used to deliver a nucleic acid encoding Cas9 or Cpfl and at least one gRNA to a cell. In some embodiments, Cas9 or Cpfl is provided to a cell using a first viral vector and at least one gRNA is provided to the cell using a second viral vector. In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. The cell may be a muscle cell, a satellite cell, a mesangioblast, a bone marrow derived cell, a stromal cell or a mesenchymal stem cell. In embodiments, the cell is a cardiac muscle cell, a skeletal muscle cell, or a smooth muscle cell. In embodiments, the cell is a cell in the tibialis anterior, quadriceps, soleus, diaphragm or heart. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or inner cell mass cell (iCM). In further embodiments, the cell is a human iPSC or a human iCM. In some embodiments, human iPSCs or human iCMs of the disclosure may be derived from a cultured stem cell line, an adult stem cell, a placental stem cell, or from another source of adult or embryonic stem cells that does not require the destruction of a human embryo. Delivery to a cell may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.

[0251] Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. 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.

[0252] 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.

[0253] 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. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

[0254] 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.

[0255] In some embodiments, the expression construct is delivered directly to the liver, skin, and/or muscle tissue of a subject. This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, /. e.. ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.

[0256] 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.

[0257] 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.

[0258] In certain embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ) 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-l). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-L 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 for the present disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

[0259] 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.

[0260] 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, has been used as a gene delivery vehicle and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells. Duchenne Muscular Dystrophy

[0261] 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 (see GenBank Accession NO. NC_000023.l 1), located on the human X chromosome, which codes for the protein dystrophin (GenBank Accession No. AAA53189; SEQ ID NO: 5).

[0262] In humans, dystrophin mRNA contains 79 exons. Dystrophin mRNA is known to be alternatively spliced, resulting in various isoforms. Exemplary dystrophin isoforms are listed in Table 1.

[0263] The murine dystrophin protein has the following amino acid sequence (Uniprot

Accession No. Pl 1531, SEQ ID. NO: 869):

1 MWWVDCYRDV KKTTKWNASK GKHDNSDDGK RDGTGKKKGS TRVHANNVNK ARVKNNVDVN

61 GSTDVDGNHK TGWNHWVKNV MKTMAGTNSK SWVRSTRNYV NVNTSSWSDG ANAHSHRDDW

121 NSWSHSATR HANAKCGKDD VATTYDKKSM YTSWSAVMR TSSKVTRHHH MHYSTVSAGY

181 TSSSKRKSYA TAAYVATSDS TSYSHARDKS DSSMTVNDSY TAVSWSADTR AGSNDWKHA

241 HGMMDTSHGV GNVGSVGKGK SDAVMNNSRW CRVASMKSKH KVMDNKKDDW TKTRTKKMGD

301 DKCVHKVDVR VNSTHMWW DSSGDHATAA KVGDRWANCR WTDRWVDKWH TCSTWSKDAM

361 KNTSGKDNMM SSHKSTKDKK KTMKSSNDSA KNKSVTKMWM NARWDNTKKS SASAVTTTST

421 TTVMTVTMVT TRMVKHAKKR TVDSRKRDVD THSWTRSAVS SAVYRKGNSD KVNAARKAKR

481 KDASRSAAVM ANGVNASRAS NSRWTCSRVN WYTNTYNMTT TANKTSTTST AKSKCKDVNR

541 SAKSKKGGMD ADVATNHNHD GVRAKKTDTM RYTMSSRTWS SKSVYSVTYM RGKASSKNGN

601 YSDTVKMAKK ASCKYSGHWK KSSVSCKHMN KRKNHKTKWM AVDVKWAGDA KKKCRVGDTS

661 NSVNGGKKSA ASRTRNTWDH CRVYTRKAKA GDKTVSKDSM HWMTAYRDYK TDTAVMKRAK

721 AKTKVKTTVN SVAHASAAKK TTTNYWCTRN GKCKTVWACW HSYKANKWNV KKTMNVAGTV

781 SNMHHSNNRA TTDGGVMDNT NSRWRHAVRK KSSAKSHSDK AAYTDKVDAA MAKSDTSHSM

841 KKHNGKDANR VSDVAKKDVS MKRKANRSKM DVKMHATKSV VSSHCVNYKS SVKSVMVKTG

901 RVKKTNKDRV TAKHYNGAKV TRKKCKSRKM RKMNVTWAAT DTTKRSAVGM SNDSVAWGKA

961 TKKKAHKSVT GSKMVGKKTV DKSNSNWAVT SRWNYKHMT DNTKWHADDS KKKKDKRKAM

1021 NDMRKVDSTR DAAKMANRGD HCRKWSNRR AASHRKTGKA SKNSDKAGVN KDNKDMSDNG

1081 TVNRGDNRTD RKRKKTKHNA KDRSRRKKAS HWYYKRADDK CDKKASRDRK KDRKKKNAVR

1141 RAGSNGAAMA VTSKRWRSNA RRNAHTHTMV VTTDMDVSYV STYTSHASVD HNTCAKDDKS

1201 KNKDNSGRDH KKKTAASATS MKVKVAVAMD GKHRMYKRGR DRSVKWRHHY DMKVNWNVKK

1261 TNNWHAKYKW YKDGGRAWR TNATGSSKTD VNKGSSRWHD CKARRKRKNV SRDNWADNA

1321 TGDKVKARGK NTGGAWSAR DKKKKTNWKV SRAKGVHKDR DHWSRNYNSA GDKVTVHGKA

1381 DVRSKGHYKK STVKRKDRSW AVNHRRTKDR AGSTTGASAS TVTVTSWTK TVSKMSSVAA

1441 DNRAWTTDWS DRVKSRVMVG DDNMKKATDR RTAANKNKTS NARTTDRRWD VNRRNMKDST

1501 WAKAVGVRGK DSWKGHTVDA KKTTKAKDRR SVDVANDAKR DYSADDTRKV HMTNNTSWGN

1561 HKRVSAATHR DKSWTATTAN VDASRKKDSR GVRMKWDGTH TDYHNDNGKR SGSDARRDNM

1621 NKWSKKSNRS HASSDWKRHS WKDDSRAGG DAVKNDHRAK RKTKVMSTTV RTGKYRRANV

1681 TRRKAVNAWD KNRSADWRKD ARAADDKRAV KGSWVGDDSD HKVKARGAKN VNRVNDAHTT

1741 GSYNSTDNTR WRVAVDRVRH AHRDGASHST SVGWRASNKV YYNHTTTCWD HKMTYSADNN

1801 VRSAYRTAMK RRKACDSSAA CDADHNKNDM DNCTTYDRHN NVNVCVDMCN WNVYDTGRTG

1861 RRVSKTGSCK AHDKYRYKVA SSTGCDRRGH DSRGVASGGS NSVRSCANNK AADWMRSMVW

1921 VHRVAAATAK HAKCNCKCGR YRSKHNYDCS CSGRVAKGHK MHYMVYCTTT SGDVRDAKVK

1981 NKRTKRYAKH RMGYVTVGDN MTVTNWVDSA ASSSHDDTHS RHYASRAMNS NGSYNDSSNS

2041 DDHHYCSNDS SRSASSRGRA DNRNAYDRKH HKGSSMMTSS RDAAAKRHKG RARMDHNKSH

2101 RRAAKVNGTT VSSSTSRSDS SMRWGSTSS MGDSDTSTGV MNNSSSRGRN AGKMRDTM. [0264] 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.

[0265] 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. Bladen et al. (2015), who examined 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).

DMD Subject Characteristics and Clinical Presentation

[0266] 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 develomment 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.

[0267] 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:

1. 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.)

2. Frequent falls.

3. Fatigue.

4. Difficulty with motor skills (running, hopping, jumping).

5. 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.

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

7. Progressive difficulty walking.

8. Muscle fiber deformities.

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

10. 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.

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

12. Skeletal deformities (including scoliosis in some cases).

13. Trouble getting up from lying or sitting position.

[0268] 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 develomment of congestive heart failure or arrhythmia (irregular heartbeat) is only occasional. [0269] 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 Xp2l 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.

[0270] DMD patients may suffer from:

1. Abnormal heart muscle (cardiomyopathy).

2. Congestive heart failure or irregular heart rhythm (arrhythmia).

3. Deformities of the chest and back (scoliosis).

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

5. Loss of muscle mass (atrophy).

6. Muscle contractures in the heels, legs.

7. Muscle deformities.

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

[0271] Duchenne muscular dystrophy (DMD) is caused by a mutation of the dystrophin gene at locus Xp2l, 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.

[0272] 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.

[0273] 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.

[0274] Exon deletions preceding exon 51 of the human DMD gene, which disrupt the open reading frame (ORF) by juxtaposing out of frame exons, represent the most common type of human DMD mutation. Skipping of exon 51 can, in principle, restore the DMD ORF in 13% of DMD patients with exon deletions.

[0275] Duchenne muscular dystrophy has an incidence of 1 in 5000 male infants. Mutations within the dystrophin gene can either be inherited or occur spontaneously during germline transmission. A table of exemplary but non-limiting mutations and corresponding models are set forth below:

Table 2: Dystrophin mutations and corresponding mouse models

[0276] DMD Diagnosis: 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 performed during pregnancy.

[0277] 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.

[0278] 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. [0279] 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.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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.

Combination Therapies

[0284] Beyond the gene therapy disclosed herein, there is no current cure for DMD. Prior to the develomment of the gene therapy of the disclosure, treatment was generally aimed at controlling the onset of symptoms to maximize the quality of life. These therapies may be used in combinatoin with the gene therapies of the disclopsure and may include the following:

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

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

4. Physical therapy is helpful to maintain muscle strength, flexibility, and function.

5. 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.

6. Appropriate respiratory support as the disease progresses is important.

[0285] 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.

[0286] DMD generally progresses through five stages, as outlined in Bushby et al, Lancet Neurol., 9(1): 77-93 (2010) and Bushby et al, Lancet Neurol., 9(2): 177-198 (2010), incorporated by reference in their entireties. During the presymptomatic stage, patients typically show develommental delay, but no gait disturbance. During the early ambulatory stage, patients typically show the Gowers’ sign, waddling gait, and toe walking. During the late ambulatory stage, patients typically exhibit an increasingly labored gait and begin to lose the ability to climb stairs and rise from the floor. During the early non-ambulatory stage, patients are typically able to self-propel for some time, are able to maintain posture, and may develop scoliosis. During the late non-ambulatory stage, upper limb function and postural maintenance is increasingly limited.

[0287] In some embodiments, treatment is initiated in the presymptomatic stage of the disease. In some embodiments, treatment is initiated in the early ambulatory stage. In some embodiments, treatment is initiated in the late ambulatory stage. In embodiments, treatment is initiated during the early non-ambulatory stage. In embodiments, treatment is initiated during the late non-ambulatory stage.

Physical Therapy

[0288] Physical therapists are concerned with enabling patients to reach their maximum physical potential. Their aim is to:

1. minimize the develomment of contractures and deformity by developing a program of stretches and exercises where appropriate,

2. anticipate and minimize other secondary complications of a physical nature by recommending bracing and durable medical equimment, and 3. monitor respiratory function and advise on techniques to assist with breathing exercises and methods of clearing secretions.

Respiration Assistance

[0289] 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 equimment may easily fit on a ventilator tray on the bottom or back of a power wheelchair with an external battery for portability.

[0290] 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. Prognosis

[0291] Duchenne muscular dystrophy is a progressive disease which, left untreated, 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.”

[0292] 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.

[0293] 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 amechanistic insight for their unique pathophysiological properties. The ILM may facilitate the develomment of novel strategies for the prevention and treatment of muscle wasting in a variety of clinical scenarios.

Animal Models

[0294] The disclosure provides mouse and canine models of DMD to provide not only proof of concept data but also to demonstrate the safety and efficacy of the pharmaceutical compositions of the disclosure when used in vivo.

Humanized Mouse Models of DMD

AEx50 Mouse

Rationale

[0295] The most common hot spot mutation region in DMD patients is the region between exon 45 to 51. Reframing and/or skipping of exon 51 may be used to treat the largest group (13-14%) of patients.

Recapitulation of the Human Condition

[0296] To investigate CRISPR/Cas9-mediated exon 51 skipping in vivo, a mouse model was generated that mimics the human“hot spot” region by deleting exon 50 using the

CRISPR/Cas9 system directed by 2 sgRNAs (FIG. 1A). The deletion of exon 50 was confirmed by DNA sequencing (FIG. 1B). Deletion of exon 50 placed the dystrophin gene out of frame leading to the absence of dystrophin protein in skeletal muscle and heart (FIGS. 1C-1E). Mice lacking exon 50 showed pronounced dystrophic muscle changes by 2 months of age (FIG. 1E). Serum analysis of delta-exon 50 mice showed a significant increase in creatine kinase (CK) levels, indicative of muscle damage (FIG. 1F). Taken together, dystrophin protein expression, muscle histology and serum CK levels validated the dystrophic phenotype of the DEc50 mouse model.

Proof of Concept - Efficacious correction of the dystrophin reading frame in

AEx50 mice by a single DNA cut.

[0297] The sgRNA-ex51 -S A2 was delivered to mice in triple copy (AAV-Tri-SA2), along with a Cas9 (AAV-Cas9), by intra-muscular (IM) injection. Following the injection, muscle tissues were analyzed. In vivo targeting efficiency was estimated by RT-PCR with primers for sequences in exons 48 and 53 and the T7E1 assay for the targeted genomic regions. To investigate whether efficient target cleavage was achieved, the inventors amplified a 771 bp region spanning the target site and analyzed it using the T7EI assay (FIG. 10A). The activity of SpCas9 with the corresponding sgRNA was analyzed on the target site. T7EI assays revealed mutagenesis of the Dmd locus after delivery of AAV-Cas9 and AAV9-sgRNA-5l- SA2 (FIG. 10A). To investigate the type of mutations generated in DEc50 mice injected with Cas9 and sgRNA-expressing AAV9s, genomic PCR amplification products spanning the target site were analyzed by amplicon deep-sequencing analysis. Deep sequencing of the targeted region indicated that 27.9% of total reads contained changes at the targeted genomic site (FIG. 10B). On average, 15% of the identified mutations contained the same A insertion seen in mouse 10T1/2 and human 293 cells in vitro. The deletions identified using this method encompassed a highly -predicted exonic splicing enhancer site for exon 51 (FIG.

10B).

[0298] RT-PCR products of RNA from muscle of AEx50 mice injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l showed that deletion of exon 51 (DEc50-51) allowed splicing from exon 49 to 52 (FIG. 11 A, lower band). By sequencing RT-PCR products of the DEc50-51 band, it was confirmed that exon 49 was spliced to exon 52. To further define the mutations introduced by our gene editing strategy, RT-PCR amplification products from 4 samples were directly subjected to topoisomerase-based thymidine to adenosine (TOPO-TA) cloning without gel purification, then sequenced. Surprisingly, sequence analysis of 40 clones from each sample showed that in addition to exon 5l-skipped cDNA products (DEc50-51) identified in 15% of sequenced clones, DEc50 mice injected with AAV9-Cas9 and AAV9- sgRNA-5l showed a high frequency of reframing events. Of sequenced clones, 63% contained a single nucleotide insertion in the sequence of exon 51 (FIGS. 11 B-C). The most dominant insertion mutation seen was an A insertion. [0299] On gels, the A insertion was indistinguishable in size from non-edited cDNA products, so deep-sequencing analysis was performed to determine the abundance of this insertion compared to other small insertions. Deep-sequencing of the upper band containing the non-edited cDNA product and reframed cDNA products indicated that 69.22% of total reads contained refrained cDNA products with an A insertion, 17.71% contained non-edited cDNA product, and the rest contained small deletions and insertions (FIG. 11D). The deep sequencing analysis of uninjected AEx50 mice confirmed that the A insertion is a result of Cas9-generated editing. These amplicon deep-sequencing results confirmed the results from TOPO-TA cloning and sequencing. Taken together, RT-PCR analysis revealed that AEx50 mice injected with AAV9-Cas9 and AAV9-sgRNA-5l-SA2 showed a high frequency of reframing events with cDNA products containing an A insertion in the sequence of exon 51 in addition to exon 51 skipping events resulting from deletion in a highly conserved exonic splicing enhancer region.

Proof of Concept— Restoration of dystrophin expression after intramuscular AAV9 delivery of Cas9 and sgRNA-51-SA2.

[0300] Remarkably, dystrophin immunostaining of muscle cryosections from AEx50 mice injected with AAV-Tri-SA2 revealed significantly higher numbers of dystrophin-positive fibers with an average of 99% restoration of normal fibers (FIG. 12A, FIG. 13). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle. (FIG. 12C, FIG. 12D). Hematoxylin and eosin (H&E) staining of muscle showed that histopathologic hallmarks of muscular dystrophy, such as necrotic myofibers, were corrected in TA muscle at 3-weeks post-AAV delivery (FIG. 12B and FIG. 14). This method, using a distinct sgRNA design, represents a major advance in efficiency of DMD correction with direct applicability to the patients with the most common dystrophin mutations.

Proof of Concept— Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 combined with different AAV9s expressing single copy or triple copy of sgRNA.

[0301] To evaluate the benefit of triple promoter expression of sgRNA-ex5l-SA2 in vivo, different constructs were investigated, where sgRNA expression was driven by a single RNA polymerase III promoter (U6 or Hl or 7SK) and, separately, by three RNA polymerase III promoters (U6, Hl and 7SK) (FIG. 15A). The inventors delivered the sgRNA-ex5l-SA2 in single copy driven separately by the U6 promoter (AAV9-U6-sgRNA-5l-SA2), the Hl promoter (AAV9-Hl-sgRNA-5l-SA2), the 7SK promoter (AAV9-7SK-sgRNA-5l-SA2) and triple copy (AAV9-Triple-sgRNA-5l-SA2). Following intra-muscular (IM) injection of P12 mice with AAV9s, muscle tissues were analyzed. Unexpectedly, dystrophin immunostaining of muscle cryosections from AEx50 mice injected with AAV9-Triple-sgRNA-5l-SA2 revealed significantly higher numbers of dystrophin-positive fibers with an average of 95% restoration of normal fibers compared to AEx50 mice injected with AAV9-U6-sgRNA-5l- SA2, AAV -H 1 -sgRNA-51 -S A2 and AAV-7SK-sgRNA-5l-SA2 with an average of 70%; 40% and 50% restoration of normal fibers respectively (FIGS. 15B).

Superior Properties - Triple guide RNA strategy. Compared to the efficacious activity of the single gRNA, the triple gRNA provides superior and unexpected restoration of dystrophin-positive fibers.

Proof of Concept— Rescue of muscle structure and function following systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2.

[0302] Systemic delivery of AAV9-Cas9 and AAV9-sgRNA-5l-SA2 to P4 AEx50 mice yielded widespread dystrophin expression in the heart, triceps, tibialis anterior (TA) muscle, and diaphragm in gene-edited AEx50 mice at 4 and 8 weeks post-injection (FIG. 16A and FIG. 17A). Western blot analysis confirmed the restoration of dystrophin expression in skeletal and heart muscles (FIG. 16B and FIG. 17B). Grip strength testing also showed a significant increase in muscle strength of AEx50 mice at 4 weeks post-intraperitoneal AAV9 injection compared to AEx50 control mice (wildtype control 92.6±l.63; AEx50 control 50.5±1.85; AEx50-AAV9-sgRNA-5l 79.7±2.63) (FIG. 18A). Consistently, AAV9-sgRNA- 51-SA2 gene-edited AEx50 mice also showed significant reductions in serum CK

concentrations compared to AEx50 control mice (FIG. 18B).

Proof of Concept— Tailoring the AAV dose for single DNA cut genome editing strategy.

