Recombinant AAV vectors for treating muscular dystrophy

Abstract

The present disclosure provides gene therapy vectors, such as recombinant adeno-associated viruses (rAAVs), for expressing the human microdystrophin gene. The present disclosure also provides compositions and methods for using these rAAVs to treat muscular dystrophies, such as Duchenne muscular dystrophy. The present disclosure also provides for genotyping a subject's DMD gene to determine whether rAAV gene therapy should be contraindicated. The present disclosure is directed to gene therapy vectors, such as AAV vectors, produced by the floating seed process described herein that express the human microdystrophin gene in skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis.

Description

[Technical Field]

[0001] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing (Name: 4140_062PC04_Seqlisting_ST26.xml; Size: 55,294 bytes; Creation Date: September 18, 2023) submitted with this application are incorporated herein by reference in their entirety.

[0002] Field The present disclosure is in the field of gene therapy. More specifically, the present disclosure provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, for expressing a miniaturized human micro-dystrophin gene. The present disclosure also provides methods of using these vectors to express micro-dystrophin in skeletal muscles, including the diaphragm and cardiac muscles, and to protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy. [Background technology]

[0003] background It is clear that muscle mass and strength are important for daily activities, such as locomotion and respiration, as well as for whole-body metabolism. Deficits in muscle function result in muscular dystrophies (MDs), which are characterized by muscle weakness and wasting and have a significant impact on quality of life. The best-characterized MDs result from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs result from membrane fragility associated with the lack of DAPC linking the sarcolemma to the cytoskeleton. Duchenne muscular dystrophy (DMD) is one of the most serious muscle diseases, affecting 1 in 5,000 newborn boys.

[0004] DMD is caused by mutations in the DMD gene, which result in reduced mRNA and the absence of dystrophin, a 427-kD sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51: 919-28, 1987). The DAPC is composed of several proteins present in the sarcolemma that form structural links between the extracellular matrix (ECM) and the cytoskeleton via dystrophin, actin-binding proteins, and alpha-dystroglycan, a laminin-binding protein. These structural links stabilize the muscle membrane during contraction and protect it from contraction-induced damage. Loss of dystrophin leads to membrane fragility, resulting in sarcolemmal rupture and calcium influx, which triggers calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol. 10: 168-75(1997)). This uncontrolled cycle of muscle degeneration and regeneration ultimately depletes the muscle stem cell population (Sacco et al., Cell 143: 1059-1071(2010); Wallace et al., Annu Rev Physiol 71: 37-57(2009)), resulting in progressive muscle weakness, endomysial inflammation, and fibrotic scarring. Without membrane stabilization by dystrophin or microdystrophin, DMD manifests as an uncontrolled cycle of tissue injury and repair, ultimately replacing lost muscle fibers with fibrous scar tissue through connective tissue proliferation. Fibrosis is characterized by the excessive deposition of extracellular matrix proteins, including collagen and elastin. ECM proteins are primarily produced from cytokines, such as TGFβ, released by activated fibroblasts in response to stress and inflammation. While myofiber degeneration and necrosis are the primary pathological hallmarks of DMD, fibrosis as a pathological consequence is equally impactful. Excessive production of fibrous tissue limits muscle regeneration and contributes to the progressive muscle weakness in DMD patients. In one study, the presence of fibrosis in initial DMD muscle biopsies was highly correlated with poor motor outcome at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol 68: 762-767 (2009)). These results indicate that fibrosis is a major contributor to muscle dysfunction in DMD and emphasize the need for early intervention before fibrosis becomes apparent. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hoffman et al., Cell 51: 919-28, 1987 [Non-patent document 2] Straub et al., Curr Opin. Neurol. 10: 168-75(1997) [Non-patent document 3] Sacco et al., Cell 143: 1059-1071(2010) [Non-patent document 4] Wallace et al., Annu Rev Physiol 71: 37-57(2009) [Non-patent document 5] Desguerre et al., J Neuropathol Exp Neurol 68: 762-767(2009) Summary of the Invention [Means for solving the problem]

[0006] International Publication No. WO2019 / 245973A1, the entirety of which is incorporated herein by reference, describes AAV vector delivery of the microdystrophin gene for treating muscular dystrophy (e.g., DMD) in human subjects. International Application No. PCT / US2022 / 029328, filed May 13, 2022, the entirety of which is incorporated herein by reference, describes the production of recombinant AAV vectors for treating muscular dystrophy (e.g., DMD) in human subjects. However, there remains a need in the art for improved methods of treating DMD using AAV vectors, such as by genotyping the patient's human dystrophin (DMD) gene prior to treatment to identify patient populations likely to benefit from or be contraindicated for the AAV gene therapy described herein.

[0007] overview The present disclosure is directed to gene therapy vectors, e.g., AAV vectors, produced by the floating seed process described herein, which express the human microdystrophin gene in skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis. The present disclosure is also directed to compositions and methods using these AAV vectors to treat muscular dystrophy, e.g., Duchenne muscular dystrophy.

[0008] The present disclosure provides a method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin in adherent mammalian cells by a suspension seeding process, the method comprising: (a) culturing the cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container that is serum-free or contains serum at a lower concentration than the first medium; (d) culturing the cells in the N-1 container under suspension conditions; and (e) inoculating a third medium in a bioreactor with the cells from step (d).

[0009] In some embodiments, the rAAV used in the methods described herein comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1. In some embodiments, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 7. In some embodiments, the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 7.

[0010] In some aspects, the suspension seeding process further comprises (f) transfecting the adherent cells with a transgene plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct, a plasmid comprising the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

[0011] In some embodiments, the transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct comprises the nucleic acid sequence of SEQ ID NO:9; nucleotides 55-5021 of SEQ ID NO:3; or nucleotides 1-4977 of SEQ ID NO:8. In some embodiments, the plasmid containing the AAV rep gene and AAV cap gene comprises the AAV2 rep gene and the rAAVrh74 cap gene. In some embodiments, the adenovirus helper plasmid comprises the adenovirus 5 E2A, E4ORF6, and VA RNA genes.

[0012] In some embodiments, the suspension seeding process further comprises (g) lysing the adherent cells, hi some embodiments, the adherent cells are lysed by freeze-thawing, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

[0013] In some aspects, the floating seed process further comprises purifying the (h)rAAV by at least one column chromatography step, hi some aspects, the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0014] In some embodiments, the suspension seed process further comprises culturing the cells in a first growth medium in an N-3 container. In some embodiments, the suspension seed process further comprises culturing the cells in a first growth medium in an N-4 container.

[0015] In some embodiments, the bioreactor is an adherent bioreactor. In some embodiments, the rAAV is purified from the culture produced in the adherent bioreactor.

[0016] In some embodiments, the third culture medium in the bioreactor comprises at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third culture medium in the bioreactor comprises DMEM and 10% FBS.

[0017] In some embodiments, the adherent cells are cultured in suspension for about 48 to 72 hours.

[0018] In some embodiments, the N-1 container is a suspension shake flask.

[0019] In some embodiments, the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells. In some embodiments, the adherent cells are HeLa cells or HEK-293 cells. In some embodiments, the adherent cells are HEK-293 cells. In some embodiments, the adherent cells are not adapted to a suspension system. In some embodiments, culturing the cells in suspension does not alter the adhesion dependency of the cells. In some embodiments, culturing does not alter the cells to create a new cell line.

[0020] The present disclosure also provides compositions comprising the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is produced by any of the methods described herein. In some embodiments, the composition comprises: a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8.

[0021] In some embodiments, the present disclosure provides a composition comprising a recombinant adeno-associated virus (rAAV) rAAV.rh74MHCK7.micro-dystrophin for treating muscular dystrophy in a human subject in need thereof, wherein the rAAV is produced in adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container. In some embodiments, the rAAV comprises the human micro-dystrophin nucleotide sequence of SEQ ID NO: 1. In some embodiments, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 7. In some embodiments, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 7 and the human micro-dystrophin nucleotide sequence of SEQ ID NO: 1.

[0022] In some embodiments, the composition comprises: (a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; (b) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; (c) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; (d) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; (e) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8; and / or (f) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8.

[0023] The present disclosure also provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering to the human subject a composition comprising an rAAV described herein.

[0024] In some embodiments, the rAAV is administered using a systemic route, at a dose of about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 15 In some embodiments, the systemic route of administration is intravenous, and the dose of rAAV administered is about 2×10 14 In some embodiments, the systemic administration route is intravenous and the dose of rAAV administered is about 1.33 x 10 14 vg / kg.

[0025] In some embodiments, the dose of rAAV is administered at a concentration of about 10 mL / kg. In some embodiments, the rAAV is administered by injection, infusion, or implantation. In some embodiments, the rAAV is administered by infusion over approximately 1 hour. In some embodiments, the rAAV is administered intravenously through a peripheral vein in the limb.

[0026] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0027] In some embodiments, the level of micro-dystrophin gene expression in target cells increases after administration of rAAV compared with the level of micro-dystrophin gene expression before administration of rAAV.In some embodiments, the expression of micro-dystrophin gene in cells is detected by measuring micro-dystrophin protein level by Western blot for muscle biopsy before and after administration of rAAV.In some embodiments, after administration of rAAV, expression is at least 55.4% compared with before administration.

[0028] In some embodiments, the average percentage of microdystrophin-positive fibers in the muscle tissue of the subject increases after administration of rAAV compared with the number of microdystrophin-positive fibers before administration of rAAV.In some embodiments, the average percentage of microdystrophin-positive fibers is at least 70.5%, and the average intensity detected by immunofluorescence (IF) for muscle biopsy specimens before and after administration of rAAV is at least 116.9%.In some embodiments, the microdystrophin transduction by vector genome counting is at least 3.87 average vector genome copies per nucleus.

[0029] In some embodiments, the compositions described herein are administered to a subject who has been genotyped for at least one mutation in the human dystrophin (DMD) gene. In some embodiments, the mutation in the at least one DMD gene is a frameshift deletion, a frameshift duplication, a premature termination, or other pathogenic variant that results in the absence of expression of human dystrophin protein. In some embodiments, the subject has been genotyped for at least one mutation in exons 18-79 of the DMD gene.

[0030] In some embodiments, the subject has been genotyped for at least one mutation in exons 9-13 of the DMD gene. In some embodiments, the at least one mutation in exons 9-13 is a deletion. In some embodiments, the deletion completely encompasses exons 9-13 of the DMD gene.

[0031] In some embodiments, the subject has been genotyped for at least one mutation in exon 8 and / or 9 of the DMD gene. In some embodiments, the at least one mutation in exon 8 and / or 9 of the DMD gene is a deletion. In some embodiments, the deletion is in exon 8 of the DMD gene. In some embodiments, the deletion is in exon 9 of the DMD gene. In some embodiments, the deletion is in exon 8 and exon 9 of the DMD gene.

[0032] In some aspects, the method of treating muscular dystrophy further comprises genotyping the DMD gene in the human subject prior to administering the composition to said human subject.

[0033] In some embodiments, genotyping detects at least one mutation in exons 18-79 of the DMD gene. In some embodiments, the at least one mutation is a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the absence of expression of the human dystrophin protein.

[0034] In some embodiments, genotyping detects at least one mutation in exons 9-13 of the DMD gene. In some embodiments, the at least one mutation in exons 9-13 is a deletion. In some embodiments, the deletion completely encompasses exons 9-13 of the DMD gene.

[0035] In some embodiments, genotyping detects at least one mutation in exon 8 and / or 9 of the DMD gene. In some embodiments, the at least one mutation in exon 8 and / or 9 is a deletion. In some embodiments, the deletion is in exon 8 of the DMD gene. In some embodiments, the deletion is in exon 9 of the DMD gene. In some embodiments, the deletion is in exon 8 and exon 9 of the DMD gene.

[0036] The present disclosure also provides for the use of the compositions described herein for the treatment of muscular dystrophy in a human subject in need thereof. In some aspects, the present disclosure also provides for the use of the compositions described herein in the manufacture of a medicament for the treatment of muscular dystrophy.

[0037] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0038] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles encapsidating an expression cassette comprising a human microdystrophin transgene, provided that the subject does not have a deletion that completely encompasses exons 9-13 of the DMD gene.

[0039] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles encapsidating an expression cassette comprising a human microdystrophin transgene, provided that the subject does not have a deletion in exons 8 and / or 9 of the human dystrophin (DMD) gene.

[0040] In some embodiments, the subject's DMD gene is genotyped prior to treatment.

[0041] In some embodiments, the AAV viral particles are of serotype rh74. In some embodiments, the rAAV vector is administered as a composition comprising: a) rh74 serotype AAV viral particles encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rh74 serotype AAV viral particles encapsidating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rh74 serotype AAV viral particles encapsidating nucleotides 1-4977 of SEQ ID NO:8.

[0042] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, the method comprising: i) genotyping the subject's human dystrophin (DMD) gene prior to treatment; and ii) providing that genotyping does not identify a deletion entirely encompassing exons 9-13 of the DMD gene, administering to the human subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the composition comprises: a) rAAV particles encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsidating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsidating nucleotides 1-4977 of SEQ ID NO:8.

[0043] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, the method comprising: i) genotyping the subject's human dystrophin (DMD) gene prior to treatment; and ii) providing that genotyping does not identify a deletion in exons 8 and / or 9 of the DMD gene, administering to the subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the composition comprises: a) rAAV particles encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsidating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsidating nucleotides 1-4977 of SEQ ID NO:8.

[0044] In some aspects, the human subject treated according to any of the methods described herein is ambulatory.

[0045] In some aspects, the human subject treated according to any of the methods described herein is non-ambulatory.

[0046] In some embodiments, the human subject treated according to any of the methods described herein is between 2 and 3 years old. In some embodiments, the human subject treated according to any of the methods described herein is between 4 and 5 years old.

[0047] In some embodiments, the human subject treated according to any of the methods described herein has been non-ambulatory for at least 9 months.

[0048] In some embodiments, the human subject treated according to any of the methods described herein further has a stable forced vital capacity (FVC) of less than 40% of predicted and / or a need for nocturnal ventilator support.

[0049] In some aspects, the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin used in the treatment methods described herein is produced by any of the methods described herein. [Brief explanation of the drawings]

[0050] [Figure 1]Figure 1 illustrates the rAAV.MHCK7.microdystrophin construct. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). This construct features an in-frame rod deletion (R4-R23) while retaining hinges 1, 2, and 4 (H1, H2, and H4) and the cysteine-rich domain, resulting in the production of a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) is driven by the MHCK7 promoter (795 bp). The intron and 5' UTR were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contained a consensus Kozak sequence immediately before the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contained the (R4-R23 / Δ71-78) domain as previously described by Harper et al. (Nature Medicine 8: 253-261 (2002)).

[0051] [Figure 2-1] FIG. 2 presents the nucleic acid sequence of AAVrh74.MHCK7.microdystrophin (SEQ ID NO: 3). [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above.

[0052] [Figure 3] Figure 3 presents the pNLREP2-Caprh74 AAV helper plasmid map.

[0053] [Figure 4] FIG. 4 presents the Ad helper plasmid pHELP.

[0054] [Figure 5] Figure 5 illustrates the rAAV.MCK.microdystrophin plasmid construct.

[0055] [Figure 6-1] FIG. 6 presents the nucleic acid sequence of rAAVrh74.MCK.microdystrophin (SEQ ID NO: 5). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0056] [Figure 7] FIG. 7 demonstrates micro-dystrophin gene expression in muscle fibers of gastrocnemius muscle biopsies as measured by immunocytochemistry.

[0057] [Figure 8A] Figures 8A-8C present Western blots demonstrating microdystrophin protein expression at the correct molecular weight. For Figures 8A and 8B, Western blot analysis detected microdystrophin protein expression in Subject 1 (5 years old), Subject 2 (4 years old), and Subject 3 (6 years old). For Figure 8C, Subject 4 samples (*) exceeded the ULDQ (>80%) in the initial analysis and were diluted 1:4 (linear range), and the mean values ​​were multiplied by a dilution correction factor to obtain the final values ​​compared to normal. Mean microdystrophin expression relative to normal was 182.7% for Method 1 and 222.0% for Method 2. [Figure 8B] Same as above. [Figure 8C] Same as above.

[0058] [Figure 9A] Figures 9A-9C demonstrate that administration of rAAVrh74.MHCK7.microdystrophin upregulates the expression of DAPC protein, alpha-sarcoglycan, and beta-sarcoglycan in subject 1 (Figure 9A), subject 2 (Figure 9B), and subject 3 (Figure 9C). [Figure 9B] Same as above. [Figure 9C] Same as above.

[0059] [Figure 10] FIG. 10 presents a graph showing the sustained dramatic reduction in creatine kinase (CK) levels associated with administration of rAAVrh74.MHCK7.microdystrophin.

[0060] [Figure 11] Figure 11 presents a graph showing the mean creatine kinase (CK) change from baseline to day 270. The data demonstrate that CK significantly decreased over time following administration of rAAVrh74.MHCK7.microdystrophin.

[0061] [Figure 12] Figure 12 presents graphs showing the mean NSAA change and mean CK change from baseline to day 270. The data demonstrate that NSAA significantly increased over time following administration of rAAVrh74.MHCK7.microdystrophin.

[0062] [Figure 13] Figure 13 presents the 4977 base nucleic acid sequence (SEQ ID NO: 9) of the AAVrh74.MHCK7.microdystrophin construct. The following molecular elements are shown: 5'ITR (bases 1-145); MHCK7 promoter (190-981 (792 bases)); intron (991-1140 (150 bases)); human microdystrophin sequence (1151-4729 (3579 bases)); polyA tail (4732-4784 (53 bases)); and 3'ITR (4833-4977 (145 bases)).

[0063] [Figure 14] Figure 14 illustrates the AAVrh74.MHCK7.microdystrophin plasmid construct.

[0064] [Figure 15-1]Figure 15 presents the nucleic acid sequence (SEQ ID NO: 8) of the AAVrh74.MHCK7.microdystrophin plasmid construct containing the kanamycin resistance gene. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above.

[0065] [Figure 16] FIG. 16 is a visual representation of the hybrid seed train expansion method described herein.

[0066] [Figure 17] Figure 17 is a visual representation of the production of AAV particles using the hybrid seed train expansion method described herein.

[0067] [Figure 18] Figures 18A-18B present graphs showing the viability of HEK-293 cells cultured according to the hybrid seed train expansion method described herein (Figure 18A) and the viability of HEK-293 cells cultured only under adherent conditions (Figure 18B).

[0068] [Figure 19] 19A-19B present graphs showing the viable cell density of HEK-293 cells cultured according to the hybrid seed train expansion method described herein (FIG. 19A) and HEK-293 cells cultured only under adherent conditions (FIG. 19B).

[0069] [Figure 20] Figure 20 is a graph showing the mean NSAA scores from Cohort 1 (first 11 patients treated with rAAVrh74.MHCK7.microdystrophin) described in Example 7. The first 11 patients improved by 3 points from baseline. The 6-7 year olds (n=9) improved by 2.9 points from baseline. Each time point represents 11 patients.

[0070] [Figure 21] Figures 21A-21C demonstrate microdystrophin expression (immunofluorescence) in skeletal and cardiac muscle of DMDmdx rats after 12 weeks (Figure 21B) and 24 weeks (Figure 21C) of treatment with delandistrogene moxeparvovec compared to saline (Figure 21A), as discussed in Example 11. Abbreviations: LTA = left tibialis anterior; HRT = heart.

[0071] [Figure 22] 22A-22B are bar graphs showing quantification of microdystrophin expression (immunofluorescence) ( FIG. 22A ) and vector transduction (vector genome copy number) ( FIG. 22B ) in muscle tissue of DMDmdx rats after 12 and 24 weeks of treatment with the delan dystrogen moxeparvovec, as discussed in Example 11. Abbreviations: TA = tibialis; HRT = heart; MG = medial head of gastrocnemius; LG = lateral head of gastrocnemius; DIA = diaphragm; TRI = triceps; PSO = psoas major.

[0072] [Figure 23] 23A-23B are bar graphs showing increased ambulation (FIG. 23A) and vertical activity (FIG. 23B) in DMDmdx rats after 12 and 24 weeks of treatment with the delanystrogen moxeparvovec compared to saline, as discussed in Example 11. Rats' movement (ambulation and vertical activity) in activity cages was measured by laser light beam interruptions per hour. Each point represents the value for an individual animal. Data are reported as mean ± SD; ***=p<0.001; **=p<0.01. SD=standard deviation.

[0073] [Figure 24A]Figures 24A-24B show significant reductions in muscle degeneration by central nucleation analysis in skeletal muscle after 12 and 24 weeks of delan-dystrogen moxeparvovec gene transfer compared to saline in DMDmdx rats, as discussed in Example 11. Figure 24A shows hematoxylin and eosin (H&E) staining of gastrocnemius muscle. Figure 24B is a bar graph showing the percentage of fibers with central nuclei. Bars are reported as mean ± SD; **** = p < 0.0001. SD = standard deviation. [Figure 24B] Same as above.

[0074] [Figure 25A] Figures 25A-25B show analysis of collagen deposition in skeletal and cardiac muscle demonstrating reduced fibrosis at 12 and 24 weeks after treatment with the delan dystrogen moxeparvovec compared to saline in DMDmdx rats, as discussed in Example 11. Figure 25A shows Masson's trichrome staining after 12 weeks of treatment. Figure 25B presents bar graphs quantifying collagen deposition in skeletal and cardiac muscle after 12 and 24 weeks of treatment. Abbreviations: HRT = heart; MG = medial head of gastrocnemius; DIA = diaphragm. Data are reported as mean ± SD; **** = p<0.0001; * = p<0.05. SD = standard deviation. [Figure 25B] Same as above.

[0075] [Figure 26] 26 is a bar graph showing that serum troponin I levels in blood do not change significantly after 1 week and 12 weeks of treatment with delanystrogen moxeparvovec compared to saline in DMDmdx rats, as discussed in Example 11. Bars represent the mean value + SD. Each point represents the value for an individual animal.

[0076] [Figure 27]

[0023] Figures 27A-27C are bar graphs analyzing cardiac function as determined by echocardiography after 24 weeks of treatment with delan-dystrogen moxeparvovec compared to saline in DMDmdx rats, as discussed in Example 11. Figure 27A shows data for left ventricular end-systolic dimension (LVESD). Figure 27B shows data for ejection fraction (%) (EF). Figure 27C shows data for fractional shortening (%) (FS).

[0077] [Figure 28] FIG. 28 shows histology images showing sarcolemmal localization of microdystrophin in DMDMDX rats after 12, 24, and 52 weeks of treatment with the delan dystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11.

[0078] [Figure 29] FIG. 29 is a bar graph showing the percentage of microdystrophin-positive fibers (PDPF) in DMDMDX rats treated with delan dystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11, after 12 and 24 weeks of treatment.

[0079] [Figure 30] FIG. 30 is a bar graph showing the distribution of transgene across skeletal and cardiac muscle and liver of DMDMDX rats treated with the delanystrogen moxeparvovec, as discussed in Example 11, after 12, 24, and 52 weeks of treatment.

[0080] [Figure 31A]Figure 31A is a Western blot showing microdystrophin expression in the heart muscle (HRT), triceps muscle (TRI), tibialis anterior muscle (TA), and gastrocnemius muscle (GAS) after 12, 24, and 52 weeks of treatment in DMDMDX rats following a single dose of the delan dystrogen moxeparvovec, as discussed in Example 11. Figure 31B is a bar graph showing quantification of microdystrophin expression in the gastrocnemius, tibialis anterior, and triceps muscles (skeletal muscle) (left) and heart muscle (right) after 12, 24, and 52 weeks of treatment in DMDMDX rats following a single dose of the delan dystrogen moxeparvovec, as discussed in Example 11. [Figure 31B] Same as above.

[0081] [Figure 32] FIG. 32 is a graph showing the survival probability of DMDMDX rats treated with delan dystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11.

[0082] [Figure 33] FIG. 33 shows H&E histology images showing the gastrocnemius muscle of DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11, after 12, 24, and 52 weeks of treatment.

[0083] [Figure 34] FIG. 34 is a bar graph showing percent CN (centrally nucleated) positive fibers in DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11, after 12, 24, and 52 weeks of treatment.

[0084] [Figure 35] FIG. 35 shows histology images showing skeletal and cardiac muscle stained for collagen in DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11, after 12, 24, and 52 weeks of treatment.

[0085] [Figure 36] FIG. 36 is a bar graph showing the percentage of fibrosis in skeletal and cardiac muscle of DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11, after 12, 24, and 52 weeks of treatment.

[0086] [Figure 37] FIG. 37 is a bar graph showing improvement in cardiac performance after 52 weeks of treatment in DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11.

[0087] [Figure 38] FIG. 38 is a graph showing recovery of cardiomyocyte contractility and Ca kinetics after 52 weeks of treatment in DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11.

[0088] [Figure 39] FIG. 39 is a bar graph showing horizontal activity, ambulation, in DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline after 12, 24, and 52 weeks of treatment, as discussed in Example 11.

