Production of recombinant AAV vectors for treating muscular dystrophy

Abstract

The present disclosure provides gene therapy vectors such as recombinant adeno-associated virus (rAAV) produced in mammalian adherent cells cultured in suspension to express human micro-dystrophin gene.The present disclosure also provides compositions of these rAAV and methods of using them to treat muscular dystrophy (such as Duchenne muscular dystrophy).The rAAV can be produced from adherent cells cultured in suspension by the suspension seeding process described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 253,998, filed October 8, 2021, U.S. Provisional Application No. 63 / 243,944, filed September 14, 2021, U.S. Provisional Application No. 63 / 209,733, filed June 11, 2021, and U.S. Provisional Application No. 63 / 189,676, filed May 17, 2021, which are incorporated by reference in their entireties. Reference to Electronically Submitted Sequence Listing

[0002] The contents of the Sequence Listing submitted electronically in an ASCII text file with this application (filename: 4140_052PC04_Seqlisting_ST25.txt; size: 60,194 bytes; and creation date: May 13, 2022) are incorporated by reference in their entirety herein. Field

[0003] The present disclosure belongs to the field of gene therapy.More specifically, the present disclosure provides gene therapy vectors, such as adeno-associated virus (AAV) vectors for expressing miniaturized human micro-dystrophin gene, wherein AAV is produced from adherent cells cultured in suspension.The present disclosure also provides the method of using these vectors to express micro-dystrophin in skeletal muscle (including diaphragm and cardiac muscle) in subjects suffering from muscular dystrophy, protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis. [Background technology]

[0004] background There is no doubt that muscle mass and strength are important for daily activities such as locomotion and breathing, as well as for whole-body metabolism. Deficits in muscle function result in muscular dystrophies (MDs) characterized by muscle weakness and muscle wasting, severely impacting 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 loss of sarcolemma-cytoskeleton anchoring by DAPC. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in 5,000 newborn boys.

[0005] DMD is caused by mutations in the DMD gene that lead to reduced mRNA and absence of dystrophin, a 427 kD sarcolemmal protein that binds to the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51:919-28, 1987). The DAPC is composed of several proteins that form linkages between the extracellular matrix (ECM) and the cytoskeleton via dystrophin, actin-binding proteins, and alpha-dystroglycan (a laminin-binding protein) in the sarcolemma. These linkages act to stabilize the sarcolemma during contraction and protect it from contraction-induced damage. Loss of dystrophin leads to membrane fragility, resulting in sarcolemmal tearing, calcium influx, triggering the action of calcium-activated proteases, and focal fiber necrosis (Straub et al., Curr Opin. Neurol. 10:168-75 (1997)). This uncontrolled cycle of muscle degeneration and regeneration eventually exhausts 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. In the absence of membrane stabilization derived from dystrophin or micro-dystrophin, DMD would manifest as an uncontrolled tissue damage and repair cycle, eventually replacing lost muscle fibers with fibrous scar tissue via proliferation of connective tissue. Fibrosis is characterized by the excessive deposition of ECM 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. Although the primary pathological feature of DMD is degeneration and necrosis of muscle fibers, the impact of fibrosis as a pathological outcome is comparable. Overproduction of fibrous tissue limits muscle regeneration, which contributes to the progressive muscle weakness in DMD patients. In one study, the presence of fibrosis in the initial DMD muscle biopsy was highly correlated with impaired athletic performance outcomes 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 DMD muscle dysfunction and highlight the need for early intervention before the development of overt fibrosis. International Publication No. WO2019 / 245973A1 (incorporated herein in its entirety by reference) describes the AAV vector delivery of micro-dystrophin gene for treating muscular dystrophy (e.g., DMD) in human subjects.However, there is still a need in the art for improved methods of producing such AAV vectors, particularly methods suitable for large-scale production of such gene therapy vectors. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2019 / 245973 [Non-patent literature]

[0007] [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

[0008] Abstract The present disclosure relates to gene therapy vectors (e.g., AAV vectors) produced by the suspension seed process described herein that express the human micro-dystrophin gene in skeletal muscles (including diaphragm and cardiac muscle) to protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis.

[0009] The present disclosure provides a method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin in mammalian adherent cells by a suspension seed process, comprising the steps of: (a) culturing cells in an N-2 container with a first growth medium comprising serum; (b) removing the cells from said first medium; (c) inoculating the cells from said step (b) in an N-1 container into a second medium comprising no serum or a lower concentration of serum than said first medium; (d) culturing the cells in said N-1 container under suspension conditions; and (e) inoculating the cells from said step (d) into a third medium in a bioreactor.

[0010] In some embodiments, the rAAV used in the methods described herein 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 human micro-dystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 7.

[0011] In some embodiments, the suspension seed process further comprises (f) transfecting the adherent cells with a transgene plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct, a plasmid comprising the AAVrep and AAVcap genes, and an adenovirus helper plasmid.

[0012] 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 AAVrep and AAVcap genes comprises the AAV2rep and rAAVrh74cap genes. In some embodiments, the adenovirus helper plasmid comprises the E2A, E4ORF6, and VA RNA genes of adenovirus type 5.

[0013] In some embodiments, the suspension seed process further comprises the step of (g) lysing the adherent cells, hi some embodiments, the adherent cells are lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

[0014] In some embodiments, the suspension seed process further comprises (h) purifying the rAAV by at least one column chromatography step. In some embodiments, the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0015] In some embodiments, the suspension seed process further comprises culturing the cells in an N-3 container with the first growth medium, hi some embodiments, the suspension seed process further comprises culturing the cells in an N-4 container with the first growth medium.

[0016] In some embodiments, the bioreactor is an adherent bioreactor. In some embodiments, the rAAV is purified from the culture produced in the adherent bioreactor.

[0017] In some embodiments, the third 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, proteoglycan or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third medium in the bioreactor comprises DMEM and 10% FBS.

[0018] In some embodiments, the adherent cells are cultured under suspension conditions for about 48 to 72 hours.

[0019] In some embodiments, the N-1 container is a suspension shake flask.

[0020] 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 suspension. In some embodiments, culturing the cells under suspension conditions does not alter the adhesion dependency of the cells. In some embodiments, culturing does not alter the cells to create a new cell line.

[0021] The disclosure also provides a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein said rAAV is produced by any of the methods described herein. In some embodiments, the composition comprises: a) an rAAV particle comprising the nucleic acid sequence of SEQ ID NO:9; b) an rAAV particle comprising nucleotides 55-5021 of SEQ ID NO:3; and / or c) an rAAV particle comprising nucleotides 1-4977 of SEQ ID NO:8.

[0022] In some embodiments, the present disclosure provides a composition for treating muscular dystrophy in a human subject in need of such treatment, the composition comprising a recombinant adeno-associated virus (rAAV) rAAV.rh74MHCK7.micro-dystrophin, 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.

[0023] In some embodiments, the composition comprises (a) an rAAV comprising the nucleic acid sequence of SEQ ID NO:9; (b) an rAAV particle comprising the nucleic acid sequence of SEQ ID NO:9; (c) an rAAV comprising nucleotides 55-5021 of SEQ ID NO:3; (d) an rAAV particle comprising nucleotides 55-5021 of SEQ ID NO:3; (e) an rAAV comprising nucleotides 1-4977 of SEQ ID NO:8; and / or (f) an rAAV particle comprising nucleotides 1-4977 of SEQ ID NO:8.

[0024] The present disclosure also provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering to said human subject a composition comprising an rAAV as described herein. In some embodiments, the rAAV is administered using a systemic route, at a dose of about 5.0×10 12 vg / kg ~ approx. 1.0×10 15 In some embodiments, the systemic route of administration is intravenous and the administered dose of rAAV is about 2×10 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 by intravenous route via a peripheral vein in a limb.

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

[0027] In some embodiments, the expression level of micro-dystrophin gene in cells of subject increases after administration of rAAV compared to the expression level of micro-dystrophin gene 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 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.

[0028] In some embodiments, the average percentage of micro-dystrophin positive fibers in muscle tissue of a subject increases after administration of rAAV compared to the number of micro-dystrophin positive fibers before administration of rAAV.In some embodiments, the average percentage of micro-dystrophin positive fibers is at least 70.5%, and the average intensity is at least 116.9%, as detected by immunofluorescence (IF) in muscle biopsies before and after administration of rAAV.In some embodiments, micro-dystrophin transduction by vector genome count is at least 3.87 average vector genome copy number per nucleus.

[0029] In some embodiments, the composition is administered to a genotyped patient. In some embodiments, the patient's human dystrophin (DMD) gene is genotyped. In some embodiments, the genotyped patient is genotyped for at least one mutation in exons 18-79 of the human dystrophin (DMD) gene.

[0030] In some embodiments, the method of treating muscular dystrophy further comprises genotyping the DMD gene of said human subject prior to administration of said composition to said human subject. In some embodiments, the 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 stop, or other pathogenic variant that results in the absence of expression of human dystrophin protein.

[0031] The present disclosure also provides for the use of the compositions described herein to treat 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.

[0032] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. [Brief description of the drawings]

[0033] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the rAAV.MHCK7.micro-dystrophin construct. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). The construct features an in-frame rod deletion (R4-R23), while the hinges 1, 2, and 4 (H1, H2, and H4) and cysteine-rich domains still produce a 138 kDa protein. Expression of the micro-dystrophin protein (3579 bp) is guided by the MHCK7 promoter (795 bp). The intron and 5'UTR are derived from the plasmid pCMVβ (Clontech). The micro-dystrophin cassette had a consensus Kozak immediately before the ATG start codon and a small 53 bp synthetic polyA signal for mRNA termination. The human micro-dystrophin cassette contained the (R4-R23 / Δ71-78) domain previously described by Harper et al. (Nature Medicine 8:253-261 (2002)).

[0034] [Figure 2-1] FIG. 2 provides the nucleic acid sequence of AAVrh74.MHCK7.micro-dystrophin (SEQ ID NO:3). [Figure 2-2]Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above.

[0035] [Diagram 3] Figure 3 provides the pNLREP2-Caprh74 AAV helper plasmid map.

[0036] [Figure 4] FIG. 4 provides the Ad helper plasmid pHELP.

[0037] [Diagram 5] FIG. 5 shows the rAAV.MCK.micro-dystrophin plasmid construct.

[0038] [Figure 6-1] FIG. 6 provides the nucleic acid sequence of rAAVrh74.MCK.micro-dystrophin (SEQ ID NO:5). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0039] [Figure 7] FIG. 7 demonstrates micro-dystrophin gene expression in muscle fibers of gastrocnemius biopsies as measured by immunocytochemistry.

[0040] [Figure 8-1]Figures 8A-8C provide Western blots demonstrating micro-dystrophin protein expression at the correct molecular weight. In Figures 8A and 8B, Western blot analysis detected micro-dystrophin protein expression in subject 1 (5 years old), subject 2 (4 years old), and subject 3 (6 years old). In Figure 8C, the sample (*) from subject 4 was diluted 1:4 (to the linear range) since it exceeded the ULDQ (>80%) in the first analysis, and the average value was multiplied by the dilution correction factor to obtain the final value compared to normal. The average micro-dystrophin expression relative to normal was 182.7% in method 1 and 222.0% in method 2. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0041] [Figure 9-1] Figures 9A-9C demonstrate that rAAVrh74.MHCK7.micro-dystrophin upregulates expression of DAPC proteins (alpha-sarcoglycan and beta-sarcoglycan) in subject 1 (Figure 9A), subject 2 (Figure 9B), and subject 3 (Figure 9C). [Figure 9-2] Same as above. [Figure 9-3] Same as above.

[0042] [Figure 10] FIG. 10 provides a graph showing a sustained dramatic reduction in creatine kinase (CK) levels upon administration of rAAVrh74.MHCK7.micro-dystrophin.

[0043] [Figure 11] Figure 11 provides a graph showing the mean creatine kinase (CK) change from baseline to day 270. The data demonstrates that CK was significantly reduced over time following administration of rAAVrh74.MHCK7.micro-dystrophin.

[0044] [Figure 12]Figure 12 provides graphs showing the change in mean NSAA and the change in mean CK from baseline to day 270. The data demonstrates that NSAA increased significantly over time following administration of rAAVrh74.MHCK7.micro-dystrophin.

[0045] [Figure 13] Figure 13 provides the 4977 base nucleic acid sequence of the AAVrh74.MHCK7.micro-dystrophin construct (SEQ ID NO:9). The molecular elements are described below: 5'ITR (bases 1-145); MHCK7 promoter (190-981 (792 bases)); intron (991-1140 (150 bases)); human micro-dystrophin sequence (1151-4729 (3579 bases)); polyA tail (4732-4784 (53 bases)); and 3'ITR (4833-4977 (145 bases)).

[0046] [Figure 14] FIG. 14 shows the AAVrh74.MHCK7.micro-dystrophin plasmid construct.

[0047] [Figure 15-1] FIG. 15 provides the nucleic acid sequence of the AAVrh74.MHCK7.micro-dystrophin plasmid construct containing the kanamycin resistance gene (SEQ ID NO:8). [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above.

[0048] [Figure 16] FIG. 16 is a visual representation of the hybrid seed train expansion method described herein.

[0049] [Figure 17] FIG. 17 is a visual representation of AAV particle production using the hybrid seed train expansion method described herein.

[0050] [Figure 18] 18A-18B provide graphs showing the viability of HEK-293 cells when cultured according to the hybrid seed train expansion method disclosed herein (FIG. 18A) and when cultured only under adherent conditions (FIG. 18B).

[0051] [Figure 19] 19A-19B provide graphs showing the viable cell density of HEK-293 cells when cultured according to the hybrid seed train expansion method disclosed herein (FIG. 19A) and when cultured only under adherent conditions (FIG. 19B).

[0052] [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 3 points from baseline. The 6-7 year old (n=9) improved 2.9 points from baseline. Each time point represents 11 patients.

