Low dose AAV administration

Abstract

The invention described herein provides methods and reagents to treat muscular dystrophy (such as DMD), using an nNOS-binding domain containing microdystrophin coding sequence delivered by a modified AAV capsid (SLB-101) having superior expression profile in target tissues.

Description

[0001]Attorney Docket No.: 129159-02620 LOW DOSE AAV ADMINISTRATION REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of the filing date of U.S. Provisional Application Number 63 / 636,083, filed on April 18, 2024, which is hereby incorporated by reference in its entirety. STATEMENT REGARDING SEQUENCE LISTING The application contains a Sequence Listing XML file which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. The Sequence Listing XML file, created on April 11, 2025, is named 129159-02620-SL.xml and is 10,120 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTION With the advent of gene therapy, research and clinical trials for gene therapy treatment has focused on gene replacement or other genetic therapies aimed to at least partially restore function of compensate for the mutated gene. These include supplying a functional copy of the gene, such as a minigene or microgene, or repairing a defective gene product by exon skipping and nonsense mutation suppression. Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length, including 145 nucleotide inverted terminal repeat (ITRs). AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are -1- ME152715650v.1 Attorney Docket No.: 129159-02620 contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection. Multiple studies have demonstrated long-term (> 1.5 years) recombinant AAV- mediated protein expression in muscle. See, Clark et al., Hum Gene Ther 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. U.S.A.93:14082-14087 (1996); and Xiao et al., J Virol 70: 8098-8108 (1996). See also, Chao et al., Mol Ther 2:619-623 (2000) and Chao et al., Mol Ther 4:217-222 (2001). Moreover, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation following intramuscular injection as described in Herzog et al., Proc Natl Acad Sci U.S.A. 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci U.S.A.94: 13921-13926 (1997). Moreover, Lewis et al., J Virol 76: 8769-8775 (2002) demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. There is rising demand for improved AAV-mediated gene therapy with lower dosage and greater efficiency. SUMMARY OF THE INVENTION The invention described herein is partly based on the discovery that genetic diseases, disorder and / or condition related to muscle tissue, such as DMD (Duchenne Muscular Dystrophy) or BMD (Becker Muscular Dystrophy), can be effectively treated using a recombinant AAV vector comprising a vector genome encoding a gene of interest, such as an nNOS-containing microdystrophin, encapsidated in a modified AAV capsid having enhanced muscle tropism (SLB101), at a surprisingly low dose of between 2-90 × 1012vg / kg, for example, in a mouse model of the muscular dystrophy (such as the mdx mouse model of DMD). The invention described herein is summarized in the numbered paragraphs below. 2 ME152715650v.1 Attorney Docket No.: 129159-02620 1. A method of treating a disease, disorder, and / or condition related to muscle tissue (e.g., muscular dystrophy) in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV), wherein the therapeutically effective amount is about 2-90 × 1012vg / kg, wherein the rAAV comprises a gene of interest (GOI) related to treating said muscle tissue, optionally wherein the GOI is flanked by a pair of AAV ITR sequences, and, wherein the rAAV comprises a modified adeno-associate virus (mAAV) capsid comprising an amino acid sequence at least about 80% (e.g., at least about 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1, and wherein the mAAV capsid comprises the amino acid sequence of RGDLGLS (SEQ ID NO: 4). 2. A method of treating muscular dystrophy in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV), wherein the therapeutically effective amount is about 2-90 × 1012vg / kg, wherein the rAAV comprises a gene of interest (GOI) defective in said muscular dystrophy, flanked by a pair of AAV ITR sequences, and, wherein the rAAV comprises a modified adeno-associate virus (mAAV) capsid having the amino acid sequence of SEQ ID NO: 1. 3. The method of paragraph 1 or 2, wherein the GOI is LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 2I), or a gene or coding sequence for NAGLU (α-N- acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain- 3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin. 4. The method of paragraph 1 or 2, wherein the muscular dystrophy is DMD (Duchenne Muscular Dystrophy) or BMD (Becker Muscular Dystrophy), and wherein the GOI encodes a microdystrophin. 3 ME152715650v.1 Attorney Docket No.: 129159-02620 5. The method of paragraph 4, wherein the microdystrophin is one described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; PCT / US2016 / 013733; or US10,166,272. 6. The method of paragraph 5, wherein the microdystrophin comprises a coding sequence for R16 and R17 spectrin-like repeats for the full-length dystrophin protein (such as one described in US7,892,824). 7. The method of paragraph 6, wherein the microdystrophin comprises a coding sequence for the R1, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein; or a microdystrophin gene described in PCT / US2016 / 013733 or US10,479,821. 8. The method of any one of paragraphs 1-7, wherein the GOI is operatively linked to a transcriptional regulatory cassette, such as a muscle specific promoter (e.g., a CK8 promoter or a cardiac troponin T (cTnT) promoter). 9. The method of any one of paragraphs 1-8, wherein the GOI is a micro-dystrophin gene encoding a protein comprising, from N- to C-terminus, an amino-terminal actin- binding (AB1) domain, a β-dystroglycan binding domain, a Hinge 1 domain (H1), a spectrin-like repeat domain consisting of five spectrin-like repeats that include spectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16), spectrin-like repeat 17 (SR17), spectrin-like repeat 23 (SR23), and spectrin-like repeat 24 (SR24), and a Hinge 4 domain (H4), wherein the micro-dystrophin gene is operatively linked to a muscle-specific human muscle creatine kinase CK8 promoter (e.g., SEQ ID NO:19 of US10,479,821), and wherein the GOI is flanked by a pair of AAV2 ITR (inverted terminal repeat) sequence. 10. The method of any one of paragraphs 1-9, wherein the GOI is codon-optimized for mammalian expression. 