[0303] To optimize the in vivo efficiency of gene editing, the inventors tested different doses and delivery strategies for AAV9-Cas9 and AAV9-sgRNA-5l. The inventors systemically delivered by intraperitoneal injection the AAV9-Cas9 and AAV9-sgRNA-5l to P4 AEx50 mice using 2 different doses (2.6xl0¹³ vg/kg of each AAV9 referred as Dose 1 and 6xl0¹² vg/kg of each AAV9 referred as Dose 2). The systemic delivery yielded widespread dystrophin expression in the heart, triceps, tibialis anterior muscle, and diaphragm in gene- edited AEx50 mice at 4 weeks post-injection (FIG. 22A). Western blot analysis confirmed the restoration of dystrophin expression in muscles (FIG. 22B). The AAV dose 2 (6xl0¹² vg/kg) lead to lower dystrophin correction in diaphragm and triceps muscle compared to AAV dose 1 (2.6xl0¹³ vg/kg), suggesting that the efficient AAV dose should not be lower than 2.6xl0¹³ vg/kg.

Proof of Concept -Tailoring the AAV dosage ratio for single DNA cut genome editing strategy.

[0304] To further optimize the in vivo efficiency of gene editing, the inventors tested different ratios of AAV9-Cas9 and AAV9-sgRNA-5l. The inventors delivered the AAV9- Cas9 and AAV9-sgRNA-ex5l in different doses and ratios (1: 1 and 1:2) using IP injection P4 DEc50 mice (FIG. 75) using the following conditions: 5xl0¹³vg/kg AAV9-Cas9 and

5xl0¹³vg/kg AAV 9-sgRNA-51 (Ex-5l-SA2 SEQ. No: 708), 5xl0¹³vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg AAV9-sgRNA-51 and 1xl0¹⁴vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg AAV9- sgRNA-5l. Unexpectedly, dystrophin immunostaining of muscle cryosections from AEx50 mice injected mice injected with 5xl0¹³vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg (1:2) of AAV9- Cas9 to AAV9-sgRNA-ex5l displayed better dystrophin correction then 5xl0¹³vg/kg AAV9- Cas9 and 5xl0¹³ vg/kg AAV9-sgRNA-5l. Moreover, dystrophin immunostaining of muscle cryosections from AEx50 mice injected mice injected with 5xl0¹³ vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg showed comparable correction to the mice injected with 1x10¹⁴ vg/kg AAV9- sgRNA-51 and 1x10¹⁴ vg/kg AAV9-Cas9 and 1xl 0¹⁴ vg/kg AAV9-sgRNA-5l (FIG. 75).

Proof of Concept— Evaluation of rescue of dystrophin expression following intravenous injections of AAV9-Cas9 with AAV9-sgRNA-51 at later stages of DMD disease.

[0305] To investigate in vivo efficiency of the gene editing and dystrophin correction at later stages of Duchenne muscular dystrophy disease, the inventors treated 1 month old AEx50 mice with AAV9-Cas9 and AAV9-sgRNA-5l. The inventors systemically delivered 2.6xl0¹³ vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l via a tail vein injection to 1 month old AEx50 mice. The systemic delivery resulted in widespread dystrophin expression in the heart, triceps, tibialis anterior muscle, and diaphragm in gene-edited AEx50 mice at 4 weeks and 8 weeks post-injection (FIG. 23 A and FIG. 24 A). Western blot analysis confirmed the restoration of dystrophin expression in muscles (FIG. 23B and FIG. 24B). Grip strength testing also showed a significant increase in muscle strength of AEx50 mice at 4 weeks and 8 weeks post-intravenous injection compared to AEx50 control mice (FIG. 23C and FIG. 24C). Superior Properties - Therapeutic compositions show efficacious resue of dystrophin expression following systemic administration to a subject modeling late-stage DMD disease.

[0306] Contrary to expectation that treatment of a subject with more advanced DMD would be less efficacious, the therapeutic compositions of the disclosure restore dystrophin expression following administration to these subjects. Moreover, in this example, the administration is systemic as opposed to local. Local administration would be expected to provide even greater efficacy because the AAV constructs would not have to be delivered from the injection point to the treatment site, which is likely a farther distance and which involves traversing the subject’s blood stream, where the AAV constructs are exposed to the immune system.

Proof of concept - Luciferase model demonstrates the ability to monitor in vivo in a non-invasive way the correction of DMD mutations and restoration of DMD expression.

[0307] A luciferase reporter was introduced in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. The inventors evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of

CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. These results provide a sensitive non-invasive means of monitoring of dystrophin correction in mouse models of DMD and provide a platform for testing different strategies for amelioration of DMD pathogenesis.

[0308] To correct the dystrophin reading frame and evaluate the bioluminescence signal in \Ex50-Dmd-Luc mice, an sgRNA targeting a region adjacent to the exon 51 splice acceptor site (referred to as sgRNA-5l) was used. For the in vivo delivery of Cas9 and sgRNA-5l to skeletal muscle and the heart, the inventors used AAV9, which displays preferential tropism for these tissues. Muscle-specific expression of the AAV9-Cas9 vector was further ensured by incorporating the muscle creatine kinase (CK8e) promoter, which is highly specific for expression in muscle and heart. Expression of the sgRNA in a separate AAV9 vector was driven by three RNA polymerase III promoters (U6, Hl and 7SK). [0309] Following intra-muscular (IM) injection of the left tibialis anterior (TA) muscle of \Ex50-Dmd-Luc mice at postnatal day (P) 12 with a total of 5xl0¹⁰ AAV9 viral genomes (vg), muscles were analyzed by dystrophin immunostaining and bioluminescence for 4 weeks (FIG. 35A). Bioluminescence signal was apparent in the injected leg within 1 week after injection and increased in intensity thereafter, ultimately reaching a level comparable to that of WT-Dmd-Luc mice within 4 weeks (FIG. 35B, 35E). Histological analysis of AAV9- injected TA muscle was performed to evaluate the number of fibers that expressed dystrophin and the correlation with the bioluminescence signal. Dystrophin immunohistochemistry of muscle from \Ex50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA-5l revealed restoration of dystrophin expression throughout the entire muscle (FIG. 35C, 35D).

[0310] To further evaluate the sensitivity of the Luciferase reporter to in vivo, the inventors administered AAV9-Cas9 and AAV9-sgRNA-5l intraperitoneally to \Ex50-Dmd-Luc mice at P4 and monitored the signal over time (FIG. 36A). Widespread bioluminescence was observed 3 weeks after injection and continued to increase to a level -70% of wild-type by 10 weeks. (Fig. 36B). Histological analysis revealed widespread dystrophin expression in the diaphragm, heart, TA and triceps muscles of gene-edited AEx50-Dmd-Luc mice at 10 weeks post-injection (FIG. 36C). Western blot analysis revealed a close correlation between expression of Cas9, dystrophin and Luciferase in skeletal muscles and heart following systemic IP delivery of AAV9-encoded gene editing components to AEx50-Dmd-Luc mice (FIG. 37A, B).

Proof of concept and unexpected result— a 1:2 ratio of AAV9-Cas9 to AAV9- sgRNA may provide superior efficacy to when compared with a 1:1 ratio of AAV9-Cas9 to AAV9-sgRNA.

[0311] To further optimize the in vivo efficiency of gene editing, the inventors tested different ratios of AAV9-Cas9 and AAV9-sgRNA-5l. The inventors delivered the AAV9- Cas9 and AAV9-sgRNA-ex5l in different ratios (1 :1 and 1 :2) using intravenous injection in the tail vein of 1 month old \ Ex50-/9m/-l uci ferase mice (FIG. 25 A). The in vivo

bioluminescence analysis showed appearance of signal 2 weeks after injection. Unexpectedly, \ Ex5 ( )-l)md-\ uci ferase mice injected with a 1 :2 ratio of AAV9-Cas9 to AAV9-sgRNA-ex51 displayed higher bioluminescence signal than the mice injected with a 1: 1 ratio of AAV9- Cas9 to AAV 9-sgRNA-ex51 (FIG. 25B). Superior Properties - efficiency of gene modification following systemic

(intravenious) injection is at least 30% when all muscle types are considered but over 60% for core/essential body muscles of the diagraphram and heart.

[0312] In vivo targeting efficiency was assessed within muscle biopsies by tracking indels by decomposition (TIDE) analysis of RT-PCR products with primers for sequences in exons 48 and 53 (FIG. 40). TIDE analysis showed 68.6%, 87.08%, 29.6% and 66.5% frequencies of indels for diaphragm, heart, tibialis anterior and triceps muscle respectively (FIG. 40).

AEx44 Mouse

Rationale

[0313] Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in -12% of patients by skipping surrounding exons, which restores the dystrophin open reading frame.

Recapitulation of Human Condition

[0314] As described in greater detail in Example 2, AEx44 DMD mice were generated in the C57/BL6J background using the CRISPR/Cas9 system. Two sgRNAs specific to the intronic regions surrounding exon 44 of the mouse Dmd locus were cloned into vector PX458 (Addgene plasmid #48138) using the primers from Table 16.

[0315] Deletion of exon 44 was confirmed by RT-PCR analysis (Fig. 62A). Sequencing of the RT-PCR products using primers for sequences in exons 43 and 46 confirmed the removal of exon 44 in these mice (Fig. 62B). At 4 weeks of age, immunostaining of tibialis anterior (TA) muscle, diaphragm, and heart in the AEx44 DMD mice showed complete absence of dystrophin protein expression (Fig. 26F). Western blot analysis confirmed loss of dystrophin protein (Fig. 26E). Necrotic fibers, inflammatory infiltration, and regenerative fibers with centralized nuclei were observed in 4-week old AEx44 DMD mice, indicative of a severe muscular dystrophy phenotype (Fig. 26G). Serum creatine kinase levels in the AEx44 DMD mice were elevated 22-fold compared to WT littermates, similar to mdx mice, an established DMD mouse model (Fig. 26D).

[0316] Shear force generated during muscle contraction leads to muscle membrane tearing in muscle lacking dystrophin, eventually causing myofiber degeneration and muscle fibrosis. Fibrotic tissue increases muscle stiffness and compromises contractility of muscles. To further analyze muscle function of DEc44 DMD mice, maximal tetanic force was measured in the extensor digitorum longus (EDL) muscle ex vivo. Compared with WT littermates at 4 weeks of age, DEc44 DMD mice showed an about 50% decrease in the specific and absolute tetanic force in the EDL muscle (Fig. 27, C and D). A similar decrease of muscle strength was observed by grip strength analysis in 8-week old DEc44 DMD mice (Fig. 27E).

Proof of Concept - Efficacious correction of DMD exon 44 deletion in mice by intramuscular AAV9 delivery of gene editing components.

[0317] To deliver 5/;Cas9 and sgRNA in vivo, AAV9 was utilized to package the gene editing components. AAV9 is a single-stranded DNA virus 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 was used, which combines key elements of the enhancer and promoter regions of the muscle creatine kinase gene to drive SpCas9 expression in skeletal muscle and heart. For delivery of sgRNA, three RNA polymerase III promoters (U6, Hl, and 7SK) were used to express three copies of a sgRNA (Fig. 9A).

[0318] First, the efficiency of the AAV construct was tested by comparing the editing efficiency of PX458 and AAV expression constructs encoding gene editing components that targeted exon 45 in mouse C2C12 muscle cells. By T7E1 assay, comparable editing efficiency was observed with both constructs (Fig. 66A). Among the two sgRNAs tested, G6 showed better cutting efficiency than G5, consistent with the observations in mouse 10T1/2 cells and human 293 cells (Fig. 64C).

[0319] To validate the efficacy of the single cut gene editing strategy in the DEc44 DMD mouse model, localized intramuscular (IM) injection of AAV9 expressing 5'/;Cas9 (AAV- Cas9) and AAV9 expressing sgRNA (AAV-G5 or AAV-G6) was performed in TA muscle of postnatal day 12 (P12) mice. As a control group, WT and DEc44 DMD mice were injected with AAV-Cas9 without AAV-sgRNA. In initial studies, 50 pl of AAV9 (1 ^c 10¹² vg/ml) was injected per leg, containing equal amounts of AAV-Cas9 and AAV9-G5 or AAV-G6. Three weeks after IM injection, TA muscles were collected for analysis. In vivo gene editing by AAV-G5 and AAV-G6 was compared by the T7E1 assay and RT-PCR of the targeted region (Fig. 28E and Fig. 66B). Gene editing with AAV-G6 showed higher efficiency based on DNA cutting in vivo (Fig. 66B). RT-PCR with primers that amplify the region from exon 43 to exon 46 revealed deletion of exon 45 in TA muscle injected with AAV-Cas9 and AAV- G6 (Fig. 28E). This allows exon 43 to skip exon 45 and directly splice to exon 46 when processing the pre-mRNA. As a result, the alternate mRNA enables the production of a truncated dystrophin protein in corrected TA muscle of AEx44 DMD mice.

[0320] 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. Sequencing results demonstrated that 7% of sequenced clones represented exon 45-skipped cDNA products, and 42% of sequenced clones contained a single adenosine (A) insertion in exon 45 that resulted in reframing of dystrophin protein (Fig. 28F, Fig. 28G, and Fig. 66B). The predominance of reframing explains the high abundance of the RT-PCR band at 355 bp and the lower abundance of the smaller RT-PCR product of 179 bp that reflects exon skipping (Fig. 28E).

[0321] Genomic and cDNA amplicon deep sequencing on the target region of the TA muscles with AAV-G6 IM injection also confirmed that 9.8% of mutations at the genomic level and 35.7% of mutations at the mRNA level contain a single A insertion at the cutting site after gene editing with AAV-G6 (Fig. 66C and 66D). This single A insertion leads to refraining of exon 45, and restores the dystrophin protein reading frame. Minor AAV integration events were also observed at the cutting site, with 0.2% at the genomic level (Fig. 66C), and 1.2% at the mRNA level (Fig. 66D). The integrated sequence is from the ITR region of AAV and prevents production of functional dystrophin protein from those transcripts and, thus, has neither positive nor negative effects on the dystrophic muscle phenotype.

[0322] To evaluate dystrophin protein restoration after IM injection with AAV-Cas9 and AAV-G5 or AAV-G6, we performed Western blot analysis on TA muscle and the heart (Fig. 28C). We observed restoration of dystrophin protein expression to 74% of the WT level in edited TA muscles of AEx44 DMD mice. Interestingly, although the injection was localized to the TA muscle, we observed expression of dystrophin in the heart at 21% of WT level (Fig. 28C). This suggests transfer of AAV into the circulation and delivery of the gene editing components to the heart. Immunostaining showed that dystrophin protein expression was restored in 99% of the myofibers in TA muscle injected with AAV-Cas9 and AAV-G6 (Fig. 28A and Fig. 28A). Histological analysis and hematoxylin and eosin (H&E) staining showed a pronounced reduction in fibrosis, necrotic myofibers and regenerating fibers with central nuclei, indicating amelioration of the abnormalities associated with muscular dystrophy in the TA muscle 3 weeks after AAV9-Cas9 and AAV-G6 injection (Fig. 28B). Superior Properties— no detectable off-target activity

[0323] Based on CRISPR design tools (crispr.mit.edu/ and benchling.com/), the top 10 potential off-target sites were determined and, based on sequencing analysis, no off-target effects were detected at these sites (Fig. 67A-C). T7E1 analysis confirmed the absence of off-target cutting in the top 10 potential off target sites, and DNA sequencing of the isolated genomic PCR amplification products spanning the potential off-target sites confirmed the absence of sgRNA/Cas9-mediated mutations at the predicted sites (Fig. 67 A). In addition, genomic amplicon deep sequencing of the top 10 predicted off-target sites within protein coding exons was performed. None of these sites showed significant sequence alterations (Fig. 67B and 67C).

Proof of Concept - Efficacious systemic delivery of AAV9 expressing gene editing components rescues dystrophin expression in AEx44 mice.

[0324] To achieve body-wide rescue of the disease phenotype in AEx44 DMD mice, AAV- Cas9 and AAV-G6 was delivered systemically by intraperitoneal (IP) injection. AAV-Cas9 was injected at a dosage of 5 ^c 10¹³ vg/kg. Multiple ratios of AAV-G6 to AAV-Cas9 were tested to determine whether there might be an optimal ratio of the viruses for maximal systemic editing efficiency. Four weeks after injection, dystrophin protein expression in several muscle tissues was assessed, including TA muscle of the hindlimb, triceps of the forelimb, diaphragm, and cardiac muscle. By immunostaining, dystrophin expression was observed in 94%, 90% and 95% of myofibers in the TA, triceps, and diaphragm, respectively, and in 94% of cardiomyocytes when AEx44 mice were injected with a 1 : 10 ratio of AAV- Cas9:AAVG6 (Fig. 29B and Fig. 68). The restoration of dystrophin protein in skeletal muscles correlated with the dosage of AAV-G6 delivered through IP injection. In contrast, in the heart, dystrophin positive cardiomyocytes were seen at a low dosage of AAV-G6 and remained consistent at higher dosages. Western blot analysis of the same muscle groups after systemic delivery showed similar trends of dystrophin correction (Fig. 29 A, and Fig. 69). At every ratio of AAV-Cas9:AAV-G6 tested by systemic delivery, cardiac muscle showed higher dystrophin restoration than skeletal muscle. Correction of cardiac muscle reached 82% when injected at a 1 : 1 ratio of AAV-Cas9:AAV-G6 and increased an additional 12% at a 1 : 10 ratio. In contrast, an increase of dystrophin-expressing hallmarks of muscular dystrophy, such as necrotic myofibers and regenerated fibers with central nuclei, were diminished in the TA, diaphragm, and triceps muscles at 4 weeks after AAV-Cas9/AAV-G6 delivery (Fig. 70 and Fig. 71). [0325] To further assess systemic delivery of AAV-Cas9 in the presence of different amounts of AAV-G6, Western blot analysis was performed to evaluate the amount of Cas9 protein expressed in the muscles. Although the total AAV-Cas9 dosage was kept constant (5 ^c 10¹³ vg/kg), the mice that received higher doses of AAV-G6 showed greater expression of Cas9 protein in corrected muscles (Fig. 29A and Fig. 69). qPCR analysis of the skeletal and cardiac muscle groups comparing the low doses and high doses of AAV-G6 also revealed increased Cas9 mRNA expression in the presence of high doses of AAV-G6 (Fig. 72). These results indicate that Cas9 expression is affected by the amount of sgRNA present, and thus sgRNA is limiting for optimal gene editing in vivo. These results also suggest that the extent of dystrophin restoration and muscle recovery may provide an environment that favors Cas9 expression.

[0326] To examine the effect of dystrophin restoration on muscle function in systemically corrected AEx44 DMD mice, electrophysiology was performed on EDL muscle of AEx44 DMD mice at 4 weeks post-injection with AAV-Cas9 and AAV-G6.

[0327] Rescue of maximal tetanic force was observed in the EDL of the corrected AEx44 DMD mice (Fig. 63 A). Improvement of muscle function correlated with increased dystrophin expression and decreased muscle degeneration and was associated with administration of increasing amounts of AAV-G6 relative to AAV-Cas9 (Fig. 73). For measurement of muscle- specific force, which is calibrated with the muscle cross sectional area, an increase in force from 59% to 89% was observed for a 1 :5 ratio and to 107% for a 1 : 10 ratio of AAV- Cas9:AAV-G6 in EDL of systemically corrected AEx44 DMD mice (Fig. 63B). This data demonstrates that systemic delivery of AAV-Cas9 and AAV-G6 efficiently restores dystrophin expression and improves muscle function in corrected AEx44 DMD mice, and the amount of sgRNA delivered to muscle is critical to the efficiency of genome editing in vivo.

Proof of Concept - Ratios of AAV-nuclease (e.g., Cas9) to AAV-gRNA may be varied to achieve optimal therapeutic efficacy.

[0328] The required ratio may vary by route of administration, type of tissue targeted (skeletal muscle, cadiac muscle or smooth muscle), or type of genetic modification

(refraining, exon skipping, and/or deletion) or any combination thereof.

[0329] In some embodiments of the disclosure, the amount of the AAV-gRNA delivered to the subject, the amount of the AAV-gRNA delivered to a target cell, the amount of AAV- gRNA expressed (including copy number) and/or the amount of gRNA operably linked to a nuclease may affect the activity of the nuclease. [0330] In some embodiments of the disclosure, the amount of the AAV -nuclease delivered to the subject, the amount of the AAV-nuclease delivered to a target cell, the amount of AAV- nuclease expressed (including copy number) and/or the amount of nuclease operably linked to a gRNA may affect the ability of a gRNA to selectively and specifically bind to a target sequence.