[0053] [Figure 21] Figures 21A-21C demonstrate micro-dystrophin expression (immunofluorescence) in skeletal and cardiac muscles of DMDmdx rats after 12 weeks (Figure 21B) and 24 weeks (Figure 21C) of treatment with the derandistrogen moxeparvovec compared to saline (Figure 21A), as discussed in Example 11. Abbreviations: LTA = left tibialis anterior; HRT = heart.

[0054] [Figure 22]22A-22B are bar graphs showing quantification of micro-dystrophin 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 delandistrogene moxeparvove, as discussed in Example 11. Abbreviations: TA=tibialis; HRT=heart; MG=medial gastrocnemius; LG=lateral medial gastrocnemius; DIA=diaphragm; TRI=triceps; PSO=psoas.

[0055] [Figure 23] 23A-23B are bar graphs showing increased ambulation (FIG. 23A) and rearing (FIG. 23B) in DMDmdx rats after 12 and 24 weeks of treatment with the derandistrogen moxeparvovec compared to saline, as discussed in Example 11. Rats' movement (ambulation and rearing) in activity cages was measured by the number of laser beam breaks per hour. Each point represents a value for each animal. Data are reported as mean ± SD; ***=p<0.001; **=p<0.01. SD=standard deviation.

[0056] [Figure 24-1] Figures 24A-24B show that muscle degeneration was significantly reduced by central nucleation analysis in skeletal muscle 12 and 24 weeks after derandistrogen moxeparvovec gene transfer in DMDmdx rats compared to saline, 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 24-2] Same as above.

[0057] [Figure 25-1]Figures 25A-25B show analysis of collagen deposition in skeletal and cardiac muscle showing reduced fibrosis in DMDmdx rats after 12 and 24 weeks of treatment with derandistrogen moxeparvovec compared to saline, as discussed in Example 11. Figure 25A shows Masson's Trichrome staining 12 hours after treatment. Figure 25B provides a bar graph quantifying collagen deposition in skeletal and cardiac muscle for 12 and 24 weeks after treatment. Abbreviations: HRT=heart; MG=medial gastrocnemius; DIA=diaphragm. Data are reported as mean ± SD; ****=p<0.0001; *=p<0.05. SD=standard deviation. [Figure 25-2] Same as above.

[0058] [Figure 26] Figure 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 derandistrogen moxeparvovec in DMDmdx rats compared to saline, as discussed in Example 11.Bars indicate mean + SD.Each point indicates the value for each animal.

[0059] [Figure 27] Figures 27A-27C are bar graphs analyzing cardiac function determined by echocardiography in DMDmdx rats after 24 weeks of treatment with derandistrogen moxeparvovec compared to saline, 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). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] Detailed Description The present disclosure provides a gene therapy vector (e.g., rAAV) that expresses human micro-dystrophin, where the rAAV is produced in mammalian adherent cells and the adherent cells are cultured under suspension conditions in N-1 containers.

[0061] The present disclosure provides a method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin in mammalian adherent cells by a suspension seed process, comprising the steps of: (a) culturing cells in an N-2 container with a first growth medium comprising serum; (b) removing the cells from said first medium; (c) inoculating the cells from said step (b) in an N-1 container into a second medium comprising no serum or a lower concentration of serum than said first medium; (d) culturing the cells in said N-1 container under suspension conditions; and (e) inoculating the cells from said step (d) into a third medium in a bioreactor.

[0062] The present disclosure also provides compositions (e.g., pharmaceutical compositions) comprising the rAAV disclosed herein and methods of treating muscular dystrophy (e.g., DMD) using the compositions disclosed herein.

[0063] Muscle biopsies performed at the earliest stage of DMD diagnosis reveal significant connective tissue proliferation. Muscle fibrosis is often detrimental. It reduces the normal passage of endomysial nutrients through the connective tissue barrier, reducing blood flow and depriving the muscle of vascular derived nutrients, contributing functionally to early loss of ambulation due to limb contractures. Significant muscle fibrosis results in multiple treatment attempts over time. This can be observed in muscle biopsies comparing connective tissue proliferation at successive time points. This process continues to worsen, leading to loss of ambulation and accelerating loss of control, especially in wheelchair users.

[0064] Without early treatment, including parallel approaches to reduce fibrosis, it is unlikely that exon skipping, stop codon read-through, or gene replacement therapy can fully benefit. Even small molecule or protein replacement strategies are likely to fail without using approaches to reduce muscle fibrosis. In a previous study in aged mdx mice with existing fibrosis treated with AAV.micro-dystrophin, it was demonstrated that we could not achieve complete functional recovery (Liu, M. et al., Mol Ther 11:245-256 (2005)). It is also known that the progression of DMD cardiomyopathy involves scarring and fibrosis in the ventricular wall. definition

[0065] Unless otherwise defined, all technical and scientific terms used herein have the meaning 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 the plural and plural terms shall include the singular.

[0066] Throughout this disclosure, the terms "a" or "an" entity refer to one or more entities; for example, a "polynucleotide" is understood to refer to one or more polynucleotides. As such, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0067] Furthermore, "and / or" as used herein shall be construed as a specific disclosure of each of the two specified features or components with or without the other feature or component. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases 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 (single); B (single); and C (single).

[0068] The term "about" is used herein to mean approximately, roughly, roughly, or within a region thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by expanding the boundaries above and below the numerical values ​​set forth. Unless otherwise indicated, the term "about" is generally used herein to modify (up or down (higher or lower)) a numerical value above and below the set forth value by a variance of 10 percent.

[0069] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included, if clear 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 is present before a series of numbers or ranges, it is understood that "at least" can modify each number in the series or range. Also, "at least" is not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, without considering the number of significant digits).

[0070] Unless specifically indicated otherwise, nucleotide sequences are depicted herein in a right-to-left, 5' to 3' orientation as a single strand only. Nucleotides and amino acids are depicted herein in the form recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either single-letter or three-letter code, both in accordance with 37 CFR § 1.822 and established usage.

[0071] As used herein, "polynucleotide" or "nucleic acid" refers to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are depicted herein in the 5' to 3' direction. Polynucleotides of the present disclosure can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Nucleotide bases are depicted herein by the following single letter notations: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0072] As used herein, the term "polypeptide", unless otherwise indicated, includes both peptides and proteins.

[0073] The terms "coding sequence" or "encoding" sequence are used herein to mean a DNA or RNA region (transcribed region) that "encodes" a particular protein, such as, for example, insulin or glucokinase. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide in vitro or in vivo when placed under the control of an appropriate regulatory region (such as a promoter). 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.

[0074] A gene can include several operably linked fragments, such as a promoter, a 5' leader sequence, introns, coding sequences, and 3' untranslated sequences (including, for example, a polyadenylation site or 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.

[0075] As used herein, the term "promoter" refers to a nucleic acid sequence or fragment that functions to regulate the transcription of one or more genes (or coding sequences) located upstream in the transcription direction of the transcription start site of the gene, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including but not limited to, a transcription factor binding site, a suppressor and activator protein binding site, and any other nucleotide sequence known to those skilled in the art to directly or indirectly control 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 controlled depending on physiological and developmental conditions. A "tissue-specific" promoter is preferentially active in a specific differentiated cell / tissue type.

[0076] 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 (e.g., associate with coding sequences) in either direction over distances up to several kilobase pairs (kb) from downstream positions of coding sequences and transcribed regions.

[0077] The term "operably linked" refers to the positioning of a nucleotide sequence of a control element (eg, a nucleotide sequence of a promoter) for expression of said nucleotide sequence by said control element.

[0078] As used herein, the term "transgene" refers to a gene (e.g., micro-dystrophin) or a nucleic acid molecule that is transferred into a cell. One example of a transgene is a nucleic acid that encodes a therapeutic polypeptide. In some embodiments, the gene may be present, 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 aforementioned gene is normally expressed in the cell, but symptoms and / or disease may still develop. In certain embodiments, the transgene is capable of increasing or overexpressing the expression of the gene. The transgene may include sequences derived from the cell, may include sequences that do not naturally occur in the cell, or may include a combination of both. In certain embodiments, the transgene may include sequences that can be operably linked to the appropriate control sequences for gene expression. In some embodiments, the transgene is not integrated into the genome of the host cell.

[0079] 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, 13 serotypes of AAV have been characterized. 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, it is well known that various serotypes are very closely related in both structure and function, even at the genetic level, so it is fully expected that these same principles can be applied to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R. Puttson, ed.; and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; all possess three related capsid proteins (such as those expressed in AAV2). This similarity is further suggested by extensive cross-hybridization between serotypes along the length of the genome; and heteroduplex analysis, which reveals the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0080] As used herein, the term "adeno-associated vector" or "AAV vector" refers to a vector that contains one or more polynucleotides of interest (or transgenes such as micro-dystrophin) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector that encodes and expresses the rep and cap gene products.

[0081] 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 encapsidated polynucleotide AAV vector.When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), the particle is typically referred to as "AAV vector particle" or simply "AAV vector".Therefore, the production of AAV vector particle necessarily includes the production of AAV vector.As such, the vector is contained within the AAV vector particle.

[0082] The term "muscle-specific regulatory element" refers to a nucleotide sequence that controls the expression of a coding sequence specific for expression in muscle tissue. These regulatory elements include enhancers and promoters. The present disclosure provides constructs that include muscle-specific regulatory elements MCKH7 promoter, MCK promoter, and MCK enhancer.

[0083] "Muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle, such as skeletal muscle and smooth muscle (e.g., derived from the gastrointestinal tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated (such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts).

[0084] As used herein, the term "transduction" refers to the administration / delivery of the coding region for micro-dystrophin to a recipient cell either in vivo or in vitro via a replication-deficient rAAV of the present disclosure, resulting in expression of micro-dystrophin by the recipient cell.

[0085] As used herein, the term "transfection" of a cell means the introduction of genetic material into a cell for the purpose of genetically modifying the cell. Transfection can be accomplished by a variety of means known in the art, such as transduction or electroporation.

[0086] As used herein, "vector" refers to a recombinant plasmid or virus containing a polynucleotide to be delivered to a host cell either in vitro or in vivo. "Recombinant" means different from that normally found in nature.

[0087] "Serotypes" for vectors or viral capsids are defined by differences in immunological profiles based on capsid protein sequences and capsid structure.

[0088] "AAV Cap" refers to the AAV Cap proteins, VP1, VP2, and VP3, and analogs thereof.

[0089] "AAV Rep" means the AAV Rep protein or an analog thereof.

[0090] As used herein, "adjacent" with respect to a sequence adjacent to another element indicates the presence of one or more adjacent elements upstream and / or downstream (i.e., 5' and / or 3') relative to the sequence. The term "adjacent" is not intended to 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) that is "adjacent" to two other elements (e.g., an ITR) indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be an intervening sequence between them.

[0091] 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 polynucleotide encoding a transgene (e.g., micro-dystrophin)) into cells and / or tissues of an individual 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 to be treated.

[0092] As used herein, the term "genotyping" refers to the process of determining the composition of specific alleles 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. In some embodiments, the human dystrophin gene (DMD) of a subject is genotyped to characterize the mutations of the gene that may be particularly feasible for treatment with the compositions disclosed herein. Many genotyping techniques are known to those skilled in the art.

[0093] The term "stringent" is used to refer to conditions that are generally understood in the art as stringent. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturing agents (such as formamide). Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate (65-68°C) or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide (42°C). See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may also be used, but will affect the rate of hybridization. When considering hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6xSSC (containing 0.05% sodium pyrophosphate) at 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). Other agents may be included in the hybridization and washing buffers to reduce non-specific and / or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 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 also be used. The concentrations and types of these additives may be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, under typical ionic strength conditions, the hybridization rate is largely pH dependent.See Anderson, ML Met al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England) (1998). One skilled in the art can adjust hybridisation conditions to accommodate these variations and allow hybridisation to DNAs that differ in sequence relatedness.

[0094] 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 nourish growing cultured eukaryotic cells. 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. The solutions can also contain components that enhance growth and / or survival beyond the minimal rate, including hormones and growth factors. The solutions are formulated to an optimal pH and salt concentration for cell survival and proliferation. The media can also be "defined media" or "chemically defined media" (serum-free media that does not contain proteins, hydrolysates, or components of unknown composition). Defined media are free of animal-derived components, and all components have a known chemical structure. Those skilled in the art will appreciate that defined media can contain recombinant glycoproteins or proteins, including, but not limited to, hormones, cytokines, interleukins, and other signaling molecules.

[0095] As used herein, the term "basal medium formulation" or "basal medium" refers to any cell culture medium used for cell culture that has not been modified either by supplementation or by the selective removal of certain components.

[0096] As used herein, the terms "culture," "cell culture," and "eukaryotic cell culture" refer to a population of eukaryotic cells, either 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 eukaryotic cell population. As will be apparent to one of skill in the art, these terms as used herein can refer to a combination that includes a mammalian cell population and the medium in which the population is suspended.

[0097] As used herein, the term "batch culture" refers to a cell culture method in which all components that will ultimately be used in the cell culture, 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.

[0098] As used herein, the term "fed-batch culture" refers to a cell culture method 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 that provides additional components to the culture at some time after the start of the culture process is a feed medium. The components provided typically include nutritional supplements for cells that are exhausted 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 optionally purified.

[0099] As used herein, the term "perfusion culture" refers to a cell culture method in which additional components are continuously or semi-continuously provided to the culture after the start of the culture process. The components provided typically include nutritional supplements for the cells that are depleted during the culture process. A portion of the cells and / or components in the medium are typically continuously or semi-continuously harvested and optionally purified.

[0100] The "growth phase" of a cell culture refers to the exponential cell growth phase (log phase) during which cells generally divide rapidly. During this phase, cells are cultured for a period of time (usually 1-4 days) under conditions that maximize cell growth. The host cell growth cycle can be determined for a particular host cell without undue experimentation. "The period of time and under such conditions that maximize cell growth" and the like refer to medium conditions that are 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 for optimal growth of the particular cell line, generally in a humidified controlled atmosphere at about 25°C to 40°C.