11. The method of paragraph 10, wherein the GOI comprises a polynucleotide having the sequence at least about 80% (e.g., at least about 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 2. 12. The method of any one of paragraphs 1-11, wherein the therapeutically effective amount is between about 2-50×1012vg / kg, between about 5-50×1012vg / kg, between about 6-30×1012vg / kg, about 2×1012vg / kg, about 6×1012vg / kg, about 1×1013vg / kg, about 15×1012vg / kg, about 20×1012vg / kg, about 25×1012vg / kg, about 30×10124 ME152715650v.1 Attorney Docket No.: 129159-02620 vg / kg, about 40×1012vg / kg, about 50×1012vg / kg, about 60×1012vg / kg, about 70×1012vg / kg, or about 80×1012vg / kg. 13. The method of any one of paragraphs 1-12, wherein the subject is a human, and wherein the therapeutically effective amount is for a mouse (such as an mdx mouse) before conversion to an equivalent dose in said human. 14. The method of paragraph 13, wherein said human is a neonate (from birth through the first 28 days of life), an infant (from 29 days to less than 2 years), a child (from 2 years to less than 12 years), or an adolescent (aged 12 through 21). 15. The method of paragraph 14, wherein the child is between 4-8 years old. 16. The method of paragraph 14 or 15, wherein the child is a boy. It should be understood that any one embodiment of the invention described herein can be combined with any one or more additional embodiments of the invention, including those embodiments described only in the examples or only described in one of the sections above or below, or one aspect of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a representative schematic (not to scale) diagram showing the selected domains in a microdystrophin protein construct (referred to herein as “MD-5”), including the N-terminal actin-binding domain (ABD), Hinge 1 region, spectrin-like repeats R1, R16, R17, R23 and R24 that together comprise the nNOS-binding domain (nNOS), Hinge 4 region, and the dystroglycan binding domain. This compact design - intended to act as a functional surrogate of the full-length dystrophin - is small enough to be packaged into an AAV vector (e.g., SLB101); uniquely includes the nNOS binding domain, which presence correlated with milder phenotypes of the Becker muscular dystrophy (BMD) patient and is potentially important for prevention of activity-induced ischemia and associated muscle injury. The term “SGT” is used herein to refer to SLB101 encapsidating MD-5. FIG.2 shows the study design for demonstrating the effects of SLB101 encapsidating MD-5 in the mdx mouse model of DMD, at various dosing levels, including the functional assays used to demonstrate efficacy. FIG.3 shows improved serum biomarkers (e.g., CK, titin and AST) of muscle membrane integrity in mdx Mice treated with SLB101 encapsidating MD-5 at doses ≥3.0E13 vg / kg. 5 ME152715650v.1 Attorney Docket No.: 129159-02620 FIG.4 shows that biodistribution dose response was observed with treatment of SLB101 encapsidating MD-5. ****All groups statistically significant in comparison with each other. The lowest dose used was 2E12 vg / kg. FIG.5 shows that microdystrophin protein expression dose response was observed with treatment of SLB101 encapsidating MD-5 (referred to as “SGT”). *** Statistically significant between mdx vehicle, mdx-SGT 2.0E12, mdx-SGT 6.0E12; * Statistically significant between mdx vehicle; % Normal Dystrophin was calculated by dividing each sample concentration by the average concentration of five individual human adult muscle samples. The lowest dose used was 2E12 vg / kg. FIG.6 shows that microdystrophin protein and functionally active of nNOS were properly localized to the Sarcolemma with treatment of SLB101 encapsidating MD-5. The lowest dose of the SLB101 encapsidating MD-5 (referred to as “SGT”) that produced a differential response was 2E12 vg / kg. FIG.7 shows dose response in microdystrophin-positive myofibers in heart (cardiac muscle), diaphragm (smooth muscle) and quadriceps (skeletal muscle), produced by treatment of SLB101 encapsidating MD-5. The lowest dose inducing a change was 2E12 vg / kg. FIG.8 shows dose response in myofibers positive for active nNOS, observed produced by treatment of SLB101 encapsidating MD-5. The lowest dose inducing a change was 2E12 vg / kg. FIG.9 shows functional improvements (as measured by forelimb grip strength test, treadmill exhaustion test, and EDL specific force measurement) at doses ≥6.0E12 vg / kg, observed after treatment of SLB101 encapsidating MD-5. The lowest dose inducing a change was 2E12 vg / kg. FIG.10 shows improvements in serum biomarkers (e.g., CK, titin, and ALT) of muscle membrane integrity, observed after treatment of SLB101 encapsidating MD-5. The lowest dose inducing a change was 2E12 vg / kg. DETAILED DESCRIPTION OF THE INVENTION In some aspects, disclosed herein are compositions and methods for treating a diseases, disorder and / or condition related to muscle tissue (e.g., a diseases, disorder and / or condition related to skeletal, cardiac, and / or smooth muscle tissue) in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount 6 ME152715650v.1 Attorney Docket No.: 129159-02620 of a recombinant AAV (rAAV), wherein the therapeutically effective amount is about 2-90 × 1012vg / kg, wherein the rAAV comprises a gene of interest (GOI) related to treating said muscle tissue, optionally flanked by a pair of AAV ITR sequences, and wherein the rAAV comprises a modified adeno-associate virus (mAAV) capsid having the amino acid sequence at least about 80% (e.g., at least about 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1, and wherein the mAAV capsid comprises the amino acid sequence of RGDLGLS (SEQ ID NO: 4). In some examples, the disease, disorder, and / or condition related to muscle tissue is muscular dystrophy, a group of diseases that cause progressive weakness and loss of muscle mass where abnormal genes (mutant genes) produce no functional wild-type proteins needed to form healthy muscle, such as Duchenne type muscular dystrophy (DMD), one of the most devastating muscle diseases affecting 1 in 5,000 newborn males. DMD results from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These mutations result in loss of structural links that act to stabilize the muscle cell membrane during contraction, and protect against contraction-induced damage. Loss of these structural links leads to increased muscle membrane fragility and progressive muscle degeneration. Disease