Microdystrophin gene therapy administration for treatment of dystrophinopathies

Abstract

Provided are methods of treating or ameliorating the symptoms of dystrophinopathies, such as Duchenne muscular dystrophy and Becker muscular dystrophy by administration of therapeutically effective doses of recombinant adeno-associated viruses (rAAV) containing a transgene encoding a microdystrophin comprising a CT domain.

Description

Atty. Docket No.38013.0045P1 MICRODYSTROPHIN GENE THERAPY ADMINISTRATION FOR TREATMENT OF DYSTROPHINOPATHIES 0. SEQUENCE LISTING

[0001] The contents of the electronic sequence listing submitted herewith as file 38013_0045P1.xml, Size: 204,800 bytes; and Date of Creation: June 23, 2025, is hereby incorporated by reference in its entirety. 1. FIELD OF THE INVENTION

[0002] The present invention relates to treatment of dystrophinopathies by administration of doses of gene therapy vectors, such as AAV gene therapy vectors in which the transgene encodes a microdystrophin comprising a CT domain. 2. BACKGROUND

[0003] A group of neuromuscular diseases called dystrophinopathies are caused by mutations in the DMD gene. Each dystrophinopathy has a distinct phenotype, with all patients suffering from muscle weakness and ultimately cardiomyopathy with ranging severity. Duchenne muscular dystrophy (DMD) is a severe, X-linked, progressive neuromuscular disease affecting approximately one in 3,600 to 9,200 live male births. The disorder is caused by mutations in the dystrophin gene, including deletions, duplications, frameshift mutations, nonsense mutations, splicesite mutations and missense mutations in the DMD gene, abolishing the expression of the dystrophin protein. Onset of DMD usually appears at age 3 to 5 years of age, beginning with pelvic girdle weakness, tight heel cords, and creatine kinase (CK) values of 50 to 100 times normal. Thus, progressive weakness and muscle atrophy begin in childhood with loss of ambulation usually occurring by age 12. Due to the lack of the dystrophin protein, skeletal muscle, and ultimately heart and respiratory muscles (e.g., intercostal muscles and diaphragm), degenerate causing premature death. Affected individuals experience breathing difficulties, respiratory infections, and swallowing problems. Almost all DMD patients will develop cardiomyopathy. Pneumonia compounded by cardiac involvement is the most frequent cause of death, which frequently occurs before the third decade.

[0004] Becker muscular dystrophy (BMD) has less severe symptoms than DMD, but still leads to premature death. BMD is generally caused by mutations that result in an in-frame mutation, - 1 - 40222320338013.0045P1 such that translation of a protein with partial function occurs, whereas DMD mutations result in no dystrophin protein (“out-of-frame” mutation). Compared to DMD, BMD is characterized by later-onset skeletal muscle weakness, usually loss of ambulation beginning after age 12 to 15. Whereas DMD patients are wheelchair dependent before age 13, those with BMD lose ambulation and require a wheelchair after age 16. Use of steroids in modern treatment protocols delays the loss of ambulation. BMD patients also exhibit preservation of neck flexor muscle strength, unlike their counterparts with DMD. Despite milder skeletal muscle involvement, heart failure from DMD-associated dilated cardiomyopathy (DCM) is a common cause of morbidity and the most common cause of death in BMD, may occur on average in the mid-40s, although certain forms of BMD have seen patients continue as ambulatory well passed their 50s.

[0005] With advances in establishing a clinical diagnosis for dystrophinopathies using genetic screening and the use of adeno-associated virus (AAV) mediated gene therapy, there exists a need in the art for methods of administering AAV vectors encoding microdystrophins at dosages therapeutically effective for treatment or prophylactic reduction of symptoms of dystrophinopathies, including DMD or BMD in younger children. 3. SUMMARY OF THE INVENTION

[0006] Provided are methods of treating or ameliorating the symptoms of dystrophinopathies by administration of rAAV vector particles containing nucleic acid genomes (generated from vectors, where “constructs” as used herein generally describe arrangement of the subunits of the dystrophin protein that form the microdystrophin and may include the regulatory elements that control expression of the microdystrophin, including in cis plasmids used to produce recombinant AAV particles and the recombinant genomes packaged in the AAV particles) encoding microdystrophins, such as those recombinant genomes exemplified in FIG. 2 and the microdystrophin proteins described herein, sequences of which are provided in Table 2. Based upon pharmacology studies in mice, for example, as described herein in Examples 5, 7, and 8 (Sections 6.5, 6.7, and 6.8, infra) (Section 6.6, infra) and in PCT International Publication No WO 2022 / 232141 A1, published November 3, 2022, which is incorporated herein by reference, and in results from human clinical studies, as described in Examples 2 and 6 herein (Sections 6.2 and 6.6, infra). Provided are methods of treatment of subjects in need thereof by administration, including peripheral administration, such as intravenous administration, of therapeutically effective dosages - 2 - 40928727338013.0045P1 and uses for gene constructs that encode a microdystrophin protein for use in gene therapy. Based on the mouse study results and human clinical trial results provided, the methods of administering the microdystrophin gene therapies of the present disclosure result in, at least 12 weeks, 26 weeks or 52 weeks after administration, including to subjects 1-3 years old (i.e., less than 4 years old at dosing), 4 to 5 years old, 6 to 7 years old, and greater than 8 years old, including subjects who may be in the Decline Phase (Muntoni 2019 and FIG.10), the presence of RGX-202 microdystrophin protein (% normal dystrophin as measured by capillary-based Western assay) representative of a form of shortened dystrophin anchoring the dystrophin-associated protein complex (DAPC), detection of the microdystrophin in muscle fibers, improvements (or stability) in symptoms and biomarkers of dystrophinopathy disease, such as, improvements (or stability) in timed function tests (TFTs) and North Star Ambulatory Assessment (NSAA) or PDMS-3 results relative to both before administration of the therapeutic (the patient baseline measurements) and / or to matched natural history control subjects (matched for age and baseline disease), including without any treatment emergent serious adverse events, including central or peripheral neurotoxicity, drug- induced liver injury, thrombocytopenia, myocarditis, or myositis.

[0007] Embodiments described herein are methods of treating dystrophinopathy in a subject comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle comprises a transgene that encodes a microdystrophin protein a microdystrophin protein having from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, CR is the cysteine-rich region of dystrophin or at least a portion thereof which binds β-dystroglycan, and CT is at least a portion of a C-terminal region of dystrophin, wherein the portion comprises an α1-syntrophin binding site and / or an α-dystrobrevin binding site. In certain embodiments, the CT domain comprises or consists of a) the proximal 194 amino acids of the C- terminus of dystrophin (the amino acid sequence of SEQ ID NO: 16) or b) at least the proximal - 3 - 40928727338013.0045P1 portion of the C-terminus corresponding to human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 92 (UniProtKB-P11532) or c) at least the proximal portion of the C-terminus encoded by exons 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75. Alternatively, the CT domain is truncated and comprises an α1- syntrophin binding site but not an α-dystrobrevin binding site, such as, the amino acid sequence of SEQ ID NO: 83. In embodiments, the microdystrophin comprises a CT domain comprising an α1-syntrophin binding site and does not have the amino acid sequence DTM at the C-terminus of the microdystrophin. In embodiments, provided is a microdystrophin protein comprising a means for enhancing DAPC restoration or a means for stabilizing interaction of the microdystrophin with α-dystrobrevin and / or nNOS, or a means for protecting muscle against contraction-induced damage, or means for enabling muscle recovery from contraction-induced damage, including a microdystrophin having the first 194 amino acids (the N-terminal amino acids) of the CT domain. In some embodiments, the microdystrophin may have the amino acid sequence of SEQ ID NO: 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. In some embodiments, the microdystrophin may have the amino acid sequence of SEQ ID NO: 79. The constructs include regulatory sequences, such as muscle-specific promoter sequences, including the SPc5-12 promoter (SEQ ID NO: 39), or alternatively, a truncated SPc5-12 promoter (SEQ ID NO: 40) or a SPc5-12 promoter variant, mutant or transcriptionally active portion thereof (such as, modified Spc5-12 promoters Spc5v1 (SEQ ID NO: 93) or Spc5v2 (SEQ ID NO: 94)), and polyadenylation signal sequences, such as, the small polyA signal sequence (SEQ ID NO: 42), which are operably linked to the coding sequence for the microdystrophin. Specific constructs include RGX-DYS1 and RGX- DYS5 (see FIG. 2) having microdystrophin encoding nucleotide sequences of SEQ ID NO: 20 and SEQ ID NO: 81, respectively, operably linked to regulatory sequences and flanked by AAV2 ITR sequences, where the entire construct, including the recombinant genome, has the nucleotide sequence of SEQ ID NO: 53 or 82, respectively. The rAAV particles containing the recombinant genomes are, in embodiments, AAV8. For example, the rAAV particle or gene therapy vector is AAV8-RGX-DYS1 (recombinant AAV8 comprising a polynucleotide with the nucleotide sequence of SEQ ID NO: 53).

[0008] Preclinical studies in mice (see, e.g., Examples 3 and 9 (Sections 6.3 and 6.9), herein) show that administration of a microdystrophin comprising a CT domain or the first 194 amino - 4 - 40928727338013.0045P1 acids of the CT comprising an α1-syntrophin binding site i) significantly enhanced restoration of the DAPC in mdx mice, similarly to natural dystrophin; ii) increased the half-life of microdystrophin protein DYS1, which remains in muscle fibers longer to strengthen muscle; and iii) protected against contraction-induced damage enabling better recovery relative to administration of a microdystrophin that does not have a CT domain with an α1-syntrophin binding site (DYS3). Accordingly, provided are methods of enhancing restoration of the DAPC, methods of increasing the half-life of AAV8-RGX-DYS1 (RGX-202) administered microdystrophin, and methods of protecting against contraction-induced muscle damage, improving muscle resilience and increasing recovery after contraction-induced damage in subjects having a dystrophinopathy and / or DMD mutation.

[0009] In certain embodiments, the therapeutically effective amount of an rAAV particle comprising a transgene encoding a microdystrophin disclosed herein, including in embodiments, AAV8-RGX-DYS1, is administered intravenously or intramuscularly at a dose of 5×1013to 1x1015genome copies / kg, including 1x1014genome copies / kg, 2×1014genome copies / kg or 3x1014genome copies / kg. In certain embodiments, the therapeutically effective amount of an rAAV particle comprising a transgene encoding a microdystrophin disclosed herein, including AAV8- RGX-DYS1, is administered intravenously or intramuscularly at a dose of 1x1014, 1.1x1014, 1.2x1014, 1.3x1014, 1.4x1014, 1.5x1014, 1.6x1014, 1.7x1014, 1.8x1014, 1.9x1014, 2x1014, 2.1x1014, 2.2x1014, 2.3x1014, 2.4x1014, 2.5x1014, 2.6x1014, 2.7x1014, 2.8x1014, 2.9x1014, or 3x1014genome copies / kg. In certain embodiments, the therapeutically effective amount of an rAAV particle (including AAV8-RGX-DYS1) is administered intravenously at a dose 1x1014genome copies / kg. In other embodiments, the therapeutically effective amount of an rAAV particle (including AAV8- RGX-DYS1) is administered intravenously at a dose 2x1014genome copies / kg. In still other embodiments, the therapeutically effective amount of an rAAV particle (including AAV8-RGX- DYS1) is administered intravenously at a dose 3x1014genome copies / kg. In embodiments, subjects administered the therapeutic are prophylactically administered an immunosuppressant either prior to, concomitantly with and / or subsequent to, including as maintenance therapy after, administration of the rAAV particle having a transgene encoding a microdystrophin disclosed herein. Immunosuppressants include corticosteroids, anti-complement agents, such as anti-C3 and C5 antibodies, anti-cytokine agents, such as anti-cytokine antibodies, such as anti-IL-6 and anti- - 5 - 40928727338013.0045P1 IL6R antibodies, anti-CD20 antibodies, combinations of anti-C5 and anti-CD20 antibodies, rapamycin, or anti-IgG therapies, such as imlifidase. In embodiments, the concomitant immunosuppression regimen includes a daily dose of oral prednisolone, and / or doses of eculizumab (anti-C5 antibody; SOLIRIS®) prior to and subsequent to administration of microdystrophin and, optionally, administration of oral sirolimus (also known as rapamycin; RAPAMUNE®).

[0010] In certain embodiments, the pharmaceutically acceptable carrier comprises a modified Dulbecco’s phosphate buffered saline (DPBS) with sucrose buffer comprising 0.2 g / L potassium chloride, 0.2 g / L potassium phosphate monobasic, 1.2 g / L sodium phosphate dibasic (anhydrous or hydrate), 5.8 g / L sodium chloride, 40 g / L sucrose, and 0.01 g / L poloxamer 188, pH 7.4.

[0011] Also provided are pharmaceutical compositions comprising the recombinant vectors encoding the microdystrophins provided herein, including with a pharmaceutically acceptable excipient and methods of treatment for any dystrophinopathy, such as for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, as well as DMD or BMD female carriers, by administration of the gene therapy vectors described herein (including AAV8-RGX-DYS1) to a subject in need thereof, including administration intravenously at dosages of 5×1013to 1x1015genome copies / kg, including 1x1014genome copies / kg, 2×1014genome copies / kg or 3x1014genome copies / kg genome copies / kg. Provided are methods of treating, ameliorating the symptoms of or managing a dystrophinopathy, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy by administration of an rAAV containing a transgene or gene cassette described herein (including AAV8-RGX-DYS1), by administration to a subject in need thereof such that the microdystrophin is delivered to the muscle (including skeletal muscle, cardiac muscle, and / or smooth muscle). In embodiments, the subject has a form of DMD with a mutation (including a deletion, duplication, point mutation, including a nonsense mutation) of the DMD gene. In embodiments, the subject has a form of DMD where the mutation is in any exon except those with deletions or point mutations in exons 8, 9, and / or 10 of the DMD gene. In embodiments, the subject has a form of DMD where the mutation is in exon 18 or higher (i.e., in exons 18 to 79) of the gene encoding DMD. In other embodiments, the subject has a form of DMD with a mutation within exons 12 to 17 the DMD gene. In other embodiments, the subject has a form of DMD with a - 6 - 40928727338013.0045P1 mutation in exon 12 to 79 of the DMD gene. In embodiments, the subject has a form of DMD where the mutation is outside exons 1 to 11 or outside exons 1 to 17 of the DMD gene. In particular embodiments, the rAAV is administered systemically, including intravenously or intramuscularly. In embodiments, the subjects are, at dosing, 1-3 years old (i.e., less than 4 years old at dosing), 4 to 5 years old, 6 to 7 years old, and greater than 8 years old.

[0012] Also provided are methods of decreasing inflammation or fibrosis and / or muscle degeneration and / or muscle damage, including contraction-induced muscle damage, in a muscle of a subject in need thereof comprising administering one or more of the disclosed pharmaceutical compositions.