Dog Models of DMD

AEx50 Dog

Rationale

[0331] The AE50-MD dog model harbors a missense mutation in the 5’ donor splice site of exon 50 that results in deletion of exon 50 ((Walmsley et al, 2010)). Thus, this represents an ideal canine model for the investigation of gene-editing as an approach to permanently correct the most common DMD mutations in humans.

Recapitulation of the Human Condition

[0332] Expression of Cas9 and a single guide RNA (sgRNA) targeting a genomic sequence adjacent to the intron-exon junction of exon 51, using adeno-associated virus serotype 9 (AAV 9), creates refraining mutations and allows skipping of exon 51. This leads to highly efficient restoration of dystrophin expression in skeletal and cardiac muscles of these dogs. These results demonstrate for the first time the applicability of a relatively simple, but effectively permanent, gene editing strategy for preventing DMD progression in a large mammal.

Proof of Concept - Restoration of dystrophin expression in a large mammal by intramuscular injection of therapeutic composition.

[0333] Restoration of dystrophin expression in dystrophic dogs by a single genomic cut. To correct the dystrophin reading frame in the deltaE50-MD canine model (henceforth referred to as AEx50) (Fig. 41A), the inventors used S. pyogenes Cas9 coupled with a sgRNA to target a region adjacent to the exon 51 splice acceptor site (referred to as sgRNA-5l) (Fig. 41B).

The sgRNA-5l corresponded to a highly conserved sequence that differs by only one nucleotide between the human and dog genomes (Fig. 43 A, Table 4). The inventors evaluated the specificity of Cas9 activity by testing the sgRNA-5l sequences in human and dog cell lines. Cas9 coupled with each of these sgRNA-5l sequences only introduced a genomic cut in each respective species’ DNA, highlighting the specificity of CRISPR cutting (Fig. 43B). [0334] For the in vivo delivery of Cas9 and sgRNA-5l to skeletal muscle and heart tissue in dogs, the inventors used AAV9, which displays preferential tropism for these tissues. A muscle-specific creatine kinase (CK) regulatory cassette was used to drive expression of Cas9; three RNA polymerase III promoters (U6, Hl and 7SK) directed expression of the sgRNA, as described previously in mice (Fig. 3D).

[0335] Correction of dystrophin expression in a dog model of Duchenne muscular dystrophy by intramuscular delivery of Cas9 and sgRNA. AAV9-Cas9 and AAV9-sgRNA-51 were initially introduced into the cranial tibialis muscles of two 1 -month-old dogs by intramuscular (IM) injection with l.2xl0¹³ AAV9 viral genomes (vg) of each virus. Muscles were analyzed 6 weeks after injection. To evaluate dystrophin correction at the protein level, the inventors performed histological analysis of AAV9-injected cranial tibialis muscles 6 weeks after viral injection. Dystrophin immunohistochemistry of muscle from AEx50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l revealed widespread expression: the majority of fibers within the injected muscles expressed sarcolemmal dystrophin, albeit to varying levels (Fig. 41D). A considerable number of corrected fibers were detected in the uninjected contralateral muscles, far more than could be attributed to rare revertant events (typically represent fewer than 0.001% of fibers in AEx50 muscle). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle (Fig. 20A and 20B) to -60% of wildtype levels. Injected muscles also appeared markedly healthier via H&E staining, with fewer

hypercontracted or necrotic fibers, reduced edema and fibrosis, and fewer regions of inflammatory cellular infiltration (Fig. 21). Immunohistochemistry for develommental myosin heavy chain (dMHC), a marker of regenerating fibers, revealed a marked reduction in develommental myosin (dMHC)-positive fibers within injected muscles (Fig. 47).

[0336] Dystrophin nucleates a series of proteins into the dystrophin-associated glycoprotein complex (DCG) to link the cytoskeleton and extracellular matrix. In AEx50 mice, dogs, and DMD patients, these proteins are destabilized and fail to appropriately localize to the sub- sarcolemmal region. Muscles injected with AAV9-Cas9 and AAV9-sgRNA-5l showed recovery of the DCG protein beta-dystroglycan compared to contralateral uninjected muscles (Fig. 48). In conclusion, single-cut genomic editing using AAV9-Cas9 and AAV9-sgRNA-5l is highly efficient in restoration of dystrophin expression and assembly of the DGC in dystrophic muscles.

[0337] In vivo targeting efficiency was estimated within muscle biopsy samples by RT-PCR with primers for sequences in exons 48 and 53, and genomic PCR amplification products spanning the target site were subjected to amplicon deep-sequencing. The latter indicated that 9.96% of total reads contained changes at the targeted genomic site (Fig. 44). The most common identified mutations contained an adenosine (A) insertion immediately 3’ to the Cas9 genomic cutting site. The deletions identified using this method encompassed a highly- predicted exonic splicing enhancer (ESE) site for exon 51 (Fig. 44A). However, this method does not identify larger deletions that might occur beyond the annealing sites of the primers used for PCR. Since these tissue samples contain a mixture of muscle and non-muscle cells, the method probably under-estimates the actual efficiency of gene editing within muscle cells.

[0338] Sequencing of RT-PCR products of RNA from muscle of AEx50 dogs injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l showed that deletion of exon 51

(AEx50-5l) allowed splicing from exon 49 to 52, which restores the dystrophin open reading frame (Fig. 44B). On gels, the PCR product with the A insertion was indistinguishable in size from non-edited cDNA products, so the inventors performed deep sequencing analysis to quantify its abundance compared to other small insertions. Deep sequencing of the upper band containing the non-edited cDNA product and refrained cDNA products indicated that 73.19% of total reads contained refrained cDNA products with an A insertion, 26.81% contained non-edited cDNA product, and the rest contained small deletions and insertions (Fig. 44C). Taken together, our RT-PCR analysis revealed that AEx50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l had a high frequency of refraining events (with cDNA products containing an A insertion in the sequence of exon 51) and exon 51 skipping events resulting from deletion of the highly conserved ESE region.

Superior Property - no detectable off-target activity of the therapeutic composition following intramuscular delivery to a large mammal.

[0339] Defining the off-target activity of the Cas9 single-cut DNA strategy in vivo. To evaluate the specificity of our gene editing approach, the inventors analyzed predicted off- target genomic sites for possible promiscuous editing. A total of 3 potential genome-wide off target sites (OT1 to OT3) (Fig. 45) were predicted in coding exons and 4 in non-coding regions (Fig. 45) by the CRISPR design tool (http://crispr.mit.edu/). Deep sequencing was performed at the top predicted off-target sites within protein-coding exons. None of these sites revealed significantly more sequence alterations than the background analysis performed with other regions of the amplicons (Fig. 46). Proof of Concept - Dystrophin and muscle structure correction in a large mammal following systemic delivery.

[0340] Dystrophin and muscle structure correction in DEc50 dogs by systemic delivery of Cas9 and sgRNA. Based on the high dystrophin-correction efficiency observed following IM injection of AAV9-Cas9 and AAV9-sgRNA-5l, the inventors tested for rescue of dystrophin expression in DEc50 dogs following systemic delivery of gene editing components. Dogs at 1 month of age were injected with the viruses and analyzed 8 weeks later. The inventors tested two doses (2xl0¹³ vg/kg and 1x10¹⁴ vg/kg) of each of the two viruses AAV9-Cas9 and AAV9-sgRNA-5l. Systemic delivery of 2xl0¹³ vg/kg in AEx50-Dog-#2A allowed expression of virus in peripheral skeletal muscle samples, and to a lower extent in heart samples, as shown by qPCR analysis (Fig. 51A). The delivery of 1x10¹⁴ vg/kg of each virus (AAV9-Cas9 and AAV9-sgRNA-5l) in AEx50-Dog-#2B (via infusion) allowed more widespread expression of viral constructs in the peripheral skeletal nuscle samples and in heart samples (Fig. 51B). Systemic delivery of AAV9-Cas9 and AAV9-sgRNA-5l led to dystrophin expression in a broad range of muscles, including the heart, in gene-edited DEc50 dogs at 8 weeks post-injection, and to a markedly greater extent than that achieved with the lower dose (Fig. 30). Sequencing of RT-PCR products of the DEc50-51 band confirmed that exon 49 was spliced to exon 52. Western blot analysis confirmed the restoration of dystrophin expression in skeletal and heart muscles (FIG. 31). H&E staining of multiple skeletal muscles showed that histopathologic hallmarks of muscular dystrophy, such as necrotic myofibers, were also largely corrected 8 weeks after AAV9 delivery (FIG. 32).

Blood samples were collected the day before and then at least weekly for CK assessment (FIG. 33) the inventors did detect a modest decline in serum CK activity in treated dog with 1x10¹⁴ vg/kg.

[0341] To investigate the proportions of various indels generated by systemic delivery of

AAV9-Cas9 and AAV9-sgRNA-5l, the inventors performed amplicon deep-sequencing analysis of the genomic DNA from heart, triceps and biceps muscles. The genomic deep sequencing analysis revealed an increase of percentage of reads containing changes at the targeted genomic site, especially of the 1 A insertion mutation in the samples from Dog-#2B compared to Dog-#2A (FIG. 58). Proof of concept - efficacious gene editing in a variety of muscle cell types following systemic administration.

[0342] Additionally, the inventors performed TIDE analysis, which showed an increase in numbers of indels in the samples from Dog-#2B compared to the samples from Dog-#2A (FIG. 52). Testes analysis and western blot analysis showed no activity of Cas9 and confirmed muscle specific expression of gene editing machinery (FIG. 59). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle (FIG. 31 A and B) to levels -50%, 20%, 3% of wild-type levels for the cranial tibialis, triceps, and biceps, respectively, after systemic delivery of 2xl0¹³ vg/kg. For Dog-#2B, which received 1x10¹⁴ vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l (total virus 2xl0¹⁴ vg/kg), western blot analysis showed restoration of dystrophin expression (FIG. 31, C and D and FIG. 60) to levels -70%, 25%, 64%, 58%, 92% and 5% of wild-type levels for the cranial tibialis, triceps, biceps, diaphragm, heart and tongue muscles, respectively. Similar to IM injection, muscles appeared visibly healthier via H&E staining (Fig. 32). Immunostaining of muscle sections from treated AEx50 dogs also showed recovery of beta-dystroglycan expression (FIG. 53) and widespread reduction in markers of muscle regeneration (FIG. 54).

Proof of Concept - Adminstration of therapeutic compositions lead to

improvement of clinically-relevant behavior.

[0343] To evaluate the clinical improvement following systemic administration of AAV9-

Cas9 and AAV9-sgRNA-5l the inventors recorded the movement and behavior of the dogs in this study. AEx50 dogs of this age typically exhibit prominent pelvic limb paresis, displaying a distinctive“bunny -hopping” phenotype when walking and trotting. Additionally, AEx50 dogs demonstrate marked reluctance to jump or rear up. The untreated AEx50 dog displayed all these clinical signs. The AEx50 dog that received 2xl0¹³ vg/kg showed a mild

improvement of the“bunny -hopping” gait, while the dog receiving 1xl 0¹⁴ vg/kg displayed a dramatic improvement in movement, using the pelvic limbs in a manner comparable with healthy dogs, and moreover, readily jumping, rearing and running whilst playing, without apparent difficulty.

Superior Property - therapeutic compositions demonstrate low immunogenicity.

[0344] Assessment of immune response. To investigate possible immune responses, the inventors performed immunohistochemistry on sections from injected muscles, using canine- specific CD4 and CD8 T cell markers (Fig. 49). No evidence of an enhanced mononuclear cellular infiltration in the treated muscles was observed, compared to muscles from untreated DEc50 dogs. Additionally, hematological evaluation of treated dogs up to 6 weeks post injection (Fig. 50) revealed no significant abnormalities in comparison with untreated controls or reference ranges.

[0345] To determine T-cell reactivity to Cas9, antigen-induced T-cell secretion of IFN-g and

IL-2 was measured, as a marker of T-cell reactivity in peripheral blood mononuclear cells using a canine IFN-gamma/IL-2 Dual-Color ELISpot Kit. Blood samples were collected the day before injection and then at 1, 2, 4, 6 and 8 weeks post-injection for peripheral blood mononuclear cell (PBMC) isolation. No increase of immune response over time or compared to blood samples collected before the day of injection was seen (Fig. 55). Furthermore, hematological evaluation of treated dogs revealed no significant abnormalities in comparison with untreated controls or reference ranges (Fig. 56A). Biochemical and hematological evaluation of blood samples from all 4 dogs (healthy untreated, DEc50 untreated, and DEc50 dogs receiving 2xl0¹³ vg/kg and 1x10¹⁴ vg/kg) before and after injection were largely unremarkable (Fig. 56B): with hematology counts, serum electrolytes and kidney /liver function parameters remained within the normal ranges in all dogs. Additionally, blood samples were collected weekly for CK assessment: a modest decline in serum CK activity in Dog-#2B treated with 1x10¹⁴ vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l was observed (Fig. 33).

Proof of Concept - Correction of dystrophin expression in human derived iPS cells with a large deletion mutation.

[0346] As shown in Example 4, to evaluate the efficiency of the equivalent single cut strategy using sgRNA-5l to correct human DMD mutations, a DMD iPSC line carrying a deletion from exon 48 to 50 was used. Deletion of exons 48 to 50 leads to a frameshift mutation and appearance of a premature stop codon in exon 51. To correct the dystrophin reading frame, the inventors introduced Cas9 and sgRNA-5l into cells using nucleofection. Two concentrations of Cas9 and sgRNA were tested (26 ng/pl, referred to as high, and l3ng/pl, referred to as low). First, the inventors evaluated the DNA cutting activity of Cas9 coupled with the human sgRNA-5l sequence at different concentrations in DMD-iPSCs using the mismatch-specific T7 endonuclease I (T7E1) assay (Fig. 57A). Indel analysis showed 55.8% and 31.9% of indels for the high and low concentrations, respectively (Fig. 57B). Genomic deep-sequencing analysis revealed that 27.94% of mutations contained a single A insertion 3’ to the PAM sequence for the high concentration condition and 19.03% for the low concentration condition (Fig. 42 A), as observed in mouse and dog cells with a similar sgRNA directed against exon 51. The inventors also observed genomic sequences that contained deletions covering the splice acceptor site and ESE site for exon 51 (Fig. 42A).

[0347] Next, the inventors differentiated the mixed DMD iPSCs into induced cardiomyocytes (iCMs) to investigate the restoration of dystrophin protein by immunocytochemistry and Western blot analysis. The DMD-iCMs treated with Cas9 and sgRNA-5l were dystrophin positive (Fig. 42B). Dystrophin protein expression levels of the corrected DMD-iCMs were comparable to WT cardiomyocytes (67 to 100%) by Western blot analysis (Fig. 42C and 42D).

Human Cells

Rationale

[0348] As described in Example 2, patient-derived induced pluripotent stem cells (iPSCs) were generated from a DMD patient lacking exon 44 of the dystrophin gene {DMD) and from the patient’s brother as a heathy control (Fig. 61 A). Deletion of exon 44 (AEx44) disrupts the open reading frame of dystrophin by causing splicing of exon 43 to exon 45 and introducing a premature termination codon (Fig. 61B). The reading frame can be restored using

CRISPR/Cas9 gene editing by skipping exon 43, which allows splicing between exons 42 and 45, or by skipping exon 45, which allows splicing between exons 43 and 46. Alternatively, reframing of exon 43 or 45 can restore the protein reading frame by inserting one nucleotide (+3n+l insertion) or deleting two nucleotides (+3n-2 deletion).

Proof of Concept— Efficacious specific and selective gene editing in human cells.

[0349] sgRNAs that target the splice acceptor or donor sites for exon 43 and 45 were selected, thereby allowing splicing between surrounding exons to recreate in-frame dystrophin. For editing exon 43, four sgRNAs (Gl, G2, G3 and G4) directed against sequences near the 5’ and 3’ boundaries of the splice junctions of exon 43 (Fig. 61 C) were designed. For exon 45, it was observed that the intr on-exon junction of the splice acceptor site is contained within a 33-base pair (bp) region that is identical in the human and mouse genomes, allowing exon skipping strategies to be interchanged between the two species (Fig. 64A). Four sgRNAs (G5, G6, G7, and G8) were generated to target the 5’ boundary of exon 45, within the conserved region of the human and mouse genomes (Fig. 61D). By the mismatch-specific T7 endonuclease I (T7E1) assay, the sgRNAs were compared for their ability to direct Cas9-mediated gene editing in human 293 cells (Fig. 64B). Two out of four sgRNAs for exon 43 efficiently edited the targeted region, and all four sgRNAs for exon 45 generated precise cuts at the conserved region (Fig. 67C). The editing activity of the same four sgRNAs for exon 45 in mouse 10T1/2 cells was concurrently tested, and confirmed the effectiveness of the four sgRNAs in both the human and mouse genomes (Fig. 67C).

[0350] sgRNAs with the highest gene editing activity were then tested for the ability to efficiently edit the corresponding exons in patient-derived iPSCs lacking exon 44 (referred to as AEx44). A plasmid encoding optimized sgRNAs (G3 or G4 for exon 43, or G6 for exon 45) along with SpCas9 was introduced into AEx44 patient-derived iPSCs by electroporation, and the edited iPSCs were differentiated into cardiomyocytes (CMs). Dystrophin expression was assessed by Western blot analysis and immunostaining, confirming restoration of dystrophin protein expression in edited AEx44 iPSC-derived CMs (Fig. 61E and 61F). Levels of dystrophin protein expression in AEx44 iPSC-derived CMs edited with sgRNAs G4 and G6 were approximately comparable to those seen in healthy control iPSC-derived CMs (Fig. 61E).

Validation of AEx44 DMD mouse model.

[0351] Due to the high efficiency of editing and the complete conservation of sequence between human and mouse genomes, sgRNA G6 was used to derive single clones of AEx44 iPSCs that were edited within exon 45. Thirty-four single clones were isolated by flow cytometry and expanded. Sequence analysis of the clones showed exon skipping events in 3 out of 34 clones, and dystrophin refraining by either +3n+l or +3n-2 in 13 out of 34 clones (Fig. 64D). Western blot analysis confirmed the restoration of dystrophin expression in the three CRISPR/Cas9 corrected clones (Fig. 64E). Proof of Concept— Correction of dystrophin expression in human iPSCs with a large deletion.

[0352] As shown in Example 4, to evaluate the efficiency of the equivalent single cut strategy using sgRNA-5l to correct human DMD mutations, a DMD iPSC line carrying a deletion from exon 48 to 50 was used. Deletion of exons 48 to 50 leads to a frameshift mutation and appearance of a premature stop codon in exon 51. To correct the dystrophin reading frame, the inventors introduced Cas9 and sgRNA-5l into cells using nucleofection. Two concentrations of Cas9 and sgRNA were tested (26 ng/pl, referred to as high, and l3ng/pl, referred to as low). First, the inventors evaluated the DNA cutting activity of Cas9 coupled with the human sgRNA-5l sequence at different concentrations in DMD-iPSCs using the mismatch-specific T7 endonuclease I (T7E1) assay (Fig. 57A). Indel analysis showed 55.8% and 31.9% of indels for the high and low concentrations, respectively (Fig. 57B). Genomic deep-sequencing analysis revealed that 27.94% of mutations contained a single A insertion 3’ to the PAM sequence for the high concentration condition and 19.03% for the low concentration condition (Fig. 42 A), as observed in mouse and dog cells with a similar sgRNA directed against exon 51. The inventors also observed genomic sequences that contained deletions covering the splice acceptor site and ESE site for exon 51 (Fig. 42A).

Proof of Concept - Correction of dystrophin expression in human induced cardiomyocyte cells.

[0353] The inventors differentiated the mixed DMD iPSCs into induced cardiomyocytes (iCMs) to investigate the restoration of dystrophin protein by immunocytochemistry and Western blot analysis. The DMD-iCMs treated with Cas9 and sgRNA-5l were dystrophin positive (Fig. 42B). Dystrophin protein expression levels of the corrected DMD-iCMs were comparable to WT cardiomyocytes (67 to 100%) by Western blot analysis (Fig. 42C and 42D).