[0101] In some embodiments, the cells are maintained in the growth phase for a period of between approximately 1 and 7 days (e.g., between 2 and 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 will be a period sufficient to regenerate a particular cell to a viable cell density within about 20%-80% of the maximum viable cell density possible if the culture is maintained under growth conditions. In some embodiments, "maximal growth rate" refers to the growth rate of a particular cell line / clone measured in its exponential growth phase while the cells are in fresh culture medium (e.g., measured at a time during culture when nutrients are adequately present and there is no significant growth inhibition from any component of the culture).

[0102] As used herein, the term "cell viability" refers to the ability of cells to survive in culture under a given set of culture conditions or a variety of experimental conditions. Also, as used herein, the term refers to the fraction of cells that are viable at a particular time relative to the total number of live and dead cells in the culture at that time.

[0103] As used herein, the term "cell density" refers to the number of cells present in a given volume of medium.

[0104] 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.

[0105] As used herein, the term "bioreactor operation" can include one or more of the lag phase, log phase, or plateau phase growth phases during a cell culture cycle.

[0106] As used herein, the terms "N1 culture vessel", "N1 seed train culture vessel", "N1 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. The cell culture grown in the N1 culture vessel may be obtained after cell culture in several vessels (such as N-4, N-3, and N-2 vessels) preceding the N1 culture vessel.

[0107] 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 N1 bioreactor. The N culture is used in the production of AAV.

[0108] As used herein, the term "seeding" or "inoculation" refers to the process of providing a cell culture to a bioreactor or another vessel. In one embodiment, the cells have been previously grown in another bioreactor or vessel. In another embodiment, the cells have been frozen and thawed immediately prior to providing to the bioreactor or vessel. The term refers to any number of cells, including single cells. rAAV and methods for producing rAAV (or compositions comprising rAAV) (e.g., rAAVrh74.MHCK7.microdystrophin)

[0109] The present disclosure provides a composition comprising a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.micro-dystrophin, wherein the rAAV is produced in a mammalian adherent cell and the adherent cell is cultured under suspension conditions in an N-1 container. In some embodiments, the rAAV is of serotype AAVrh.74 (e.g., rAAV.MHCK7.micro-dystrophin).

[0110] The present disclosure also provides a method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin in mammalian adherent cells by a suspension seed process, comprising the steps of: (a) culturing cells in an N-2 container with a first growth medium comprising serum; (b) removing said cells from said first medium; (c) inoculating cells from said step (b) in an N-1 container into a second medium comprising no serum or a lower concentration of serum than said first medium; (d) culturing said cells in said N-1 container under suspension conditions; and (e) inoculating cells from said step (d) into a third medium in a bioreactor:

[0111] In some embodiments, the rAAV used in the methods described herein 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 human micro-dystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 7.

[0112] In some embodiments, the suspension seed process further comprises (f) transfecting the adherent cells with a transgene plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct, a plasmid comprising the AAVrep and AAVcap genes, and an adenovirus helper plasmid.

[0113] 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 AAVrep and AAVcap genes comprises the AAV2rep and rAAVrh74cap genes. In some embodiments, the adenovirus helper plasmid comprises the E2A, E4ORF6, and VA RNA genes of adenovirus type 5.

[0114] In some embodiments, the suspension seed process further comprises the step of (g) lysing the adherent cells, hi some embodiments, the adherent cells are lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

[0115] In some embodiments, the suspension seed process further comprises (h) purifying the rAAV by at least one column chromatography step. In some embodiments, the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0116] In some embodiments, the suspension seed process further comprises culturing the cells in an N-3 container with the first growth medium, hi some embodiments, the suspension seed process further comprises culturing the cells in an N-4 container with the first growth medium.

[0117] In some embodiments, the bioreactor is an adherent bioreactor. In some embodiments, the rAAV is purified from the culture produced in the adherent bioreactor.

[0118] In some embodiments, the third 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, proteoglycan or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some embodiments, the third medium in the bioreactor comprises DMEM and 10% FBS.

[0119] In some embodiments, the adherent cells are cultured under suspension conditions for about 48 to 72 hours.

[0120] In some embodiments, the N-1 container is a suspension shake flask.

[0121] 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 suspension. In some embodiments, culturing the cells under suspension conditions does not alter the adhesion dependency of the cells. In some embodiments, culturing does not alter the cells to create a new cell line.

[0122] In some embodiments, the suspension seed process used to produce rAAVrh74.MHCK7.microdystrophin in mammalian adherent cells includes: (a) culturing cells in an N-2 container with a first growth medium containing serum; (b) removing said cells from said first medium; (c) inoculating the cells from step (b) in an N-1 container into a second medium that is serum-free or contains a lower concentration of serum than the first medium; (d) culturing said cells under suspension conditions in said N-1 container; (e) inoculating the third medium in a bioreactor with the cells from step (d) above; (f) transfecting the cells with a transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAVrep and AAVcap genes, and an adenovirus helper plasmid; (g) lysing the cells; and (h) purifying the rAAV by at least one column chromatography step.

[0123] In some embodiments, the rAAV is described in International Publication No. WO2019 / 245973A1, the entirety of which is expressly incorporated by reference herein.

[0124] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains 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 shown in Srivastava et al., J Virol. 45:555-564 (1983) (as amended by Ruffing et al., J Gen Virol. 75:3385-3392 (1994)). As other examples, the entire genome of AAV-1 is provided in Genbank accession number NC_002077; the entire genome of AAV-3 is provided in Genbank accession number NC_1829; the entire genome of AAV-4 is provided in Genbank accession number NC_001829; the AAV-5 genome is provided in Genbank accession number AF085716; the entire genome of AAV-6 is provided in Genbank accession number NC_00 1862; at least a portion of the AAV-7 genome and the AAV-8 genome are provided in 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 described in Gao et al. al., J. Virol. 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); 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). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 from their relative positions on the map) drive expression of two AAV internal reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19) in conjunction 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 are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology 158:97-129 (1992).

[0125] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cultured cells is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. In addition, AAV is capable of infecting many mammalian cells and targeting many different tissues in vivo. Furthermore, AAV can transduce slow-dividing and non-dividing cells and persist essentially for the life of said cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, allowing the construction of recombinant genomes. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the approximately 4.3 kb internal to 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, DNA of interest, and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another important feature of AAV is that it is a very stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making refrigeration less important for AAV. AAV can even be lyophilized. Finally, cells infected with AAV do not resist superinfection.

[0126] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. 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, so recombinant AAV transduction resulted in the appearance of transgene products in the systemic circulation after intramuscular injection. Furthermore, Lewis et al., J Virol. 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors required for the correct glycosylation, folding, and secretion of antibodies, indicating that muscle can stably express secreted protein therapeutics.

[0127] 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 recombinant virus, including but not limited to AAV serotypes AAVrh.74, 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 (e.g., rAAV with capsid mutations) are also contemplated. See, for example, Marsic et al., Molecular Therapy 22(11):1900-1909(2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. AAV1, AAV6, AAV8, or AAVrh.74 can be used to promote skeletal muscle-specific expression.

[0128] The DNA plasmid of the present disclosure comprises the rAAV genome of the present disclosure. The DNA plasmid is introduced into a cell that is permissive for infection with a helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) to assemble the rAAV genome into an infectious virus particle. The technology of producing rAAV particles, in which the AAV genome to be packaged, the rep and cap genes, and the helper virus functions are provided to the cell, is standard in the art. The production of rAAV requires the following components to be present in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes that are separate from the rAAV genome (i.e., not in the rAAV genome), and the helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype that can induce recombinant virus, and may be derived from an AAV serotype different from the rAAV genome ITR. These serotypes include, but are 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. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated by reference in its entirety.

[0129] The method for generating packaging cells is to create a cell line that stably expresses all the components necessary for AAV particle production, for example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selection marker such as a neomycin resistance gene is integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid 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 are suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0130] The general principles of rAAV production have been reviewed, for example, in Carter, Current Opinions in Biotechnology 1533-539 (1992); and Muzyczka, N., Curr. Topics Microbial. Immunol. 158:97-129 (1992). Various approaches have been reviewed in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al. al., J. Virol., 63:3822-3828 (1989); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 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 incorporated by reference in their entireties, with particular emphasis being placed on the sections of the documents relating to rAAV production.

[0131] Thus, the present disclosure provides a packaging cell that produces infectious rAAV. In one embodiment, the packaging cell can be a stably transformed cancer cell, such as HeLa cell, 293 cell, and PerC.6 cell (synonymous 293 line). In another embodiment, the packaging cell is a cell that is not a transformed cancer cell, such as low-passage 293 cell (human fetal kidney cell transformed with adenovirus E1), MRC-5 cell (human fetal fibroblast), WI-38 cell (human fetal fibroblast), Vero cell (monkey kidney cell), and FRhL-2 cell (embryo rhesus lung cell).

[0132] The recombinant AAV of the present disclosure (i.e., infectious encapsidated rAAV particle) comprises a rAAV genome. In an exemplary embodiment, the genome of both rAAVs lacks AAVrepDNA and AAVcapDNA (i.e., there is no AAVrepDNA or AAVcapDNA between the ITRs of the genome). An example of a rAAV that can be constructed to comprise the nucleic acid molecule of the present disclosure is described in International Patent Publication No. PCT / US2012 / 047999 (WO2013 / 016352), which is incorporated herein by reference in its entirety.

[0133] In an exemplary embodiment, the recombinant AAV vector of the present invention is produced by a triple transfection method (Xiao et al., J Virol 72:2224-2232 (1998)) using the AAV vector plasmids rAAV.MHCK7.micro-dystrophin, pNLRep2-Caprh74, and pHELP. The rAAV contains a micro-dystrophin gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is encapsidated into the AAVrh74 virion. The plasmid contains the micro-dystrophin sequence and the MHCK7 enhancer and muscle-specific promoter core promoter elements to drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) to promote high levels of gene expression, and the bovine growth hormone polyadenylation signal is used for efficient transcription termination.

[0134] pNLREP2-Caprh74 is an AAV helper plasmid encoding the 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.

[0135] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. This plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4ORF6, and VA RNA (adenovirus E1 function is provided by 293 cells). The adenovirus sequences present in this plasmid represent only about 40% of the adenovirus genome and do not contain cis elements important 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.

[0136] rAAV can be purified by standard methods in the art, such as by column chromatography or cesium chloride gradients, as disclosed herein. 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 WO 98 / 09657.

[0137] In one embodiment, the present disclosure provides an rAAV comprising a nucleotide sequence of a muscle-specific regulatory element 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 to 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.

[0138] In one aspect, the rAAV is rAAVrh74.MHCK7.microdystrophin (i.e., a viral particle form of rAAV serotype rh74 capsid encapsidating a nucleic acid expression cassette or genome containing a microdystrophin transgene driven by the MHCK7 promoter / enhancer), also referred to by the non-proprietary drug name derandistrogen moxeparvovec in the context of administration to a subject. In some aspects, when referring to a study of subjects in an embodiment administered derandistrogen moxeparvovec, the data (e.g., bar graphs) may be more simply indicated as "treated."

[0139] In one embodiment, the rAAVrh74.MHCK7.microdystrophin is rAAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, or 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 under suspension conditions in an N-1 container. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the rAAVrh74.MCK.microdystrophin is rAAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0140] 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.

[0141] In one embodiment, the rAAV is a non-replicating recombinant adeno-associated virus (AAV) designated rAAVrh74.MHCK7.micro-dystrophin 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, where the rAAV is produced in adherent cells and the adherent cells are cultured under suspension conditions in an N-1 container. The vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeats (ITRs), micro-dystrophin, SV40 introns (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 FIG. 1. The AAVrh74 serotype can be used for efficient gene transfer into skeletal and cardiac muscles following IV administration.

[0142] 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 fast skeletal troponin c gene element, a slow cardiac troponin c gene element, a slow troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0143] For example, the muscle-specific regulatory element is the MHCK7 promoter nucleotide sequence (SEQ ID NO:2 or SEQ ID NO:7) or the muscle-specific regulatory element is the MCK nucleotide sequence (SEQ ID NO:4). Additionally, in any rAAV vector of the present disclosure, the nucleotide sequence of the muscle-specific regulatory element (e.g., the MHCK7 or MCK nucleotide sequence) is operably linked to a nucleotide sequence encoding a micro-dystrophin protein. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO:2 or SEQ ID NO:7) is operably linked to a human micro-dystrophin coding sequence (SEQ ID NO:1) as set forth in the construct provided in FIG. 1 or FIG. 2 (SEQ ID NO:3) or FIG. 13 (SEQ ID NO:9). In another example, the MCK promoter (SEQ ID NO:4) is operably linked to a human micro-dystrophin coding sequence (SEQ ID NO:1) as shown in the construct provided in FIG. 5 or FIG. 6 (SEQ ID NO:5). In another aspect, the present disclosure provides a 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 disclosure also provides an rAAV vector comprising the nucleotide sequence of SEQ ID NO:1 and SEQ ID NO:4.

[0144] 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.micro-dystrophin vector comprises the nucleotide sequence depicted in FIG. 2, within and including the ITR of SEQ ID NO:3. In another aspect, the rAAV vector comprises a 5'ITR, a MHCK7 promoter, a chimeric intron sequence, a coding sequence of the human micro-dystrophin gene, 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.

[0145] In another embodiment, 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.micro-dystrophin vector construct comprises the nucleotide sequence of SEQ ID NO:9 and is shown in FIG. 13. This rAAV vector construct comprises the MHCK7 promoter, a chimeric intron sequence, a coding sequence of the human micro-dystrophin gene, and polyA. In one embodiment, the rAAV vector construct further comprises an ITR 5' to the promoter and an ITR 3' to the polyA. In one embodiment, the rAAV is AAVrh74.

[0146] In another embodiment, the rAAVrh74.MHCK7.micro-dystrophin vector (i.e., viral vector) comprises the nucleotide sequence depicted in Figure 15 within and including the ITRs of SEQ ID NO:8. The rAAV vector comprises the 5'ITR, MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human micro-dystrophin 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.