[0013] Also provided are methods of reducing and / or preventing contraction-induced muscle damage in a subject in need thereof comprising administering one or more of the disclosed pharmaceutical compositions. Also provided are methods of increasing muscle resilience in a subject in need thereof comprising administering one or more of the disclosed pharmaceutical compositions. In embodiments, the microdystrophin comprises at least the portion of the C- terminal domain (CT) comprising an α1-syntrophin binding site, including the initial 194 amino acids of the CT domain. In embodiments, the microdystrophin comprises a means for restoring DAPC in muscle cells. In embodiments, the microdystrophin comprises a means for stabilization of a microdystrophin protein in muscle cells by interaction between a microdystrophin C-terminal domain (CT) and alpha-syntrophin. In embodiments, the microdystrophin comprises a means for protecting muscle against contraction-induced damage. In embodiments, the microdystrophin comprises a means for enabling muscle recovery from contraction-induced damage. In embodiments, improvement in PDMS-3 (e.g. Body Control and Body Transport subtests) for the subjects 1-3 years old (i.e., less than 4 yrs years old) at dosing is indicative of effectiveness following administration of microdystrophin vectors. The present inventions are illustrated by way of examples infra describing the construction and making of microdystrophin vectors and in vitro and in vivo assays demonstrating effectiveness as well as clinical data obtained from administering a microdystrophin encoding viral particle to patients resulting in improvement in results of timed function tests, NSAA, and caregiver reported improved function reports (PODCI) without any reported serious adverse events, including central or peripheral neurotoxicity, drug-induced liver injury, thrombocytopenia, myocarditis, or myositis. Data show persistent improvement in subjects - 7 - 40928727338013.0045P1 over 12 months from dosing, including subjects at 4 to 5 years old, 6 to 7 years old, and greater than 8 years old (including 8 to 12 years of age). 3.1 Embodiments of the Invention 1. A method of treating a dystrophinopathy and / or delaying the progression of the symptoms of dystrophinopathy in a human subject 1 year of age to less than 4 years of age, or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8 years of age, including subjects in the Decline Phase or whose natural history peers are in the Decline Phase, in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle comprises a transgene that encodes a microdystrophin protein consisting of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD- H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, and wherein the therapeutically effective amount of the rAAV particle is administered intravenously at dose of 1×1014to 2×1014genome copies / kg; and wherein i) microdystrophin levels and / or dystrophin levels in muscle tissue are greater than about 5%, greater than about 10 %, greater than about 15%, or greater than about 20%, or greater than about 40%, or greater than about 60%, or greater than about 80% or greater than about 80% relative to a reference standard as determined by a method for quantitating microdystrophin or dystrophin levels at 12 weeks after administration of said pharmaceutical composition, and / or ii) PDMS-3 Total Motor Score Index is equal to or greater than the average age-equivalent PDMS-3 Total Motor Score Index or is stable relative to baseline for the patient, and / or iii) results from a timed function test are stable or improved relative to baseline for the patient or relative to natural history matched subjects. - 8 - 40928727338013.0045P1 2. The method of embodiment 1, wherein the CT comprises or consists of the proximal 194 amino acids of the C-terminus of dystrophin or at least the proximal portion of the C-terminus corresponding to human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 92 (UniProtKB-P11532) or at least the proximal portion of the C-terminus encoded by exons 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75. 3. The method of embodiment 1 or 2, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 1. 4. The method of embodiment 3, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 20. 5. The method of embodiment 1, wherein the CT comprises or consists of the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence which comprises the α1-syntrophin binding site but not the dystrobrevin binding site. 6. The method of embodiment 1 or 5, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. 7. The method of embodiment 6, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 81. 8. The method of any one of embodiments 1-7, wherein the transgene further comprises a transcription regulatory element that promotes expression in muscle operably linked to the nucleic acid sequence that encodes the microdystrophin protein. 9. The method of embodiment 8, wherein the transcription regulatory element comprises a muscle-specific promoter. 10. The method of embodiment 9, wherein the muscle-specific promoter is a skeletal, smooth, or cardiac muscle specific promoter. 11. The method of any one of embodiments 9 or 10, wherein the muscle specific promoter is SPc5-12 or a transcriptionally active portion or mutant thereof. 12. The method of embodiment 11, wherein the promoter consists of the nucleic acid sequence of SEQ ID NO: 39. - 9 - 40928727338013.0045P1 13. The method of any one of embodiments 1-12, wherein the transgene comprises a polyadenylation signal 3’ of the nucleic acid sequence encoding the microdystrophin protein. 14. The method of any one of embodiments 1-13, wherein the transgene comprises an intron sequence between the promoter and the microdystrophin coding sequence. 15. The method of embodiment 14, wherein the intron sequence is a VH4 intron sequence (SEQ ID NO: 41) 16. The method of any one of embodiments 1-4 and 8-13, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 53 17. The method of any one of embodiments 1, and 5-13, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 82. 18. The method of any of embodiments 1-17, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77. 19. The method of embodiment 18, wherein the rAAV is an AAV8 serotype. 20. The method of any one of embodiments 1-19, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy, is a DMD with a mutation of the DMD gene, including with a mutation in exon 18 or higher (exon 18 to 79) of the DMD gene, or a mutation in exon 12 to 17 of the DMD gene, or has a mutation in exon 12 to 79 of the DMD gene, or is a mutation in any exon except in exons 8, 9, and / or 10 of the DMD gene. 21. The method of any one of embodiments 1-20, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014genome copies / kg, 2x1014genome copies / kg or 3x1014genome copies / kg. 22. The method of any one of embodiments 1-21 wherein the pharmaceutical composition is administered intravenously. 23. The method of any one of embodiments 1-22, wherein by 12 weeks, 24 weeks, 1 year,2 years or 3 years after the administration, the creatine kinase activity decreased in the subject relative to the level prior to said administration. - 10 - 40928727338013.0045P1 24. The method of embodiment 1-22, wherein the creatine kinase activity is maintained between 0.5 fold to 1.5 fold of baseline CK activity. 25. The method of embodiment 23, wherein the decrease in creatine kinase activity is 3000 to 10000 creatine kinase units / liter. 26. The method of any one of embodiments 1-25, wherein by 12 weeks, 24 weeks, 1 year,2 years or 3 years after the administration of the pharmaceutical compositions, lesions in gastrocnemius muscle of the subject decreased compared to the lesions in the gastrocnemius muscle prior to said administration. 27. The method of embodiment 26, wherein the lesions in gastrocnemius muscle of the subject are assessed using magnetic resonance imaging (MRI). 28. The method of any one of embodiments 26-27, wherein the decrease of lesions in gastrocnemius muscle after administration is about 3-10% compared to the lesions in the gastrocnemius muscle prior to said administration. 29. The method of any one of embodiments 1-28, wherein by 12 weeks, 24 weeks, 1 year, 2 years or 3 years after the administration of the pharmaceutical compositions, gastrocnemius muscle volume of the subject decreased compared to the gastrocnemius muscle volume prior to said administration. 30. The method of embodiment 29, wherein the gastrocnemius muscle volume decrease is 20 to 100 mm3. 31. The method of any one of embodiments 1-30, wherein by 12 weeks, 24 weeks, 1 year, 2 years or 3 years after the administration of the pharmaceutical compositions, T2-relaxation time of lesions in muscle decreased compared to the T2-relaxation time prior to said administration. 32. The method of embodiment 31, wherein the decrease is 2 to 8 milliseconds. 33. The method of any one of embodiments 31-32, wherein the lesions in muscle are lesions in gastrocnemius muscle. 34. The method of any one of embodiments 1-33, wherein by 12 weeks, 24 weeks, 1 year, 2 years or 3 years after the administration of the pharmaceutical composition, the subject exhibited a gait score of about -1 to 2. - 11 - 40928727338013.0045P1 35. The method of embodiment 34, wherein by 12 weeks after the administration of the pharmaceutical composition, the subject exhibited a gait score of about 1. 36. The method of any one of embodiments 1-35, wherein by 12 weeks, 24 weeks, 1 year, 2 years, 3 years after the administration of the pharmaceutical composition, the North Star Ambulatory Assessment (NSAA) score or PDMS-3 increased or is stable compared to the NSAA score or PDMS-3 prior to said administration or compared to natural history matched controls that were not administered the composition and / or wherein the subject did not experience any serious adverse event after said administration, including central or peripheral neurotoxicity, drug-induced injury, thrombocytopenia, myocarditis or myositis. 37. The method of embodiment 36, wherein i) the increase in NSAA score is more than 2 in a human subject 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8 years of age at time of administration, or ii) the increase or stability in PDMS-3 score is in a human subject 1 year of age to less than 4 years of age at time of administration. 38. The method of any one of embodiments 1-37, wherein by 12 weeks, 24 weeks, 1 year, 2 years or 3 years after the administration of the pharmaceutical composition, there was a decrease in the amount of time it takes the subject to stand, run / walk a determined distance, climb a set number of stairs relative to the subject prior to administration or compared to natural history matched controls. 39. The method of embodiment 38, wherein the determined distance is 10 meters. 40. The method of any one of embodiments 38-39, wherein the set number of stairs is 4. 41. The method of any one of embodiments 38-40, wherein the decrease in the amount of time it takes to stand is an at least 5%, 10%, 20% or 30% decrease compared to before said administration or relative to a natural history matched control. 42. The method of any one of embodiments 38-41, wherein the decrease in the amount of time it takes to run / walk a determined distance is an at least 5%, 10%, 20% or 30% decrease compared to before said administration or relative to a natural history matched control. - 12 - 40928727338013.0045P1 43. The method of any one of embodiments 38-42, wherein the decrease in the amount of time it takes to climb a set number of stairs is an at least 5%, 10%, 20% or 30% decrease compared to before said administration or relative to a natural history matched control. 44. The method of any one of embodiments 1-43, wherein by 12 weeks, 24 weeks, 1 year, 2 years or 3 years after the administration of the pharmaceutical composition, the subject exhibited improved cardiac function compared to the cardiac function prior to said administration or compared to a natural history matched control that were not administered the composition. 45. The method of any one of embodiments 1-44, wherein by 12 weeks, 24 weeks, 1 year, 2 years or 3 years after the administration of the pharmaceutical composition, the subject exhibited improved pulmonary function compared to the pulmonary function prior to said administration or compared to age-equivalent subjects that were not administered the composition. 46. A method of preventing inflammation and / or fibrosis in a muscle of a subject 1 year of age to less than 4 years of age or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8, including subjects in the Decline Phase, and having a confirmed mutation in the DMD gene comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier; wherein the rAAV particle comprises a gene expression cassette comprising a nucleic acid sequence that encodes a microdystrophin protein; wherein the microdystrophin protein consists of dystrophin domains arranged from amino- terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin of, CT comprises the entire C-terminus of dystrophin or a portion of the CT comprising an α1-syntrophin binding site and a dystrobrevin binding site or the first 194 amino acids of the CT domain; - 13 - 40928727338013.0045P1 wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 5×1013to 1x1015, or at a dose of 1x1014or 2x1014, genome copies per kilogram (GC / kg); and wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject. 47. A method of decreasing and / or preventing muscle degeneration in a subject 1 year of age to less than 4 years of age or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8, including subjects in the Decline Phase, and having a confirmed mutation in the DMD gene comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier; wherein the rAAV particle comprises a gene expression cassette that comprises a nucleic acid sequence that encodes a microdystrophin protein, wherein the microdystrophin protein comprises or consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, CT comprises the entire C-terminus of dystrophin or a portion of the CT comprising an α1-syntrophin binding site and a dystrobrevin binding site or the initial 194 amino acids of the CT domain; wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of at a dose of 1x1014or 2x1014, genome copies per kilogram (GC / kg); and wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject. 48. The method of embodiment 46 or 47, wherein the muscle is a subject’s diaphragm. 49. A method of delaying the onset and progression of muscle weakness in a subject 1 year of age to less than 4 years of age or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years - 14 - 40928727338013.0045P1 of age or greater than 8, including subjects in the Decline Phase, and having a confirmed mutation in the DMD gene comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle comprises a gene expression cassette comprising a nucleic acid sequence that encodes a microdystrophin protein; wherein the microdystrophin protein comprises or consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, wherein the therapeutically effective amount of a rAAV particle is administered intravenously at a dose of at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg); and wherein the microdystrophin levels and / or dystrophin levels in muscle tissue are greater than about 20%, or greater than about 30%, or greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80% or greater than about 90% relative to a reference standard as determined by a method for quantitating microdystrophin or dystrophin levels at 12 weeks after said administration of the composition. 50. The method of embodiment 49, wherein delaying the onset and progression of muscle weakness comprises an increase in balance, change in stride length, decrease in head movement, or a combination thereof and / or a composite PDMS-3 score equal to or greater than that of an average age equivalent composite PDMS-3 score. 51. The method of any one of embodiments 46-50, wherein the CT comprises or consists of the proximal 194 amino acids of the C-terminus of dystrophin or at least the proximal portion of the C-terminus corresponding to human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 92 (UniProtKB-P11532) or at least the proximal portion of the C-terminus encoded by exons - 15 - 40928727338013.0045P1 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75. 52. The method of any one of embodiments 46-51, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 1. 53. The method of embodiment 52, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 20. 54. The method of any one of embodiments 46-53, wherein the CT comprises or consists of the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence which comprises the α1- syntrophin binding site but not the dystrobrevin binding site. 55. The method of any one of embodiments 46-54, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. 56. The method of embodiment 55, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 81. 57. The method of any one of embodiments 46-56, wherein the transgene further comprises a transcription regulatory element that promotes expression in muscle operably linked to the nucleic acid sequence that encodes the microdystrophin protein. 58. The method of embodiment 57, wherein the transcription regulatory element comprises a muscle-specific promoter. 59. The method of embodiment 58, wherein the muscle-specific promoter is a skeletal, smooth, or cardiac muscle specific promoter. 60. The method of any one of embodiments 58 or 59, wherein the muscle specific promoter is SPc5-12 or a transcriptionally active portion or mutant thereof. 61. The method of embodiment 58, wherein the promoter consists of the nucleic acid sequence of SEQ ID NO: 39. 62. The method of any one of embodiments 46-61, wherein the transgene comprises a polyadenylation signal 3’ of the nucleic acid sequence encoding the microdystrophin protein. - 16 - 40928727338013.0045P1 63. The method of any one of embodiments 46-62, wherein the transgene comprises an intron sequence between the promoter and the microdystrophin coding sequence. 64. The method of embodiment 63, wherein the intron sequence is a VH4 intron sequence (SEQ ID NO: 41) 65. The method of any one of embodiments 46-64, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 53 66. The method of any one of embodiments 46-65, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 82. 67. The method of any of embodiments 46-66, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77. 68. The method of embodiment 67, wherein the rAAV is an AAV8 serotype. 69. The method of any one of embodiments 46-68, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014genome copies / kg or 2x1014genome copies / kg. 70. The method of any one of embodiments 46-69 wherein the pharmaceutical composition is administered intravenously. 71. The method of any one of embodiments 1 to 70 further comprising prophylactically administering an immunosuppressant therapy to said subject prior to, concomitantly with and / or after said administration of the AAV particle. 72. The method of any one of embodiment 71, wherein the immunosuppressant therapy is a corticosteroid, an anti-C5 antibody, an anti-IL6 or anti-IL6R antibody, and anti-CD20 antibody, a combination of an anti-C5 and anti-CD20 antibody, rapamycin, imlifidase, or a combination thereof. 73. A pharmaceutical composition for use in treating a dystrophinopathy in a subject in need thereof, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, - 17 - 40928727338013.0045P1 wherein the rAAV particle comprises a gene expression cassette comprising a nucleic acid sequence that encodes a microdystrophin protein; wherein the microdystrophin protein comprises or consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site; and wherein the therapeutically effective amount of a rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies / kg, and wherein the subject is 1 year of age to less than 4 years of age or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8, including subjects in the Decline Phase, and has a confirmed mutation in the DMD gene. 74. The composition of embodiment 73, wherein the CT comprises or consists of the proximal 194 amino acids of the C-terminus of dystrophin or at least the proximal portion of the C-terminus corresponding to human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 92 (UniProtKB-P11532) or at least the proximal portion of the C-terminus encoded by exons 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75. 75. The composition of embodiment 73 or 74, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 1. 76. The composition of embodiment 75, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 20. 77. The composition of embodiment 73, wherein the CT comprises or consists of the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence which comprises the α1-syntrophin binding site but not the dystrobrevin binding site. - 18 - 40928727338013.0045P1 78. The composition of embodiment 73 or 77, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. 79. The composition of embodiment 78, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 81. 80. The composition of any one of embodiments 73-79, wherein the transgene further comprises a transcription regulatory element that promotes expression in muscle operably linked to the nucleic acid sequence that encodes the microdystrophin protein. 81. The composition of embodiment 80, wherein the transcription regulatory element comprises a muscle-specific promoter. 82. The composition of embodiment 81, wherein the muscle-specific promoter is a skeletal, smooth, or cardiac muscle specific promoter. 83. The composition of any one of embodiments 81 or 82, wherein the muscle specific promoter is SPc5-12 or a transcriptionally active portion or mutant thereof. 84. The composition of embodiment 83, wherein the promoter consists of the nucleic acid sequence of SEQ ID NO: 39. 85. The composition of any one of embodiments 73-84, wherein the transgene comprises a polyadenylation signal 3’ of the nucleic acid sequence encoding the microdystrophin protein. 86. The composition of any one of embodiments 73-85, wherein the transgene comprises an intron sequence between the promoter and the microdystrophin coding sequence. 87. The composition of embodiment 86, wherein the intron sequence is a VH4 intron sequence (SEQ ID NO: 41). 88. The composition of any one of embodiments 73-76 and 80-85, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 53. 89. The composition of any one of embodiments 73, and 77-85, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 82. 90. The composition of any of embodiments73-89, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77. - 19 - 40928727338013.0045P1 91. The composition of embodiment 90, wherein the rAAV is an AAV8 serotype. 92. The composition of any one of embodiments 73-91, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy, is a DMD with a mutation of the DMD gene, including with a mutation in exon 18 or higher (exon 18 to 79) of the DMD gene, or a mutation in exon 12 to 17 of the DMD gene, or has a mutation in exon 12 to 79 of the DMD gene, or is a mutation in any exon except in exons 8, 9, and / or 10 of the DMD gene. 93. The composition of any one of embodiments 73-92, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014genome copies / kg or 2x1014genome copies / kg. 94. The composition of any one of embodiments 73-93 wherein the pharmaceutical composition is administered intravenously. 95. The composition of any one of embodiments 73-94, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration, the creatine kinase activity decreased in the subject relative to the level prior to said administration. 96. The composition of embodiment 95, wherein the decrease in creatine kinase activity is 0.5 fold to 1.5 fold. 97. The composition of embodiment 95, wherein the decrease in creatine kinase activity is 3000 to 10000 creatine kinase units / liter. 98. The composition of any one of embodiments 73-97, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical compositions, lesions in gastrocnemius muscle of the subject decreased compared to the lesions in the gastrocnemius muscle prior to said administration. 99. The composition of embodiment 98, wherein the lesions in gastrocnemius muscle of the subject are assessed using magnetic resonance imaging (MRI). 100. The composition of any one of embodiments 98-99, wherein the decrease of lesions in gastrocnemius muscle after administration is about 3-10% compared to the lesions in the gastrocnemius muscle prior to said administration. - 20 - 40928727338013.0045P1 101. The composition of any one of embodiments 73-100, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical compositions, gastrocnemius muscle volume of the subject decreased compared to the gastrocnemius muscle volume prior to said administration. 102. The composition of embodiment 101, wherein the gastrocnemius muscle volume decrease is 20 to 100 mm3. 103. The composition of any one of embodiments 73-102, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical compositions, T2-relaxation time of lesions in muscle decreased compared to the T2-relaxation time prior to said administration. 104. The composition of embodiment 103, wherein the decrease is 2 to 8 milliseconds. 105. The composition of any one of embodiments 103-104, wherein the lesions in muscle are lesions in gastrocnemius muscle. 106. The composition of any one of embodiments 73-105, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical composition, the subject exhibited a gait score of about -1 to 2. 107. The composition of embodiment 106, wherein by 12 weeks after the administration of the pharmaceutical composition, the subject exhibited a gait score of about 1. 108. The composition of any one of embodiments 73-107, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical composition, the North Star Ambulatory Assessment (NSAA) score, PDMS-3 or timed function test or caregiver reported improved function results increased or stable compared to the NSAA score or timed function test or PDMS-3 or caregiver reported improved function results prior to said administration or relative to a natural history matched control. 109. The composition of embodiment 108, wherein the increase is in score is more than 2. 110. The composition of any one of embodiments 73-109, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical composition, there was a decrease in the amount of time it takes the subject to stand, run / walk a determined distance, climb a set number - 21 - 40928727338013.0045P1 of stairs compared to the subject prior to said administration or relative to a natural history matched control. 111. The composition of embodiment 110, wherein the determined distance is 10 meters. 112. The composition of any one of embodiments 110-111, wherein the set number of stairs is 4. 113. The composition of any one of embodiments 110-112, wherein the decrease in the amount of time it takes to stand is wherein the decrease in the amount of time it takes to stand is an at least 5%, 10%, 20% or 30% decrease compared to before said administration or relative to a natural history matched control. 114. The composition of any one of embodiments 110-113, wherein the decrease in the amount of time it takes to run / walk a determined distance is an at least 5%, 10%, 20% or 30% decrease compared to before said administration or relative to a natural history matched control. 115. The composition of any one of embodiments 110-114, wherein the decrease in the amount of time it takes to climb a set number of stairs is an at least 5%, 10%, 20% or 30% decrease compared to before said administration or relative to a natural history matched control. 116. The composition of any one of embodiments 73-115, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical composition, the subject exhibited improved cardiac function compared to the cardiac function prior to said administration. 117. The composition of any one of embodiments 73-116, wherein by 12 weeks, 24 weeks, 1 year or 2 years after the administration of the pharmaceutical composition, the subject exhibited improved pulmonary function compared to the pulmonary function prior to said administration. 118. The composition of any one of embodiments 73 to 117 further comprising prophylactically administering an immunosuppressant therapy to said subject prior to, concomitantly with and / or after said administration of the AAV particle. 119. The composition of embodiment 118, wherein the immunosuppressant therapy is a corticosteroid, an anti-C5 antibody, an anti-IL6 or anti-IL6R antibody, and anti-CD20 antibody, a combination of an anti-C5 and anti-CD20 antibody, rapamycin, imlifidase, or a combination thereof. - 22 - 40928727338013.0045P1 120. The method or composition of any one of embodiments 1 to 119, wherein said subject is administered a prophylactic immunosuppression regimen. 121. The method or composition of embodiment 120 wherein the prophylactic immunosuppression regimen comprises (1) a daily dose of oral prednisolone from Day 1 to week 8; (2) infusions of eculizumab prior to and subsequent to administration of the rAAV; and (3) daily oral sirolimus from Day -7 to week 8, where Day 1 is the day of rAAV administration. 122. The method or composition of embodiment 121, wherein oral prednisolone is administered at 1 mg / kg / day from Day 1 until the end of Week 8, where Day 1 is the day of rAAV administration, and then, if no safety concerns identified, lowering the dose to 0.5 mg / kg / day from Week 9 to Week 10, and then if no safety concerns are identified, lowering the dose to 0.25 mg / kg / day from Week 11 to Week 12. 123. The method or composition of embodiments 121 or 122, wherein eculizumab is administered by infusion, (1) for subjects weighing 10 to <20 kg, 600 mg eculizumab on Day -9, Day -2, Day 4 and Day 12; (2) for subjects weighing 20 kg to <30 kg, 800 mg eculizumab on Day -16, Day -9, Day -2 and Day 12; (3) for subjects weighing 30 kg to <40 kg, 900 mg eculizumab on Day -16, Day -9, Day -2 and Day 12; and (4) for subjects weighing greater than or equal to 40 kg, 1200 mg eculizumab on Day -30, Day -23, Day -16, Day -9, Day -2 and Day 12, where Day 1 is the day of rAAV administration. 124. The composition or method of any of embodiments 121 to 123, wherein the sirolimus is administered at 3 mg / m2at Day -7, each day of Day -6 to Week 8 a dose of 1 mg / m2 / day divided into 2 doses, to achieve target blood levels of 8-12 ng / ml, reducing the dose to 0.5 mg / m2 / day for Weeks 9-10 if safety tests remain stable, and reducing the dose to 0.25 mg / m2 / day for Weeks 11- 12 if safety tests remain stable. 125. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle comprises a transgene that encodes a microdystrophin protein consisting of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD- H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region - 23 - 40928727338013.0045P1 of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, and wherein the therapeutically effective amount of the rAAV particle is administered intravenously at dose of 1x1014or 2x1014genome copies / kg. 126. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle comprises a transgene that encodes a microdystrophin protein, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 1. 127. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle is an AAV8 particle and comprises an artificial genome having a nucleotide sequence of SEQ ID NO: 53. 128. The method of embodiments 125 to 127, wherein the pharmaceutically acceptable carrier comprises a modified Dulbecco’s phosphate buffered saline (DPBS) with sucrose buffer comprising 0.2 g / L potassium chloride, 0.2 g / L potassium phosphate monobasic, 1.2 g / L sodium phosphate dibasic (anhydrous or hydrate), 5.8 g / L sodium chloride, 40 g / L sucrose, and 0.01 g / L poloxamer 188, pH 7.4. 129. A method of treating a dystrophinopathy in a subject in need thereof, comprising administering intravenously to the subject the pharmaceutical composition of any one of embodiments 125 to 128. 130. The method of embodiment 129, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy. 131. The method of any one of embodiments 129-130, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014, 1.1x1014, 1.2x1014, 1.3x1014, 1.4x1014, 1.5x1014, 1.6x1014, 1.7x1014, 1.8x1014, 1.9x1014, or 2x1014genome copies / kg. - 24 - 40928727338013.0045P1 132. The method of any one of embodiments 129-130, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014genome copies / kg. 133. The method of any one of embodiments 129-130, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 2x1014genome copies / kg. 134. A pharmaceutical composition for use in treating a dystrophinopathy, decreasing inflammation and / or fibrosis in a muscle, decreasing muscle degeneration or altering gait in a subject in need thereof comprising a therapeutically effective amount of a rAAV particle and a pharmaceutically acceptable carrier: wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes a microdystrophin protein consisting of dystrophin domains arranged from amino- terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, operably linked to a regulatory element that promotes expression in muscle, and flanked by AAV ITR sequences; wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg); and wherein the administering results in greater than 50 ng / mg microdystrophin protein to the muscle of the subject. 135. A method of treating a dystrophinopathy, decreasing inflammation and / or fibrosis in a muscle, decreasing muscle degeneration or altering gait in a subject in need thereof in a subject in need thereof, said method comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, - 25 - 40928727338013.0045P1 wherein the rAAV particle comprises an artificial genome comprising a transgene that encodes a microdystrophin protein consisting of dystrophin domains arranged from amino- terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, operably linked to a regulatory element that promotes expression in muscle, and flanked by AAV ITR sequences; wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg); and wherein the administering results in greater than 50 ng / mg microdystrophin protein in the muscle of the subject. 136. The composition of method of embodiments 134 or 135, wherein the subject is 1 year of age to less than 4 years of age or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8, including subjects in the Decline Phase, and has a confirmed mutation in the DMD gene. 137. The composition or method of embodiments 134 or 136 wherein the amount of microdystrophin in the muscle of the subject is measured by capillary-based Western assay method at 5 weeks, 10 weeks, 12 weeks, 20 weeks, or 26 weeks after the administration. 138. The composition or method of embodiments 134 to 137, wherein the CT comprises or consists of the proximal 194 amino acids of the C-terminus of dystrophin or at least the proximal portion of the C-terminus corresponding to human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 92 (UniProtKB-P11532) or at least the proximal portion of the C-terminus encoded by exons 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75. 139. The composition or method of any one of embodiments 134-138, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 1. - 26 - 40928727338013.0045P1 140. The composition or method of embodiment 139, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 20. 141. The composition or method of embodiments 134-137, wherein the CT comprises or consists of the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence which comprises the α1-syntrophin binding site but not the dystrobrevin binding site. 142. The composition or method of embodiments 134, 135, 136, 137 or 141, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. 143. The composition or method of embodiment 142, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 81. 144. The composition or method of any of embodiments 134-143, wherein the transcription regulatory element comprises a muscle-specific promoter. 145. The composition or method of embodiment 144, wherein the muscle-specific promoter is a skeletal, smooth, or cardiac muscle specific promoter. 146. The composition or method of any one of embodiments 144 or 145, wherein the muscle specific promoter is SPc5-12 or a transcriptionally active portion or mutant thereof. 147. The composition or method of embodiment 146, wherein the promoter consists of the nucleic acid sequence of SEQ ID NO: 39. 148. The composition or method of any one of embodiments 134-147, wherein the transgene comprises a polyadenylation signal 3’ of the nucleic acid sequence encoding the microdystrophin protein. 149. The composition or method of any one of embodiments 134-148, wherein the transgene comprises an intron sequence between the promoter and the microdystrophin coding sequence. 150. The composition or method of embodiment 149, wherein the intron sequence is a VH4 intron sequence (SEQ ID NO: 41) 151. The composition or method of any one of embodiments 134-140 and 144-150, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO:53 - 27 - 40928727338013.0045P1 152. The composition or method of any one of embodiments 134-137 and 141-150, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 82. 153. The composition or method of any of embodiments 134-152, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77. 154. The composition or method of embodiment 153, wherein the rAAV is an AAV8 serotype. 155. The composition or method of any one of embodiments 134-154, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy. 156. The composition or method of any one of embodiments 134-155, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014genome copies / kg or 2x1014genome copies / kg. 157. The composition or method of any one of embodiments 134-156 wherein the pharmaceutical composition is administered intravenously. 158. The composition or method of any one of the foregoing wherein the subject after said administration does not experience any serious adverse event after said administration, including central or peripheral neurotoxicity, drug-induced injury, thrombocytopenia, myocarditis or myositis. 159. A method of reducing and / or preventing contraction-induced muscle damage or to increase muscle resilience in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising: administering a therapeutically effective amount of a microdystrophin comprising at least the portion of the C-terminal domain (CT) comprising an α1-syntrophin binding site wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject, and the muscles of the subject exhibit greater than 80%, or 85%, or 90%, or 95% of the force generation of the muscles of a subject that does not have a DMD mutation or dystrophinopathy. 160. A method of reducing and / or preventing contraction-induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising: - 28 - 40928727338013.0045P1 administering to the subject a therapeutically effective amount of a microdystrophin comprising at least the portion of the C-terminal domain (CT) comprising an α1-syntrophin binding site wherein the administering results in delivery of microdystrophin protein to the muscle of the subject, and the muscles of the subject generate about 50% more force relative to a subject administered a microdystrophin without the CT portion comprising an α1-syntrophin binding site. 161. A method of reducing and / or preventing muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising administering a therapeutically effective amount of a microdystrophin comprising at least the portion of the C- terminal domain (CT) comprising an α1-syntrophin binding site wherein the administering results in delivery of microdystrophin protein to the muscle of the subject, and the muscles of the subject generate about 50% more force relative to the muscles of a natural history matched control subject. 162. A method of reducing or preventing contraction-induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising administering a therapeutically effective amount of a microdystrophin comprising a means for restoring DAPC in muscle cells of the subject, wherein the administering results in delivery of microdystrophin protein to the muscle of the subject and the subject has improved timed function test or NSAA results or PDMS-3 or caregiver reported improved function results relative to a natural history matched control subject. 163. A method of reducing or preventing contraction induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising administering a therapeutically effective amount of a microdystrophin comprising a means for stabilization of a microdystrophin protein in muscle cells by interaction between a microdystrophin C-terminal domain (CT) and alpha-syntrophin, or a means for protecting muscle against contraction-induced damage or a means for enabling muscle recovery from contraction-induced damage, wherein the administering results in delivery of microdystrophin protein to the muscle of the subject and the subject has improved timed function test or NSAA results or PDMS-3 or caregiver reported improved functional results relative to a natural history matched control subject. - 29 - 40928727338013.0045P1 164. The method of any one of embodiments 159 to 161 wherein the subject has improved or stable timed function test or NSAA results or PDMS-3 or caregiver reported improved function results relative to a natural history matched control subject. 165. The method of any one of embodiments 159 to 164 wherein the subject has improved or stable timed test function or NSAA results or PDMS-3 or caregiver reported improved function results at 12 weeks, 24 weeks, 1 year, 2 years, or 3 years after administration relative to results prior to administration. 166. The method of any one of embodiments 162 to 165, wherein the increase in NSAA score is more than 2. 167. The method of any one of embodiments 162 to 166, wherein the improvement is greater than the minimal clinically important difference (MCID) relative to a natural history matched control of MCID standard. 168. The method of any one of embodiments 159 to 167, wherein the microdystrophin consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2- R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site 169. The method of any one of embodiments 159 to 168, wherein the CT comprises or consists of the proximal 194 amino acids of the C-terminus of dystrophin or at least the proximal portion of the C-terminus corresponding to human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 92 (UniProtKB-P11532) or at least the proximal portion of the C-terminus encoded by exons 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75. 170. The method of any one of embodiments 159 to 169, wherein the CT comprises or consists of the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence which comprises the α1-syntrophin binding site but not the dystrobrevin binding site. - 30 - 40928727338013.0045P1 171. The method of any one of embodiments 159 to 170, wherein the CT domain does not comprise a C-terminal DTM amino acid sequence. 172. The method of any one of embodiments 159 to 171, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 1. 173. The method of embodiment 172, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 20. 174. The method of any one of embodiments 159 to 171, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. 175. The method of embodiment 174, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 81. 176. The method of any one of embodiments 159 to 175, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or X-linked dilated cardiomyopathy, is a DMD with a mutation of the DMD gene, including with a mutation in exon 18 or higher (exon 18 to 79) of the DMD gene, or a mutation in exon 12 to 17 of the DMD gene, or has a mutation in exon 12 to 79 of the DMD gene, or is a mutation in any exon except in exons 8, 9, and / or 10 of the DMD gene. 177. The method of any one of embodiments 159 to 176, wherein the subject is 1 to 13 years of age. 178. The method of embodiment 177, wherein the subject is 1 to 4 years of age. 179. The method of embodiment 177, wherein the subject is 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age, including subjects in the Decline Phase. 180. The method of any one of embodiments 159 to 179, wherein the microdystrophin is administered by a recombinant adeno-associated virus (rAAV) particle, wherein the rAAV particle comprises i) a transgene that encodes a microdystrophin protein and ii) a capsid protein. 181. The method of embodiment 180, wherein the transgene further comprises a transcription regulatory element that promotes expression in muscle operably linked to the nucleic acid sequence that encodes the microdystrophin protein. - 31 - 40928727338013.0045P1 182. The method of embodiment 181, wherein the transcription regulatory element comprises a muscle-specific promoter. 183. The method of embodiment 182, wherein the muscle-specific promoter is a skeletal, smooth, or cardiac muscle specific promoter. 184. The method of embodiment 183, wherein the muscle specific promoter is SPc5-12 or a transcriptionally active portion or mutant thereof. 185. The method of embodiment 184, wherein the SPc5-12 consists of the nucleic acid sequence of SEQ ID NO: 39. 186. The method of any one of embodiments 180 to 185, wherein the transgene comprises a polyadenylation signal 3’ of the nucleic acid sequence encoding the microdystrophin protein. 187. The method of any one of embodiments 180 to 186, wherein the transgene comprises an intron sequence between the promoter and the microdystrophin coding sequence. 188. The method of any one of embodiments 180 to 187, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 53 189. The method of any one of embodiments 180 to 188, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 82. 190. The method of any one of embodiments 180 to 189, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77. 191. The method of any one of embodiments 180 to 190, wherein the rAAV is an AAV8 serotype. 192. The method of any one of embodiments 180 to 191, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014genome copies / kg or 2x1014genome copies / kg. 193. The method of any one of embodiments 180 to 192, wherein the pharmaceutical composition is administered intravenously. - 32 - 40928727338013.0045P1 194. The method of any one of embodiments 180 to 189 or 192 or 193, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is at least 95% identical to AAV9, AAVrh74, AAVhu37, or AAVhu32. 195. The method of any one of embodiments 180 to 189 or 192 or 193, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is identical to AAV9, AAVrh74, AAVhu37, or AAVhu32. 196. A pharmaceutical composition for use in treating or ameliorating symptoms of a dystrophinopathy in a subject suffering from a dystrophinopathy, including DMD, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle is an AAV8 particle and comprises an artificial genome having a nucleotide sequence of SEQ ID NO: 53 (AAV8-RGX-DYS1), wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg) to a subject of 4 to 12 years of age, wherein the subject does not experience a serious adverse event after administration, and wherein the subject has improved or stable timed test function results, NSAA results, or PODCI results relative to results prior to administration or a matched natural history control. 197. A pharmaceutical composition for use in treating or ameliorating symptoms of a dystrophinopathy, including DMD, in a subject suffering from a dystrophinopathy, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle is an AAV8 particle and comprises an artificial genome having a nucleotide sequence of SEQ ID NO: 53 (AAV8-RGX-DYS1), wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg) to a subject of 1 to less than 4 years of age, wherein the subject does not experience a serious adverse event after administration, and wherein the subject has improved or stable timed test function results, PDMS-3 results, or PODCI results relative to results prior to administration or a matched natural history control. - 33 - 40928727338013.0045P1 198. A pharmaceutical composition for use in treating or ameliorating symptoms of a dystrophinopathy in a subject suffering from a dystrophinopathy, including DMD, said pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle is an AAV8 particle and comprises an artificial genome having a nucleotide sequence of SEQ ID NO: 53 (AAV8-RGX-DYS1), wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg) to a subject in the Decline Phase or whose matched natural history controls are in the Decline Phase, wherein the subject does not experience a serious adverse event after administration, and wherein the subject has improved or stable timed test function results, NSAA results, or PODCI results relative to results prior to administration or a matched natural history control. 199. A method of treating or ameliorating symptoms of a dystrophinopathy in a subject suffering from a dystrophinopathy, including DMD, comprising administering a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle is an AAV8 particle and comprises an artificial genome having a nucleotide sequence of SEQ ID NO: 53 (AAV8-RGX-DYS1), wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg) to a subject of 4 to 12 years of age, wherein the subject does not experience a serious adverse event after administration, and wherein the subject has improved or stable timed test function results, NSAA results, or PODCI results relative to results prior to administration or a natural history control. 200. A method of treating or ameliorating symptoms of a dystrophinopathy in a subject suffering from a dystrophinopathy, including DMD, said method comprising administering a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle is an AAV8 particle and comprises an artificial genome having a nucleotide sequence of SEQ ID NO: 53 (AAV8-RGX-DYS1), wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg) to a subject of 1 to less than 4 years of age, wherein the subject does not experience a serious adverse event after administration, - 34 - 40928727338013.0045P1 and wherein the subject has improved or stable timed test function results, NSAA results, PDMS- 3 score or PODCI results relative to results prior to administration or a matched natural history control. 201. A method of treating or ameliorating symptoms of a dystrophinopathy in a subject suffering from a dystrophinopathy, including DMD, said method comprising administering a therapeutically effective amount of a recombinant adeno-associated vector (rAAV) particle and a pharmaceutically acceptable carrier, wherein the rAAV particle is an AAV8 particle and comprises an artificial genome having a nucleotide sequence of SEQ ID NO: 53 (AAV8-RGX-DYS1), wherein the therapeutically effective amount of the rAAV particle is administered intravenously at a dose of 1x1014or 2x1014genome copies per kilogram (GC / kg) to a subject in the Decline Phase or whose matched natural history controls are in the Decline Phase, wherein the subject does not experience a serious adverse event after administration, and wherein the subject has improved or stable timed test function results, NSAA results, or PODCI results relative to results prior to administration or a matched natural history control. 4. BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGs.1A-1B. Illustration of the sarcolemma showing interaction among (A) the RGX- DYS1 microdystrophin (which has a C Terminal domain containing dystrobrevin and ^^- syntrophin binding sites (as well as ^^^syntrophin binding sites), and (B) the wild-type dystrophin protein and the dystrophin-associated protein complex (DAPC) with the actin cytoskeleton. It is envisioned that RGX-DYS1 having at least dystrobrevin and ^^^syntrophin binding sites, will partly recruit and anchor nNOS to the sarcolemma through α1-syntrophin. Syn: Syntrophin; Dbr: Dystrobrevin; CR: Cysteine rich domain; nNOS: Neuronal nitric oxide synthase; DG: Dystroglycan; H: hinge; R: spectral-like repeat; SG: Sarcoglycan.

[0015] FIG. 2. illustrates vector gene expression cassettes, otherwise referred to as microdystrophin constructs or transgenes, for use in a cis plasmid for gene therapy that result in AAV recombinant genomes. DNA length for each component and complete transgene are listed for each construct. SPc5-12: synthetic muscle-specific promoter; CT1.5: truncated / minimal CT domain containing 140 amino acids of the CT domain (SEQ ID NO: 83) including an α1- syntrophin binding site but not a dystrobrevin binding site; VH4: human immunoglobin heavy - 35 - 40928727338013.0045P1 chain variable region intron; ABD: actin binding domain; H: hinge; R: rod; CR: cysteine rich domain; CT: C-terminal domain; smPA: small polyA; ABD: Actin Binding Domain 1 (ABD1).

[0016] FIG. 3 shows RGX- DYS1 microdystrophin protein expression at Week 6 in the diaphragm, gastrocnemius and cardiac muscles collected from wild-type and mdx mice. The protein levels in the tissues were measured by a capillary-based Western immunoassay. Bars show mean percent microdystrophin ±SEM. Wild-type mice (n = 3) and vehicle control mdx mice (n = 5) did not show any quantifiable RGX- DYS1 microdystrophin protein level and all below level of quantitation (BLQ) values (< level of quantitation (LOQ )of 5 ng / mg protein) were treated as zero in the calculation of mean; RGX- DYS1 administered mdx mice (n = 4-5 per dose group).

[0017] FIGs. 4A-4C illustrate microdystrophin stability of RGX-DYS1 (μDys-CT194) compared to RGX-DYS3 (μDys-CT48) as measured by half-life determination according to (A) live cell fluorescent pulse-chase imaging, (B) protein gel fluorescence, and (C) cycloheximide- chase methods.