[0089] [Figure 40] FIG. 40 is a bar graph showing serum troponin I levels after 12 and 52 weeks of treatment in DMDMDX rats treated with delanystrogen moxeparvovec compared to control DMDMDX rats treated with saline, as discussed in Example 11.

[0090] [Figure 41] Figures 41A-41B are Western blots showing the expression of microdystrophin and alpha-actinin in the tibialis anterior muscle, heart, and diaphragm of a non-human primate after a single dose of the delan dystrogen moxeparvovec, as discussed in Example 11. Figure 41A is a Western blot showing the expression of microdystrophin and alpha-actinin in the heart, diaphragm, and tibialis anterior muscle of a non-human primate after plasmapheresis and re-dosing with the delan dystrogen moxeparvovec, as discussed in Example 11.

[0091] [Figure 42A] Figure 42A is a phase contrast microscopy image showing human cardiomyocytes in vitro from day 1 to day 5 (when the cardiomyocytes were transduced with the AAVrh74-MHCK7-GFP vector) and after day 9 (fixation day), as discussed in Example 11. Figure 42B is an immunofluorescence image showing human cardiomyocytes (day 9) (4 days after transduction) transduced with the AAVrh74-MHCK7-GFP vector in vitro, as discussed in Example 11. [Figure 42B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0092] Detailed Description The present disclosure provides a gene therapy vector, e.g., rAAV, that expresses human microdystrophin, wherein the rAAV is produced in mammalian adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container. The present disclosure also provides a composition (e.g., a pharmaceutical composition) comprising the rAAV described herein, as well as a method for treating muscular dystrophy (e.g., DMD) using the composition described herein. The present disclosure also provides genotyping of a subject's DMD gene before administration of rAAV to identify DMD gene mutations that are suitable for AAV gene therapy and DMD gene mutations that may contraindicate AAV gene therapy (e.g., due to an increased risk of severe immune responses). The present disclosure provides a method for treating DMD when treated with the rAAV described herein. MDX It also provides successful improvement of skeletal and heart muscle function in a rat model.

[0093] Muscle biopsies obtained at the earliest age at diagnosis of DMD reveal significant connective tissue proliferation. Muscle fibrosis is detrimental in multiple ways. It reduces the normal transfer of endomysial nutrients across the connective tissue barrier, reducing blood flow and starving the muscle of angiogenic nutrients, contributing to early loss of ambulation due to limb contractures. Over time, significant fibrosis in the muscle results in increased treatment challenges. This can be observed in muscle biopsies comparing connective tissue proliferation at successive time points. The process continues to worsen, leading to loss of ambulation and uncontrollable acceleration, especially in wheelchair-dependent patients.

[0094] Without early intervention, including parallel approaches to reduce fibrosis, the full benefits of exon skipping, stop codon readthrough, or gene replacement therapy are unlikely to be realized. Furthermore, small molecule or protein replacement strategies are likely to fail without approaches to reduce muscle fibrosis. Previous studies in aged mdx mice with pre-existing fibrosis have shown that treatment with AAV.microdystrophin failed to achieve sufficient functional repair (Liu, M. et al., Mol Ther 11: 245-256 (2005)). Progression of DMD cardiomyopathy is also known to be accompanied by scarring and fibrosis in the ventricular wall. definition

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0096] Throughout this disclosure, the terms "a" or "an" entity refer to one or more of that entity. For example, "a polynucleotide" is understood to represent one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0097] Furthermore, "and / or," when used herein, should be construed as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term "and / or," when used herein in a phrase such as "A and / or B," is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," when used in a phrase such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0098] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above or below (greater than or less than) the stated value by a difference of 10 percent, unless otherwise specified.

[0099] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that may be logically included as is apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" appears before a series of numbers or range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18%, regardless of the number of significant digits).

[0100] Nucleotide sequences are presented herein as single strands only, left to right, in 5' to 3' orientation, unless otherwise indicated. Nucleotides and amino acids are represented herein by the one-letter or three-letter code in the format recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) in accordance with 37 CFR § 1.822 and established usage.

[0101] "Polynucleotide" or "nucleic acid," as used herein, refers to a sequence of nucleotides joined by phosphodiester linkages. Polynucleotides are presented herein in a 5' to 3' orientation. Polynucleotides of the present disclosure can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are designated herein by the single-letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0102] As used herein, the term "polypeptide" encompasses both peptides and proteins, unless otherwise specified.

[0103] The terms "coding sequence" or "encoding" sequence are used herein to refer to a DNA or RNA region (transcribed region) that "encodes" a particular protein, e.g., insulin or glucokinase. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide when placed under the control of appropriate regulatory regions, such as a promoter, in vitro or in vivo. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic sources, genomic DNA from prokaryotic or eukaryotic sources, and synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0104] A gene may include several operably linked fragments, such as a promoter, a 5' leader sequence, introns, a coding sequence, and a 3' nontranslated sequence containing, for example, a polyadenylation site or a signal sequence. As used herein, "expression of a gene" refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0105] As used herein, the term "promoter" refers to a nucleic acid sequence or fragment that functions to control the transcription of one or more genes (or coding sequences) located upstream in the direction of transcription of the gene's transcription start site and is structurally identified by the presence of binding sites for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequence, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated in response to physiological or developmental conditions. A "tissue-specific" promoter is preferentially active in specific types of differentiated cells / tissues.

[0106] As used herein, the term "enhancer" refers to a cis-acting element that stimulates or inhibits the transcription of adjacent genes. Enhancers that inhibit transcription are also called "silencers." Enhancers can function in either direction (e.g., they can be associated with coding sequences) over distances of up to several kilobase pairs (kb) from the coding sequence and downstream of the transcribed region.

[0107] The term "operably linked" refers to the positioning of a nucleotide sequence of a regulatory element, such as a nucleotide sequence of a promoter, such that the regulatory element effects the expression of the nucleotide sequence.

[0108] As used herein, the term "transgene" refers to a gene (e.g., microdystrophin) or nucleic acid molecule that is introduced into a cell. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide. In some embodiments, a gene may be present in a cell, but in some cases, the gene is not normally expressed in the cell or is expressed at an insufficient level. In this context, "insufficient" means that the gene is normally expressed in the cell, but a condition and / or disease may still occur. In certain embodiments, a transgene allows for increased expression or overexpression of a gene. A transgene may include a sequence native to the cell, a sequence that does not naturally occur in the cell, or a combination of both. In certain embodiments, a transgene may include a sequence that may be operably linked to suitable regulatory sequences for expression of the gene. In some embodiments, a transgene is not integrated into the genome of a host cell.

[0109] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 characterized AAV serotypes. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that the various serotypes are closely related, both structurally and functionally, even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes (see, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization along the length of the genome between serotypes; and the presence of similar self-annealing segments at the termini, corresponding to "inverted terminal repeats" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0110] As used herein, the term "adeno-associated vector" or "AAV vector" refers to a vector containing one or more polynucleotides of interest (or "transgenes," such as microdystrophin) flanked by AAV interterminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with the vector, which encodes and expresses the rep and cap gene products.

[0111] As used herein, the term " AAV virion ", " AAV virus particle " or " AAV vector particle " refers to a virus particle that is composed of at least one AAV capsid protein and an AAV vector that contains a polynucleotide that is enclosed in the capsid.When particle contains heterologous polynucleotide (i.e., a polynucleotide other than wild-type AAV genome, such as a transgene that is delivered to mammalian cells), it is typically referred to as " AAV vector particle ", or in some cases, simply as " AAV vector ".Therefore, the production of AAV vector particle necessarily includes the production of AAV vector.Therefore, vector is contained in AAV vector particle.

[0112] As used herein, the term "expression cassette" refers to any type of genetic construct containing a nucleic acid from which part or all of a nucleic acid coding sequence can be transcribed. Typically, an expression cassette includes a promoter operably linked to a nucleic acid (e.g., a transgene of interest). In some aspects, an "expression cassette" includes a polynucleotide sequence encoding human microdystrophin.

[0113] The term "muscle-specific control element" refers to a nucleotide sequence that regulates the expression of a coding sequence that is specific for expression in muscle tissue. These control elements include enhancers and promoters. The present disclosure provides constructs containing the muscle-specific control elements MCKH7 promoter, MCK promoter, and MCK enhancer.

[0114] "Muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, e.g., gastrointestinal, bladder, vascular, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiac myocytes, and cardiomyoblasts.

[0115] As used herein, the term "transduction" refers to the administration / delivery of the coding region for microdystrophin to a recipient cell either in vivo or in vitro by a replication-deficient rAAV of the present disclosure, thereby resulting in the expression of microdystrophin by the recipient cell.

[0116] As used herein, the term "transfection" of a cell means introducing genetic material into a cell in order to genetically modify the cell. Transfection can be achieved by various means known in the art, for example, by transduction or electroporation.

[0117] As used herein, a "vector" refers to a recombinant plasmid or virus containing a polynucleotide that is delivered to a host cell either in vitro or in vivo. "Recombinant" generally means separate from that found in nature.

[0118] "Serotypes," with respect to vector or viral capsids, are defined by distinct immunological profiles based on capsid protein sequence and capsid structure.

[0119] "AAV Cap" refers to the AAV Cap proteins, VP1, VP2, and VP3 and analogs thereof.

[0120] "AAV Rep" refers to the AAV Rep protein and analogs thereof.

[0121] As used herein, "adjacent," with respect to a sequence flanked by other elements, indicates that one or more adjacent elements are located upstream and / or downstream, i.e., 5' and / or 3', relative to the sequence. The term "adjacent" does not indicate that the sequences are necessarily contiguous. For example, there may be an intervening sequence between the nucleic acid encoding the transgene and the adjacent element. A sequence (e.g., a transgene) "adjacent" to two other elements (e.g., ITRs) indicates that one element is located 5' and the other is located 3' relative to the sequence, although there may be an intervening sequence between them.

[0122] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a nucleic acid comprising a promoter operably linked to a transgene, e.g., a polynucleotide encoding micro-dystrophin) into an individual's cells and / or tissues to treat a disease or condition. Such a transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that is not normally present in the cells, tissues, and / or individual being treated.

[0123] As used herein, the term "genotyping" refers to the process of determining the specific allele composition of a cell and / or subject at one or more positions in the genome, for example, by determining the nucleic acid sequence at that position.Genotyping refers to nucleic acid analysis and / or analysis at the nucleic acid level.Many genotyping techniques are known to those skilled in the art.

[0124] In some embodiments, the subject's human dystrophin gene (DMD) is genotyped to characterize mutations in the gene that may be particularly suitable for treatment with the compositions described herein.

[0125] In some embodiments, the subject's DMD gene is genotyped to characterize mutations in the DMD gene that would contraindicate treatment with the compositions described herein.

[0126] The term "stringent" is used to refer to conditions that are generally understood in the art to be stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ndSee, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). More stringent conditions (e.g., higher temperature, lower ionic strength, higher formamide, or other denaturing agents) can also be used, but will affect the hybridization rate. For deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos), 6xSSC, and washing with 0.05% sodium pyrophosphate. Other agents can be included in the hybridization and washing buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4 (SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents may be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4, and under typical ionic strength conditions, the hybridization rate is largely pH-dependent. See Anderson, MLM et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England) (1998). Those skilled in the art can adapt these variables and adjust hybridization conditions to allow DNAs with different sequence relatedness to form hybrids.

[0127] As used herein, the terms "media," "medium," "cell culture medium," "culture medium," "tissue culture medium," "tissue culture media," and "growth medium" refer to a solution containing nutrients that support the growth of eukaryotic cells in culture. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. Solutions may also contain components that enhance growth and / or survival beyond the minimal rate, including hormones and growth factors. Solutions are formulated to an optimal pH and salt concentration for cell survival and proliferation. Media may also be "defined media" or "synthetic media," which are serum-free media that contain no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components, and all components have known chemical structures. Those skilled in the art will understand that defined media may contain recombinant glycoproteins or proteins, such as, but not limited to, hormones, cytokines, interleukins, and other signaling molecules.

[0128] As used herein, the term "basal medium formulation" or "basal medium" refers to any cell culture medium used to culture cells that has not been modified by either supplementation or selective removal of certain components.

[0129] As used herein, the terms "culture," "cell culture," and "eukaryotic cell culture" refer to a population of eukaryotic cells, attached to a surface (i.e., adherent) or in suspension, maintained or grown in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, these terms, as used herein, can refer to a combination comprising a mammalian cell population and the medium in which the population is suspended.

[0130] As used herein, the term "batch culture" refers to a method of culturing cells in which all of the components that will ultimately be used to culture the cells, including the medium and the cells themselves, are provided at the beginning of the culture process. Batch cultures are typically stopped at some point, and the cells and / or components in the medium are harvested and optionally purified.

[0131] As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture at some time after the start of the culture process. Fed-batch culture can be initiated using a basal medium. A culture medium in which additional components are provided to the culture at some time after the start of the culture process is a fed-batch medium. The provided components typically include nutrient supplements for the cells that are depleted during the culture process. Fed-batch culture is typically stopped at some time, and the cells and / or components in the medium are harvested and, if necessary, purified.

[0132] As used herein, the term "perfusion culture" refers to a method of culturing cells in which additional components are continuously or semi-continuously provided to the culture after the start of the culture process. The provided components typically include nutrient supplements to the cells that are depleted during the culture process. Typically, a portion of the cells and / or components in the medium are continuously or semi-continuously harvested and optionally purified.

[0133] The "growth phase" of a cell culture refers to the period of exponential cell growth (log phase) during which cells generally divide rapidly. During this phase, cells are cultured for a period of time, typically 1-4 days, under conditions that maximize cell growth. The determination of the host cell growth cycle can be determined without undue experimentation for a particular host cell under consideration. "For a period of time and under conditions that maximize cell growth" and the like refer to culture conditions determined to be optimal for cell growth and division for a particular cell line. In some embodiments, during the growth phase, cells are cultured in a nutrient medium containing necessary additives to achieve optimal growth for the particular cell line, typically at about 25°C to 40°C, in a humidified, controlled atmosphere.

[0134] In some embodiments, cells are maintained in the growth phase for a period of about 1 to 7 days, e.g., 2 to 6 days, e.g., 6 days. The length of the growth phase for a particular cell can be determined without undue experimentation. For example, the length of the growth phase is a period sufficient for the particular cells to reproduce to a viable cell density within about 20% to 80% of the maximum viable cell density possible when the culture is maintained under growth conditions. In some embodiments, "maximum growth rate" refers to the growth rate of a particular cell line / clone measured during its exponential growth phase but when the cells are in fresh culture medium (e.g., measured at a point in the culture when nutrients are sufficient and there is no significant growth inhibition from any component of the culture).

[0135] As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term as used herein also refers to the proportion of surviving cells at a particular time point relative to the total number of live and dead cells in the culture at that time point.

[0136] As used herein, the term "cell density" refers to the number of cells present in a given volume of medium.

[0137] As used herein, the term "bioreactor" or "culture vessel" refers to any vessel used for the growth of mammalian cell cultures. Bioreactors can be of any size so long as they are useful for culturing mammalian cells.

[0138] As used herein, the term "bioreactor operation" can include one or more of a lag phase, log phase, or stationary phase growth period during a cell culture cycle.

[0139] As used herein, the terms "N-1 culture vessel," "N-1 seed train culture vessel," "N-1 vessel," "N1 culture," or "N1 container" refer to the culture vessel immediately preceding the N culture vessel (production culture vessel) and are used to grow cell cultures to high viable cell densities for inoculation into the subsequent N (production) culture vessel. Cell cultures grown in N-1 culture vessels can be obtained after cells are cultured in several vessels prior to the N-1 culture vessel, e.g., N-4, N-3, and N-2 vessels.

[0140] As used herein, the terms "N culture vessel," "production culture vessel," "N vessel," "N bioreactor," or "production bioreactor" refer to the cell culture in the bioreactor after the N-1 bioreactor. The N culture is used for the production of AAV.

[0141] As used herein, the terms "seeding" or "inoculating" refer to the process of providing a cell culture to a bioreactor or another vessel. In one aspect, the cells have been previously expanded in another bioreactor or vessel. In another aspect, the cells have been frozen and are thawed immediately prior to providing to the bioreactor or vessel. The terms refer to any number of cells, including a single cell. rAAV and methods for producing rAAV (or compositions comprising rAAV) (e.g., rAAVrh74.MHCK7.microdystrophin)

[0142] The present disclosure provides a composition comprising a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the rAAV is produced in adherent mammalian cells and the adherent cells are cultured in an N-1 container under suspension conditions. In some embodiments, the rAAV is of the AAVrh.74 serotype (e.g., rAAV.MHCK7.microdystrophin).

[0143] The present disclosure also provides a method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin in adherent mammalian cells by a suspension seeding process, the method comprising: (a) culturing the cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container that is serum-free or contains serum at a lower concentration than the first medium; (d) culturing the cells in the N-1 container under suspension conditions; and (e) inoculating a third medium in a bioreactor with the cells from step (d).

[0144] In some embodiments, the rAAV used in the methods described herein comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1. In some embodiments, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 7. In some embodiments, the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 7.

[0145] In some aspects, the suspension seeding process further comprises (f) transfecting the adherent cells with a transgene plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct, a plasmid comprising the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

[0146] In some embodiments, the transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct comprises the nucleic acid sequence of SEQ ID NO:9; nucleotides 55-5021 of SEQ ID NO:3; or nucleotides 1-4977 of SEQ ID NO:8. In some embodiments, the plasmid containing the AAV rep gene and AAV cap gene comprises the AAV2 rep gene and the rAAVrh74 cap gene. In some embodiments, the adenovirus helper plasmid comprises the adenovirus 5 E2A, E4ORF6, and VA RNA genes.

[0147] In some embodiments, the suspension seeding process further comprises (g) lysing the adherent cells, hi some embodiments, the adherent cells are lysed by freeze-thawing, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

[0148] In some aspects, the floating seed process further comprises purifying the (h)rAAV by at least one column chromatography step, hi some aspects, the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0149] In some embodiments, the suspension seed process further comprises culturing the cells in a first growth medium in an N-3 container. In some embodiments, the suspension seed process further comprises culturing the cells in a first growth medium in an N-4 container.

[0150] In some embodiments, the bioreactor is an adherent bioreactor. In some embodiments, the rAAV is purified from the culture produced in the adherent bioreactor.

[0151] In some embodiments, the third culture medium in the bioreactor comprises at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third culture medium in the bioreactor comprises DMEM and 10% FBS.

[0152] In some embodiments, the adherent cells are cultured in suspension for about 48 to 72 hours.

[0153] In some embodiments, the N-1 container is a suspension shake flask.

[0154] In some embodiments, the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells. In some embodiments, the adherent cells are HeLa cells or HEK-293 cells. In some embodiments, the adherent cells are HEK-293 cells. In some embodiments, the adherent cells are not adapted to a suspension system. In some embodiments, culturing the cells in suspension does not alter the adhesion dependency of the cells. In some embodiments, culturing does not alter the cells to create a new cell line.

[0155] In some aspects, the suspension seed process used to produce rAAVrh74.MHCK7.micro-dystrophin in adherent mammalian cells includes: (a) culturing the cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing serum at a lower concentration than the first medium; (d) culturing the cells in the N-1 container under suspension conditions; (e) inoculating the cells from step (d) into a third medium in a bioreactor; (f) transfecting the cells with a transgene plasmid containing the rAAVrh74.MHCK7.micro-dystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid; (g) lysing the cells; and (h) purifying the rAAV by at least one column chromatography step.

[0156] In some aspects, rAAV is described in International Publication No. WO2019 / 245973A1, which is expressly incorporated by reference in its entirety.

[0157] Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of about 4.7 kb in length, containing 145 nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol. 45: 555-564 (1983), revised by Ruffing et al., J Gen Virol. 75: 3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided under GenBank accession number NC_002077, the complete genome of AAV-3 is provided under GenBank accession number NC_1829, the complete genome of AAV-4 is provided under GenBank accession number NC_001829, the AAV-5 genome is provided under GenBank accession number AF085716, the complete genome of AAV-6 is provided under GenBank accession number NC_001862, at least portions of the AAV-7 genome and the AAV-8 genome are provided under GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 regarding AAV-8), the AAV-9 genome is provided in Gao et al., J. Virol. 78: 6381-6388 (2004), and the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac et al., Journal of Translational Medicine 5: 45 (2007).

[0158] Any AAV serotype can be used in accordance with the present disclosure. In some embodiments, the term "AAV" as used herein includes, but is not limited to, AAV1, AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAVrh74, the AAV serotypes and clades disclosed by Gao et al., J. Virol. 78: 6381 (2004)) and Morris et al., Virol. 33: 375 (2004), as well as any other AAV now known or later discovered. See, for example, Knipe et al. (ed.), Fields Virology, vol. 2, chapter 69 (4th ed., Lippincott Williams & Wilkins Publishers) (2001).

[0159] In some embodiments, an "AAV viral particle" is an AAV viral particle of any of the AAV serotypes listed above.

[0160] In some embodiments, the AAV viral particles are AAV viral particles of serotype rh74.

[0161] The ITRs contain cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and integration into host cell chromosomes. Three AAV promoters (designated p5, p19, and p40 based on their relative mapping locations) drive expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), produce four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. The production of the three related capsid proteins involves alternative splicing and non-consensus translation start sites. A single consensus polyadenylation site is located in the AAV genome at map position 95. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology 158: 97-129 (1992).

[0162] AAV has 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 not cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cell types, thus offering the potential for targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially as transcriptionally active nuclear episomes (extrachromosomal elements) throughout the life of those cells. The AAV proviral genome is infectious as cloned DNA in a plasmid, enabling the construction of recombinant genomes. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can 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 can be provided in trans. Another important feature of AAV is that it is a very stable and viable virus. AAV readily resists the conditions used to inactivate adenovirus (56°C to 65°C for several hours), and therefore cryopreservation is less important for AAV. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0163] Several studies have demonstrated long-term (more than 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. USA 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). Furthermore, as described by Herzog et al., Proc Natl Acad Sci USA 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA 94: 13921-13926 (1997), muscle is highly vascularized, and recombinant AAV transduction results in the appearance of the transgene product in the systemic circulation after intramuscular injection. Furthermore, Lewis et al., J Virol. 76: 8769-8775 (2002) demonstrated that skeletal muscle fibers (myofibers) possess the cellular factors necessary for correct antibody glycosylation, folding, and secretion, thereby indicating that muscle is capable of stably expressing secreted protein therapeutics.

[0164] The recombinant AAV genome of the present disclosure comprises the nucleic acid molecule of the present disclosure and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype that can derive a recombinant virus, including, but not limited to, AAV serotypes AAVrh74, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy 22 (11): 1900-1909 (2014). As noted above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle-specific expression, AAV1, AAV6, AAV8, or AAVrh74 can be used.

[0165] The DNA plasmid of the present disclosure contains the rAAV genome of the present disclosure. The DNA plasmid is transferred into cells permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles that provide cells with the packaged AAV genome, rep and cap genes, and helper virus functions are standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not within the rAAV genome), and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype capable of eliciting recombinant virus, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13, and may be derived from an AAV serotype different from the rAAV genome ITRs. The production of pseudotyped rAAVs is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0166] A method for generating packaging cells is to create a cell line that stably expresses all of the components necessary for AAV particle production, for example, by integrating into the cell's genome a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., Proc. Natl. Acad. S6. USA 79: 2077-2081 (1982)), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., Gene 23: 65-73 (1983)), or by direct blunt-end ligation (Senapathy & Carter, J. Biol. Chem. 259: 4661-4666 (1984)). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. In another example of a suitable method, adenovirus or baculovirus, rather than a plasmid, is used to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0167] The general principles of rAAV production are reviewed, for example, in Carter, Current Opinions in Biotechnology 1533-539 (1992); and Muzyczka, N., Curr. Topics Microbial. Immunol. 158: 97-129 (1992). Various techniques have been used. Ratschin et al., Mol. Cell. Biol. 4: 2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. 62: 1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7: 349 (1988), Samulski et al., J. Virol., 63: 3822-3828 (1989); U.S. Patent No. 5,173,414; WO95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO95 / 13392; WO96 / 17947; PCT / US98 / 18600; WO97 / 09441 (PCT / US96 / 14423); WO97 / 08298 (PCT / US96 / 13872); WO97 / 21825 (PCT / US96 / 20777); WO97 / 06243 (PCT / FR96 / 01064); WO99 / 11764; Perrin et al. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4: 609-615 (1993); Clark et al. Gene Therapy 3: 1124-1132 (1996); U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entireties, with particular emphasis being placed on the sections thereof relating to rAAV production.

[0168] Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (an allied 293 lineage). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (embryonic rhesus lung cells).

[0169] The recombinant AAV (i.e., infectious capsid-enclosed rAAV particle) of the present disclosure comprises an rAAV genome. In an exemplary embodiment, the genomes of both rAAVs lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAVs that can be constructed to contain the nucleic acid molecules of the present disclosure are described in International Patent Application No. PCT / US2012 / 047999 (WO2013 / 016352), the entire contents of which are incorporated herein by reference.