[0147] In another embodiment, the present disclosure provides a plasmid comprising the AAVrh74.MHCK7.micro-dystrophin construct. In one embodiment, the plasmid comprises a 5'ITR, a MHCK7 promoter, a chimeric intron sequence, a coding sequence of the human micro-dystrophin gene, polyA, and a 3'ITR. In one embodiment, the plasmid comprises kanamycin resistance and optionally comprises a pGEX plasmid backbone with a pBR322 origin of replication. In a particular embodiment, the plasmid is set forth in SEQ ID NO:8 and shown in Figures 14 and 15.

[0148] The present disclosure provides a recombinant AAV vector comprising a human micro-dystrophin nucleotide sequence of SEQ ID NO:1 and an 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.

[0149] The disclosure also provides an rAAV comprising the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct within and including the ITRs in SEQ ID NO:3, the nucleotide sequence within and including the ITRs in 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. The rAAV vector is of the AAV serotype AAVrh.74.

[0150] The rAAV vectors of the disclosure can be of any AAV serotype, such as serotypes AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0151] 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.

[0152] In another aspect, the present disclosure provides a method of producing rAAV vector particles, 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

[0153] In another aspect, the present disclosure contemplates a composition comprising the rAAV of the present disclosure. The composition of the present disclosure comprises rAAV and a pharma- ceutically acceptable carrier. The composition may also comprise other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are preferably non-toxic to recipients and inert at the dosages and concentrations used, and include buffers and surfactants (such as Pluronics).

[0154] The present disclosure provides a composition for treating muscular dystrophy (e.g., DMD) in a human subject in need thereof comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin, wherein the rAAV is produced in adherent cells and the adherent cells are cultured under suspension conditions in an N-1 container.

[0155] In some embodiments, the disclosure provides a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is produced in a mammalian adherent cell by a suspension seed process described herein. In some embodiments, the composition comprises: a) an rAAV particle comprising the nucleic acid sequence of SEQ ID NO:9; b) an rAAV particle comprising nucleotides 55-5021 of SEQ ID NO:3; and / or c) an rAAV particle comprising nucleotides 1-4977 of SEQ ID NO:8.

[0156] The titer of the rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the targeted cell type(s). Titers can be determined by standard methods in the art. The titer of rAAV is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 The dosage may range from 0.1 to 1.5 mg / mL, or more than DNase resistant particles (DRP). The dosage may also be expressed in units of viral genome (vg). One exemplary method for determining the encapsulated vector genome titer is to use quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25:855-869 (2017)).

[0157] Methods of transducing rAAV into target cells in vivo or in vitro are contemplated by 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 the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. In aspects of the present disclosure, an effective dose is a dose that relieves (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) of the disease, and / or prolongs life. One example of a disease contemplated for prevention or treatment using the methods of the present disclosure is DMD.

[0158] Combination therapy is also contemplated by the present disclosure.Combination as used herein includes both simultaneous and sequential treatment.The combination of the method of the present disclosure with standard medical treatment (e.g., corticosteroids) is particularly contemplated, as well as the combination with new therapeutic methods.

[0159] Administration of an effective dose of the composition may be by a route standard in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The administration route(s) and serotype(s) of the AAV components of the rAAV of the present disclosure (particularly the AAV ITRs and capsid proteins) may be selected and / or adapted by those skilled in the art in consideration of the infectious disease and / or disease state being treated and the target cell / tissue(s) that will express the micro-dystrophin protein.

[0160] The present disclosure provides for local and systemic administration of the rAAV and composition of the present disclosure in effective dose.For example, systemic administration is administered 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 by injection, infusion or implantation.

[0161] In particular, the actual administration of the rAAV of the present disclosure can be accomplished 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, injection into muscle and injection into the bloodstream. It has been demonstrated that resuspension of rAAV in phosphate buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although in the usual manner with rAAV, compositions that degrade DNA should be avoided). 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, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a topical formulation that is delivered to muscle by transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in the practice of the present disclosure. The rAAV can be used with any pharma- ceutically acceptable carrier for ease of administration and handling.

[0162] In one embodiment of the disclosure, the 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.

[0163] The dose of rAAV to be administered in the methods disclosed herein will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the target cell type. The aforementioned doses can be determined by standard methods in the art. The titer of each rAAV administered is about 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×1012 , about 1×10 13 , about 1×10 14 , about 2×10 14 , or approximately 1 × 10 15 or more of DNase resistant particles (DRP). Dosages may also be expressed in units of viral genomes (vg) (i.e., 1×10 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, 1×10 15 Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1×10 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, 1.5×10 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, 3.0×10 14 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 A method for measuring AAV titer is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0164] In particular, the actual administration of the rAAV of the present disclosure can be accomplished 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, injection into muscle and injection into the bloodstream. It has been demonstrated that resuspension of rAAV in phosphate buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although in the usual manner with rAAV, compositions that degrade DNA should be avoided). 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, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a topical formulation that is delivered to muscle by transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in the practice of the present disclosure. The rAAV can be used with any pharma- ceutically acceptable carrier for ease of administration and handling.

[0165] For the purpose of 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 necessary, and the liquid diluent can be first made isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains an acidic phosphate group) or pharmacologically acceptable salt 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 connection, the sterile aqueous media employed are all readily available by standard techniques well known to those skilled in the art.

[0166] Pharmaceutical carriers, diluents, or excipients suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or suspensions for injection. 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, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof, as well as 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. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0167] Sterile injection is prepared by incorporating rAAV in the required amount in a suitable solvent together with various other components as listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle that contains basic dispersion medium and other components from the above-listed components.For the preparation of sterile powder for sterile injection, the preferred preparation method is vacuum drying and freeze-drying technology, which can obtain powder of active ingredient and any additional desired ingredients from the solution of active ingredient that has been previously sterilized by filtration.

[0168] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells that do not generate an inappropriate immune response in the subject can be used.

[0169] Suitable methods for transduction and reintroduction of transduced cells into subjects 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 screening for cells carrying the DNA of interest using conventional techniques such as Southern blot and / or PCR, or by using a selection marker.The transduced cells can then be formulated into pharmaceutical compositions, and the compositions can be introduced into subjects by various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or injection into smooth and cardiac muscles, for example, using a catheter.

[0170] When cells are transduced with the rAAV of the present disclosure, micro-dystrophin protein is persistently expressed.Therefore, the present disclosure provides a method for administering / delivering the rAAV that expresses micro-dystrophin protein to animals, preferably humans.These methods include transducing one or more rAAV of the present disclosure into tissues, including but not limited to tissues such as muscle, organs such as liver and brain, and glands such as salivary glands.Transduction can be carried out using gene cassettes that contain tissue-specific regulatory elements. For example, one embodiment of the disclosure provides a method for the identification and characterization of muscle specific regulatory elements (actin and myosin gene families such as 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)), human skeletal actin gene (Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)), cardiac actin gene, muscle creatine kinase sequence elements (Johnson et al., Mol Cell Biol, 1999, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2040, 2041, 2042, 2043, 2044, 2050, 2051, 2052, 2060, 2061, 2070, 2071, 2072, 2080, 2081, 2073, 2074, 2075, 2076, 2077, 2078, 2082, 2083, 2084, 2085, 2086, 20 9:3393-3399 (1989)), and regulatory elements derived from the mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow 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 regulatory elements.

[0171] Muscle tissue is an attractive target for in vivo DNA delivery because it is a non-essential organ and is easily accessible. The present disclosure contemplates sustained expression of micro-dystrophin from transduced muscle fibers.

[0172] Thus, the present disclosure provides a method of administering an effective dose (or doses administered essentially simultaneously or at intervals) of a rAAV encoding micro-dystrophin to a subject in need thereof (a subject having muscular dystrophy).

[0173] The disclosure provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, 8, or 9. The disclosure also provides 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.

[0174] Another aspect of the disclosure provides compositions comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, 8, or 9, a 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 a 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 disclosed herein may be practiced using these compositions. Hybrid seed train expansion of adherent cells

[0175] Some embodiments of the present disclosure relate to a method for expanding cells, comprising the steps of: (a) culturing cells in an N-2 container with a first medium containing serum; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) in an N-1 container with a second medium 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) in a third medium in a bioreactor. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains serum at a lower concentration than the serum concentration in the first medium.

[0176] Some embodiments of the present disclosure relate to a seed train expansion method comprising the steps of: (a) culturing cells in an N-3 container with a first medium containing serum; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) in an N-2 container with a second medium that is serum-free or contains serum at a lower concentration than the first medium; (d) culturing the cells in the N-2 container under suspension conditions; (e) and inoculating the second medium in an N-1 vessel with cells from step (d); and (f) inoculating a third medium in a bioreactor with cells from step (d). In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains serum at a lower concentration than the serum concentration in the first medium.

[0177] Some embodiments of the present disclosure relate to a cell expansion method of adherent cells, comprising the steps of: (a) culturing 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 containing no serum or a lower concentration of serum 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 embodiments, the method can further comprise passaging the adherent cells of step (a) at least once under adherent conditions. In some embodiments, the method can further comprise passaging the adherent cells of step (d) at least once under suspension conditions. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains a lower concentration of serum than the first medium in the N-1 container.

[0178] The first medium, the second medium, and the third medium can be any medium suitable for the particular cells to be cultured. In some embodiments, the medium comprises, 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., compositions comprising 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 and animal sources), and combinations thereof. Growth media can 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).

[0179] In some embodiments, the serum-free secondary growth medium, which is serum-free or contains a lower concentration of serum than the first serum, is substantially free (including ultra-trace or less) of calcium ions, fetal bovine serum (FBS), fibronectin, collagen, laminin, proteoglycans of the extracellular matrix, or non-proteoglycan polysaccharides, which are believed to support cell anchoring. In one embodiment, the second medium is a serum-free medium. In another embodiment, the second medium contains a lower concentration of serum than the serum concentration in the first medium.

[0180] In some embodiments, the growth medium has a pH of 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. 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, between about 6 The composition may have a pH of between about 7.9 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.

[0181] In some embodiments, the 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 37.5°C, between about 34°C and about 37°C, between about 35°C and about 37°C, between about 35.5°C and about 37.5°C, between about 36°C and about 37°C, or about 36.5°C. In some embodiments, the cells can be cultured at a temperature of about 37°C from the beginning to the end of the culture period. In some embodiments, the temperature can be changed or fluctuated slightly (e.g., by hours or days) during the culture period. In some embodiments, the temperature can be changed or transitioned (e.g., increased or decreased) about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, or 15 days 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, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 degrees Celsius. In some embodiments, the temperature can be shifted downward by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 degrees Celsius.

[0182] In some embodiments, cells can be cultured using an atmosphere containing about 1% to about 15% CO2. In some embodiments, cells can be cultured using an atmosphere containing about 14% CO2, 12% CO2, 10% CO2, 8% CO2, 6% CO2, 5% CO2, 4% CO2, 3% CO2, 2% CO2, or about 1% CO2.

[0183] In some embodiments, the dissolved oxygen (dO2) in the cell culture is 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%, between about 5% and about 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 1 Between 5% 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%, between about 30% and 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 50%, or between about 50% and about 55%.

[0184] In some embodiments, the pH of the cell culture can be maintained at a specific pH value by the addition of a base solution (such as an alkaline base solution). The pH of the cell culture can be 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 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, between about 6.9 and about 7 The pH of the solution may be maintained at 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.

[0185] In some embodiments, cell culture can be performed under suspension conditions in any type of cell culture flask suitable for static or mixed / shaking suspension cell expansion (e.g., using T-flasks, roller bottles, spinner flasks, or shake flasks; or combinations thereof). In some embodiments, the N-1 container is a shake flask.

[0186] In some embodiments, the suspension conditions can include several forms of stirring. In some embodiments, the stirring can be rotary stirring. In some embodiments, the stirring can 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 280 RPM, between about 25 RPM and about 26 ... 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 to 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, PM, 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 to 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 to 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 to 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 may be performed at a speed 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 may be performed continuously or periodically.

[0187] In some embodiments, the cells are passaged no more than twice under suspension conditions.

[0188] In some embodiments, the cells are cultured in suspension culture for about 24 to about 96 hours. In some embodiments, the cells are cultured in suspension culture for about 36 to about 84 hours. In some embodiments, the cells are cultured in suspension culture for about 48 to about 72 hours. In some embodiments, the cells are cultured in suspension culture for about 54 to about 66 hours. In some embodiments, the cells are cultured in suspension culture for about 24, about 30, about 36, about 42, about 48, about 54, about 60, about 66, about 72, about 78, about 84, about 90, or about 96 hours.

[0189] In some embodiments of the present disclosure, the cell is an adherent cell. In some embodiments, the adherent cell is a HeLa cell, a CHO cell, a HEK-293 cell, a VERO cell, a BHK cell, an MDCK cell, an MDBK cell, or a COS cell. In some embodiments, the adherent cell is human. In some embodiments, the adherent cell is a HeLa cell or a HEK-293 cell. In some embodiments, the adherent cell is a HEK-293 cell.

[0190] In some embodiments, the adherent cells are not adapted to suspension. In some embodiments, culturing the cells under suspension conditions does not change the adhesion dependency of the cells. In some embodiments, the method does not alter the cells to create new cell lines. The methods disclosed herein do not change the genomic or transcriptomic profile of the cells. The methods disclosed herein do not change the phenotype of the cells.

[0191] In some embodiments, the cells are passaged multiple times in serum-supplemented growth medium under adherent conditions before inoculating 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 inoculating 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.

[0192] In some embodiments, the bioreactor is an adherent bioreactor. In some embodiments, the adherent cells are purified from a culture produced in the adherent bioreactor.