[0018] FIGs. 5A-5B illustrate both RGX-DYS1 doses (1x1014and 2x1014GC / kg) were significantly different from mdx vehicle treated animals for both (A) forelimb grip strength and (B) treadmill exhaustion; by comparison RGX-DYS1 treated animals were not significantly different from wild type vehicle treated controls. Data was checked for normality; a Two-way ANOVA with a Tukey post-hoc analysis (α = 0.05) was performed to correct for multiple comparisons.

[0019] FIG. 6 shows detectable microdystrophin by a Capillary Western blot (JESS) from muscle biopsies of two clinical trial participants.

[0020] FIGs. 7A-7B show the LC-MS analyte peaks of microdystrophin in (A) Participant 1 and (B) Participant 2.

[0021] FIGS.8A-8B illustrate the detectable microdystrophin by (A) a Capillary Western blot (JESS) and (B) LC-MS analyte peaks of expressed microdystrophin from a muscle biopsy of a third clinical trial participant at week 12 post administration of RGX-DYS1 at 1x1014GC / kg. Note the RGX-DYS1 microdystrophin (µDys) as expressed in tissue at 3 months measures ~153kDa (lane 4) and control µDys (tagged recombinant protein) measures at ~165kDa (lane 1 and 2) as detected by JESS and shown in blot A. - 36 - 40928727338013.0045P1

[0022] FIGS. 9A-9B illustrates (A) the domain structure of RGX-DYS1-encoded microdystrophin protein compared to a natural shortened dystrophin such as found in individuals with a Becker muscular dystrophy phenotype. (B) Another view illustrates a microdystrophin of the RGX-DYS1 structure having a functional C-terminus of 194 amino acids compared to a C- terminus of 48 amino acids overlapped with the exon numbers encoding each domain. An illustration of full-length normal dystrophin is also depicted in each illustration for comparison.

[0023] FIG. 10 illustrates mean NSAA trajectory over time in patients with DMD as adapted from Muntoni et al.2019, PLoS ONE 14(9): e0221097.

[0024] FIGS.11A-11C illustrate NSAA scores for (A) dose level 1 participants (n=3) 12 months post-RGX-202 administration relative to natural history external control (n=4) and (B) dose level 2 participants (n=5) 9 months post-RGX-202 administration relative to natural history external control (n=35, 12-year-old patient did not have matching natural history external controls) and (C) dose level 2 participants (n=4) 12 months post-RGX-202 administration relative to natural history external control (n=26).

[0025] FIGS. 12A-12C illustrate the results of timed function tests TTStand, 10MWR, and TTClimb for (A) dose level 1 participants 12 months post-RGX-202 administration relative to natural history external control and (B) dose level 2 participants 9 months post-RGX-202 administration relative to natural history external control and (C) dose level 2 participants 12 months post-RGX-202 administration relative to natural history external control. TTStand: dose level 1 participants n=3, natural history external controls n=17; dose level 2 participants at 9 months n=5, natural history external controls, n=65; dose level 2 participants at 12 months n=4, natural history external controls, n=73. 10MWR: dose level 1 participants n=3, natural history external controls n=18; dose level 2 participants at 9 months n=5, natural history external controls, n=65; dose level 2 participants at 12 months n=4, natural history external controls, n=73. TTClimb: dose level 1 participants n=2, natural history external controls n=15; dose level 2 participants at 9 months n=5, natural history external controls, n=65; dose level 2 participants at 12 months n=4, natural history external controls, n=73.

[0026] FIGS.13A-13B illustrate timed task velocity changes for (A) dose level 1 participants 12 months post-RGX-202 administration compared to MCID benchmarks described in Duong et al. TTStand velocity, n=3, 10MWR velocity n=3, TTClimb velocity, n=2 and (B) dose level 2 - 37 - 40928727338013.0045P1 participants 12 months post-RGX-202 administration compared to MCID benchmarks described in Duong et al. TTStand velocity, n=4, 10MWR velocity n=4, TTClimb velocity, n=4.

[0027] FIGS.14A-14F illustrate force-frequency or eccentric contraction (ECC) measurements. Plots for (A) in vivo plantarflexion force measured 4 weeks post-AAV.hu32-Microdystrophin vector administration, (B) force drop following repeated eccentric contractions, and (C) force following 20 ECCs relative to maximal force prior to ECC [Bl10 Vehicle, n = 6; mdx Vehicle; n = 5, μDysCT48 (RGX-DYS3) n = 12; μDysCT194 (RGX-DYS1), n= 9. *, p < 0.05; **, p < 0.01; ***, p < 0.001]. (D) Quantification of Evans blue-positive areas in naïve or ECC challenged gastrocnemius muscles. (E) Relationship between percentage of muscle fibers expressing μDys and level of Evans blue infiltration following ECC protocol. (F) Relationship between force drop between eccentric contractions 1 and 20 and Evans blue dye infiltration [regression performed on grouped data for D-F from mdx Vehicle (light gray) and mdx + μDysCT194; n = 4, mdx vehicle; n = 8, mdx + μDysCT194].

[0028] FIGS.15A-15B illustrate open field activity and treadmill data as represented in Volcano plots (A), with the change in the recorded value relative to wild-type (WT) controls shown on the x-axis and the p-value associated with that change shown on the y-axis. Dotted lines show a p- value < 0.05 and relative parameter values <0.5 and >2.0. Red boxes (#1) show activities significantly reduced relative to WT, while green boxes (#2) show activities significantly increased. Treadmill running fatigue and exhaustion distances were also measured at 6 or 12 weeks post-vector administration (B). *, p < 0.05; **, p < 0.01. n = 5, WT vehicle; n = 7, mdx vehicle; n = 7, μDysCT194 (RGX-DYS1).

[0029] FIGS. 16A-16D illustrate µDys expression 12-weeks post vector administration as represented in (A) Capillary Western analysis of dystrophin or µDys protein in gastrocnemius and (B) quadriceps muscles. Levels of µDys were quantified relative to dystrophin levels averaged from C57Bl10 controls. (C) Normalized mRNA expression and (D) AAV biodistribution from skeletal muscle and heart of mice administered AAV-µDys. Data are presented as mean ± S.E.M. n = 13, µDysCT48; n = 15, µDysCT194.

[0030] FIGS. 17A-17C illustrate quantification of (A) α-Syntrophin, (B) α-Dystrobrevin, and (C) nNOS fluorescent intensity specifically at the plasma membrane in mdx + µDysCT48 and mdx + µDysCT194 gastrocnemius. Data are presented as mean ± S.E.M. *, p < 0.05, ***, p<0.001. n - 38 - 40928727338013.0045P1 = 8, C57Bl10; n = 8, mdx; n = 13, µDysCT48; n = 15, µDysCT194. In all images, scale bar = 200µm.

[0031] FIG. 18 illustrates the design of studies used to compare µDysCT48 and µDysCT194. In Study 1 (top) force and force drop following eccentric contractions (5 total) were measured from isolated EDL muscles in vitro during the 13thstudy week. n = 8, C57Bl10 Vehicle; n = 8, mdx Vehicle; n = 13, mdx + µDysCT48; n = 15, mdx + µDysCT194. In Study 2 (middle), plantarflexion force was measured bi-weekly except for Study Weeks 5 and 11, where grip strength and treadmill running were evaluated. At the beginning of Study Week 13, plantarflexion force was measured before and after eccentric contractions (20 total). Starting one day after the eccentric contraction protocol, force was measured daily for an additional 4 days. n = 8, C57Bl10 Vehicle; n = 8, mdx Vehicle; n = 16, mdx + µDysCT48; n = 16, mdx + µDysCT194. In Study 3 (bottom), mice were injected with Evans blue dye solution on Study Day 28, assessed for plantarflexion force and subjected to eccentric contraction (20 total) on Study Day 29, with tissue harvest occurring on Study Day 30. n = 6, C57Bl10 Vehicle; n = 6, mdx Vehicle; n = 12, mdx + µDysCT48; n = 12, mdx + µDysCT194.

[0032] FIGS. 19A-19E illustrates functional outcomes and recovery from eccentric exercise. (A) Force, expressed as a percentage of initial force, following a series of eccentric contractions in vivo 12-weeks post-dosing. (B) Maximal force generated by plantar flexors following 20 eccentric contractions, normalized to bodyweight. (C) Bodyweight-normalized maximal force generated by plantar flexors immediately before, immediately after, and daily for 4 days post eccentric contraction protocol. (D) Distance to exhaustion on an accelerating treadmill test 4- and 10-weeks post-dosing. (E) Grip strength measurements taken 4- and 10-weeks post-dosing, normalized to mouse bodyweight. n = 8, C57Bl10; n = 7-8, mdx; n = 16, µDysCT48; n = 16, µDysCT194. In all panels, *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0033] FIGS. 20A-20F illustrate plantarflexion force and Evans blue dye permeability in gastrocnemius muscles with or without eccentric contraction. (A) Maximal in vivo plantarflexion force normalized to mouse bodyweight 4 weeks post-AAV administration. n = 5, C57Bl10; n = 5, mdx; n = 12, µDysCT48; n = 9, µDysCT194. (B) Force, expressed as a percentage of initial force, following a series of eccentric contractions in vivo. n = 5, C57Bl10; n = 6, mdx; n = 12, µDysCT48; n = 9, µDysCT194. (C) Maximal force generated by plantar flexors following 20 eccentric - 39 - 40928727338013.0045P1 contractions, normalized to bodyweight. n = 5, C57Bl10; n = 6, mdx; n = 12, µDysCT48; n = 9, µDysCT194. (D) Representative whole tissue images showing permeation of Evans blue dye into muscle fibers of naïve (uncontracted, top) or eccentrically contracted (middle) gastrocnemius muscle. High resolution insets showing dye infiltration are also shown (bottom). (E) Percentage of Evans blue-positive muscle fibers in naïve (uncontracted, left) or eccentrically contracted (right) gastrocnemius muscle. n = 4, C57Bl10; n = 4, mdx; n = 9, µDysCT48; n = 8, µDysCT194. (F) Comparison of percentages of mdx muscle fibers expressing dystrophin / µDys and percentages of Evans blue-positive fibers following eccentric contraction protocol. Asterisk denotes a significantly non-zero slope for µDysCT194. n = 9, µDysCT48; n = 8, µDysCT194. Pearson correlation coefficients and associated p-values are also shown. Data are presented as mean ± S.E.M. In all panels, *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0034] FIG. 21 illustrates improvements in caregiver reported function in dose level 2 participants (n=4) 12 months post-RGX-202 administration relative to natural history external control, based on Transfer and Basic Mobility, Sports and Physical Functioning, and Global Function. 5. DETAILED DESCRIPTION

[0035] Provided are methods of administering gene therapy vectors, particularly AAV vectors, comprising recombinant genomes (and the cis plasmid for producing the rAAV with the recombinant genome) with transgenes encoding microdystrophin proteins operably linked to regulatory elements for expression, for example, in muscle cells, for treatment of dystrophinopathies, including but not limited to Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and X-linked dilated cardiomyopathy. The microdystrophin proteins encoded by the transgene consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, and may comprise or consist of at least the proximal portion of the C-terminus encoding human dystrophin amino acid residues 3361-3354 of SEQ ID - 40 - 40928727338013.0045P1 NO: 92 (UniProt-KB-P111532), or the amino acid sequence of SEQ ID NO: 16, or alternatively, the CT is a truncated CT comprising or consisting of SEQ ID NO: 83, and the microdystrophins may have amino acid sequences of SEQ ID NO: 1 (RGX-DYS1) or SEQ ID NO: 79 (RGX-DYS5). In some embodiments, the microdystrophins may have amino acid sequence of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. In embodiments, the microdystrophin comprises a CT domain or portion thereof comprising an α1-syntrophin binding domain but does not comprise at the C-terminus the amino acid sequence DTM. Also provided are microdystrophins, and constructs and viral particles encoding microdystrophins comprising a means for enhancing DAPC restoration or a means for stabilizing interaction of the microdystrophin with α-dystrobrevin and / or nNOS or a means for protecting muscle against contraction-induced damage or a means for enabling muscle recovery from contraction-induced damage. The transgenes further comprise regulatory sequences, including, for example, a muscle specific promoter, such as SPc5-12 (SEQ ID NO: 39) and a polyadenylation signal, such as the small polyA signal (SEQ ID NO: 42). Exemplary constructs (including cis plasmids and AAV genomes) are depicted, for example, in FIG.2 and may have nucleotide sequences of (SEQ ID NO: 53 for RGX-DYS1 and SEQ ID NO: 82 for RGX-DYS5) and, may include ITR sequences (including, AAV2 ITR sequences such as found in the nucleotide sequences of SEQ ID NO: 53 and SEQ ID NO: 82). The gene therapy vectors may be AAV8 or AAV9 serotype vectors. In particular embodiments, the gene therapy vector is an AAV8 comprising an artificial genome having the nucleotide sequence of SEQ ID NO: 53 (RGX-DYS1), and may be referred to as AAV8-RGX-DYS1. The gene therapy vectors may be administered to subjects 1 year of age to less than 4 years of age or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8 years of age, including subjects in the Decline Phase or whose natural history peers are in the Decline Phase.

[0036] Based upon pharmacology studies conducted in mdx mice (E.g., Examples 39 infra and those disclosed in PCT International Publication No WO 2022 / 232141 A1, published November 3, 2022, which is incorporated herein by reference,) and human dosing studies (Example 6, infra), therapeutically effective single doses for peripheral (including intravenous) administration of the rAAVs containing the transgenes described herein (including RGX-DYS1) (including, in embodiments, AAV8-RGX-DYS1 and other microdystrophins with an extended CT domain) are between 5x1013GC / kg to 1x1015GC / kg and include dosages within that range, including 1x1014, - 41 - 40928727338013.0045P1 1.1×1014, 1.2x1014, 1.3x1014, 1.4x1014, 1.5x1014, 1.6x1014, 1.7x1014, 1.8x1014, 1.9x1014, 2x1014, 2.1x1014, 2.2x1014, 2.3x1014, 2.4x1014, 2.5x1014, 2.6x1014, 2.7x1014, 2.8x1014, 2.9x1014, or 3x1014GC / kg. Administration of such therapeutically effective dosages of the rAAVs comprising transgenes described herein (including AAV8-RGX-DYS1) results in amelioration of one or more indicators of dystrophinopathy disease, such as, reduction in creatine kinase activity, reduction in muscle volume, muscle lesions, improvement in gait or ambulatory score (such as NSAA or PDMS-3 score) or other measure of strength or mobility within 12 weeks, 26 weeks, 52 weeks or longer from the administration. Preclinical studies in mice show that administration of a microdystrophin comprising the portion of the CT domain comprising the α1-syntrophin binding site i) significantly enhanced restoration of the DAPC in mdx mice, similarly to natural dystrophin; ii) increased the half-life of DYS1 microdystrophin protein, which remains in muscle fibers longer to strengthen muscle; and iii) protected against contraction-induced damage enabling better recovery relative to a microdystrophin that does not comprise the CT domain. Accordingly, the pharmaceutical compositions provided herein can be administered to a human subject suffering from a dystrophinopathy and / or having a DMD mutation to protect against and enable recovery from contraction-inducted muscle damage. Human clinical trial results provided herein show that administration of a microdystrophin encoding-AAV particle therapeutic as disclosed herein to a human subject results expression of the microdystrophin in the muscles of the subject and presence in muscle fibers (and sarcolemma localization), at 12 weeks, and in improvement in timed function tests (such as TTStand Velocity, 10MWR Velocity and TTClimb Velocity) and NSAA results relative to a natural history matched external control (matched for age and baseline disease, see for example subjects from D-RSC Data platform of the Critical Path Institute, and like natural history studies of DMD patients) and improvements that exceed the minimal clinically important difference (MCID) for DMD subjects (see, Duong et al., 2021, J. Neuromuscular Diseases 202:939-948) at 12 months after administration of the gene therapy. The results further show that the subjects, after administration, did not experience any serious adverse event after said administration, including central or peripheral neurotoxicity, drug-induced injury, thrombocytopenia, myocarditis or myositis. Subjects, including subjects from 1 to less than 4 years of age at administration can, within 12 months of administration, exhibit stable or improvement in - 42 - 40928727338013.0045P1 PDMS-3 relative to patients baseline score or natural history control, including within 2 standard deviations of the normative mean.

[0037] Accordingly, provided are methods of reducing and / or preventing contraction-induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising: administering to the subject a therapeutically effective amount of a microdystrophin comprising at least the portion of the CT comprising an α1-syntrophin binding site wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject, and the muscles of the subject generate about 50% more force relative to a subject administered a microdystrophin without the CT portion comprising an α1-syntrophin binding site.

[0038] Also provided are methods of reducing and / or preventing muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising administering a therapeutically effective amount of a microdystrophin comprising at least the portion of the CT comprising an α1-syntrophin binding site wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject and / or localization of the microdystrophin to the sarcolemma, and the muscles of the subject generate about 50% more force relative to the muscles of a natural history matched control subject.

[0039] Also provided are methods of improving symptoms of a dystrophin, including improvements or stability in time to function tests and NSAA score or PDMS-3, relative to patient baseline at administration or natural history peers, or caregiver reported improved function results and / or reducing or preventing contraction-induced muscle damage and promoting recovery from contraction-induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising administering a therapeutically effective amount of a microdystrophin protein comprising a means for restoring DAPC in muscle cells of the subject, wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject and the subject has improved or stable timed function test or NSAA results or PDMS-3 or caregiver reported improved function results relative to a natural history matched control subject.

[0040] Also provided are methods of improving symptoms of a dystrophin, including improvements or stability in time to function tests and NSAA score or PDMS-3 or caregiver reported improved function results, and / or reducing or preventing contraction induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method - 43 - 40928727338013.0045P1 comprising administering therapeutically effective amount of a microdystrophin protein comprising a means for stabilization of a microdystrophin protein in muscle cells by interaction between a microdystrophin C-terminal domain and alpha-syntrophin, wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject and / or localization of the microdystrophin to the sarcolemma and the subject has improved timed function test or NSAA results or caregiver reported improved function results relative to the subject prior to the administration and / or relative to a natural history matched control subject.

[0041] Also provided are methods of improving symptoms of a dystrophin, including improvements in time to function tests and NSAA score, or PDMS-3 or caregiver reported improved function results and / or reducing or preventing contraction induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising administering therapeutically effective amount of a microdystrophin protein comprising a means for protecting muscle against contraction-induced damage, wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject and / or localization of the microdystrophin to the sarcolemma and the subject has improved timed function test or NSAA results or caregiver reported improved function results relative to the subject prior to the administration and / or relative to a natural history matched control subject.

[0042] Also provided are methods of improving symptoms of a dystrophin, including improvements in time to function tests and NSAA score, or PDMS-3 or caregiver reported improved function results and / or reducing or preventing contraction induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said method comprising administering therapeutically effective amount of a microdystrophin protein comprising a means for enabling muscle recovery from contraction-induced damage, wherein the administering results in delivery of the microdystrophin protein to the muscle of the subject and / or localization of the microdystrophin to the sarcolemma and the subject has improved timed function test or NSAA results or caregiver reported improved function results relative to the subject prior to the administration and / or relative to a natural history matched control subject.

[0043] The methods provided herein result in the subjects experiencing no serious adverse events after administration, including central or peripheral neurotoxicity, drug-induced liver injury, thrombocytopenia, myocarditis, or myositis. - 44 - 40928727338013.0045P1

[0044] Accordingly, provided and described herein are methods of administering an rAAV, including an rAAV8, comprising a recombinant genome comprising a transgene encoding a microdystrophin, including the RGX-DYS1 construct, to a subject, including a human subject, in need thereof, wherein the administration is intravenous or other peripheral administration at a dosage of between 1×1014GC / kg to 2×1014GC / kg and include dosages within that range, including 1×1014, 1.1×1014, 1.2×1014, 1.3×1014, 1.4×1014, 1.5×1014, 1.6×1014, 1.7×1014, 1.8×1014, 1.9×1014, 2×1014GC / kg. Also provided are pharmaceutical compositions formulated for peripheral, including, intravenous, administration of the microdystrophin-encoding rAAV described herein.

[0045] Subjects in the foregoing methods are, in embodiments, 1 to less than 4 years of age, 4 to 5 years of age, 6 to 7 years of age, or 8 to 12 years of age, or greater than 8 years of age or in the Decline Phase (or where natural history peers are in the Decline Phase) at time of administration. 5.1. Definitions

[0046] The term “AAV” or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses. The AAV can be an AAV derived from a naturally occurring “wild- type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and / or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene. An example of the latter includes a rAAV having a capsid protein having a modified sequence and / or a peptide insertion into the amino acid sequence of the naturally-occurring capsid.

[0047] The term “rAAV” refers to a “recombinant AAV.” In some embodiments, a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.

[0048] The term “rep-cap helper plasmid” refers to a plasmid that provides the viral rep and cap genes and aids the production of AAVs from rAAV genomes lacking functional rep and / or the cap gene sequences.

[0049] The term “cap gene” refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus. For AAV, the capsid protein may be VP1, VP2, or VP3. - 45 - 40928727338013.0045P1

[0050] The term “rep gene” refers to the nucleic acid sequences that encode the non-structural protein needed for replication and production of virus.

[0051] The terms “nucleic acids” and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA / RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes. The nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double- stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.

[0052] Amino acid residues as disclosed herein can be modified by conservative substitutions to maintain, or substantially maintain, overall polypeptide structure and / or function. As used herein, “conservative amino acid substitution” indicates that: hydrophobic amino acids (i.e., Ala, Cys, Gly, Pro, Met, Val, lie, and Leu) can be substituted with other hydrophobic amino acids; hydrophobic amino acids with bulky side chains (i.e., Phe, Tyr, and Trp) can be substituted with other hydrophobic amino acids with bulky side chains; amino acids with positively charged side chains (i.e., Arg, His, and Lys) can be substituted with other amino acids with positively charged side chains; amino acids with negatively charged side chains (i.e., Asp and Glu) can be substituted with other amino acids with negatively charged side chains; and amino acids with polar uncharged side chains (i.e., Ser, Thr, Asn, and Gln) can be substituted with other amino acids with polar uncharged side chains.

[0053] The terms “subject”, “host”, and “patient” are used interchangeably. A subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), most preferably a human.

[0054] The term “therapeutically functional microdystrophin” means that the microdystrophin exhibits therapeutic efficacy in one or more of the assays for therapeutic utility described in Section 5.4 herein or in assessment of methods of treatment described in Section 5.5 herein. - 46 - 40928727338013.0045P1

[0055] The terms “subject”, “host”, and “patient” are used interchangeably. A subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), most preferably a human.

[0056] The terms “therapeutic agent” refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. A “therapeutically effective amount” refers to the amount of agent, (e.g., an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom. Further, a therapeutically effective amount with respect to an agent of the invention means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder. In embodiments, the therapeutic benefit is reducing the incidence of a disease condition, e.g., muscle damage, exhibiting improvement in timed functional tests or other tests of muscle function, or preventing the incidence of a disease condition relative to an age matched subject(s) who has not been treated, including natural history matched controls.

[0057] The term “prophylactic agent” refers to any agent which can be used in the prevention, reducing the likelihood of, delay, or slowing down of the progression of a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. A “prophylactically effective amount” refers to the amount of the prophylactic agent (e.g., an amount of product expressed by the transgene) that provides at least one prophylactic benefit in the prevention or delay of the target disease or disorder, when administered to a subject predisposed thereto. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent, reduce the likelihood of, or delay the occurrence of the target disease or disorder; or slow the progression of the target disease or disorder; the amount sufficient to delay or minimize the onset of the target disease or disorder; or the amount sufficient to prevent or delay the recurrence or spread thereof. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the exacerbation of symptoms of a target disease or disorder. Further, a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or when in combination with other agents, that provides at least one prophylactic benefit in the prevention or delay of the disease or disorder. - 47 - 40928727338013.0045P1

[0058] A prophylactic agent of the invention can be administered to a subject “pre-disposed” to a target disease or disorder. A subject that is “pre-disposed” to a disease or disorder is one that shows symptoms associated with the development of the disease or disorder, or that has a genetic makeup, environmental exposure, or other risk factor for such a disease or disorder, but where the symptoms are not yet at the level to be diagnosed as the disease or disorder. For example, a patient with a family history of a disease associated with a missing gene (to be provided by a transgene) may qualify as one predisposed thereto. Further, a patient with a dormant tumor that persists after removal of a primary tumor may qualify as one predisposed to recurrence of a tumor.

[0059] The term “CpG islands” means those distinctive regions of the genome that contain the dinucleotide CpG (e.g. C (cytosine) base followed immediately by a G (guanine) base (a CpG)) at high frequency, thus the G+C content of CpG islands is significantly higher than that of non-island DNA. CpG islands can be identified by analysis of nucleotide length, nucleotide composition, and frequency of CpG dinucleotides. CpG island content in any particular nucleotide sequence or genome may be measured using the following criteria: island size greater than 100, GC Percent greater than 50.0 %, and ratio greater than 0.6 of observed number of CG dinucleotides to the expected number on the basis of the number of Gs and Cs in the segment (Obs / Exp greater than 0.6). Obs / Exp CpG = Number of CpG * N / (Number of C * Number of G) where N = length of sequence.

[0060] Various software tools are available for such calculations, such as world-wide- web.urogene.org / cgi-bin / methprimer / methprimer.cgi, world-wide-web.cpgislands.usc.edu / , world-wide-web.ebi.ac.uk / Tools / emboss / cpgplot / index.html and world-wide- web.bioinformatics.org / sms2 / cpg_islands.html. (See also Gardiner-Garden and Frommer, J Mol Biol. 1987 Jul 20;196(2):261-82; Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics.2002 Nov;18(11):1427-31). In one embodiment the algorithm to identify CpG islands is found at www.urogene.org / cgi-bin / methprimer / methprimer.cgi. 5.2. Microdystrophin Transgenes

[0061] Dystrophin is a cytoplasmic protein encoded by the DMD gene, and functions to link cytoskeletal actin filaments to membrane proteins. Normally, the dystrophin protein, located primarily in skeletal and cardiac muscles, with smaller amounts expressed in the brain, acts as a - 48 - 40928727338013.0045P1 shock absorber during muscle fiber contraction by linking the actin of the contractile apparatus to the layer of connective tissue that surrounds each muscle fiber. In muscle, dystrophin is localized at the cytoplasmic face of the sarcolemma membrane.

[0062] The DMD gene is the largest known human gene. The most common mutations that cause DMD or BMD are large deletion mutations of one or more exons (60-70%), but duplication mutations (5-10%), and single nucleotide variants (including small deletions or insertions, single- base changes, and splice site changes accounting for approximately 25-35% of pathogenic variants in males with DMD and about 10-20% of males with BMD), can also cause pathogenic dystrophin variants. In DMD, mutations often lead to a frame shift resulting in a premature stop codon and a truncated, non-functional or unstable protein. Nonsense point mutations can also result in premature termination codons with the same result. While mutations causing DMD can affect any exon, exons 2-20 and 45-55 are common hotspots for large deletion and duplication mutations. In- frame deletions result in the less severe Becker muscular dystrophy (BMD), in which patients express a truncated, partially functional dystrophin.

[0063] Full-length dystrophin is a large (427 kDa) protein comprising a number of subdomains that contribute to its function. These subdomains include, in order from the amino-terminus toward the carboxy-terminus, the N-terminal actin-binding domain, a central so-called “rod” domain, a cysteine-rich domain, and lastly a carboxy-terminal domain or region. The rod domain is comprised of 4 proline-rich hinge domains (abbreviated H), and 24 spectrin-like repeats (abbreviated R) in the following order: a first hinge domain (H1), 3 spectrin-like repeats (R1, R2, R3), a second hinge domain (H2), 16 more spectrin-like repeats (R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19), a third hinge domain (H3), 5 more spectrin-like repeats (R20, R21, R22, R23, R24), and a fourth hinge domain (H4) (including the WW domain). Following the rod domain are the cysteine-rich domain, and the COOH (C)-terminal (CT) domain.

[0064] A shortened dystrophin, or microdystrophin, may ideally contain those domains that enhance or improve its ability to associate with the sarcolemma and Dystrophin Associated Protein Complex (DAPC). Several Becker phenotypes have been studied and elucidated the corresponding mutated dystrophin gene sequences that result in a shortened dystrophin of approximately half the normal size of dystrophin (FIG. 9A), however the patient presented with a very mild muscular phenotype (England, SB, et al., 1990, Nature 343:180-182). - 49 - 40928727338013.0045P1 5.2.1 Microdystrophins Encoded by the Transgenes

[0065] Encoded by the transgenes provided herein for the methods of the invention are microdystrophins that consist of dystrophin domains arranged amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is a hinge 4 region of dystrophin, CR is a cysteine-rich region of dystrophin and CT is the C terminal domain (and comprises at least the portion of the CT domain containing the α1-syntrophin binding site, including SEQ ID NO: 84). Table 1 below has the amino acid sequences for these components, in particular from the full length human DMD protein (UniProtDB-11532, which is incorporated by reference herein) and they are encoded by the nucleotide sequences in Tables 3 and 4 (including the wild type and codon optimized sequences).