Translation to Human Therapy

[0354] As shown by the animal models and human cell lines provided by the disclosure, the therapeutic or pharmaceutical compositions described herein comprising at least one gRNA and at least one nuclease specifically and selectively target mutations in the DMD gene, effectively induce breaks in the target sequence, and by a variety of mechanisms (including reframing and/or exon skipping depending on the DMD mutation targeted), restore DMD expression and function. The therapeutic and/or pharmaceutical compositions described herein induce a reframing of the DMD gene and exon skipping to restore DMD gene expression in each of the mouse models, dog model and human cells lines. Accordingly, the proof of concept studies provided in this disclosure recapitulate the in vivo activity of these compositions when used as a human therapeutic. The compositions described herein are shown in vivo to have no detectable off-target activity, demonstrating that these compositions not only efficacious, but also safe. Moreover, the compositions described herein are shown in vivo to have no detectable immune response from a host with a functional immune system, demonstrating that these compositions not only efficacious, but also well-tolerated. Data presented herein demonstrate the composition of the disclosure are efficacious for treating and/or curing late-staged or advanced DMD in individuals with substantial muscle deterioration and functional losses.

[0355] Dosages provided herein may be scaled to human adults or to children of various ages using known equivalents, for example, as shown below in Table A or B (Reproduced from “Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), July 2005, Pharmacology and Toxicology):

5

10

5

10

EXAMPLES

[0356] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. EXAMPLE 1 - Exon 51 Skipping

Materials and Methods

[0357] Study Approval. All experimental procedures involving animals in this study were reviewed and approved by the University of Texas Southwestern Medical Center’s Institutional Animal Care and Use Committee.

[0358] CRISPR/Cas9-mediated exon 50 deletion in mice. Two single-guide RNA (sgRNA) specific to the intronic region surrounding exon 50 sequence of the mouse Dmd locus were cloned into vector px330 using the following primers: Dmd exon 50 F1 : 5’- CACCGAAATGATGAGTGAAGTTATAT-3’ (SEQ ID NO: 926); Dmd exon 50 R1 : 5’- AAAC ATATAACTTCACTCATCATTTC-3’ (SEQ ID NO: 927); Dmd exon 50 F2: 5’

CACCGGTTTGTTCAAAAGCGTGGCT-3’ (SEQ ID NO: 928); Dmd exon 50 R2: 5’-

AAAC AGCC ACGCTTTTGAACAAAC-3’ (SEQ ID NO: 929).

[0359] For the in vitro transcription of sgRNA, T7 promoter sequence was added to the sgRNA template by PCR using the following primers: Dmd exon 50 T7-F1 :

G A AT TGT A AT AC G AC T C AC T AT AGG A AT GAT G AGT G A AGT T AT AT (SEQ ID NO: 930); Dmd exon 50 T7-F2:

GAATTGTAATACGACTCACTATAGGGTTTGTTCAAAAGCGTGGCT (SEQ ID NO: 931); Dmd exon 50_T7-Rv: AAAAGCACCGACTCGGTGCCAC (SEQ ID NO: 932).

[0360] The gel purified PCR products were used as template for in vitro transcription using the MEGAshortscript T7 Kit (Life Technologies). sgRNA were purified by MEGAclear kit (Life Technologies) and eluted with nuclease-free water (Ambion). The concentration of guide RNA was measured by a NanoDrop instrument (Thermo Scientific). [0361] Plasmids. The pSpCas9(BB)-2A-GFP (PX458) plasmid containing the human codon optimized SpCas9 gene with 2A-EGFP and the backbone of sgRNA was purchased from

Addgene (Plasmid #48138). AAV TRIPSR plasmids were obtained from Dr. Dirk Grimm (Heidelberg University Hospital). Cloning of sgRNA was done using a Bbsl site. pGL3-CK8e plasmid was obtained from Dr. Stephen Hauschka (Department of Biochemistry, University of Washington, Seattle, USA). AAV-miniCMV-Cas9-shortPolyA plasmid was obtained from Dr. Dirk Grimm (Heidelberg University Hospital). To generate the final AAV9-CK8-CRISPR/Cas9 vector used in this manuscript, AAV-miniCMV-Cas9-short-PolyA was digested with Pad and Nhel enzyme to remove the miniCMV promoter. CK8 promoter was amplified from pGL3- CK8e plasmid using primers containing PacI and Nhel site sequence and cloned into digested vector to generate AAV-CK8-Cas9-shortPolyA plasmid.

[0362] sgRNA Identification and Cloning for skipping exon 51. Dmd exon 51 guide RNAs were defined using crispr.mit.edu. Guide sequences were cloned into Addgene plasmid #42230, a gift from Feng Zhang, using the following primers: Dmd exon 51 _F 1 : 5'- CACCGAGAGTAACAGTCTGACTGG -3’ (SEQ ID NO: 942); Dmd exon 51 R1 : 5'- AAACGTC AGACTGTTACTCTAGTGC-3 ' (SEQ ID NO: 943); Dmd exon 51 F2: 5'- CACCGCACTAGAGTAACAGTCTGAC -3' (SEQ ID NO: 944); Dmd exon 51 R2: 5'- AAACCC AGTC AGACTGTT ACTCTC -3' (SEQ ID NO: 945). Guide sequences were tested in culture using 10T1/2 cells before cloning into the AAV backbone.

[0363] Triplicate sgRNA assembly in AAV backbone using Golden Gate system. The assembly of the AAV TRISPR backbone cloning system relies on two consecutive steps of Golden Gate Assembly. First step assembly of gRNA into donor plasmid. Annealing of oligonucleotides is performed by heating a reaction containing 2.5 pl of each oligo (0.5 mM), 5 mΐ NEBuffer 2 (NEB) and 40 mE ddH₂0 to 95 °C for 5 minutes using heating block. For the assembly reaction into donor plasmid mix 40 fmol (-100 ng) destination backbone, 1 pL annealed, diluted oligos, 0.75 pL of Esp3I, 1 pL buffer tango (both Thermo Scientific), 1 pL of T4 DNA Ligase (400 U/pL) (NEB) as well as ATP and DTT at a final concentration of 1 mM in 10 pL total volume. Using a thermocycler, conduct 25 to 50 cycles of 37°C/3 min followed by 20 °C/5 minutes. Denature restriction enzyme and ligase by heating to 80 °C for 20 minutes. Use 3 pL of this reaction for transformation of chemo-competent bacteria, recover in SOC (37 °C,

800 rmm, 40 min) and spread on LB-Agar plates containing chloramphenicol (25 pg/mL). Annealed oligonucleotides encoding for the sgRNA are cloned into donor plasmids that carry the negative selection marker ccdB (to reduce background during cloning) as well as the

chloramphenicol resistance gene. To test the correct assembly the plasmid are sequenced using the primer D ono-R-5’ -GT AT GTT GT GT GGA ATT GT GAG-3’ (SEQ ID NO: 948). Second step is that three of these donor plasmids driving expression of one sgRNA under transcriptional control of U6, Hl or 7SK promoter are pooled in a second Golden Gate assembly along with a recipient plasmid that carries AAV ITRs. The assembly reaction will contain all four plasmids: donor plasmid-# l-U6-sgRNA, donor plasmid-#2-Hl -sgRNA, donor plasmid-#3-7SK-sgRNA and recipient plasmid containing the ITR. Digest with Bbsl will generate unique overhangs for each fragment (U6, Hl, 7SK, recipient backbone). During the ligation procedure, these overhangs anneal a circularized plasmid is only obtained, when the three cassettes match each other.

[0364] Serum creatine kinase (CK) measurement. Mouse serum CK was measured by the Metabolic Phenotyping Core at UT Southwestern Medical Center. Blood was collected from the submandibular vein and serum CK level was measured by VITROS Chemistry 7 Products CK Slides to quantitatively measure CK activity using VITROS 250 Chemistry System.

Results

[0365] A humanized model of DMD. The most common hot spot mutation region in DMD patients is the region between exon 45 to 51, and skipping of exon 51 could be used to treat the largest group (13-14%) of patients. To investigate CRISPR/Cas9-mediated exon 51 skipping in vivo , the inventors generated a mouse model that mimics the human“hot spot” region by deleting exon 50 using the CRISPR/Cas9 system directed by 2 sgRNAs (FIG. 1 A). The deletion of exon 50 was confirmed by DNA sequencing (FIG. 1B). Deletion of exon 50 placed the dystrophin gene out of frame leading to the absence of dystrophin protein in skeletal muscle and heart (FIGS. 1C-1E). Mice lacking exon 50 showed pronounced dystrophic muscle changes by 2 months of age (FIG. 1E). Serum analysis of delta-exon 50 mice showed a significant increase in creatine kinase (CK) levels, indicative of muscle damage (FIG. 1F). Taken together, dystrophin protein expression, muscle histology and serum CK levels validated the dystrophic phenotype of the AEx50 mouse model.

[0366] Restoration of dystrophin expression using a single cut strategy to skip exon 51. S. pyogenes Cas9 requires NAG/NGG as a PAM sequence to generate a double-strand DNA break. Interestingly, the universal splice acceptor and donor sites of exons contain NAG or NGG (FIG. 3B). Therefore, to correct the reading frame and dystrophin expression in the DEc50 mouse model, the inventors generated sgRNA that targeted splice acceptor and donor sites of exon 51 to delete it, thereby recreating the in-frame dystrophin protein. To test whether the sgRNA guides were able to efficiently cut, the inventors first evaluated their effectiveness in mouse and human cell lines (FIG. 5). To determine the most efficient way to correct the DMD reading frame, the inventors compared 2 different strategies: (1) double-guide strategy in which one copy of a first sgRNA targeting splice acceptor site (sgRNA-SA) and one copy of a second sgRNA targeting donor acceptor site (sgRNA-SD), were cloned into the rAAV9-sgRNA vector; (2) triplicate strategy in which the inventors cloned 3 copies of the same sgRNA (sgRNA-SA) into the rAAV9-sgRNA vector (FIG. 3C). Expression of each copy of sgRNA-SA was driven by a different RNA promoter (U6, Hl and 7SK). The inventors generated AAV-Cas9 using an AAV- Cas9 vector (CK8-Cas9-shortPolyA), which employs a CK8 promoter to drive expression of the humanized SpCas9 specifically in skeletal muscle and heart tissues. Following intra-muscular (IM) injection of P12 mice with AAVs, muscle tissues were analyzed. RT-PCR of RNA from DEc50 mice injected with AAV-Tri-SA and AAV-SA+SD showed that deletion of exon 51 (DEc50-51) allowed splicing from exon 49 to 52 (FIG. 2A, lower band). Sequencing of RT-PCR products of the DEc50-51 band confirmed that exon 49 spliced to exon 52 (FIG. 2B).

ETnexpectedly, the RT-PCR analysis showed that a single cut strategy using a triplicate version of sgRNA-SA (AAV-Tri-SA) is as efficient as using two sgRNAs - sgRNA-SA and sgRNA-SD (AAV-SA+SD).

[0367] To further assess the efficiency of the AAV-Tri-SA editing strategy, the inventors performed histological analysis of injected muscle to evaluate the number of fibers that express dystrophin throughout entire muscle sections. Interestingly, dystrophin immunostaining of muscle cryosections from mice injected with AAV-Tri-SA revealed significantly higher numbers of dystrophin-positive fibers (average of 43 ± 0.9%) compared to the muscle from DEc50 mice injected with AAV-SA+SD (average of 31 ± 0.1%) (FIGS. 3D-3E, FIG. 6). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle. (FIGS. 3F-3G). Hematoxylin and eosin (H&E) staining of muscle showed that histopathologic hallmarks of muscular dystrophy, such as necrotic myofibers, were diminished in TA muscle at 3-weeks post- AAV delivery (FIG. 4A). Quantitative analysis of the distribution of myofiber areas showed a clear increase in fiber size for both AAV-Tri-SA and AAV-SA-SD treated muscles compared to DEc50 muscles (FIG. 4B). However, AAV-Tri-SA treated muscles revealed a higher decrease in the frequency of small fibers (<500 mm) compared to AAV-SA+SD treated muscles. Together, these results demonstrate that targeting the splice acceptor site of exon 51 with one single cut using AAV-Tri-SA is highly efficient in restoring dystrophin expression in DMD. This approach has usefulness for many disorders that can be corrected by exon skipping.

[0368] Tailoring of the single DNA cut genome editing strategy. S. pyogenes Cas9 guided by sgRNAs binds to the targeted genomic locus next to a PAM and generates a double-strand DNA break (DSB) 3 nucleotides preceding the PAM sequence. To further assess the efficiency of the method by targeting the splice acceptor site, the inventors designed a second sgRNA triple guide construct (sgRNA-ex5l-SA2), targeting a region adjacent to the exon 51 splice acceptor site.

This gRNA uses a PAM sequence 3 nucleotides further into the exon in order to generate the DSB close to the splice acceptor site for exon 51 (FIG. 7A-FIG. 7B). Cutting in the vicinity of the splice acceptor region and within the exon sequence resulted in reframing events and exon skipping events. Moreover, designing the sgRNA in the exon sequence that shows higher conservation than intron sequence across species facilitates translation of the sgRNA to other species.

[0369] The DNA cutting activity of Cas9 coupled with sgRNA-ex5l-SA2 was evaluated in 10T1/2 mouse fibroblasts using the mismatch-specific T7 endonuclease 1 (T7E1) assay (FIG. 8A). To investigate the type of mutations generated by Cas9 coupled with sgRNA-5l-SA2, genomic deep-sequencing analysis was performed. The sequencing analysis revealed that 9.3% of mutations contained a single adenosine (A) insertion located 3 nucleotides 3’ of the PAM sequence. In addition, 7.3% of mutations contained deletions covering the splice acceptor site and a highly-predicted exonic splicing enhancer site for exon 51 (FIG. 8B). The sgRNA-ex5l- SA2 corresponds to a highly conserved region of the Dmd gene (FIGS. 8C-D), and the inventors tested the ability of Cas9 and human sgRNA-51 to cut the human Dmd locus in 293T cells. The T7E1 assay revealed clear cleavage at the predicted site (FIG. 8E). Similarly, sequence analysis revealed that Cas9 coupled with human sgRNA-ex5l-SA2 generated the same adenosine (A) insertion and a different range of deletions around the cleavage site (FIG. 8F).

[0370] For the in vivo delivery of Cas9 and sgRNA-ex5l-SA2 to skeletal muscle and heart tissue, adeno-associated virus 9 (AAV9) was used, which displays preferential tropism for these tissues. To further enhance muscle-specific expression, an AAV9-Cas9 vector (CK8e-Cas9- shortPolyA) was employed, which contains a muscle-specific CK regulatory cassette, referred to as the CK8e promoter, which is highly specific for expression in muscle and heart (FIG. 9A). Together, this 436 bp muscle-specific cassette and the 4101 bp Cas9 cDNA are within the packaging limit of AAV9. Expression of each sgRNA was driven by one of three RNA polymerase III promoters (U6, Hl and 7SK) (FIG. 9B).

[0371] Correction of the dystrophin reading frame in DEc50 mice by a single DNA cut.

The sgRNA-ex5l-SA2 was delivered to mice in triple copy (AAV-Tri-SA2), along with a Cas9 (AAV-Cas9), by intra-muscular (IM) injection. Following the injection, muscle tissues were analyzed. In vivo targeting efficiency was estimated by RT-PCR with primers for sequences in exons 48 and 53 and the T7E1 assay for the targeted genomic regions. To investigate whether efficient target cleavage was achieved, the inventors amplified a 771 bp region spanning the target site and analyzed it using the T7EI assay (FIG. 10 A). The activity of SpCas9 with the corresponding sgRNA was analyzed on the target site. T7EI assays revealed mutagenesis of the Dmd locus after delivery of AAV-Cas9 and AAV9-sgRNA-5l-SA2 (FIG. 10A). To investigate the type of mutations generated in DEc50 mice injected with Cas9 and sgRNA-expressing AAV9s, genomic PCR amplification products spanning the target site were analyzed by amplicon deep-sequencing analysis. Deep sequencing of the targeted region indicated that 27.9% of total reads contained changes at the targeted genomic site (FIG. 10B). On average, 15% of the identified mutations contained the same A insertion seen in mouse 10T1/2 and human 293 cells in vitro. The deletions identified using this method encompassed a highly-predicted exonic splicing enhancer site for exon 51 (FIG. 10B).

[0372] RT-PCR products of RNA from muscle of DEc50 mice injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l showed that deletion of exon 51 (DEc50-51) allowed splicing from exon 49 to 52 (FIG. 11 A, lower band). By sequencing RT-PCR products of the DEc50-51 band, it was confirmed that exon 49 was spliced to exon 52. To further define the mutations introduced by our gene editing strategy, RT-PCR amplification products from 4 samples were directly subjected to topoisomerase-based thymidine to adenosine (TOPO-TA) cloning without gel purification, then sequenced. Surprisingly, sequence analysis of 40 clones from each sample showed that in addition to exon 5 l-skipped cDNA products (DEc50-51) identified in 15% of sequenced clones, DEc50 mice injected with AAV9-Cas9 and AAV9- sgRNA-5l showed a high frequency of refraining events. Of sequenced clones, 63% contained a single nucleotide insertion in the sequence of exon 51 (FIGS. 11 B-C). The most dominant insertion mutation seen was an A insertion.

[0373] On gels, the A insertion was indistinguishable in size from non-edited cDNA products, so deep-sequencing analysis was performed to determine the abundance of this insertion compared to other small insertions. Deep-sequencing of the upper band containing the non-edited cDNA product and refrained cDNA products indicated that 69.22% of total reads contained reframed cDNA products with an A insertion, 17.71% contained non-edited cDNA product, and the rest contained small deletions and insertions (FIG. 11D). The deep-sequencing analysis of uninjected DEc50 mice confirmed that the A insertion is a result of Cas9-generated editing. These amplicon deep-sequencing results confirmed the results from TOPO-TA cloning and sequencing. Taken together, RT-PCR analysis revealed that DEc50 mice injected with AAV9-Cas9 and AAV9- sgRNA-5l-SA2 showed a high frequency of reframing events with cDNA products containing an A insertion in the sequence of exon 51 in addition to exon 51 skipping events resulting from deletion in a highly conserved exonic splicing enhancer region.

[0374] Restoration of dystrophin expression after intramuscular AAV9 delivery of Cas9 and sgRNA-51-SA2. Remarkably, dystrophin immunostaining of muscle cryosections from DEc50 mice injected with AAV-Tri-SA2 revealed significantly higher numbers of dystrophin positive fibers with an average of 99% restoration of normal fibers (FIG. 12A, FIG. 13). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle. (FIG. 12C, FIG. 12D). Hematoxylin and eosin (H&E) staining of muscle showed that histopathologic hallmarks of muscular dystrophy, such as necrotic myofibers, were corrected in TA muscle at 3- weeks post- AAV delivery (FIG. 12B and FIG. 14). This method, using a distinct sgRNA design, represents a major advance in efficiency of DMD correction with direct applicability to the patients with the most common dystrophin mutations.

[0375] Rescue of dystrophin expression following intramuscular injections of AAV9-Cas9 combined with different AAV9s expressing single copy or triple copy of sgRNA. To evaluate the benefit of triple promoter expression of sgRNA-ex51-SA2 in vivo , different constructs were investigated, where sgRNA expression was driven by a single RNA polymerase III promoter (EG6 or Hl or 7SK) and, separately, by three RNA polymerase III promoters (EG6,

Hl and 7SK) (FIG. 15A). The inventors delivered the sgRNA-ex5l-SA2 in single copy driven separately by the U6 promoter (AAV9-U6-sgRNA-5l-SA2), the Hl promoter (AAV9-H1- sgRNA-5l-SA2), the 7SK promoter (AAV9-7SK-sgRNA-5l-SA2) and triple copy (AAV9- Triple-sgRNA-5l-SA2). Following intra-muscular (IM) injection of P12 mice with AAV9s, muscle tissues were analyzed. Unexpectedly, dystrophin immunostaining of muscle cryosections from DEc50 mice injected with AAV9-Triple-sgRNA-5l-SA2 revealed significantly higher numbers of dystrophin-positive fibers with an average of 95% restoration of normal fibers compared to DEc50 mice injected with AAV9-U6-sgRNA-5l-SA2, AAV-Hl-sgRNA-5 l-SA2 and AAV-7SK-sgRNA-5l-SA2 with an average of 70%; 40% and 50% restoration of normal fibers respectively (FIGS. 15B).