[0193] In some embodiments, the bioreactor comprises at least one, more preferably a plurality of carriers, on which the expanded cells tend to adhere and can be suspended or fixed in the bioreactor. Preferably, the aforementioned carriers can be made using, for example, polyethylene terephthalate, polystyrene, polyester, polypropylene, DEAE-dextran, collagen, glass, alginate, or acrylamide. In some embodiments, the bioreactor can be a bioreactor comprising a bead-type microcarrier (e.g., Cytodex® brand beads, sold by GE Healthcare Inc. division of General Electric Corp.) or a matrix-type carrier (e.g., Fibra-Cell™ brand disks, sold by Eppendorf Corp.). In some embodiments, the bioreactor uses polyester fiber carriers, such as those used in iCELLis® nano or iCELLis® 500 bioreactors (commercially available from Advanced Technology Materials Inc. (Brussels, Belgium) and Pall corporation (Fall River, Mass)).

[0194] In some embodiments, the third 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 FBS, fibronectin, collagen, laminin, calcium ions, proteoglycan 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 medium immediately before, during, or after inoculation of the suspension cells into the bioreactor.

[0195] 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.

[0196] In some embodiments, the suspension expanded cells from step (d) can be inoculated directly into a bioreactor. In some embodiments, the amount of cells inoculated into the bioreactor varies based on the size of the bioreactor. In some embodiments, a 4 m2 bioreactor (e.g., an iCELLis® nanobioreactor) is used. In some embodiments, about 1×10 cells are inoculated into a 4 m2 bioreactor. 8 1 x 10 cells 9 In some embodiments, a 4 m bioreactor is inoculated with between about 3×10 8 cells and 7 × 10 8 In some embodiments, a 4 m bioreactor is inoculated with between about 4×10 cells. 8 cells and 6 × 10 8 In some embodiments, a 4 m bioreactor is inoculated with between about 5×10 cells. 8 Inoculate the cells. In some embodiments, equivalent cell densities are used for other size bioreactors.

[0197] In some aspects, the methods of the present disclosure can further comprise culturing the cells in a bioreactor. In some aspects, the cell culture comprises batch culture. In some aspects, the cell culture comprises fed-batch culture. In some aspects, the cell culture comprises perfusion culture.

[0198] Fed-batch culture involves 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 culture can be placed in a bioreactor (e.g., a production bioreactor, such as 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 either liquid or a dry powder. In some embodiments, the feed culture medium is a concentrated form of 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., added continuously) 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 about the same time. In some embodiments, the total volumes of the first liquid feed culture medium and the second liquid feed culture medium added to the culture over the entire culture period can be about the same.

[0199] When feed culture medium is added continuously, the rate of addition of the feed culture medium can be kept constant or increased (e.g., steadily increased) over the culture period. Continuous addition of the feed culture medium can be started at a particular time point during the culture period, for example, when the cells reach a target viable cell density (e.g., a viable cell density of about 1×106 cells / mL, about 1.1×106 cells / mL, about 1.2×106 cells / mL, about 1.3×106 cells / mL, about 1.4×106 cells / mL, about 1.5×106 cells / mL, about 1.6×106 cells / mL, about 1.7×106 cells / mL, about 1.8×106 cells / mL, about 1.9×106 cells / mL, or about 2.0×106 cells / mL). In some embodiments, continuous addition of the feed culture medium can be started on the second, third, fourth, or fifth day of the culture period.

[0200] In some embodiments, incremental (periodic) addition of feed culture medium can be initiated when the cells reach a target cell density (e.g., about 1×106 cells / mL, about 1.1×106 cells / mL, about 1.2×106 cells / mL, about 1.3×106 cells / mL, about 1.4×106 cells / mL, about 1.5×106 cells / mL, about 1.6×106 cells / mL, about 1.7×106 cells / mL, about 1.8×106 cells / mL, about 1.9×106, or about 2.0×106 cells / mL). In some embodiments, incremental addition of feed culture medium can be performed at regular intervals (e.g., every day, every second 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 increased incrementally between the first incremental addition of feed culture medium and subsequent additions of feed culture medium. In some embodiments, the volume of feed culture medium of a liquid culture 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.

[0201] In some embodiments, the addition (continuous or periodic) of liquid feed culture medium is increased for between 6 and 7 days, between about 6 and about 6 days, between about 6 and about 5 days, between about 6 and about 4 days, between about 6 and about 3 days, between about 6 and about 2 days, between about 6 and about 1 day, between about 12 and about 7 days, between about 12 and about 6 days, between about 12 and about 5 days, between about 12 and about 4 days, between about 12 and about 3 days, between about 12 and about 2 days, between about 1 day and about 7 days, between about 1 day and about 6 days, between about 1 day and about 1 day ... between about 1 and about 5 days, between about 1 and about 4 days, between about 1 and about 3 days, between about 1 and about 2 days, between about 2 and about 7 days, between about 2 and about 6 days, between about 2 and about 5 days, between about 2 and about 4 days, between about 2 and about 3 days, between about 3 and about 7 days, between about 3 and about 6 days, between about 3 and about 5 days, between about 3 and about 4 days, between about 4 and about 7 days, between about 4 and about 6 days, between about 4 and about 5 days, between about 5 and about 7 days, or between about 5 and about 6 days.

[0202] 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 fractions may be between about 0.01 and about 0.28, between about 0.01 and about 0.26, between about 0.01 and about 0.24, between about 0.01 and about 0.22, between about 0.01 and about 0.20, between about 0.01 and about 0.18, between about 0.01 and about 0.16, between about 0.01 and about 0.14, between about 0.01 and about 0.12, between about 0.01 and about 0.10, between about 0.01 and about 0.08, between about 0.01 and about 0.06, between about 0.0 Between 1 and about 0.04, between about 0.02 and about 0.3, between about 0.02 and about 0.28, between about 0.02 and about 0.26, between about 0.02 and about 0.24, between about 0.02 and about 0.22, between about 0.02 and about 0.20, between about 0.02 and about 0.18, between about 0.02 and about 0.16, between about 0.02 and about 0.14, between about 0.02 and about 0.12, between about 0.02 and about 0.10, between about 0.02 and about 0.08, between about 0.02 and about Between about 0.06 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. between about 0.025 and about 0.12, between about 0.025 and about 0.10, between about 0.025 and about 0.08, between about 0.025 and about 0.06, between about 0.025 and about 0.04, between about 0.05 and about 0.3, between about 0.05 and about 0.28, between about 0.05 and about 0.26, between about 0.05 and about 0.24, between about 0.05 and about 0.22, between about 0.05 and about 0.20, between about 0.05 and about 0.18, between about 0.05 and about 0.16, between about 0.between about 0.05 and about 0.14, between about 0.05 and about 0.12, between about 0.05 and about 0.10, between about 0.1 and about 0.3, between about 0.1 and about 0.28, between about 0.1 and about 0.26, between about 0.1 and about 0.24, between about 0.1 and about 0.22, between about 0.1 and about 0.20, between about 0.1 and about 0.18, between about 0.1 and about 0.16 It may be between about 0.1 and about 0.14, between about 0.1, between about 0.15 and about 0.3, between about 0.15 and about 0.2, between about 0.2 and about 0.3, or between about 0.25 and about 0.3.

[0203] 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 about 0.02 and about 1.0 times the volume of the initial cell culture, between about 0.02 and about 0.9 times, between about 0.02 and about 0.8 times, between about 0.02 and about 0.7 times, between about 0.02 and about 0.6 times, between about 0.02 and about 0.5 times, between about between 0.02 times and about 0.4 times, between about 0.02 times and about 0.3 times, between about 0.02 times and about 0.2 times, between about 0.02 times and about 0.1 times, between about 0.02 times and about 0.08 times, between about 0.02 times and about 0.06 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 0.05 times and about 1.0 times, between about 0.05 times and about 0.8 times, Between about 0.05 and about 0.7, between about 0.05 and about 0.6, between about 0.05 and about 0.5, between about 0.05 and about 0.4, between about 0.05 and about 0.3, between about 0.05 and about 0.2, between about 0.05 and about 0.1, between about 0.1 and about 1.0, between about 0.1 and about 0.9, between about 0.1 and about 0.8, between about 0.1 and about 0.7, between about 0.1 and about 0 The antibody may be between about 0.6-fold, between about 0.1-fold and about 0.5-fold, between about 0.1-fold and about 0.4-fold, between about 0.1-fold and about 0.3-fold, between about 0.1-fold and about 0.2-fold, between about 0.2-fold and about 1.0-fold, between about 0.2-fold and about 0.9-fold, between about 0.2-fold and about 0.8-fold, between about 0.2-fold and about 0.7-fold, between about 0.2-fold and about 0.6-fold, between about 0.2-fold and about 0.5-fold, or between about 0.2-fold and about 0.4-fold.

[0204] In some embodiments, the total amount of feed culture medium added (continuously or periodically) over the entire culture period is between about 1% and about 40% (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% of the volume of the initial culture. 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%, Between 4% and about 20%, 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%, between about 35% and about 40%).

[0205] In some embodiments, two different feed culture media are added (continuously or incrementally) during the fed batch culture. In some embodiments, the amount or volume of the first feed culture medium and the second feed culture medium added can be substantially the same or different. In some embodiments, the first feed culture medium can be in liquid form and the second feed culture medium can be in solid form. In some embodiments, the first feed culture medium and the second feed culture medium can be liquid feed culture media.

[0206] Perfusion culture involves 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, where 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 done simultaneously or sequentially, or some combination of the two. In some embodiments, removal and addition can be performed continuously, such as at rates that remove and replace volume 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.

[0207] In some embodiments, the first volume of the first growth medium removed and the second volume of the second growth medium added can be kept approximately the same over each 24 hour period. In some embodiments, the rate at which the first volume of the first growth medium is removed (volume / unit time) and the rate at which the second volume of the second growth medium is added (volume / unit time) can be varied and will depend on the conditions of the particular cell culture system. In some embodiments, the rate at which the first volume of the first growth medium is removed (volume / unit time) and the rate at which the second volume of the second growth medium is added (volume / unit time) can be approximately the same or can be different.

[0208] In some embodiments, the volumes removed and added can be varied by incremental increases over each 24 hour period. In some embodiments, the volume of the first growth medium removed and the volume of the second growth medium added in each 24 hour period can be increased over the culture period. In some embodiments, the volume can be increased by a volume fraction that is between 0.5% and about 20% of the volume of the bioreactor over a 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 the volume of the first liquid culture medium over a 24 hour period.

[0209] In some embodiments, after the first 48 to 96 hours of the culture period, during each 24 hour 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.

[0210] In some embodiments, the first and second growth media can be the same type of media. In some embodiments, the first and second growth media can be different. In some embodiments, the second liquid culture medium can have a higher concentration of one or more media components.

[0211] In some embodiments, the first volume of the first growth medium can be removed by 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 permeation of the first volume of the first growth medium through a sterile membrane with a molecular weight cutoff that excludes cells or by gravity flow. In some embodiments, the first volume of the first growth medium can be removed by stopping agitation or significantly reducing the agitation speed for 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.

[0212] 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, such as by pipetting or pouring the second volume of the second liquid culture medium directly onto the first liquid culture medium, or can be added in an automated manner.

[0213] In some embodiments, the method further comprises contacting the 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 prior to inoculating the cells in a bioreactor. In some embodiments, the cells are transfected after inoculating the cells in a bioreactor. In some embodiments, the cells are contacted or transfected with a second polynucleotide, comprising a nucleic acid encoding a transgene. In some embodiments, the cells are cultured under conditions to produce a viral vector. In some embodiments, the method further comprises isolating the produced viral vector.

[0214] In some embodiments, the polynucleotide is a viral vector. In some embodiments, the viral vector is an adenoviral vector and an adeno-associated viral (AAV) vector. These vectors infect many dividing and non-dividing cell types, including synovial cells and hepatocytes. As shown above, the adenoviral vector and the AAV vector after cell entry are episomal, so these vectors are suitable for therapeutic application (Russell, J.Gen.Virol.81:2573-2604(2000)); Goncalves, Virol J.2(1):43 2005)). AAV vectors are capable of stable expression of transgenes for very long periods of time (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). Gene therapy methods using AAV vectors have been described by Wang et al., 2005, J Gene Med. March 9 (advance online publication); 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).

[0215] 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.

[0216] In some aspects, the cells are cultured under conditions to produce recombinant viral particles, hi some aspects, the method further comprises isolating the produced recombinant viral particles.

[0217] In some embodiments, the viral vector comprises a transgene operably linked to a suitable control sequence. The term "control sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that regulate protein transcription or translation. Such control sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990). In some embodiments, the control sequence can include a promoter sequence. In some embodiments, the promoter sequence can 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.

[0218] In some embodiments, the viral vector comprises an additional nucleotide sequence encoding an additional polypeptide. In some embodiments, the additional polypeptide can be a (selectable) marker polypeptide that allows identification, selection, and / or screening of cells containing the viral vector. In some embodiments, the marker polypeptide can be the fluorescent protein GFP, and the selectable marker genes HSV thymidine kinase (for selection in HAT medium), bacterial hygromycin B phosphotransferase (for selection in hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection in G418), and dihydrofolate reductase (DHFR) (for selection in methotrexate), CD20, a low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are provided in Sambrook and Russel (2001)''Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. Methods for Producing Viral Vectors (e.g., AAV)

[0219] Some embodiments of the present disclosure relate to a method of producing a viral vector (e.g., an rAAV as disclosed herein), comprising expanding cells according to any one of the seed train expansion methods disclosed herein, inoculating the cells into a growth medium in a bioreactor, transfecting the cells with a polynucleotide sequence encoding a viral particle, and culturing the cells in the bioreactor under conditions such that said viral particles are produced. In some embodiments, the production of viral vectors is disclosed in U.S. Patent Application Serial No. 63 / 123,602, expressly incorporated herein by reference.

[0220] The method of transferring exogenous nucleic acid into host cell is well known in the art and will vary according to the host cell used.Techniques include, but are not limited to, dextran-mediated transfection, calcium sulfate precipitation, calcium chloride treatment, polyethyleneimine-mediated transfection, polybrene-mediated transfection, protoplast fusion, electroporation, virus or phage infection, encapsulation of polynucleotide(s) in liposome, and direct microinjection of DNA into nucleus.Transfection can be either transient or stable.