[0066] To overcome the packaging limitation that is typical of AAV vectors, many of the microdystrophin genes developed for clinical use are lacking the CT domain. Several researchers have indicated that the DAPC does not even require the C-terminal domain in order to assemble or that the C-terminus is non-essential [Crawford, et al., J Cell Biol, 2000, 150(6):1399–1409; and Ramos, J.N, et al. Molecular Therapy 2019, 27(3):1-13]. However, overexpression of a microdystrophin gene containing helix 1 of the coiled-coil motif of the CT domain in skeletal muscle of mdx mice increased the recruitment α1-syntrophin and α-dystrobrevin, which are members of DAP complex, serving as modular adaptors for signaling proteins recruited to the sarcolemma membrane (Koo, T., et al., Delivery of AAV2 / 9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of ^^-syntrophin and ^-dystrobrevin in skeletal muscles of mdx mice. Hum Gene Ther, 2011.22(11): p. 1379-88). Overexpression of the longer version of microdystrophin also improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice as compared with the shorter version (Koo, T., et al.2011, supra). The CT domain does play a role in the formation of the Dystrophin Associated Protein Complex (DAPC) (see FIG.1B). - 50 - 40928727338013.0045P1

[0067] The CT domain of dystrophin contains two polypeptide stretches that are predicted to form α-helical coiled coils similar to those in the rod domain (see H1 indicated by single underlining and H2 indicated by double underlining in SEQ ID NO: 16 in Table 1 below). Each coiled coil has a conserved repeating heptad (a,b,c,d,e,f,g)n similar to those found in leucine zippers where leucine predominates at the “d” position. This domain has been named the CC (coiled coil) domain. The CC region of dystrophin forms the binding site for dystrobrevin and may modulate the interaction between ^^^syntrophin and other dystrophin-associated proteins.

[0068] Both syntrophin isoforms, ^^^syntrophin and ^^^syntrophin, are thought to interact directly with dystrophin through more than one binding site in dystrophin exons 73 and 74 (Yang et al, JBC 270(10):4975-8 (1995)). ^1- and ^1-syntrophin bind separately to the dystrophin C- terminal domain, and the binding site for ^1-syntrophin reportedly resides at least within the amino acid residues 3447 to 3481, while that for ^1-syntrophin has been reported to reside within the amino acid residues 3495 to 3535 (as numbered in the DMD protein of UniProtDB-11532 (SEQ ID NO: 92), see also Table 1, SEQ ID NO: 16, italic). Alpha1- (^1-) syntrophin and alpha- syntrophin are used interchangeably throughout.

[0069] Microdystrophins disclosed herein were found to bind to and recruit nNOS, as well as alpha-syntrophin, alpha-dystrobrevin and beta-dystroglycan. Binding to nNOS, in the context of a microdystrophin including a C-terminal domain of dystrophin binding to nNOS, means that the microdystrophin expressed in muscle tissue was determined by immunostaining with appropriate antibodies to identify each of alpha-syntrophin, alpha-dystrobrevin, and nNOS in or near the sarcolemma in a section of the transduced muscle tissue. In certain embodiments, the microdystrophin protein has a C-terminal domain (including a portion thereof) that “increases binding” to ^^^syntrophin, β-syntrophin and / or dystrobrevin compared to a comparable microdystrophin that does not contain the C-terminal domain (but has the same amino acid sequence otherwise, that is a “reference microdystrophin protein”), meaning that the DAPC is stabilized or anchored to the sarcolemma, to a greater extent than a reference microdystrophin that does not have the C-terminal domain (but has the same amino acid sequence otherwise as the microdystrophin), as determined by greater levels of one or more DAPC components in the muscle membrane by immunostaining of muscle sections or western blot analysis of muscle tissue lysates or muscle membrane preparations for one of more DAPC components, including ^^-syntrophin, - 51 - 40928727338013.0045P1 β-syntrophin, ^-dystrobrevin, ^-dystroglycan or nNOS in mdx mouse muscle treated with the microdystrophin having the C-terminal domain, as compared to the mdx mouse muscle treated with the reference microdystrophin protein (having the same sequence and dystrophin components except not having the C-terminal domain or having a minimal 48 amino acids proximal to the CR domain) (see Example 9, Section 6.9, infra).

[0070] In some embodiments, the microdystrophin including a C-terminal domain of dystrophin comprises an α1-syntrophin binding site and / or a dystrobrevin binding site in the C-terminal domain. In some embodiments, the C-terminal domain comprising an ^^^syntrophin binding site is a truncated C-terminal domain. The ^^^syntrophin binding site functions in part to recruit and anchor nNOS to the sarcolemma through α1-syntrophin (See FIGs.1A and 1B).

[0071] The embodiments described herein can comprise all or a portion of the CT domain comprising the Helix 1 of the coiled-coil motif. The C Terminal sequence may be defined by the coding sequence of the exons of the DMD gene, including at least exons 70 to 74, and a portion of exon 75 (in particular, the nucleotide sequence encoding the first 36 amino acids of the amino acid sequence encoded by exon 75, or by the sequence of the human DMD protein, for example, the sequence of UniProtKB-P11532 (SEQ ID NO: 92) (the CT of DYS1 is or corresponds to amino acids 3361 to 3554 of the UniProtKB-P11532 sequence), or comprising binding sites for dystrobrevin and / or ^^^syntrophin (indicated in Table 1, SEQ ID NO: 16). In certain embodiments, the CT domain comprises the 194 C-terminal amino acids of the DMD protein, for example, residues 3361 to 3554 of the amino acid sequence of UniProtKB-P11532 (SEQ ID NO: 92), the amino acids encoded by exons 70 to 74, and the nucleotide sequence encoding the first 36 nucleotides of the nucleotide sequence of exon 75 of the DMD gene, or the amino acid sequence of SEQ ID NO: 16 (see Table 1). For example, RGX-DYS1 (also μDys-CT194) has the 194 amino acid CT sequence of SEQ ID NO: 16, RGX-DYS20 (CT2) has the 243 amino acid CT sequence of SEQ ID NO: 111, RGX-DYS21 (CT3) has the 278 amino acid CT sequence of SEQ ID NO: 112, and RGX-DYS22 (CT4) has the 325 amino acid CT sequence of SEQ ID NO: 113. Microdystrophin proteins including a CT domain having at least exons 70 to 74, and a portion of exon 75 and administered via AAV are shown herein to provide several benefits in vivo, including behavior, muscle force, and eccentric contraction assays at 12 weeks post dosing in mdx mice. - 52 - 40928727338013.0045P1

[0072] In other embodiments, the amino acid sequence of the C-terminal domain is truncated and comprises at least the binding sites for dystrobrevin and / or ^^^syntrophin. In certain embodiments, the truncated C-terminal domain comprises the amino acid sequence MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ (^^^syntrophin binding site) (SEQ ID NO: 84). In certain embodiments, the truncated C-terminal domain comprises an ^^^syntrophin binding site, wherein the binding site has amino acid sequence MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ (SEQ ID NO: 84). In particular embodiments, the CT domain sequence has the amino acid sequence of SEQ ID NO: 83 or amino acids 3361 to 3500 of the UniProtKB-P11532 human DMD sequence (referred to as CT140 or CT1.5). In alternative embodiments, the microdystrophin lacks a CT domain (or includes a minimal 48 amino acids of the CT domain, referred to as CT48, which is or corresponds to amino acids 3361 to 3408 of the UniProtKB-P11532 human DMD sequence; SEQ ID NO: 91), and may have the domains arranged as follows: ABD1-L1-H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR, for example RGX-DYS3 (μDys-CT48) (FIG. 2; SEQ ID NO: 2). In embodiments the microdystrophin, such as, for example, RGX-DYS1 has a half-life that is greater than 1.5 fold, 2 fold, 2.5 fold, or 3 fold greater than a microdystrophin, such as RGS-DYS3, with a CT sequence of 48 amino acids (SEQ ID NO: 91) as determined by a pulse-chase assay in tissue culture, for example, as described in Example 3, herein.

[0073] The NH2terminus and a region in the rod domain of dystrophin bind directly to but do not cross-link cytoskeletal actin. The rod domain of wild type dystrophin is composed of 24 repeating units that are similar to the triple helical repeats of spectrin. This repeating unit accounts for the majority of the dystrophin protein and is thought to give the molecule a flexible rod-like structure similar to β-spectrin. These α-helical coiled-coil repeats are interrupted by four proline- rich hinge regions. At the end of the 24th repeat is the fourth hinge region that is immediately followed by the WW domain [Blake, D. et al, Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle. Physiol. Rev. 82: 291–329, 2002]. Microdystrophins disclosed herein do not include R4 to R23, and only include 3 of the 4 hinge regions or portions thereof. In some embodiments, no new amino acid residues or linkers are introduced into the microdystrophin. - 53 - 40928727338013.0045P1

[0074] In some embodiments, microdystrophin comprises H3 (e.g., SEQ ID NOS: 1, 2, or 79). In embodiments, H3 can be a full endogenous H3 domain from N-terminal to C-terminal, e.g., SEQ ID NO: 11. Stated another way, some microdystrophin embodiments do not contain a fragment of the H3 domain but contain the entire H3 domain. In some embodiments, the C- terminal amino acid of the R3 domain is coupled directly (or covalently bonded to) the N-terminal amino acid of the H3 domain. In some embodiments, the C-terminal amino acid of the R3 domain coupled to the N-terminal amino acid of the H3 domain is Q. In some embodiments, the 5' amino acid of the H3 domain coupled to the R3 domain is Q.

[0075] Without being bound by any one theory, a full hinge domain may be appropriate in any microdystrophin in order to convey full activity upon the derived microdystrophin protein. Hinge segments of dystrophin have been recognized as being proline-rich in nature and may therefore confer flexibility to the protein product (Koenig and Kunkel, 265(6):4560-4566, 1990). Any deletion of a portion of the hinge, especially removal of one or more proline residues, may reduce its flexibility and therefore reduce its efficacy by hindering its interaction with other proteins in the DAP complex.

[0076] Further, the microdystrophin may be advantageously similar in domain structure to the naturally occurring dystrophin expressed in Becker’s dystrophin (see FIG. 9 for comparison of wild type, Becker phenotype dystrophin and microdystrophin expressed from RGX-DYS1 (RGX- 202)). Similar to the dystrophin from Becker phenotype subjects, microdystrophins provided herein, including RGX-DYS1 have a C terminal domain with functional elements, including the binding sites for dystrobrevin and ^^^syntrophin. In addition, the microdystrophins provided herein, including RGS-DYS1, have a deletion of the H2 and R4-R19 domains, similar to that found in the dystrophin from the Becker phenotype. The microdystrophins further link the H3 domain to an R24 domain just upstream of the CR region to form a stabilized, active, shortened form of dystrophin

[0077] Microdystrophins disclosed herein comprise the wild-type dystrophin H4 sequence (which contains the WW domain) to and including the CR domain (which contains the ZZ domain, represented by a single underline (UniProtKB-P11532 aa 3307-3354) in SEQ ID NO: 15). The WW domain is a protein-binding module found in several signaling and regulatory molecules. The WW domain binds to proline-rich substrates in an analogous manner to the src homology-3 (SH3) - 54 - 40928727338013.0045P1 domain. This region mediates the interaction between β-dystroglycan and dystrophin, since the cytoplasmic domain of β-dystroglycan is proline rich. The WW domain is in the Hinge 4 (H4 region). The CR domain contains two EF-hand motifs that are similar to those in α-actinin and that could bind intracellular Ca2+. The ZZ domain contains a number of conserved cysteine residues that are predicted to form the coordination sites for divalent metal cations such as Zn2+. The ZZ domain is similar to many types of zinc finger and is found both in nuclear and cytoplasmic proteins. The ZZ domain of dystrophin binds to calmodulin in a Ca2+-dependent manner. Thus, the ZZ domain may represent a functional calmodulin-binding site and may have implications for calmodulin binding to other dystrophin-related proteins.

[0078] Microdystrophin embodiments can further comprise linkers (L1, L2, L3, L4, L4.1 and / or L4.2) or portions thereof connected the domains as shown as follows: ABD1-L1-H1-L2-R1-R2- L3-R3-H3-L4-R24-H4-CR-CT (e.g., SEQ ID NO: 1, 79, or 91) or ABD1-L1-H1-L2-R1-R2-L3- R3-H3-L4-R24-H4-CR (e.g., SEQ ID NO: 2) L1 can be an endogenous linker L1 (e.g., SEQ ID NO: 4) that can couple ABD1 to H1. L2 can be an endogenous linker L2 (e.g., SEQ ID NO: 6) that can couple H1 to R1. L3 can be an endogenous linker L3 (e.g., SEQ ID NO: 9) that can couple R2 to R3.

[0079] L4 can also be an endogenous linker that can couple H3 and R24. In some embodiments, L4 is 3 amino acids, e.g. TLE (SEQ ID NO: 12) that precede R24 in the native dystrophin sequence. In other embodiments, L4 can be the 4 amino acids that precede R24 in the native dystrophin sequence (SEQ ID NO: 17) or the 2 amino acids that precede R24 (SEQ ID NO: 18). In other embodiments, there is no linker, L4 or otherwise, in between H3 and R24. On the 5’ end of H3, as mentioned above, no linker is present, but rather R3 is directly coupled to H3.

[0080] The above described components of microdystrophin other domains not specifically described can have the amino acid sequences as provided in Table 1 below. The amino acid sequences for the domains provided herein correspond to the dystrophin isoform of UniProtKB- P11532 (DMD_HUMAN) (SEQ ID NO: 92), which is herein incorporated by reference. Other embodiments can comprise the domains from naturally-occurring functional dystrophin isoforms known in the art, such as UniProtKB-A0A075B6G3 (A0A075B6G3_HUMAN), (incorporated by reference herein) wherein, for example, R24 has an R substituted for the Q at amino acid 3 of SEQ ID NO: 13. - 55 - 40928727338013.0045P1

[0081] Additional embodiments are disclosed in International Application PCT / US2020 / 062484, filed November 27, 2020, which is hereby incorporated by reference in its entirety. Table 1: Microdystrophin segment amino acid sequences Structure SEQ Sequence ID L L S E YTN E Q K S LSNK P S C- 56 - 40928727338013.0045P1 Structure SEQ Sequence ID N E A N E L V N F E A E L M L R L C Q F S E K A S L- 57 - 40928727338013.0045P1 Structure SEQ Sequence ID T E M L E R K K S L V E T R E K L I Y G D D S L L Q K V V I R D K D L A V L R Q N I K E R H V S- 58 - 40928727338013.0045P1 Structure SEQ Sequence ID S P P Q N E A M N E A M R N E A M R T- 59 - 40928727338013.0045P1

[0082] The present disclosure also contemplates variants of these sequences so long as the function of each domain and linker is substantially maintained and / or the therapeutic efficacy of microdystrophin comprising such variants is substantially maintained. Functional activity includes (1) binding to one of, a combination of, or all of actin, β-dystroglycan, α1-syntrophin, α- dystrobrevin, and nNOS; (2) improved muscle function in an animal model (for example, in the mdx mouse model described herein) or in human subjects; and / or (3) cardioprotective or improvement in cardiac muscle function in animal models or human patients. In particular, microdystrophin can comprise ABD consisting of SEQ ID NO: 3 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3; H1 consisting of SEQ ID NO: 5 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5; R1 consisting of SEQ ID NO: 7 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7; R2 consisting of SEQ ID NO: 8 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8; H3 consisting of SEQ ID NO: 11 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11; R24 consisting of SEQ ID NO: 13 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13; H4 consisting of SEQ ID NO: 14 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14; CR consisting of SEQ ID NO: 15 or 90 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 or 90; CT consisting of SEQ ID NO: 16 or 83 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 or 83, or CT comprising SEQ ID NO: 84. In embodiments, the CT does not consist of the last three amino acids (DTM; aspartic acid – threonine - methionine) present in the 3′ portion of natural human dystrophin as in SEQ ID NO: 92. In addition to the foregoing, - 60 - 40928727338013.0045P1 microdystrophin can comprise linkers in the locations described above that comprise or consist of sequences as follows: L1 consisting of SEQ ID NO: 4 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4; L2 consisting of SEQ ID NO: 6 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6; L3 consisting of SEQ ID NO: 9 or an amino acid sequence with at least 50% identity to SEQ ID NO: 9 or a variant with conservative substitutions for both L3 residues; and L4 consisting of SEQ ID NO: 12, 17, or 18 or an amino acid sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 12, 17, or 18.

[0083] Table 2A and 2B provides the amino acid sequences of the microdystrophin embodiments in accordance with the present disclosure. It is also contemplated that other embodiments are substituted variant of microdystrophin as defined by SEQ ID NOs: 1 (RGX- DYS1), 2 (RGX-DYS3), or 79 (RGX-DYS5) or any of the microdystrophins defined by SEQ ID NOs 100-109. For example, conservative substitutions can be made to SEQ ID NOs: 1, 2, or 79 and substantially maintain its functional activity. In embodiments, microdystrophin may have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109 and maintain functional microdystrophin activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed in Section 5.4, infra. RGX-DYS2 and RGX-DYS4 (Example 1) of the disclosure also encode microdystrophin proteins comprising SEQ ID NO: 1. In embodiments, the microdystrophin protein comprises a portion of the C terminal domain which comprises an α1-syntrophin binding domain and has enhanced DAPC restoration, half-life or protection against contraction-induced damage relative to a comparable microdystrophin which does not comprise the portion of the C terminal domain. Not to be bound by theory, the presence of hinge 2 or other sequences in certain microdystrophins may result in destabilization or improper folding of the microdystrophin protein (see, Boehler et al., 2023, Neuromuscular Disorders 33:4049).

[0084] Accordingly provided are microdystrophins that may lack these sequences yet include a proximal portion of the C-terminus of dystrophin. Provided are microdystrophins including but - 61 - 40928727338013.0045P1 not limited to microdystrophins that i) lack hinge 2 and comprise or consist of at least the proximal portion of the C-terminus (SEQ ID NO: 16 or 83); ii) lack hinge 2, comprise or consist of at least the proximal portion of the C-terminus (SEQ ID NO: 16 or 83) and lack a DTM amino acid sequence at the 3′ end of the C-terminus of the microdystrophin; and iii) comprise or consist of a hinge 2 domain and at least the proximal portion of the C-terminus (SEQ ID NO: 16 or 83) that lacks a DTM amino acid sequence at the 3′ end of the C-terminus of the microdystrophin. Table 2A: Amino acid sequences of RGX-DYS proteins Structure SEQ Amino Acid Sequence ID G W R F R W S R- 62 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID E F R S E R R L K A Q V G W R F R W S R E F R S E R R L K A Q V N G W R F R W S R E F R P- 63 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID L E N Y Y I D Y S G W R F R W S R E F R P L E N Y Y I D Y S G W R F R W S R E F R P A- 64 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID F S Q V F C T S G W R F R W S R E F R P A F S Q V F C T S G W R F R W S R E S K K A- 65 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID E I L P M I V I R Y E G W R F R W S R E S K K A E I L P M I V I R Y E G W R F R W S N L E- 66 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID K A E I L P M I H P N R G W R F R W S N L E K A E I L P M I H P N R G W R F R W S R E- 67 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID F R P A F S Q V F C T S G W R F R W S R E F R P A F S Q V F C T S G W R F R W S R E F- 68 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID R S E R R L K A Q V N E A G W R F R W S R E F R S E R R L K A Q V N E A G G W R F R W S R- 69 - 40928727338013.0045P1 Structure SEQ Amino Acid Sequence ID E F R S E R R L K A Q V N E A GTable 2B: Amino acid sequences of microdystrophin proteins Structure Microdystrophin constructs having a functional C Terminal domain- 70 - 40928727338013.0045P1 Table 2C: Amino acid sequences of microdystrophin proteins without a CT Microdyst SEQ Amino Acid Sequence rophin ID G W R F R W S R E F R P L E N Y Y I D G W R F R W S R E F R P A F S Q V F C- 71 - 40928727338013.0045P1 Microdyst SEQ Amino Acid Sequence rophin ID G W R F R W S R E S K K A E I L P M I V I G W R F R W S N L E K A E I L P M I H G W R- 72 - 40928727338013.0045P1 Microdyst SEQ Amino Acid Sequence rophin ID F R W S R E F R P A F S Q V F C5.2.2 Nucleic Acid Compositions encoding Microdystrophin

[0085] Another aspect of the present disclosure are nucleic acids comprising a nucleotide sequence encoding a microdystrophin as described herein. Such nucleic acids comprise nucleotide sequences that encode the microdystrophin that has the domains arranged N-terminal to C-terminal as follows: ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT as detailed in Section 5.2.1, supra. The nucleotide sequence can be any nucleotide sequence that encodes the domains. The nucleotide sequence may be codon optimized and / or depleted of CpG islands for expression in the appropriate context. In particular embodiments, the nucleotide sequences encode a microdystrophin having an amino acid sequence of SEQ ID NO: 1, 2, or 79. Some embodiments include the nucleotide sequences encoding a microdystrophin having an amino acid sequence of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. The nucleotide sequence can be any sequence that encodes the microdystrophin, including the microdystrophin of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 79, or encodes the microdystrophin of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109, which nucleotide sequence may vary due to the degeneracy of the code. Tables 3 and 4 provide exemplary nucleotide sequences that encode the DMD domains. Table 3 - 73 - 40928727338013.0045P1 provides the wild type DMD nucleotide sequence for the component and Table 4 provides the nucleotide sequence for the DMD component used in the constructs (transgenes, expression constructs, cis plasmids, and recombinant AAV genomes) herein, including sequences that have been codon optimized and / or CpG depleted of CpG islands as follows: Table 3: Dystrophin segment nucleotide sequences Structure SEQ Nucleic Acid Sequence ID T- 74 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID A- 75 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID CStructure SEQ Nucleic Acid Sequence ID G T A A C C G G C C C C A C C G G C A G C C G A G G G G A- 76 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID T C G C G C G G A C G G G G G A T C G T A T G C C A C C C C G T C C G T G- 77 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID C G G C A T T C T A G G T G G C A T T C T p G G C A T T C T A G G T C G T- 78 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID G G C A T T C T A G G T C G T C G G G C A T T C T A G G T C G T C G A C G

[0086] n some embod ments, suc compos t ons compr se a nuc e c ac d sequence encoding ABD1 that consists of SEQ ID NO: 22 or 57, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22 or 57; a nucleic acid sequence encoding H1 that consists of SEQ ID NO: 24 or 59, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% - 79 - 40928727338013.0045P1 identity to SEQ ID NO: 24 or 59; a nucleic acid sequence encoding R1 that consists of SEQ ID NO: 26 or 61, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26 or 61; a nucleic acid sequence encoding R2 that consists of SEQ ID NO: 27 or 62, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27 or 62; a nucleic acid sequence encoding R3 that consists of SEQ ID NO: 29 or 64, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 29 or 64; a nucleic acid sequence encoding H3 that consists of SEQ ID NO: 30 or 65, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 30 or 65; a nucleic acid sequence encoding R24 that consists of SEQ ID NO: 32 or 67, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32 or 67; a nucleic acid sequence encoding H4 that consists of SEQ ID NO: 33 or 68, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33 or 68; a nucleic acid sequence encoding CR that consists of SEQ ID NO: 34, 47, or 69 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34, 47 or 69; and / or a nucleic acid sequence encoding CT that consists of SEQ ID NO: 35 or 70, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35 or 70, encoding a microdystrophin that has functional activity.

[0087] In some embodiments, such compositions comprise a nucleic acid sequence encoding ABD1 that consists of SEQ ID NO: 22 or 57, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22 or 57, and encodes for the ABD1 domain of SEQ ID NO: 3; a nucleic acid sequence encoding H1 that consists of SEQ ID NO: 24 or 59, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 24 or 59, and encodes for the H1 domain of SEQ ID NO: 5; a nucleic acid sequence encoding R1 that consists of SEQ ID NO: 26 or 61, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26 or 61, and encodes for the R1 domain of SEQ ID NO: 7; a nucleic acid sequence encoding R2 that consists of SEQ ID NO: 27 or 62, or a sequence - 80 - 40928727338013.0045P1 with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27 or 62, and encodes for the R2 domain of SEQ ID NO: 8; a nucleic acid sequence encoding R3 that consists of SEQ ID NO: 29 or 64, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 29 or 64, and encodes for the R3 domain of SEQ ID NO: 10; a nucleic acid sequence encoding H3 that consists of SEQ ID NO: 30 or 65, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 30 or 65, and encodes for the H3 domain of SEQ ID NO: 11; a nucleic acid sequence encoding R24 that consists of SEQ ID NO: 32 or 67, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32 or 67, and encodes for the R24 domain of SEQ ID NO: 13; a nucleic acid sequence encoding H4 that consists of SEQ ID NO: 33 or 68, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33 or 68, and encodes for the H4 domain of SEQ ID NO: 14; a nucleic acid sequence encoding CR that consists of SEQ ID NO: 34.47 or 69, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34, 47 or 69, and encodes for the CR domain of SEQ ID NO: 15 or 89; and / or a nucleic acid sequence encoding CT that consists of SEQ ID NO: 35, 70 or 80, or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35, 70 or 80, and encodes for the CT domain of SEQ ID NO: 16 or 83.

[0088] In addition to the foregoing, the nucleic acid compositions can optionally comprise nucleotide sequences encoding linkers in the locations described above that comprise or consist of sequences as follows: a nucleic acid sequence encoding L1 consisting of SEQ ID NO: 23 or 58, or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23 or 58 (e.g. encoding the L1 domain of SEQ ID NO: 4); a nucleic acid sequence encoding L2 consisting of SEQ ID NO: 25 or 60, or sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 or 60 (e.g. encoding the L2 domain of SEQ ID NO: 6); a nucleic acid sequence encoding L3 consisting of SEQ ID NO: 28 or 63, or a sequence with at least 50% identity to SEQ ID NO: 28 or 63, encoding the L3 domain of SEQ ID NO: 9 or a variant with conservative - 81 - 40928727338013.0045P1 substitutions for both L3 residues; and a nucleic acid sequence encoding L4 consisting of SEQ ID NO: 19, 31, 36, 37, 38, 46, or 66, or a sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 19, 31, 36, 37, 38, 46, or 66 (e.g. encoding the L4 domain of SEQ ID NO: 12, 17, or 18 or a variant with conservative substitutions for any of the L4 residues).

[0089] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding the microdystrophin having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 79. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the microdystrophin having the amino acid sequence of microdystrophin having an amino acid sequence of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. In embodiments, the nucleic acid comprises a nucleotide sequence which is SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 81, (encoding the microdystrophins of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 79, respectively). In various embodiments, the nucleotide sequence encoding a microdystrophin may have at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 20, 21, or 83 (Table 5) or the reverse complement thereof and encode a therapeutically effective microdystrophin. Table 5: RGX-DYS Nucleotide Sequences Structure SEQ ID Nucleic Acid Sequence DYS120ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAAGATGTGCAA G G C G A A C T A C A A G A C C T C A- 82 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence GTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AAT A AAT AT T AA T T AA A A TTT A A AT A G G A C G C G T T A A A A G T A C A C C A A C A C T G C T A G A A A T A A C T G T G G A A A A G- 83 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence ATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGC TT A T A AA AT AAA T T A A T AT AATTT T A A A T C G C A C A A G G C G A A C T A C A A G A C C T C A A G G A C G C G T T A A A A G T A- 84 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTTTGCTAGATGC T A AA T T A AA T AAAA T TA A A AT A A C C A A C A C T G C T A G A A A T A A C T G T G G A A A A G C A A G G C G A A C T A C A A G- 85 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence GTTGAGATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAACACTTCCA T A A A AT A TA T T A A AT A A T T T T C C T C A A G G A C G C G T T A A A A G T A C A C C A A C A C T G C T A G A A A T A A C T G T G- 86 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence GATGAGACTTGAGCCTCAGAGCATGGTCTGGCTGCCTGTGCTTCATAGAG T T T T A A T AA A A AA T AA AT T AAA A A A G C A A A T C T A A G G C G A A C T A C A A G A C C T C A A G G A C G C G T T A A A A- 87 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence AGATCCATACCACAGGCTTCAAGGATCAGAATGAGATGCTCAGCTCCCTG A AAA T T T T AA T A T AAAA AAAAA A T AT A C A C C A A C A C T G C T A G A A A T A A C T G T G G A A A A G C A A A T C G C A C G A C- 88 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence DYS21 122ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAAGATGTGCAAA AAAA TT A AAAT T AAT A TT A AA TTT A G G C G A A C T A C A A G A C C T C A A G G A C G C G T T A A A A G T A C A C C A A C A C T G- 89 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence AGTCAGGCAGCTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGC A TTT T T TA T T T AA T A A A TAT T T T A G A A A T A A C T G T G G A A A A G C A A A T C G C A C G A C C G A A G G C G A A C T A C- 90 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence AACATTGCCAGATACCAGCTGGGAATTGAGAAACTGCTGGACCCTGAGGA T T A A A TAT T A AA AAAT AT T AT TA AT A A G A C C T C A A G G A C G C G T T A A A A G T A C A C C A A C A C T G C T A G A A A T A A C T- 91 - 40928727338013.0045P1 Structure SEQ ID Nucleic Acid Sequence GGGCCTGCTCCTGCATGACAGCATTCAGATCCCTAGACAGCTGGGAGAAG T TT TTT A A AATATT A AT A T A T T TTTT G G A A A A G C A A A T C G C A C G A C C G G C C

[0090] In one aspect the nucleotide sequence encoding the microdystrophin cassette is modified by codon optimization and CpG dinucleotide and CpG island depletion. Immune response against microdystrophin transgene is a concern for human clinical application, as evidenced in the first Duchenne Muscular Dystrophy (DMD) gene therapy clinical trials and in several adeno-associated vial (AAV)-minidystrophin gene therapy in canine models [Mendell, J.R., et al., Dystrophin immunity in Duchenne's muscular dystrophy. N Engl J Med, 2010. 363(15): p. 1429-37; and Kornegay, J.N., et al., Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs. Mol Ther, 2010.18(8): p.1501- 8].