[0376] Rescue of muscle structure and function following systemic delivery of AAV9-Cas9 and AAV9-sgRNA-51-SA2. Systemic delivery of AAV9-Cas9 and AAV9-sgRNA-5l-SA2 to P4 DEc50 mice yielded widespread dystrophin expression in the heart, triceps, tibialis anterior (TA) muscle, and diaphragm in gene-edited DEc50 mice at 4 and 8 weeks post-injection (FIG. 16A and FIG. 17A). Western blot analysis confirmed the restoration of dystrophin expression in skeletal and heart muscles (FIG. 16B and FIG. 17B). Grip strength testing also showed a significant increase in muscle strength of DEc50 mice at 4 weeks post-intraperitoneal AAV9 injection compared to DEc50 control mice (wildtype control 92.6± 1.63; DEc50 control

50.5±l.85; DEc50-A A V9-sgRNA-5 1 79.7±2.63) (FIG. 18A). Consistently, AAV9-sgRNA-51- SA2 gene-edited DEc50 mice also showed significant reductions in serum CK concentrations compared to DEc50 control mice (FIG. 18B).

[0377] Tailoring the AAV dose for single DNA cut genome editing strategy. To optimize the in vivo efficiency of gene editing, the inventors tested different doses and delivery strategies for AAV9-Cas9 and AAV9-sgRNA-5l. The inventors systemically delivered by intraperitoneal injection the AAV9-Cas9 and AAV9-sgRNA-5l to P4 DEc50 mice using 2 different doses (2.6xl0¹³ vg/kg of each AAV9 referred as Dose 1 and 6xl0¹² vg/kg of each AAV9 referred as

Dose 2). The systemic delivery yielded widespread dystrophin expression in the heart, triceps, tibialis anterior muscle, and diaphragm in gene-edited DEc50 mice at 4 weeks post-injection (FIG. 22A). Western blot analysis confirmed the restoration of dystrophin expression in muscles (FIG. 22B). The AAV dose 2 (6x10¹² vg/kg) lead to lower dystrophin correction in diaphragm and triceps muscle compared to AAV dose 1 (2.6xl0¹³ vg/kg), suggesting that the efficient AAV dose should not be lower than 2.6xl0¹³ vg/kg. [0378] Tailoring the AAV dosage ratio for single DNA cut genome editing strategy. To further optimize the in vivo efficiency of gene editing, the inventors tested different ratios of AAV9-Cas9 and AAV9-sgRNA-5l. The inventors delivered the AAV9-Cas9 and AAV9- sgRNA-ex5l in different doses and ratios (1 : 1 and 1 :2) using IP injection P4 DEc50 mice (FIG. 75) using the following conditions: 5xl0¹³vg/kg AAV9-Cas9 and 5xl0¹³vg/kg AAV9-sgRNA-5l

(Ex-5l-SA2 SEQ. No: 708), 5xl0¹³vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg AAV9-sgRNA-51 and 1xl0¹⁴vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg AAV9-sgRNA-5l. ETnexpectedly, dystrophin immunostaining of muscle cryosections from DEc50 mice injected mice injected with

5xl0¹³vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg (1 :2) of AAV9-Cas9 to AAV9-sgRNA-ex5l displayed better dystrophin correction then 5xl0¹³vg/kg AAV9-Cas9 and 5xl0¹³ vg/kg AAV9- sgRNA-5l. Moreover, dystrophin immunostaining of muscle cryosections from DEc50 mice injected mice injected with 5xl0¹³ vg/kg AAV9-Cas9 and 1xl0¹⁴vg/kg showed comparable correction to the mice injected with 1x10¹⁴ vg/kg AAV9-sgRNA-5l and 1x10¹⁴ vg/kg AAV9- Cas9 and 1x10¹⁴ vg/kg AAV9-sgRNA-51 (FIG. 75).

[0379] Evaluation of rescue of dystrophin expression following intravenous injections of

AAV9-Cas9 with AAV9-sgRNA-51 at later stages of DMD disease. To investigate in vivo efficiency of the gene editing and dystrophin correction at later stages of Duchenne muscular dystrophy disease, the inventors treated 1 month old DEc50 mice with AAV9-Cas9 and AAV9- sgRNA-5l. The inventors systemically delivered 2.6xl0¹³ vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l via a tail vein injection to 1 month old DEc50 mice. The systemic delivery resulted in widespread dystrophin expression in the heart, triceps, tibialis anterior muscle, and diaphragm in gene-edited DEc50 mice at 4 weeks and 8 weeks post-injection (FIG. 23A and FIG. 24A). Western blot analysis confirmed the restoration of dystrophin expression in muscles (FIG. 23B and FIG. 24B). Grip strength testing also showed a significant increase in muscle strength of DEc50 mice at 4 weeks and 8 weeks post-intravenous injection compared to DEc50 control mice (FIG. 23 C and FIG. 24C).

EXAMPLE 2 - Correction of an Exon 44 Mutation

[0380] Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in -12% of patients by skipping surrounding exons, which restores the dystrophin open reading frame. In this example, a simple and efficient strategy is presented for correction of exon 44 deletion mutations by CRISPR/Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, the importance of the dosages of these gene editing components for optimal gene correction in vivo is also demonstrated.

Materials and Methods

[0381] Study details. 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 was 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 grip strength tests, histology validation, immunostaining analysis, CK analysis, and muscle electrophysiology. For each experiment, sample size reflects the number of independent biological replicates and was provided in the figure legend.

[0382] Plasmids and cloning. The pSpCas9(BB)-2A-GFP (PX458) plasmid contained the human codon optimized SpCas9 gene with 2A-EGFP. pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138). Cloning of sgRNA was done using Bbs I sites. The sgRNAs in this study, listed in table 21, were selected using prediction of crispr.mit.edu. sgRNA sequences were cloned into PX458, then tested in tissue culture using HEK 293 and 10T cells. The AAV TRISPR-sgRNAs-CK8e-GFP plasmid contained three sgRNAs driven by the U6, Hl or 7SK promoters and GFP driven by the CK8e regulatory cassette. TRISPR backbone cloning system relies on two consecutive steps of the Golden Gate Assembly (New England Biolabs).

[0383] Human iPSCs maintenance and nucleofection. Human iPSCs were cultured in mTeSR TM1 media (STEMCELL Technologies) and passaged approximately every 4 days (1 : 12 to 1 : 18 split ratio depending on the cell lines). One hour before nucleofection, iPSCs were treated with 10 mM ROCK inhibitor (Y-27632) and dissociated into single cells using Accutase (Innovative Cell Technologies, Inc.). 1 x 10⁶ iPSCs were mixed with 5 pg of PX458-sgRNA-2A-GFP plasmid and nucleofected using the P3 Primary Cell 4D-Nucleofector X kit (Lonza) according to manufacturer's protocol. After nucleofection, iPSCs were cultured in mTeSR TM1 media supplemented with 10 mM ROCK inhibitor and changed to mTeSR TM1 media the next day. Three days post-nucleofection, media were changed into mTeSR TM1 media supplemented with 10 mM ROCK inhibitor and 100 pg/ml Primosin (InvivoGen) one hour before FACS sorting. GFP(+) and (-) cells were sorted by FACS and subjected to T7E1 assay. Single clones derived from GFP(+) iPSCs were picked and sequenced.

[0384] Mice. Mice were housed in a barrier facility with a l2-hour light/dark cycle and maintained on standard chow (2916 Teklad Global). DEc44 DMD mice were generated in the C57/BL6J background using the CRISPR/Cas9 system. Two sgRNAs specific to the intronic regions surrounding exon 44 of the mouse Dmd locus were cloned into vector PX458 (Addgene plasmid #48138) using the primers from Table 16. For the in vitro transcription of sgRNA, T7 promoter sequence was added to the sgRNA template by PCR using the primers from Table 17. The gel purified PCR products were used as template for in vitro transcription using the MEGAshortscript T7 Kit (Life Technologies). sgRNA were purified by MEGAclear kit (Life Technologies) and eluted with nuclease-free water (Ambion). The concentration of guide RNA was measured by a NanoDrop instrument (Thermo Scientific). AEx44 DMD mice were backcrossed with C57/BL6J mice for more than three generations. DEc44 DMD mice and WT littermates were genotyped using primers encompassing the targeted region from Table 18. Tail biopsies were digested in 100 pl of 25 mMNaOH, 0.2 mMEDTA (pH 12) for 20 min at 95 °C. Tails were briefly centrifuged followed by addition of 100 pl of 40 mM Tris-HCl (pH 5) and mixed to homogenize. Two milliliters of this reaction was used for subsequent PCR reactions with the primers in Table 18, followed by gel electrophoresis.

[0385] Genomic DNA isolation, PCR amplification and T7E1 analysis of PCR products. Genomic DNA of mouse 10T1/2 fibroblasts, mouse C2C12 myoblasts, human HEK 293 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 (Thermo Fisher Scientific) according to manufacturer’s protocol. Genomic DNA was PCR-amplified using GoTaq DNA polymerase (Promega) or with primers. PCR products were gel purified and 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 21. Mismatched duplex DNA was obtained by denaturing/renaturing of 25 mΐ of the genomic PCR product using the following conditions: 95 °C for 5 min, 95 °C to 85 °C (-2.0 °C /seconds), 85 °C to 25 °C (-0.1 °C /seconds), hold at 4 °C. Then 25 mΐ of the mismatched duplex DNA was incubated with 2.7 mΐ of 10X NEB buffer 2 and 0.3 mΐ of T7E1 (New England BioLabs) at 37 °C for 90 minutes. The T7E1 digested PCR product was analyzed by 2% agarose gel electrophoresis.

[0386] Human cardiomyocyte differentiation. Human iPSCs were cultured in mTeSR TM1 media for 3 days until they reached 90-95% confluence. To differentiate the iPSCs into cardiomyocytes, the cells were cultured in CDM3-C media for 2 days, followed by CDM3-WNT media for 2 days, followed by BASAL media for 6 days, followed by SELECTIVE media for 10 days and lastly by BASAL media for 2 days. Then, the cardiomyocytes were dissociated using TrypLExpress media and re-plated at 2 x 10⁶ cells per well in a 6-well dish. The contents of the differentiation medium can be found in the Table below. Media for iPSC-CMs differentiation

[0387] AAV9 delivery to DEc44 DMD mice. Before AAV9 injections, the DEc44 DMD mice were anesthetized. For intramuscular injection, the TA muscle of P12 male DEc44 DMD mice was injected with 50 mΐ of 22 AAV9 (1 x 10¹² vg/ml) preparations or with saline solution. For intraperitoneal injection, the P4 DEc44 DMD mice were injected using an ultrafme needle (31 gauge) with 80 mΐ of AAV9 preparations with a dosage of 5 x 10¹³ vg/kg of AAV-Cas9 and a corresponding ratio of AAV-G6 indicated in the figure legend or with saline solution.

[0388] Dystrophin Western blot analysis. For Western blot of iPSC-derived cardiomyocytes, 2 x 10⁶ cardiomyocytes were harvested and lysed with lysis buffer (10% SDS, 62.5 mM Tris pH6.8, 1 mM EDTA, and protease inhibitor). For Western blot of skeletal or heart 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 one. Protein concentration was determined by BCA assay and 50 pg of total protein was loaded onto a 4-20% acrylamide gel. Gels were run at 100V (20 mA) for 5 hours followed by 1 hour 20 min transfer to a PVDF membrane at 35V (200 mA) at 4°C. The blot was incubated with mouse anti-dystrophin antibody (MANDYS8, Sigma- Aldrich, D8168), anti-Cas9 antibody (Clone 7A9, Millipore, MAC133) at 4°C overnight and 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 antivinculin antibody (Sigma-Aldrich, V9131).

[0389] Amplicon deep-sequencing analysis. PCR of genomic DNA and cDNA from muscles was performed using primers designed against the respective target region and the top 10 off-target sites. A second round of PCR was used to add Illumina flow cell binding sequences and target specific barcodes on the 5’ end of the primer sequence. All primer sequences are listed in Table 24 and 25. Before sequencing, DNA libraries were analyzed using a Bioanalyzer High Sensitivity

DNA Analysis Kit (Agilent). Library concentration was then determined by qPCR using a KAPA Library Quantification Kit for Illumina platforms. The resulting PCR products were pooled and sequenced with 300 bp paired-end reads on an Illumina MiSeq instrument. Samples were demultiplexed according to assigned barcode sequences. FASTQ format data was analyzed using the CRISPResso software package version 1.0.8.

[0390] Histological analysis of muscles. Skeletal muscles from WT and DEc44 DMD mice were individually dissected and cryoembedded 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 -l55°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 MANDYS8 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.

[0391] Isolated EDL muscle preparation and electrophysiology stimulation. Muscles were surgically isolated from 4-week-old mice and mounted on Grass FT03.C force transducers connected to Powerlab 8/SP data acquisition unit (AD Instruments, Colorado Springs, CO), bathed in physiological salt solution at 37°C, and gassed continuously with 95% 02-5% CO2. After calibration, muscles were adjusted to initial length at which the passive force was 0.5 grams and then stimulated with two platinum wire electrodes to establish optimal length (Lo) for obtaining maximal isometric tetanic tension step by step following the protocol (at 150 Hz for 2 s). Specific force (mN/mm²) was calculated to normalize contraction responses to tissue cross-sectional area.

[0392] Statistics. All data are presented as mean ± S.E.M. One-way ANOVA was performed followed by Newman-Keuls post hoc test for multiple comparisons. Unpaired two-tailed Student’s t-tests were performed for comparison between the respective two groups (wild-type and DEc44 DMD mice, wild-type and DEc44 DMD-AAV9 treated mice, and DEc44 DMD control and DEc44 DMD-AAV9 treated mice). Data analyses were performed with statistical software (GraphPad Prism Software, San Diego, CA, ETSA). P values less than 0.05 were considered statistically significant.

[0393] Correction of a DMD exon 44 deletion in patient-derived iPSCs. Patient-derived induced pluripotent stem cells (iPSCs) were generated from a DMD patient lacking exon 44 of the dystrophin gene {DMD) and from the patient’s brother as a heathy control (Fig. 61A). Deletion of exon 44 (DEc44) disrupts the open reading frame of dystrophin by causing splicing of exon 43 to exon 45 and introducing a premature termination codon (Fig. 61B). The reading frame can be restored using CRISPR/Cas9 gene editing by skipping exon 43, which allows splicing between exons 42 and 45, or by skipping exon 45, which allows splicing between exons 43 and 46. Alternatively, refraining of exon 43 or 45 can restore the protein reading frame by inserting one nucleotide (+3n+l insertion) or deleting two nucleotides (+3n-2 deletion). sgRNAs that target the splice acceptor or donor sites for exon 43 and 45 were selected, thereby allowing splicing between surrounding exons to recreate in-frame dystrophin. For editing exon 43, four sgRNAs (Gl, G2, G3 and G4) directed against sequences near the 5’ and 3’ boundaries of the splice junctions of exon 43 (Fig. 61C) were designed. For exon 45, it was observed that the intron-exon junction of the splice acceptor site is contained within a 33-base pair (bp) region that is identical in the human and mouse genomes, allowing exon skipping strategies to be interchanged between the two species (Fig. 64A). Four sgRNAs (G5, G6, G7, and G8) were generated to target the 5’ boundary of exon 45, within the conserved region of the human and mouse genomes (Fig. 61D). By the mismatch- specific T7 endonuclease I (T7E1) assay, the sgRNAs were compared for their ability to direct Cas9-mediated gene editing in human 293 cells (Fig. 64B). Two out of four sgRNAs for exon 43 efficiently edited the targeted region, and all four sgRNAs for exon 45 generated precise cuts at the conserved region (Fig. 64B, C). The editing activity of the same four sgRNAs for exon 45 in mouse 10T1/2 cells was concurrently tested, and confirmed the effectiveness of the four sgRNAs in both the human and mouse genomes (Fig. 64C).

[0394] sgRNAs with the highest gene editing activity were then tested for the ability to efficiently edit the corresponding exons in patient-derived iPSCs lacking exon 44 (referred to as DEc44). A plasmid encoding optimized sgRNAs (G3 or G4 for exon 43, or G6 for exon 45) along with SpCas9 was introduced into DEc44 patient-derived iPSCs by electroporation, and the edited iPSCs were differentiated into cardiomyocytes (CMs). Dystrophin expression was assessed by Western blot analysis and immunostaining, confirming restoration of dystrophin protein expression in edited DEc44 iPSC-derived CMs (Fig. 61E and 61F). Levels of dystrophin protein expression in DEc44 iPSC-derived CMs edited with sgRNAs G4 and G6 were approximately comparable to those seen in healthy control iPSC-derived CMs (Fig. 61E).

[0395] Due to the high efficiency of editing and the complete conservation of sequence between human and mouse genomes, sgRNA G6 was used to derive single clones of DEc44 iPSCs that were edited within exon 45. Thirty-four single clones were isolated by flow cytometry and expanded. Sequence analysis of the clones showed exon skipping events in 3 out of 34 clones, and dystrophin refraining by either +3n+l or +3n-2 in 13 out of 34 clones (Fig. 64D). Western blot analysis confirmed the restoration of dystrophin expression in the three CRISPR/Cas9 corrected clones (Fig. 64E).

[0396] Generation of mice with a DMD exon 44 deletion. To optimize gene editing for correction of an exon 44 deletion in vivo , a mouse model bearing an exon 44 deletion in the Dmd gene was generated by CRISPR/Cas9 gene editing (Fig. 26A). Zygotes of C57BL/6 mice were injected with two sgRNAs that target the introns flanking exon 44, and the zygotes were injected into surrogate female mice (fig. 65). An F0 founder with a 957 bp deletion that eliminated exon 44 was chosen for further studies. These DEc44 DMD mice contain one of the most common deletions responsible for DMD in humans. In principle, correction of exon 44 deletions by gene editing could potentially restore the reading frame of dystrophin in -12% of DMD patients. Deletion of exon 44 was confirmed by RT-PCR analysis (Fig. 62A). Sequencing of the RT-PCR products using primers for sequences in exons 43 and 46 confirmed the removal of exon 44 in these mice (Fig. 62B). At 4 weeks of age, immunostaining of tibialis anterior (TA) muscle, diaphragm, and heart in the DEc44 DMD mice showed complete absence of dystrophin protein expression (Fig. 26F). Western blot analysis confirmed loss of dystrophin protein (Fig. 26E). Necrotic fibers, inflammatory infiltration, and regenerative fibers with centralized nuclei were observed in 4-week old DEc44 DMD mice, indicative of a severe muscular dystrophy phenotype (Fig. 26G). Serum creatine kinase levels in the DEc44 DMD mice were elevated 22-fold compared to WT littermates, similar to mdx mice, an established DMD mouse model (Fig. 26D). [0397] Shear force generated during muscle contraction leads to muscle membrane tearing in muscle lacking dystrophin, eventually causing myofiber degeneration and muscle fibrosis. Fibrotic tissue increases muscle stiffness and compromises contractility of muscles. To further analyze muscle function of DEc44 DMD mice, maximal tetanic force was measured in the extensor digitorum longus (EDL) muscle ex vivo. Compared with WT littermates at 4 weeks of age, DEc44 DMD mice showed an about 50% decrease in the specific and absolute tetanic force in the EDL muscle (Fig. 27, C and D). A similar decrease of muscle strength was observed by grip strength analysis in 8-week old DEc44 DMD mice (Fig. 27E).