[0170] In an exemplary embodiment, the recombinant AAV vector of the present invention is produced by triple transfection using the AAV vector plasmids rAAV.MHCK7.microdystrophin, pNLRep2-Caprh74, and pHELP (Xiao et al., J Virol 72: 2224-2232 (1998)). The rAAV contains a microdystrophin gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs). This sequence is encapsidated into the AAVrh74 virion. The plasmid contains the microdystrophin sequence and the MHCK7 enhancer and core promoter elements of the muscle-specific promoter to drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) to promote high-level gene expression and uses the bovine growth hormone polyadenylation signal for efficient transcription termination.

[0171] pNLREP2-Caprh74 is an AAV helper plasmid encoding four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from serotype rh74. A schematic map of the pNLREP2-Caprh74 plasmid is shown in Figure 3.

[0172] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. The plasmid contains regions of the adenovirus genome important for AAV replication, namely, E2A, E4 ORF6, and VA RNA (the adenovirus E1 function is provided by 293 cells). The adenovirus sequences present in this plasmid represent only approximately 40% of the adenovirus genome and do not contain cis elements critical for replication, such as the adenovirus terminal repeats. Therefore, infectious adenovirus is not expected to be generated from such a production system. A schematic map of the pHELP plasmid is shown in Figure 4.

[0173] The rAAV described herein can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther. 10 (6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med. 69 427-443 (2002); U.S. Patent No. 6,566,118 and WO98 / 09657.

[0174] In one aspect, the present disclosure provides an rAAV comprising a muscle-specific regulatory element nucleotide sequence and a nucleotide sequence encoding a micro-dystrophin protein.For example, the nucleotide sequence encodes a functional micro-dystrophin protein, where the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, and the protein retains micro-dystrophin activity.The micro-dystrophin protein provides stability to muscle membranes during muscle contraction, for example, micro-dystrophin acts as a shock absorber during muscle contraction.

[0175] In one embodiment, the rAAV is in the form of a viral particle, rAAVrh74.MHCK7.microdystrophin, i.e., a nucleic acid expression cassette or genome containing a microdystrophin transgene driven by an MHCK7 promoter / enhancer, enclosed in an rAAV serotype rh74 capsid, and is also referred to by the generic drug name delan dystrogen moxeparvovec in the context of administration to a subject. In some embodiments, when the examples refer to test subjects administered delan dystrogen moxeparvovec, the data (e.g., bar graphs) may be more succinctly designated as "treatment."

[0176] In one embodiment, the rAAVrh74.MHCK7.microdystrophin is the rAAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, or the rAAVrh74.MHCK7.microdystrophin of nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, wherein the rAAV is produced in adherent cells and the adherent cells are cultured in an N-1 container under suspension conditions. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the rAAVrh74.MCK.microdystrophin is the rAAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0177] The present disclosure also provides an rAAV, wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 1 or its complement, and encodes a functional micro-dystrophin protein.

[0178] In one embodiment, the rAAV is a non-replicating recombinant adeno-associated virus (AAV) designated rAAVrh74.MHCK7.microdystrophin, which is SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, where the rAAV is produced in adherent cells, which are cultured in suspension in an N-1 container. This vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeats (ITRs), microdystrophin, an SV40 intron (SD / SA), and a synthetic polyadenylation (polyA) signal, all under the control of the MHCK7 promoter / enhancer. A schematic diagram of the vector genome and expression cassette is shown in Figure 1. The AAVrh74 serotype can be used to achieve efficient gene transfer to skeletal and cardiac muscle following IV administration.

[0179] In one aspect, the disclosure provides an rAAV wherein the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF), a muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), a hybrid alpha-myosin heavy chain enhancer / MCK enhancer-promoter (MHCK7), C5-12, a mouse creatine kinase enhancer element, a skeletal fast-twitch troponin c gene element, a slow-twitch cardiac troponin c gene element, a slow-twitch troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0180] For example, the muscle-specific control element is the MHCK7 promoter nucleotide sequence SEQ ID NO:2 or SEQ ID NO:7, or the muscle-specific control element is the MCK nucleotide sequence SEQ ID NO:4. Furthermore, with respect to any of the rAAV vectors of the present disclosure, the muscle-specific control element nucleotide sequence, e.g., the MHCK7 or MCK nucleotide sequence, is operably linked to a nucleotide sequence encoding a microdystrophin protein. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO:2 or SEQ ID NO:7) is operably linked to the human microdystrophin coding sequence (SEQ ID NO:1) set forth in the construct presented in Figure 1 or Figure 2 (SEQ ID NO:3) or in Figure 13 (SEQ ID NO:9). In another example, the MCK promoter (SEQ ID NO:4) is operably linked to the human microdystrophin coding sequence (SEQ ID NO:1) set forth in the construct presented in Figure 5 or Figure 6 (SEQ ID NO:5). In another aspect, the present disclosure provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:1 and SEQ ID NO:7. The present disclosure also provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:4.

[0181] In a further aspect, the disclosure provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8. For example, the AAVrh74.MHCK7.microdystrophin vector comprises the nucleotide sequence within, including the ITRs of SEQ ID NO:3, as shown in Figure 2. In another aspect, the rAAV vector comprises a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, a coding sequence for the human microdystrophin gene, a polyA, and a 3' ITR. In one aspect, the vector comprises nucleotides 55-5021 of SEQ ID NO:3. The plasmid set forth in SEQ ID NO:3 further comprises a pGEX plasmid backbone with ampicillin resistance and a pBR322 origin of replication.

[0182] In another aspect, the present disclosure provides an rAAV comprising the nucleotide sequence of SEQ ID NO:9, wherein the rAAV is produced in adherent cells and the adherent cells are cultured under suspension conditions in an N-1 container. For example, the AAVrh74.MHCK7.microdystrophin vector construct comprises the nucleotide sequence of SEQ ID NO:9 shown in Figure 13. This rAAV vector construct comprises an MHCK7 promoter, a chimeric intron sequence, a coding sequence for the human microdystrophin gene, and a polyA. In one aspect, the rAAV vector construct further comprises an ITR 5' to the promoter and an ITR 3' to the polyA. In one aspect, the rAAV is AAVrh74.

[0183] In another embodiment, the rAAVrh74.MHCK7.microdystrophin vector (i.e., viral vector) comprises the nucleotide sequence within, and including the ITRs of SEQ ID NO:8, as shown in Figure 15. The rAAV vector comprises the 5' ITR, the MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, polyA, and the 3' ITR. In one embodiment, the vector comprises nucleotides 1-4977 of SEQ ID NO:9. The plasmid set forth in SEQ ID NO:3 further comprises a pGEX plasmid backbone with kanamycin resistance and a pBR322 origin of replication.

[0184] In another aspect, the present disclosure provides a plasmid comprising the AAVrh74.MHCK7.microdystrophin construct. In one aspect, the plasmid comprises a 5'ITR, an MHCK7 promoter, a chimeric intron sequence, a coding sequence of the human microdystrophin gene, polyA, and a 3'ITR. In one aspect, the plasmid comprises a kanamycin resistance and, optionally, a pGEX plasmid backbone with a pBR322 origin of replication. In a specific aspect, the plasmid is set forth in SEQ ID NO:8 and is depicted in Figures 14 and 15.

[0185] The present disclosure provides a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7, wherein the rAAV is produced in adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container. The rAAV vector is of the AAV serotype AAVrh.74.

[0186] The present disclosure also provides an rAAV comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence within and including the ITRs of SEQ ID NO:3, the nucleotide sequence within and including the ITRs of SEQ ID NO:8, or the nucleotide sequence set forth in SEQ ID NO:9, wherein the rAAV is produced in adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container. This rAAV vector is of the AAV serotype AAVrh.74.

[0187] The rAAV vectors of the present disclosure can be of any AAV serotype, such as serotype AAVrh74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0188] The present disclosure also provides pharmaceutical compositions (or sometimes simply referred to herein as "compositions") comprising any of the rAAV vectors of the present disclosure.

[0189] In another aspect, the present disclosure provides a method for producing rAAV vector particles, the method comprising culturing cells transfected with any of the rAAV vectors of the present disclosure and recovering the rAAV particles from the supernatant of the transfected cells. The present disclosure also provides viral particles comprising any of the recombinant AAV vectors of the present disclosure. Compositions Comprising rAAV and Their Administration

[0190] In another aspect, the present disclosure provides a composition comprising the rAAV of the present disclosure. The composition of the present disclosure comprises the rAAV and a pharmaceutically acceptable carrier. The composition may also contain other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients, preferably inert at the dosages and concentrations used, and include buffers and surfactants such as Pluronic®.

[0191] The present disclosure provides a composition for treating muscular dystrophy (e.g., DMD) in a subject in need thereof, the composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is produced in adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container.

[0192] In some embodiments, the present disclosure provides a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is produced in adherent mammalian cells by the suspension seed process described herein. In some embodiments, the composition comprises: a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8.

[0193] The titer of the rAAV administered in the methods of the present disclosure varies depending, for example, on the particular rAAV, the mode of administration, the goal of treatment, the individual, and the targeted cell type(s), and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per ml. 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 From about 1 x 10 14 The dosage may range from 100 or more DNase-resistant particles (DRP). The dosage may also be expressed in units of viral genomes (vg). One exemplary method for determining the encapsulated vector genome titer uses quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25: 855-869(2017)).

[0194] Methods for transducing target cells with rAAV in vivo or in vitro are contemplated in the present disclosure. In vivo methods include administering an effective dose or effective multiple doses of a composition comprising the rAAV of the present disclosure to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present disclosure, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / condition being treated, slows or prevents progression to the disorder / condition, slows or prevents progression of the disorder / condition, reduces the extent of the disease, causes remission (partial or complete remission) of the disease, and / or prolongs survival. An example of a disease contemplated for prevention or treatment using the methods of the present disclosure is DMD.

[0195] Combination therapy is also contemplated by the present disclosure. "Combination," as used herein, includes both simultaneous and sequential treatment. Combination of the methods of the present disclosure with standard medical treatments (e.g., corticosteroids) is specifically contemplated, as is combination with novel therapies.

[0196] Administration of an effective dose of the composition may be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route(s) of administration and the serotype(s) of the AAV components of the rAAV (particularly the AAV ITRs and capsid proteins) of the present disclosure can be selected and / or coordinated by one skilled in the art by taking into consideration the infection and / or condition to be treated and the target cell / tissue(s) expressing the micro-dystrophin protein.

[0197] The present disclosure provides for local administration and systemic administration of effective doses of the rAAV and compositions of the present disclosure.For example, systemic administration is administration to the circulatory system, so that the whole body is affected.Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration through injection, infusion or implantation.

[0198] In particular, the actual administration of the rAAV of the present disclosure can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present disclosure includes, but is not limited to, intramuscular injection and injection into the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations regarding carriers or other components that can be co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner of using rAAV). The capsid protein of rAAV can be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or topical formulations delivered to muscle via transdermal delivery. Numerous formulations have previously been developed for both intramuscular injection and transdermal delivery and can be used in the practice of the present disclosure. For ease of administration and handling, the rAAV can be used with any pharmaceutically acceptable carrier.

[0199] In one embodiment of the disclosure, AAVrh74.MHCK7.microdystrophin described herein is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0200] The dose of rAAV administered in the methods described herein varies depending, for example, on the particular rAAV, the mode of administration, the goal of treatment, the individual, and the targeted cell type(s), and can be determined by standard methods in the art. The titer of each rAAV administered is approximately 1 x 10 per ml. 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, about 1×10 11pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, approximately 2×10 14 From about 1 x 10 15 Dosages can range from 1 x 10 or more DNase-resistant particles (DRP). Dosages can also be expressed in units of viral genomes (vg) (e.g., 1 x 10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 2 × 10 14 vg, 4×10 14 vg, 6×10 14 vg, 8×10 14 vg, 1×10 15 vg, 2 × 10 15 vg, 3 × 10 15 vg, 4×10 15 vg, 5 × 10 15 vg, 6×10 15 vg, 7×10 15 vg, 8×10 15 vg, 9×10 15 vg, 1×10 16 Dosage can also be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1 x 10 10 vg / kg, 1 × 10 11 vg / kg, 1 × 10 12 vg / kg, 1 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.25 × 10 14 vg / kg, 1.33 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.75 × 10 14 vg / kg, 2.0 × 10 14 vg / kg, 2.25 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.75 × 10 14 vg / kg, 3.0 × 1014 vg / kg, 3.25 × 10 14 vg / kg, 3.5 × 10 14 vg / kg, 3.75 × 10 14 vg / kg, 4.0 × 10 14 vg / kg, 1 × 10 15 Dosages can also be expressed as a fixed total dose (e.g., 9.31 x 10 15 vg). A "fixed total dose" is a dose administered to a subject weighing at or above a certain weight, where the dose is not adjusted for the subject's weight. For example, in some embodiments, a subject weighing less than 70 kg may receive a 1.33 x 10 14 vg / kg, and subjects weighing 70 kg or more received a dose of 9.31 × 10 15 A fixed total dose of 1.33 × 10 vg was administered. 14 vg × 70kg is 9.31 × 10 15 vg. This means that subjects weighing over 70 kg are administered the same dose ("fixed dose") as subjects weighing 70 kg. A method for titrating AAV is described in Clark et al., Hum. Gene Ther., 10: 1031-1039 (1999).

[0201] In particular, the actual administration of the rAAV of the present disclosure can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present disclosure includes, but is not limited to, intramuscular injection and injection into the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations regarding carriers or other components that can be co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner of using rAAV). The capsid protein of rAAV can be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or topical formulations delivered to muscle via transdermal delivery. Numerous formulations have previously been developed for both intramuscular injection and transdermal delivery and can be used in the practice of the present disclosure. For ease of administration and handling, the rAAV can be used with any pharmaceutically acceptable carrier.

[0202] For intramuscular injection, solutions in adjuvants such as sesame or peanut oil, or in aqueous propylene glycol, as well as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered, if desired, and the liquid diluent is first rendered isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared in water, suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed can be readily obtained by standard techniques well known to those skilled in the art.

[0203] 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 dispersion. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. 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 dispersions, and by the use of surfactants. 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 is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0204] Sterile injectable solution is prepared by incorporating the required amount of rAAV into a suitable solvent with various other ingredients as listed above, and then, if necessary, filter sterilization.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technique, which obtains a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0205] Transduction using rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from a subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used, in which case they do not generate an inappropriate immune response in the subject.

[0206] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art.In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in a suitable medium, and cells carrying the DNA of interest can be screened using conventional techniques such as Southern blot and / or PCR, or by using selectable markers.The transduced cells can then be formulated into pharmaceutical compositions, and the compositions can be introduced into a subject by various techniques, for example, by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle, for example, using a catheter.

[0207] Transduction of cells with the rAAV of the present disclosure results in the expression of persistent micro-dystrophin protein.Therefore, the present disclosure provides a method for administering / delivering rAAV that expresses micro-dystrophin protein to animals, preferably humans.These methods include transducing one or more rAAVs of the present disclosure into tissues (including but not limited to tissues such as muscle, cardiac muscle, organs such as liver and brain, and glands such as salivary glands).Transduction can be carried out using a gene cassette that contains tissue-specific control elements. For example, one embodiment of the present disclosure provides a method for the detection of mitochondrial endothelial cells, including, but not limited to, those derived from the actin and myosin gene families, such as those derived from the myoD gene family (see Weintraub et al., Science, 251: 761-766 (1991)), muscle cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11: 4854-4862 (1991)), a regulatory element derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7: 4089-4099 (1987)), the cardiac actin gene, and the muscle creatine kinase sequence element (Johnson et al., Mol Cell Biol 9: 3393-3399 (1987)). (1989)) and mouse creatine kinase enhancer (mCK) element, control elements from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene, and the slow-twitch troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA 88: 5680-5684 (1991)), steroid-inducible elements and promoters, including glucocorticoid response elements (GREs) (Mader and White, Proc. Natl. Acad. Sci. USA 90: 5603-5607 (1993)), and other control elements.

[0208] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present disclosure contemplates sustained expression of micro-dystrophin from transduced myofibers.

[0209] Heart muscle tissue is an attractive target for in vivo DNA delivery because it is a vital organ and sustained expression of microdystrophin by transduced cardiac muscle fibers can prolong survival in patients with muscular dystrophy.

[0210] Thus, the present disclosure provides methods of administering an rAAV encoding micro-dystrophin at an effective dose (or doses administered essentially simultaneously or at intervals) to a subject in need thereof (e.g., a subject with muscular dystrophy).

[0211] The present disclosure provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, 8, or 9. The present disclosure also provides an rAAV comprising the nucleic acid sequence of SEQ ID NO: 9, or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3, and an rAAV particle comprising the nucleic acid sequence of SEQ ID NO: 9, or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3, wherein the rAAV is produced in adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container.

[0212] Another aspect of the present disclosure provides compositions comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, 8, or 9, an rAAV comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 55-5021 of SEQ ID NO:3, and an rAAV particle comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 55-5021 of SEQ ID NO:3, wherein the rAAV is produced in adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container. Any of the methods described herein can be practiced using these compositions. Hybrid seed train expansion of adherent cells

[0213] Some aspects of the present disclosure relate to a method of cell expansion, comprising: (a) culturing cells in a first medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container that is serum-free or contains serum at a lower concentration than the first medium; (d) culturing the cells in the N-1 container under suspension conditions; and (e) inoculating the cells from step (d) into a third medium in a bioreactor. In one aspect, the second medium is serum-free. In another aspect, the second medium contains serum at a lower concentration than the serum concentration in the first medium.

[0214] Some aspects of the present disclosure relate to a method of seed train expansion, comprising: (a) culturing cells in a first medium containing serum in an N-3 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-2 container that is serum-free or contains serum at a lower concentration than the first medium; (d) culturing the cells under suspension conditions in the N-2 container; (e) inoculating the second medium in an N-1 vessel with the cells from step (d); and (f) inoculating a third medium in a bioreactor with the cells from step (d). In one aspect, the second medium is serum-free. In another aspect, the second medium contains serum at a concentration lower than the serum concentration in the first medium.

[0215] Some aspects of the present disclosure relate to a method for cell expansion of adherent cells, comprising: (a) culturing the adherent cells under adherent conditions in a first medium containing serum; (b) removing the adherent cells from the first medium; (c) suspending the adherent cells in a second medium that is serum-free or contains serum at a lower concentration than the first medium; (d) culturing the adherent cells under suspension conditions; and (e) inoculating a third medium in a bioreactor with the adherent cells from step (d). In some aspects, the method may further comprise passaging the adherent cells of step (a) at least once under adherent conditions. In some aspects, the method may further comprise passaging the adherent cells of step (d) at least once under suspension conditions. In one aspect, the second medium is a serum-free medium. In another aspect, the second medium contains serum at a lower concentration than the first medium in the N-1 container.

[0216] The first medium, the second medium, and the third medium may be any medium suitable for the particular cells to be cultured. In some embodiments, the medium contains, for example, inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin, and fetuin), serum (e.g., a composition containing albumin, growth factors, and growth inhibitors, such as fetal bovine serum, newborn calf serum, and horse serum), trace elements (e.g., zinc, copper, selenium, and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins from plant or animal sources), and combinations thereof. Growth media may be commercially available media such as 5x concentrated DMEM / F12 (Invitrogen), CD OptiCHO feed (Invitrogen), CD EfficientFeed (Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (Irvine Scientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMD Millipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria), ActiCHO (PAA / GE Healthcare), Ham's F10 (Sigma), Minimum Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma).

[0217] In some embodiments, the second medium is a serum-free growth medium that does not contain serum or contains serum at a lower concentration than the first serum, and is substantially free (more than trace levels) of calcium ions, fetal bovine serum (FBS), fibronectin, collagen, laminin, or proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix that support cell fixation. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains serum at a concentration lower than the serum concentration in the first medium.

[0218] In some embodiments, the growth medium is between about 6.5 and about 7.5, between about 6.5 and about 7.4, between about 6.5 and about 7.3, between about 6.5 and about 7.2, between about 6.5 and about 7.1, between about 6.5 and about 7.0, between about 6.5 and about 6.9, between about 6.5 and about 6.8, between about 6.5 and about 6.7, between about 6.6 and about 7.5, between about 6.6 and about 7.4 between about 6.6 and about 7.3, between about 6.6 and about 7.2, between about 6.6 and about 7.1, between about 6.6 and about 7.0, between about 6.6 and about 6.9, between about 6.6 and about 6.8, between about 6.7 and about 7.5, between about 6.7 and about 7.4, between about 6.7 and about 7.3, between about 6.7 and about 7.2, between about 6.7 and about 7.1, between about 6.7 and about Between about 7.0, between about 6.7 and about 6.9, between about 6.8 and about 7.5, between about 6.8 and about 7.4, between about 6.8 and about 7.3, between about 6.8 and about 7.2, between about 6.8 and about 7.1, between about 6.8 and about 7.0, between about 6.9 and about 7.5, between about 6.9 and about 7.4, between about 6.9 and about 7.3, between about 6.9 and about 7.2, It may have a pH between 6.9 and about 7.1, between about 7.0 and about 7.5, between about 7.0 and about 7.4, between about 7.0 and about 7.3, between about 7.0 and about 7.2, between about 7.1 and about 7.5, between about 7.1 and about 7.4, between about 7.1 and about 7.3, between about 7.2 and about 7.5, between about 7.2 and about 7.4, or between about 7.3 and about 7.5.

[0219] In some embodiments, cells can be cultured at a temperature of 32°C to about 39°C, about 32°C to about 37°C, between about 32°C and about 37°C, between about 32°C and about 37.5°C, between about 34°C to about 37°C, between about 35°C to about 37°C, between about 35.5°C to about 37.5°C, between about 36°C to about 37°C, or about 36.5°C. In some embodiments, cells can be incubated at a temperature of about 37°C throughout the culture period. In some embodiments, the temperature can be changed or slightly fluctuated, for example, hourly or daily, during the culture period. In some embodiments, the temperature can be changed or shifted (e.g., raised or lowered) on about day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, or 15 after the start of the culture period, or at any time during the culture period. In some embodiments, the temperature can be shifted upward by about 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C, 7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5, or 10.0°C. In some embodiments, the temperature can be shifted downward by about 0.1°C, 0.2°C, 0.3°C, 0.4°C, 0.5°C, 0.6°C, 0.7°C, 0.8°C, 0.9°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C, 7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5°C, or 10°C.

[0220] In some embodiments, cell culture can be performed using an atmosphere containing about 1% to about 15% CO. In some embodiments, cells can be cultured using an atmosphere containing about 14% CO, 12% CO, 10% CO, 8% CO, 6% CO, 5% CO, 4% CO, 3% CO, 2% CO, or about 1% CO.

[0221] In some embodiments, the cell culture is maintained at a dissolved oxygen (dO2) concentration in the cell culture of between about 3% and about 55%, between about 3% and about 50%, between about 3% and about 45%, between about 3% and about 40%, between about 3% and about 35%, between about 3% and about 30%, between about 3% and about 25%, between about 3% and about 20%, between about 3% and about 15%, between about 5% and about 55%, between about 5% and about 50%, between about 5% and about 45%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, or about 5%. between about 25%, between about 5% and about 20%, between about 5% and about 15%, between about 5% and about 10%, between about 10% and about 55%, between about 10% and about 50%, between about 10% and about 45%, between about 10% and about 40%, between about 10% and about 35%, between about 10% and about 30%, between about 10% and about 25%, between about 10% and about 20%, between about 15% and about 55%, between about 15% and about 50%, between about 15% and about 45%, between about 15% and about 40%, between about 15% and about 35% , between about 15% and about 30%, between about 15% and about 25%, between about 15% and about 20%, between about 20% and about 55%, between about 20% and about 50%, between about 20% and about 45%, between about 20% and about 40%, between about 20% and about 35%, between about 20% and about 30%, between about 20% and about 25%, between about 25% and about 55%, between about 25% and about 50%, between about 25% and about 45%, between about 25% and about 40%, between about 25% and about 35%, between about 25% and about 30%, about 30% to about 55%, between about 30% and about 50%, between about 30% and about 45%, between about 30% and about 40%, between about 30% and about 35%, between about 35% and about 55%, between about 35% and about 50%, between about 35% and about 45%, between about 35% and about 40%, between about 40% and about 55%, between about 40% and about 50%, between about 40% and about 45%, between about 45% and about 55%, between about 45% and about 50%, or between about 50% and about 55%.