[0091] AAV-directed immune responses can be inhibited by reducing the number of CpG di- nucleotides in the transgene [de Wolf, H. K. et al. Pharmaceutical Research, Vol.25, No.7, July - 92 - 40928727338013.0045P1 2008]. Depleting the transgene sequence of CpG motifs may diminish the role of TLR9 in activation of innate immunity upon recognition of the transgene as non-self, and thus provide stable and prolonged transgene expression. [See also Wang, D., P.W.L. Tai, and G. Gao, Adeno- associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov, 2019.18(5): p.358-378; and Rogers GL et al. Innate immune responses to AAV vectors. Front. Microbiol 2, 194 (2011).]. In embodiments, the microdystrophin cassette is human codon-optimized with CpG depletion. Codon-optimized and CpG depleted nucleotide sequences may be designed by any method known in the art, including for example, by Thermo Fisher Scientific GeneArt Gene Synthesis tools utilizing GeneOptimizer (Waltham, MA USA)). Nucleotide sequences SEQ ID NOs: 20, 21, 57-72, 80, 81, and 101-103 described herein represent codon-optimized and CpG depleted sequences.

[0092] Provided are microdystrophin transgenes that have reduced numbers of CpG dinucleotide sequences and, as a result, have reduced number of CpG islands. In certain embodiments, the microdystrophin nucleotide sequence has fewer than two (2) CpG islands, or one (1) CpG island or zero (0) CpG islands. In embodiments, provided are microdystrophin transgenes having fewer than 2, or 1 CpG islands, or 0 CpG islands that have reduced immunogenicity, as measured by anti-drug antibody titer compared to a microdystrophin transgene having more than 2 CpG islands. In certain embodiments, the microdystrophin nucleotide sequence consisting essentially of SEQ ID NO: 20, 21, or 81 has zero (0) CpG islands. In other embodiments, the microdystrophin transgene nucleotide sequence consisting essentially of a microdystrophin gene operably linked to a promoter, wherein the microdystrophin coding sequence consists of SEQ ID NO: 20, 21, or 81, has less than two (2) CpG islands. In still other embodiments, the microdystrophin transgene nucleotide sequence consisting essentially of a microdystrophin gene operably linked to a promoter, wherein the microdystrophin coding sequence consists of SEQ ID NO: 20, 21, or 81, has one (1) CpG island. 5.3. Gene Cassettes and Regulatory Elements

[0093] Another aspect of the present invention relates to nucleic acid expression cassettes comprising regulatory elements designed to confer or enhance expression of the microdystrophins. The invention further involves engineering regulatory elements, including promoter elements, and optionally enhancer elements and / or introns, to enhance or facilitate expression of the transgene. - 93 - 40928727338013.0045P1 In some embodiments, the rAAV vector also includes such regulatory control elements known to one skilled in the art to influence the expression of the RNA and / or protein products encoded by nucleic acids (transgenes) within target cells of the subject. Regulatory control elements and may be tissue-specific, that is, active (or substantially more active or significantly more active) only in the target cell / tissue. 5.3.1 Promoters 5.3.1.1 Tissue-specific promoters

[0094] In specific embodiments, the expression cassette of an AAV vector comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues. The promoter may be a muscle promoter. In certain embodiments, the promoter is a muscle-specific promoter. The phrase “muscle-specific”, “muscle-selective” or “muscle-directed” refers to nucleic acid elements that have adapted their activity in muscle cells or tissue due to the interaction of such elements with the intracellular environment of the muscle cells. Such muscle cells may include myocytes, myotubes, cardiomyocytes, and the like. Specialized forms of myocytes with distinct properties such as cardiac, skeletal, and smooth muscle cells are included. Various therapeutics may benefit from muscle-specific expression of a transgene. In particular, gene therapies that treat various forms of muscular dystrophy delivered to and enabling high transduction efficiency in muscle cells have the added benefit of directing expression of the transgene in the cells where the transgene is most needed. Cardiac tissue will also benefit from muscle-directed expression of the transgene. Muscle-specific promoters may be operably linked to the transgenes of the invention. In some embodiments, the muscle-specific promoter is selected from an SPc5-12 promoter (SEQ ID NO: 39), a muscle creatine kinase myosin light chain (MLC) promoter, a myosin heavy chain (MHC) promoter, a desmin promoter (SEQ ID NO: 75), a MHCK7 promoter (SEQ ID NO: 76), a CK6 promoter, a CK8 promoter (SEQ ID NO: 51), a MCK promoter (or a truncated form thereof) (SEQ ID NO: 86), an alpha actin promoter, an beta actin promoter, an gamma actin promoter, an E-syn promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, or a muscle-selective promoter residing within intron 1 of the ocular form of Pitx3. - 94 - 40928727338013.0045P1

[0095] Synthetic promoter c5-12 (Li, X. et al. Nature Biotechnology Vol. 17, pp. 241-245, MARCH 1999), known as the SPc5-12 promoter, has been shown to have cell type restricted expression, specifically muscle-cell specific expression. At less than 350 bp in length, the SPc5- 12 promoter is smaller in length than most endogenous promoters, which can be advantageous when the length of the nucleic acid encoding the therapeutic protein is relatively long.

[0096] In order to further reduce the length of a vector, regulatory elements can be a reduced or shortened version (referred to herein as a “minimal promoter”) of any one of the promoters described herein. A minimal promoter comprises at least the transcriptionally active domain of the full-length version and is therefore still capable of driving expression. For example, in some embodiments, an AAV vector can comprise the transcriptionally active domain of a muscle- specific promoter, e.g., a minimal SPc5-12 promoter (e.g., SEQ ID NO: 40), operably linked to a therapeutic protein transgene. In embodiments, the therapeutic protein is microdystrophin as described herein. A minimal promoter of the present disclosure may or may not contain the portion of the promoter sequence that contributes to regulating expression in a tissue-specific manner.

[0097] Accordingly, in embodiments, provided are gene therapy cassettes with a SPc5-12 promoter (SEQ ID NO: 39) or SPc5-12 promoter variants, mutants or fragments thereof. For example, RGX-DYS1 and RGX-DYS5 (FIG.2) have the Spc5-12 promoter. Sequences of these promoters are provided in Table 6.

[0098] In some aspects, provided are modified SPc5-12 promoters that are altered or mutated. Mutant SPc5-12 promoters can comprise the nucleic acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94. These unique SPc5-12 promoter sequences promote muscle specific expression and can increase the yield of capsids produced with full genomes. Accordingly, in embodiments, provided are gene therapy vectors comprising a mutant SPc5-12 promoter (SEQ ID NO: 93 or 94).

[0099] In some aspects, variants of SEQ ID NO: 93 are provided. In some aspects, the disclosed nucleic acids can comprise a nucleotide sequence having muscle specific promoter activity, at least 80% sequence identity to SEQ ID NO: 93, and, in certain embodiments, 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NO: 93. In some aspects, the disclosed nucleic acids can comprise a nucleotide sequence having muscle specific promoter activity, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 93, and, in some embodiments, 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NO: 93. - 95 - 40928727338013.0045P1 A variant of SEQ ID NO: 93 can be the sequence of SEQ ID NO: 93 but having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleic acid substitutions. In some aspects, any of the variants retain 100% sequence identity over nucleotides 121-129 and 197-209 of SEQ ID NO: 93 and retain muscle specific promoter activity. In some aspects, variants of SEQ ID NO: 94 are provided. In some aspects, the disclosed nucleic acid can comprise a nucleotide sequence having muscle-specific promoter activity, at least 80% sequence identity to SEQ ID NO: 94, and, in some embodiments, 100% sequence identity over nucleotides 113-131 and 191-212 of SEQ ID NO: 94. In some aspects, the disclosed nucleic acid can comprise a nucleotide sequence having muscle-specific promoter activity, at least 85, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 94, and, in some embodiments, 100% sequence identity over nucleotides 113-131 and 191-212 of SEQ ID NO: 94. A variant of SEQ ID NO: 94 can be the sequence of SEQ ID NO: 94 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleic acid substitutions. In some aspects, any of the variants retain 100% sequence identity over nucleotides 113-131 and 191-212 of SEQ ID NO: 94 and retain muscle specific promoter activity.

[0100] In some aspects are small promoters to allow for transgenes that are larger in size. In embodiments, the promoter may be ACTA core promoter, alone or in combination with one or more enhancers, particular muscle enhancers. In some embodiments, the promoter is an MCK enhancer, or a modified MCK enhancer, in combination with an ACTA2 promoter, for example SEQ ID NO: 124. Examples of such promoters are found in PCT International Publication No. WO2024 / 081746 A2, published April 18, 2024, and are incorporated herein by reference.

[0101] Alternatively, the promoter may be a constitutive promoter, for example, the CB7 promoter. Additional promoters include: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter (SEQ ID NO: 54), UB6 promoter, chicken beta-actin promoter, CAG promoter (SEQ ID NO: 52), RPE65 promoter, opsin promoter, the TBG (Thyroxine-binding Globulin) promoter, the APOA2 promoter, SERPINA1 (hAAT) promoter, or MIR122 promoter. In some embodiments, particularly where it may be desirable to turn off - 96 - 40928727338013.0045P1 transgene expression, an inducible promoter is used, e.g., hypoxia-inducible or rapamycin- inducible promoter. Table 6: Promoter sequences Promoter SEQ Nucleic Acid Sequence ID T G G T C C T C C T T T A T A A T G G T G G T G T G A C A C C A C T G C C G A G G A C- 97 - 40928727338013.0045P1 Promoter SEQ Nucleic Acid Sequence ID G G G G A A G T C G C C A T C T A G A C G A- 98 - 40928727338013.0045P1 Promoter SEQ Nucleic Acid Sequence ID A T A C C A G C G G T T T A anexpress on cassette can comprse a promoter se ected rom a promoter so ated rom t e genes of neuron specific enolase (NSE), any neuronal promoter such as the promoter of Dopamine-1 receptor or Dopamine-2 receptor, the synapsin promoter, CB7 promoter (a chicken β-actin promoter and CMV enhancer), RSV promoter, GFAP promoter (glial fibrillary acidic protein), MBP promoter (myelin basic protein), MMT promoter, EF-1α, U86 promoter, RPE65 promoter or opsin promoter, an inducible promoter, for example, a hypoxia-inducible promoter, and a drug inducible promoter, such as a promoter induced by rapamycin and related agents.

[0103] In still other embodiments, expression cassettes can comprise multiple promoters which may be placed in tandem in the expression cassette comprising a microdystrophin transgene. As such, tandem or hybrid promoters may be employed in order to enhance expression and / or direct expression to multiple tissue types, (see, e.g. PCT International Publication No. WO2019154939A1, published Aug.15, 2019, incorporated herein by reference) and, in particular, LMTP6, LMTP13, LMTP14, LMTP15, LMTP18, LMTP19, or LMTP20 as disclosed in PCT International Application No. PCT / US2020 / 043578, filed July 24, 2020, hereby incorporated by reference). - 99 - 40928727338013.0045P1 5.3.2 Introns

[0104] Certain gene expression cassettes further include an intron, for example, 5’ of the microdystrophin coding sequence which may enhances proper splicing and, thus, microdystrophin expression. Accordingly, in some embodiments, an intron is coupled to the 5’ end of a sequence encoding a microdystrophin protein. In particular, the intron nucleotide sequence can be linked to the nucleotide sequence attached to the actin-binding domain. In other embodiments, the intron is less than 100 nucleotides in length.

[0105] In embodiments, the intron is a VH4 intron. The VH4 intron nucleic acid can comprise SEQ ID NO: 41 as shown in Table 7 below. Table 7: Nucleotide sequences for different introns Structure SEQ ID Sequence VH4 intron 41GTGAGTATCTCAGGGATCCAGACATGGGGATATGGGAGGTGCCTCTGATCGC Gnd Ig heavy chain (also known as β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, or β-globin / IgG chimeric intron) (Table 7, SEQ ID NO: 75). Other introns well known to the skilled person may be employed, such as the chicken β-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin splice acceptor intron, SV40 late splice donor / splice acceptor (19S / 16S) intron (Table 7, SEQ ID NO: 76). 5.3.3 Other regulatory elements 5.3.3.1 polyA

[0107] Another aspect of the present disclosure relates to expression cassettes comprising a polyadenylation (polyA) site downstream of the coding region of the microdystrophin transgene. Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is - 100 - 40928727338013.0045P1 suitable for use in AAV vectors of the present disclosure. Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit β-globin gene, the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, and the synthetic polyA (SPA) site. In one embodiment, the polyA signal comprises SEQ ID NO: 42 as shown in Table 8. Table 8: Nucleotide sequence of the polyA signal Structure SEQ Sequence ID T

[0108] The microdystrophin transgene in accordance with the present disclosure can be included in an AAV vector for gene therapy administration to a human subject. In some embodiments, recombinant AAV (rAAV) vectors can comprise an AAV viral capsid and a viral or artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs) wherein the expression cassette comprises a microdystrophin transgene, operably linked to one or more regulatory sequences that control expression of the transgene in human muscle or CNS cells to express and deliver the microdystrophin. The provided methods are suitable for use in the production of any isolated recombinant AAV particles for delivery of a microdystrophins described herein, in the production of a composition comprising any isolated recombinant AAV particles encoding a microdystrophin, or in the method for treating a disease or disorder amenable for treatment with a microdystrophin in a subject in need thereof comprising the administration of any isolated recombinant AAV particles encoding a microdystrophin described herein. As such, the rAAV can be of any serotype, variant, modification, hybrid, or derivative thereof, known in the art, or any combination thereof (collectively referred to as “serotype”). In particular embodiments, the AAV serotype has a tropism for muscle tissue. And, in other embodiments, the AAV serotype has a tropism for the liver, in which case the liver cells transduced with the AAV form a depot of microdystrophin secreting cells, secretin the microdystrophin into the circulation.

[0109] In some embodiments, rAAV particles have a capsid protein from an AAV8 or AAV9 serotype. Provided herein are the RGX-DYS1 construct (recombinant AAV genome, including the polynucleotide with a nucleotide sequence of SEQ ID NO: 53) in an rAAV particle having an AAV8 capsid and the RGX-DYS1 construct (recombinant AAV genome) in an rAAV particle - 101 - 40928727338013.0045P1 having an AAV9 capsid. In some embodiments, the rAAV particles comprise a capsid protein that is AAV7, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, and AAV.hu37. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV1 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV4 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV5 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9 or a derivative, modification, or pseudotype thereof. As such, modified capsids may be bonded to a binding peptide, such as a 7 amino acid peptide or a peptide including the amino acids RGD. Examples of peptide insertions including peptides inserted in variable region 8 (VR8) of the capsid protein are found in PCT International Publication No. WO2019207132, Weinmann, J. et al. Nature Comm 11 Article number: 5432 (2020), PCT International Publication No. WO2021072197, PCT International Publication No. WO2021050974, PCT International Publication No. WO2021077000, and PCT International Publication No. WO2022020616, each of which is incorporated herein by reference in its entirety.

[0110] In some embodiments, rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV8 or AAV9 capsid protein. In some embodiments, rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of AAV8 capsid protein (amino acid sequence of VP3 is SEQ ID NO: 77). In some embodiments, rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV9 capsid protein (amino acid sequence SEQ ID NO: 78). In some embodiments, rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, - 102 - 40928727338013.0045P1 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of AAV9 capsid protein.

[0111] In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identity, to the VP1, VP2 and / or VP3 sequence of AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, or AAV.7m8 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identity, to the VP1, VP2 and / or VP3 sequence of an AAV capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, and AAV.hu37.

[0112] Alternatively, the rAAV particles have a capsid protein of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and / or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof. - 103 - 40928727338013.0045P1

[0113] For example, a population of rAAV particles can comprise two or more serotypes, e.g., comprising two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof.)

[0114] In some embodiments, rAAV particles comprise the capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep.12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the rAAV particles comprise the capsid with one of the following amino acid insertions: LGETTRP (SEQ ID NO: 87) or LALGETTRP (SEQ ID NO: 88), as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no.2016 / 0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV.7m8, as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no.2016 / 0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,585,971, such as AAVPHP.B. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No.9,840,719 and WO 2015 / 013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014 / 172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2 / 5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017 / 070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med.29(9): 418, which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US - 104 - 40928727338013.0045P1 Pat Nos.8,628,966; US 8,927,514; and WO 2016 / 049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.

[0115] In some embodiments, rAAV particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0116] In some embodiments, rAAV particles have a capsid protein disclosed in Intl. Appl. Publ. No. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2 of ´051), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of ´321), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of ´397), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of ´888), WO 2006 / 110689, (see, e.g., SEQ ID NOs: 5-38 of ´689) WO2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of ´964), WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38 of ´097), and WO 2015 / 191508 (see, e.g., SEQ ID NOs: 80-294 of ´508), and U.S. Appl. Publ. No.20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of ´924), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of an AAV capsid disclosed in Intl. Appl. Publ. No. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2 of ´051), WO 2005 / 033321 (see, e.g., - 105 - 40928727338013.0045P1 SEQ ID NOs: 123 and 88 of ´321), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of ´397), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of ´888), WO 2006 / 110689 (see, e.g., SEQ ID NOs: 5-38 of ´689) WO2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of 964), WO 2010 / 127097 (see, e.g., SEQ ID NOs: 5-38 of ´097), and WO 2015 / 191508 (see, e.g., SEQ ID NOs: 80-294 of ´508), and U.S. Appl. Publ. No.20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of ´924).

[0117] Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos.7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335; WO 2003 / 052051, WO 2005 / 033321, WO 03 / 042397, WO 2006 / 068888, WO 2006 / 110689, WO2009 / 104964, W0 2010 / 127097, and WO 2015 / 191508, and U.S. Appl. Publ. No.20150023924.

[0118] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet.10:3075- 3081, (2001)).

[0119] In certain embodiments, a single-stranded AAV (ssAAV) can be used. In certain embodiments, a self-complementary vector, e.g., scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol.8, Number 16, Pages 1248- 1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0120] In additional embodiments, rAAV particles comprise a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, - 106 - 40928727338013.0045P1 AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[0121] In some embodiments, rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0122] In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle. In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from AAVx (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16). In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle comprised of a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle containing AAV8 capsid protein. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle is comprised of AAV9 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet.10:3075-3081, (2001)).

[0123] In additional embodiments, rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In further embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, - 107 - 40928727338013.0045P1 AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In further embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0124] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[0125] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. - 108 - 40928727338013.0045P1

[0126] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10.

[0127] In some embodiments the rAAV particles comprises a Clade A, B, E, or F AAV capsid protein. In some embodiments, the rAAV particles comprises a Clade F AAV capsid protein. In some embodiments the rAAV particles comprises a Clade E AAV capsid protein.

[0128] Table 9 below provides examples of amino acid sequences for an AAV8, AAV9, AAV.rh74, AAV.hu31, AAV.hu32, and AAV.hu37 capsid proteins and the nucleic acid sequence of AAV25’- and 3’ ITRs. Table 9: AAV ITR and Capsid Sequences Structure SEQ Sequence ID T C A C- 109 - 40928727338013.0045P1 Structure SEQ Sequence ID- 110 - 40928727338013.0045P1 Structure SEQ Sequence IDAAV encoding a transgene. In certain embodiments, the transgene is a microdystrophin as described herein. In some embodiments, the rAAV genome (or cis plasmid) comprises the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter / enhancers, b) a poly A signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the described transgene. In a specific embodiment, the constructs (cis plasmid or recombinant AAV genome sequences) described herein comprise the following components: (1) AAV2 or AAV8 inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a muscle-specific SPc5-12 promoter and - 111 - 40928727338013.0045P1 a small poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid encoding microdystrophin as described herein, including the microdystrophin coding sequence of the RGX- DYS1 transgene (SEQ ID NO: 20). In a specific embodiment, the constructs (cis plasmid or recombinant AAV genome) described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle- specific SPc5-12 promoter, b) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus, ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, including the CT having an amino acid sequence of SEQ ID NO:16 or 83.

[0130] In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle-specific SPc5-12 promoter, and b) a small poly A signal; and (3) the nucleic acid encoding the RGX-DYS1 microdystrophin having an amino acid sequence of SEQ ID NO: 1, including encoded by a nucleotide sequence of SEQ ID NO: 20. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle-specific SPc5-12 promoter, and b) a small poly A signal; and (3) the nucleic acid encoding the RXG-DYS5 microdystrophin having an amino acid sequence of SEQ ID NO: 79, including encoded by a nucleotide sequence of SEQ ID NO: 81. In some embodiments, constructs described herein comprising AAV ITRs flanking a microdystrophin expression cassette, which includes from the N-terminus to the C- terminus ABD1-H1-R1-R2-R3-H2-R24-H4-CR-CT, wherein the CT comprises at least the portion of the CT comprising an α1-syntrophin binding site, including the CT having an amino acid sequence of SEQ ID NO: 16 or 83, can be between 4000 nucleotides and 5000 nucleotides in length. In some embodiments, such constructs (recombinant AAV genomes including ITR sequences)) are less than 4900 nucleotides, 4800 nucleotides, 4700 nucleotides, 4600 nucleotides, 4500 nucleotides, 4400 nucleotides, or 4300 nucleotides in length.

[0131] Some nucleic acid embodiments of the present disclosure comprise rAAV vectors (cis plasmids or recombinant AAV genomes) encoding microdystrophin comprising or consisting of a nucleotide sequence of SEQ ID NO: 53, 55, or 82 provided in Table 10 below. In various embodiments, an rAAV vector comprising a nucleotide sequence that has at least 50%, at least - 112 - 40928727338013.0045P1 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 53, 55, or 82 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective microdystrophin in muscle cells. In embodiments, the constructs having the nucleotide sequence of SEQ ID NO: 53, 55 or 82 are in a recombinant rAAV8 or rAAV9 particle. In embodiments, the recombinant AAV vector or particle is AAV8-RGX-DYS1. Table 10: RGX-DYS cassette and genome nucleotide sequences Structure SEQ Nucleic Acid Sequence ID c c C T A T G C G T G C C G C A A C A G A G C A A A G G T T C C A G G G G G C A- 113 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID A A T G G T G G A C C C C G G C G T C A C T C T A G T A C A G G G G T C G C T G t c g c c C T A T G C G- 114 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID A T A T C T T G G C T A A C A T A A T C A T A T A G A T T A A A T A G C C T T T A C A T A A T G T T A T G- 115 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID A A T C T G A A C T T T g c c c C T A T G C G T G C C G C A A C A G A G C A A A G G T T C C A G G G G- 116 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID G C A A A T G G T G G A C C C C G G C G T C A C T C T A G T A C A G G G G T C G t a g c c G A T T G C T- 117 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID C G C C A G C G C C A G A G G T T C G T G G G A A G G G G T G G T T G G G G G A G T G C C G C T C C A C A- 118 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID T G C T C C C C C G C C G T A G T T c g c c c C T A T G C G T G C C G C A A C A G A G C A A A G G T T- 119 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID C C A G G G G G C A A A T G G T G G A C C C C G G C G T C A C T C T A G T A C A G G G G T C G C T G t c- 120 - 40928727338013.0045P1 Structure SEQ Nucleic Acid Sequence ID g. . e o s o a ng r ar c es

[0132] Another aspect of the present invention involves making molecules disclosed herein. In some embodiments, a molecule according to the invention is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein. Such capsid proteins are described in Section 5.3.4, supra. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of a capsid protein molecule described herein and retains (or substantially retains) biological function of the capsid protein and the inserted peptide from a heterologous protein or domain thereof. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the AAV8 capsid protein, while retaining (or substantially retaining) biological function of the AAV8 capsid protein and the inserted peptide.

[0133] The capsid protein, coat, and rAAV particles may be produced by techniques known in the art. In some embodiments, the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector. In some embodiments, the viral genome further comprises a cap gene and / or a rep gene for expression and splicing of the cap gene. In embodiments, the cap and rep genes are provided by a packaging cell and not present in the viral genome.

[0134] In some embodiments, the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap plasmid in place of the existing capsid gene. When introduced together into host cells, this plasmid helps package an rAAV genome into the engineered capsid protein as the capsid coat. Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging.

[0135] Numerous cell culture-based systems are known in the art for production of rAAV particles, any of which can be used to practice a method disclosed herein. The cell culture-based - 121 - 40928727338013.0045P1 systems include transfection, stable cell line production, and infectious hybrid virus production systems which include, but are not limited to, adenovirus-AAV hybrids, herpesvirus-AAV hybrids and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV virus particles require: (1) suitable host cells, including, for example, human-derived cell lines, mammalian cell lines, or insect-derived cell lines; (2) suitable helper virus genes, provided by wild type or mutant adenovirus (such as temperature-sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper genes; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences and optionally regulatory elements; and (5) suitable media and media components (nutrients) to support cell growth / survival and rAAV production.

[0136] Nonlimiting examples of host cells include: A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293 and their derivatives (HEK293T cells, HEK293F cells), Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, myoblast cells, CHO cells or CHO-derived cells, or insect-derived cell lines such as SF-9 (e.g. in the case of baculovirus production systems). For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054, which is incorporated by reference herein in its entirety for manufacturing techniques.

[0137] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising an insect cell; (b) introducing into the cell one or more baculovirus vectors encoding at least one of: i. an rAAV genome to be packaged, ii. an AAV rep protein sufficient for packaging, and iii. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the method comprises using a first baculovirus vector encoding the rep and cap genes and a second baculovirus vector encoding the rAAV genome. In some embodiments, the method comprises using a baculovirus encoding the rAAV genome and an insect cell expressing the rep and cap genes. In some embodiments, the method comprises using a baculovirus vector encoding the rep and cap genes and the rAAV genome. In some embodiments, the insect cell is an Sf-9 cell. In some embodiments, the insect cell is an Sf-9 cell comprising one or more stably integrated heterologous polynucleotide encoding the rep and cap genes. - 122 - 40928727338013.0045P1

[0138] In some embodiments, a method disclosed herein uses a baculovirus production system. In some embodiments the baculovirus production system uses a first baculovirus encoding the rep and cap genes and a second baculovirus encoding the rAAV genome. In some embodiments the baculovirus production system uses a baculovirus encoding the rAAV genome and a host cell expressing the rep and cap genes. In some embodiments the baculovirus production system uses a baculovirus encoding the rep and cap genes and the rAAV genome. In some embodiments, the baculovirus production system uses insect cells, such as Sf-9 cells.