[0398] Correction of DMD exon 44 deletion in mice by intramuscular AAV9 delivery of gene editing components. To deliver SpCas9 and sgRNA in vivo , AAV9 was utilized to package the gene editing components. AAV9 is a single-stranded DNA virus 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 was used, which combines key elements of the enhancer and promoter regions of the muscle creatine kinase gene to drive ripCas9 expression in skeletal muscle and heart. For delivery of sgRNA, three RNA polymerase III promoters (U6, Hl, and 7SK) were used to express three copies of a sgRNA (Fig. 9A).

[0399] First, the efficiency of the AAV construct was tested by comparing the editing efficiency of PX458 and AAV expression constructs encoding gene editing components that targeted exon 45 in mouse C2C12 muscle cells. By T7E1 assay, comparable editing efficiency was observed with both constructs (Fig. 66A). Among the two sgRNAs tested, G6 showed better cutting efficiency than G5, consistent with the observations in mouse 10T1/2 cells and human 293 cells (Fig. 64C).

[0400] To validate the efficacy of the single cut gene editing strategy in the DEc44 DMD mouse model, localized intramuscular (IM) injection of AAV9 expressing ripCas9 (AAV-Cas9) and AAV9 expressing sgRNA (AAV-G5 or AAV-G6) was performed in TA muscle of postnatal day 12 (P12) mice. As a control group, WT and DEc44 DMD mice were injected with AAV-Cas9 without AAV-sgRNA. In initial studies, 50 pl of AAV9 (1 x 10¹² vg/ml) was injected per leg, containing equal amounts of AAV-Cas9 and AAV9-G5 or AAV-G6. Three weeks after IM injection, TA muscles were collected for analysis. In vivo gene editing by AAV-G5 and AAV- G6 was compared by the T7E1 assay and RT-PCR of the targeted region (Fig. 28E and Fig.

66B). Gene editing with AAV-G6 showed higher efficiency based on DNA cutting in vivo (Fig. 66B). RT-PCR with primers that amplify the region from exon 43 to exon 46 revealed deletion of exon 45 in TA muscle injected with AAV-Cas9 and AAV-G6 (Fig. 28E). This allows exon 43 to skip exon 45 and directly splice to exon 46 when processing the pre-mRNA. As a result, the alternate mRNA enables the production of a truncated dystrophin protein in corrected TA muscle of DEc44 DMD mice.

[0401] 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. Sequencing results demonstrated that 7% of sequenced clones represented exon 45-skipped cDNA products, and 42% of sequenced clones contained a single adenosine (A) insertion in exon 45 that resulted in reframing of dystrophin protein (Fig. 28F, Fig. 28G, and Fig. 66B). The predominance of refraining explains the high abundance of the RT-PCR band at 355 bp and the lower abundance of the smaller RT-PCR product of 179 bp that reflects exon skipping (Fig. 28E).

[0402] Genomic and cDNA amplicon deep sequencing on the target region of the TA muscles with AAV-G6 IM injection also confirmed that 9.8% of mutations at the genomic level and

35.7% of mutations at the mRNA level contain a single A insertion at the cutting site after gene editing with AAV-G6 (Fig. 66C and 66D). This single A insertion leads to reframing of exon 45, and restores the dystrophin protein reading frame. Minor AAV integration events were also observed at the cutting site, with 0.2% at the genomic level (Fig. 66C), and 1.2% at the mRNA level (Fig. 66D). The integrated sequence is from the ITR region of AAV and prevents production of functional dystrophin protein from those transcripts and, thus, has neither positive nor negative effects on the dystrophic muscle phenotype.

[0403] To evaluate dystrophin protein restoration after IM injection with AAV-Cas9 and AAV- G5 or AAV-G6, we performed Western blot analysis on TA muscle and the heart (Fig. 28C). We observed restoration of dystrophin protein expression to 74% of the WT level in edited TA muscles of DEc44 DMD mice. Interestingly, although the injection was localized to the TA muscle, we observed expression of dystrophin in the heart at 21% of WT level (Fig. 28C). This suggests transfer of AAV into the circulation and delivery of the gene editing components to the heart. Immunostaining showed that dystrophin protein expression was restored in 99% of the myofibers in TA muscle injected with AAV-Cas9 and AAV-G6 (Fig. 28 A and Fig. 28 A).

Histological analysis and hematoxylin and eosin (H&E) staining showed a pronounced reduction in fibrosis, necrotic myofibers and regenerating fibers with central nuclei, indicating amelioration of the abnormalities associated with muscular dystrophy in the TA muscle 3 weeks after AAV9-Cas9 and AAV-G6 injection (Fig. 28B).

[0404] Based on CRISPR design tools (http://crispr.mit.edu/ and https://benchling.com/), the top 10 potential off-target sites were determined and, based on sequencing analysis, no off-target effects were detected at these sites (Fig. 67A-C). T7E1 analysis confirmed the absence of off- target cutting in the top 10 potential off target sites, and DNA sequencing of the isolated genomic PCR amplification products spanning the potential off-target sites confirmed the absence of sgRNA/Cas9-mediated mutations at the predicted sites (Fig. 67A). In addition, genomic amplicon deep sequencing of the top 10 predicted off-target sites within protein-coding exons was performed. None of these sites showed significant sequence alterations (Fig. 67B and 67C).

[0405] Systemic delivery of AAV9 expressing gene editing components rescues dystrophin expression in DEc44 mice. To achieve body -wide rescue of the disease phenotype in DEc44 DMD mice, AAV-Cas9 and AAV-G6 was delivered systemically by intraperitoneal (IP) injection. AAV- Cas9 was injected at a dosage of 5 ^c 10¹³ vg/kg. Multiple ratios of AAV-G6 to AAV-Cas9 were tested to determine whether there might be an optimal ratio of the viruses for maximal systemic editing efficiency. Four weeks after injection, dystrophin protein expression in several muscle tissues was assessed, including TA muscle of the hindlimb, triceps of the forelimb, diaphragm, and cardiac muscle. By immunostaining, dystrophin expression was observed in 94%, 90% and 95% of myofibers in the TA, triceps, and diaphragm, respectively, and in 94% of cardiomyocytes when DEc44 mice were injected with a 1 : 10 ratio of AAV-Cas9:AAVG6 (Fig. 29B and Fig. 68). The restoration of dystrophin protein in skeletal muscles correlated with the dosage of AAV-G6 delivered through IP injection. In contrast, in the heart, dystrophin positive cardiomyocytes were seen at a low dosage of AAV-G6 and remained consistent at higher dosages. Western blot analysis of the same muscle groups after systemic delivery showed similar trends of dystrophin correction (Fig. 29A, and Fig. 69). At every ratio of AAV-Cas9: AAV-G6 tested by systemic delivery, cardiac muscle showed higher dystrophin restoration than skeletal muscle. Correction of cardiac muscle reached 82% when injected at a 1 : 1 ratio of AAV-Cas9: AAV-G6 and increased an additional 12% at a 1 :10 ratio. In contrast, an increase of dystrophin-expressing hallmarks of muscular dystrophy, such as necrotic myofibers and regenerated fibers with central nuclei, were diminished in the TA, diaphragm, and triceps muscles at 4 weeks after AAV-Cas9/AAV-G6 delivery (Fig. 70 and Fig. 71).

[0406] To further assess systemic delivery of AAV-Cas9 in the presence of different amounts of AAV-G6, Western blot analysis was performed to evaluate the amount of Cas9 protein expressed in the muscles. Although the total AAV-Cas9 dosage was kept constant (5 ^c 10¹³ vg/kg), the mice that received higher doses of AAV-G6 showed greater expression of Cas9 protein in corrected muscles (Fig. 29A and Fig. 69). qPCR analysis of the skeletal and cardiac muscle groups comparing the low doses and high doses of AAV-G6 also revealed increased Cas9 mRNA expression in the presence of high doses of AAV-G6 (Fig. 72). These results indicate that Cas9 expression is affected by the amount of sgRNA present, and thus sgRNA is limiting for optimal gene editing in vivo. These results also suggest that the extent of dystrophin restoration and muscle recovery may provide an environment that favors Cas9 expression.

[0407] To examine the effect of dystrophin restoration on muscle function in systemically corrected DEc44 DMD mice, electrophysiology was performed on EDL muscle of AEx44 DMD mice at 4 weeks post-injection with AAV-Cas9 and AAV-G6.

[0408] Rescue of maximal tetanic force was observed in the EDL of the corrected AEx44 DMD mice (Fig. 63 A). Improvement of muscle function correlated with increased dystrophin expression and decreased muscle degeneration and was associated with administration of increasing amounts of AAV-G6 relative to AAV-Cas9 (Fig. 73). For measurement of muscle- specific force, which is calibrated with the muscle cross sectional area, an increase in force from 59% to 89% was observed for a 1 :5 ratio and to 107% for a 1 : 10 ratio of AAV-Cas9: AAV-G6 in EDL of systemically corrected DEc44 DMD mice (Fig. 63B). This data demonstrates that systemic delivery of AAV-Cas9 and AAV-G6 efficiently restores dystrophin expression and improves muscle function in corrected AEx44 DMD mice, and the amount of sgRNA delivered to muscle is critical to the efficiency of genome editing in vivo.

EXAMPLE 3 - Generation of a AEx50-KI-Luciferase reporter mouse, and in vivo

monitoring of correction of the dystrophin reading frame

[0409] To enable the non-invasive analysis of DMD correction strategies in vivo , the inventors introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. The inventors evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. These results provide a sensitive non-invasive means of monitoring of dystrophin correction in mouse models of DMD and provide a platform for testing different strategies for amelioration of DMD pathogenesis.

Materials and Methods

[0410] Study approval: All experimental procedures involving animals in this study were reviewed and approved by the University of Texas Southwestern Medical Center’s Institutional Animal Care and Use Committee.

[0411] Mice: Mice were housed in a barrier facility with a l2-hour light/dark cycle and maintained on standard chow (2916 Teklad Global). A single-guide RNA (sgRNA) specific to the exon 79 sequence of the mouse Dmd locus was cloned into vector px330 using the primers from Table 22. A donor vector containing the protease 2A and luciferase reporter sequence was constructed by incorporating short 5’ and 3’ homology arms specific to the Dmd gene locus.

[0412] To generate AEx50-Dmd-Luc mice 2 single-guide RNA (sgRNA) specific intronic regions surrounding exon 50 sequence of the mouse Dmd locus were cloned into vector px330 using the primers from Table 22. For the in vitro transcription of sgRNA, T7 promoter sequence was added to the sgRNA template by PCR using the primers from Table 22. The gel purified PCR products were used as template for in vitro transcription using the MEGAshortscript T7 Kit (Life Technologies). sgRNA was purified by MEGAclear kit (Life Technologies) and eluted with nuclease-free water (Ambion). The concentration of sgRNA was measured by a NanoDrop instrument (Thermo Scientific).

CRISPR/Cas9-mediated Homologous Recombination in Mice

[0413] Genotypins ofDmd-Luc and AEx50-Dmd-Luc Mice: WT-Dmd-Luc and AEx50-Dmd-Luc mice were genotyped using primers encompassing the targeted region shown in Table 22. Tail biopsies were digested in 100 pL of 25-mM NaOH, 0.2-mM EDTA (pH 12) for 20 min at 95 °C. Tails were briefly centrifuged followed by addition of 100 pL of 40-mM Tris-HCl (pH 5) and mixed to homogenize. Two microliters of this reaction were used for subsequent PCR reactions with the primers below, followed by gel electrophoresis. [0414] Plasmids: The pSpCas9(BB)-2A-GFP (PX458) plasmid containing the human codon optimized SpCas9 gene with 2A-EGFP and the backbone of sgRNA was purchased from

Addgene (Plasmid #48138). Cloning of sgRNA was performed using Bbs I site.

[0415] Cas9 Plasmid and sgRNA assembly in the AAV9 backbone: The AAV9-CK8-Cas9 vector has been previously described. Cloning of sgRNA in three copies under transcriptional control of three different promoters U6, Hl, or 7SK has also been previously described. To test for correct assembly, the plasmid was sequenced using the primer Dono-R-5’- GTATGTTGTGTGGAATTGTGAG-3’ (SEQ ID NO: 1064).

[0416] A A V9 strategy and delivery to AEx50-Dmd-Luc mice: Dmd exon 51 sgRNAs were selected using crispr.mit.edu. sgRNA sequences were cloned into px330 using primers in Table 22. sgRNAs were tested in tissue culture using 10T1/2 cells as previously described before cloning into the rAAV9 backbone. Prior to AAV9 injections, AEx50-Dmd-Luc mice were anesthetized by intraperitoneal (IP) injection of ketamine and xylazine anesthetic cocktail. For intramuscular (IM) injection, tibialis anterior (TA) muscle of P12 male AEx50-Dmd-Luc mice was injected with 50 mΐ of AAV9 (1E12 vg/ml) preparations. For IP injection, P4 AEx50-Dmd- Luc mice were injected using an ultrafme needle (31G) with 80 mΐ of 1E14 vg/kg for AAV9- Cas9 and 2E14 vg/kg AAV9-sgRNA.

[0417] Bioluminescence Imaging: Bioluminescence imaging was performed using the IVIS Spectrum Imaging System from Xenogen (Caliper Life Sciences). The hair was removed using Nair® hair removal lotion prior to imaging. The mice were anesthetized using 2% isoflurane and 100% oxygen with a flow rate of 2.5 L/min. Sterile D-luciferin at a concentration of 40 mg/mL was administered by intraperitoneal injection at 100 pL per mouse. The images were collected for thirty seconds at the maximum light collection. The images were saved for analysis. The image analysis was performed using Living Image 4.5.2 (Caliper Life Sciences). A manually - generated circle (ROI function) was placed upon the region of interest of the mouse.

Bioluminescence values are indicated as radiance (photons/cm Vs ¹).

[0418] Histolosical analysis of muscles: Histological analysis of muscles was performed according to standard methods.

[0419] Western blot analysis. Western blot was performed according to standard methods.

Antibodies to dystrophin (1 : 1000, D8168, Sigma-Aldrich), vinculin (1 : 1000, V9131, Sigma- Aldrich), luciferase (1 : 1000, Ab2l l76, Abeam), goat anti-mouse and goat-anti rabbit HRP- conjugated secondary antibodies (1 :3000, Bio-Rad) were used for the described experiments.

[0420] TIDE analysis: In the first step of tracking indels by decomposition (TIDE), RT-PCR products around the editing site from muscles were generated using primers designed against the respective target region (Table 22). The PCR products were then directly subjected to

sequencing. The sequencing results were analyzed using TIDE software package (tide.nki.nl). TIDE first aligns the sgRNA sequence to the control sequence to determine the position of the expected Cas9 break site. Then, the control sequence region upstream of the break site is aligned to the experimental sample sequence in order to determine any offset between the two sequence reads. Alignments were done using standard Smith-Waterman local alignment implemented in the BioStrings package in Bioconductor. The software uses the peak heights for each base, as determined by the sequence analysis software using 3730 Series Data Collection Software V4 and Sequencing Analysis Software V6. TIDE uses these peak heights to determine the relative abundance of aberrant nucleotides over the length of the whole sequence trace.

[0421] Statistics: Values are presented as mean ± S.E.M. Differences between respective groups were assessed using unpaired two-tailed Student’s t-tests. P<0.05 was regarded as significant. Statistical analysis was performed in Excel (Microsoft).

Results

[0422] Dystroyhin-Luciferase reporter mice: In an effort to facilitate the analysis of dystrophin correction strategies in vivo in a non-invasive way, reporter mice were generated by insertion of a Luciferase expression cassette into the 3’ end of the Dmd gene such that Luciferase would be translated in-frame with exon 79 of dystrophin (FIG. 34A). To avoid the possibility that

Luciferase might destabilize the dystrophin protein or perturb its various protein interactions, a protease 2A cleavage site was engineered between the proteins, allowing auto-catalytic cleavage and release from dystrophin after translation (FIG. 34A). The Dmd-Luciferase reporter line (WT- Dmd-Luc) was validated by DNA sequencing. Bioluminescence imaging of mice showed high, muscle-specificity of Luciferase expression (FIG. 34B).

[0423] The most prevalent hot spot region for dystrophin mutations in DMD patients lies between exons 45 and 51 where skipping of exon 51 could potentially correct the largest group of 13-14% of patients. The inventors deleted exon 50 in WT-Dmd-Luc mice using CRISPR/Cas9 with 2 single guide RNAs (sgRNAs) to create a reporter line of mice referred to as AEx50-Dmd- Luc (FIG. 34C). The deletion of exon 50 was confirmed by DNA sequencing (FIG. 38) and placed the dystrophin gene out of frame, preventing dystrophin protein expression in skeletal muscle and heart (FIG. 34D, 34E, 34F). Because expression of Luciferase is linked to the translation of dystrophin, the deletion of exon 50 prevents Luciferase expression in AEx50-Dmd- Luc mice (FIG. 34E). Residual background bioluminescence detected in these mice is likely attributable to the expression of a smaller isoform of dystrophin (Dp7l), which is expressed in tissues other than skeletal muscle. Dp7l isoform expression is initiated from a downstream promoter located in intron 62.

[0424] AEx50-Dmd-Luc mice showed pronounced dystrophic muscle with necrotic myofibers, fibrosis, and centralized myonuclei, indicative of degeneration and regeneration (FIG. 39). Thus, based on the absence of dystrophin protein expression and muscle histology, the AEx50-Dmd- Luc mice represent a faithful model of DMD.

[0425] In vivo noninvasive monitoring of dystrophin correction in AEx50-Dmd-Luc mice by a single DNA cut: To correct the dystrophin reading frame and evaluate the bioluminescence signal in AEx50-Dmd-Luc mice, an sgRNA targeting a region adjacent to the exon 51 splice acceptor site (referred to as sgRNA-5l) was used. For the in vivo delivery of Cas9 and sgRNA- 51 to skeletal muscle and the heart, the inventors used AAV9, which displays preferential tropism for these tissues. Muscle-specific expression of the AAV9-Cas9 vector was further ensured by incorporating the muscle creatine kinase (CK8e) promoter, which is highly specific for expression in muscle and heart. Expression of the sgRNA in a separate AAV9 vector was driven by three RNA polymerase III promoters (U6, Hl and 7SK).

[0426] Following intra-muscular (IM) injection of the left tibialis anterior (TA) muscle of AEx50-Dmd-Luc mice at postnatal day (P) 12 with a total of 5xl0¹⁰ AAV9 viral genomes (vg), muscles were analyzed by dystrophin immunostaining and bioluminescence for 4 weeks (FIG. 35 A). Bioluminescence signal was apparent in the injected leg within 1 week after injection and increased in intensity thereafter, ultimately reaching a level comparable to that of WT-Dmd-Luc mice within 4 weeks (FIG. 35B, 35E). Histological analysis of AAV9-injected TA muscle was performed to evaluate the number of fibers that expressed dystrophin and the correlation with the bioluminescence signal. Dystrophin immunohistochemistry of muscle from AEx50-Dmd-Luc mice injected with AAV9-Cas9 and AAV9-sgRNA-51 revealed restoration of dystrophin expression throughout the entire muscle (FIG. 35C, 35D). [0427] To further evaluate the sensitivity of the Luciferase reporter to in vivo , the inventors administered AAV9-Cas9 and AAV9-sgRNA-5l intraperitoneally to AEx50-Dmd-Luc mice at P4 and monitored the signal over time (FIG. 36A). Widespread bioluminescence was observed 3 weeks after injection and continued to increase to a level -70% of wild-type by 10 weeks. (Fig. 36B). Histological analysis revealed widespread dystrophin expression in the diaphragm, heart,

TA and triceps muscles of gene-edited AEx50-Dmd-Luc mice at 10 weeks post-injection (FIG. 36C). Western blot analysis revealed a close correlation between expression of Cas9, dystrophin and Luciferase in skeletal muscles and heart following systemic IP delivery of AAV9-encoded gene editing components to AEx50-Dmd-Luc mice (FIG. 37A, B).

[0428] In vivo targeting efficiency was assessed within muscle biopsies by tracking indels by decomposition (TIDE) analysis of RT-PCR products with primers for sequences in exons 48 and 53 (FIG. 40). TIDE analysis showed 68.6%, 87.08%, 29.6% and 66.5% frequencies of indels for diaphragm, heart, tibialis anterior and triceps muscle respectively (FIG. 40).