progression is characterized by increasing muscle necrosis, fibrosis, and fatty tissue replacement and a greater degree of fiber size variation seen in subsequent muscle biopsies. Currently there is no cure for DMD. The standard of care includes administering corticosteroids (such as prednisone or deflazacort) to stabilize muscle strength and function, prolong independent ambulation, and delay scoliosis and cardiomyopathy; or to administer bisphosphonates, denosumab, or recombinant parathyroid hormones. With the advent of gene therapy, research and clinical trials for DMD treatment focuses on gene replacement or other genetic therapies aimed to at least partially restore dystrophin function. These include supplying a functional copy of the dystrophin gene, such as a dystrophin minigene, or repairing a defective dystrophin gene product by exon skipping and nonsense mutation suppression. One aspect of the invention provides a method of treating muscular dystrophy in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV), wherein the therapeutically effective amount is about 2-90 × 1012vg / kg, wherein the rAAV comprises a gene of interest (GOI) defective in said muscular dystrophy, flanked by a pair of AAV ITR sequences, and, 7 ME152715650v.1 Attorney Docket No.: 129159-02620 wherein the rAAV comprises a modified adeno-associate virus (mAAV) capsid having the amino acid sequence of SEQ ID NO: 1. MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKG EPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKR LLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQ PIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWA LPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRP KRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVF MIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDR LMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQN NNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKV MITNEEEIKTTNPVATESYGQVATNHQSAQRGDLGLSAQAQTGWVQNQGILPGMVWQDRDVY LQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYS TGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 1) In certain embodiments, the GOI defective in the muscular dystrophy is LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb- girdle muscular dystrophy type 2I), or a gene or coding sequence for NAGLU (α-N- acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha- glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin. In certain embodiments, the muscular dystrophy is DMD (Duchenne Muscular Dystrophy) or BMD (Becker Muscular Dystrophy), and wherein the GOI encodes a microdystrophin. In certain embodiments, the microdystrophin is one described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; PCT / US2016 / 013733; or US10,166,272 (all incorporated herein by reference). In certain embodiments, the microdystrophin comprises a coding sequence for R16 8 ME152715650v.1 Attorney Docket No.: 129159-02620 and R17 spectrin-like repeats for the full-length dystrophin protein (such as one described in US7,892,824, incorporated herein by reference). In certain embodiments, the microdystrophin comprises a coding sequence for the R1, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein; or a microdystrophin gene described in PCT / US2016 / 013733 or US10,479,821 (all incorporated herein by reference). In certain embodiments, the GOI is operatively linked to a transcriptional regulatory cassette, such as a muscle specific promoter (e.g., a CK8 promoter or a cardiac troponin T (cTnT) promoter). In certain embodiments, the GOI is a micro-dystrophin gene encoding a protein comprising, from N- to C-terminus, an amino-terminal actin-binding (AB1) domain, a β- dystroglycan binding domain, a Hinge 1 domain (H1), a spectrin-like repeat domain consisting of five spectrin-like repeats that include spectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16), spectrin-like repeat 17 (SR17), spectrin-like repeat 23 (SR23), and spectrin-like repeat 24 (SR24), and a Hinge 4 domain (H4), wherein the micro-dystrophin gene is operatively linked to a muscle-specific human muscle creatine kinase CK8 promoter (e.g., SEQ ID NO:19 of US10,479,821, incorporated herein by reference), and wherein the GOI is flanked by a pair of AAV2 ITR (inverted terminal repeat) sequence. In certain embodiments, the GOI is codon-optimized for mammalian expression. As used herein, “codon-optimized” polynucleotide coding sequence refers to a polynucleotide sequence that has been altered / changed in some respect, such that the resulting codons are optimal for expression in a particular cell, host, or system, such as in a specific mammalian (human) cell type, e.g., muscle cells. Codon optimization does not alter the amino acid sequence of the encoded protein, i.e., the codon optimized polynucleotide coding sequence, and the native sequence based on which codon optimization was performed, encode the same amino acid sequence. In certain embodiments, the GOI comprises a polynucleotide having the sequence of SEQ ID NO: 2. atgctgtggtgggaggaagtggaagattgctacgagcgcgaggacgtgcagaagaaaaccttcaccaa atgggtcaacgcccagttcagcaagttcggcaagcagcacatcgagaacctgttcagcgacctgcagg acggcagacggctgctggatctgctggaaggcctgaccggacagaagctgcccaaagagaagggcagc accagagtgcacgccctgaacaacgtgaacaaggccctgcgggtgctgcagaacaacaaTgtggacct GgtgaacatTggcagcacAgacatTgtggaTggcaaccacaagctgaccctgggcctgatctggaaca tcatcctgcactggcaagtgaagaacgtgatgaagaacatcatggccggcctgcagcagaccaacagc gagaagatcctgctgagctgggtgcgccagagcaccagaaactacccccaagtgaacgtgatcaactt 9 ME152715650v.1 Attorney Docket No.: 129159-02620 caccacctcttggagcgacggcctggccctgaatgccctgatccacagccacagacccgacctgttTg actggaacagTgtGgtgtgtcagcagagcgccacccagaggctggaacacgccttcaatatcgccaga taccagctgggcatTgagaagctgctggaccccgaggatgtggacaccacctaccccgacaagaaatc catcctgatgtatatcaccagcctgttccaggtgctgcctcagcaggtgtccatcgaggccatccagg aagtggaaatgctgcccagaccccccaaagtgaccaaagaggaacacttccagctgcaccaccagatg cactactctcagcagatcaccgtgtccctggcccagggctacgagagaaccagcagccccaagccccg gttcaagagctacgcctatacccaggccgcctacgtgaccaccagcgaccctaccagaagcccattcc ccagccagcatctggaagcccccgaggacaagagcttcggcagcagcctgatggaaagcgaagtgaac ctggatagataccagaccgccctggaagaggtgctgtcctggctgctgagcgccgaggatacactgca ggctcagggcgagatcagcaaTgaTgtggaagtGgtgaaggaccagttccacacccacgagggctaca tgatggacctgacagcccaccagggcagagtgggcaacattctgcagctgggctccaagctgatcggc accggcaagctgagcgaggacgaagagacagaggtgcaggaacagatgaacctgctgaacagcagatg ggagtgcctgagagtggccagcatggaaaagcagagcaacctgcacagctacgtgcccagcacctacc tgaccgagatcacccatgtgtcccaggccctgctggaagtggaacagctgctgaacgcccccgatctg