[0139] A skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV. The phrase “adenovirus helper functions” or “adenovirus helper genes” refers to a number of viral helper coding sequences or genes that when expressed in a cell (as RNA or protein) help the AAV grow efficiently in the cell. The skilled artisan understands that helper viruses, including adenovirus and herpes simplex virus (HSV), promote AAV replication and certain genes have been identified that provide the essential functions, e.g. the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication. In some embodiments of a method disclosed herein, AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, AAV rep and cap genes, helper genes, and rAAV genomes can be introduced into cells by transduction with viral vectors, for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, one or more of AAV rep and cap genes, helper genes, and rAAV genomes are introduced into the cells by transduction with an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes and the rAAV genome. In some embodiments, the rHSV vector encodes the helper genes and the AAV rep and cap genes. - 123 - 40928727338013.0045P1

[0140] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising a host cell; (b) introducing into the cell one or more rHSV vectors encoding at least one of: i. an rAAV genome to be packaged, ii. helper genes necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper genes. In some embodiments, the rHSV vector comprises helper genes. In some embodiments, the rHSV vector comprises one or more endogenous helper genes. In some embodiments, the rHSV vector comprises one or more heterogeneous helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper genes and the rAAV genome. In some embodiments, the rHSV vector encodes helper genes and the AAV rep and cap genes. In some embodiments, the cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[0141] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising a mammalian cell; (b) introducing into the cell one or more polynucleotides encoding at least one of: i. an rAAV genome to be packaged (including, for example, the recombinant AAV genome having the nucleotide sequence of SEQ ID NO: 53), ii. helper genes necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the helper genes are encoded by adenovirus genes. In some embodiments, the mammalian cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[0142] Molecular biology techniques to develop plasmid or viral vectors encoding the AAV rep and cap genes, helper genes, and / or rAAV genome are commonly known in the art. In some embodiments, AAV rep and cap genes are encoded by one plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene) are encoded by one plasmid vector. In some embodiments, the E1a gene or E1b gene is stably - 124 - 40928727338013.0045P1 expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the E1a gene and E1b gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, AAV rep and cap genes are encoded by one viral vector. In some embodiments, AAV helper genes (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector. In some embodiments, the E1a gene or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the E1a gene and E1b gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one viral vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the AAV rep and cap genes, the adenovirus helper genes necessary for packaging, and the rAAV genome to be packaged are introduced to the cells by transfection with one or more polynucleotides, e.g., vectors. In some embodiments, a method disclosed herein comprises transfecting the cells with a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding adenovirus helper genes necessary for packaging (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is an AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is an AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the vector encoding the rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs. In some embodiments, the AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, - 125 - 40928727338013.0045P1 AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes.

[0143] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and rAAV genome to a cell in which rAAV particles are to be produced or packaged. In some embodiments of a method disclosed herein, a first plasmid vector encoding an rAAV genome comprising a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding AAV rep and cap genes, and a third vector encoding helper genes can be used. In some embodiments, a mixture of the three vectors is co-transfected into a cell. In some embodiments, a combination of transfection and infection is used by using both plasmid vectors as well as viral vectors.

[0144] In some embodiments, one or more of rep and cap genes, and AAV helper genes are constitutively expressed by the cells and does not need to be transfected or transduced into the cells. In some embodiments, the cell constitutively expresses rep and / or cap genes. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses E1a. In some embodiments, the cell comprises a stable transgene encoding the rAAV genome.

[0145] In some embodiments, AAV rep, cap, and helper genes (e.g., Ela gene, E1b gene, E4 gene, E2a gene, or VA gene) can be of any AAV serotype. Similarly, AAV ITRs can also be of any AAV serotype. For example, in some embodiments, AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV cap gene is from AAV8 or AAV9 cap gene. In some embodiments, an AAV cap gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, - 126 - 40928727338013.0045P1 AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.rh74, AAV.hu31, AAV.hu32, or AAV.hu37 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV rep and cap genes for the production of a rAAV particle are from different serotypes. For example, the rep gene is from AAV2 whereas the cap gene is from AAV8. In another example, the rep gene is from AAV2 whereas the cap gene is from AAV9.

[0146] In some embodiments, the rep gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In other embodiments, the rep and the cap genes are from the same serotype. In still other embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises at least one modified protein domain or modified promoter domain. In certain embodiments, the at least one modified domain comprises a nucleotide sequence of a serotype that is different from the capsid serotype. The modified domain within the rep gene may be a hybrid nucleotide sequence consisting fragments different serotypes.

[0147] Hybrid rep genes provide improved packaging efficiency of rAAV particles, including packaging of a viral genome comprising a microdystrophin transgene greater than 4 kb, greater than 4.1 kb, greater than 4.2 kB, greater than 4.3 kb, greater than 4.4 kB, greater than 4.5 kb, or greater than 4.6 kb. AAV rep genes consist of nucleic acid sequences that encode the non-structural proteins needed for replication and production of virus. Transcription of the rep gene initiates from the p5 or p19 promoters to produce two large (Rep78 and Rep68) and two small (Rep52 and Rep40) nonstructural Rep proteins, respectively. Additionally, Rep78 / 68 domain contains a DNA- binding domain that recognizes specific ITR sequences within the ITR. All four Rep proteins have - 127 - 40928727338013.0045P1 common helicase and ATPase domains that function in genome replication and / or encapsidation (Maurer AC, 2020, DOI: 10.1089 / hum.2020.069). Transcription of the cap gene initiates from a p40 promoter, which sequence is within the C-terminus of the rep gene, and it has been suggested that other elements in the rep gene may induce p40 promoter activity. The p40 promoter domain includes transcription factor binding elements EF1A, MLTF, and ATF, Fos / Jun binding elements (AP-1), Sp1-like elements (Sp1 and GGT), and the TATA element (Pereira and Muzyczka, Journal of Virology, June 1997, 71(6):4300–4309). In some embodiments, the rep gene comprises a modified p40 promoter. In some embodiments, the p40 promoter is modified at any one or more of the EF1A binding element, MLTF binding element, ATF binding element, Fos / Jun binding elements (AP-1), Sp1-like elements (Sp1 or GGT), or the TATA element. In other embodiments, the rep gene is of serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, rh8, rh10, rh20, rh39, rh.74, RHM4-1, or hu37, and the portion or element of the p40 promoter domain is modified to serotype 2. In still other embodiments, the rep gene is of serotype 8 or 9, and the portion or element of the p40 promoter domain is modified to serotype 2.

[0148] ITRs contain A and A’ complimentary sequences, B and B’ complimentary sequences, and C and C’ complimentary sequences; and the D sequence is contiguous with the ssDNA genome. The complimentary sequences of the ITRs form hairpin structures by self-annealing (Berns KI. The Unusual Properties of the AAV Inverted Terminal Repeat. Hum Gene Ther 2020). The D sequence contains a Rep Binding Element (RBE) and a terminal resolution site (TRS), which together constitute the AAV origin of replication. The ITRs are also required as packaging signals for genome encapsidation following replication. In some embodiments, the ITR sequences and the cap genes are from the same serotype, except that one or more of the A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence may be modified to contain sequences from a different serotype than the capsid. In some embodiments, the modified ITR sequences are from the same serotype as the rep gene. In other embodiments, the ITR sequences and the cap genes are from different serotypes, except that one or more of the ITR sequences selected from A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence are from the same serotype as the capsid (cap gene), and one or more of the ITR sequences are from the same serotype as the rep gene. - 128 - 40928727338013.0045P1

[0149] In some embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises a modified Rep78 domain, DNA binding domain, endonuclease domain, ATPase domain, helicase domain, p5 promoter domain, Rep68 domain, p5 promoter domain, Rep52 domain, p19 promoter domain, Rep40 domain or p40 promoter domain. In other embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises at least one protein domain or promoter domain from a different serotype. In one embodiment, an rAAV comprises a transgene flanked by AAV2 ITR sequences, an AAV8 cap, and a hybrid AAV2 / 8 rep. In another embodiment, the AAV2 / 8 rep comprises serotype 8 rep except for the p40 promoter domain or a portion thereof is from serotype 2 rep. In other embodiments, the AAV2 / 8 rep comprises serotype 2 rep except for the p40 promoter domain or a portion thereof is from serotype 8 rep. In some embodiments, more than two serotypes may be utilized to construct a hybrid rep / cap plasmid.

[0150] Any suitable method known in the art may be used for transfecting a cell may be used for the production of rAAV particles according to a method disclosed herein. In some embodiments, a method disclosed herein comprises transfecting a cell using a chemical based transfection method. In some embodiments, the chemical-based transfection method uses calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)), lipofection. In some embodiments, the chemical-based transfection method uses cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)). In some embodiments, the chemical-based transfection method uses polyethylenimine (PEI). In some embodiments, the chemical-based transfection method uses DEAE dextran. In some embodiments, the chemical-based transfection method uses calcium phosphate.

[0151] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which - 129 - 40928727338013.0045P1 is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0152] Nucleic acid sequences of AAV-based viral vectors, and methods of making recombinant AAV and AAV capsids, are taught, e.g., in US 7,282,199; US 7,790,449; US 8,318,480; US 8,962,332; and PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety.

[0153] Provided are host cell lines for production of the rAAV particles containing the constructs (genomes) encoding the microdystrophins as disclosed herein, including the constructs of SEQ ID NO: 53 or 82 (RGX-DYS1 or RGX-DYS5).

[0154] In preferred embodiments, the rAAVs provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below. 5.4. Therapeutic Utility

[0155] Provided are methods of assaying the constructs, including recombinant gene therapy vectors and recombinant AAV genomes, encoding microdystrophins, as disclosed herein, for therapeutic efficacy. Methods include both in vitro and in vivo tests in animal models as described herein or using any other methods known in the art for testing the activity and efficacy of microdystrophins. 5.4.1 In vitro assays 5.4.1.1 In vitro infection system for muscle cells

[0156] Provided are methods of testing of the infectivity of a recombinant vector disclosed herein, for example rAAV particles. For example, the infectivity of recombinant gene therapy vectors in muscle cells can be tested in C2C12 myoblasts. Several muscle or heart cell lines may be utilized, including but not limited to T0034 (human), L6 (rat), MM14 (mouse), P19 (mouse), G-7 (mouse), G-8 (mouse), QM7 (quail), H9c2 (2-1) (rat), Hs 74.Ht (human), and Hs 171.Ht (human) cell lines. Vector copy numbers may be assessed using polymerase chain reaction - 130 - 40928727338013.0045P1 techniques and level of microdystrophin expression may be tested by measuring levels of microdystrophin mRNA in the cells. 5.4.2 Animal Models

[0157] The efficacy of a viral vector containing a transgene encoding a microdystrophin as described herein may be tested by administering to an animal model to replace mutated dystrophin, for example, by using the mdx mouse and / or the golden retriever muscular dystrophy (GRMD) model and to assess the biodistribution, expression and therapeutic effect of the transgene expression. The therapeutic effect may be assessed, for example, by assessing change in muscle strength in the animal receiving the microdystrophin transgene. Animal models using larger mammals as well as non-mammalian vertebrates and invertebrates can also be used to assess pre- clinical therapeutic efficacy of a vector described herein. Accordingly, provided are compositions and methods for therapeutic administration comprising a dose of a microdystrophin encoding vector disclosed herein in an amount demonstrated to be effective according to the methods for assessing therapeutic efficacy disclosed here. 5.4.2.1 Murine Models

[0158] The efficacy of gene therapy vectors may be assessed in murine models of DMD. The mdx mouse model (Yucel, N., et al, Humanizing the mdx mouse model of DMD: the long and the short of it, Regenerative Medicine volume 3, Article number: 4 (2018)), carries a nonsense mutation in exon 23, resulting in an early termination codon and a truncated protein (mdx). Mdx mice have 3-fold higher blood levels of pyruvate kinase activity compared to littermate controls. Like the human DMD disease, mdx skeletal muscles exhibit active myofiber necrosis, cellular infiltration, a wide range of myofiber sizes and numerous centrally nucleated regenerating myofibers. This phenotype is enhanced in the diaphragm, which undergoes progressive degeneration and myofiber loss resulting in an approximately 5-fold reduction in muscle isometric strength. Necrosis and regeneration in hind-limb muscles peaks around 3–4 weeks of age, but plateaus thereafter. In mdx mice and mdx mice crossed onto other mouse backgrounds (for example DBA / 2J), a mild but significant decrease in cardiac ejection fraction is observed (Van Westering, Molecules 2015, 20, 8823-8855). Such DMD model mice with cardiac functional defects may be used to assess the cardioprotective effects or improvement or maintenance of cardiac function or - 131 - 40928727338013.0045P1 attenuation of cardiac dysfunction of the gene therapy vectors described herein. Examples 5 and 7-9 herein details use of the mdx mouse model to assess gene therapy vectors encoding microdystrophins.

[0159] Additional mdx mouse models: A number of alternative versions in different genetic backgrounds have been generated including the mdx2cv, mdx3cv, mdx4cv, and mdx5cv lines (C57BL / 6 genetic background). These models were created by treating mice with N-ethyl-N- nitrosourea, a chemical mutagen. Each strain carries a different point mutation. As a whole, there are few differences in the presentation of disease phenotypes in the mdxcv models compared to the mdx mouse. Additional mouse models have been created by crossing the mdx line to various knock- out mouse models (e.g. Myod1- / -, ^-Integrin7- / -, ^-Dystrobrevin- / -, and Utrophin- / -). All mouse models which are currently used to study DMD have been described in detail by Yucel, N., et al, Humanizing the mdx mouse model of DMD: the long and the short of it, npj Regenerative Medicine volume 3, Article number: 4 (2018), which is incorporated herein by reference.

[0160] Cardiac function: Assessment of efficacy on cardiac function can be measured in mice, including mdx mice. To measure the blood pressure (BP) mice are sedated using 1.5% isofluorane with constant monitoring of the plane of anesthesia and maintenance of the body temperature at 36.5–37.58 C. The heart rate is maintained at 450–550 beats / min. A BP cuff is placed around the tail, and the tail is then placed in a sensor assembly for noninvasive BP monitoring during anesthesia. Ten consecutive BP measurements are taken. Qualitative and quantitative measurements of tail BP, including systolic pressure, diastolic pressure and mean pressure, are made offline using analytic software. See, for example, Wehling-Henricks et al, Human Molecular Genetics, 2005, Vol.14, No.14; Uaesoontrachoon et al, Human Molecular Genetics, 2014, Vol. 23, No.12.

[0161] To monitor ECG wave heights and interval durations in awake, freely moving mice, radio telemetry devices are used. Transmitter units are implanted in the peritoneal cavity of anesthetized mice and the two electrical leads are secured near the apex of the heart and the right acromion in a lead II orientation. Mice are housed singly in cages over antenna receivers connected to a computer system for data recording. Unfiltered ECG data is collected for 10 seconds each hour for 35 days. Thefirst 7 days of data are discarded to allow for recovery from the surgical procedure and ensure any effects of anesthesia has subsided. Data waveforms and parameters are analyzed - 132 - 40928727338013.0045P1 with the DSI analysis packages (ART 3.01 and Physiostat 4.01) and measurements are compiled and averaged to determine heart rates, ECG wave heights and interval durations. Raw ECG waveforms are scanned for arrhythmias by two independent observers.

[0162] Picro-Sirius red staining is performed to measure the degree of fibrosis in the heart of trial mice. In brief, at the end of trial, directly following euthanasia, the heart muscle is removed and fixed in 10% formalin for later processing. The heart is sectioned and paraffin sections are deparaffinized in xylene followed by nuclear staining with Weigert’s hematoxylin for 8 min. They are then washed and then stained with Picro-Sirius red (0.5 g of Sirius red F3B, saturated aqueous solution of picric acid) for an additional 30 min. The sections are cleared in three changes of xylene and mounted in Permount. Five random digital images are taken using an Eclipse E800 (Nikon, Japan) microscope, and blinded analysis is done using Image J (NIH). Blood samples are taken via cardiac puncture when the animals are euthanized, and the serum collected is used for the measurement of muscle CK levels. 5.4.2.2 Canine

[0163] Most canine studies are conducted in the golden retriever muscular dystrophy (GRMD) model (Korneygay, J.N., et al, The golden retriever model of Duchenne muscular dystrophy. Skelet Muscle. 2017; 7: 9, which is incorporated by reference in its entirety). Dogs with GRMD are afflicted with a progressive, fatal disease with skeletal and cardiac muscle phenotypes and selective muscle involvement - a severe phenotype that more closely mirrors that of DMD. GRMD dogs carry a single nucleotide change that leads to exon skipping and an out-of-frame DMD transcript. Phenotypic features in dogs include elevation of serum CK, CRDs on EMG, and histopathologic evidence of grouped muscle fiber necrosis and regeneration. Phenotypic variability is frequently observed in GRMD, as in humans. GRMD dogs develop paradoxical muscle hypertrophy which seems to play a role in the phenotype of affected dogs, with stiffness at gait, decreased joint range of motion, and trismus being common features. Objective biomarkers to evaluate disease progression include tetanic flexion, tibiotarsal joint angle, % eccentric contraction decrement, maximum hip flexion angle, pelvis angle, cranial sartorius circumference, and quadriceps femoris weight. - 133 - 40928727338013.0045P1 5.5. Methods of Treatment

[0164] Provided are methods of treating human subjects for any muscular dystrophy disease that can be treated by providing a functional dystrophin. DMD is the most common of such disease, but the gene therapy vectors that express microdystrophin provided herein can be administered to treat Becker muscular dystrophy (BMD), myotonic muscular dystrophy (Steinert’s disease), Facioscapulohumeral disease (FSHD), limb-girdle muscular dystrophy, X-linked dilated cardiomyopathy, or oculopharyngeal muscular dystrophy. The subjects are, in embodiments, from 1 to less than 4 years of age, 5 to 6 years of age, 7 to 8 years of age, or greater than 8 years of age (including up to 12 years of age). The microdystrophin of the present disclosure may be any microdystrophin described herein, including those that have the domains in an N-terminal to C- terminal order of ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, CR is a cysteine-rich region of dystrophin and CT is at least a portion of a C-terminal region of dystrophin comprising a α1- syntrophin binding site, in certain embodiments SEQ ID NO: 16 or SEQ ID NO: 83. In embodiments, the microdystrophin has an amino acid sequence of SEQ ID NO: 1, 2, or 79. In embodiments, the microdystrophin has an amino acid sequence of SEQ ID NO: 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109. In embodiments, provided are microdystrophins comprising a means for enhancing DAPC restoration or stabilizing interaction of the microdystrophin with α- dystrobrevin and / or nNOS. In embodiments, the microdystrophin does not comprise the amino acid sequence DTM at the C-terminus of the microdystrophin. In embodiments, the microdystrophin has the first 194 amino acids of the CT domain. The vectors encoding the microdystrophin include those having a nucleic acid sequence of SEQ ID NO: 20, 21, or 81, in certain embodiments, operably linked to regulatory elements for constitutive, muscle-specific (including skeletal, smooth muscle and cardiac muscle-specific) expression, or CNS specific expression, and other regulatory elements such as poly A sites. Such nucleic acids may be in the context of an rAAV genome, for example, flanked by ITR sequences, particularly, AAV2 ITR sequences. In certain embodiments, the methods and compositions comprising administering to a subject in need thereof, an rAAV comprising the construct (recombinant genome) having a nucleic - 134 - 40928727338013.0045P1 acid sequence of SEQ ID NO: 53, 55, or 82. In embodiments, the constructs or recombinant genomes are in an rAAV8 or rAAV9 particle. In embodiments, the recombinant AAV is AAV8- RGX-DYS1. In embodiments, the patient has been diagnosed with and / or has symptom(s) associated with DMD.

[0165] Also provided are methods of reducing and / or preventing contraction-induced muscle damage or methods of increasing muscle resilience in a subject suffering from a dystrophinopathy or having a DMD mutation. As shown in Example 9 and FIG. 19, the extended CT domain in µDys supports greater force development and protects against force loss following lengthening contraction. Accordingly, in the methods provided herein, muscles of the subject exhibit greater than 80%, or 85%, or 90%, or 95% of the force generation of the muscles of a subject that does not have a DMD mutation or dystrophinopathy. Further, in the methods provided herein, the muscles of a subject having a DMD mutation and / or dystrophinopathy are protected against force loss following a lengthening contraction. In the methods provided herein, the muscles of the subject generate about 50% more force relative to a subject administered a microdystrophin without the CT portion comprising an α1-syntrophin binding site. Methods of measuring force development and force loss can be performed by any method known in the art, including those described in Examples 7 and 9 and Moorwood et al. Provided are microdystrophins comprising a means for protecting muscle against contraction-induced damage, or means for enabling muscle recovery from contraction-induced damage

[0166] Also provided are methods of reducing or preventing contraction-induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation relative to the subject prior to administration of a therapeutic described herein or a natural history matched control (either with no microdystrophin administered or administered a microdystrophin without the extended CT domain, such as Elevidys®). As shows in Example 9, there was a reduction in Evans blue dye permeability post ECC in mice treated with microdystrophin comprising an extended CT domain compared to mice treated with microdystrophin without an extended CT domain, showing that muscle treated with the extended CT domain microdystrophin has better resilience of muscle fibers to contraction-associated stress. Measuring contraction-induced muscle damage can be done by any method known in the art, including but not limited to those described in Example 17. - 135 - 40928727338013.0045P1

[0167] Human clinical trial results (Example 6) provided herein show that administration of a microdystrophin encoding-AAV particle therapeutic as disclosed herein (AAV8-RGX-DYS1) to a human subject results in improvement in timed function tests (such as Time to Stand (TTSTAND), Time to Run / Walk 10 meters (TTRW or 10MWR), Time to Climb four stairs (TTCLIMB)) and NSAA results and caregiver reported improved function relative to a natural history matched external control. In the methods provided herein, the subject has improved timed function test (such as TTStand Velocity, 10MWR Velocity and / or TTClimb Velocity) or NSAA results relative to a natural history matched control subject (or relative to a matched control subject who has been administered a microdystrophin without an extended CT domain). The phrase “improved timed function” refers to improved ambulation. In embodiments, the mean change in improved time function is more than 0.5 seconds. In embodiments, the mean change in improved time function is more than 0.5 seconds, more than 1 second, more than 1.5 seconds, more than 2 seconds, more than 2.5 seconds or more than 3 seconds. In embodiments, the subject has improved timed test function or improved ambulation or NSAA results at 12 weeks, 24 weeks, 1 year, 2 years, or 3 years after administration relative to results prior to administration. In embodiments, the increase in NSAA score is more than 2. In embodiments, the increase in NSAA score is more than 2, more than 3, more than 4, more than 5, or more than 6. In embodiments, the improvement is greater than the minimal clinically important difference (MCID) relative to a natural history matched control of MCID standard. The phrase ‘minimal clinically important difference’ refers to the minimal amount of change to a clinical outcome that may make a difference in clinical care or quality of life to a patient (Duong et al 2021). By ‘natural history matched control subject’ is a subject or subjects matched for age and baseline disease, see for example subjects from D-RSC Data platform of the Critical Path Institute, and like natural history studies of DMD patients. In embodiments, the improvement is greater than age and baseline disease matched patients administered a microdystrophin without an extended CT domain, such as Elevidys. In embodiments, the subject caregiver-reported functional abilities are evaluated using Pediatric Outcomes Data Collection Instrument (PODCI) questionnaire, which quantifies functional abilities of a subject before and after treatment. In embodiments, the PODCI scale score mean change is more than 8. In embodiments, the PODCI scale score mean change is more than 8, more - 136 - 40928727338013.0045P1 than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, or more than 18.

[0168] Human clinical trial results provided herein show that administration of a microdystrophin encoding-AAV particle therapeutic as disclosed herein to a human subject who has a DMD mutation and / or a dystrophinopathy results in expression of the microdystrophin in the muscles of the subject and presence in muscle fibers (and sarcolemma localization) at 12 weeks. Studies in mice (Example 9) also show that microdystrophin comprising an extended CT domain is stabilized in the sarcolemma, in the context of the DAPC, leading to an observed greater steady- state protein level. In embodiments, provided are microdystrophins comprising a means for restoring DAPC in muscle cells of the subject. The phrase ‘restoring DAPC in muscle cells’ refers to restoring the stabilization of DAPC proteins in the sarcolemma of muscle fibers. Restoring DAPC can be detected by any method known in the art, including immunofluorescence of muscles with, for example, anti-α1-syntrophin, dystrobrevin, nNOS-1, and ß-dystroglycan antibodies as described in Example 9. In embodiments, microdystrophin comprising an extended CT domain have about 5%, about 10%, about 15%, about 20%, about 25% or about 30% increased levels of α1-syntrophin, α-dystrobrevin and / or nNOS at the sarcolemma compared to a microdystrophin that does not comprise an extended CT domain.

[0169] The “Decline Phase” of DMD can be defined the period of development during childhood where DMD patients experience progressive muscle weakness, which impairs walking, motor function, posture, breathing, which ultimately leads to a progressive decline in respiratory and cardiac function. This phase is generally characterized by loss of motor function accompanied by decreased NSAA scores, starting around the age of 7. However clinical variability is documented and recognized amongst patients. FIG. 10 illustrates the Decline Phase according to age and mean NSAA score. See also, Muntoni et al. 2019, PLoS ONE 14(9): e0221097, which is incorporated by reference in its entirety. Decline Phase may also refer to a subject whose natural history peers would experience progressive decline.

[0170] In embodiments, patients who are younger than 4 years of age, will be administered the PDMS-3 Body Control and Body Transport subtests at several timepoints, for example 12 weeks, 6 months, 9 months, 12 months, 18 months and 24 months following treatment with AAV8-RGX- DYS1. These subtests of the PDMS-3 provide raw, scaled, and age-equivalent (AEq) scores, which - 137 - 40928727338013.0045P1 scores are summarized using descriptive statistics. Number and percentage of participants who demonstrate stability in subtest scaled scores for patients with developmental function within 2 standard deviations (SD) from the normative mean are tabulated at the assessed time points. Stability (2 SD from the normative mean) at timepoints 12 weeks, 6 months, 9 months, 12 months, 18 months and / or 24 months in the PDMS-3 subtests is indicative of therapeutic efficacy. As such, patients who are younger than 4 years of age and treated with AAV8-RGX-DYS1 are not expected to enter the Decline Phase as time progresses.

[0171] In embodiments, treatment efficacy is determined by results of functional stability for patients that were previously considered in the Decline Phase. However, patients previously considered in the Decline Phase no longer exhibit progressive decline, and exhibit improved or stable function as indicated by timed function tests, NSAA stabilization, caregiver reported improved function reports (PODCI), and PDMS-3 Total Motor Score Index, which are indicative of treatment efficacy. In further embodiments, treatment efficacy is measured, for example, 12 weeks, 6 months, 9 months, 12 months, 18 months and 24 months after administration of AAV8- RGX-DYS1 and compared to natural history peers, including those in the Decline Phase, or pre- administration baseline.

[0172] In embodiments, provided are microdystrophins comprising a means for stabilization of a microdystrophin protein in muscle cells by interaction between a microdystrophin C-terminal domain (CT) and alpha-syntrophin. The phrase ‘stabilization of a microdystrophin’ refers to a greater steady-state protein level of microdystrophin in the muscle. Not wishing to be bound by one particular theory, said stabilization of the microdystrophin by and between the C terminal domain and DAPC proteins also provides for a stable muscle membrane, whereas the lack of a functional dystrophin in patients with DMD gene mutations caused a reduction in muscle cells’ membrane stability. Also provided are microdystrophins comprising a means for protecting muscle against contraction-induced damage, or means for enabling muscle recovery from contraction- induced damage.

[0173] Based upon pharmacology studies in mice, see, Example 9 herein (Section 6.9, infra) and human clinical results (Example 6, Section 6.6, infra), provided are methods of treatment of human patients having a dystrophinopathy amenable to treatment with functional dystrophin, such as DMD or BMD by peripheral, including intravenous administration, of an rAAV particle, including - 138 - 40928727338013.0045P1 rAAV8 or rAAV9 particle, containing a recombinant genome encoding a microdystrophin described herein (for example, AAV8-RGX-DYS1) at a dosage of 5×1013to 1x1015GC / kg, including a dose of 1×1014GC / kg or 2×1014GC / kg. Doses can range from 1×108vector genomes copies per kg (GC / kg) to 1×1015GC / kg. The gene therapy vectors may be administered to subjects 1 year of age to less than 4 years of age or 4 years to 5 years of age, or 6 to 7 years of age, or 8 to 12 years of age or greater than 8 years of age, including subjects in the Decline Phase or whose natural history peers are in the Decline Phase. In some embodiments, the dose can be 3.0×1013, 1.0×1014, 2.0×1014, 3.0×1014, or 5.0×1014GC / kg. In some embodiments, the dose can be 1.0×1014, 1.1×1014, 1.2×1014, 1.3×1014, 1.4×1014, 1.5×1014, 1.6×1014, 1.7×1014, 1.8×1014, 1.9×1014, 2.0×1014, 2.1×1014, 2.2×1014, 2.3×1014, 2.4×1014, 2.5×1014, 2.6×1014, 2.7×1014, 2.8×1014, 2.9×1014, or 3.0×1014GC / kg. Therapeutically effective dosages are administered as a single dosage and may be administered intravenously or intramuscularly. Alternatively, multiple doses may be administered during the course of a treatment regimen (i.e., days, weeks, months, etc.).