[0221] In some embodiments, the polynucleotide sequence is a plasmid. In some embodiments, the plasmid encodes an AAV-derived viral particle.

[0222] In some embodiments, the polynucleotide sequence is a viral vector. In some embodiments, the viral vector encodes a viral particle. In a preferred embodiment, the viral particle is derived from AAV. In some embodiments, the rAAV comprises a nucleic acid sequence of SEQ ID NO:9. In some embodiments, the present disclosure provides an rAAV particle comprising a nucleic acid sequence of SEQ ID NO:9. In some embodiments, the present disclosure provides an rAAV particle 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.

[0223] In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector can include a recombinant AAV vector (rAAV). As used herein, "rAAV vector" refers to a recombinant vector that includes a portion of the AAV genome encapsidated in a protein shell of capsid proteins from an AAV serotype disclosed herein. In some embodiments, the AAV vector can include an inverted terminal repeat (ITR) from an adeno-associated virus serotype (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVRH10, AAV11, AAV12, etc.).

[0224] Typically, the vector genome is required to use adjacent 5' and 3' ITR sequences to allow efficient packaging of the vector genome into the rAAV capsid. In some embodiments, the rAAV genome present in the rAAV vector comprises at least the nucleotide sequence of the inverted terminal repeat region (ITR) of one of the AAV serotypes, or a nucleotide sequence substantially identical to the aforementioned nucleotide sequence, and a nucleic acid sequence encoding a transgene under the control of a suitable control element (e.g., a promoter), wherein the control element and modified nucleic acid sequence(s) are inserted between the two ITRs.

[0225] The entire genome of several AAV serotypes and the corresponding ITRs have been sequenced (Chiorini et al. J. of Virology 73:1309-1319 (1999)). They can be cloned or produced by chemical synthesis known in the art (e.g., using an oligonucleotide synthesizer, e.g., provided by Applied Biosystems Inc., Fosters, Calif., USA) or by standard molecular biology techniques. ITRs can be cloned from the AAV viral genome or excised from a vector containing AAV ITRs. Either end of the ITR nucleotide sequence can be ligated 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.

[0226] In some embodiments, the viral capsid component of the packaged viral vector can be a parvovirus capsid (e.g., AAV Cap) and / or a chimeric capsid. An example of a suitable parvovirus viral capsid component is a capsid component from the Parvoviridae family (such as an autonomous parvovirus or a dependovirus). For example, the viral capsid can 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 will know that there are likely other variants that have not yet been identified that perform the same or similar functions), or can include components from two or more AAV capsids. The full complement of AAV Cap proteins includes VP1, VP2, and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein can comprise less than a complete complement of the AAV Cap protein or can provide a complete complement of the AAV Cap protein.

[0227] In some embodiments, one or more of the AAV Cap proteins can be chimeric proteins that contain the amino acid sequence of AAV Cap from two or more viruses, preferably two or more AAVs. For example, a chimeric viral capsid can 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 nucleotide sequences that encode any viral proteins (such as the AAV rep (replication) or cap (capsid) genes). In some embodiments, the rAAV genome can further include a marker or reporter gene (such as an antibiotic resistance gene, a gene encoding a fluorescent protein (e.g., gfp) or a gene encoding a chemical, enzymatic, or other detectable and / or selectable product (e.g., lacZ, aph, etc.) known in the art).

[0228] In some embodiments, the rAAV genome present in the above-described rAAV vectors can further comprise a promoter sequence operably linked to the nucleotide sequence encoding the transgene.

[0229] In some embodiments, a suitable 3' untranslated sequence can also be operably linked to the modified nucleic acid sequence encoding the transgene. A suitable 3' untranslated region can be one naturally associated with the nucleotide sequence or can be derived from a different gene, such as, for example, the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal, and enhancer sequence).

[0230] Except as otherwise indicated, methods known to those skilled in the art can be used for the construction of recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvovirus Rep and / or Cap sequences, and transiently or stably engineered packaging cells. Such techniques are 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).

[0231] In some embodiments, a suitable 3' untranslated sequence can also be operably linked to the nucleic acid sequence encoding the transgene. A suitable 3' untranslated region can be one naturally associated with the nucleotide sequence or can be derived from a different gene, such as, for example, the bovine growth hormone 3' untranslated region (e.g., the bGH polyadenylation signal, the SV40 polyadenylation signal, the SV40 polyadenylation signal, and an enhancer sequence).

[0232] In some embodiments, additional nucleotide sequences can be operably linked to the nucleic acid sequence encoding the transgene (such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like).

[0233] Unless otherwise indicated, methods known to those skilled in the art can be used for constructing lentiviral constructs, vectors, and transiently and stably engineered packaging cells. Such techniques are known to those skilled in the art. See, for example, 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).

[0234] In some embodiments, the method of the present disclosure includes transfecting a cell with a transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAVrep and AAVcap genes, 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 AAVrep and AAVcap genes comprises the AAV2rep and rAAVrh74cap genes. In some embodiments, the adenovirus helper plasmid comprises the E2A, E4ORF6, and VA RNA genes of adenovirus type 5.

[0235] In some embodiments, the method further comprises isolating the produced viral particles. The viral vector is amplified by replicating inside the cell to produce viral particles. Viral infection causes the transfected cell to lyse. Therefore, the lytic properties of viral vectors such as AAV allow two different modes of viral particle production and isolation. The first mode is to use an external agent to lyse cells, and collect viral particles before cell lysis. The second mode is to collect viral particles from the supernatant after almost complete cell lysis by the produced virus.

[0236] Methods that can be used for active cell lysis are known to those skilled 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, and combinations of the above, etc.

[0237] In some embodiments, the cells can be lysed using at least one detergent. In some embodiments, detergents can include anionic, cationic, zwitterionic, and non-ionic 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.

[0238] 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 RNase commonly used in the art.

[0239] Methods for harvesting or isolating viral vectors from transfected cells are broadly disclosed in WO2005 / 080556, which is incorporated herein by reference in its entirety.

[0240] In some embodiments, the time of harvesting or isolating the viral vector is between about 24 and 120 hours, between about 36 and 108 hours, between about 48 and about 96 hours, between about 60 and about 84 hours after transfection, hi some embodiments, the time of harvesting or isolating the vector is about 72 hours after transfection.

[0241] In some embodiments, the isolated virus particles can be further purified. In some embodiments, the purification of the virus particles can be carried out in several steps, including clarification, ultrafiltration, diafiltration, or separation using chromatography. Such methods are described in WO2005 / 080556, which is incorporated by reference in its entirety. In some embodiments, clarification can be carried out by a filtration step that removes cell debris and other contaminants from the cell lysate. In some embodiments, ultrafiltration is used to concentrate the virus solution. In some embodiments, diafiltration, buffer exchange, or ultrafiltration membranes can be used to remove and exchange salts and sugars, etc. Those skilled in the art know how to find the optimal conditions for each purification step.

[0242] In some embodiments, the purification can be performed by density gradient centrifugation. In some embodiments, the purification uses at least one chromatography step. In some embodiments, the viral vector can be purified by anion exchange chromatography, size exclusion chromatography, or a combination thereof. Methods of Treating Muscular Dystrophy (eg, DMD)

[0243] 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 at a dose of about 5.0×10 12 vg / kg ~ approx. 1.0×1015 The present invention provides a method for producing rAAV in mammalian adherent cells, and culturing the adherent cells in suspension in an N-1 container. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0244] The present disclosure also provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering to said human subject a composition comprising an rAAV as described herein. In some embodiments, the rAAV is administered using a systemic route, at a dose of about 5.0×10 12 vg / kg ~ approx. 1.0×10 15 In some embodiments, the systemic route of administration is intravenous and the administered dose of rAAV is about 2×10 14 vg / kg.

[0245] 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 by intravenous route via a peripheral vein in a limb.

[0246] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0247] In some embodiments, the expression level of micro-dystrophin gene in cells of subject increases after administration of rAAV compared to the expression level of micro-dystrophin gene 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 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.

[0248] In some embodiments, the average percentage of micro-dystrophin positive fibers in muscle tissue of a subject increases after administration of rAAV compared to the number of micro-dystrophin positive fibers before administration of rAAV.In some embodiments, the average percentage of micro-dystrophin positive fibers is at least 70.5%, and the average intensity is at least 116.9%, as detected by immunofluorescence (IF) in muscle biopsies before and after administration of rAAV.In some embodiments, micro-dystrophin transduction by vector genome count is at least 3.87 average vector genome copy number per nucleus.

[0249] In some embodiments, in a method of treating a patient with DMD, the composition is administered to a genotyped patient. In some embodiments, the patient's human dystrophin (DMD) gene is genotyped. In some embodiments, the genotyped patient is genotyped for at least one mutation in exons 18-79 of the human dystrophin (DMD) gene.

[0250] In some embodiments, the method of treating muscular dystrophy further comprises genotyping the DMD gene of said human subject prior to administration of said composition to said human subject. In some embodiments, the 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 stop, or other pathogenic variant that results in the absence of expression of human dystrophin protein.

[0251] In some embodiments, detection of a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the abolition of expression of human dystrophin protein confirms that the subject is suitable to be administered the compositions disclosed herein.

[0252] In one embodiment, a subject is identified as not suitable to receive a composition disclosed herein if at least one mutation in the DMD gene is a mutation in exons 1-17, an in-frame deletion, an in-frame duplication, a mutation of unknown significance (VUS), or a mutation entirely contained within exon 45.

[0253] The present disclosure also provides for the use of the compositions described herein for treating muscular dystrophy in a human subject in need of such treatment. 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.

[0254] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0255] For example, the dose of rAAV is 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 14vg / 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 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 × 1013 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. 3.0×1014 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 about 1.5 × 10 14 vg / kg~5.0×10 14 , or about 1.5 × 10 14 vg / kg~4.0×10 14 , or about 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 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 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg.

[0256] In some embodiments, the human subject is between about 4 and less than 8 years of age. 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 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.

[0257] In some embodiments, the human subject is between about 8 years old and less than 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. ... 2.75 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.

[0258] In one embodiment, the method of the disclosure includes systemic administration of rAAV, where the systemic administration route is intravenous and the administered dose of rAAV is about 2.0×10 14 In another embodiment, the methods of the disclosure include systemic administration of rAAV, where the systemic administration route is intravenous and the administered dose of rAAV is about 5.0×10 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 × 1013 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 1.5 × 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 15vg / 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 embodiment, the rAAV is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0259] In any of the methods of the 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 or 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 embodiment, the rAAV is AAVrh74.MCK.microdystrophin, nucleotides 56-4820 of SEQ ID NO:5.

[0260] 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 is administered by infusion over approximately 1 hour. Additionally, the dose of rAAV is administered by intravenous route via a peripheral vein of a limb (such as a peripheral vein of an arm or a peripheral vein of a leg). Alternatively, the dose of rAAV can be administered by infusion over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or 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 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.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0261] The rAAV administered by any of the methods of the disclosure can comprise the human micro-dystrophin 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 can comprise the human micro-dystrophin 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 can comprise the AAVrh74.MHCK7.micro-dystrophin construct of the 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.micro-dystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is the AAVrh74.MHCK7.microdystrophin 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.

[0262] In one 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.

[0263] In any of the methods of the disclosure, the rAAV administered is of serotype AAVrh7.4.

[0264] In some embodiments, the disclosed methods treat Duchenne or Becker muscular dystrophy. An exemplary embodiment is a method of treating Duchenne or Becker muscular dystrophy in a human subject in need of such treatment, comprising administering a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the route of administration is intravenous infusion, and the administered dose of rAAV is about 2×10 14 vg / kg and the rAAV vector comprises an AAVrh74.MHCK7.microdystrophin construct of the nucleotide sequence of SEQ ID NO:9 or 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 or 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 from nucleotides 56 to 4820 of SEQ ID NO:5.

[0265] In any of the methods for treating muscular dystrophy, the expression level of the micro-dystrophin gene in the cells of the subject is increased after administration of rAAV. The expression of the micro-dystrophin gene in the cells is detected by measuring the micro-dystrophin protein level by Western blot in the muscle biopsied before and after administration of rAAV. In particular, the micro-dystrophin protein level 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 with the micro-dystrophin level before administration of rAAV. For example, micro-dystrophin 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 rAAV compared to micro-dystrophin levels before administration of the rAAV.

[0266] Furthermore, the expression of the micro-dystrophin gene in cells is detected by measuring micro-dystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of rAAV. The micro-dystrophin protein level 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 micro-dystrophin level before administration of rAAV. For example, the micro-dystrophin protein level 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 rAAV compared to the micro-dystrophin level before administration of rAAV.

[0267] In any of the methods of treating muscular dystrophy, serum CK levels in the subject are reduced after administration of the rAAV compared to serum CK levels before administration of the rAAV. For example, serum CK levels in the subject are 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 serum CK levels 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. Alternatively, in any of the methods of treating muscular dystrophy of the present disclosure, the serum CK level in the subject is reduced by about 95% by 60 days after administration of rAAV, compared to the serum CK level before administration of rAAV.In any of the methods of treating muscular dystrophy, the number of micro-dystrophin positive fibers in the muscle tissue of the subject is increased after administration of rAAV, compared to the number of micro-dystrophin positive fibers before administration of rAAV.For example, the number of micro-dystrophin positive fibers is detected by measuring micro-dystrophin protein level by Western blot or immunohistochemistry on muscle biopsy before or after administration of rAAV.

[0268] In any of the methods for treating muscular dystrophy, as described herein, administration of rAAV upregulates the expression of DAPC protein (such as alpha-sarcoglycan or beta-sarcoglycan).For example, the alpha-sarcoglycan level in a subject increases after administration of rAAV compared to the alpha-sarcoglycan level before administration of rAAV.Furthermore, the beta-sarcoglycan level in a subject increases after administration of rAAV compared to the beta-sarcoglycan level before administration of rAAV.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 before or after administration of rAAV.