tgcgccaaggacttcgaggatctgttcaagcaggaagagagcctgaagaatatcaaggactctctgca gcagtccagcggcagaatcgacatcatccacagcaagaaaacagccgccctgcagtccgccacccccg tggaaagagtgaagctgcaggaagccctgtcccagctggacttccagtgggagaaagtgaacaagatg tacaaggaccggcagggcagattTgaccgcagTgtggaaaagtggAggAggttccactacgacatcaa gatcttcaaccagtggctgacAgaggccgagcagttcctgagaaagacccagatccccgagaactggg agcacgccaagtacaagtggtatctgaaagaactgcaggatggcatTggccagagacagacAgtGgtg cggacactgaatgccaccggcgaggaaatcatccagcagagcagcaagaccgacgccagtattctgca ggaaaagctgggcagcctgaacctgagatggcaggaagtgtgcaagcagctgtccgaccggaagaaga gactggaagaacagagTgaccagtggaagcggctgcatctgtcactgcaggaactgctGgtgtggctg cagctgaaggaTgaTgagctgagcagacaggcccctatTggcggcgattttcccgcAgtgcagaaaca gaacgaTgtgcaccgggccttcaagagagagctgaaaacaaaagaaccAgtgatcatgagcaccctgg aaacAgtgcggatctttctgaccgagcagcccctggaaggactggaaaaactgtaccaggaacccaga gagctgccccctgaagaacgggcccagaacgtgaccagactgctgAggaagcaggccgaggaagtgaa cacAgaatgggagaagctgaacctgcactcTgcTgactggcagAggaagatTgaTgagacactggaac ggctgcaggaactgcaggaggccacAgacgagctggacctgaaactgagacaggccgaagtgatcaag ggcagctggcagccagtgggcgacctgctgatcgacagcctgcaggatcacctggaaaaagtgaaagc cctgagaggcgagatTgcccccctgaaagaaaaTgtgtcccaTgtgaacgacctggcccggcagctga caacactgggcatccagctgagcccctacaacctgtccacactggaagatctgaacacccggtggaaa ctgctgcaggtggccgtggaagatagagtgcggcagctgcacgaggcccacagagattttggccctgc ctcccagcacttcctgagcacatctgtgcagggcccctgggagagagccatctcccccaacaaggtgc cctactacatcaaccacgagacacagaccacctgttgggaccaccccaagatgacAgagctgtaccag agcctggccgacctgaacaatgtgAggttcagTgcctacAggaccgccatgaagctgcggagactgca gaaagctctgtgcctggacctgctgtccctgtccgccgcttgtgatgccctggaccagcacaacctga agcagaacgaccagcccatggatatcctgcagatcatcaactgcctgaccaccatctacgaccgcctg gaacaggaacacaacaacctGgtgaatgtgcccctgtgTgtggacatgtgcctgaattggctgctgaa tgtgtacgacaccggccggacaggccggatcagagtgctgagcttcaagaccggcatcatcagcctgt gcaaggcccacctggaagataagtaccgctacctgttcaaacaggtggccagctccaccggcttttgc gaccagagaaggctgggcctgctgctgcacgacagcatccagatccctagacagctgggcgaggtggc ctctttTggcggcagcaatatTgagcctagTgtgcggagctgcttccagttTgccaacaacaagcccg agatTgaggccgccctgttcctggactggatgcggctggaaccccagagcatggtgtggctgcctgtg ctgcatagagtggccgctgccgagacagccaagcaccaggccaagtgcaacatctgcaaagagtgccc catcatcggcttccggtacagaagcctgaagcacttcaactacgatatctgccagagctgctttttca gcggacgggtggccaagggccacaaaatgcactaccccatggtggaatactgcacccccaccacctcc ggggaggatgtgcgggattttgccaaggtgctgaaaaacaagttccggaccaagcgctacttTgccaa acacccccggatgggctatctgcccgtgcagacagtgctggaaggcgacaacatggaaaccgacacca tgtag (SEQ ID NO: 2) SEQ ID NO: 2 is a codon optimized polynucleotide sequence encoding a microdystrophin protein of SEQ ID NO: 3 (also known as “microD5,” “MD5,” or “µD5”). The codon optimized polynucleotide sequence of SEQ ID NO: 2 has reduced number of CpG 10 ME152715650v.1 Attorney Docket No.: 129159-02620 sites / islands, or has substantially eliminated CpG islands. MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGS TRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNS EKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIAR YQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQM HYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVN LDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIG TGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHSYVPSTYLTEITHVSQALLEVEQLLNAPDL CAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKM YKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVV RTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQSDQWKRLHLSLQELLVWL QLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPR ELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIK GSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWK LLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQ SLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRL EQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFC DQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPV LHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTS GEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETDTM (SEQ ID NO: 3) In SEQ ID NO: 2 shown above, certain nucleotides are marked up as capital letters, and these nucleotides are collectively referred to herein as “capitalized nucleotides of SEQ ID NO: 2.” Specifically, the capitalized nucleotides of SEQ ID NO: 2 includes nucleotides 264, 273, 282, 291, 297, 303, 543, 555, 558, 627, 1110, 1113, 1122, 1656, 1665, 1678, 1681, 1722, 1815, 1830, 1833, 1989, 2031, 2052, 2055, 2079, 2097, 2115, 2157, 2181, 2290, 2316, 2343, 2346, 2356, 2364, 2367, 2406, 2532, 2550, 2559, 2844, 2881, 2889, 2896, 3081, 3099, 3339, 3354, 3363, 3384, 3405, and 3735 of SEQ ID NO: 2. In certain embodiments, the codon optimized polynucleotide sequence of the invention not only encode the same protein (i.e., SEQ ID NO: 3), but also share the same set of capitalized nucleotides of SEQ ID NO: 2, yet they differ from SEQ ID NO: 2 at nucleotide positions other than the capitalized nucleotides of SEQ ID NO: 2. In certain embodiments, the codon optimized polynucleotide sequence of the invention not only encode the same protein (i.e., SEQ ID NO: 3), but are also substantially identical to SEQ ID NO: 2 at the capitalized nucleotides of SEQ ID NO: 2, despite additional sequence changes (e.g., to result in 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, or 99.9% overall sequence identity) in positions of SEQ ID NO: 2 other than the capitalized nucleotides. In certain embodiments, the codon optimized polynucleotide sequence of the invention is identical to SEQ ID NO: 2 at each capitalized nucleotides, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides. 11 ME152715650v.1 Attorney Docket No.: 129159-02620 In certain embodiments, the codon optimized polynucleotide sequence of the invention comprises, consists essentially of, or consists of a nucleotide sequence at least 95% identical to SEQ ID NO: 2. That is, the polynucleotide of the invention encodes the microdystrophin of SEQ ID NO: 3, and further, the polynucleotide of the invention is (1) identical to SEQ ID NO: 2 at each capitalized nucleotides, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides; and / or (2) substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpg-plot analysis). In certain embodiments, the polynucleotide of the invention comprises, consists essentially of, or consists of a nucleotide sequence at least 97% identical to SEQ ID NO: 2. That is, the polynucleotide of the invention encodes the microdystrophin of SEQ ID NO: 3, and further, the polynucleotide of the invention is (1) identical to SEQ ID NO: 2 at each capitalized nucleotide, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides; and / or (2) substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpg-plot analysis). In certain embodiments, the codon optimized polynucleotide sequence of the invention comprises, consists essentially of, or consists of a nucleotide sequence at least 99% identical to SEQ ID NO: 2. That is, the polynucleotide of the invention encodes the microdystrophin of SEQ ID NO: 3, and further, the polynucleotide of the invention is (1) identical to SEQ ID NO: 2 at each capitalized nucleotides, or differ by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 capitalized nucleotides; and / or (2) substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpg-plot analysis). Sequence percentage identity between any two or more related or unrelated polynucleotides, or between any two or more related or unrelated protein sequences, can be aligned and the percentage of the matches between the nucleotides or amino acid residues, respectively, can be calculated using any art recognized methods, such as the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215:403-10, 1990), which is available from online sources, such as the National Center for Biological Information (NCBI) website, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx, depending on the type of query and database. Similar web-based tools can be found at the EMBL-EBI website. 12 ME152715650v.1 Attorney Docket No.: 129159-02620 In certain embodiments, the polynucleotide of the invention substantially lacks CpG islands (e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 CpG islands based on EMBOSS Cpg- plot analysis). In certain embodiments, the therapeutically effective amount is between about 2- 50×1012vg / kg, between about 5-50×1012vg / kg, between about 6-30×1012vg / kg, about 2×1012vg / kg, about 6×1012vg / kg, about 1×1013vg / kg, about 15×1012vg / kg, about 20×1012vg / kg, about 25×1012vg / kg, about 30×1012vg / kg, about 40×1012vg / kg, about 50×1012vg / kg, about 60×1012vg / kg, about 70×1012vg / kg, or about 80×1012vg / kg. Methods for tittering AAV are described in Clark et al., Hum. Gene Ther.10:1031-1039, 1999 (incorporated by reference). In certain embodiments, the subject is a human, and wherein the therapeutically effective amount is for a mouse (such as an mdx mouse) before conversion to an equivalent dose in said human. In certain embodiments, the human is a neonate (from birth through the first 28 days of life), an infant (from 29 days to less than 2 years), a child (from 2 years to less than 12 years), or an adolescent (aged 12 through 21). In certain embodiments, the child is between 4-8 years old. In certain embodiments, the child is a boy. Methods of transducing a target cell with rAAV, in vivo or in vitro, are contemplated by the invention. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the invention to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder / disease, the administration is prophylactic. If the dose is administered after the development of a disorder / disease, the administration is therapeutic. In embodiments of the invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the muscular dystrophy being treated, that slows or prevents progression to a more advanced disorder / disease state, that slows or prevents progression of a disorder / disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and / or that prolongs survival. For administration, effective amounts and therapeutically effective amounts (also referred to herein as doses) may be initially estimated based on results from in vitro assays and / or animal model studies, such as doses effective to treat a mouse model of human disease (e.g., the mdx model of DMD). For example, a dose may be formulated in animal models to 13 ME152715650v.1 Attorney Docket No.: 129159-02620 achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information may be used to more accurately determine or extrapolate to useful doses in subjects of interest, such as human. Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration and serotype(s) of AAV components of the rAAV (in, the AAV ITRs and capsid protein) of the invention may be chosen and / or matched by those skilled in the art taking into account the infection and / or disease state being treated and the target cells / tissue(s) that are to express the one or more coding sequences and / or micro-dystrophin. Specifically, the formulations described herein may be administered by, without limitation, injection, infusion, perfusion, inhalation, lavage, and / or ingestion. Routes of administration may include, but are not limited to, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topically, intratumoral, intramuscular, intravesicular, intrapericardial, intraumbilical, intraocularal, mucosal, oral, subcutaneous, and / or subconjunctival. The invention provides for local administration or systemic administration of an effective dose of rAAV and compositions of the invention including combination therapy of the invention. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parental administration through injection, infusion or implantation. In particular, actual administration of rAAV of the present invention may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal, such as the skeletal muscles. Administration according to the invention includes, but is not limited to, injection into muscle, and / or the bloodstream. Simply re-suspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). Pharmaceutical compositions can be prepared as injectable formulations or as topical 14 ME152715650v.1 Attorney Docket No.: 129159-02620 formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling. The dose of rAAV to be administered in methods disclosed herein may vary depending, for example, on the particular mode of administration, the treatment goal, the individual, and the cell type(s) being targeted. The actual dose amount administered to a particular subject may also be determined by a physician, a veterinarian, or a researcher, taking into account parameters such as, but not limited to, physical and physiological factors including body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject, and / or route of administration. In some embodiments, the composition and / or vector disclosed herein is formulated in a typical formulation known in the art. In some embodiments, the pharmaceutical composition is in a dosage form of 10 mL of aqueous solution having at least 1-2×1013vector genomes. In some embodiments, the