[0222] In some embodiments, the pH of the cell culture can be maintained at a particular pH value by the addition of a base solution, such as an alkaline base solution, such as by adjusting the pH of the cell culture to between about 6.5 and about 7.5, between about 6.5 and about 7.4, between about 6.5 and about 7.3, between about 6.5 and about 7.2, between about 6.5 and about 7.1, between about 6.5 and about 7.0, between about 6.5 and about 6.9, between about 6.5 and about 6.8, between about 6.5 and about 6.7, between about 6.6 and about 7.5, between about 6.6 and about 7.4, between about between 6.6 and about 7.3, between about 6.6 and about 7.2, between about 6.6 and about 7.1, between about 6.6 and about 7.0, between about 6.6 and about 6.9, between about 6.6 and about 6.8, between about 6.7 and about 7.5, between about 6.7 and about 7.4, between about 6.7 and about 7.3, between about 6.7 and about 7.2, between about 6.7 and about 7.1, between about 6.7 and about 7.0 between about 6.7 and about 6.9, between about 6.8 and about 7.5, between about 6.8 and about 7.4, between about 6.8 and about 7.3, between about 6.8 and about 7.2, between about 6.8 and about 7.1, between about 6.8 and about 7.0, between about 6.9 and about 7.5, between about 6.9 and about 7.4, between about 6.9 and about 7.3, between about 6.9 and about 7.2, The pH can be maintained at between about 7.1, between about 7.0 and about 7.5, between about 7.0 and about 7.4, between about 7.0 and about 7.3, between about 7.0 and about 7.2, between about 7.1 and about 7.5, between about 7.1 and about 7.4, between about 7.1 and about 7.3, between about 7.2 and about 7.5, between about 7.2 and about 7.4, or between about 7.3 and about 7.5.

[0223] In some embodiments, cell culture under suspension conditions can be carried out in any type of cell culture flask suitable for stationary or mixed / shaken suspension cell expansion, such as a T-flask, roller bottle, spinner flask, or shaker flask; or a combination thereof. In some embodiments, the N-1 container is a shaker flask.

[0224] In some embodiments, the flotation conditions may include some form of agitation. In some embodiments, the agitation may be rotary agitation. In some embodiments, the agitation may be between about 25 RPM and about 500 RPM, between about 25 RPM and about 480 RPM, between about 25 RPM and about 460 RPM, between about 25 RPM and about 440 RPM, between about 25 RPM and about 420 RPM, between about 25 RPM and about 400 RPM, between about 25 RPM and about 380 RPM, between about 25 RPM and about 360 RPM, between about 25 RPM and about 340 RPM, between about 25 RPM and about 320 RPM, between about 25 RPM and about 300 RPM, between about 25 RPM and about 28 ... Between 60 RPM, between about 25 RPM and about 240 RPM, between about 25 RPM and about 220 RPM, between about 25 RPM and about 200 RPM, between about 25 RPM and about 180 RPM, between about 25 RPM and about 160 RPM, between about 25 RPM and about 140 RPM, between about 25 RPM and about 120 RPM, between about 25 RPM and about 100 RPM, between about 25 RPM and about 80 RPM, between about 25 RPM and about 60 RPM, between about 25 RPM and about 40 RPM, between about 25 RPM and about 35 RPM, between about 25 RPM and about 30 RPM between about 50 RPM and about 500 RPM, between about 50 RPM and about 480 RPM, between about 50 RPM and about 460 RPM, between about 50 RPM and about 440 RPM, between about 50 RPM and about 420 RPM, between about 50 RPM and about 400 RPM, between about 50 RPM and about 380 RPM, between about 50 RPM and about 360 RPM, between about 50 RPM and about 340 RPM, between about 50 RPM and about 320 RPM, between about 50 RPM and about 300 RPM, between about 50 RPM and about 280 RPM, between about 50 RPM and about 260 RPM between about 50 RPM and about 240 RPM, between about 50 RPM and about 220 RPM, between about 50 RPM and about 200 RPM, between about 50 RPM and about 180 RPM, between about 50 RPM and about 160 RPM, between about 50 RPM and about 140 RPM, between about 50 RPM and about 120 RPM, between about 50 RPM and about 100 RPM, between about 50 RPM and about 80 RPM, between about 50 RPM and about 60 RPM, between about 75 RPM and about 500 RPM, between about 75 RPM and about 480 RPM, between about 75 RPM and about 460 RPM,Between about 75 RPM and about 440 RPM, between about 75 RPM and about 420 RPM, between about 75 RPM and about 400 RPM, between about 75 RPM and about 380 RPM, between about 75 RPM and about 360 RPM, between about 75 RPM and about 340 RPM, between about 75 RPM and about 320 RPM, between about 75 RPM and about 300 RPM, between about 75 RPM and about 280 RPM, between about 75 RPM and about 260 RPM, between about 75 RPM and about 240 RPM, between about 75 RPM and about 220 RPM, between about 75 RPM and about 200 RPM, between about 75 RPM and about 180 RPM, between about 75 RPM and about 160 RPM, between about 75 RPM and about 140 RPM, between about 75 RPM and about 120 RPM, between about 75 RPM and about 100 RPM, between about 75 RPM and about 80 RPM, between about 100 RPM and about 500 RPM, between about 100 RPM and about 480 RPM, between about 100 RPM and about 460 RPM, between about 100 RPM and about 440 RPM, between about 100 RPM and about 420 RPM, between about 100 RPM and about 400 RPM, between about 100 RPM and about 380 RPM, between about 100 RPM and about 360 RPM, Between about 100 RPM and about 340 RPM, between about 100 RPM and about 320 RPM, between about 100 RPM and about 300 RPM, between about 100 RPM and about 280 RPM, between about 100 RPM and about 260 RPM, between about 100 RPM and about 240 RPM, between about 100 RPM and about 220 RPM, between about 100 RPM and about 200 RPM, between about 100 RPM and about 180 RPM, between about 100 RPM and about 160 RPM, between about 100 RPM and about 140 RPM, between about 100 RPM and about 120 RPM, between about 150 RPM and about 500 RPM, Between about 150 RPM and about 480 RPM, between about 150 RPM and about 460 RPM, between about 150 RPM and about 440 RPM, between about 150 RPM and about 420 RPM, between about 150 RPM and about 400 RPM, between about 150 RPM and about 380 RPM, between about 150 RPM and about 360 RPM, between about 150 RPM and about 340 RPM, between about 150 RPM and about 320 RPM, between about 150 RPM and about 300 RPM, between about 150 RPM and about 280 RPM, between about 150 RPM and about 260 RPM, between about 150 RPM and about 240 RPM,Between about 150 RPM and about 220 RPM, between about 150 RPM and about 200 RPM, between about 150 RPM and about 180 RPM, between about 150 RPM and about 160 RPM, between about 200 RPM and about 500 RPM, between about 200 RPM and about 480 RPM, between about 200 RPM and about 460 RPM, between about 200 RPM and about 440 RPM, between about 200 RPM and about 420 RPM, between about 200 RPM and about 400 RPM, between about 200 RPM and about 380 RPM, between about 200 RPM and about 360 RPM, between about 200 RPM and about 340 RPM, between about 200 RPM and about 320 RPM, between about 200 RPM and about 300 RPM, between about 200 RPM and about 280 RPM, between about 200 RPM and about 260 RPM, between about 200 RPM and about 240 RPM, between about 200 RPM and about 220 RPM, between about 240 RPM and about 500 RPM, between about 240 RPM and about 480 RPM, between about 240 RPM and about 460 RPM, between about 240 RPM and about 440 RPM, between about 240 RPM and about 420 RPM, between about 240 RPM and about 400 RPM, between about 240 RPM and about 380 RPM, between about 240 RPM and about 360 RPM, between about 240 RPM and about 340 RPM, between about 240 RPM and about 320 RPM, between about 240 RPM and about 300 RPM, between about 240 RPM and about 280 RPM, between about 240 RPM and about 260 RPM, between about 260 RPM and about 500 RPM, between about 260 RPM and about 480 RPM, between about 260 RPM and about 460 RPM, between about 260 RPM and about 440 RPM, between about 260 RPM and about 420 RPM, between about 260 RPM and about 400 RPM, between about 260 RPM and about 380 RPM, between about 260 RPM and about 360 RPM, between about 260 RPM and about 340 RPM, between about 260 RPM and about 320 RPM, between about 260 RPM and about 300 RPM, between about 260 RPM and about 280 RPM, between about 280 RPM and about 500 RPM, between about 280 RPM and about 480 RPM, between about 280 RPM and about 460 RPM, between about 280 RPM and about 440 RPM, between about 280 RPM and about 420 RPM, between about 280 RPM and about 400 RPM, between about 280 RPM and about 380 RPM, between about 280 RPM and about 360 RPM,The stirring can be performed at a frequency of between about 280 RPM and about 340 RPM, between about 280 RPM and about 320 RPM, between about 280 RPM and about 280 RPM, between about 300 RPM and about 500 RPM, between about 380 RPM and about 480 RPM, between about 380 RPM and about 460 RPM, between about 380 RPM and about 440 RPM, between about 380 RPM and about 420 RPM, between about 380 RPM and about 400 RPM, between about 400 RPM and about 500 RPM, between about 400 RPM and about 480 RPM, between about 400 RPM and about 460 RPM, between about 400 RPM and about 440 RPM, or between about 400 RPM and about 420 RPM. The stirring can be performed continuously or periodically.

[0225] In some embodiments, the cells are passaged in suspension up to two times.

[0226] In some embodiments, the cells are cultured in suspension for about 24 to about 96 hours. In some embodiments, the cells are cultured in suspension for about 36 to about 84 hours. In some embodiments, the cells are cultured in suspension for about 48 to about 72 hours. In some embodiments, the cells are cultured in suspension for about 54 to about 66 hours. In some embodiments, the cells are cultured in suspension for about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours.

[0227] In some embodiments of the present disclosure, the cells are adherent cells. In some embodiments, the adherent cells are HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, or COS cells. In some embodiments, the adherent cells are human. In some embodiments, the adherent cells are HeLa cells or HEK-293 cells. In some embodiments, the adherent cells are HEK-293 cells.

[0228] In some aspects, the adherent cells are not adapted to suspension systems. In some aspects, culturing the cells in suspension conditions does not alter the adhesion dependency of the cells. In some aspects, the methods do not alter the cells to create a new cell line. The methods described herein do not alter the genomic or transcriptomic profile of the cells. The methods described herein do not alter the phenotype of the cells.

[0229] In some embodiments, the cells are passaged multiple times under adherent conditions in serum-supplemented growth medium before being inoculated into an N-1 container. In some embodiments, the cells are cultured in an N-2, N-3, N-4, N-5, N-6, N-7, N-8, N-9, or N-10 container before being inoculated into an N-1 container. In some embodiments, the cells are cultured in N-3 and N-2 containers. In some embodiments, the cells are cultured in N-4, N-3, and N-2 containers.

[0230] In some aspects, the bioreactor is an adherent bioreactor. In some aspects, adherent cells are purified from a culture produced in the adherent bioreactor.

[0231] In some embodiments, the bioreactor includes at least one, and more preferably multiple, carriers onto which expanded cells are intended to adhere, which may be floating or fixed in the bioreactor. Preferably, the carriers may be made using, for example, polyethylene terephthalate, polystyrene, polyester, polypropylene, DEAE-dextran, collagen, glass, alginate, or acrylamide. In some embodiments, the bioreactor may be a bioreactor containing bead-type microcarriers (e.g., Cytodex® brand beads commercially available from GE Healthcare Inc., a General Electric Corp. company) or matrix-type carriers (e.g., Fibra-Cell™ brand discs commercially available from Eppendorf Corp.). In some embodiments, the bioreactor uses polyester fiber carriers, such as those used in iCELLis® nanobioreactors or iCELLis® 500 bioreactors (commercially available from Advanced Technology Materials Inc., Brussels, Belgium, and Pall Corporation, Fall River, Massachusetts).

[0232] In some embodiments, the third culture medium in the bioreactor contains at least one factor that promotes cell adhesion. In some embodiments, the at least one factor that promotes cell adhesion is selected from the group consisting of FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the at least one factor that promotes cell adhesion can be added to the third culture medium immediately before, during, or after inoculation of suspension cells into the bioreactor.

[0233] In some embodiments, the growth medium comprises DMEM and about 10% FBS by weight. In some embodiments, the growth medium comprises about 2% to about 20% FBS by weight. In some embodiments, the growth medium comprises about 3% to about 19% FBS by weight. In some embodiments, the growth medium comprises about 4% to about 18% FBS by weight. In some embodiments, the growth medium comprises about 5% to about 17% FBS by weight. In some embodiments, the growth medium comprises about 6% to about 16% FBS by weight. In some embodiments, the growth medium comprises about 7% to about 15% FBS by weight. In some embodiments, the growth medium comprises about 8% to about 14% FBS by weight. In some embodiments, the growth medium comprises about 9% to about 13% FBS by weight. In some embodiments, the growth medium comprises about 10% to about 12% FBS by weight. In some embodiments, the growth medium comprises about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% FBS by weight.

[0234] In some embodiments, the expanded suspension cells from step (d) can be directly inoculated into a bioreactor. In some embodiments, the number of cells inoculated into the bioreactor varies based on the size of the bioreactor. In some embodiments, a 4 m bioreactor (e.g., an iCELLis® nanobioreactor) is used. In some embodiments, approximately 1 x 10 cells are inoculated into a 4 m bioreactor. 8 pieces~1×10 9 In some embodiments, a 4 m bioreactor is inoculated with between about 3 x 10 cells. 8 pieces~7×10 8 In some embodiments, a 4 m bioreactor is inoculated with between about 4 x 10 cells. 8 pieces~6×10 8 In some embodiments, a 4 m bioreactor is inoculated with between about 5 x 10 cells. 8 In some embodiments, comparable cell densities are used for other sized bioreactors.

[0235] In some aspects, the methods of the present disclosure may further comprise culturing the cells in a bioreactor. In some aspects, the cell culturing comprises batch culturing. In some aspects, the cell culturing comprises fed-batch culturing. In some aspects, the cell culturing comprises perfusion culturing.

[0236] Fed-batch culturing involves the incremental (periodic) or continuous addition of a feed culture medium to an initial cell culture without substantial or significant removal of growth medium from the cell culture. The cell culture in fed-batch culturing can be placed in a bioreactor (e.g., a production bioreactor, e.g., a 10,000 L production bioreactor). In some embodiments, the feed culture medium can be the same as the growth medium. The feed culture medium can be in liquid form or as a dry powder. In some embodiments, the feed culture medium is a concentrated form of the growth medium and / or is added as a dry powder. In some embodiments, both a first liquid feed culture medium and a different second liquid feed culture medium can be added (e.g., continuously added) to the growth medium. In some embodiments, the addition of the first liquid feed culture medium and the second liquid feed culture medium to the culture can begin at approximately the same time. In some embodiments, the total volumes of the first and second liquid feed culture media added to the culture over the entire culture period can be about the same.

[0237] When the feed culture medium is continuously added, the rate of addition of the feed culture medium can be kept constant or increased (e.g., gradually increased) over the culture period. The continuous addition of the feed culture medium can be initiated at a specific time point during the culture period (e.g., when the cells reach a target viable cell density, e.g., a viable cell density of about 1×10 cells / mL, about 1.1×10 cells / mL, about 1.2×10 cells / mL, about 1.3×10 cells / mL, about 1.4×10 cells / mL, about 1.5×10 cells / mL, about 1.6×10 cells / mL, about 1.7×10 cells / mL, about 1.8×10 cells / mL, about 1.9×10 cells / mL, or about 2.0×10 cells / mL). In some embodiments, the continuous addition of the feed culture medium can be initiated on the second, third, fourth, or fifth day of the culture period.

[0238] In some embodiments, incremental (periodic) addition of feed culture medium can begin when the cells reach a target cell density (e.g., about 1×10 cells / mL, about 1.1×10 cells / mL, about 1.2×10 cells / mL, about 1.3×10 cells / mL, about 1.4×10 cells / mL, about 1.5×10 cells / mL, about 1.6×10 cells / mL, about 1.7×10 cells / mL, about 1.8×10 cells / mL, about 1.9×10 cells, or about 2.0×10 cells / mL). In some embodiments, incremental addition of feed culture medium can occur at regular intervals (e.g., daily, every other day, or every third day) or when the cells reach a particular target cell density (e.g., a target cell density that increases over the culture period). In some embodiments, the amount of feed culture medium added can be gradually increased between a first incremental addition of feed culture medium and subsequent additions of feed culture medium. In some embodiments, the volume of liquid culture feed culture medium added to the initial cell culture over any 24 hour period during the culture period can be some fraction of the initial volume of the bioreactor containing the culture or some fraction of the volume of the initial culture.

[0239] In some embodiments, the addition (continuous or periodic) of liquid feed culture medium is between 6 hours and 7 days after the start of the culture period, between about 6 hours and about 6 days, between about 6 hours and about 5 days, between about 6 hours and about 4 days, between about 6 hours and about 3 days, between about 6 hours and about 2 days, between about 6 hours and about 1 day, between about 12 hours and about 7 days, between about 12 hours and about 6 days, between about 12 hours and about 5 days, between about 12 hours and about 4 days, between about 12 hours and about 3 days, between about 12 hours and about 2 days, between about 12 hours and about 7 days, between about 1 day and about 7 days. The incubation can be performed at a time point between about 1 day and about 6 days, between about 1 day and about 5 days, between about 1 day and about 4 days, between about 1 day and about 3 days, between about 1 day and about 2 days, between about 2 days and about 7 days, between about 2 days and about 6 days, between about 2 days and about 5 days, between about 2 days and about 4 days, between about 2 days and about 3 days, between about 3 days and about 7 days, between about 3 days and about 6 days, between about 3 days and about 5 days, between about 3 days and about 4 days, between about 4 days and about 7 days, between about 4 days and about 6 days, between about 4 days and about 5 days, between about 5 days and about 7 days, or between about 5 days and about 6 days.

[0240] In some embodiments, the volume of liquid feed culture medium added (continuously or periodically) to the initial cell culture over any 24 hour period can be between 0.01 and about 0.3 times the volume of the bioreactor. The ratio is between about 0.01 times and about 0.28 times, between about 0.01 times and about 0.26 times, between about 0.01 times and about 0.24 times, between about 0.01 times and about 0.22 times, between about 0.01 times and about 0.20 times, between about 0.01 times and about 0.18 times, between about 0.01 times and about 0.16 times, between about 0.01 times and about 0.14 times, between about 0.01 times and about 0.12 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.08 times, between about 0.01 times and about 0.06 ...0 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 times, between about 0.01 times and about 0.10 between about 0.02 and about 0.04 times, between about 0.02 and about 0.3 times, between about 0.02 and about 0.28 times, between about 0.02 and about 0.26 times, between about 0.02 and about 0.24 times, between about 0.02 and about 0.22 times, between about 0.02 and about 0.20 times, between about 0.02 and about 0.18 times, between about 0.02 and about 0.16 times, between about 0.02 and about 0.14 times, between about 0.02 and about 0.12 times, between about 0.02 and about 0.10 times, between about 0.02 and about 0.08 times, between about 0.02 and about 0.06 times times, between about 0.02 times and about 0.05 times, between about 0.02 times and about 0.04 times, between about 0.02 times and about 0.03 times, between about 0.025 times and about 0.3 times, between about 0.025 times and about 0.28 times, between about 0.025 times and about 0.26 times, between about 0.025 times and about 0.24 times, between about 0.025 times and about 0.22 times, between about 0.025 times and about 0.20 times, between about 0.025 times and about 0.18 times, between about 0.025 times and about 0.16 times, between about 0.025 times and about 0.14 times, between about 0.025 times and about Between 0.12 times, between about 0.025 times and about 0.10 times, between about 0.025 times and about 0.08 times, between about 0.025 times and about 0.06 times, between about 0.025 times and about 0.04 times, between about 0.05 times and about 0.3 times, between about 0.05 times and about 0.28 times, between about 0.05 times and about 0.26 times, between about 0.05 times and about 0.24 times, between about 0.05 times and about 0.22 times, between about 0.05 times and about 0.20 times, between about 0.05 times and about 0.18 times, between about 0.05 times and about 0.16 times, between about 0.05 times and about 0.The ratio may be between about 0.14 times, between about 0.05 times and about 0.12 times, between about 0.05 times and about 0.10 times, between about 0.1 times and about 0.3 times, between about 0.1 times and about 0.28 times, between about 0.1 times and about 0.26 times, between about 0.1 times and about 0.24 times, between about 0.1 times and about 0.22 times, between about 0.1 times and about 0.20 times, between about 0.1 times and about 0.18 times, between about 0.1 times and about 0.16 times, between about 0.1 times and about 0.14 times, between about 0.1 times, between about 0.15 times and about 0.3 times, between about 0.15 times and about 0.2 times, between about 0.2 times and about 0.3 times, or between about 0.25 times and about 0.3 times.

[0241] In some embodiments, the volume of liquid feed culture medium added (continuously or periodically) to the initial cell culture over any 24 hour period during the culture period is between 0.02x and about 1.0x, between about 0.02x and about 0.9x, between about 0.02x and about 0.8x, between about 0.02x and about 0.7x, between about 0.02x and about 0.6x, between about 0.02x and about 0.5x, between about 0.02x and about 0.6x, between about 0.02x and about 0.8x, between about 0.02x and about 0.9x, between about 0.02x and about 0.9x, between about 0.02x and about 0.9x, between about 0.02x and about 0.9x, between about 0.02x and about 0.9x, between about 0.02x and about 0.9x, between about 0.02x and about 0.1 ... Between approximately 0.02 and approximately 0.4 times, between approximately 0.02 and approximately 0.3 times, between approximately 0.02 and approximately 0.2 times, between approximately 0.02 and approximately 0.1 times, between approximately 0.02 and approximately 0.08 times, between approximately 0.02 and approximately 0.06 times, between approximately 0.02 and approximately 0.05 times, between approximately 0.02 and approximately 0.04 times, between approximately 0.02 and approximately 0.03 times, between approximately 0.05 and approximately 1.0 times, between approximately 0.05 and approximately 0.8 times, Between 0.05x and approximately 0.7x, between approximately 0.05x and approximately 0.6x, between approximately 0.05x and approximately 0.5x, between approximately 0.05x and approximately 0.4x, between approximately 0.05x and approximately 0.3x, between approximately 0.05x and approximately 0.2x, between approximately 0.05x and approximately 0.1x, between approximately 0.1x and approximately 1.0x, between approximately 0.1x and approximately 0.9x, between approximately 0.1x and approximately 0.8x, between approximately 0.1x and approximately 0.7x, between approximately 0.1x and approximately The amount may be between about 0.6 times, between about 0.1 times and about 0.5 times, between about 0.1 times and about 0.4 times, between about 0.1 times and about 0.3 times, between about 0.1 times and about 0.2 times, between about 0.2 times and about 1.0 times, between about 0.2 times and about 0.9 times, between about 0.2 times and about 0.8 times, between about 0.2 times and about 0.7 times, between about 0.2 times and about 0.6 times, between about 0.2 times and about 0.5 times, or between about 0.2 times and about 0.4 times.

[0242] In some embodiments, the total amount of feed culture medium added (continuously or periodically) throughout the culture period is between about 1% and about 40% of the volume of the initial culture (e.g., between about 1% and about 35%, between about 1% and about 30%, between about 1% and about 25%, between about 1% and about 20%, between about 1% and about 15%, between about 1% and about 10%, between about 1% and about 5%, between about 1% and about 4%, between about 2% and about 40%, between about 2% and about 35%, between about 2% and about 30%, Between about 2% and about 25%, Between about 2% and about 20%, Between about 2% and about 15%, Between about 2% and about 10%, Between about 2% and about 5%, Between about 3% and about 40%, Between about 3% and about 35%, Between about 3% and about 30%, Between about 3% and about 25%, Between about 3% and about 20%, Between about 3% and about 15%, Between about 3% and about 10%, Between about 3% and about 5%, Between about 4% and about 40%, Between about 4% and about 35%, Between about 4% and about 30%, Between about 4% and about 25%, About 4% between about 4% and about 15%, between about 4% and about 10%, between about 4% and about 8%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% and about 25%, between about 5% and about 20%, between about 5% and about 15%, between about 5% and about 10%, between about 10% and about 40%, between about 10% and about 35%, between about 10% and about 30%, between about 10% and about 25%, between about 10% and about 20%, between about 10% and about 15% , between about 15% and about 40%, between about 15% and about 35%, between about 15% and about 30%, between about 15% and about 25%, between about 15% and about 20%, between about 20% and about 40%, between about 20% and about 35%, between about 20% and about 30%, between about 20% and about 25%, between about 25% and about 40%, between about 25% and about 35%, between about 25% and about 30%, between about 30% and about 40%, between about 30% and about 35%, or between about 35% and about 40%.

[0243] In some embodiments, two different feed culture media are added (continuously or incrementally) during fed-batch culture. In some embodiments, the amounts or volumes of the first and second feed culture media added may be substantially the same or different. In some embodiments, the first feed culture medium may be in liquid form, and the second feed culture medium may be in solid form. In some embodiments, the first and second feed culture media may be liquid feed culture media.