[0269] In any of the methods of treating muscular dystrophy, disease progression in the subject is delayed following administration of the rAAV as measured by any of the 6-minute walk test, time to stand, 4-step climb, 4-step climb, North Star Ambulation Assessment (NSAA), Timed 10-meter test, Timed 100-meter test, Handheld Dynamometry (HHD), Timed Up and Go, and / or Gross Motor Coordination subtest (Bayley-III) score.

[0270] For example, in any of the methods, the subject improves NSAA score 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 rAAV, compared to the NSAA score before administration of rAAV. Furthermore, in any of the methods, the subject improves stand-up time by at least about 0.8 seconds at least 270 days after administration of rAAV, compared to the stand-up time before administration of rAAV. Furthermore, in any of the methods, the subject improves 4-stair climbing test by at least about 1.2 seconds at least 270 days after administration of rAAV, compared to the 4-stair climbing test before administration of rAAV. Further, in any of the methods, the subject improves in the 100 m timed test by at least about 7 seconds at least 270 days after rAAV administration compared to the 100 m timed test prior to rAAV administration.

[0271] In another aspect, the disclosure provides a method of expressing a micro-dystrophin gene in cells of a patient, comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct of the 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, expression of the micro-dystrophin gene in cells of the patient 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, expression of the micro-dystrophin gene is measured by detecting a higher number of vector genomes per nucleus in the patient, where one vector genome per nucleus is about 50% micro-dystrophin expression, and greater than one copy per nucleus is consistent with micro-dystrophin expression levels. For example, the cell has 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.

[0272] In some embodiments, the disclosure provides a method 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, serum CK levels in the patient are 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 rAAV, compared to serum CK levels prior to administration of the rAAV. In particular, 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.

[0273] The disclosure also provides a method of increasing micro-dystrophin positive fibers in muscle tissue of a patient, 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, the number of micro-dystrophin positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry on muscle biopsies before or after administration of rAAV. Additionally, expression of the micro-dystrophin gene is measured by detecting a higher number of vector genomes per nucleus in the patient, where one vector genome per nucleus is about 50% micro-dystrophin expression, and greater than one copy per nucleus is consistent with micro-dystrophin expression levels. For example, the cell has 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.

[0274] 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 or after administration of rAAV.

[0275] Additionally, the 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 or after administration of rAAV.

[0276] 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, such that disease progression in the patient is slowed as measured by any of the 6-minute walk test, time to stand, 4-step climb, 4-step climb, North Star Ambulation Assessment (NSAA), Timed 10-meter test, Timed 100-meter test, Handheld Dynamometry (HHD), Timed Up and Go, and / or Gross Motor Coordination (Bayley-III) score.

[0277] For example, in any of the methods, the subject improves NSAA score 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 rAAV, compared to the NSAA score before administration of rAAV. Furthermore, in any of the methods, the subject improves stand-up time by at least about 0.8 seconds at least 270 days after administration of rAAV, compared to the stand-up time before administration of rAAV. Furthermore, in any of the methods, the subject improves 4-stair climbing test by at least about 1.2 seconds at least 270 days after administration of rAAV, compared to the 4-stair climbing test before administration of rAAV. Further, in any of the methods, the subject improves in the 100 m timed test by at least about 7 seconds at least 270 days after rAAV administration compared to the 100 m timed test prior to rAAV administration.

[0278] "Fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components in tissues (including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas) upon injury and abnormal repair processes. The deposited ECM components include fibronectin and collagen (e.g., collagen 1, collagen 2, or collagen 3).

[0279] The 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 comprising the human micro-dystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or an rAAV vector 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. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, 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 aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 56-5066 of SEQ ID NO: 6. In a further 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.

[0280] In another aspect, the 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 micro-dystrophin 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 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 disclosure can be administered to a subject suffering from muscular dystrophy to prevent fibrosis, e.g., a rAAV of the disclosure expressing human micro-dystrophin protein is administered before fibrosis is observed in the subject. Additionally, rAAV of the present disclosure expressing the human micro-dystrophin gene can be administered to subjects at risk of developing fibrosis, such as subjects suffering from or diagnosed with muscular dystrophy (e.g., DMD). rAAV of the present disclosure can be administered to subjects suffering from muscular dystrophy to prevent new fibrosis in these subjects.

[0281] The present disclosure contemplates administering rAAV before fibrosis is observed in a subject.Furthermore, rAAV can be administered to subjects at risk of developing fibrosis, such as subjects suffering from or diagnosed with muscular dystrophy (e.g., DMD).rAAV can be administered to subjects suffering from muscular dystrophy who have already developed fibrosis, to prevent new fibrosis in these subjects.

[0282] The disclosure also provides a method of increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of a rAAV comprising the human micro-dystrophin 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.

[0283] The present disclosure contemplates administering an rAAV vector to a subject diagnosed with DMD before fibrosis is observed in the subject, before muscle weakness, or before muscle mass is lost.

[0284] The present disclosure also contemplates administering an rAAV comprising the human micro-dystrophin 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 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 suffering from muscular dystrophy who has already developed fibrosis to prevent new fibrosis in such subjects or to reduce fibrosis in such subjects. The disclosure also provides for administering an rAAV vector comprising the human micro-dystrophin 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.micro-dystrophin construct of the 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 suffering from muscular dystrophy who already has reduced muscle strength or reduced muscle mass to protect the muscle from further injury.

[0285] The present disclosure also provides a method of treating cardiomyopathy in a human subject with muscular dystrophy (e.g., DMD), comprising administering any of the compositions described herein (e.g., derandistrogen moxeparvovec) to said human subject. In some embodiments, the method is used to improve cardiac function in a subject with DMD. In some embodiments, cardiac function is improved by, for example, increasing ejection fraction (EF); increasing fractional shortening (FS); decreasing left ventricular internal diameter in diastolic (LVIDd); decreasing left ventricular end systolic diameter (LVESD); and / or maintaining or decreasing serum troponin blood levels. 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.

[0286] In other embodiments of any of the disclosed methods described herein, 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 by a percentage level selected from the group consisting of: a) at least 78% by 90, 180, or 270 days after dosing; 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 after dosing; 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 after dosing; f) 70–95% by 90, 180, or 270 days after dosing; 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 dosing; 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% by 90, 180, or 270 days after dosing.

[0287] In another aspect, the disclosure provides a composition for treating muscular dystrophy in a human subject in need thereof, the composition comprising a dose of a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, the composition being formulated for a systemic route of administration, the dose of the rAAV being about 1×10 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 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.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0288] 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 12vg / 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 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. 5.0×1014 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 × 1014 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 about 1.5 × 10 14 vg / kg~5.0×10 14 , or about 1.5 × 10 14 vg / kg~4.0×10 14 , or about 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 14vg / 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 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14The rAAV comprises a dose of rAAV of 1000-150 ...

[0289] In one embodiment, the composition of the present disclosure is formulated for intravenous administration and is administered in a dose of about 2.0×10 14 In another embodiment, the composition of the present disclosure is formulated for intravenous administration and contains a dose of rAAV that is about 5.0×10 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 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 13vg / 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 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. In another 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.

[0290] 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.

[0291] 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.Furthermore, the composition of the present disclosure is formulated for intravenous administration via peripheral veins of the limbs (such as peripheral veins of the arm or peripheral veins of the leg).Alternatively, it can be administered by infusion over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours, or approximately 3 hours.

[0292] Any of the compositions of the disclosure includes a rAAV comprising the human micro-dystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7, or a rAAV vector comprising the AAVrh74.MHCK7.microdystrophin construct of the 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.

[0293] In particular, the composition of the present disclosure is 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 of such treatment, the composition comprising a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, the composition being formulated for administration by intravenous infusion over approximately 1 hour, and the administered dose of rAAV is approximately 2×10 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.

[0294] In another aspect, the present disclosure also provides compositions comprising rAAV vectors for reducing fibrosis in a subject in need thereof. Additionally, the present disclosure provides compositions comprising rAAV vectors for preventing fibrosis in a subject suffering from muscular dystrophy.

[0295] The present disclosure also provides compositions comprising rAAVs for increasing muscle strength and / or muscle mass in subjects 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.

[0296] In other embodiments of any of the compositions of the present disclosure, following administration of said composition to a human subject in need of treatment for muscular dystrophy, serum CK levels in the subject are reduced by a percentage level selected from the group consisting of the following, as compared to serum CK levels prior to administration of the composition: a) at least 78% by 90, 180, or 270 days after dosing; 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 after dosing; e) at least 70% by 270 days after dosing; f) 70–95% by 90, 180, or 270 days after dosing; 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 dosing; 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 dosing.

[0297] In another aspect, the disclosure provides a use of a dose of a 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 the rAAV is about 1×10 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 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.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0298] For example, the drug is 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 13 vg / kg ~ approx. 1.0×1014 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 × 1014 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 about 1.5 × 10 14 vg / kg~5.0×10 14 , or about 1.5 × 10 14 vg / kg~4.0×10 14 , or about 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. 2.0×1014 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 14 vg / kg, or approximately 2.0 × 1014 vg / kg ~ approx. 3.25×10 14 The rAAV comprises a dose of rAAV of 1000-150 ...

[0299] In one embodiment, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, where the systemic administration route is intravenous and the administered dose of rAAV is about 2.0×10 14 In another embodiment, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, where the systemic administration route is intravenous and the dose of rAAV is about 5.0×10 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 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 1013 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 vg / kg. 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. 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.

[0300] 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 or rAAV 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.

[0301] 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 via a peripheral vein of a limb (such as a peripheral vein of an arm or a peripheral vein of a leg).Alternatively, it can be administered by infusion over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours, or approximately 3 hours.

[0302] In any of the uses of the disclosure, the medicament comprises a rAAV comprising the human micro-dystrophin 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.

[0303] A particular application 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 relates to 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 of such treatment, wherein the medicament is formulated for administration by intravenous infusion over approximately one hour, and the administered dose of rAAV is about 2×10 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.

[0304] In a further aspect, the present disclosure provides the use of rAAV for preparing a medicament for reducing fibrosis in a subject in need of reducing fibrosis.For example, the subject in need may suffer from muscular dystrophy, such as DMD or any other dystrophin-related muscular dystrophy.

[0305] In another aspect, the disclosure provides a use of rAAV for preparing a medicament for preventing fibrosis in a subject suffering from muscular dystrophy.

[0306] Additionally, the present disclosure provides the use of rAAV for preparing a medicament for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy.

[0307] The present disclosure also provides the use of rAAV for preparing a medicament for the treatment of muscular dystrophy.

[0308] The disclosure provides for the use of an rAAV vector comprising the human micro-dystrophin 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 of the 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.

[0309] In other embodiments of any of the uses of the present disclosure, the serum CK level in the subject, after administration of the rAAV to the subject, is reduced compared to the serum CK level before administration of the rAAV by a percentage level selected from the group consisting of: a) at least 78% by 90, 180, or 270 days after dosing; 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 after dosing; f) 70–95% by 90, 180, or 270 days after dosing; 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 dosing; 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 dosing.

[0310] In any of the compositions for treating muscular dystrophy or the uses of the medicaments for treating muscular dystrophy, the expression level of the micro-dystrophin gene in the cells of the subject is increased after administration of the compositions or medicaments. The expression of the micro-dystrophin gene in the cells is detected by measuring the micro-dystrophin protein level by Western blot in the muscle biopsied before and after administration of the compositions or medicaments. In particular, the micro-dystrophin protein level 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 compositions or medicaments, compared to the micro-dystrophin level before administration of the compositions or medicaments. For example, micro-dystrophin 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 compared to the micro-dystrophin levels before administration of the composition or medicament.

[0311] Furthermore, the expression of the micro-dystrophin gene in cells is detected by measuring micro-dystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of the composition or medicament. The micro-dystrophin protein level 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 rAAV compared to the micro-dystrophin level before administration of the composition or medicament. For example, the micro-dystrophin protein level 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 or medicament compared to the micro-dystrophin level before administration of the composition or medicament.

[0312] In any of the compositions for treating muscular dystrophy, serum CK levels in the subject are reduced after administration of the rAAV compared to serum CK levels before administration of the composition or medicament. For example, serum CK levels in the subject are 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 serum CK levels before administration of the composition or medicament. In particular, in any of the compositions for treating muscular dystrophy disclosed herein, 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 disclosed herein, 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 disclosed herein, 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. In either of the disclosed compositions for treating muscular dystrophy or uses of the medicament for treating muscular dystrophy, the serum CK level in the subject 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 in either of the disclosed compositions for treating muscular dystrophy or uses of the medicament for treating muscular dystrophy, the serum CK level in the subject 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 in either of the disclosed compositions for treating muscular dystrophy or uses of the medicament for treating muscular dystrophy, the serum CK level in the subject 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. In either of the disclosed compositions for treating muscular dystrophy or uses of the medicament for treating muscular dystrophy, the number of micro-dystrophin positive fibers in the muscle tissue of the subject is increased after administration of the composition or medicament, compared to the number of micro-dystrophin positive fibers before administration of the composition or medicament.For example, the number of micro-dystrophin positive fibers is detected by measuring micro-dystrophin protein levels by Western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament.

[0313] In any of the compositions for treating muscular dystrophy or the uses of the medicine for treating muscular dystrophy, the expression of DAPC protein (such as alpha-sarcoglycan or beta-sarcoglycan) is upregulated when the composition or medicine is administered.For example, the alpha-sarcoglycan level in the subject increases after the composition or medicine is administered, compared to the alpha-sarcoglycan level before the composition or medicine is administered.Furthermore, the beta-sarcoglycan level in the subject increases after the composition or medicine is administered, compared to the beta-sarcoglycan level before the composition or medicine is administered.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 before or after the composition or medicine is administered.