dosage has a potency of at least 2×1012vector genomes per milliliter. In some embodiments, the dosage comprises a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride. In some embodiments, the pharmaceutical composition is in a dosage form of 10 mL of a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride; and having at least 1.6×1013vector genomes. In some embodiments, the pharmaceutical composition may be a dosage comprising between 1×1010and 1×1015vector genomes in 10 mL aqueous solution; between 1×1011and 1×1014vector genomes in 10 mL aqueous solution; between 1×1012and 2×1013vector genomes in 10 mL aqueous solution; or greater than or equal to about 1.6×1013vector genomes in 10 mL aqueous solution. In some embodiments the aqueous solution is a sterile aqueous solution comprises about 10 mM L histidine pH 6.0, with 150 mM sodium chloride, and 1 mM magnesium chloride. In some embodiments, the dosage has a potency of greater than about 1×1011vector genomes per milliliter (vg / mL), greater than about 1×1012vg / mL, greater than about 2×1012vg / mL, greater than about 3×1012vg / mL, or greater than about 4×1012vg / mL. In some embodiments, at least one AAV vector is provided as part of a 15 ME152715650v.1 Attorney Docket No.: 129159-02620 pharmaceutical composition. The pharmaceutical composition may comprise, for example, at least 0.1% w / v of the AAV vector. In some other embodiments, the pharmaceutical composition may comprise between 2% to 75% of compound per weight of the pharmaceutical composition, or between 25% to 60% of compound per weight of the pharmaceutical composition. In some embodiments, the dosage is in a kit. The kit may further include directions for use of the dosage. For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art. In some embodiments, for injection, formulations may be made as aqueous solutions, such as in buffers including, but not limited to, Hanks' solution, Ringer's solution, and / or physiological saline. The solutions may contain excipients or formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the formulation may be in lyophilized and / or powder form for constitution with a suitable vehicle control (e.g., sterile pyrogen-free water) before use. Any formulation disclosed herein may advantageously comprise any other pharmaceutically acceptable carrier or carriers which comprise those that do not produce significantly adverse, allergic, or other untoward reactions that may outweigh the benefit of administration, whether for research, prophylactic, and / or therapeutic treatments. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, which is incorporated by reference herein for its teachings regarding the same. Moreover, formulations may be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the United States FDA’s Division of Biological Standards and Quality Control and / or other 16 ME152715650v.1 Attorney Docket No.: 129159-02620 relevant U.S. and foreign regulatory agencies. Exemplary, generally used pharmaceutically acceptable carriers may comprise, but are not limited to, bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, and vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and / or lubricants. Exemplary buffering agents may comprise, but are not limited to, citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts. Exemplary preservatives may comprise, but are not limited to, phenol, benzyl alcohol, meta-cresol, methylparaben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, and / or 3-pentanol. Exemplary isotonic agents may comprise polyhydric sugar alcohols comprising, but not limited to, trihydric or higher sugar alcohols, (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and / or mannitol). Exemplary stabilizers may comprise, but are not limited to, organic sugars, polyhydric sugar alcohols, polyethylene glycol, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, and / or polysaccharides. Formulations may also be depot preparations. In some embodiments, such long- acting formulations may be administered by, without limitation, implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, compounds may be formulated with suitable polymeric and / or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). Additionally, in various embodiments, the AAV vectors may be delivered using sustained-release systems, such as semipermeable matrices of solid polymers comprising the AAV vector. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release capsules may, depending on their chemical nature, release the vector following administration for a few weeks up to over 100 days. 17 ME152715650v.1 Attorney Docket No.: 129159-02620 The pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof. Transduction with rAAV may also be carried out in vitro. In one embodiment, desired target muscle cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used where those cells will not generate an inappropriate immune response in the subject. Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and / or 18 ME152715650v.1 Attorney Docket No.: 129159-02620 PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle, using e.g., a catheter. Transduction of cells with rAAV of the invention results in sustained co-expression of said one or more additional coding sequences and micro-dystrophin. The present invention thus provides methods of administering / delivering rAAV which co-expresses said one or more additional coding sequences and micro-dystrophin to an animal, preferably a human being. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more rAAV of the present invention. Transduction may be carried out with gene cassettes comprising tissue specific control elements. For example, one embodiment of the invention provides methods of transducing muscle cells and muscle tissues directed by muscle specific control elements, including, but not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family (See Weintraub et al., Science 251:761-766, 1991), the myocyte-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11:4854-4862, 1991), control elements derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol 7:4089-4099, 1987), the cardiac actin gene, muscle creatine kinase sequence elements (Johnson et al., Mol Cell Biol 9:3393-3399, 1989), and the murine creatine kinase enhancer (mCK) element, control elements derived from the skeletal fast- twitch troponin C gene, slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene: hypoxia-inducible nuclear factors (Semenza