[0244] Perfusion culturing includes removing a first volume of growth medium from a bioreactor and adding a second volume of a second growth culture medium to a production bioreactor, wherein the first volume and the second volume are approximately equal. The cells are retained in the bioreactor by a cell retention device or by techniques such as cell sedimentation in a settling cone. In some embodiments, the removal and addition of growth medium can be performed simultaneously or sequentially, or some combination of the two. In some embodiments, removal and addition can be carried out continuously, for example, at a rate where between 0.1% and 800%, between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 350%, between 1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and 30% of the volume of the bioreactor is removed and replaced.

[0245] In some embodiments, the first volume of the first growth medium is removed and the second volume of the second growth medium is added can be kept approximately the same over each 24-hour period. In some embodiments, the flow rate (volume / time unit) at which the first volume of the first growth medium is removed and the flow rate (volume / time unit) at which the second volume of the second growth medium is added can be varied depending on the conditions of the particular cell culture system. In some embodiments, the flow rate (volume / time unit) at which the first volume of the first growth medium is removed and the flow rate (volume / time unit) at which the second volume of the second growth medium is added can be approximately the same or different.

[0246] In some embodiments, the volumes removed and added can be varied by gradually increasing them over each 24-hour period. In some embodiments, the volume of first growth medium removed and the volume of second growth medium added within each 24-hour period can be increased over the culture period. In some embodiments, the volume can be increased by volumetric increments of between 0.5% and about 20% of the volume of the bioreactor over the 24-hour period. In some embodiments, the volume can be increased over the culture period to a volume that is about 25% to about 150% of the volume of the bioreactor, or to the first liquid culture medium volume over the 24-hour period.

[0247] In some embodiments, in each 24-hour period, after the first 48 to 96 hours of the culture period, the first volume of the first growth medium removed and the second volume of the second growth medium added is about 10% to about 95%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 85% to about 95%, about 60% to about 80%, or about 70% of the volume of the first growth medium.

[0248] In some embodiments, the first and second growth media can be the same type of medium. In some embodiments, the first and second growth media can be different. In some embodiments, the second liquid culture medium can be more concentrated with respect to one or more media components.

[0249] In some embodiments, the first volume of the first growth medium can be removed using any automated system. In some embodiments, alternating tangential flow filtration can be used. In some embodiments, the first volume of the first growth medium can be removed by percolation or gravity flow, passing the first volume of the first growth medium through a sterile membrane with a molecular weight cutoff that excludes cells. In some embodiments, the first volume of the first growth medium can be removed by stopping agitation or significantly reducing the agitation rate for a period of at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, and removing or aspirating the first volume of growth medium from the top of the production bioreactor.

[0250] In some embodiments, the second volume of the second liquid culture medium can be added to the first liquid culture medium by a pump. In some embodiments, the second liquid culture medium can be added to the first liquid culture medium manually, for example, by pipetting or injecting the second volume of the second liquid culture medium directly into the first liquid culture medium, or in an automated manner.

[0251] In some embodiments, the method further comprises contacting cells with a first polynucleotide sequence. In some embodiments, the method further comprises transfecting the cells with the polynucleotide sequence. In some embodiments, the polynucleotide sequence is a plasmid. In some embodiments, the plasmid encodes a capsid of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpesvirus, polyomavirus, and vaccinia virus. In some embodiments, the cells are transfected before the cells are inoculated into a bioreactor. In some embodiments, the cells are transfected after the cells are inoculated into a bioreactor. In some embodiments, the cells are contacted with or transfected with a second polynucleotide comprising a nucleic acid encoding a transgene. In some embodiments, the cells are cultured under conditions in which a viral vector is produced. In some embodiments, the method further comprises isolating the produced viral vector.

[0252] In some embodiments, the polynucleotide is a viral vector. In some embodiments, the viral vector is an adenoviral or adeno-associated viral (AAV) vector. These vectors infect many dividing and non-dividing cell types, including synovial cells and hepatocytes. As mentioned above, adenoviral and AAV vectors are episomal after entering cells, making these vectors suitable for therapeutic applications (Russell, J. Gen. Virol. 81: 2573-2604 (2000)); Goncalves, Virol J. 2 (1): 43 2005)). AAV vectors can provide very stable, long-term transgene expression (up to 9 years in dogs (Niemeyer et al., Blood 113 (4): 797-806 (2009)) and up to 2 years in humans (Nathwani et al., N Engl J Med. 365 (25): 2357-2365 (2011); Simonelli et al., Mol Ther. 18 (3): 643-50 (2010), Epub 2009 Dec. 1)). In some embodiments, adenoviral vectors are modified to reduce the host response, as reviewed by Russell (2000, supra). Methods for gene therapy using AAV vectors have been described by Wang et al., 2005, J Gene Med. March 9 (Epub ahead of print); Mandel et al., Curr Opin Mol Ther. 6: 482-90 (2004); Martin et al., Eye 18: 1049-55 (2004); Nathwani et al., N Engl J Med. 22;365: 2357-65 (2011); and Apparailly et al., Hum Gene Ther. 16 (4): 426-34 (2005).

[0253] In some embodiments, the first polynucleotide sequence comprises one or more of an inverted terminal repeat, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, a nucleic acid encoding at least one AAV structural capsid protein, or a combination thereof.

[0254] In some aspects, the cells are cultured under conditions whereby recombinant viral particles are produced. In some aspects, the method further comprises isolating the produced recombinant viral particles.

[0255] In some embodiments, the viral vector comprises a transgene operably linked to an appropriate regulatory sequence. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control protein transcription or translation. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). In some embodiments, the regulatory sequence may comprise a promoter sequence. In some embodiments, the promoter sequence may be a cytomegalovirus (CMV) intermediate-early promoter, a viral long terminal repeat promoter (LTR), such as those derived from Moloney murine leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1, a simian virus 40 (SV40) early promoter, or a herpes simplex virus thymidine kinase promoter.

[0256] In some embodiments, the viral vector comprises an additional nucleotide sequence encoding an additional polypeptide. In some embodiments, the additional polypeptide may be a (selectable) marker polypeptide that allows for identification, selection, and / or screening of cells containing the viral vector. In some embodiments, the marker polypeptide may be the fluorescent protein GFP, as well as the selectable marker genes HSV thymidine kinase (for selection in HAT medium), bacterial hygromycin B phosphotransferase (for selection with hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection with G418), and dihydrofolate reductase (DHFR) (for selection with methotrexate), CD20, low-affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are described in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. Methods for Producing Viral Vectors (e.g., AAV)

[0257] Some aspects of the present disclosure are directed to a method of producing a viral vector (e.g., an rAAV described herein), comprising expanding cells according to any one of the seed train expansion methods described herein, inoculating the cells into a growth medium in a bioreactor, transfecting the cells with a polynucleotide sequence encoding viral particles, and culturing the cells in the bioreactor under conditions in which viral particles are produced. In some aspects, the production of viral vectors is disclosed in U.S. Application No. 63 / 123,602, expressly incorporated herein by reference.

[0258] The method of introducing exogenous nucleic acid into host cell is well known in the art and varies depending on the host cell used.Techniques include but are not limited to dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polyethyleneimine-mediated transfection, polybrene-mediated transfection, protoplast fusion, electroporation, virus infection or phage infection, polynucleotide (s) encapsulated in liposome, and DNA directly microinjected into nucleus.Transfection can be either transient or stable.

[0259] In some aspects, the polynucleotide sequence is a plasmid. In some aspects, the plasmid encodes an AAV-derived viral particle.

[0260] In some embodiments, the polynucleotide sequence is a viral vector. In some embodiments, the viral vector encodes a viral particle. In preferred embodiments, the viral particle is derived from AAV. In some embodiments, the rAAV comprises the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the present disclosure provides an rAAV particle comprising the nucleic acid sequence of SEQ ID NO:9. In some embodiments, the present disclosure provides an rAAV comprising nucleotides 55-5021 of SEQ ID NO:3. In some embodiments, the present disclosure provides an rAAV particle comprising nucleotides 55-5021 of SEQ ID NO:3. In some embodiments, the rAAV comprises nucleotides 1-4988 of SEQ ID NO:8. In some embodiments, the present disclosure provides an rAAV particle comprising nucleotides 1-4977 of SEQ ID NO:8.

[0261] In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector may comprise a recombinant AAV vector (rAAV). As used herein, "rAAV vector" refers to a recombinant vector that comprises a portion of the AAV genome encapsulated in a protein shell of a capsid protein derived from an AAV serotype described herein. In some embodiments, the AAV vector may comprise an inverted terminal repeat (ITR) derived from an adeno-associated virus serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVRH10, AAV11, AAV12, and others.

[0262] Typically, the vector genome requires the use of flanking 5' and 3' ITR sequences to enable efficient packaging of the vector genome into an rAAV capsid. In some embodiments, the rAAV genome present in the rAAV vector comprises the nucleotide sequence of the inverted terminal repeat region (ITR) of at least one of the AAV serotypes, or a nucleotide sequence substantially identical thereto, and a nucleic acid sequence encoding a transgene under the control of an appropriate regulatory element (e.g., a promoter), wherein the regulatory element and modified nucleic acid sequence(s) are inserted between the two ITRs.

[0263] The complete genomes and corresponding ITRs of several AAV serotypes have been sequenced (Chiorini et al. J. of Virology 73: 1309-1319 (1999)). They can be produced by cloning or chemical synthesis using an oligonucleotide synthesizer, such as those supplied by Applied Biosystems Inc. (Fosters, Calif., USA), or by standard molecular biology techniques, as is known in the art. ITRs can be cloned from the AAV viral genome or excised from a vector containing AAV ITRs. The ITR nucleotide sequence can be ligated at either end to a nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the wild-type AAV sequence between the ITRs can be replaced with a desired nucleotide sequence.

[0264] In some embodiments, the viral capsid component of the packaged viral vector may be a parvovirus capsid, such as an AAV Cap and / or a chimeric capsid. Examples of suitable parvovirus viral capsid components include those derived from Parvoviridae, such as self-replicating parvoviruses or Dependoviruses. For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAV9, AAV10, AAVRH10, AAV11, or AAV12 capsid; those skilled in the art know that there may be other variants that have not yet been identified that perform the same or similar functions), or may contain components from two or more AAV capsids. The complete AAV Cap protein components include VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein can contain less than the complete component of the AAV Cap protein, or the complete component of the AAV Cap protein can be provided.

[0265] In some embodiments, one or more of the AAV Cap proteins may be chimeric proteins containing the amino acid sequences of AAV Cap from two or more viruses, preferably two or more AAVs. For example, a chimeric viral capsid may contain an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. In some embodiments, the rAAV genome present in the rAAV vector does not contain any nucleotide sequences encoding viral proteins such as the AAV rep (replication) or cap (capsid) genes. In some embodiments, the rAAV genome may further contain a marker or reporter gene, such as a gene encoding an antibiotic resistance gene, a gene encoding a fluorescent protein (e.g., gfp), or a gene encoding a chemically, enzymatically, or otherwise detectable and / or selectable product known in the art (e.g., lacZ, aph, etc.).

[0266] In some aspects, the rAAV genome present in the rAAV vector may further comprise a promoter sequence operably linked to the nucleotide sequence encoding the transgene.

[0267] In some embodiments, a suitable 3' untranslated sequence may be operably linked to the modified nucleic acid sequence encoding the transgene. The suitable 3' untranslated region may be one naturally associated with the nucleotide sequence or may be from a different gene, such as the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal and enhancer sequence).

[0268] Unless otherwise specified, methods known to those skilled in the art can be used to construct recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvoviral Rep and / or Cap sequences, and transient and stable trans-acting packaging cells. Such techniques are well known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); AUSUBEL el al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0269] In some embodiments, a suitable 3' untranslated sequence may be operably linked to the nucleic acid sequence encoding the transgene. The suitable 3' untranslated region may be one naturally associated with the nucleotide sequence or may be from a different gene, such as the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal and enhancer sequence).

[0270] In some aspects, additional nucleotide sequences, such as those encoding signal sequences, nuclear localization signals, expression enhancers, etc., may be operably linked to the nucleic acid sequence encoding the transgene.

[0271] Unless otherwise specified, methods known to those skilled in the art can be used to construct lentiviral constructs, vectors, and transiently and stably trans-acting packaging cells. Such techniques are known to those skilled in the art. For example, see SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); AUSUBEL et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0272] In some embodiments, methods according to the present disclosure include transfecting a cell with a transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and AAV cap gene, and an adenovirus helper plasmid. In some embodiments, the transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct comprises the nucleic acid sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, or nucleotides 1-4977 of SEQ ID NO:8. In some embodiments, the plasmid containing the AAV rep gene and AAV cap gene comprises the AAV2 rep gene and the rAAVrh74 cap gene. In some embodiments, the adenovirus helper plasmid comprises the adenovirus 5 E2A, E4ORF6, and VA RNA genes.

[0273] In some embodiments, the method further comprises isolating the produced viral particles.The viral vector replicates inside the cell, thereby amplifying and producing viral particles.Virus infection causes the lysis of transfected cells.Therefore, the lytic properties of viral vectors such as AAV allow two different ways of producing and isolating viral particles.The first way is to collect viral particles before cell lysis, and then use external factors to lyse cells.The second way is to collect viral particles from the supernatant after the produced virus has almost completely lysed cells.

[0274] Methods that can be used for active cell lysis are known to those of skill in the art, in some embodiments, cells can be lysed by freeze-thawing, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, combinations of the above, etc.

[0275] In some embodiments, cells can be lysed using at least one detergent. In some embodiments, detergents can include anionic, cationic, zwitterionic, and nonionic detergents. In some embodiments, the detergent concentration can be about 0.1% to 5% (w / w). In some embodiments, the detergent can be Triton® X-100.

[0276] In some embodiments, a nuclease can be used to remove contaminating nucleic acid, i.e., native nucleic acid from the transfected cells. In some embodiments, the nuclease can be BENZONASE®, PULMOZYME®, or any other DNase and / or ribonuclease (RNase) commonly used in the art.

[0277] Methods for harvesting or isolating viral vectors from transfected cells are extensively disclosed in WO2005 / 080556, which is incorporated herein by reference in its entirety.

[0278] In some embodiments, the viral vector is collected or isolated about 24 to 120 hours, about 36 to 108 hours, about 48 to 96 hours, or about 60 to 84 hours after transfection. In some embodiments, the vector is collected or isolated about 72 hours after transfection.

[0279] In some embodiments, the isolated virus particles can be further purified. In some embodiments, the purification of virus particles can be carried out in several steps, including clarification, ultrafiltration, diafiltration, or chromatographic separation. Such methods are described in WO2005 / 080556, the entire contents of which are incorporated herein by reference. In some embodiments, clarification can be carried out by a filtration step to remove cell debris and other impurities from the cell lysate. In some embodiments, ultrafiltration is used to concentrate the virus solution. In some embodiments, diafiltration, buffer exchange, or ultrafiltration can be used to remove and exchange salts, sugars, etc. Those skilled in the art know how to find the optimal conditions for each purification step.

[0280] In some embodiments, purification can be achieved by density gradient centrifugation. In some embodiments, purification involves at least one chromatography step. In some embodiments, viral vectors can be purified by anion exchange chromatography, size exclusion chromatography, or a combination thereof. Methods of Treating Muscular Dystrophy (e.g., DMD)

[0281] The present disclosure provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the rAAV is administered in an amount of about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 15The method includes administering the rAAV to a mammalian animal via a systemic route at a dose of about 1.33×10 vg / kg, wherein the rAAV is produced in mammalian adherent cells, and the adherent cells are cultured under suspension conditions in an N-1 container. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some embodiments, the rAAV is administered .... 14 In some embodiments, the rAAV is administered by systemic administration at a dose of about 1.33 x 10 vg / kg. 14 In some embodiments, the rAAV is administered by intravenous (IV) infusion at a dose of 1000 mg / kg. In some embodiments, the rAAV is administered as a single infusion by a systemic route. In some embodiments, the rAAV is administered as multiple infusions by a systemic route. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. The terms "human subject" and "human patient" are used interchangeably herein.

[0282] The present disclosure also provides a method for treating muscular dystrophy in a human subject in need thereof, comprising administering to the human subject a composition comprising an rAAV described herein. In some aspects, the composition is produced by the methods described herein. In some aspects, the rAAV is administered systemically, at a dose of about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 15 In some embodiments, the systemic route of administration is intravenous, and the dose of rAAV administered is about 2×10 14 In some embodiments, the systemic administration route is intravenous and the dose of rAAV administered is about 1.33 x 10 14 vg / kg.

[0283] In some embodiments, the rAAV is administered to a human subject weighing less than 70 kg. In some embodiments, the rAAV is administered to a human subject weighing more than 70 kg. In some embodiments, the rAAV is administered to a human subject weighing less than 70 kg at a dose of about 1.33 x 10 14In some embodiments, the rAAV is administered intravenously at a single dose of about 9.31 x 10 vg / kg to a human subject weighing more than 70 kg. 15 It is administered by the intravenous route at a fixed total dose of vg.

[0284] In some embodiments, the dose of rAAV is administered at a concentration of about 10 mL / kg. In some embodiments, the rAAV is administered by injection, infusion, or implantation. In some embodiments, the rAAV is administered by infusion over approximately 1 hour. In some embodiments, the rAAV is administered intravenously through a peripheral vein in the limb.

[0285] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0286] In some embodiments, after rAAV is administered, the micro-dystrophin gene expression level in the cell of object is increased compared with the micro-dystrophin gene expression level before rAAV is administered.In some embodiments, after rAAV is administered, the micro-dystrophin gene expression level in the muscle cell of object is increased compared with the micro-dystrophin gene expression level before rAAV is administered.In some embodiments, after rAAV is administered, the micro-dystrophin gene expression level in the cardiomyocyte of object is increased compared with the micro-dystrophin gene expression level before rAAV is administered.

[0287] In some embodiments, the expression of the microdystrophin gene in cells is detected by measuring microdystrophin protein levels by Western blot in muscle biopsied before and after administration of rAAV. In some embodiments, the expression is at least 55.4% after administration of rAAV compared to before administration.

[0288] In some embodiments, the average percentage of microdystrophin-positive fibers in the muscle tissue of the subject increases after administration of rAAV compared with the number of microdystrophin-positive fibers before administration of rAAV.In some embodiments, the average percentage of microdystrophin-positive fibers is at least 70.5%, and the average intensity detected by immunofluorescence (IF) for muscle biopsy specimens before and after administration of rAAV is at least 116.9%.In some embodiments, the microdystrophin transduction by vector genome counting is at least 3.87 average vector genome copies per nucleus.

[0289] In some embodiments, the average percentage of centrally nucleated positive fibers in the subject's muscle tissue is decreased after administration of the rAAV compared to the number of centrally nucleated positive fibers before administration of the rAAV.

[0290] In some embodiments, the average percentage of microdystrophin-positive fibers in the subject's myocardial tissue is increased after administration of the rAAV compared to the number of microdystrophin-positive fibers before administration of the rAAV. Glucocorticoids

[0291] In some embodiments, subjects treated with the rAAVs described herein have been receiving a stable weekly oral glucocorticoid equivalent dose for at least 12 weeks prior to rAAV treatment (e.g., daily or on weekends, as opposed to 10 days on / 10 days off). In some embodiments, the glucocorticoid dose after rAAV administration remains constant (except for modifications to accommodate changes in body weight). In some embodiments, subjects treated with the rAAVs described herein are non-ambulatory, and the subjects are not receiving glucocorticoids. In some embodiments, subjects treated with the rAAVs described herein may not yet require long-term steroid use for the treatment of DMD. Pre-infusion glucocorticoids

[0292] In some embodiments, subjects treated with the rAAVs described herein are treated with 1 mg / kg / day of oral glucocorticoids (prednisone or prednisolone) for immunosuppression in addition to the subject's stable baseline oral glucocorticoid for DMD prior to treatment with the rAAVs described herein.

[0293] In some embodiments, subjects treated with the rAAV described herein are steroid-naive for DMD and are started on oral glucocorticoids (prednisone or prednisolone) at 1.5 mg / kg / day for immunosuppression one week prior to rAAV infusion. Post-infusion glucocorticoid

[0294] In some embodiments, subjects treated with the rAAV described herein maintain a stable baseline oral glucocorticoid dose for DMD and additionally receive 1 mg / kg / day of oral glucocorticoid (prednisone or prednisolone) for immunosuppression. In some embodiments, subjects receive an additional 1 mg / kg / day of oral glucocorticoid for the first 60 days after infusion of the rAAV.

[0295] In some embodiments, subjects treated with the rAAVs described herein have not previously been treated with glucocorticoids, are initiated on oral glucocorticoids at 1.5 mg / kg / day prior to rAAV treatment, and are continued on oral glucocorticoids at a dose of 1.5 mg / kg / day for the first 60 days after rAAV infusion for immunosuppression.

[0296] In some embodiments, if the GGT level is confirmed to be 150 U / L or higher after infusion or if other clinically significant liver function abnormalities are observed, the oral glucocorticoid dose is adjusted. In some embodiments, if the subject is receiving 1 mg / kg of additional steroids for immunosuppression in addition to steroids for DMD, this dose can be increased to 2 mg / kg of additional steroids for immunosuppression in addition to steroids for DMD. In some embodiments, if the subject is receiving a fixed dose of 60 mg / day, this dose can be increased to 120 mg / day. In some embodiments, if the subject is receiving 1.5 mg / kg / day of immunosuppression, this dose can be increased to 2.5 mg / kg / day.

[0297] In some embodiments, if a subject develops severe or severe elevations in liver biochemistry (including GGT, bilirubin, and ALT compared to baseline) or elevations that are unresponsive to increasing oral steroids, the subject receives an IV bolus of steroids. Genotyping the DMD gene in the subject

[0298] In some embodiments, the subject has been genotyped for at least one mutation in the DMD gene. In some embodiments, the subject's human dystrophin (DMD) gene is genotyped prior to treatment (i.e., prior to administering an rAAV described herein). In some embodiments, the subject has been genotyped for at least one mutation in exons 18-79 of the DMD gene. In some embodiments, the at least one mutation is a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the absence of expression of human dystrophin protein. In some embodiments, detecting at least one of these mutations in the DMD gene identifies the subject as eligible to receive a composition described herein.

[0299] In some embodiments, genotyping of a subject's DMD gene identifies subjects who should be contraindicated from undergoing rAAV gene therapy as described herein. For example, subjects with a deletion that completely encompasses exons 9-13 of the DMD gene should be contraindicated from undergoing rAAV gene therapy. In another example, subjects with a deletion (i.e., any deletion) in exons 8 and / or 9 of the DMD gene should be contraindicated from undergoing rAAV gene therapy.

[0300] In some embodiments, the at least one mutation in the DMD gene is a mutation in exons 1-17, an in-frame deletion, an in-frame duplication, a variant of unknown clinical significance (VUS), or a mutation entirely contained within exon 45, thereby identifying the subject as ineligible to receive the compositions described herein.

[0301] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles encapsidating an expression cassette comprising a human microdystrophin transgene, provided that the subject does not have a deletion that completely encompasses exons 9-13 of the DMD gene.

[0302] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles encapsidating an expression cassette comprising a human microdystrophin transgene, provided that the subject does not have a deletion in exons 8 and / or 9 of the DMD gene.

[0303] In some embodiments, the subject's DMD gene is genotyped prior to treatment.

[0304] In some embodiments, the AAV viral particles are of serotype rh74.

[0305] In some embodiments, the rAAV vector is administered as a composition comprising: a) rh74 serotype AAV viral particles encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rh74 serotype AAV viral particles encapsidating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rh74 serotype AAV viral particles encapsidating nucleotides 1-4977 of SEQ ID NO:8.

[0306] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, the method comprising: i) genotyping the subject's human dystrophin (DMD) gene prior to treatment; and ii) providing that genotyping does not identify a deletion entirely encompassing exons 9-13 of the DMD gene, administering to the subject a composition comprising the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the composition comprises: a) rAAV particles encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsidating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsidating nucleotides 1-4977 of SEQ ID NO:8.

[0307] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, the method comprising: i) genotyping the subject's human dystrophin (DMD) gene prior to treatment; and ii) providing that genotyping does not identify a deletion in exons 8 and / or 9 of the DMD gene, administering to the subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the composition comprises: a) rAAV particles encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsidating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsidating nucleotides 1-4977 of SEQ ID NO:8.

[0308] In some embodiments, the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin administered to the subject is produced by any of the methods described herein.

[0309] The present disclosure also provides for the use of the compositions described herein for the treatment of muscular dystrophy in a human subject in need thereof. In some aspects, the present disclosure also provides for the use of the compositions described herein in the manufacture of a medicament for the treatment of muscular dystrophy.