[0174] The dosages are therapeutically effective, which can be assessed at appropriate times after the administration, including 12 weeks, 26 weeks, 52 weeks or more, and include assessments for improvement, stability or amelioration of symptoms and / or biomarkers of the dystrophinopathy as known in the art and detailed herein, including the presence of the microdystrophin and localization to the sarcolemma in muscle tissue of the subject, and improvement or stability of performance in timed function tests and / or other tests such as the NSAA or PDMS-3 relative to the subject prior to administration to the subject or relative to a natural history matched control or relative to a matched control subject having been administered a microdystrophin without an extended CT domain, such as ELEVIDYS. Recombinant vectors used for delivering the transgene encoding the microdystrophin are described herein. Such vectors should have a tropism for human muscle cells (including skeletal muscle, smooth muscle and / or cardiac muscle) and can include non-replicating rAAV, particularly those bearing an AAV8 capsid. The recombinant vectors, including vectors having the recombinant construct or genome of RGX-DYS1 (see FIG. 2) (including in AAV8 or AAV9 recombinant vectors, e.g., AAV8-RGX-DYS1) can be administered in any manner such that the recombinant vector enters the muscle tissue or CNS, preferably by introducing the recombinant vector into the bloodstream, including intravenous administration. - 139 - 40928727338013.0045P1

[0175] In embodiments of the disclosed methods, the subject does not experience a significant or serious adverse event such as central neurotoxicity, peripheral neurotoxicity, drug-induced liver injury, thrombocytopenia, myocarditis, or myositis.

[0176] Subjects to whom such gene therapy is administered can be those responsive to gene therapy mediated delivery of a microdystrophin to muscles. In particular embodiments, the methods encompass treating patients who have been diagnosed with DMD or other muscular dystrophy disease, such as, Becker muscular dystrophy (BMD), myotonic muscular dystrophy (Steinert’s disease), Facioscapulohumeral disease (FSHD), limb-girdle muscular dystrophy, X- linked dilated cardiomyopathy, or oculopharyngeal muscular dystrophy, or have one or more symptoms associated therewith, and identified as responsive to treatment with microdystrophin, or considered a good candidate for therapy with gene mediated delivery of microdystrophin. In some embodiments, subjects have mutations (including a deletion, duplication, point mutation, including a nonsense mutation) in any exon. In embodiments, the subject has a form of DMD where the mutation is in any exon except those with deletions or point mutations in exons 8, 9, and / or 10. In embodiments, the subject has a form of DMD where the mutation is in exons 18 and above (i.e., in exons 18 to 79) in the DMD gene. In other embodiments, the subject has a form of DMD with a mutation within exons 12 to 17 the DMD gene. In other embodiments, the subject has a form of DMD with a mutation in any one or more of exons 12 to 79 of the DMD gene. In embodiments, the subject has a form of DMD where the mutation is outside exons 1 to 11 or outside exons 1 to 17 of the DMD gene. In specific embodiments, the patients have previously been treated with synthetic version of dystrophin and have been found to be responsive to one or more of synthetic versions of dystrophin. To determine responsiveness, the synthetic version of dystrophin (e.g., produced in human cell culture, bioreactors, etc.) may be administered directly to the subject.

[0177] Subjects to be administered the microdystrophin gene therapy are subjects 1-3 years old (i.e., less than 4 years old at dosing), 4 to 5 years old, 6 to 7 years old, and greater than 8 years old, including 8 to 12 years old. Subjects to be treated may include subjects in the decline phase of the disease or whose natural history peers are in the Decline Phase of the disease (see, e.g., Muntoni et al.2019, PLoS ONE 14(9): e0221097 and FIG.10). - 140 - 40928727338013.0045P1

[0178] Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the muscle (e.g., skeletal muscle or cardiac muscle), preferably by introducing the recombinant vector into the bloodstream. In specific embodiments, the vector is administered subcutaneously, intramuscularly or intravenously. Intramuscular, subcutaneous, or intravenous administration should result in expression of the soluble transgene product in cells of the muscle (including skeletal muscle, cardiac muscle, and / or smooth muscle). The expression of the transgene product results in delivery and maintenance of the transgene product in the muscle. Alternatively, the delivery may result in gene therapy delivery and expression of the microdystrophin in the liver, and the soluble microdystrophin product is then carried through the bloodstream to the muscles where it can impart its therapeutic effect.

[0179] Administration of gene therapy vectors described herein, including AAV8-RGX-DYS1, results in microdystrophin expression in tissues of the subject, including muscle tissue, including, for example within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 10 weeks, 12 weeks, 20 weeks, or 26 weeks after administration. The amount of microdystrophin in the muscle tissue may be measured by any method known in the art (see, e.g., Aardsma-Rus et al. Report of a TREAT- NMD / World Duchenne Organisation Meeting on Dystrophin Quantification Methodology. J Neuromuscul Dis. 2019;6(1):147-159), including a capillary-based Western immune assay, for example, as described in Example 4 herein. The amount of microdystrophin in the muscle tissue may also be measured by liquid chromatography-mass spectroscopy (LC-MS) as described in WO2023 / 064947 or WO2022 / 091025, precision mass spectrometry proteomics methods as described in US 2015 / 0168425 or immunodetection and / or immunostaining. In embodiments, administering the gene therapy results in greater than 10 ng / mg, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, or 150 ng / mg microdystrophin protein in the muscle of a subject administered a microdystrophin encoding gene therapy vector, including within 5 weeks, 6 weeks, 10 weeks, 12 weeks, 20 weeks or 26 weeks after the administration. In addition, microdystrophin may also be localized to the sarcolemma, for example as determined by detection of positive muscle fibers in a muscle same by immunofluorescence. The dose may be administered intravenously at 5×1013GC / kg to 1×1015GC / kg, including 1×1014GC / kg or 2×1014GC / kg.

[0180] In embodiments, microdystrophin levels, in the muscle tissue, for example a sample of bicep tissue, at 12 weeks after administration is greater than about 5%, greater than about 10 %, - 141 - 40928727338013.0045P1 greater than about 15%, or greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80% or greater than about 90% as determined by any method for quantitating dystrophin levels, for example, JESS (e.g., as described in Example 4), LCMS, western blot analysis, immunoprecipitation, immunostaining, using an antibody that detects microdystrophin or dystrophin and microdystrophin levels relative to a reference standard, such as compared to a microdystrophin or dystrophin control in the same assay. In embodiments, the microdystrophin levels at 12 weeks after administration are from about 10% to 90%, 20% to 90%, 40% to 90%, 50% to 90%, 40% to 80%, 50% to 80%, 40% to 70%, or about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% microdystrophin relative to a standard control. The values may be adjusted for the percent muscle content present in the sample. In embodiments, microdystrophin levels or dystrophin, including microdystrophin, in the muscle tissue is greater than about 130 fmol / mg, or greater than about 260 fmol / mg, greater than about 390 fmol / mg, or greater than about 522 fmol / mg microdystrophin, including up to 520 fmol / mg, 600 fmol / mg, 700 fmol / mg, 800 fmol / mg in a sample taken 12 weeks after administration as measured by a capillary Western or immunoaffinity liquid chromatography-mass spectroscopy (IA-LC / MS) assay, also referred to as hybrid ligand binding assay (LBA) / liquid chromatography–tandem mass spectrometry (LBA- LC / MS-MS, or LC-MS). In some embodiments, the capillary Western or IA-LC / MS assay specifically captures RGX-DYS1 protein with an antibody that binds RGX-DYS1 protein or a RGX-DYS1 protein fragment with high affinity.

[0181] In embodiments, the level of microdystrophin in a muscle sample taken from the subject at 6 months, one year, 18 months or 24 months after administration is stable (within 10%, 20%, 30%, 40% or 50%) relative to the value at 12 weeks after administration, as determined by JESS and / or LC-MS analysis. Without being bound solely by one theory, the stability of microdystrophin protein expressed by the vector genome and durability of the vector genome are factors that provide long-term efficacy for gene therapies, including an AAV-microdystrophin gene therapy such as AAV8-RGX-DYS1. Functional and imaging assessments, amongst other assessments and markers, are taken at regular intervals following administration of AAV8-RGX- DYS1 and compared to baseline assessments and the earliest post-dose assessment. Such assessments taken from the subject at 6 months, one year, 18 months, 24 months, 36 months, 48 - 142 - 40928727338013.0045P1 months or 60 months after administration indicate that the microdystrophin is stable (within 10%, 20%, 30%, 40% or 50%) relative to the value or score at baseline or the earliest post-dose assessment. Functional assessments include North Star Ambulatory Assessment (NSAA), Time to Stand (TTSTAND), Time to Run / Walk 10 meters (TTRW), Time to Climb four stairs (TTCLIMB), (TTSTAND, TTRW and TTCLIMB collectively being timed function tests (TFTs), and Peabody Developmental Motor Scale 3 (PDMS-3). Efficacy may be shown by improvement or stability in these assessments relative to pre-administration baseline or natural history peers as described herein. Actigraphy measurements (measuring human rest / activity cycles) can also be used to assess gross motor activity in young children. An actimitry sensor, including SV95C and the wearable Syde device (Sysnav Health Care) can be used to take actigraphy measurements. See Poleur, M. et al. 2021. Performance of Upper Limb 2.0, Range of Motion, Pulmonary function tests, and / or myometry. Imaging assessments include Doppler echocardiogram, Cardiac MRI imaging, and / or MRI of skeletal muscle. Other assessments include creatine kinase levels, and patient-reported outcomes (such as PedsQL™: Generic Core, Multidimensional Fatigue, Neuromuscular, and FIM; and / or POSNA PODCI).

[0182] Pharmaceutical compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding microdystrophin in a formulation buffer comprising a physiologically compatible aqueous buffer. The formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil. The disclosed pharmaceutical compositions can comprise any of the microdystrophins, particularly the rAAV vectors comprising a transgene encoding the microdystrophins, disclosed herein and can be used in the disclosed methods.

[0183] For example, a pharmaceutical composition comprising a rAAV, including an rAAV8 comprising a transgene encoding RGX-DYS1, including the RGX-DYS1 recombinant genome having the nucleotide sequence of SEQ ID NO: 53 can be used in the disclosed methods. In some embodiments, a pharmaceutical composition can comprise a recombinant adeno-associated virus serotype 8 (AAV8) that contains a vector (recombinant AAV genome encoding a microdystrophin). The rAAV particles containing recombinant genomes encoding the microdystrophins disclosed herein, including RGX-DYS1, and in embodiments AAV8-RGX- DYS1, can be formulate in modified Dulbecco’s phosphate buffered saline (DPBS) with sucrose - 143 - 40928727338013.0045P1 buffer, which comprises 0.2 g / L potassium chloride, 0.2 g / L potassium phosphate monobasic, 1.2 g / L sodium phosphate dibasic (anhydrous or hydrate), 5.8 g / L sodium chloride, 40 g / L sucrose, and 0.01 g / L poloxamer 188, pH 7.4. The pharmaceutical composition can be supplied as a frozen suspension in sterile, single-use vials for intravenous (IV) administration. In some embodiments, the pharmaceutical composition can be filled into Crystal Zenith® (CZ) vials sealed with latex- free rubber stoppers and flip-off aluminum seals. In some embodiments, the pharmaceutical composition can be available in one configuration: 5.0 mL deliverable volume in a 10 mL vial.

[0184] In embodiments, immunosuppressant prophylaxis is administered with the therapeutic, such as AAV8-RGX-DYS1 (or other microdystrophin encoding vector disclosed herein). See, for example, Chu and Ng, Frontiers in Immunology, 12:, article 658038 (April 2021), which is incorporated herein by reference in its entirety). In embodiments, a corticosteroid, such as prednisone, prednisolone, methylprednisolone, dexamethasone, or betamethasone is administered starting at least 1 day prior, and up to 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks prior to gene therapy delivery, including RGX-DYS1. Including daily administration for that period of time and including, in embodiments, corticosteroid administration the day of, including concomitantly with, the gene therapy administration, and then, in embodiments, continued administration, including daily administration, for up to 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months 5 months, 6 months or up to one year where in dose of the corticosteroid may be held constant or may be tapered down to zero over time. In certain embodiments, the patient is on oral corticosteroid, such as prednisone or prednisolone (at a dose, for example, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 1.5 mg / kg dosage) for 12 weeks prior to gene therapy delivery and then is continued for a year after at the same dose or the dose is tapered over 4 weeks, 8 weeks or 12 weeks. In embodiments, a prophylactic immunosuppressant regimen is administered in addition to a subject’s baseline glucocorticoid dose 1 mg / kg / day from day -1 (the day prior to AAV8-RGX- DYS1 administration) to end of week 8, if there are no safety concerns at week 8, then 0.5 mg / kg / day from week 9 to week 10, and if there are no safety concerns at week 10, then 0.25 mg / kg / day, and if there are no safety concerns at week 12, no additional prednisolone. In embodiments, the total daily steroid dose (baseline regimen dose and immunosuppressant dose) does not exceed a dose equivalent to 60 mg per day. Patients may also be pre-treated with - 144 - 40928727338013.0045P1 acetaminophen and an H1-antihistamine the day of, including within 2 hours or 1 hour, of gene therapy administration. Day 0 is the day of gene therapy administration.

[0185] Alternatively or in addition to the corticosteroid prophylaxis, patients may be administered prophylactically a non-steroidal immunosuppressant. The immunosuppressant may be administered before, concomitantly with and / or after administration of the gene therapy, e.g., AAV8-RGX- DYS1, for example, at least 1 day prior, and up to 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks prior to gene therapy delivery, including on a regular basis prior to gene therapy delivery and / or is administered after the gene therapy delivery, for example, for up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months or even indefinitely as maintenance therapy. Such immunosuppressants include, but are not limited to cyclosporine, rapamycin, an anti-cytokine antibody treatment, such as anti-IL-6 or IL-6 receptor antibodies, such as, satralizumab, sarilumab, siltuximab, clazakizumab, sirukumab, olokizumab, gerilimzumab, and tocilizumab; or anti-complement antibodies, including anti-C5 antibodies, such as, but not limited to eculizumab, ravulizumab or tsidolumab or anti-C3 antibodies, such as NGM621. Other immunosuppressants that may be used include, for example, anti-CD20 antibodies, such as rituximab (including biosimilar forms thereof, such as rutixumab- abbs and rituximab-arrx) and obinutuzumab, and anti-TNF-α antibodies, such as, but not limited to, etanercept, adalimumab, infliximab, daclizumab or golimumab. In an embodiment, the prophylactic regimen is a combination of an anti-CD-20 antibody and an anti-C5 antibody, for example, a combination of rituximab and eculizumab or ravulizumab. In one embodiment, the combination of rituximab and eculizumab or ravulizumab are administered after gene therapy administration Other prophylactic agents include imlifidase. In embodiments, provided in combination with the microdystrophin therapy is an anti-complement (anti-C5) immunosuppressive regimen with administration of eculizumab prior to administration of the AAV vector containing the microdystrophin transgene and then through day 12 after administration. Eculizumab is administered by IV infusion depending upon the subject’s body weight. For subjects 10 kg to less than 20 kg, a dose of 600 mg is administered at day -9, day -2, day 4 and day 12; for subjects of 20 kg to less than 30 kg, 800 mg is administered at day -16, day -9, day -2, and day 12; for subjects from 30 kg to less than 40 kg, a dose of 900 mg at day -16, day - 145 - 40928727338013.0045P1 -9, day -2 and day 12; and for subjects over 40 kg, a dose of 1200 mg is administered at day -30, day -23, day -16, day -9, day -2 and day 12, where day 0 is the day of administration of the microdystrophin gene therapy.

[0186] In other embodiments, the gene therapy administration is in combination with an immunosuppressive regimen of administration of sirolimus (also known as rapamycin), which inhibits the ability of cytokines to promoter T cell expansion and maturation by blocking intracellular signaling and metabolic pathways. In embodiments, the gene therapy administration is administered in combination with oral sirolimus with a loading dose of 3 mg / m2at day -7, then from day -6 to week 8, oral sirolimus 1 mg / m2 / day divided into twice daily (BID) dosing, with target blood level of 8-12 mg / ml using a chromatography assay. Trough monitoring may be carried out on day -2, day 2, day 6, day 12 and day 14, and then as needed (day 0 being the day of gene therapy administration). If liver function tests, platelets and any other relevant safety laboratory measurements remain stable, the daily dose of sirolimus is decreased by 50 % (to 0.5 mg / m2 / day) for weeks 9 to 10, and if liver function tests, platelets and any other relevant safety laboratory measurements remain stable, the daily dose of sirolimus can be decreased by 50% (to 0.25 mg / m2 / day) for weeks 11 to 12, and then, if liver function tests, platelets and any other relevant safety laboratory measurements remain stable, after week 12, sirolimus dosing can be discontinued. In embodiments, the immunosuppressive regimen administered with the gene therapy can be a combination of the prednisolone dosing regimen and / or the eculizumab dosing regimen and / or the sirolimus dosing regimen as detailed above. Generally, patients will be pre- treated with the prophylactic immunosuppressant and immunosuppressant therapy may be continued after gene therapy administration, including for days, weeks, months or years.

[0187] The gene therapy vectors provided herein may be administered in combination with other treatments for muscular dystrophy, including corticosteroids, beta blockers and ACE inhibitors.

[0188] Gene therapy administration as described herein can result in improvement in disease parameters and / or symptoms associated with muscular dystrophy, including but not limited to, increase in or slowing in reduction of muscle strength, improvement in or slowing in the rate or extent of muscle degeneration, inflammation, fibrosis, muscle lesions, and other clinical endpoints discussed below. - 146 - 40928727338013.0045P1

[0189] The disclosed methods of treatment can result in one of many endpoints indicative of therapeutic efficacy described herein. In some embodiments, the endpoints can be monitored 6 weeks, 12 weeks, 24 weeks, 30 weeks, 36 weeks, 42 weeks, 48 weeks, 1 year, 2 years, 3 years, 4 years or 5 years after the administration of a rAAV particle comprising a transgene that encodes one of the disclosed microdystrophins.

[0190] In some embodiments, creatine kinase activity can be used as an endpoint for therapeutic efficacy of the methods of treatment and administration disclosed herein. The creatine kinase activity can decrease in the subject relative to the level (of creatine kinase activity) prior to said administration. In some embodiments, the creatine kinase activity can decrease in the subject relative to the level (of creatine kinase activity) in the subject prior to treatment or relative to the level (of creatine kinase activity) in a non-treated subject having a dystrophinopathy (for example, a reference level identified in a natural history study). The creatine kinase activity measured in the human subject after administration of a rAAV with a transgene encoding microdystrophin can be to a control value which can be the creatine kinase activity in the subject prior to administration, creatine kinase activity in a subject with a dystrophinopathy that has not be treated, creatine kinase activity in a subject that does not have a dystrophinopathy, creatine kinase activity in a standard.

[0191] In some embodiments, provided are methods of treating a dystrophinopathy, including DMD and BMD, by peripheral, including intravenous administration of an rAAV vector containing a microdystrophin recombinant genome disclosed herein, including AAV8-RGX- DYS1, at dosages, including intravenous administration dosages, disclosed herein, including dosages of 5×1013genome copies / kg to 1x1015genome copies / kg, including 1x1014genome copies / kg, 2x1014genome copies / kg or 3x1014genome copies / kg genome copies / kg, wherein the in creatine kinase activity is reduced by 0.5 fold to 1.5 fold at least 12 weeks, 26 weeks, or 52 weeks after administration of the rAAV therapeutic. In some embodiments, a decrease in creatine kinase activity can be a decrease of 1000 to 10,000 units / liter compared to a control or the value measured in the subject amount prior to administration of the therapeutic. In some embodiments, an amount of 1000, 2000, 3000, 4000, or 5000 units / liter in the after administration endpoint is indicative of a decrease.

[0192] In some embodiments, serum creatine kinase levels are decreased by about 20% to about 60%, 70%, 80% or 90% in patients compared to average baseline levels prior to administration of - 147 - 40928727338013.0045P1 the rAAV therapeutic at least 10 weeks after administration of the rAAV therapeutic. In some embodiments, serum creatine kinase levels are decreased by about 40% to about 50%, or from about 70% to about 90% in patients compared to average baseline levels prior to administration of the rAAV therapeutic at least 10 weeks after administration of the rAAV therapeutic. In some embodiments, serum creatine kinase levels are decreased by about 40% to about 45% in patients compared to average baseline levels prior to administration of the rAAV therapeutic at least 10 weeks after administration of the rAAV therapeutic. In some embodiments, serum creatine kinase levels are decreased by about 77% in patients compared to average baseline levels prior to administration of the rAAV therapeutic at least 10 weeks after administration of the rAAV therapeutic. In some embodiments, serum creatine kinase levels are decreased by about 93% in patients compared to average baseline levels prior to administration of the rAAV therapeutic at least 10 weeks after administration of the rAAV therapeutic.

[0193] In some embodiments, reduction in lesions in a gastrocnemius muscle (or other muscle) can be used as an endpoint measure for therapeutic efficacy for the methods of treatment and administration disclosed herein. The lesions in a gastrocnemius muscle can decrease in the subject relative to the level (of lesions in the gastrocnemius muscle) prior to said administration of rAAV with a transgene encoding microdystrophin. In some embodiments, the lesions in the gastrocnemius muscle can decrease in the subject relative to the level (of lesions in the gastrocnemius muscle) in a non-treated subject having a dystrophinopathy. The comparison of lesions in the gastrocnemius muscle can be to a standard, wherein the standard is a number or set of numbers that represent the lesions in a subject that does not have a dystrophinopathy or the lesions in a non-treated subject having a dystrophinopathy. Thus, in some embodiments, the comparison of lesions in the gastrocnemius muscle after administration of a rAAV with a transgene encoding microdystrophin can be to a control subject. The control can be the lesions in the gastrocnemius muscle in the subject prior to administration lesions in the gastrocnemius muscle in a subject with a dystrophinopathy that has not be treated, lesions in the gastrocnemius muscle in a subject that does not have a dystrophinopathy, or lesions in the gastrocnemius muscle in a standard.

[0194] In some embodiments, the lesions in the gastrocnemius muscle of the subject are assessed using magnetic resonance imaging (MRI). MRI can be a good tool for imagine muscles, ligaments, and tendons, therefore, muscle disorders can be detected and / or characterized using MRI. In some - 148 - 40928727338013.0045P1 embodiments, provided are methods of treating a dystrophinopathy, including DMD and BMD, by peripheral, including intravenous administration of an rAAV vector containing a microdystrophin construct disclosed herein, including AAV8-RGX-DYS1, at dosages disclosed herein, including dosages of 5×1013genome copies / kg to 1x1015genome copies / kg including 1x1014genome copies / kg, 2x1014genome copies / kg or 3x1014genome copies / kg, resulting in a decrease of lesions in gastrocnemius muscle after administration is about 1-100%, 2-50%, or 3- 10% compared a control, for example, compared to the lesions in the gastrocnemius muscle of the subject prior to said administration. For example, a subject treated with a rAAV with a transgene encoding microdystrophin can have 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or greater decrease in lesions compared to a control.

[0195] In some embodiments, gastrocnemius muscle volume (or muscle volume of any other muscle) can be used as an endpoint for treatment efficacy. The gastrocnemius muscle volume can decrease in the subject relative to the level (of gastrocnemius muscle volume) prior to said administration of rAAV with a transgene encoding microdystrophin (for example, AAV8-RGX- DYS1). In some embodiments, the gastrocnemius muscle volume can decrease in the subject relative to the level (of gastrocnemius muscle volume) in a subject that does not have a dystrophinopathy. In some embodiments, the gastrocnemius muscle volume can decrease in the subject relative to the level (of gastrocnemius muscle volume) in a non-treated subject having a dystrophinopathy. The comparison of gastrocnemius muscle volume can be to a standard, wherein the standard is a number or set of numbers that represent the volume in a subject that does not have a dystrophinopathy or the volume in a non-treated subject having a dystrophinopathy. Thus, in some embodiments, the comparison of gastrocnemius muscle volume after administration of a rAAV with a transgene encoding microdystrophin can be to a control. The control can be the gastrocnemius muscle volume in the subject prior to administration, gastrocnemius muscle volume in a subject with a dystrophinopathy that has not be treated, gastrocnemius muscle volume in a subject that does not have a dystrophinopathy, or gastrocnemius muscle volume in a standard.

[0196] In some embodiments, the gastrocnemius muscle volume of the subject can be assessed using MRI. In some embodiments, provided are methods of treating a dystrophinopathy, including DMD and BMD, by peripheral, including intravenous administration, of an rAAV vector containing a microdystrophin construct disclosed herein, including AAV8-RGX-DYS1, at dosages - 149 - 40928727338013.0045P1 disclosed herein, including dosages of 5×1013genome copies / kg to 1x1015genome copies / kg including 1x1014genome copies / kg, 2x1014genome copies / kg or 3x1014genome copies / kg, resulting in a decrease in gastrocnemius muscle volume of about 1-100%, 2-50%, or 3-20% compared a control, for example, compared to the gastrocnemius muscle volume prior to said administration. In some embodiments, a decrease of gastrocnemius muscle volume after administration of a rAAV comprising a transgene that encodes microdystrophin can be about 2- 400 mm3, 5-200 mm3, or 20-100 mm3compared a control. For example, a subject treated with a rAAV with a transgene encoding microdystrophin can have 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mm3or greater decrease in gastrocnemius muscle volume compared to a control.

[0197] In some embodiments, a fat fraction of muscle can be used as an endpoint for therapeutic efficacy of the methods of administering rAAV microdystrophin-encoding therapeutics disclosed herein. The muscle can be muscles in the pelvic girdle and thigh (gluteus maximus, adductor magnus, rectus femoris, vastus lateralis, vastus medialis, biceps femoris, semitendinosus, and gracilis). The fat fraction of muscle can decrease in the subject relative to the level (of fat fraction of muscle) prior to said administration of rAAV with a transgene encoding microdystrophin as disclosed herein. In some embodiments, the fat fraction of muscle can decrease in the subject relative to the level (of fat fraction of muscle) in a non-treated subject having a dystrophinopathy. The comparison of fat fraction of muscle can be to a standard, wherein the standard is a number or set of numbers that represent the amount or percent of fat fraction of muscle in a subject that does not have a dystrophinopathy or the amount or percent in a non-treated subject having a dystrophinopathy. Thus, in some embodiments, the comparison of fat fraction of muscle after administration of a rAAV with a transgene encoding microdystrophin can be to a control. The control can be the fat fraction of muscle in the subject prior to administration, fat fraction of muscle in a subject with a dystrophinopathy that has not be treated, fat fraction of muscle in a subject that does not have a dystrophinopathy, or fat fraction of muscle of a standard.

[0198] In some embodiments, the fat fraction of muscle of the subject are assessed using magnetic resonance imaging (MRI). In some embodiments, provided are methods of treating a dystrophinopathy, including DMD and BMD, by peripheral, including intravenous administration of an rAAV vector containing a microdystrophin construct disclosed herein, including AAV8- - 150 - 40928727338013.0045P1 RGX-DYS1, at dosages disclosed herein, including dosages of 5×1013genome copies / kg to 1×1015genome copies / kg including 1×1014genome copies / kg, 2×1014genome copies / kg, and 3×1014genome copies / kg, results in a decrease of fat fraction of muscle after administration of a rAAV comprising a transgene that encodes microdystrophin can be about 1-100%, 2-50%, or 3-10% compared a control, for example, compared to the fat fraction of muscle prior to said administration. For example, a subject treated with a rAAV with a transgene encoding microdystrophin can have 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or greater decrease in fat fraction of muscle compared to a control.

[0199] In some embodiments, T2-relaxation time of lesions in muscle can be used as an endpoint for treatment. The muscle can be any muscle, for example, gastrocnemius. The T2-relaxation time of lesions in muscle can decrease in the subject relative to the level (of T2-relaxation time of lesions in muscle) prior to said administration of rAAV with a transgene encoding microdystrophin. In some embodiments, the T2-relaxation time of lesions in muscle can decrease in the subject relative to the level (of T2-relaxation time of lesions in muscle) in a subject that does not have a dystrophinopathy. In some embodiments, the T2-relaxation time of lesions in muscle can decrease in the subject relative to the level (of T2-relaxation time of lesions in muscle in a non-treated subject having a dystrophinopathy. The comparison of T2-relaxation time of lesions in muscle can be to a standard, wherein the standard is a number or set of numbers that represent the T2-relaxation time of lesions in muscle in a subject that does not have a dystrophinopathy or the T2-relaxation time of lesions in muscle in a non-treated subject having a dystrophinopathy. Thus, in some embodiments, the comparison of T2-relaxation time of lesions in muscle after administration of a rAAV with a transgene encoding microdystrophin can be to a control. The control can be the T2-relaxation time of lesions in muscle in the subject prior to administration, T2-relaxation time of lesions in muscle in a subject with a dystrophinopathy that has not be treated, T2-relaxation time of lesions in muscle in a subject that does not have a dystrophinopathy, or T2- relaxation time of lesions in muscle in a standard.