et al., Proc Natl Acad Sci U.S.A.88:5680- 5684, 1991), steroid-inducible elements and promoters including the glucocorticoid response element (GRE) (See Mader and White, Proc. Natl. Acad. Sci. U.S.A.90:5603-5607, 1993), and other control elements. Muscle tissue is an attractive target for in vivo DNA delivery, because it is not a vital organ and is easy to access. As used herein, “muscle cell” or “muscle tissue” is meant a cell or group of cells derived from muscle of any kind (for example, skeletal muscle and smooth muscle, e.g., from the digestive tract, urinary bladder, blood vessels or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts. The term “transduction” is used to refer to the administration / delivery of the one or 19 ME152715650v.1 Attorney Docket No.: 129159-02620 more additional coding sequences and the coding region of the micro-dystrophin to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the invention resulting in co-expression of the one or more additional coding sequences and micro- dystrophin by the recipient cell. Thus, the invention provides methods of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV that encode said one or more additional coding sequences and a GOI to a patient in need thereof at a lower dose than conventionally understood, in particular for systemically delivered gene therapy or gene therapy delivered to a subject in need thereof requiring more than specifically localized delivery (e.g., direct delivery to the eye). With the general aspect of the invention described above, the following examples further illustrate the specific, non-limiting embodiments of the invention. EXAMPLES Example 1 Systemic Delivery of Microdystrophin Gene Therapy Using Capsid AAV- SLB101 in the mdx Mouse Model of Duchenne Muscular Dystrophy Duchenne type muscular dystrophy (DMD) is one of the most devastating muscle diseases with serious debilitating impacts on quality of life of affected patients, mostly in boys. It has been thought that the nature of these diseases requires high dose gene therapy to reach target tissues for appropriate transgene expression. The invention described herein provides the surprising finding that a significantly lower dose of AAV viral particles (as delivery vehicles for the microdystrophin transgene) than doses previously thought necessary can in fact impart significant therapeutic efficacy while further lowering potential treatment side effects at said lower dose. Specifically, 3 months post-treatment in mdx mice, functional efficacy was observed at doses ≥ 6.0E12 vg / kg as measured by forelimb grip strength test, EDL specific force production, and treadmill exhaustion. These data demonstrated the effectiveness and safety of the subject AAV viral particle comprising two salient features: (1) the proprietary SLB101 capsid having enhanced muscle tropism, and (2) the nNOS-containing microdystrophin transgene. FIG.5 is a schematic (not drawn to scale) showing the domain structures of the nNOS-containing microdystrophin transgene that retains several key dystrophin protein 20 ME152715650v.1 Attorney Docket No.: 129159-02620 functional domains (also referred to herein as “MD-5”). Specifically, the five spectrin-like repeats from the wild-type human dystrophin, R1, R16, R17, R23, and R24, together forms a microdystrophin encompassing a functional nNOS (neuronal nitric oxide synthase)-binding domain, which is potentially important for prevention of activity-induced ischemia and associated muscle injury (see Lai et al., J Clin Invest.2009; and Ramos et al., Mol Ther. 2019); and its presence was correlated with milder phenotypes of Becker muscular dystrophy (BMD) (Gentil et al., Hum Mol Genet.2012). An in vivo study was performed in the mdx mice model of human DMD to ascertain functional measurements at different dosing levels of the subject AAV-SLB101 rAAV. The study design is summarized in FIG.2. Functional improvements were associated with improvements in several serum biomarkers of muscle membrane integrity (including serum CK level, serum titin level, and serum AST level) in treated mdx mice vs. control mice (mdx vehicle), at doses as low as about 2E12 vg / kg, 6E12 vg / kg, and 3.0E13 vg / kg (FIG.3). A dose response relationship was observed in skeletal muscles (quadriceps), smooth muscles (diaphragm), and cardiac muscles (heart), among the three treatment groups, based on the detected vg copies per unit (μg) weight (FIG.4). Dose response in microdystrophin expression was also observed after treatment (FIG. 5). Further, dose response in microdystrophin-positive myofiber percentage was observed after treatment (FIG.7). The expressed microdystrophin was found to be functional, as microdystrophin protein and functionally active nNOS were found to be properly localized to the sarcolemma in all doses of the treatment groups (FIG.6). Dose response was also observed in diaphragm and quadriceps myofibers positive for nNOS activity, in the treatment groups (FIG.8). Additional functional testing, including forelimb grip strength test, treadmill exhaustion test, and EDL specific force measurement, showed that these functional improvements were achieved at treatment doses as low as about 6.0E12 vg / kg (FIG.9). Again, at these lower doses, improvements in serum biomarkers of muscle membrane integrity (including CK, titin, and ALT), were maintained in all treatment groups (FIG.10). Collectively, these data shows that microdystrophin protein expression, as well as nNOS enzymatic activity, were detected at the correct subcellular localization in sarcolemma. 21 ME152715650v.1 Attorney Docket No.: 129159-02620 Microdystrophine expression was accompanied by improvements in muscle function. Meanwhile, serum biomarkers showed improvements in sarcolemmal membrane integrity at treatment doses as low as 6.0E12 vg / kg. The data presented herein show that, in mdx mice, treatment-related changes noted in clinical chemistry included minimally to markedly decreased ALT, AST, and creatine kinase at ≥ 3.0E13 vg / kg on Days 4, 29, and 92. Further, a decrease in degeneration / necrosis was observed in mdx mice treated with SLB101 encapsidating MD-5 compared to vehicle controls. Overall, these data provided experimental evidence to use the subject recombinant AAV viral vector with enhanced biodistribution to muscles, to deliver an effective dose of nNOS-containing microdystrophin for treating muscular dystrophy such as DMD at a lower vg / kg than previously known or expected. 22 ME152715650v.1