[0429] To further optimize the in vivo efficiency of gene editing, the inventors tested different ratios of AAV9-Cas9 and AAV9-sgRNA-5l. The inventors delivered the AAV9-Cas9 and

AAV9-sgRNA-ex51 in different ratios (1 : 1 and 1 :2) using intravenous injection in the tail vein of 1 month old D Ex 50-I)mc/-\ uci ferase mice (FIG. 25A). The in vivo bioluminescence analysis showed appearance of signal 2 weeks after injection. Unexpectedly, AEx50-/9/m/-luciferase mice injected with a 1 :2 ratio of AAV9-Cas9 to AAV9-sgRNA-ex5l displayed higher

bioluminescence signal than the mice injected with a 1 : 1 ratio of AAV9-Cas9 to AAV9-sgRNA- ex5l (FIG. 25B).

EXAMPLE 4 - Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

[0430] To further assess the efficiency and the therapeutic potential of this new approach, gene editing in AE50-MD dog model was investigated. The AE50-MD dog model harbors a missense mutation in the 5’ donor splice site of exon 50 that results in deletion of exon 50 ((Walmsley et al., 2010)). Thus, this represents an ideal canine model for the investigation of gene-editing as an approach to permanently correct the most common DMD mutations in humans. In this example, it is demonstrated that expression of Cas9 and a single guide RNA (sgRNA) targeting a genomic sequence adjacent to the intron-exon junction of exon 51, using adeno-associated virus serotype 9 (AAV9), creates reframing mutations and allows skipping of exon 51. This leads to highly efficient restoration of dystrophin expression in skeletal and cardiac muscles of these dogs.

These results demonstrate for the first time the applicability of a relatively simple, but effectively permanent, gene editing strategy for preventing DMD progression in a large mammal.

[0431] Restoration of dystrophin expression in dystrophic dogs by a single genomic cut. To correct the dystrophin reading frame in the deltaE50-MD canine model (henceforth referred to as DEc50) (Fig. 41 A), the inventors used S. pyogenes Cas9 coupled with a sgRNA to target a region adjacent to the exon 51 splice acceptor site (referred to as sgRNA-5l) (Fig. 41B). The sgRNA-5l corresponded to a highly conserved sequence that differs by only one nucleotide between the human and dog genomes (Fig. 43 A, Table 4). The inventors evaluated the specificity of Cas9 activity by testing the sgRNA-5l sequences in human and dog cell lines. Cas9 coupled with each of these sgRNA-51 sequences only introduced a genomic cut in each respective species’ DNA, highlighting the specificity of CRISPR cutting (Fig. 43B).

[0432] For the in vivo delivery of Cas9 and sgRNA-51 to skeletal muscle and heart tissue in dogs, the inventors used AAV9, which displays preferential tropism for these tissues. A muscle- specific creatine kinase (CK) regulatory cassette was used to drive expression of Cas9; three RNA polymerase III promoters (EG6, Hl and 7SK) directed expression of the sgRNA, as described previously in mice (Fig. 3D).

[0433] Correction of dystrophin expression in a dog model of Duchenne muscular dystrophy by intramuscular delivery of Cas9 and sgRNA. AAV9-Cas9 and AAV9-sgRNA-5l were initially introduced into the cranial tibialis muscles of two 1 -month-old dogs by intramuscular (IM) injection with l.2xl0¹³ AAV9 viral genomes (vg) of each virus. Muscles were analyzed 6 weeks after injection. To evaluate dystrophin correction at the protein level, the inventors performed histological analysis of AAV9-injected cranial tibialis muscles 6 weeks after viral injection. Dystrophin immunohistochemistry of muscle from DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA-5l revealed widespread expression: the majority of fibers within the injected muscles expressed sarcolemmal dystrophin, albeit to varying levels (Fig. 41D). A considerable number of corrected fibers were detected in the uninjected contralateral muscles, far more than could be attributed to rare revertant events (typically represent fewer than 0.001% of fibers in DEc50 muscle). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle (Fig. 20A and 20B) to -60% of wildtype levels. Injected muscles also appeared markedly healthier via H&E staining, with fewer hypercontracted or necrotic fibers, reduced edema and fibrosis, and fewer regions of inflammatory cellular infiltration (Fig. 21).

Immunohistochemistry for develommental myosin heavy chain (dMHC), a marker of

regenerating fibers, revealed a marked reduction in develommental myosin (dMHC)-positive fibers within injected muscles (Fig. 47).

[0434] Dystrophin nucleates a series of proteins into the dystrophin-associated glycoprotein complex (DCG) to link the cytoskeleton and extracellular matrix. In DEc50 mice, dogs, and DMD patients, these proteins are destabilized and fail to appropriately localize to the sub- sarcolemmal region. Muscles injected with AAV9-Cas9 and AAV9-sgRNA-5l showed recovery of the DCG protein beta-dystroglycan compared to contralateral uninjected muscles (Fig. 48). In conclusion, single-cut genomic editing using AAV9-Cas9 and AAV9-sgRNA-5l is highly efficient in restoration of dystrophin expression and assembly of the DGC in dystrophic muscles.

[0435] In vivo targeting efficiency was estimated within dog muscle biopsy samples by RT-PCR with primers for sequences in exons 48 and 53, and genomic PCR amplification products spanning the target site were subjected to amplicon deep-sequencing. The latter indicated that 9.96% of total reads contained changes at the targeted genomic site (Fig. 44). The most common identified mutations contained an adenosine (A) insertion immediately 3’ to the Cas9 genomic cutting site. The deletions identified using this method encompassed a highly -predicted exonic splicing enhancer (ESE) site for exon 51 (Fig. 44A). However, this method does not identify larger deletions that might occur beyond the annealing sites of the primers used for PCR. Since these tissue samples contain a mixture of muscle and non-muscle cells, the method probably under-estimates the actual efficiency of gene editing within muscle cells.

[0436] Sequencing of RT-PCR products of RNA from muscle of DEc50 dogs injected intramuscularly with AAV9-Cas9 and AAV9-sgRNA-5l showed that deletion of exon 51 (DEc50-51) allowed splicing from exon 49 to 52, which restores the dystrophin open reading frame (Fig. 44B). On gels, the PCR product with the A insertion was indistinguishable in size from non-edited cDNA products, so the inventors performed deep sequencing analysis to quantify its abundance compared to other small insertions. Deep sequencing of the upper band containing the non-edited cDNA product and refrained cDNA products indicated that 73.19% of total reads contained refrained cDNA products with an A insertion, 26.81% contained non-edited cDNA product, and the rest contained small deletions and insertions (Fig. 44C). Taken together, our RT-PCR analysis revealed that DEc50 dogs injected with AAV9-Cas9 and AAV9-sgRNA- 51 had a high frequency of reframing events (with cDNA products containing an A insertion in the sequence of exon 51) and exon 51 skipping events resulting from deletion of the highly conserved ESE region.

[0437] Defining the off-target activity of the Cas9 single-cut DNA strategy in vivo. To evaluate the specificity of our gene editing approach, the inventors analyzed predicted off-target genomic sites for possible promiscuous editing. A total of 3 potential genome-wide off target sites (OT1 to OT3) (Fig. 45) were predicted in coding exons and 4 in non-coding regions (Fig.

45) by the CRISPR design tool (http://crispr.mit.edu/). Deep sequencing was performed at the top predicted off-target sites within protein-coding exons. None of these sites revealed significantly more sequence alterations than the background analysis performed with other regions of the amplicons (Fig. 46).

[0438] Dystrophin and muscle structure correction in DEc50 dogs by systemic delivery of Cas9 and sgRNA. Based on the high dystrophin-correction efficiency observed following IM injection of AAV9-Cas9 and AAV9-sgRNA-51, the inventors tested for rescue of dystrophin expression in DEc50 dogs following systemic delivery of gene editing components. Dogs at 1 month of age were injected with the viruses and analyzed 8 weeks later. The inventors tested two doses (2xl0¹³ vg/kg and 1x10¹⁴ vg/kg) of each of the two viruses AAV9-Cas9 and AAV9- sgRNA-5l. Systemic delivery of 2xl0¹³ vg/kg in AEx50-Dog-#2A allowed expression of virus in peripheral skeletal muscle samples, and to a lower extent in heart samples, as shown by qPCR analysis (Fig. 51 A). The delivery of 1x10¹⁴ vg/kg of each virus (AAV9-Cas9 and AAV9- sgRNA-5l) in AEx50-Dog-#2B (via infusion) allowed more widespread expression of viral constructs in the peripheral skeletal nuscle samples and in heart samples (Fig. 51B). Systemic delivery of AAV9-Cas9 and AAV9-sgRNA-5l led to dystrophin expression in a broad range of muscles, including the heart, in gene-edited DEc50 dogs at 8 weeks post-injection, and to a markedly greater extent than that achieved with the lower dose (Fig. 30). Sequencing of RT-PCR products of the DEc50-51 band confirmed that exon 49 was spliced to exon 52. Western blot analysis confirmed the restoration of dystrophin expression in skeletal and heart muscles (FIG. 31). H&E staining of multiple skeletal muscles showed that histopathologic hallmarks of muscular dystrophy, such as necrotic myofibers, were also largely corrected 8 weeks after AAV9 delivery (FIG. 32). Blood samples were collected the day before and then at least weekly for CK assessment (FIG. 33) the inventors did detect a modest decline in serum CK activity in treated dog with 1xl0¹⁴vg/kg.

[0439] To investigate the proportions of various indels generated by systemic delivery of AAV9- Cas9 and AAV9-sgRNA-5l, the inventors performed amplicon deep-sequencing analysis of the genomic DNA from heart, triceps and biceps muscles. The genomic deep-sequencing analysis revealed an increase of percentage of reads containing changes at the targeted genomic site, especially of the 1 A insertion mutation in the samples from Dog-#2B compared to Dog-#2A (FIG. 58).

[0440] Additionally, the inventors performed TIDE analysis, which showed an increase in numbers of indels in the samples from Dog-#2B compared to the samples from Dog-#2A (FIG. 52). Testes analysis and western blot analysis showed no activity of Cas9 and confirmed muscle specific expression of gene editing machinery (FIG. 59). Western blot analysis confirmed the restoration of dystrophin expression in skeletal muscle (FIG. 31 A and B) to levels -50%, 20%, 3% of wild-type levels for the cranial tibialis, triceps, and biceps, respectively, after systemic delivery of 2xl0¹³ vg/kg. For Dog-#2B, which received 1x10¹⁴ vg/kg of each of AAV9-Cas9 and AAV9-sgRNA-5l (total virus 2xl0¹⁴ vg/kg), western blot analysis showed restoration of dystrophin expression (FIG. 31, C and D and FIG. 60) to levels -70%, 25%, 64%, 58%, 92% and 5% of wild-type levels for the cranial tibialis, triceps, biceps, diaphragm, heart and tongue muscles, respectively. Similar to IM injection, muscles appeared visibly healthier via H&E staining (Fig. 32). Immunostaining of muscle sections from treated DEc50 dogs also showed recovery of beta-dystroglycan expression (FIG. 53) and widespread reduction in markers of muscle regeneration (FIG. 54).

[0441] To evaluate the clinical improvement following systemic administration of AAV9-Cas9 and AAV9-sgRNA-5l the inventors recorded the movement and behavior of the dogs in this study. DEc50 dogs of this age typically exhibit prominent pelvic limb paresis, displaying a distinctive“bunny-hopping” phenotype when walking and trotting. Additionally, DEc50 dogs demonstrate marked reluctance to jump or rear up. The untreated DEc50 dog displayed all these clinical signs. The DEc50 dog that received 2xl0¹³ vg/kg showed a mild improvement of the “bunny-hopping” gait, while the dog receiving 1x10¹⁴ vg/kg displayed a dramatic improvement in movement, using the pelvic limbs in a manner comparable with healthy dogs, and moreover, readily jumping, rearing and running whilst playing, without apparent difficulty. [0442] Assessment of immune response. To investigate possible immune responses, the inventors performed immunohistochemistry on sections from injected muscles, using canine- specific CD4 and CD8 T cell markers (Fig. 49). No evidence of an enhanced mononuclear cellular infiltration in the treated muscles was observed, compared to muscles from untreated DEc50 dogs. Additionally, hematological evaluation of treated dogs up to 6 weeks post-injection (Fig. 50) revealed no significant abnormalities in comparison with untreated controls or reference ranges.

[0443] To determine T-cell reactivity to Cas9, antigen-induced T-cell secretion of IFN-g and IL- 2 was measured, as a marker of T-cell reactivity in peripheral blood mononuclear cells using a canine IFN-gamma/IL-2 Dual-Color ELISpot Kit. Blood samples were collected the day before injection and then at 1, 2, 4, 6 and 8 weeks post-injection for peripheral blood mononuclear cell (PBMC) isolation. No increase of immune response over time or compared to blood samples collected before the day of injection was seen (Fig. 55). Furthermore, hematological evaluation of treated dogs revealed no significant abnormalities in comparison with untreated controls or reference ranges (Fig. 56A). Biochemical and hematological evaluation of blood samples from all 4 dogs (healthy untreated, DEc50 untreated, and DEc50 dogs receiving 2xl0¹³ vg/kg and 1x10¹⁴ vg/kg) before and after injection were largely unremarkable (Fig. 56B): with hematology counts, serum electrolytes and kidney /liver function parameters remained within the normal ranges in all dogs. Additionally, blood samples were collected weekly for CK assessment: a modest decline in serum CK activity in Dog-#2B treated with 1x10¹⁴ vg/kg of each of AAV9- Cas9 and AAV9-sgRNA-5l was observed (Fig. 33).

[0444] Correction of dystrophin expression in human derived iPS cells with a large deletion mutation. To evaluate the efficiency of the equivalent single cut strategy using sgRNA-5l to correct human DMD mutations, a DMD iPSC line carrying a deletion from exon 48 to 50 was used. Deletion of exons 48 to 50 leads to a frameshift mutation and appearance of a premature stop codon in exon 51. To correct the dystrophin reading frame, the inventors introduced Cas9 and sgRNA-5l into cells using nucleofection. Two concentrations of Cas9 and sgRNA were tested (26ng/pl, referred to as high, and l3ng/pl, referred to as low). First, the inventors evaluated the DNA cutting activity of Cas9 coupled with the human sgRNA-51 sequence at different concentrations in DMD-iPSCs using the mismatch-specific T7 endonuclease I (T7E1) assay (Fig. 57A). Indel analysis showed 55.8% and 31.9% of indels for the high and low concentrations, respectively (Fig. 57B). Genomic deep-sequencing analysis revealed that 27.94% of mutations contained a single A insertion 3’ to the PAM sequence for the high concentration condition and 19.03% for the low concentration condition (Fig. 42A), as observed in mouse and dog cells with a similar sgRNA directed against exon 51. The inventors also observed genomic sequences that contained deletions covering the splice acceptor site and ESE site for exon 51 (Fig. 42A).

[0445] Next, the inventors differentiated the mixed DMD iPSCs into induced cardiomyocytes (iCMs) to investigate the restoration of dystrophin protein by immunocytochemistry and Western blot analysis. The DMD-iCMs treated with Cas9 and sgRNA-5l were dystrophin-positive (Fig. 42B). Dystrophin protein expression levels of the corrected DMD-iCMs were comparable to WT cardiomyocytes (67 to 100%) by Western blot analysis (Fig. 42C and 42D). Taken together, our results demonstrated the efficiency of sgRNA-51 in human DMD iCMs for the correction of dystrophin with a high frequency of refraining events containing an A insertion in the sequence of exon 51 in addition to exon 51 skipping events, as seen in the dog model.

Material and methods

[0446] Study design. The objective of the present study was to test the therapeutic potential of single cut gene editing in the deltaE50-MD (DEc50) dog model, which harbors a naturally- occurring missense mutation in the 5’ donor splice site of exon 50 that results in deletion of exon 50, as in our mice. Male dogs were used in all experiments. 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 non-blinded fashion.

[0447] Animals. Dogs were housed at the Royal Veterinary College, in large pens with daily human interaction and access to outdoor runs: conditions exceeding those in conditions that exceed the minimum stipulated by the ETC, Animal (Scientific Procedures) Act 1987 and according to local Animal Welfare Ethical Review Board approval. Carrier female Beagle (RCC strain)-cross (F3 generation) dogs derived from an original founder Bichon-Frise cross Cavalier King Charles Spaniel female were mated with male Beagles (RCC strain) to produce offspring. Adult dogs were group housed (12 hour light/dark cycle; l5-24°C) until females were close to whelping; thereafter, pregnant females (singly housed) were allowed to whelp naturally and all puppies within a litter (including those on trials) were kept with their mother in a large pen, to enable nursing with access to a bed under a heat lamp (~28°C). From 4 weeks of age, puppies were also allowed puppy feed (Burns) (ad lib) until weaning at 12 weeks, whereupon dogs not required for studies were rehomed.

[0448] Genotyping. Genotyping was performed on DNA isolated from cheek swabs, or from whole blood buffy coat extracts by PCR and sequencing of the mutation site and corroborated with measurement of serum CK activity within the first week post-natally.

[0449] Study approval. All experimental procedures involving animals in this study were conducted according to UK legislation, within a project license assigned under the Animal (Scientific Procedures) Act 1986 and approved by the local Animal Welfare Ethical Review Board.

[0450] sgRNA identification, evaluation and cloning. Dmd exon 51 sgRNAs were selected using crispr.mit.edu. These are listed in Table 22. sgRNA sequences were cloned into Addgene plasmid #48138, a gift from Feng Zhang. sgRNAs were tested in tissue culture using primary myoblasts and MDCK cells, as previously described, before cloning into the rAAV9 backbone.

[0451] Cas9 Plasmid and sgRNA assembly in AAV9 backbone. The AAV9-CK8-Cas9 vector has been previously described. Cloning of sgRNA in three copies under transcriptional control of three different promoters U6, Hl, or 7SK has been previously described. To test for correct assembly, the plasmid was sequenced using the primer Dono-R-5’- GTATGTTGTGTGGAATTGTGAG-3’ (SEQ ID NO: 1065).

[0452] AAV9 delivery to DEc50 dogs. For intramuscular (IM) injection, two 1 month old male

DEc50 dogs (~ 1.1 kg) were premedicated with methadone (0.2mg/kg, IV), induced (propofol 4mg/kg IV) and maintained under ventilated general anesthesia (sevoflurane in oxygen) with occasional IV boluses of 0.5ml/kg 50% glucose; the left cranial tibialis muscle was injected with AAV9 (1x10¹³ vg/ml) preparations in 4, equally separated sites along the length of the muscle belly (300pl per site). For the systemic injection of 2xl0¹³ vg/kg dose for each virus, a single 1 month old DEc50 dog was injected (cephalic vein), via a preplaced intravenous cannula, with 3ml of AAV9a preparations.

[0453] For the systemic injection of 1x10¹⁴ vg/kg each, a single 1 month old DEc50 dog was infused (via cannula) with 12 ml of the AAV9s preparation in a cephalic vein over 30 minutes. A short term immunosuppression regimen was implemented for the systemic 1 x10¹⁴ vg/kg study. Prednisolone was administered at a reducing dose, starting with 4mg/kg PO SID for 3 days before AAV treatment and continued at this dose for 1 week post-injection. Then, prednisolone administration was continued at 2mg/kg PO SID for 1 week; then 1 mg/kg PO SID for 1 week; then 0.5mg/kg SID for 1 week and 0.5mg/kg PO every other day for a week.

[0454] Tissue isolation. Muscles were collected rapidly after IV pentobarbital euthanasia, cut into l-2cm sections and mounted on cork blocks in cryoMbed (Bright) before freezing under liquid nitrogen cooled isopentane for histological and immunofluorescence analysis. Remaining unmounted portions of the muscles were snap frozen in liquid nitrogen and then ground to a powder under liquid nitrogen using a metal tissue pulverizer for extraction of DNA, RNA and protein. For intramuscular injections, muscles were analyzed at 6 weeks postinjection. For systemic rAAV9 administrations, muscle tissues were harvested and analyzed at 8 weeks post- injection.