[0310] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0311] For example, the dose of rAAV administered is about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 13 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 1.0 × 10 13vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 5.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×1015 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg~6.0×10 14 , or approximately 1.25 × 10 14 vg / kg~5.0×10 14 , or approximately 1.25 × 10 14 vg / kg~4.0×10 14 , or approximately 1.25 × 10 14 vg / kg~1.0×10 15 , or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.5 × 10 14 vg / kg~6.0×10 14 , or approximately 1.5 × 10 14 vg / kg~5.0×10 14 , or approximately 1.5 × 1014 vg / kg~4.0×10 14 , or approximately 1.5 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.75 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.75 × 10 14 vg / kg~6.0×10 14 , or approximately 1.75 × 10 14 vg / kg~5.0×10 14 , or approximately 1.75 × 10 14 vg / kg~4.0×10 14 , or approximately 1.75 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.75 × 10 14vg / kg ~ approx. 2.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg~1.0×10 15 , or approximately 2.0 × 10 14 vg / kg~6.0×10 14 , or approximately 2.0 × 10 14 vg / kg~5.0×10 14 , or approximately 2.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg.

[0312] In some embodiments, the human subject is between about 2 and less than 3 years old. In some embodiments, the human subject is about 2 years old, about 2.25 years old, about 2.5 years old, or about 2.75 years old.

[0313] In some embodiments, the human subject is between 2 and 3 years old.

[0314] In some embodiments, the human subject is 2 years old. In some embodiments, the human subject is 3 years old. In some embodiments, the human subject is between about 4 and less than 8 years old. In some embodiments, the human subject is about 4 years old, about 4.25 years old, about 4.5 years old, about 4.75 years old, about 5 years old, about 5.25 years old, about 5.5 years old, about 5.75 years old, about 6 years old, about 6.25 years old, about 6.5 years old, about 6.75 years old, about 7 years old, about 7.25 years old, about 7.5 years old, or about 7.75 years old.

[0315] In some embodiments, the human subject is between 4 and 5 years old.

[0316] In some embodiments, the human subject is between about 8 years old and under 18 years old. In some embodiments, the human subject is between about 8 years old, about 8.25 years old, about 8.5 years old, about 8.75 years old, about 9 years old, about 9.25 years old, about 9.5 years old, about 9.75 years old, about 10 years old, about 10.25 years old, about 10.5 years old, about 10.75 years old, about 11 years old, about 11.25 years old, about 11.5 years old, about 11.75 years old, about 12 years old, about 12.25 years old, about 12.5 years old, about 12. 75 years old, about 13 years old, about 13.25 years old, about 13.5 years old, about 13.75 years old, about 14 years old, about 14.5 years old, about 14.75 years old, about 15 years old, about 15.25 years old, about 15.5 years old, about 15.75 years old, about 16 years old, about 16.25 years old, about 16.5 years old, about 16.75 years old, about 17 years old, about 17.25 years old, about 17.5 years old, or about 17.75 years old.

[0317] In one aspect, the method of the disclosure comprises systemically administering rAAV, wherein the systemic administration route is intravenous and the dose of rAAV administered is about 2.0 x 10 14 In some aspects, the methods of the present disclosure include systemically administering rAAV, wherein the systemic administration route is intravenous and the dose of rAAV administered is about 1.33 x 10 14 In another aspect, the method of the present disclosure includes systemically administering rAAV, wherein the systemic administration route is intravenous and the dose of rAAV administered is about 5.0 x 10 12 vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 10 12 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13 vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 10 13 vg / kg, or approximately 3.0 × 10 13vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.33 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14 vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 10 14 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14 vg / kg, or approximately 1 × 10 15 vg / kg. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0318] In any of the methods of the present disclosure, the dose of rAAV can be administered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the dose of rAAV is administered at about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.microdystrophin, nucleotides 56-4820 of SEQ ID NO:5.

[0319] In any of the methods of the present disclosure, the dose of rAAV can be administered by injection, infusion, or implantation. For example, the dose of rAAV can be administered by infusion over approximately one hour. Additionally, the dose of rAAV can be administered intravenously through a peripheral vein in a limb, such as a peripheral vein in the arm or a peripheral vein in the leg. Alternatively, the infusion can be administered over approximately 30 minutes, approximately 1.5 hours, approximately 2 hours, approximately 2.5 hours, or approximately 3 hours. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin set forth in SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0320] The rAAV administered by any of the methods of the disclosure may comprise the human microdystrophin nucleotide sequence of SEQ ID NO: 1, the MHCK7 promoter sequence of SEQ ID NO: 2, or SEQ ID NO: 7. Additionally, the rAAV administered by any of the methods of the disclosure may comprise the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. For example, the rAAV may comprise the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6.

[0321] In one aspect, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0322] In any of the methods of the disclosure, the rAAV administered is of the serotype AAVrh74.

[0323] In some embodiments, the methods of the present disclosure treat Duchenne muscular dystrophy or Becker muscular dystrophy. An exemplary embodiment is a method of treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, comprising administering a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the route of administration is intravenous infusion, and the dose of rAAV administered is about 2 x 10 over approximately one hour.14 vg / kg, and wherein the rAAV vector comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, or AAVrh74.MHCK7.microdystrophin of nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0324] In any of the methods for treating muscular dystrophy, the level of microdystrophin gene expression in the subject's cells is increased after administration of rAAV. In any of the methods for treating muscular dystrophy, the level of microdystrophin gene expression in the subject's muscle cells is increased after administration of rAAV. In any of the methods for treating muscular dystrophy, the level of microdystrophin gene expression in the subject's cardiac muscle cells is increased after administration of rAAV. Expression of the microdystrophin gene in cells, e.g., muscle cells or cardiac muscle cells, is detected by measuring microdystrophin protein levels by Western blot in muscle biopsied before and after administration of rAAV. In particular, the level of microdystrophin protein is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of rAAV compared to the level of microdystrophin before administration of rAAV. For example, the level of microdystrophin protein is increased after administration of the rAAV by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% compared to the level of microdystrophin before administration of the rAAV.

[0325] Furthermore, expression of the microdystrophin gene in cells is detected by measuring microdystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of rAAV. Microdystrophin protein levels are increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of rAAV compared to microdystrophin levels before administration of rAAV. For example, microdystrophin protein levels are increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of rAAV compared to microdystrophin levels before administration of rAAV.

[0326] In any of the methods for treating muscular dystrophy, the serum CK level in the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the rAAV. For example, the serum CK level in the subject is reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% by 60 days after administration of the rAAV compared to the serum CK level before administration of the rAAV. In particular, in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 87% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 72% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 73% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 78% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 95% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV. In any of the methods for treating muscular dystrophy, the number of microdystrophin-positive fibers in the muscle tissue of object increases after administration of rAAV compared with the number of microdystrophin-positive fibers before administration of rAAV.In any of the methods for treating muscular dystrophy described herein, the number of microdystrophin-positive fibers in the myocardial tissue of object increases after administration of rAAV compared with the number of microdystrophin-positive fibers before administration of rAAV.For example, the number of microdystrophin-positive fibers is detected by measuring microdystrophin protein level by Western blot or immunohistochemistry for muscle biopsy specimens, including myocardial biopsy specimens before and after administration of rAAV.

[0327] In any of the methods for treating muscular dystrophy, as described herein, administration of rAAV upregulates the expression of DAPC proteins, such as alpha-sarcoglycan or beta-sarcoglycan.For example, the level of alpha-sarcoglycan in a subject increases after administration of rAAV compared to the level of alpha-sarcoglycan before administration of rAAV.Furthermore, the level of beta-sarcoglycan in a subject increases after administration of rAAV compared to the level of beta-sarcoglycan before administration of rAAV.The level of alpha-sarcoglycan or beta-sarcoglycan is detected by measuring the level of alpha-sarcoglycan or beta-sarcoglycan protein by Western blot or immunohistochemistry on muscle biopsy specimens before and after administration of rAAV.

[0328] In any of the methods of treating muscular dystrophy, disease progression in the subject is slowed after administration of the rAAV, as measured by any of the following: 6-minute walk test, time to rise, 4-step stair climbing, 4-step stair climbing, North Star Ambulation Assessment (NSAA), 10 meter timed test, 100 meter timed test, Handheld Dynamometer Testing (HHD), Timed Up and Go, Gross Motor Subtest Scaled (Bayley-III) score, and / or Gross and Fine Motor scale (Bayley-IV).

[0329] For example, in any of the methods, the subject's NSAA score improves by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 points at least 270 days after administration of the rAAV compared to their NSAA score before administration of the rAAV. Additionally, in any of the methods, the subject's rise time improves by at least about 0.8 seconds at least 270 days after administration of the rAAV compared to their rise time before administration of the rAAV. Further, in any of the methods, the subject experiences an improvement of at least about 1.2 seconds on the timed 4-step climb test at least 270 days after administration of the rAAV compared to the timed 4-step climb test before administration of the rAAV. Further, in any of the methods, the subject experiences an improvement of at least about 7 seconds on the 100-m timed test at least 270 days after administration of the rAAV compared to the 100-m timed test before administration of the rAAV.

[0330] In another aspect, the disclosure provides a method of expressing a micro-dystrophin gene in patient cells, comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, micro-dystrophin gene expression in patient cells is detected by measuring micro-dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. Additionally, micro-dystrophin gene expression in the patient is measured by detecting a higher number of vector genomes per nucleus, where one vector genome per nucleus is approximately 50% micro-dystrophin expression and greater than one copy per nucleus is consistent with micro-dystrophin expression levels. For example, the cells have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0331] In some aspects, the disclosure provides methods of reducing serum CK levels in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, by 60 days after administration of the rAAV, the serum CK level in the patient is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, compared to the serum CK level before administration of the rAAV. In particular, serum CK levels in the subject are reduced by about 87% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 72% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 73% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 78% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV, or in any of the methods of treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 95% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV.

[0332] The present disclosure also provides methods for increasing microdystrophin-positive fibers in muscle tissue of a patient, the method comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In some aspects, the present disclosure provides methods for increasing microdystrophin-positive fibers in myocardial tissue of a patient, the method comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, the number of microdystrophin-positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry on muscle biopsies, including myocardial biopsies, before and after administration of the rAAV. Furthermore, the expression of micro-dystrophin gene in patients is measured by detecting a higher number of vector genomes per nucleus, where 1 vector genome per nucleus is about 50% micro-dystrophin expression, and more than 1 copy per nucleus corresponds to the micro-dystrophin expression level.For example, cells have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0333] In another aspect, the disclosure provides a method of increasing alpha-sarcoglycan expression in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, alpha-sarcoglycan levels are detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the rAAV.

[0334] Additionally, the present disclosure provides a method of increasing beta-sarcoglycan expression in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, beta-sarcoglycan levels are detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the rAAV.

[0335] The disclosure also provides a method of treating a patient having Duchenne muscular dystrophy or Becker muscular dystrophy, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, wherein disease progression in the patient is slowed as measured by any of the following: 6-minute walk test, stand time, 4-step stair climbing, 4-step stair climbing, North Star Gait Assessment (NSAA), 10-meter timed test, 100-meter timed test, Handheld Dynamometer Strength Test (HHD), Timed Up and Go, scaled gross motor subtest (Bayley-III) score, and / or gross and fine motor scale (Bayley-IV).

[0336] For example, in any of the methods, the subject's NSAA score improves by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 points at least 270 days after administration of the rAAV compared to the NSAA score before administration of the rAAV. Further, in any of the methods, the subject's rise time improves by at least about 0.8 seconds at least 270 days after administration of the rAAV compared to the rise time before administration of the rAAV. Further, in any of the methods, the subject experiences an improvement of at least about 1.2 seconds on the timed 4-step climb test at least 270 days after administration of the rAAV compared to the timed 4-step climb test before administration of the rAAV. Further, in any of the methods, the subject experiences an improvement of at least about 7 seconds on the 100-m timed test at least 270 days after administration of the rAAV compared to the 100-m timed test before administration of the rAAV.

[0337] "Fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues, including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas, upon injury. The deposited ECM components include fibronectin and collagen, such as type 1, type 2, or type 3 collagen.

[0338] The present disclosure also provides a method of reducing or preventing fibrosis in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of nucleotides 55-5021 of SEQ ID NO:3. In another aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9. In another embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin from nucleotides 1 to 4977 of SEQ ID NO:8 or nucleotides 56 to 5066 of SEQ ID NO:6. In a further embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0339] In another aspect, the present disclosure provides a method of preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or the AAV74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, any of the rAAVs of the present disclosure can be administered to a subject suffering from muscular dystrophy to prevent fibrosis, e.g., an rAAV of the present disclosure expressing a human microdystrophin protein can be administered before fibrosis is observed in the subject. Additionally, an rAAV of the present disclosure expressing a human microdystrophin gene can be administered to a subject at risk of developing fibrosis, e.g., a subject suffering from or diagnosed with muscular dystrophy, e.g., DMD. The rAAV of the present disclosure can be administered to subjects suffering from muscular dystrophy to prevent new fibrosis in these subjects.

[0340] The present disclosure contemplates administering rAAV before fibrosis is observed in subjects.In addition, rAAV can be administered to subjects at risk of developing fibrosis, such as subjects who suffer from or have been diagnosed with muscular dystrophy, such as DMD.rAAV can be administered to subjects who suffer from muscular dystrophy and have already developed fibrosis, to prevent new fibrosis in these subjects.

[0341] The present disclosure also provides a method of increasing muscular force and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0342] The present disclosure contemplates administering an rAAV vector to a subject diagnosed with DMD before fibrosis is observed in the subject or before the subject experiences reduced muscle force or muscle mass.

[0343] The present disclosure also contemplates administering an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 to subjects with muscular dystrophy in which fibrosis has already occurred to prevent new fibrosis in these subjects or to reduce fibrosis in these subjects. The disclosure also provides for administering an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 to a subject already suffering from muscular dystrophy who has reduced muscle force or muscle mass to protect the muscle from injury.

[0344] The present disclosure also provides methods of treating cardiomyopathy in a human subject with muscular dystrophy (e.g., DMD), comprising administering to the human subject any of the compositions described herein (e.g., delan dystrogen moxeparvovec). In some embodiments, the methods are used to improve cardiac function in a subject with DMD. In some embodiments, the methods are used to reduce myocardial fibrosis. In some embodiments, the methods are used to reduce myocardial central nucleation. In some embodiments, cardiac function is improved by, for example, an increase in ejection fraction (EF); an increase in fractional shortening (FS); an increase in cardiac output; a decrease in left ventricular diameter in diastolic (LVIDd); a decrease in left ventricular end-systolic diameter (LVESD); a decrease in systolic volume; an increase in systolic left ventricular anterior wall thickness; an increase in systolic left ventricular posterior wall thickness; an increase in sarcomere length peak height; an increase in sarcomere length shortening; a decrease in time to reach 90% of baseline sarcomere length; Ca2+ + Increased peak height of transients; baseline Ca2 + The improvement is achieved by shortening the time to reach 90% of the transient; and / or maintaining or reducing serum troponin blood level. In any of the methods of the present disclosure, the subject may suffer from muscular dystrophy, such as DMD, or any other dystrophin-related muscular dystrophy.

[0345] In other embodiments of any of the methods of the disclosure described herein, the serum CK level in the subject is increased after administration of the rAAV compared to the serum CK level before administration of the rAAV: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, or 85% by 270 days post-dose; c) at least 72%, 73%, 74%, or 95% by 180 days post-dose; d) at least 87%, 88%, 93%, or 95% by 90 days post-dose; e) at least 70% by 270 days post-dose; f) 70–95% by 90, 180, or 270 days after administration; g) at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% by 90, 180, or 270 days after administration; and h) At least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% improvement by 90, 180, or 270 days after administration. The decrease is at a percentage level selected from the group consisting of:

[0346] In another aspect, the disclosure provides a composition for treating muscular dystrophy in a human subject in need thereof, comprising a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, formulated for a systemic route of administration, wherein the dose of rAAV is about 1 x 10 14 vg / kg ~ approx. 4×10 14 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin at SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin at nucleotides 56-4820 of SEQ ID NO:5.

[0347] For example, the composition of the present disclosure may be about 5.0×10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 13 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 5.0 × 10 13vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 5.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg~6.0×10 14 , or approximately 1.25 × 10 14 vg / kg~5.0×10 14 , or approximately 1.25 × 10 14 vg / kg~4.0×10 14, or approximately 1.25 × 10 14 vg / kg~1.0×10 15 , or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.5 × 10 14 vg / kg~6.0×10 14 , or approximately 1.5 × 10 14 vg / kg~5.0×10 14 , or approximately 1.5 × 10 14 vg / kg~4.0×10 14 , or approximately 1.5 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.5 × 10 14vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.75 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.75 × 10 14 vg / kg~6.0×10 14 , or approximately 1.75 × 10 14 vg / kg~5.0×10 14 , or approximately 1.75 × 10 14 vg / kg~4.0×10 14 , or approximately 1.75 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg~1.0×10 15 , or approximately 2.0 × 10 14 vg / kg~6.0×10 14 , or approximately 2.0 × 10 14 vg / kg~5.0×10 14 , or approximately 2.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.5×10 14vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 The rAAV comprises a dose of rAAV of 1000 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin at nucleotides 55-5021 of SEQ ID NO:9, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin at nucleotides 56-4820 of SEQ ID NO:5.

[0348] In one embodiment, the composition of the present disclosure is formulated for intravenous administration and contains about 2.0 x 10 14 In one embodiment, the composition of the present disclosure is formulated for intravenous administration and comprises a dose of rAAV that is about 1.33 x 10 14 In another embodiment, the compositions of the present disclosure are formulated for intravenous administration and comprise a dose of rAAV that is about 5.0 x 10 12 vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 10 12 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13 vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 10 13 vg / kg, or approximately 3.0 × 10 13 vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14 vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 10 14 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14 vg / kg, or approximately 1 × 10 15 The dose of rAAV is

[0349] In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In another aspect, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0350] In any of the compositions of the present disclosure, the dose of rAAV is delivered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the composition comprises a dose of rAAV delivered at about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In another embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0351] The composition of the present disclosure is formulated for administration by injection, infusion or implantation.For example, the composition is formulated for administration by infusion over approximately 1 hour.In addition, the composition of the present disclosure is formulated for intravenous administration through peripheral veins of limbs, such as peripheral veins of the arm or peripheral veins of the leg.Alternatively, the infusion can be carried out over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours or approximately 3 hours.

[0352] Any of the compositions of the disclosure comprises an rAAV comprising an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0353] In particular, the compositions of the present disclosure are used to treat Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present disclosure provides a composition for treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, comprising a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, formulated for administration by intravenous infusion over approximately one hour, wherein the administered dose of rAAV is about 2 x 10 14 vg / kg, and the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0354] In another aspect, the present disclosure also provides a composition comprising an rAAV vector for reducing fibrosis in a subject in need thereof. Additionally, the present disclosure provides a composition comprising an rAAV vector for preventing fibrosis in a subject suffering from muscular dystrophy.

[0355] The present disclosure also provides compositions comprising an rAAV for increasing muscular force and / or muscle mass in a subject suffering from muscular dystrophy. In a further aspect, the present disclosure provides compositions comprising any of the rAAVs of the present disclosure for the treatment of muscular dystrophy.

[0356] In other embodiments of any of the compositions of the present disclosure, after administration of the composition to a human subject in need of treatment for muscular dystrophy, the serum CK level in the subject is increased, compared to the serum CK level before administration of the composition: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46%, 55%, 70%, or 85% by 270 days post-dose; c) at least 72%, 73%, 74%, or 95% by 180 days post-dose; d) at least 87%, 99%, 93%, or 95% by 90 days post-dose; e) at least 70% by 270 days post-dose; f) 70–95% by 90, 180, or 270 days after administration; g) at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% by 90, 180, or 270 days after administration; and h) 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% by 90, 180, or 270 days after administration The decrease is at a percentage level selected from the group consisting of:

[0357] In another aspect, the disclosure provides a use of a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin for the preparation of a medicament for the treatment of muscular dystrophy in a human subject in need thereof, wherein the medicament is formulated for a systemic route of administration and the dose of rAAV is about 1 x 10 14 vg / kg ~ approx. 4×101 4vg / kg. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin at nucleotides 55-5021 of SEQ ID NO:9, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin at nucleotides 56-4820 of SEQ ID NO:5.

[0358] For example, the drug is about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 13 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 5.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 1014 vg / kg ~ approx. 2.5×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg~6.0×10 14 , or approximately 1.25 × 10 14 vg / kg~5.0×10 14 , or approximately 1.25 × 10 14 vg / kg~4.0×10 14 , or approximately 1.25 × 10 14 vg / kg~1.0×10 15 , or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.5 × 10 14 vg / kg~6.0×10 14 , or approximately 1.5 × 10 14 vg / kg~5.0×10 14 , or approximately 1.5 × 10 14 vg / kg~4.0×10 14 , or approximately 1.5 × 10 14 vg / kg ~ approx. 3.75×10 14vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.75 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.75 × 10 14 vg / kg~6.0×10 14 , or approximately 1.75 × 10 14 vg / kg~5.0×10 14 , or approximately 1.75 × 10 14 vg / kg~4.0×10 14 , or approximately 1.75 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.0×10 14vg / kg, or approximately 2.0 × 10 14 vg / kg~1.0×10 15 , or approximately 2.0 × 10 14 vg / kg~6.0×10 14 , or approximately 2.0 × 10 14 vg / kg~5.0×10 14 , or approximately 2.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 The rAAV comprises a dose of rAAV of 1000 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin at nucleotides 55-5021 of SEQ ID NO:9, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin at nucleotides 56-4820 of SEQ ID NO:5.

[0359] In one aspect, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, wherein the systemic administration route is intravenous and the dose of rAAV administered is about 2.0 x 10 14 In another embodiment, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, wherein the systemic administration route is intravenous and the dose of rAAV is about 5.0 x 10 12 vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 1012 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13 vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 10 13 vg / kg, or approximately 3.0 × 10 13 vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.33 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14 vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 10 14 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14 vg / kg, or approximately 1 × 10 15 vg / kg.

[0360] In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0361] In any of the uses of the present disclosure, the medicament comprises a dose of rAAV of about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the dose of rAAV is about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0362] In any of the uses of the present disclosure, the medicament is formulated for administration by injection, infusion or implantation.For example, the medicament is formulated for administration by infusion over approximately 1 hour.Furthermore, the medicament is formulated for intravenous administration through peripheral veins of limbs, such as peripheral veins of the arm or peripheral veins of the leg.Alternatively, the infusion can be carried out over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours or approximately 3 hours.

[0363] In any of the uses of the disclosure, the medicament comprises an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0364] A particular use of the present disclosure is for the preparation of a medicament for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present disclosure provides the use of a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin for the preparation of a medicament for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the medicament is formulated for administration by intravenous infusion over approximately one hour, and the dose of rAAV administered is about 2 x 10 14 vg / kg, and the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6.

[0365] In a further aspect, the present disclosure provides the use of rAAV for the preparation of a medicament for reducing fibrosis in a subject in need thereof.For example, the subject in need thereof may suffer from muscular dystrophy, such as DMD or any other dystrophin-related muscular dystrophy.

[0366] In another aspect, the present disclosure provides a use of rAAV for the preparation of a medicament for preventing fibrosis in a subject suffering from muscular dystrophy.

[0367] Additionally, the present disclosure provides the use of rAAV for the preparation of a medicament for increasing muscular strength and / or muscle mass in a subject suffering from muscular dystrophy.

[0368] The present disclosure also provides the use of rAAV for the preparation of a medicament for the treatment of muscular dystrophy.

[0369] The present disclosure provides for the use of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7 for the preparation of a medicament for the treatment of muscular dystrophy, or an rAAV vector comprising the AAVrf74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the treatment of muscular dystrophy.

[0370] In other embodiments of any of the uses of the present disclosure, the serum CK level in the subject is increased after administration of the rAAV to the subject, compared to the serum CK level before administration of the rAAV: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46%, 55%, 70%, or 95% by 270 days post-dose; c) at least 72%, 73%, 74%, or 95% by 180 days post-dose; d) at least 87%, 88%, 93%, or 95% by 90 days post-dose; e) at least 70% by 270 days post-dose; f) 70–95% by 90, 180, or 270 days after administration; g) at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% by 90, 180, or 270 days after administration; and h) 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% by 90, 180, or 270 days after administration The decrease is at a percentage level selected from the group consisting of:

[0371] In either the use of the composition for treating muscular dystrophy or the medicament for treating muscular dystrophy, the level of microdystrophin gene expression in the subject's cells is increased after administration of the composition or medicament. Expression of the microdystrophin gene in the cells is detected by measuring microdystrophin protein levels by Western blot in muscles biopsied before and after administration of the composition or medicament. In particular, the level of microdystrophin protein is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of the composition or medicament compared to the microdystrophin level before administration of the composition or medicament. For example, the level of microdystrophin protein is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition compared to the level of microdystrophin before administration of the composition or medicament.

[0372] Furthermore, expression of the microdystrophin gene in cells is detected by measuring microdystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of the composition or medicament. Microdystrophin protein levels are increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of the rAAV compared to microdystrophin levels before administration of the composition or medicament. For example, microdystrophin protein levels are increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition or medicament compared to microdystrophin levels before administration of the composition or medicament.