Claims

Attorney Docket No.: 129159-02620 CLAIMS 1. A method of treating a disease, disorder, and / or condition related to muscle tissue (e.g., muscular dystrophy) in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV), wherein the therapeutically effective amount is about 2-90 × 1012vg / kg, wherein the rAAV comprises a gene of interest (GOI) related to treating said muscle tissue, optionally wherein the GOI is flanked by a pair of AAV ITR sequences, and, wherein the rAAV comprises a modified adeno-associate virus (mAAV) capsid comprising an amino acid sequence at least about 80% (e.g., at least about 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1, and wherein the mAAV capsid comprises the amino acid sequence of RGDLGLS (SEQ ID NO: 4).

2. A method of treating muscular dystrophy in a subject in need thereof, the method comprises administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV), wherein the therapeutically effective amount is about 2-90 × 1012vg / kg, wherein the rAAV comprises a gene of interest (GOI) defective in said muscular dystrophy, flanked by a pair of AAV ITR sequences, and, wherein the rAAV comprises a modified adeno-associate virus (mAAV) capsid having the amino acid sequence of SEQ ID NO:

1.

3. The method of claim 1 or 2, wherein the GOI is LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 2I), or a gene or coding sequence for NAGLU (α-N- acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain- 3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

4. The method of claim 1 or 2, wherein the muscular dystrophy is DMD (Duchenne 23 ME152715650v.1Attorney Docket No.: 129159-02620 Muscular Dystrophy) or BMD (Becker Muscular Dystrophy), and wherein the GOI encodes a microdystrophin.

5. The method of claim 4, wherein the microdystrophin is one described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; PCT / US2016 / 013733; or US10,166,272.

6. The method of claim 5, wherein the microdystrophin comprises a coding sequence for R16 and R17 spectrin-like repeats for the full-length dystrophin protein (such as one described in US7,892,824).

7. The method of claim 6, wherein the microdystrophin comprises a coding sequence for the R1, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein; or a microdystrophin gene described in PCT / US2016 / 013733 or US10,479,821.

8. The method of any one of claims 1-7, wherein the GOI is operatively linked to a transcriptional regulatory cassette, such as a muscle specific promoter (e.g., a CK8 promoter or a cardiac troponin T (cTnT) promoter).

9. The method of any one of claims 1-8, wherein the GOI is a micro-dystrophin gene encoding a protein comprising, from N- to C-terminus, an amino-terminal actin- binding (AB1) domain, a β-dystroglycan binding domain, a Hinge 1 domain (H1), a spectrin-like repeat domain consisting of five spectrin-like repeats that include spectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16), spectrin-like repeat 17 (SR17), spectrin-like repeat 23 (SR23), and spectrin-like repeat 24 (SR24), and a Hinge 4 domain (H4), wherein the micro-dystrophin gene is operatively linked to a muscle-specific human muscle creatine kinase CK8 promoter (e.g., SEQ ID NO:19 of US10,479,821), and wherein the GOI is flanked by a pair of AAV2 ITR (inverted terminal repeat) sequence.

10. The method of any one of claims 1-9, wherein the GOI is codon-optimized for mammalian expression.

11. The method of claim 10, wherein the GOI comprises a polynucleotide having the sequence at least about 80% (e.g., at least about 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:

2.

12. The method of any one of claims 1-11, wherein the therapeutically effective amount is 24 ME152715650v.1Attorney Docket No.: 129159-02620 between about 2-50×1012vg / kg, between about 5-50×1012vg / kg, between about 6- 30×1012vg / kg, about 2×1012vg / kg, about 6×1012vg / kg, about 1×1013vg / kg, about 15×1012vg / kg, about 20×1012vg / kg, about 25×1012vg / kg, about 30×1012vg / kg, about 40×1012vg / kg, about 50×1012vg / kg, about 60×1012vg / kg, about 70×1012vg / kg, or about 80×1012vg / kg.

13. The method of any one of claims 1-12, wherein the subject is a human, and wherein the therapeutically effective amount is for a mouse (such as an mdx mouse) before conversion to an equivalent dose in said human.

14. The method of claim 13, wherein said human is a neonate (from birth through the first 28 days of life), an infant (from 29 days to less than 2 years), a child (from 2 years to less than 12 years), or an adolescent (aged 12 through 21).

15. The method of claim 14, wherein the child is between 4-8 years old.

16. The method of claim 14 or 15, wherein the child is a boy. 25 ME152715650v.1

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