[0455] Isolation of RNA and DNA. RNA was isolated from muscle using TRIzol (Life

Technologies) according to the manufacturer's instructions. DNA was isolated from muscle tissues using GeneJET genomic DNA purification kit (Thermo Fischer K0721) according to the manufacturer's instructions.

[0456] RNA analysis. cDNA was prepared by reverse transcription and used for conventional PCR for T7 analysis and deep sequencing, and qPCR for analysis of gene expression. Primer sequences are available on request.

[0457] Targeted deep DNA sequencing. PCR of genomic DNA and cDNA from muscles was performed using primers designed against the respective target region and off-target sites (Table 22). A second round of PCR was used to add Illumina flow cell binding sequences and experiment-specific barcodes on the 5’ end of the primer sequence (Table 22). Before sequencing, DNA libraries were analyzed using a Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent). Library concentration was then determined by qPCR using a KAPA Library Quantification Kit for Illumina platforms. The resulting PCR products were pooled and sequenced with 300 bp paired-end reads on an Illumina MiSeq instrument. Samples were demultiplexed according to assigned barcode sequences. FASTQ format data was analyzed using the CRISPResso software package version 1.0.8.

[0458] Histological analysis of muscles. Histological analysis of muscles was performed as described previously. Immunohistochemistry was performed by incubating 10 mm skeletal muscle cryosections with primary antibodies (mouse monoclonal Dys 2 (Novocastra NCL-Dys2) DY8/6C5, 1 :20; rat anti canine CD8 (BioRad MCA1039GA), 1 : 100; rat anti canine CD4

(BioRad MCA1038GA), 1 : 100; mouse monoclonal develommental myosin heavy chain

(Novocastra NCL-dMHC), 1 :20; rat anti perlecan (Thermofisher A7L6), 1 : 1000; and mouse monoclonal anti-P-dystroglycan (Novocastra 43DAG/8D5), 1 :20). For single label staining, biotinylated secondary antibodies (1 in 200; GE Healthcare) were applied for 30 minutes followed by streptavidin Alexa 594 (1 in 2000; Molecular Probes; Invitrogen) for a further 30 minutes. For dual labelling, Alexa488-conjugated goat anti-mouse and Alexa594-conjugated goat anti -rat (both 1 :1000, Molecular Probes; Invitrogen) were applied for 1 hour. Hoechst 33342, Trihydrochloride, Trihydrate (1 :5000, Thermo Fischer H3570) was added for nuclear labeling. Slides were mounted using Vectashield mounting solution.

[0459] Western blot analysis. Western blot was performed as described previously. Antibodies to dystrophin Dys2 (1 :40, NCL-DYS2, Novocastra), develommental myosin heavy chain (1 : 1000, NCL-dMHC, Novocastra), vinculin (1 : 1000, V9131, Sigma-Aldrich), goat anti-mouse and goat- anti rabbit HRP-conjugated secondary antibodies (1 :3000, Bio-Rad) were used for the described experiments.

[0460] Blood Analyses. Blood samples in EDTA and in plain tubes were collected the day before and then at 1, 2, 4, 6 and 8 weeks after injections for blood hematology and serum biochemistry analyses and peripheral blood mononuclear cell (PBMC) isolation. The hematology and biochemistry parameters were evaluated by Diagnostic Laboratories, Royal Veterinary College.

[0461] For Elispot analyses, PBMCs were isolated from ~lml blood. The collected blood was mixed with PBS (1 : 1, both room temperature) and layered using an equivalent Ficoll-paque gradient volume in l5ml falcon tubes and centrifuged for 40 min (400g, min acceleration with no brake). The plasma layer was then collected and frozen for additional measurements. The PBMC layer was collected and washed twice with 10ml wash medium (PBS supplemented 2% FCS). The pellet was collected for each wash by centrifugation (400g for 5min). The final pellet was resuspended in freezing medium (FCS supplemented 10% DMSO) and frozen. For the Elispot assay, the cells were defrosted at 37°C, then washed by dropwise addition of lml prewarmed media (RPMI 1640 supplemented with 10% FCS, pen/strep and L-glutamine) followed by rapid addition of a further 10 ml media. Cells were collected by centrifugation (400g, 5mins) and washed a second time. The final cell pellet was resuspended in an appropriate volume of media for preincubation (~l.5xl0⁵ in IOOmI) and then plated into the wells of a 96-well

polylysinecoated plate. The antigen (recombinant spCas9, ESPCas9-Pro-250G, Sigma-Aldrich) dissolved in media was added to the wells to yield a final concentration of 0.5, 5 and 50pg/ml. Untreated cells or cells for PMA/Ionomycin stimulation received an equivalent volume of media alone. Cells were incubated for 24 hours (37°C, 5% C02) to allow antigen-presenting cells to process the antigen. After preincubation, cells were collected by agitation of each well and harvesting of media containing resuspended cells. To ensure maximum cell recovery, a further 300m1 of media was added to each well and mixed by gentle pipetting before collection. This was then repeated. The final 700m1 of cell suspension was pelleted (400g, 5min) to remove any secreted IL2/yIFN and resuspended in 300m1 of fresh media to provide three replicate wells of ~5xl0⁴ cells per IOOmI well. Cells were plated out into a prewetted yIFN/IL2 dual-colour canine Elispot plate (Biotechne). Positive control phorbol l2-myristate 13- acetate (PMA)/ionomycin treated cells were plated in duplicate at ~5xl0⁴ cells per well, and a further duplicate at 2.5 x10⁴ cells per well to minimize risk of well saturation. A volume of 10m1 of 10x cell stimulation cocktail (PMA/Ionomycin) was added to each positive control well and the plate was incubated without agitation in a tissue culture incubator for 6 hours before developing. Plates were developed exactly as specified in the manufacturer’s instructions and analysed using an iSpot elispot reader (AID). Total spot count and red/blue counts (respectively) were tabulated.

[0462] Human iPSC maintenance, nucleofection, and differentiation. The DMD iPSC line Del was purchased from Cell Bank RIKEN BioResource Center (cell no. ITPS0164). The WT iPSC line was a gift from D. Garry (University of Minnesota). Human iPSCs were cultured in mTeSRTMl medium (STEMCELL Technologies) and passaged approximately every 4 days. One hour before nucleofection, iPSCs were treated with 10 mM ROCK inhibitor (Y-27632) and dissociated into single cells using Accutase (Innovative Cell Technologies Inc.). Cells (1 ^c 10⁶) were mixed with 26 ng/pl (referenced as high) or 13 ng/mΐ (referenced as low) of SpCas9-2A- GFP plasmid and nucleofected using the P3 Primary Cell 4D-Nucleofector X kit (Lonza) according to manufacturer’s protocol. After nucleofection, iPSCs were cultured in mTeSRTMl medium supplemented with 10 mM ROCK inhibitor, penicillin-streptomycin (1 :100) (Thermo Fisher Scientific), and primosin (100 pg/ml; InvivoGen). Three days after nucleofection, GFP+ and GFP- cells were sorted by fluorescence-activated cell sorting, as described above, and subjected to PCR and T7E1 assay. [0463] Cardiomyocyte differentiation and purification. iPSCs were adapted and maintained in TESR-E8 (STEMCELL Technologies) on 1 : 120 Matrigel in PBS-coated plates and passaged using EDTA solution (Versene, Thermo Fisher Scientific) twice weekly. For cardiac

differentiation, iPSCs were plated at 5 ^c 10⁴ to 1 ^c 10⁵ cells/cm² and induced with RPMI, 2% B27, 200 mM l-ascorbic acid-2-phosphate sesquimagnesium salt hydrate (Asc; Sigma- Aldrich), activin A (9 ng/ml; R&D Systems), BMP4 (5 ng/ml; R&D Systems), 5mM (for WT iPS line) and 6mM (for DMD iPS) CHIR99021 (Stemgent), and FGF-2 (5 ng/ml; Miltenyi Biotec) for 3 days; following another wash with RPMI medium, cells were cultured from days 4 to 13 with 5 mM IWP4 (Stemgent) in RPMI supplemented with 2% B27 and 200 mM Asc. Cardiomyocytes were metabolically purified by glucose deprivation from days 13 to 17 in glucose-free RPMI (Thermo Fisher Scientific) with 2.2 mM sodium lactate (Sigma- Aldrich), 100 mM b-mercaptoethanol (Sigma- Aldrich), penicillin (100 U/ml), and streptomycin (100 pg/ml).

[0464] 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. REFERENCES

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Claims

1. A composition comprising:

a first vector comprising a nucleic acid comprising:

a sequence encoding a first DMD guide RNA targeting a first genomic target sequence;

a sequence encoding a second DMD guide RNA targeting a second genomic target sequence;

a sequence encoding a first promoter, wherein the first promoter drives expression of the sequence encoding the first DMD guide RNA, and

a sequence encoding a second promoter, wherein the second promoter drives expression of the sequence encoding the second DMD guide RNA, and a second vector comprising a nucleic acid comprising:

a sequence encoding a Cas9 or a nuclease domain thereof;

a sequence encoding a muscle-specific promoter, wherein the muscle-specific promoter drives expression of the sequence encoding a Cas9 or a nuclease domain thereof;

wherein a ratio of the first vector to the second vector in the composition is between 1 : 1 and 1 :30.

2 The composition of claim 1, wherein the ratio of the first vector to the second vector is

1 : 1.

3. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :2.

4. The composition of claim 1, wherein the ratio of the first vector to the second vector is

1 :3.

5. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :4.

6. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :5.

7. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :6.

8. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :7.

9. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :8.

10. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :9.

11. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 : 10.

12. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 : 15.

13. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :20.

14. The composition of claim 1, wherein the ratio of the first vector to the second vector is 1 :25.

15. The composition of any one of claims 1-14, wherein the first vector comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence; and at least one sequence encoding an additional promoter, wherein the at least one additional promoter drives expression of the at least one sequence encoding an additional DMD guide RNA.

16. The composition of any one of claims 1-15, wherein the first vector or the second vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).

17. The composition of any one of claims 1-15, wherein the first vector and the second vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).

18. The composition of any one of claims 1-17, wherein the sequence encoding the muscle- specific promoter comprises or consists of a sequence encoding a CK8 promoter.

19. The composition of any one of claims 1-18, wherein the sequence encoding the muscle- specific promoter comprises or consists of a sequence encoding a CK8e promoter.

20. The composition of any one of claims 1-18, wherein the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714.

21. The composition of any one of claims 1-18, wherein the sequence encoding the first DMD guide RNA and the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714.

22. The composition of any one of claims 15-21, wherein the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714.

23. The composition of any one of claims 16-22, wherein the composition comprises between 5 x 10¹¹ viral genomes (vg)/kilogram (kg) and 1 x 10¹⁵ vg/kg, inclusive of the endpoints, of the first vector.

24. The composition of any one of claims 16-22, wherein the composition comprises at least 5 x 10¹² viral genomes (vg)/kilogram (kg) of the first vector.

25. The composition of any one of claims 16-22, wherein the composition comprises at least 1 x 10¹³ viral genomes (vg)/kilogram (kg) of the first vector.

26. The composition of any one of claims 16-22, wherein the composition comprises at least 5 x 10¹³ viral genomes (vg)/kilogram (kg) of the first vector.

27. The composition of any one of claims 16-22, wherein the composition comprises at least 1 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector.

28. The composition of any one of claims 16-22, wherein the composition comprises at least 5 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector.

29. The composition of any one of claims 16-22, wherein the composition comprises at least 1 x 10¹⁵ viral genomes (vg)/kilogram (kg) of the first vector.

30. The composition of any one of claims 16-22, the ratio of the first vector to the second vector is 1 : 1 and wherein the composition comprises at least 5 x 10¹¹ vg/kg of the first vector and at least 5 x 10¹¹ vg/kg of the second AAV vector.

31. The composition of any one of claims 16-22, wherein the ratio of the first vector to the second vector is 1 : 1 and wherein the composition comprises at least 5 x 10¹³ vg/kg of the first vector and at least 5 x 10¹³ vg/kg of the second AAV vector.

32. The composition of any one of claims 16-22, wherein the ratio of the first vector to the second vector is 1 :2 and wherein the composition comprises at least 5 x 10¹³ vg/kg of the first vector and at least 1 x 10¹⁴ vg/kg of the second AAV vector.

33. The composition of any one of claims 16-22, wherein the ratio of the first vector to the second vector is 1 : 1 and wherein the composition comprises at least 1 x 10¹⁴ vg/kg of the first vector and at least 1 x 10¹⁴ vg/kg of the second AAV vector.

34. The composition of any one of claims 16-22, wherein the ratio of the first vector to the second vector is 1 :2 and wherein the composition comprises at least 1 x 10¹⁴ vg/kg of the first vector and at least 2 x 10¹⁴ vg/kg of the second AAV vector.

35. The composition of any one of claims 1-34, further comprising a pharmaceutically- acceptable carrier.

36. A composition comprising:

a first vector comprising a nucleic acid comprising:

a sequence encoding a Cas9 or a nuclease domain thereof;

wherein a ratio of the first vector to the second vector in the composition is between 30: 1 and 1 : 1.

37. The composition of claim 36, wherein the ratio of the first vector to the second vector is 1.7:1.

38. The composition of claim 36, wherein the ratio of the first vector to the second vector is 2: 1.

39. The composition of claim 36, wherein the ratio of the first vector to the second vector is

3: 1.

40. The composition of claim 36, wherein the ratio of the first vector to the second vector is 4: 1.

41. The composition of claim 36, wherein the ratio of the first vector to the second vector is 5: 1.

42. The composition of claim 36, wherein the ratio of the first vector to the second vector is 6: 1.

43. The composition of claim 36, wherein the ratio of the first vector to the second vector is 7: 1.

44. The composition of claim 36, wherein the ratio of the first vector to the second vector is 8: 1.

45. The composition of claim 36, wherein the ratio of the first vector to the second vector is 9: 1.

46. The composition of claim 36, wherein the ratio of the first vector to the second vector is 10: 1.

47. The composition of claim 36, wherein the ratio of the first vector to the second vector is 15: 1.

48. The composition of claim 36, wherein the ratio of the first vector to the second vector is 20: 1.

49. The composition of claim 36, wherein the ratio of the first vector to the second vector is 25: 1.

50. The composition of any one of claims 36-49, wherein the first vector comprises at least one sequence encoding an additional DMD guide RNA targeting a genomic target sequence; and at least one sequence encoding an additional promoter, wherein the at least one additional promoter drives expression of the at least one sequence encoding an additional DMD guide RNA.

51. The composition of any one of claims 36-50, wherein the first vector or the second vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).

52. The composition of any one of claims 36-50, wherein the first vector and the second vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).

53. The composition of any one of claims 36-52, wherein the sequence encoding the muscle- specific promoter comprises or consists of a sequence encoding a CK8 promoter.

54. The composition of any one of claims 36-53, wherein the sequence encoding the muscle- specific promoter comprises or consists of a sequence encoding a CK8e promoter.

55. The composition of any one of claims 36-54 wherein the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 60-371, SEQ ID NO: 383-694, SEQ ID NO: 682- 697, SEQ ID NO: 715-717, SEQ ID NO: 790-862, SEQ ID NO: 1036-1051, SEQ ID NO: 1066- 1380, SEQ ID NO: 1392-1467, or SEQ ID NO: 1484-1491.

56. The composition of any one of claims 36-55, wherein the sequence encoding the first DMD guide RNA or the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039.

57. The composition of any one of claims 36-55, wherein the sequence encoding the first DMD guide RNA and the sequence encoding the second DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039.

58. The composition of any one of claims 36-57, wherein the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 60-371, SEQ ID NO: 383-694, SEQ ID NO: 682-697, SEQ ID NO: 715-717, SEQ ID NO: 790- 862, SEQ ID NO: 1036-1051, SEQ ID NO: 1066-1380, SEQ ID NO: 1392-1467, or SEQ ID NO: 1484-1491.

59. The composition of any one of claims 36-57, wherein the sequence encoding the additional DMD guide RNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 714, SEQ ID NO: 762, SEQ ID NO: 1039.

60. The composition of any one of claims 36-59, wherein the composition comprises between 5 x 10¹¹ viral genomes (vg)/kilogram (kg) and 1 x 10¹⁵ vg/kg, inclusive of the endpoints, of the first vector.

61. The composition of any one of claims 36-60, wherein the composition comprises at least 5 x 10¹² viral genomes (vg)/kilogram (kg) of the first vector.

62. The composition of any one of claims 36-60, wherein the composition comprises at least 1 x 10¹³ viral genomes (vg)/kilogram (kg) of the first vector.

63. The composition of any one of claims 36-60, wherein the composition comprises at least 5 x 10¹³ viral genomes (vg)/kilogram (kg) of the first vector.

64. The composition of any one of claims 36-60, wherein the composition comprises at least 1 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector.

65. The composition of any one of claims 36-60, wherein the composition comprises at least 5 x 10¹⁴ viral genomes (vg)/kilogram (kg) of the first vector.

66. The composition of any one of claims 36-60, wherein the composition comprises at least 1 x 10¹⁵ viral genomes (vg)/kilogram (kg) of the first vector.

67. The composition of any one of claims 36-66, further comprising further comprising a pharmaceutically-acceptable carrier.

68. A method of treating muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-66.

69. The method of claim 68, wherein the composition is administered locally.

70. The method of claim 68 or 69, wherein the composition is administered directly to a muscle tissue.

71. The method of any one of claims 68-70, wherein the composition is administered by an intramuscular infusion or injection.

72. The method of any one of claim 70 or 72, wherein the muscle tissue comprises a tibialis anterior tissue, a quadriceps tissue, a soleus tissue, a diaphragm tissue, or a heart tissue.

73. The method of any one of claim 68-72, wherein the composition is administered by intra cardiac injection.

74. The method of claim 73, wherein the composition is administered systemically.

75. The method of claim 74, wherein the composition is administered by an intravenous infusion or injection.

76. The method of any one of claims 68-75, wherein the subject is a neonate, an infant, a child, a young adult, or an adult.

77. The method of any one of claims 68-76, wherein the subject has muscular dystrophy.

78. The method of any one of claims 68-77, wherein the subject is a genetic carrier for muscular dystrophy.

79. The method of any one of claims 68-78, wherein the subject is male.

80. The method of any one of claims 68-78, wherein the subject is female.

81. The method of any one of claims 68-79, wherein the subject is an adult.

82. The method of claim 81, wherein the adult is at least 18 years old.

83. The method of claim 81, wherein the adult is at least 25 years old.

84. The method of claim 81, wherein the adult is at least 20 kg.

85. The method of any one of claims 68-79, wherein the subject is a child.

86. The method of claims 85, wherein the child is less than 18 years of age.

87. The method of claims 85, wherein the child is 20 kg or less.

88. The method of any one of claims 68-79, wherein the subject is an infant.

89. The method of claim 88, wherein the subject is less than 2 years old.

90. The method of any one of claims 68-89, wherein upon administering the therapeutically effective amount of the composition, the subject produces a minimal immune response to the composition.

91. The method of claim 90, wherein the minimal immune response to the composition is reduced or eliminated treatment with an anti-inflammatory agent or an immune suppressive agent.

92. The method of any one of claims 68-91, wherein the composition does not induce breaks in a predicted alternative targeting site.

93. The method of claim 92, wherein the predicted alternative targeting site comprises a coding sequence of the human genome and wherein the coding sequence comprises at least 2 mismatches with respect to the first genomic target sequence or the second genomic target sequence.

94. The method of claim 93, wherein the predicted alternative targeting site comprises a coding sequence of the human genome and wherein the coding sequence comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 mismatches with respect to the first genomic target sequence or the second genomic target sequence.

95. The method of any one of claims 68-94, wherein the administration of the therapeutically effective amount of the composition is provided as a single dose or provided within a single medical procedure.

96. The method of any one of claims 68-94, wherein the administration of the therapeutically effective amount of the composition is provided as multiple doses or provided over multiple medical procedures.