[0373] In any of the compositions for treating muscular dystrophy, the serum CK level in a subject is reduced after administration of the rAAV compared to the serum CK level before administration of the composition or medicament. For example, the serum CK level in a subject is reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament. In particular, in any of the compositions for treating muscular dystrophy of the present disclosure, serum CK levels in the subject are reduced by about 87% by 60 days after administration of the composition or medicament compared to serum CK levels before administration of the composition or medicament, or in any of the compositions for treating muscular dystrophy of the present disclosure, or uses of the medicament for treating muscular dystrophy, serum CK levels in the subject are reduced by about 72% by 60 days after administration of the composition or medicament compared to serum CK levels before administration of the composition or medicament, or in any of the compositions for treating muscular dystrophy of the present disclosure, serum CK levels in the subject are reduced by about 72% by 60 days after administration of the composition or medicament compared to serum CK levels before administration of the composition or medicament. By 60 days, serum CK levels in the subject are reduced by about 73% compared to serum CK levels before administration of the composition or medicament, or in any of the compositions for treating muscular dystrophy or uses of the medicament for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 78% by 60 days after administration of the composition or medicament compared to serum CK levels before administration of the composition, or in any of the compositions for treating muscular dystrophy or uses of the medicament for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 95% by 60 days after administration of the composition or medicament compared to serum CK levels before administration of the composition or medicament. In any of the compositions for treating muscular dystrophy or uses of the medicament for treating muscular dystrophy, the number of microdystrophin-positive fibers in muscle tissue in the subject is increased after administration of the composition or medicament compared to the number of microdystrophin-positive fibers before administration of the composition or medicament.For example, the number of microdystrophin-positive fibers is detected by measuring microdystrophin protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament.

[0374] In either the use of the composition for treating muscular dystrophy or the medicine for treating muscular dystrophy, the administration of the composition or medicine upregulates the expression of DAPC protein, such as alpha-sarcoglycan or beta-sarcoglycan.For example, the level of alpha-sarcoglycan in a subject increases after the administration of the composition or medicine compared with the level of alpha-sarcoglycan before the administration of the composition or medicine.Furthermore, the level of beta-sarcoglycan in a subject increases after the administration of the composition or medicine compared with the level of beta-sarcoglycan before the administration of the composition or medicine.The level of alpha-sarcoglycan or beta-sarcoglycan is detected by measuring the protein level of alpha-sarcoglycan or beta-sarcoglycan by Western blot or immunohistochemistry on muscle biopsy specimens before and after the administration of the composition or medicine.

[0375] In either the composition for treating muscular dystrophy or the use of the medicament for treating muscular dystrophy, disease progression in the subject is slowed after administration of the composition or medicament as measured by any of the following: 6-minute walk test, stand time, 4-step stair climbing, 4-step stair climbing, North Star Gait Assessment (NSAA), 10-meter timed test, 100-meter timed test, Handheld Dynamometer Strength Test (HHD), Timed Up and Go, Scaled Gross Motor Subtest (Bayley-III) score, and / or Gross and Fine Motor Scale (Bayley-IV).

[0376] For example, after administration of any of the compositions for treating muscular dystrophy or use of the medicaments for treating muscular dystrophy, the subject's NSAA score is improved by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 points at least 270 days after administration of the composition or medicament, compared to the NSAA score before administration of the rAAV. Further, in any of the methods, the subject's rise time is improved by at least about 0.8 seconds at least 270 days after administration of the composition or medicament, compared to the rise time before administration of the composition or medicament. Further, in any of the methods or uses of the disclosure, the subject experiences an improvement of at least about 1.2 seconds in the timed 4-step climb test at least 270 days after administration of the composition or medicament, compared to the timed 4-step climb test before administration of the composition or medicament. Further, in any of the methods or uses of the disclosure, the subject experiences an improvement of at least about 7 seconds in the 100m timed test at least 270 days after administration of the composition or medicament, compared to the 100m timed test before administration of the composition or medicament.

[0377] In another aspect, the disclosure provides a composition for expressing a micro-dystrophin gene in patient cells, comprising the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In a further aspect, the disclosure provides use of a dose of the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, for the preparation of a medicament for expressing a micro-dystrophin gene in patient cells. For example, expression of the micro-dystrophin gene in patient cells is detected by measuring micro-dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. Furthermore, the expression of micro-dystrophin gene in patients is measured by detecting a higher number of vector genomes per nucleus, where 1 vector genome per nucleus is about 50% micro-dystrophin expression, and more than 1 copy per nucleus corresponds to the micro-dystrophin expression level.For example, cells have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0378] In a further aspect, the disclosure provides a composition for reducing serum CK levels in a patient in need thereof, the composition comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO: 6. Additionally, the disclosure provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for reducing serum CK levels in a patient in need thereof. For example, the serum CK level in the patient is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% by 60 days after administration of the composition or medicament, compared to the serum CK level before administration of the composition or medicament. In particular, the serum CK level in the subject is reduced by about 87% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 72% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 73% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 78% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 95% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament.

[0379] The present disclosure also provides a composition for increasing microdystrophin-positive fibers in muscle tissue of a patient, the composition comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. Additionally, the present disclosure provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for increasing microdystrophin-positive fibers in muscle tissue of a patient. For example, the number of microdystrophin-positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament. Furthermore, the expression of micro-dystrophin gene in patients is measured by detecting a higher number of vector genomes per nucleus, where 1 vector genome per nucleus is about 50% micro-dystrophin expression, and more than 1 copy per nucleus corresponds to the micro-dystrophin expression level.For example, cells have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0380] In another aspect, the disclosure provides a composition for increasing alpha-sarcoglycan expression in a patient in need thereof, comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. The disclosure also provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for increasing alpha-sarcoglycan expression in a patient in need thereof. For example, alpha-sarcoglycan levels are detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament.

[0381] Additionally, the present disclosure provides a composition for increasing beta-sarcoglycan expression in a patient in need thereof, comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. The present disclosure also provides use of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for increasing beta-sarcoglycan expression in a patient in need thereof. For example, beta-sarcoglycan levels are detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament.

[0382] The disclosure also provides for the use of a dose of an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, wherein administration of the medicament slows disease progression in the patient as measured by any of the following: 6-minute walk test, stand time, 4-step stair climbing, 4-step stair climbing, North Star Gait Assessment (NSAA), 10-meter timed test, 100-meter timed test, Handheld Dynamometer Strength Test (HHD), Timed Up and Go, scaled gross motor subtest (Bayley-III) score, and / or gross and fine motor scale (Bayley-IV).

[0383] For example, the subject's NSAA score is improved by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 points at least 270 days after administration of the composition or medicament, compared to the NSAA score before administration of the composition or medicament. Furthermore, the subject's rise time is improved by at least about 0.8 seconds at least 270 days after administration of the composition or medicament, compared to the rise time before administration of the composition or medicament. Furthermore, the subject's four-step stair climbing test is improved by at least about 1.2 seconds at least 270 days after administration of the composition or medicament, compared to the four-step stair climbing test before administration of the composition or medicament. Additionally, the subject experiences an improvement of at least about 7 seconds on the 100m timed test at least 270 days after administration of the composition or medicament compared to the 100m timed test prior to administration of the composition or medicament.

[0384] In any of the methods of treating muscular dystrophy in a human subject described herein, the human subject is ambulatory or non-ambulatory. In some embodiments, the human subject is ambulatory. In further embodiments, the ambulatory human subject is 8 years old or older and under 18 years old. In some embodiments, the human subject is non-ambulatory. In some embodiments, the human subject is 2 years old or older and under 3 years old. In some embodiments, the human subject is 2 years old or older and under 3 years old and ambulatory.

[0385] In some embodiments, a human subject treated for muscular dystrophy according to any of the methods described herein is between 4 and 5 years old (i.e., the subject is at least 4 years old and at most 6 years old. In other words, the subject is at least 4 years old and at most less than 6 years old). In some embodiments, a 4- to 5-year-old human subject is ambulatory.

[0386] In some embodiments, a human subject treated for muscular dystrophy according to any of the methods described herein is between 2 and 3 years old (i.e., the subject is at least 2 years old and at most 3 years old. In other words, the subject is at least 2 years old and at most less than 3 years old). In some embodiments, a 2- to 3-year-old human subject is ambulatory.

[0387] In some embodiments, the human subject is non-ambulatory. In some embodiments, the human subject has been non-ambulatory for at least 9 months. In some embodiments, the human subject further has a stable FVC of less than 40% and / or a need for nocturnal ventilator support.

[0388] It should be noted that the Detailed Description section, rather than the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may describe one or more, but not all, exemplary aspects of the present disclosure contemplated by the inventor(s), and are therefore not intended to limit the scope of the disclosure and the appended claims in any way.

[0389] The following examples are offered by way of illustration and not by way of limitation: Numerical ranges recited include each integer value within each range, including the lowest and highest integers specified. [Example]

[0390] Example 1 A) Generation of the AAVrh74.MHCK7.microdystrophin construct The AAVrh74.MHCK7.microdystrophin plasmid contains a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 1). The microdystrophin construct featured an in-frame rod deletion (R4-R23) while retaining hinges 1, 2, and 4 and the cysteine-rich domain, resulting in the production of a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) was driven by the MHCK7 promoter (792 bp). The plasmid was constructed by removing the MCK promoter from the rAAV.MCK.microdystrophin plasmid and inserting the MHCK7 promoter. Following the core promoter are 53 bp of endogenous mouse MCK exon 1 (untranslated) for efficient transcription initiation, followed by the SV40 late 16S / 19S splice signal (150 bp) and a small 5' UTR (61 bp). The intron and 5'UTR were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contained a consensus Kozak site immediately preceding the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contained the (R4-R23 / Δ71-78) domain previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)). Complementary DNA was codon-optimized for human use and synthesized by GenScript (Piscataway, NJ) (Mol Ther 18, 109-117 (2010)). The only viral sequences included in this vector were the AAV2 inverted terminal repeats, required for both viral DNA replication and packaging. The microdystrophin cassette contained a small 53-bp synthetic poly(A) signal for mRNA termination.

[0391] Previous studies have validated cardiac expression using the MHCK7 promoter (Salva et al., Mol Ther 15, 320-329 (2007)) and AAVrh.74 expression in skeletal muscle, diaphragm, and cardiac muscle (Sondergaard et al. Annals of Clinical and Transl Neurology 2, 256-270 (2015)). The construct sequence in Figure 1 was encapsidated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a rhesus monkey lymph node and is discussed in Rodino-Klapac et al., Journal of Translational Medicine 5, 45 (2007).

[0392] Table 1 shows the molecular features of the plasmid AAVrh74.MHCK7.microdystrophin (SEQ ID NO: 3). [Table 1] B) Generation of the AAVrh74.MHCK7.microdystrophin construct from a plasmid encoding kanamycin (Kan) resistance

[0393] Cloning of MHCK7.μDys.KAN was achieved by isolating the MHCK7.μDys fragment and kanamycin backbone from the MHCK7.μDys.AMP plasmid and annealing them using the NEBuilder cloning workflow. The MHCK7.μDys fragment was isolated by restriction enzyme digestion with SnaBI. Digestion was performed in a 50 μL total reaction in 1× CutSmart Buffer (NEB) and 1 μL of SnaBI at 37°C for 1 hour. The resulting fragment was isolated by electrophoresis on a 1% agarose gel run at 105 volts for 1.5 hours. The band corresponding to the MHCK7.μDys insert was excised and purified using a gel purification kit (Macherey-Nagel). The DNA concentration of the resulting fragment was 10 ng / μL. The Kan backbone fragment was isolated by XbaI restriction enzyme digestion in a 50 μL reaction using 1× CutSmart Buffer (NEB) and 1 μL of XbaI at 37°C for 1 hour. The resulting fragment was isolated by electrophoresis on a 1% agarose gel run at 105 volts for 1.5 hours. The band corresponding to the Kan backbone was excised and purified using a gel purification kit (Macherey-Nagel). The DNA concentration of the resulting fragment was 8.1 ng / μL. The two fragments were annealed using the NEB Builder cloning workflow, which has the ability to join two fragments with overlapping sequences. The NEBuilder cloning reaction was performed at 50°C for 15 minutes in a 20 μL total reaction volume using a 1:1 ratio of MHCK7.μDys to kanamycin backbone in 1× NEBuilder HiFi DNA Assembly Master Mix, according to the manufacturer's protocol. The resulting clones were transformed into NEB® Stable Competent E. coli (C3040) by adding 2.5 μL of the cloning product to the cells, followed by 30 minutes on ice, then 30 seconds at 42° C., and another 5 minutes on ice. After transformation, 950 μL of growth medium was added to the cells and grown for 1.5 hours at 30° C. with shaking at 225 rpm.After growth, 450 μL of these cells were plated onto 50 μg / mL kanamycin LB agar plates and incubated overnight at 30°C in a dry incubator. Colonies were picked from the plates and grown overnight in LB containing 50 μg / mL kanamycin. DNA was isolated from 3 mL of this culture using a QIAprep® Spin Miniprep Kit (Qiagen). This DNA was used to verify the cloning product. The cloning product was confirmed by restriction enzyme digestion with PmeI, MscI, and SmaI followed by gel electrophoresis. The cloning product was further verified by sequencing. The resulting plasmid is shown in SEQ ID NO:8 and is presented in Figures 14 and 15. SEQ ID NO:9 and the sequence of the construct in Figure 13, corresponding to nucleotides 1-4977 of SEQ ID NO:8, were encapsidated into AAVrh.74 virions as described above. Example 2 Clinical trials of systemic gene delivery for Duchenne muscular dystrophy

[0394] This was a single-dose controlled study using rAAVrh74.MHCK7.micro-dystrophin of SEQ ID NO: 3, nucleotides 55-5021, in DMD subjects. Cohort A included six subjects aged 3 months to 3 years, and Cohort B included six subjects aged 4-7 years. All subjects received intravenous micro-dystrophin vector (2 x 10 in 10 mL / kg). 14 rAAVrh74.MHCK7.microdystrophin was formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0395] In the study, rAAVrh74.MHCK7.micro-dystrophin was infused via a peripheral vein in the arm to allow it to reach all muscles in the body. Cohort A included six DMD subjects aged 3 months to 3 years, and Cohort B included six DMD subjects aged 4 to 7 years. All subjects received intravenous micro-dystrophin vector (2 x 10 in 10 mL / kg). 14 The vector genome titer of the administered dose was determined by quantitative PCR using primers directed to the MHCK7 promoter on a Prism 7500 Taqman detection system (PE Applied Biosystems) compared to a supercoiled DNA plasmid standard (Pozsgai et al., Mol. Ther. 25 (4): 855-869 (2017)).

[0396] Subjects received the infusion over one hour in the Pediatric Intensive Care Unit (PICU) at Nationwide Children's Hospital. Prior to gene therapy, a muscle biopsy was performed at the screening visit. Subjects underwent a second muscle biopsy 90 days after delivery to determine whether the gene had replaced the defective dystrophin protein. After gene transfer, patients were carefully monitored for any side effects of treatment. This monitoring included blood and urine tests and physical examinations during the screening visit and on days 0, 1, 7, 14, 30, 60, 90, 180, 9 months, 12 months, 18 months, 24 months, 30 months, and 36 months to ensure there were no side effects from the gene injection.

[0397] Subjects in Cohort A (n=6) were between 3 months and 3 years of age and received intravenous rAAVrh74.MHCK7.microdystrophin vector (2 x 10 in 10 mL / kg). 14Subjects in Cohort A received 1 mg / kg of prednisone or deflazacort, starting 1 day before gene transfer and maintained for 30 days while immune responses were monitored. If negative on day 30, steroids were tapered and discontinued over a week. If T cell responses to AAV or microdystrophin were >125 SFC / 106 PBMC, steroids were maintained until levels declined to this threshold.

[0398] Cohort B subjects (n = 6) were between 4 and 7 years of age and received intravenous rAAVrh74.MHCK7.microdystrophin vector (2 × 10 in 10 mL / kg). 14 These subjects received a stable dose of corticosteroids throughout the study, with short-term escalation if T cell responses to AAV or micro-dystrophin were >125 SFC / 106 PBMC. Eligibility Criteria

[0399] The study inclusion criteria were as follows: Ages included: Cohort A: 3 months to 7 years old and Cohort B: 4 to 7 years old, inclusive. Molecular characterization of the DMD gene, which contains a frameshift (deletion or duplication) mutation between exons 18 and 58 or a premature stop codon mutation. CK elevation >1000U / L Cohort A subjects: Below average for gross motor function on the Bayley-III Motor Assessment, defined as a scaled score of 9 or less. Cohort B: Below the mean for the 100-meter timed test, defined as less than 80% of predicted value. · Men of all ethnic groups. -Ability to participate in exercise evaluation tests. Cohort A subjects: had not previously been treated with corticosteroids. Cohort B subjects: were on a stable oral corticosteroid equivalent dose for at least 12 weeks prior to screening, and the dose was expected to remain constant throughout the study (except for modifications to accommodate changes in body weight).

[0400] The study exclusion criteria were as follows: · Active viral infection based on clinical observation. · Signs of cardiomyopathy, including an ejection fraction below 40% on an echocardiogram. · Serological evidence of HIV infection, hepatitis B or C infection. · Diagnosis of (or ongoing treatment for) an autoimmune disease. Abnormal laboratory test values ​​that are considered clinically significant The need for long-term drug treatments that pose unnecessary risks for complications or PI opinion regarding gene transfer. Subjects with an AAVrh74 or AAV8 antibody titer greater than 1:400 as determined by ELISA immunoassay. Any medical condition or consideration that, in the opinion of the investigator, may impair the subject's ability to comply with the test or procedure as required by the protocol or that may compromise the subject's well-being, safety, or clinical interpretability. Severe infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (enrollment may be postponed). Receiving any experimental or other investigational medication (other than corticosteroids) or exon-skipping medication (including ExonDys 51®) in the last 6 months prior to screening for this study. Have received any type of gene therapy, cell-based therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy. · Family members do not want the patient's trial participation to be disclosed to the patient's doctor and other health care providers. Outcome Measures

[0401] The primary outcome measure was safety (time frame: 3 years) based on the number of participants with adverse events. Adverse effects were monitored and scored for severity and relatedness to the study product.

[0402] Secondary outcome measures were:

[0403] Gross Motor Subtest Scaled (Bayley-III) Score (Time Frame: Screening, Day 30-3 Years): Motor development was measured by the Gross Motor Scaled Score. For Cohort A, the Bayley-III Gross Motor Subtest was scored at every follow-up visit through 3 years, starting at Day 30. Scaled scores were calculated for all subjects aged 43 to 47 months, inclusive, at screening, compared with normative data for children aged 42 months. The Bayley-III provided normative data for children aged 1 month to 42 months.

[0404] Physical therapy assessment, 100-meter timed test (100m) (time frame: screening, day 30-3 years): The 100m was the primary motor outcome for Cohort B. The 100m timed test was an exploratory outcome for Cohort A beginning as soon as the children turned 3 years old.

[0405] Physical Therapy Assessment, North Star Gait Assessment (NSAA) (Time Frame: Screening, Day 30-3 Years): The North Star Gait Assessment (NSAA) began as soon as the children turned 4 years old as an exploratory outcome for Cohort A and was also an exploratory outcome for Cohort B. The NSAA measures the quality of gait in young boys with Duchenne muscular dystrophy.

[0406] Physical therapy assessment, pediatric-modified Timed Up and Go (TUG) (time frame: screening, day 30–3 years): We included pediatric-modified Timed Up and Go (TUG) as an exploratory outcome in Cohort B.

[0407] Physical Therapy Assessment, 4-Step Stair Climbing (Time Frame: Screening, Day 30-3 Years): Exploratory outcomes for Cohort B include 4-step stair climbing.

[0408] Physical Therapy Assessment, Handheld Dynamometer Strength Testing (HHD) (Time Frame: Screening, Day 30-3 Years): Exploratory outcomes for Cohort B included handheld dynamometer strength testing (HHD) of the knee extensors and flexors, and elbow flexors and extensors.

[0409] Quantification of microdystrophin gene expression by immunofluorescence (Timeframe: screening, day 90): Microdystrophin gene expression levels were quantified by immunofluorescence and compared in pre- and post-muscle biopsies.

[0410] Quantification of microdystrophin gene expression by Western blot (Timeframe: screening, day 90): Microdystrophin gene expression levels were quantified by Western blot analysis and compared in pre- and post-muscle biopsies.

[0411] Decrease in CK after gene therapy (time frame: 3 years): Decrease in circulating CK levels.

[0412] Cardiac magnetic resonance imaging (at 1 year). Microdystrophin gene expression

[0413] Changes from baseline in microdystrophin expression were analyzed and quantified by fiber intensity using immunofluorescence (IF). As shown in Table 2, subject 1 (5 years old) demonstrated 78% microdystrophin protein expression in muscle fibers from gastrocnemius biopsies after administration of rAAVrh74.MHCK7.microdystrophin; subject 2 (4 years old) demonstrated 73.5% microdystrophin protein expression in muscle fibers from gastrocnemius biopsies after administration of rAAVrh74.MHCK7.microdystrophin; subject 3 (6 years old) demonstrated 77.0% microdystrophin protein expression in muscle fibers from gastrocnemius biopsies after administration of rAAVrh74.MHCK7.microdystrophin; and subject 4 (4 years old) demonstrated 96.2% microdystrophin expression in muscle fibers from gastrocnemius biopsies after administration of rAAVrh74.MHCK7.microdystrophin. All patients showed robust expression of the transduced microdystrophin, which was appropriately localized to the sarcolemma as determined by immunohistochemistry (Figure 7). [Table 2]

[0414] Changes in microdystrophin gene expression from baseline to day 60 were also assessed by quantifying microdystrophin protein expression as measured by Western blot of biopsied muscle tissue. As shown in Figures 8A and 8B, Western blot analysis detected microdystrophin protein expression in subject 1 (5 years old), subject 2 (4 years old), and subject 3 (6 years old). Figure 8C presents a Western blot analysis detecting microdystrophin protein expression in subject 4 (4 years old). All post-treatment biopsies demonstrated robust levels of microdystrophin as measured by Western blot, with mean values ​​for subjects 1-4 of 74.3% of normal using Method 1 and 95.8% of normal according to Method 2, adjusted for adipose and fibrous tissue.

[0415] For each subject, the vector genome copy number per muscle fiber nucleus was measured. As shown in Table 3, for each subject, the vector genome copy number per nucleus was greater than 1 after administration of rAAVrh74.MHCK7.micro-dystrophin. One copy of vector indicates approximately 50% micro-dystrophin gene expression. An average of 1.6 vector copies per cell nucleus was measured in subjects 1-3, consistent with the observed high micro-dystrophin expression levels. Including the value for subject 4, the average vector copy number / μg DNA was >10 5 , with an average of 3.3 vector copies per cell nucleus. [Table 3]

[0416] The protein levels of alpha-sarcoglycan and beta-sarcoglycan in muscle biopsy tissue were measured by immunohistochemistry before and after administration of rAAVrh74.MHCK7.microdystrophin. Similarly, administration of rAAVrh74.MHCK7 resulted in upregulation of DAPC protein in the subjects. As shown in Figure 9, for Subject 1 (Figure 9A), Subject 2 (Figure 9B), and Subject 3 (Figure 9C), the expression of alpha-sarcoglycan and beta-sarcoglycan in muscle biopsy tissue was increased compared to the levels of these proteins in muscle biopsies before administration of rAAVrh74.MHCK7. Circulating serum CK levels

[0417] rAAVrh74.MHCK7.micro-dystrophin vector (2 x 10 in 10 mL / kg) 14Blood samples were obtained every 30 days after administration of an intravenous infusion of 100 mg / kg (vg / kg). CK levels were measured at each visit and compared to baseline levels obtained before administration of rAAVrh74.MHCK7.microdystrophin (visit day 0). Baseline serum CK levels (units / liter) are presented in Table 4 below. As shown in Figure 10, circulating serum CK levels decreased by approximately 87% two months after administration of rAAVrh74.MHCK7.microdystrophin. All subjects demonstrated a significant decrease in serum creatine kinase (CK) levels, with a mean decrease in CK after two months of treatment exceeding 87% (n=3). CK is an enzyme associated with muscle damage, and patients with DMD consistently exhibit elevated levels of CK. Indeed, significantly elevated CK levels are often used as a preliminary diagnostic tool for DMD, with definitive genetic testing following a preliminary diagnosis.