[0200] In some embodiments, the T2-relaxation time of lesions in muscle of the subject is assessed using magnetic resonance imaging (MRI). In some embodiments, a decrease of T2- relaxation time of lesions in muscle after administration of a rAAV comprising a transgene that encodes microdystrophin can be about 1-100%, 5-50%, or 10-30% compared a control, for - 151 - 40928727338013.0045P1 example, compared to the T2-relaxation time of lesions in muscle prior to said administration. In some embodiments, provided are methods of treating a dystrophinopathy, including DMD and BMD, by peripheral, including intravenous administration, of an rAAV vector containing a microdystrophin construct disclosed herein, including AAV8-RGX-DYS1, at dosages disclosed herein, including dosages of 5×1013genome copies / kg to 1x1015genome copies / kg, including 1x1014genome copies / kg, 2x1014genome copies / kg, and 3x1014genome copies / kg, which results in a decrease of T2-relaxation time of lesions in muscle after administration of a rAAV comprising a transgene that encodes microdystrophin can be about 1-500 milliseconds (ms), 1-400 ms, 1-300 ms, 1-200 ms, 1-100 ms, 1-50 ms, 1-25 ms, 1-10 ms compared a control. In some embodiments, a decrease of T2-relaxation time of lesions in muscle after administration of a rAAV comprising a transgene that encodes microdystrophin can be about 2 to 8 ms. For example, a subject treated with a rAAV with a transgene encoding microdystrophin can have a decrease of T2-relaxation time of lesions in muscle compared to a control of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or more ms.

[0201] In some embodiments, gait score can be used as an endpoint for treatment. The gait score can be about -1 to 2 after administration of a rAAV comprising a transgene that encodes microdystrophin. In some embodiments, the gait score can be about 1 after administration of a rAAV comprising a transgene that encodes microdystrophin.

[0202] In some embodiments, the North Star Ambulatory Assessment (NSAA) can be used as an endpoint for treatment. The NSAA of the treated subject can be compared to NSAA prior to administration of rAAV comprising a transgene that encodes microdystrophin. The NSAA of the treated subject can be compared to NSAA in a subject that does not have a dystrophinopathy. The NSAA of the treated subject can be compared to a non-treated subject having a dystrophinopathy. The NSAA of the treated subject can be compared to a standard, wherein the standard is a score or set of scores that represent the NSAA in a subject that does not have a dystrophinopathy or the NSAA in a non-treated subject having a dystrophinopathy.

[0203] In some embodiments, the NSAA of the subject treated with rAAV comprising a transgene that encodes microdystrophin increased compared to the NSAA score prior to said administration or compared to any of the NSAA comparisons described above. In some embodiments, the increase can be an increase in score of 1, 2 or more than 2. - 152 - 40928727338013.0045P1

[0204] In some embodiments, any of the 17 items used in the NSAA can be used as an individual endpoint of treatment. For example, any of the following can be endpoints for treatment, stand, walk, stand up from chair, stand on one leg (right), stand on one leg (left), climb box step (right leg first), climb box step (left leg first), descend box step (right leg first), descend box step (left leg first), lying to sitting, rise from floor, lift head, stand on heels, jump, hop right leg, hop left leg, and run (10m). Each of these assessments are well known in the art. An improvement in one or more of these endpoints can be seen after administration of rAAV comprising a transgene that encodes microdystrophin. One of skill in the art would understand what is considered an improvement. For example, in some embodiments a decrease in the amount of time it takes the subject treated with of rAAV comprising a transgene that encodes microdystrophin to stand, run / walk a determined distance, and / or climb a set number of stairs can be achieved.

[0205] A decrease in the amount of time it takes a subject administered rAAV comprising a transgene that encodes microdystrophin to run / walk a determined distance can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more compared to a control, for example, the amount of time it took the subject prior to administration of the rAAV or a natural history matched control. In some embodiments, the determined distance to run and / or walk can be 10 meters.

[0206] A decrease in the amount of time it takes a subject administered rAAV comprising a transgene that encodes microdystrophin to stand can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more compared to a control, for example, the amount of time it took the subject prior to administration of the rAAV or a natural history matched control.

[0207] A decrease in the amount of time it takes a subject administered rAAV comprising a transgene that encodes microdystrophin to climb a set number of stairs can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more compared to a control, for example, the amount of time it took the subject prior to administration of the rAAV or a natural history matched control. In some embodiments, the set number of stairs can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0208] In some embodiments, questionnaires can be used as an endpoint for treatment. For example, Pediatric Outcomes Data Collection Instrument (PODCI) questionnaire can be used to - 153 - 40928727338013.0045P1 quantify functional abilities of a subject before and after treatment with rAAV comprising a transgene encoding microdystrophin. 5.5.1 Cardiac output

[0209] Although skeletal muscle symptoms are considered the defining characteristic of DMD, patients most commonly die of respiratory or cardiac failure. DMD patients develop dilated cardiomyopathy (DCM) due to the absence of dystrophin in cardiomyocytes, which is required for contractile function. This leads to an influx of extracellular calcium, triggering protease activation, cardiomyocyte death, tissue necrosis, and inflammation, ultimately leading to accumulation of fat and fibrosis. This process first affects the left ventricle (LV), which is responsible for pumping blood to most of the body and is thicker and therefore experiences a greater workload. Atrophic cardiomyocytes exhibit a loss of striations, vacuolization, fragmentation, and nuclear degeneration. Functionally, atrophy and scarring leads to structural instability and hypokinesis of the LV, ultimately progressing to general DCM. DMD may be associated with various ECG changes like sinus tachycardia, reduction of circadian index, decreased heart rate variability, short PR interval, right ventricular hypertrophy, S-T segment depression and prolonged QTc.

[0210] Gene therapy treatment provided herein (including administration with AAV8-RGX- DYS1) can slow or arrest the progression of DMD and other dystrophinopathies, particularly to reduce the progression of or attenuate cardiac dysfunction and / or maintain or improve cardiac function. Efficacy may be monitored by periodic evaluation of signs and symptoms of cardiac involvement or heart failure that are appropriate for the age and disease stage of the trial population, using serial electrocardiograms, and serial noninvasive imaging studies (e.g., echocardiography or cardiac magnetic resonance imaging (CMR)). CMR may be used to monitor changes from baseline in forced vital capacity (FVC), forced expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), inflammation, and fibrosis. ECG may be used to monitor conduction abnormalities and arrhythmias. In particular, ECG may be used to assess normalization of the PR interval, R waves in V1, Q waves in V6, ventricular repolarization, QS waves in inferior and / or upper lateral wall, conduction disturbances in right bundle branch, QT C, and QRS. - 154 - 40928727338013.0045P1

[0211] Accordingly, provided are recombinant AAV compositions, including compositions comprising gene expression cassettes and viral vectors, comprising a nucleic acid encoding a microdystrophin protein disclosed herein (including AAV8-RGX-DYS1), and methods of administering those compositions that improve or maintain cardiac function or slow the loss of cardiac function, for example, by preventing reductions in decreasing LVEF below 45% and / or normalization of function (LVFS ≥ 28%) as measured by serial electrocardiograms, and / or serial noninvasive imaging studies (e.g., echocardiography or cardiac magnetic resonance imaging (CMR)). Measurements may be compared to an untreated control or to the subject prior to treatment with the nucleic acid composition. Alternatively, the nucleic acid compositions described here in and the methods of administering nucleic acid compositions results in an improvement in cardiac function or reduction in the loss of cardiac function as assessed by monitoring changes from baseline in forced vital capacity (FVC), forced expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), inflammation, and fibrosis. ECG may be used to monitor conduction abnormalities and arrhythmias. In particular, ECG may be used to assess normalization of the PR interval, R waves in V1, Q waves in V6, ventricular repolarization, QS waves in inferior and / or upper lateral wall, conduction disturbances in right bundle branch, QT C, and QRS.

[0212] In some embodiments, cardiac function and / or pulmonary function can be used as an endpoint for assessment of therapeutic efficacy of the administration. The cardiac function and / or pulmonary function can improve or increase in the subject relative to the level (of cardiac function and / or pulmonary function) prior to said administration. In some embodiments, the cardiac function and / or pulmonary function can improve or increase in the subject relative to the level (of cardiac function and / or pulmonary function) in a subject that does not have a dystrophinopathy. In some embodiments, the cardiac function and / or pulmonary function can decrease in the subject relative to the level (of cardiac function and / or pulmonary function) in a non-treated subject having a dystrophinopathy. The comparison of cardiac function and / or pulmonary function can be to a standard, wherein the standard is a number or set of numbers that represent the cardiac function and / or pulmonary function in a subject that does not have a dystrophinopathy or the cardiac function and / or pulmonary function in a non-treated subject having a dystrophinopathy. Thus, in - 155 - 40928727338013.0045P1 some embodiments, the comparison of cardiac function and / or pulmonary function after administration of a rAAV with a transgene encoding microdystrophin can be to a control. The control can be the cardiac function and / or pulmonary function in the subject prior to administration, cardiac function and / or pulmonary function in a subject with a dystrophinopathy that has not be treated, cardiac function and / or pulmonary function in a subject that does not have a dystrophinopathy, cardiac function and / or pulmonary function in a standard.

[0213] In some embodiments, an improvement or increase in cardiac function and / or pulmonary function is 1 to 100% compared to a control, for example, compared to the subject prior to administration of rAAV comprising a transgene encoding microdystrophin. In some embodiments, cardiac function can be measured using impedance, electric activities, and calcium handling. 5.5.2 Motor Abilities

[0214] The Peabody Developmental Motor Scales–Third Edition (PDMS-3) (Folio and Fewell, 2023, incorporated herein by reference) is an early childhood motor development program that measures interrelated motor abilities that develop early in life. The PDMS-3 is used by occupational therapists, physical therapists, diagnosticians and other professionals in examining and assessing the motor abilities of younger children. There are five core subtests utilized in the PDMS-3: Body Control: measures the ability to move the limbs and trunk, postural reactions, standing, bending, extending, stooping, balancing, jumping upward. It is an estimate of the child's ability to sustain control of his or her body within its center of gravity and retain equilibrium. Body Transport: subtest measures the ability to make movements that propel the child from one location to another, such as rolling, crawling, creeping, walking, running, jumping forward and sideward, sliding, hopping, and skipping. Object Control: measures the ability to coordinate motor movements that require the incorporation of perception and movement, such as throwing, catching, bouncing, and kicking a ball. Hand Manipulation: measures the ability to move the hands and fingers (and arms as appropriate) to complete tasks and measure dexterity. Hand manipulation may include manipulation of objects such as blocks, cups, and drawing utensils. - 156 - 40928727338013.0045P1 Eye-Hand Coordination: measures the ability to interpret visual stimuli in coordination with hand-finger movements. This is an estimate of the child's ability to integrate and use their visual perceptual skills to perform complex eye-hand coordination tasks

[0215] Two core subtests were assessed at baseline and at intervals following administration of gene therapy: the Body Control subtest and the Body Transport subtest. Three composite index scores can be assessed per the PDMS-3, namely Gross Motor Index, Fine Motor Index and Total Motor Index. General Scoring Criteria for individual items is as follows: - Item Score of 2 – Performance of the item matches the criteria specified for mastery. - Item Score of 1 – Performance shows a clear resemblance to the item mastery criteria however does not fully meet the criteria. - Item Score of 0 – Subject cannot or will not attempt the item or the attempt does not show the skill has developed.

[0216] Total Motor Index is understood to be the best estimate of overall motor abilities. Increases or decreases in any of the Gross Motor Index, Fine Motor Index and Total Motor Index scores can be compared to Normative Sample data that was collected from over 1400 children between 1 and 71 months of age (Folio and Fewell, 2023) to make a determination of improvement or decline of motor abilities. For example, the PDMS-3 scoring system yields four types of normative scores: age equivalents, percentile ranks, subtest scaled scores, and composite index scores. Age equivalents are indexes of relative standing that translate subtest raw scores into motor ages. Thus, a subject is determined to benefit from therapy if the subject’s PDMS-3 Total Motor Score Index is equal to or greater than the average age-equivalent PDMS-3 Total Motor Score Index (within standard deviation) following administration of the therapy. In another example, the subject is within the 75 percentile (%) of the normative sample that performed the skill at the subject’s equivalent age. The Peabody Motor Development Chart provides a reference for the motor skills measured by the PDMS-3 at the ages at which 75% of the normative sample performed the skill.

[0217] The PDMS-3 was utilized to assess the subject during their development as the subject continues to acquire motor skills. Following administration of AAV8-RGX-DYS1 therapy, the subject may have an increased PDMS-3 score index and become closer to typical development. Subsequently, the subject is observed as improving in ambulation, or improving their acquisition - 157 - 40928727338013.0045P1 of motor skills, following administration of AAV8-RGX-202 relative to a subject(s) not administered the AAV8-RGX-202 therapy or relative to a natural history matched control(s) when the increased PDMS-3 score index over time is characterized as linear skill acquisition. In embodiments, the subject is from 1 to less than 4 years of age at administration, including of an intravenous dose of 1x1014or 2x1014, and at 6 months, 12 months or 18 months after administration exhibits stability when assessed using PDMS-3, including a normative score greater than or equal to baseline score or score of natural history control, including where measured function is within 2 standard deviations of the normative mean.

[0218] Early motor development in infants and young boys have also been assessed by the Bayley III Scales of Infant Development, Adaptive Behavioral Subtest of Bayley (ABS), and the Expanded Hammersmith Functional Motor Scales (HFMSE) (Connolly, et al.2013 Neuromuscul Disord.2013 July; 23(7): 529–539). 5.5.3 Central Nervous System

[0219] A portion of patients with DMD can also have epilepsy, learning and cognitive impairment, dyslexia, neurodevelopment disorders such as attention deficit hyperactive disorder (ADHD), autism, and / or psychiatric disorders, such as obsessive-compulsive disorder, anxiety or sleep disorders.

[0220] The goal of gene therapy treatments disclosed herein can be to improve cognitive function or alleviate symptoms of epilepsy and / or psychiatric disorders. Efficacy may be assessed by periodic evaluation of behavior and cognitive function that are appropriate for the age and disease stage of the trial population and or by quantifying and qualifying seizure events.

[0221] Accordingly, provided are recombinant AAV compositions and methods of administering the microdystrophin gene therapy compositions that improve cognitive function, reduce the occurrence or severity of seizures, alleviate symptoms of ADHD, obsessive-compulsive disorder, anxiety and / or sleep disorders. 5.5.4 Patient primary endpoints

[0222] The efficacy of the compositions, including the dosage of the composition, and methods described herein may be assessed in clinical evaluation of subjects being treated. Patient primary endpoints may include monitoring the change from baseline in forced vital capacity (FVC), forced - 158 - 40928727338013.0045P1 expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), change from baseline in the NSAA, change from baseline in the Performance of Upper Limp (PUL) score, and change from baseline in the Brooke Upper Extremity Scale score (Brooke score), change from baseline in grip strength, pinch strength, change in cardiac fibrosis score by MRI, change in upper arm (bicep) muscle fat and fibrosis assessed by MRI, measurement of leg strength using a dynamometer, walk test 6-minutes, walk test 10-minutes, walk analysis – 3D recording of walking, change in utrophin membrane staining via quantifiable imaging of immunostained biopsy sections, and a change in regenerating fibers by measuring (via muscle biopsy) a combination of fiber size and neonatal myosin positivity. See, for example, Mazzone E et al, North Star Ambulatory Assessment, 6-minute walk test and timed items in ambulant boys with Duchenne muscular dystrophy. Neuromuscular Disorders 20 (2010) 712– 716.; Abdelrahim Abdrabou Sadek, et al, Evaluation of cardiac functions in children with Duchenne Muscular Dystrophy: A prospective case-control study. Electron Physician (2017) Nov; 9(11): 5732–5739; Magrath, P. et al, Cardiac MRI biomarkers for Duchenne muscular dystrophy. BIOMARKERS IN MEDICINE (2018) VOL.12, NO.11.; Pane, M. et al, Upper limb function in Duchenne muscular dystrophy: 24 month longitudinal data. PLoS One. 2018 Jun 20;13(6):e0199223.

[0223] In some embodiments, patient primary endpoints may include reductions in serum creatine kinase levels, dystrophin and / or microdystrophin levels in the muscle, or muscle endurance tests such as grip strength or treadmill endurance testing. 6. EXAMPLES 6.1 Example 1 – Construction of microdystrophin (DMD) gene expression cassettes for insertion of Cis plasmids.

[0224] DMD constructs encode microdystrophins with the core backbone: 5’(N-terminus)- ABD-H1-R1-R2-R3-H3-R24-H4-CR-3’ (C-terminus) (FIG. 2), but which differ in the presence and length of the C-terminus (CT). The microdystrophin encoded by RGX-DYS1 has as the C terminus the proximal 194 amino acids of the wild type DMD protein C-terminus domain (SEQ ID NO: 16), the RGX-DYS3 encodes a microdystrophin has a short C-terminus (48 amino acids of SEQ ID NO: 91) without functional syntrophin or α-dystrobrevin binding domains, and RGX- DYS5 encodes a microdystrophin with 140 amino acids of the C-terminal domain (SEQ ID NO: - 159 - 40928727338013.0045P1 83), which contains an α1-syntrophin binding site but not a dystrobrevin binding site (see FIGs. 1A and 1B). The constructs include the Spc5-12 promoter (SEQ ID NO: 39) and smPA regulatory sequences, and RGX-DYS3 includes the VH4 intron sequence (SEQ ID NO: 41). All were cloned into Cis plasmids flanked by ITRs. All DNA sequences encoding the DMD genes are codon- optimized and CpG depleted. 6.1.1. Recombinant engineering of RGX-DYS1, RGX-DYS3 and RGX-DYS5 transgene

[0225] In brief, the human codon-optimized and CpG depleted nucleotide sequence of a microdystrophin construct in RGX-DYS1 as shown in FIG. 2 encoding N-terminal-ABD1-H1- R1-R2-R3-H3-R24-H4-CR-CT-C-terminal (having an amino acid sequence of SEQ ID NO:1) was synthesized using GeneArt Gene Synthesis (Invitrogen, Thermo Fisher, Waltham, MA). The desired C-terminus was made by site directed mutagenesis using the following two primers: 5′: TGA CTC GAG AGG CCT AAT AAA GAG C (SEQ ID NO: 43), 3′: CCT TGG AGA CTG TGG AGA GGT G (SEQ ID NO: 44). The construct includes a synthetic muscle promoter (e.g. SPc5- 12; SEQ ID NO: 39), and a small poly(A) signal sequence (sm pA (SEQ ID NO:42) and has a nucleotide sequence of SEQ ID NO: 53.

[0226] The construct RGX-DYS3 (FIG.2) was engineered encoding the microdystrophin of the RGX-DYS1 construct detailed above with a small portion (48 amino acids; SEQ ID NO: 91) of the CT domain (the microdystrophin having the amino acid sequence of SEQ ID NO: 2). This construct includes the SPc5-12 promoter the sm pA poly A sequence and the VH4 intron at the 5’ end of the microdystrophin coding sequence. The transgene construct has a nucleotide sequence of SEQ ID NO: 21. [...

Claims

38013.0045P1 We claim:

1. A pharmaceutical composition for use in treating or ameliorating symptoms of a dystrophinopathy in a subject suffering from a dystrophinopathy, said pharmaceutical composition comprising: a therapeutically effective amount of a microdystrophin comprising at least the portion of the C-terminal domain (CT) comprising an α1-syntrophin binding site, wherein administration to the subject results in delivery of the microdystrophin protein to the muscle of the subject, and wherein the muscles of the subject exhibit greater than 80%, or 85%, or 90%, or 95% of the force generation of the muscles of a subject that does not have a DMD mutation or dystrophinopathy.

2. A pharmaceutical composition for use in reducing and / or preventing contraction-induced muscle damage or to increase muscle resilience in a subject suffering from a dystrophinopathy or having a DMD mutation, said pharmaceutical composition comprising a therapeutically effective amount of a microdystrophin comprising at least the portion of the C-terminal domain (CT) comprising an α1-syntrophin binding site, wherein administration to the subject results in delivery of the microdystrophin protein to the muscle of the subject, and wherein the muscles of the subject exhibit greater than 80%, or 85%, or 90%, or 95% of the force generation of the muscles of a subject that does not have a DMD mutation or dystrophinopathy.

3. A pharmaceutical composition for use in reducing and / or preventing contraction-induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said pharmaceutical composition comprising a therapeutically effective amount of a microdystrophin comprising at least the portion of the C-terminal domain (CT) comprising an α1-syntrophin binding site, wherein administration to the subject results in delivery of microdystrophin protein to the muscle of the subject, and wherein the muscles of the subject generate about 50% more force relative to a subject administered a microdystrophin without the CT portion comprising an α1-syntrophin binding site.

4. A pharmaceutical composition for use in reducing and / or preventing muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said pharmaceutical composition comprising a therapeutically effective amount of a microdystrophin - 225 - 40928727338013.0045P1 comprising at least the portion of the C-terminal domain (CT) comprising an α1-syntrophin binding site, wherein administration to the subject results in delivery of microdystrophin protein to the muscle of the subject, and wherein the muscles of the subject generate about 50% more force relative to the muscles of a natural history matched control subject.

5. A pharmaceutical composition for use in reducing or preventing contraction-induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said pharmaceutical composition comprising a therapeutically effective amount of a microdystrophin comprising a means for restoring DAPC in muscle cells of the subject, wherein administration to the subject results in delivery of microdystrophin protein to the muscle of the subject and the subject has improved timed function test, North Star Ambulatory Assessment (NSAA) results, Peabody Developmental Motor Scale 3 (PDMS- 3) results, or caregiver reported improved function reports (PODCI) results relative to a natural history matched control subject or untreated subject.

6. A pharmaceutical composition for use in reducing or preventing contraction induced muscle damage in a subject suffering from a dystrophinopathy or having a DMD mutation, said pharmaceutical composition comprising a therapeutically effective amount of a microdystrophin comprising a means for stabilization of a microdystrophin protein in muscle cells by interaction between a microdystrophin C-terminal domain (CT) and alpha- syntrophin or a means for protecting muscle against contraction-induced damage or means for enabling muscle recovery from contraction-induced damage, wherein administration to the subject results in delivery of microdystrophin protein to the muscle of the subject and the subject has improved timed function test or NSAA results relative to a natural history matched control subject.

7. The pharmaceutical composition of any one of claims 1 to 4 wherein the subject has improved timed function test or NSAA results relative to a natural history matched control subject.

8. The pharmaceutical composition of any one of claims 5 to 7 wherein the subject has improved timed test function, NSAA results, Peabody Developmental Motor Scale 3 (PDMS-3) results, or caregiver reported improved function reports (PODCI) results at 12 - 226 - 40928727338013.0045P1 weeks, 24 weeks, 1 year, 2 years, or 3 years after administration relative to results prior to administration.

9. The pharmaceutical composition of any one of claim 5 to 8, wherein the increase in NSAA score is more than 2, more than 3, or more than 4.

10. The pharmaceutical composition of any one of claims 5 to 9, wherein the improvement is greater than the minimal clinically important difference (MCID) relative to a natural history matched control of MCID standard.

11. The pharmaceutical composition of any one of claims 5 to 10, wherein the increase in caregiver reported improved function reports (PODCI) results is more than 3, more than 4, more than 5, or more than 6.

12. The pharmaceutical composition of any one of claims 5 to 9, wherein the improvement of treated subjects is greater than the improvement of untreated subjects.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the microdystrophin consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an α1-syntrophin binding site 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the CT comprises or consists of the proximal 194 amino acids of the C-terminus of dystrophin or at least the proximal portion of the C-terminus corresponding to human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 92 (UniProtKB-P11532) or at least the proximal portion of the C-terminus encoded by exons 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75. - 227 - 40928727338013.0045P1 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the CT comprises or consists of the amino acid sequence of SEQ ID NO: 83 or an amino acid sequence which comprises the α1-syntrophin binding site but not the dystrobrevin binding site.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the CT domain does not comprise a C-terminal DTM amino acid sequence.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO:

1.

18. The pharmaceutical composition of claim 17, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO:

20.

19. The pharmaceutical composition of any one of claims 1 to 16, wherein the microdystrophin protein has the amino acid sequence of SEQ ID NO: 79, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109.

20. The pharmaceutical composition of claim 18, wherein the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO:

81.

21. The pharmaceutical composition of any one of claims 1 to 20, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, or a DMD with a mutation of the DMD gene.

22. The pharmaceutical composition of claim 21, wherein the mutation of the DMD gene comprises a mutation in any exon except in exons 8, 9, and / or 10 of the DMD gene.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the subject is 1 to 13 years of age.

24. The pharmaceutical composition of claim 23, wherein the subject is 4 to 5 years of age, 6 to 7 years of age, or 8 years of age or greater than 8 years old at administration.

25. The pharmaceutical composition of any one of claims 1 to 22, wherein the subject is 1 to less than 4 years of age at administration.

26. The pharmaceutical composition of any one of claims 1 to 22, wherein the subject is in Decline Phase or whose natural history peers are in the Decline Phase at administration. - 228 - 40928727338013.0045P1 27. The pharmaceutical composition of any one of claims 1 to 26, wherein the microdystrophin is administered by a recombinant adeno-associated virus (rAAV) particle, wherein the rAAV particle comprises i) a transgene that encodes a microdystrophin protein and ii) a capsid protein.

28. The pharmaceutical composition of claim 27, wherein the transgene further comprises a transcription regulatory element that promotes expression in muscle operably linked to the nucleic acid sequence that encodes the microdystrophin protein.

29. The pharmaceutical composition of claim 28, wherein the transcription regulatory element comprises a muscle-specific promoter.

30. The pharmaceutical composition of claim 29, wherein the muscle-specific promoter is a skeletal, smooth, or cardiac muscle specific promoter.

31. The pharmaceutical composition of claim 29, wherein the muscle specific promoter is SPc5-12 or a transcriptionally active portion or mutant thereof.

32. The pharmaceutical composition of claim 31, wherein the SPc5-12 consists of the nucleic acid sequence of SEQ ID NO:

39.

33. The pharmaceutical composition of any one of claims 27 to 32, wherein the transgene comprises a polyadenylation signal 3’ of the nucleic acid sequence encoding the microdystrophin protein.

34. The pharmaceutical composition of any one of claims 27 to 33, wherein the transgene comprises an intron sequence between the promoter and the microdystrophin coding sequence.

35. The pharmaceutical composition of any one of claims 27 to 34, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO: 53 36. The pharmaceutical composition of any one of claims 27 to 34, wherein the transgene comprises a nucleic acid sequence of SEQ ID NO:

82.

37. The pharmaceutical composition of any one of claims 27 to 36, wherein the rAAV particle has a capsid protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

77. - 229 - 40928727338013.0045P1 38. The pharmaceutical composition of any one of claims 27 to 36, wherein the rAAV is an AAV8 serotype.

39. The pharmaceutical composition of any one of claims 27 to 38, wherein the therapeutically effective amount of the rAAV particle is administered at a dose of 1x1014genome copies / kg or 2x1014genome copies / kg.

40. The pharmaceutical composition of any one of claims 27 to 39, wherein the pharmaceutical composition is administered intravenously.

41. The pharmaceutical composition of any one of claims 27 to 40, wherein microdystrophin protein levels in the muscle of the subject are increased at 5 weeks, 10 weeks, 12 weeks, 20 weeks, and / or 26 weeks after administration as measured by capillary western blot (JESS) and / or liquid chromatography-mass spectrometry (LC-MS) at 5 weeks, 10 weeks, 12 weeks, 20 weeks, or 26 weeks after the administration.

42. The pharmaceutical composition of any one of claims 27 to 41, wherein creatine kinase activity in the subject is decreased at 12 weeks, 24 weeks, 1 year, 2 years, or 3 years after administration, relative to the level prior to said administration.

43. The pharmaceutical composition of any one of claims 27 to 42, wherein the subject does not experience a serious adverse event after administration.

44. The pharmaceutical composition of claim 43, wherein the serious adverse event is central neurotoxicity, peripheral neurotoxicity, drug-induced liver injury, thrombocytopenia, myocarditis, or myositis. - 230 - 409287273

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