[0418] CK levels for each subject are presented in Table 4 and Figure 10. Figure 11 presents the mean CK levels over time and demonstrates that mean CK levels significantly decreased over time following administration of rAAVrh74.MHCK7.micro-dystrophin, with a mean baseline CK level of 27,064 U / L (mean, Table 4) decreasing by approximately 63% to a mean of 9,982 U / L (mean, Day 270, Table 5). [Table 4-1] [Table 4-2] [Table 5] Efficacy evaluation

[0419] In addition to microdystrophin and CK levels, efficacy was measured by the following functional tests: Time to Rise from the Floor, Ascend 4 Steps, North Star Ambulatory Assessment (NSAA), Time to Rise Test, 4 Stairs Up Test, 10 Meter Timed Test (10m), and 100 Meter Timed Test (100m). The data are presented in Tables 6 and 7 below and demonstrate consistent and durable improvement 9 months after administration of rAAVrh74.MHCK7.microdystrophin. Improvement in NSAA over time is also presented in Figure 12. [Table 6] [Table 7-1] [Table 7-2] Safety evaluation

[0420] No serious adverse events (SAEs) were observed in the study. Three subjects had elevated gamma-glutamyltransferase (GGT), which resolved and returned to baseline levels within one week with increased steroid doses. No other clinically significant laboratory findings were observed. Patients generally experienced transient nausea during the first week of increased steroid dosing concomitant with treatment. This did not correlate with elevated liver enzymes or any other abnormalities. Example 3 A randomized, double-blind, placebo-controlled, systemic gene delivery phase I / IIa clinical trial

[0421] This is a randomized, double-blind, single-dose study using rAAVrh74.MHCK7.micro-dystrophin in subjects with DMD. The study included 24 subjects aged 4-7 years. Subjects were randomized to treatment or placebo at enrollment. Twelve subjects received intravenous rAAVrh74.MHCK7.micro-dystrophin vector (2 x 10 in approximately 10 mL / kg). 14 12 subjects received 10 mL / kg of placebo (lactated Ringer's). 12 subjects received 10 mL / kg of placebo (lactated Ringer's). 12 subjects received placebo treatment, administered in the same manner as the 12 previously treated subjects, one year after the last subject received treatment. Subjects received an infusion of micro-dystrophin-containing rAAV or lactated Ringer's over approximately one hour. Pre- and post-treatment (day 90) muscle needle biopsies were performed on the gastrocnemius muscle.

[0422] The primary objective of this study is to evaluate the safety of intravenous administration of rAAVrh74.MHCK7.micro-dystrophin via a peripheral vein in the extremities in subjects with DMD. Safety endpoints will be assessed by changes in hematology, serum chemistry, urinalysis, immunological responses to rAAVrh74 and micro-dystrophin, and observations of reported medical history and symptoms. Dystrophin gene expression will serve as the primary outcome measure, along with safety. Quantification will be performed using validated immunofluorescence and immunoblot assays. Reduction in CK after gene therapy will serve as a secondary outcome. Efficacy will be measured by the following functional tests: stand time, four-step stair climbing, North Star Ambulation Assessment (NSAA), 10-meter timed test (10 m), and 100-meter timed test (100 m). Exploratory evaluation criteria will include handheld dynamometer strength testing (HHD) of the knee extensors and flexors, and elbow flexors and extensors.

[0423] The inclusion criteria for the studies were as follows: Age range: 4 to 7 years old, inclusive. Molecular characterization of the DMD gene, which contains a frameshift (deletion or duplication) mutation between exons 18 and 58 or a premature stop codon mutation. Signs of symptomatic muscular dystrophy: CK elevation >1000 U / L and a 100 meter walk test below the mean predicted percentage of time Men of all ethnic groups are eligible. -Ability to participate in exercise evaluation tests. ·Receiving a stable oral corticosteroid equivalent dose for at least 12 weeks prior to screening, with the dose expected to remain constant throughout the study (except for potential modifications to accommodate changes in body weight).

[0424] The study exclusion criteria were as follows: · Active viral infection based on clinical observation. · Signs of cardiomyopathy, including an ejection fraction below 40% on an echocardiogram. · Serological evidence of HIV infection, hepatitis B or C infection. Diagnosis of (or ongoing treatment for) an autoimmune disease · Clinically significant laboratory abnormalities (GGT>3×ULN, bilirubin≥3.0mg / dL, creatinine≥1.8mg / dL, Hgb<8 or >18g / Dl; WBC>18,500 / cmm), platelets ≤50,000. The need for long-term drug treatments that pose unnecessary risks for complications or PI opinion regarding gene transfer. Subjects with AAVrh74 or AAV8 antibody titers greater than 1:400 as determined by ELISA immunoassay. If endpoint titers are positive at screening, testing may be repeated before exclusion. · Have a medical condition or other consideration that, in the opinion of the investigator, may impair the subject's ability to comply with the test or procedure required by the protocol or that may compromise the subject's well-being, safety, or clinical interpretability. Severe infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (enrollment may be postponed). Receiving any experimental or other investigational medication (other than corticosteroids) or exon-skipping medication (including ExonDys 51®) in the last 6 months prior to screening for this study. Have received any type of gene therapy, cell-based therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy. · Family members do not want the patient's trial participation to be disclosed to the patient's doctor and other health care providers. Efficacy evaluation

[0425] Dystrophin gene expression will serve as the primary outcome measure, along with safety. Quantification will be performed using validated immunofluorescence and immunoblot assays. Reduction of CK after gene therapy will serve as a secondary outcome. In addition, efficacy will be measured by the following functional tests: floor rise time, 4-step stair climb, North Star Gait Assessment (NSAA), 10-meter timed test (10m), and 100-meter timed test (100m). Exploratory evaluation criteria will include handheld dynamometer strength testing (HHD) of the knee extensors and flexors, and elbow flexors and extensors.

[0426] Ultrasound-guided muscle biopsies will be used to quantify transgene expression and compare baseline and day 90. Biopsies will be performed on the same muscle as the first biopsy, but on the opposite leg. One year after all subjects have received medication, the study timeline will begin again at Visit 1 for placebo crossover subjects. The second baseline screening for placebo subjects will not include cardiac MRI and muscle biopsy. Placebo subjects will undergo a muscle biopsy on day 90 (a total of three muscle biopsies). Frozen sections will be stained for dystrophin using indirect immunofluorescence (IF). Whole slide scanning will be performed, and microdystrophin intensity and percent positive fibers will be quantified using validated image scanning and MuscleMap™ analysis algorithms. Muscle morphometry, including fiber size histograms, will be performed in a blinded manner. Quantitative protein analysis for microdystrophin will be performed using a validated Western blot method using blinded frozen muscle biopsy scrapings.

[0427] Needle muscle biopsies of the gastrocnemius (unless deemed contraindicated for a particular subject by the PI, in which case an alternative muscle for biopsy will be selected by the PI) will be used to quantify microdystrophin expression. Efficacy analysis

[0428] The primary efficacy endpoint is the change from baseline to day 90 in microdystrophin protein expression levels as measured by Western blot of biopsied muscle tissue. Treatment group differences for the primary efficacy endpoint will be assessed using an analysis of covariance (ANCOVA) model with treatment as the fixed factor and baseline values ​​as the covariate. Wilcoxon rank-sum tests will be performed as supportive analyses. Changes from baseline in microdystrophin expression by immunofluorescence (IF) fiber intensity will also be analyzed.

[0429] Supportive efficacy endpoints include changes from baseline to each scheduled assessment in floor rise time, four-step stair climbing, NSAA, 10-meter timed test (10 m), 100-meter timed test (100 m), and change in CK. Exploratory measures include HHD for knee extensors and flexors, and elbow flexors and extensors. Treatment group differences are assessed using an ANCOVA model with treatment as a fixed factor and baseline values ​​as covariates. Wilcoxon rank-sum tests are performed as supportive analyses. Example 4

[0430] The tests and studies described in Examples 2 and 3 above are optionally performed utilizing the rAAVrh74.MHCK7.microdystrophin construct set forth in SEQ ID NO:9; the rAAVrh74.MHCK7.microdystrophin construct set forth in SEQ ID NO:8, nucleotides 1-4977; or the rAAVrh74.MHCK7.microdystrophin construct set forth in SEQ ID NO:6, nucleotides 56-5022. Example 5 Generation of pAAV.MCK.micro-dystrophin construct

[0431] The pAAV.MCK.microdystrophin plasmid was constructed by inserting an MCK expression cassette driving a codon-optimized human microdystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski, RJ et al., J. Virol. 61 (10): 3096-3101 (1987)). To drive muscle-specific gene expression, the construct contained a muscle-specific regulatory element. This regulatory element comprised the mouse MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). Following the core promoter, the construct contained 53 bp of endogenous mouse MCK exon 1 (untranslated) for efficient transcription initiation, followed by the SV40 late 16S / 19S splice signal (97 bp) and a small 5' UTR (61 bp). The intron and 5' UTR were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contains a consensus Kozak sequence immediately before the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contains the (R4-R23 / Δ71-78) domain previously described by Harper et al., Nat. Med. 8 (3): 253-61 (2002).

[0432] The pAAV.MCK.microdystrophin plasmid contained a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 5). This sequence was encapsidated into the AAVrh.74 virion. A molecular clone of the AAVrh.74 serotype was cloned from a rhesus monkey lymph node as described by Rodino-Klapac et al., J Transl. Med. 5: 45 (2007). Example 6 rAAV production using hybrid seed train expansion

[0433] The following process can be used to produce the rAAV constructs described herein.

[0434] HEK-293 cells were passaged four times under adherent conditions. Prior to the penultimate expansion, cells were harvested, centrifuged (300 g for 5 minutes) to wash out the serum, and resuspended in serum-free growth medium (EXPI293) in suspension shake flasks at a seeding density of 0.5 + E6 cells / mL. The cells were then grown for 48–72 hours to expand their numbers. The suspension cells were then harvested and inoculated into shake flasks or WAVE bags depending on the number of viable cells required for inoculation of the bioreactor. After 72 hours, the viable cell concentration was determined using a cell counting instrument. The required volume containing the desired total viable cell number was then added to an adherent bioreactor containing DMEM and 10% FBS. Additional FBS was added appropriately to compensate for the addition of serum-free suspension culture volume so that the final FBS concentration remained at 10%.

[0435] Cell viability was similar in both the seed train and adherent systems, as shown in Figure 18. Regarding viable cell density (VCD) in the hybrid seed train system, the VCD after the sixth passage was higher than that after the first passage (Figure 19A) and comparable to the adherent system (Figure 19B).

[0436] After seeding into adherent bioreactors (iCELLis®), adherent cultures were transiently transfected with a transgene plasmid carrying a micro-dystrophin construct described herein, including, for example, the construct shown in SEQ ID NO:9 contained in the transgene plasmid of SEQ ID NO:8. In addition to the transgene plasmid, a rep / cap plasmid (AAV2 rep / rh74 cap) and a helper plasmid are also included. After the desired growth period, rAAV particles are harvested by cell lysis and column chromatography.

[0437] In some embodiments, the rAAV is (a) culturing cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing serum at a lower concentration than the first medium; (d) culturing the cells in the N-1 container under suspension conditions; (e) inoculating the third medium in a bioreactor with the cells from step (d); The yeast is produced by a floating seed process comprising:

[0438] In some aspects, the floating seed process comprises: (f) transfecting the cells with a transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. Further includes:

[0439] In some embodiments, the transgene plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct comprises the nucleic acid sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, or nucleotides 1-4977 of SEQ ID NO: 8. In some embodiments, the plasmid comprising the AAV rep gene and the AAV cap gene comprises the AAV2 rep gene and the rAAVrh74 cap gene.

[0440] In some embodiments, the adenovirus helper plasmid comprises the adenovirus 5 E2A, E4ORF6, and VA RNA genes.

[0441] In some embodiments, the suspension seed process further comprises (g) lysing the cells, hi some embodiments, the cells are lysed by freeze-thawing, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

[0442] In some aspects, the floating seed process further comprises purifying the (h)rAAV by at least one column chromatography step, hi some aspects, the at least one column chromatography step comprises anion exchange chromatography or size exclusion chromatography, or a combination thereof. Example 7 An open-label, systemic gene delivery study evaluating the safety and expression of the rAAVrh74.MHCK7.microdystrophin construct using commercially available materials in subjects with Duchenne muscular dystrophy

[0443] This is a Phase 1b, open-label study conducted in boys with Duchenne muscular dystrophy using commercially available material of the rAAVrh74.MHCK7.microdystrophin construct. Initially, 20 patients were enrolled. This Example 7 presents data from the first 11 patients under 8 years of age (e.g., 2 patients were 4-5 years old and 9 patients were 6-7 years old) (Cohort 1; 20 ambulatory male subjects with DMD aged 4 to 8 years) (Table 7). The study was later expanded to include Cohort 2 (approximately 6 ambulatory male subjects with DMD aged 8 to 18 years) and Cohort 3 (approximately 6 non-ambulatory male subjects with DMD), as further described in Example 8. [Table 8-1] [Table 8-2]

[0444] The primary objective was to evaluate microdystrophin expression from the rAAVrh74.MHCK7.microdystrophin construct (e.g., commercially available material) as measured by Western blot in biopsied muscle tissue at 12 weeks post-infusion (Part 1), and the corresponding endpoint was the change in quantitative microdystrophin protein expression as measured by Western blot from baseline to Week 12 (Part 1). Secondary objectives were (1) to evaluate microdystrophin protein expression by fiber intensity in immunofluorescence (IF) at Week 12; (2) to evaluate microdystrophin expression by percentage of dystrophin-positive fibers (PDPF) in IF at Week 12; and (3) to evaluate safety. The inclusion criteria for this study, applicable to Examples 7 and 8, are as follows:

[0445] Subjects must meet all of the following criteria to be eligible to participate in this study: 1. Cohort 1 only (ambulatory, <8 years): Male at birth, ambulatory, aged 4 to <8 years at screening, and NSAA score >17 and ≤26 at the screening visit. 2. Cohort 2 only (ambulatory, ≥8 years): Male at birth, ambulatory, ≥8 years old but <18 years old at screening, and NSAA score ≥15 and ≤26 at the screening visit. 3. Cohort 3 only (non-ambulatory): Male at birth and non-ambulatory for at least 9 months, with an NSAA ambulatory score of 0 at the screening visit, inability to perform the 10MWR, and a PUL introductory item score of 2 or greater. Incident loss of ambulatory ability is defined as the age of continued wheelchair use reported by the participant or caregiver to the nearest month. 4. Have a confirmed diagnosis of DMD prior to screening based on documented clinical findings and previous definitive genetic testing using clinical diagnostic genetic testing. 5. Have any of the following symptoms of symptomatic muscular dystrophy: Elevated CK, >1000U / L, and · Cohorts 1 and 2 only (ambulatory): 100 MWR less than 95 percent of the time predicted. 6. Able to cooperate with exercise evaluation tests. 7. Receiving a stable weekly oral corticosteroid equivalent dose for at least 12 weeks prior to screening, with the dose expected to remain constant throughout the first year of the study (except for modifications to accommodate changes in weight). 8. rAAVrh74 antibody titer of 1:400 or less (i.e., not elevated) as determined by ELISA. 9. Sexually potential subjects must agree to use condoms throughout the study, and female sexual partners must also use a medically acceptable form of birth control (e.g., oral contraceptives). 10. Have a parent(s) or legal guardian(s) who are able to understand and follow the study visit schedule and all other protocol requirements, or, for subjects 18 years of age or older, are able to understand and follow the study visit schedule and all other protocol requirements. 11. Have a parent(s) or legal guardian(s) who are willing to provide informed consent or assent (if applicable) and who are willing to provide written informed consent for the subject to participate in the study, or, in the case of subjects 18 years of age or older, are willing to provide written informed consent for the subject to participate in the study. The study exclusion criteria were as follows:

[0446] Subjects meeting any of the following criteria will be excluded from the study: 1. Left ventricular ejection fraction <40% on screening echocardiogram or clinical signs and / or symptoms of cardiomyopathy. 2. Cohorts 2 and 3 only (ambulatory aged 8 years and older and non-ambulatory): Forced vital capacity (FVC) <50% of predicted at screening and / or need for nocturnal ventilator support. 3. Have undergone major surgery within 3 months prior to Day 1 or are scheduled to undergo surgery at any time during the study. 4. Presence of any other significant genetic disorder other than DMD. 5. Have serologic evidence of current, chronic, or active human immunodeficiency virus infection, hepatitis C infection, or hepatitis B infection. 6. Have a diagnosis of autoimmune disease. 7.Having complications or the need for long-term drug treatment that, in the opinion of the investigator, pose unnecessary risks related to gene transfer. 8. Possess a medical condition or consideration that, in the opinion of the investigator, may impair the subject's ability to comply with the test or procedure required in the protocol or that may compromise the subject's well-being, safety, o...

Claims

1. 1. A method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by a suspension seed process, comprising: (a) culturing cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container that is serum-free or contains serum at a lower concentration than the first medium; (d) culturing the cells in the N-1 container under suspension conditions; (e) inoculating a third medium in a bioreactor with the cells from step (d); A method comprising:

2. 2. The method of claim 1, wherein the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO:

1.

3. The method of claim 2, wherein the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO:

7.

4. The method of any one of claims 1 to 3, wherein the rAAV comprises a human microdystrophin nucleotide sequence of SEQ ID NO: 1 and an MHCK7 promoter sequence of SEQ ID NO:

7.

5. The floating seed process comprises: (f) transfecting the adherent cells with a transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep and cap genes, and an adenovirus helper plasmid. The method of any one of claims 1 to 4, further comprising:

6. the transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct is The nucleic acid sequence of SEQ ID NO:9; Nucleotides 55 to 5021 of SEQ ID NO:3; or Nucleotides 1 to 4977 of SEQ ID NO:8 The method of claim 5 , comprising:

7. 7. The method of claim 5 or 6, wherein the plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and an rAAVrh74 cap gene.

8. The method of any one of claims 5 to 7, wherein the adenovirus helper plasmid comprises adenovirus 5 E2A, E4ORF6, and VA RNA genes.

9. The floating seed process comprises: (g) lysing the adherent cells The method of any one of claims 1 to 8, further comprising:

10. 10. The method of claim 9, wherein the adherent cells are lysed by freeze-thawing, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

11. The floating seed process comprises: (h) purifying the rAAV by at least one column chromatography step. The method of any one of claims 1 to 10, further comprising:

12. 12. The method of claim 11, wherein the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

13. 13. The method of any one of claims 1 to 12, wherein the suspension seeding process further comprises culturing cells in the first growth medium in an N-3 container.

14. 14. The method of claim 13, wherein the suspension seeding process further comprises culturing cells in the first growth medium in an N-4 container.

15. The method according to any one of claims 1 to 14, wherein the bioreactor is an adherent bioreactor.

16. 16. The method of claim 15, wherein the rAAV is purified from the culture produced in the adherent bioreactor.

17. 17. The method of claim 15 or 16, wherein the third culture medium in the bioreactor comprises at least one factor that promotes cell adhesion.

18. 18. The method of claim 17, wherein the at least one factor that promotes cell adhesion is selected from the group consisting of serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof.

19. 19. The method of claim 17 or 18, wherein the third culture medium in the bioreactor comprises DMEM and 10% FBS.

20. The method according to any one of claims 1 to 19, wherein the adherent cells are cultured in suspension for about 48 to 72 hours.

21. The method of any one of claims 1 to 20, wherein the N-1 container is a suspension shake flask.

22. 22. The method of any one of claims 1 to 21, wherein the adherent cells are selected from the group consisting of HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells.

23. The method of claim 22, wherein the adherent cells are HeLa cells or HEK-293 cells.

24. The method of claim 23, wherein the adherent cells are HEK-293 cells.

25. The method of any one of claims 1 to 24, wherein the adherent cells are not adapted to a suspension system.

26. The method according to any one of claims 1 to 25, wherein culturing the cells under suspension conditions does not alter the adhesion dependency of the cells.

27. 27. The method of any one of claims 1 to 26, wherein the cells are not altered by said culturing to create a new cell line.

28. 28. A composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is produced by the method of any one of claims 1 to 27.

29. a) an rAAV particle encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsidating nucleotides 55 to 5021 of SEQ ID NO:3; and / or c) rAAV particles encapsidating nucleotides 1 to 4977 of SEQ ID NO:8 29. The composition of claim 28, comprising:

30. 30. A method of treating muscular dystrophy in a human subject in need thereof, comprising administering to the human subject the composition of claim 29.

31. The rAAV is administered via a systemic route, and the dose is approximately 5.0 x 10 １２ vg / kg ~ approx. 1.0×10 １５ 31. The method of claim 30, wherein the antibody is administered at a dose of 100 mg / kg.

32. The systemic administration route is intravenous, and the dose of the rAAV administered is about 2×10 １４ 32. The method of claim 31 , wherein the saturation is 0.05 vg / kg.

33. 33. The method of any one of claims 30-32, wherein the dose of rAAV is administered at a concentration of about 10 mL / kg.

34. The method of any one of claims 30 to 33, wherein the rAAV is administered by injection, infusion, or implantation.

35. 35. The method of claim 34, wherein the rAAV is administered by infusion over approximately 1 hour.

36. 36. The method of any one of claims 30 to 35, wherein the rAAV is administered by intravenous route through a peripheral vein in a limb.

37. The method of any one of claims 30 to 36, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

38. 38. The method of claim 37, wherein the muscular dystrophy is Duchenne muscular dystrophy.

39. 39. The method of any one of claims 30-38, wherein the level of microdystrophin gene expression in cells of the subject is increased after administration of the rAAV compared to the level of microdystrophin gene expression before administration of the rAAV.

40. 40. The method of claim 39, wherein expression of the microdystrophin gene in the cells is detected by measuring the microdystrophin protein levels by Western blot on muscle biopsied before and after administration of the rAAV.

41. 41. The method of claim 40, wherein the expression is at least 55.4% after administration of the rAAV compared to before administration.

42. 42. The method of any one of claims 30-41, wherein the average percentage of microdystrophin-positive fibers in the subject's muscle tissue is increased after administration of the rAAV compared to the number of microdystrophin-positive fibers before administration of the rAAV.

43. 43. The method of claim 42, wherein the average percentage of microdystrophin-positive fibers is at least 70.5% and the average intensity detected by immunofluorescence (IF) for muscle biopsies before and after administration of the rAAV is at least 116.9%.

44. 44. The method of any one of claims 30-43, wherein the micro-dystrophin transduction by vector genome count is at least 3.87 average vector genome copies per nucleus.

45. 45. The method of any one of claims 30-44, wherein the subject has been genotyped for at least one mutation in the human dystrophin (DMD) gene.

46. 46. ​​The method of claim 45, wherein the at least one mutation is a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the absence of expression of the human dystrophin protein.

47. 30. Use of the composition of claim 28 or 29 in a human subject in need of treatment for muscular dystrophy.

48. 30. Use of a composition according to claim 28 or 29 in the manufacture of a medicament for the treatment of muscular dystrophy.

49. 49. The use according to claim 47 or 48, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

50. 50. The use of claim 49, wherein the muscular dystrophy is Duchenne muscular dystrophy.

51. 1. A method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles encapsidating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion that completely encompasses exons 9-13 of the DMD gene. A method comprising:

52. 1. A method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles encapsidating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion in exons 8 and / or 9 of the human dystrophin (DMD) gene. A method comprising:

53. 53. The method of claim 51 or 52, wherein the subject's DMD gene is genotyped prior to treatment.

54. 54. The method of any one of claims 51 to 53, wherein the AAV viral particles are of serotype rh74.

55. The rAAV vector is a) rh74 serotype AAV viral particles encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rh74 serotype AAV viral particles encapsidating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rh74 serotype AAV viral particles encapsidating nucleotides 1 to 4977 of SEQ ID NO:8 55. The method of claim 54, wherein the compound is administered as a composition comprising:

56. 1. A method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene in said subject prior to treatment; ii) if genotyping does not identify a deletion completely encompassing exons 9-13 of the DMD gene, administering to the subject a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the composition: a) an rAAV particle encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsidating nucleotides 55 to 5021 of SEQ ID NO:3; and / or c) rAAV particles comprising encapsidated nucleotides 1-4977 of SEQ ID NO:

8. Including steps and A method comprising:

57. 1. A method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene in said subject prior to treatment; ii) if genotyping does not identify a deletion in exons 8 and / or 9 of the DMD gene, administering to the subject a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the composition: a) an rAAV particle encapsidating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsidating nucleotides 55 to 5021 of SEQ ID NO:3; and / or c) rAAV particles encapsidating nucleotides 1 to 4977 of SEQ ID NO:8 Including steps and A method comprising:

58. 58. The method of any one of claims 30-46 or 51-57, wherein the human subject is ambulatory.

59. 58. The method of any one of claims 30-46 or 51-57, wherein the human subject is non-ambulatory.

60. 60. The method of any one of claims 30-46 or 51-59, wherein the human subject is between 2 and 3 years old.

61. 60. The method of any one of claims 30-46 or 51-59, wherein the human subject is between 4 and 5 years old.

62. 60. The method of claim 59, wherein the human subject has been non-ambulatory for at least 9 months.

63. 63. The method of claim 62, wherein the human subject has a stable forced vital capacity (FVC) of less than 40% of predicted and / or a need for nocturnal ventilator support.

64. 64. The method of any one of claims 56-63, wherein the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin is produced according to the method of any one of claims 1 and 7-27.

Images