[0314] In any of the compositions for treating muscular dystrophy or uses of the medicaments for treating muscular dystrophy, progression of the disease in a subject is slowed following administration of the composition or medicament as measured by any of the 6-minute walk test, time to stand, 4-step climb, 4-step climb, North Star Ambulation Assessment (NSAA), Timed 10-meter test, Timed 100-meter test, Handheld Dynamometry (HHD), Timed Up and Go, and / or Gross Motor Coordination subtest (Bayley-III) score.

[0315] For example, after administration of any of the compositions for treating muscular dystrophy or use of the medicament for treating muscular dystrophy, the subject will have an NSAA score of 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 improved at least 270 days after administration of the composition or medicament, compared to the NSAA score before administration of rAAV. Furthermore, in any of the methods, the subject will have an improvement in time to stand up of at least about 0.8 seconds at least 270 days after administration of the composition or medicament, compared to the time to stand up before administration of the composition or medicament. Further, in any of the disclosed methods or uses, the subject improves by at least about 1.2 seconds in the 4-Stage Climbing Test at least 270 days after administration of the composition or medicament, compared to the 4-Stage Climbing Test before administration of the composition or medicament. Further, in any of the disclosed methods or uses, the subject improves by 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.

[0316] In another aspect, the disclosure provides a composition for expressing a micro-dystrophin gene in cells of a patient comprising an AAVrh74.MHCK7.micro-dystrophin construct of the 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 the use of a dose of an AAVrh74.MHCK7.micro-dystrophin construct of the 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 cells of a patient. For example, expression of the micro-dystrophin gene in cells of a patient 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 is measured by detecting more vector genomes per nucleus in patients, where 1 vector genome per nucleus is about 50% micro-dystrophin expression, and more than 1 copy per nucleus is consistent with the expression level of micro-dystrophin.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.

[0317] In a further aspect, the disclosure provides a composition for reducing serum CK levels in a patient in need thereof, the composition comprising 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. Additionally, the disclosure provides use of a dose of an AAVrh74.MHCK7.microdystrophin construct of the 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, serum CK levels in a patient are 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 serum CK levels before administration of the composition or medicament. In particular, serum CK levels in the subject are 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 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 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 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 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.

[0318] The disclosure also provides a composition for increasing micro-dystrophin positive fibers in muscle tissue of a patient, comprising 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. The disclosure further provides a use of a dose of an AAVrh74.MHCK7.micro-dystrophin construct of 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 micro-dystrophin positive fibers in muscle tissue of a patient. For example, the number of micro-dystrophin positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry on muscle biopsies before or after administration of the composition or medicament. Furthermore, the expression of micro-dystrophin gene is measured by detecting more vector genomes per nucleus in patients, where 1 vector genome per nucleus is about 50% micro-dystrophin expression, and more than 1 copy per nucleus is consistent with the expression level of micro-dystrophin.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.

[0319] In another aspect, the disclosure provides a composition for increasing expression of alpha-sarcoglycan in a patient in need thereof comprising 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. The disclosure also provides a use of a dose of an AAVrh74.MHCK7.microdystrophin construct of 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 expression of alpha-sarcoglycan 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 or after administration of the composition or medicament.

[0320] Further, the disclosure provides a composition for increasing beta-sarcoglycan expression in a patient in need thereof comprising 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. The disclosure also provides a use of an AAVrh74.MHCK7.microdystrophin construct of the 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 or after administration of the composition or medicament.

[0321] The disclosure also provides for the use of a dose of an AAVrh74.MHCK7.microdystrophin construct of the 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 having Duchenne muscular dystrophy or Becker muscular dystrophy, wherein administration of said medicament slows disease progression in the patient as measured by any of the 6-minute walk test, time to stand, 4-step climb, 4-step climb, North Star Ambulation Assessment (NSAA), Timed 10-meter test, Timed 100-meter test, Handheld Dynamometry (HHD), Timed Up and Go, and / or Gross Motor Subtest Adjustment (Bayley-III) score.

[0322] For example, the subject improves NSAA score 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 improves stand-up time by at least about 0.8 seconds at least 270 days after administration of the composition or medicament, compared to the stand-up time before administration of the composition or medicament.Furthermore, the subject improves 4-step climb test by at least about 1.2 seconds at least 270 days after administration of the composition or medicament, compared to the 4-step climb test before administration of the composition or medicament. Additionally, the subject experiences an improvement in the 100m time test of at least about 7 seconds at least 270 days after administration of the composition or medicament, as compared to the 100m time test prior to administration of the composition or medicament.

[0323] It should be appreciated that the Detailed Description section, and not the Summary and Abstract sections, are intended to be used to interpret the claims. The Summary and Abstract sections describe one or more exemplary aspects of the present disclosure contemplated by the inventor(s), but may not be all-inclusive, and thus are not intended to limit the disclosure and the appended claims in any manner.

[0324] The following examples are offered by way of illustration and not by way of limitation of the present invention. The numerical ranges listed include each integer value within each range as well as the minimum and maximum integer values ​​listed. EXAMPLES

[0325] Working Example Example 1 A) Generation of the AAVrh74.MHCK7.microdystrophin construct The AAVrh74.MHCK7.micro-dystrophin plasmid contains a human micro-dystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see FIG. 1). The micro-dystrophin construct featured an in-frame rod deletion (R4-R23), while the hinges 1, 2, and 4, and the cysteine-rich domain still produce a 138 kDa protein. Expression of the micro-dystrophin protein (3579 bp) was guided by the MHCK7 promoter (792 bp). The plasmid was constructed from the rAAV.MCK.micro-dystrophin plasmid by removing the MCK promoter and inserting the MHCK7 promoter. The core promoter is followed by 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 micro-dystrophin cassette had a consensus Kozak immediately preceding the ATG start codon and a small 53bp synthetic polyA signal for mRNA termination. The human micro-dystrophin 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 contained in this vector were the inverted terminal repeats of AAV2, necessary for viral DNA replication and packaging. The micro-dystrophin cassette has a small 53bp synthetic polyA signal for mRNA termination.

[0326] Previous studies have demonstrated cardiac expression using the MHCK7 promoter (Salva et al., Mol Ther 15, 320-329 (2007) and that AAVrh74 is expressed in skeletal, diaphragm, and cardiac muscle (Sondergaard et al. Annals of Clinical and Transl Neurology 2, 256-270 (2015)), and the construct sequences in Figure 1 were 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).

[0327] Table 1 shows the characteristics of the plasmid AAVrh74.MHCK7.micro-dystrophin molecule (SEQ ID NO:3). [Table 1] B) AAVrh74.MHCK7.micro-dystrophin construct and generation of plasmids encoding kanamycin (Kan) resistance

[0328] Cloning of MHCK7.μDys.KAN was performed by isolating the MHCK7.μDys fragment from the MHCK7.μDys.AMP plasmid and the kanamycin backbone and annealing them using the NEBuilder cloning workflow. The MHCK7.μDys fragment was isolated by restriction enzyme digestion with SnaBI. Digestion was performed for 1 hour at 37°C in 1x CutSmart buffer (NEB) and 1 μL SnaBI in a total reaction volume of 50 μL. 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 resulting fragment had a DNA concentration of 10 ng / μL. The Kan backbone fragment was isolated by XbaI restriction enzyme digestion at 37°C for 1 hour using 1x CutSmart buffer (NEB) and 1 µL XbaI in a reaction volume of 50 µL. The resulting fragment was isolated by electrophoresis using a 1% agarose gel run at 105 volts for 1.5 hours. The band corresponding to the Kan backbone was excised and purified by gel purification kit (Macherey-Nagel). The resulting fragment had a DNA concentration of 8.1 ng / µL. The two fragments were annealed using the NEB Builder cloning workflow, which can join two fragments with overlapping sequences. The NEBuilder cloning reaction was performed at 50°C for 15 minutes using a 1:1 ratio of MHCK7.µDys and kanamycin backbone in 1x NEBuilder HiFi DNA assembly master mix for a total volume of 20 µL according to the manufacturer's protocol. The resulting clones were transformed into NEB® Stable Competent E. coli (C3040) by adding 2.5 μL cloning product to the cells followed by 30 seconds on ice, then 30 seconds at 42° C. and an additional 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 on 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 with 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 confirm 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 additionally confirmed by sequencing. The resulting plasmid is set forth in SEQ ID NO:8 and shown in FIGS. 14 and 15. The sequences of the constructs in FIG. 13 corresponding to the sequence in SEQ ID NO:9, and nucleotides 1 to 4977 of SEQ ID NO:8 were encapsidated into the AAVrh.74 virions described above. Example 2 Systemic Gene Delivery Clinical Trial for Duchenne Muscular Dystrophy

[0329] This was a single-dose controlled study using rAAVrh74.MHCK7.micro-dystrophin, nucleotides 55-5021 of SEQ ID NO:3, in DMD subjects. Cohort A included six subjects aged 3 months to 3 years, and Cohort B included six subjects aged 4 to 7 years. All subjects received intravenous micro-dystrophin vector (2 x 10 in 10 mL / kg). 14 rAAVrh74.MHCK7.micro-dystrophin 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.

[0330] In this study, rAAVrh74.MHCK7.micro-dystrophin was infused via a peripheral vein in the arm so that it could reach all muscles in the body. Six DMD subjects aged 3 months to 3 years in Cohort A and six DMD subjects aged 4 to 7 years in Cohort B were enrolled. All subjects received intravenous micro-dystrophin vector (2 × 10 in 10 mL / kg). 14 The titers of encapsulated vector genomes for the administered dose were determined relative to supercoiled DNA plasmid standards using quantitative PCR using the Prism7500 Taqman detection system (PE Applied Biosystems) with primers directed to the MHCK7 promoter (Pozsgai et al., Mol. Ther. 25(4):855-869(2017)).

[0331] Subjects were infused for 1 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 will undergo a second muscle biopsy 90 days after delivery to determine if the gene has replaced the missing dystrophin protein. After gene transfer, patients were carefully monitored for any side effects from the 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, and 180, and at months 9, 12, 18, 24, 30, and 36 to ensure there are no side effects from gene injection.

[0332] Subjects in cohort A (n=6) were between 3 months and 3 years of age and received the rAAVrh74.MHCK7.micro-dystrophin vector (2×10 in 10 mL / kg). 14Prednisone or deflazacort 1 mg / kg was administered intravenously to subjects in cohort A one day prior to gene transfer and maintained for 30 days while immune responses were monitored. If negative on day 30, steroids were withdrawn for one week. If T cell responses to AAV or micro-dystrophin were greater than 125 SFC / 106 PBMC, steroid levels were maintained until they fell below this threshold.

[0333] Cohort B subjects (n=6) were between 4 and 7 years of age and received the rAAVrh74.MHCK7.micro-dystrophin vector (2×10 in 10 mL / kg). 14 These subjects were maintained on stable doses of corticosteroids throughout the study, but could be briefly increased if T cell responses to AAV or micro-dystrophin exceeded 125 SFC / 106 PBMC. Eligibility Criteria

[0334] The inclusion criteria for the studies were: Participant ages: Cohort A: 3 months to 7 years old, and Cohort B: between 4 and 7 years old (inclusive). The molecule is characterized by a DMD gene with a frameshift (deletion or duplication) or a premature stop codon mutation between exons 18 and 58. Increased CK levels of over 1000 U / L Cohort A subjects: below average Bayley-III motor assessment for gross motor function defined as an adjusted score of 9 or less Cohort B: Below average 100m timed test defined as less than 80% predicted · Males of any racial group. ·Ability to cooperate with physical assessment tests. Cohort A subjects: had not been previously treated with corticosteroids. Cohort B subjects: A stable dose equivalent to oral corticosteroids for at least 12 weeks prior to screening and dosing, expected to remain constant (except for adjustments to accommodate weight changes) throughout the study.

[0335] The exclusion criteria for the study were as follows: Active viral infection based on clinical findings. · Signs of cardiomyopathy (including an echocardiogram showing an ejection fraction less than 40%). · Serological evidence of HIV infection or hepatitis B or C infection. Diagnosing (or treating) an autoimmune disease Abnormal laboratory values ​​that are considered clinically significant · Concomitant illness or need for chronic drug treatment that, in the opinion of the PI, poses unnecessary risk of gene transfer. Subjects with AAVrh74 or AAV8 antibody titers greater than 1:400 as determined by ELISA immunoassay. Any medical or extenuating medical condition that, in the opinion of the investigator, may impair the subject's ability to comply with the tests or procedures required by the protocol or that may compromise the patient's welfare, safety, or clinical interpretability. Severe infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (participation may be postponed). Have received any investigational medication (other than corticosteroids) or exon-skipping agents (including ExonDys51®) for experimental or other purposes within 6 months prior to the last screening for this study. Have undergone 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 research participation to be disclosed to their doctor and other health care providers. Outcome measures

[0336] The primary outcome measure was safety based on the number of participants with adverse events (time frame: 3 years). Adverse effects were monitored and scored for severity and relationship to study outcomes.

[0337] Secondary outcome measures were:

[0338] Gross Motor Subtest Adjustment (Bayley-III) Score (Time Frame: Screening, Day 30-3 Years): Motor development was measured by the Gross Motor Adjustment score. The Bayley-III Gross Motor Subtest was scored at every follow-up visit for Cohort A starting at Day 30 for 3 years. Any subject who was 43-47 months old (inclusive) at screening had an adjustment score calculated relative to normative data for 42-month-old children. Normative data for children aged 1-42 months were obtained from the Bayley-III.

[0339] Physiotherapy assessment timed 100 metre test (100m) (time frame: screening, 30 days to 3 years): The 100m was the primary motor outcome for Cohort B. The timed 100m test was an exploratory outcome for Cohort A starting as soon as possible after children reached the age of 3 years.

[0340] North Star Ambulation Assessment (NSAA) (Time Frame: Screening, Day 30-3 Years): The North Star Ambulation Assessment (NSAA) was an exploratory outcome beginning for Cohort A and Cohort B as early as possible after children turned 4 years of age. The NSAA measures gait quality in boys with Duchenne muscular dystrophy.

[0341] Physical Therapy Assessment: Timed Up and Go (TUG) Modified for Children (Time Frame: Screening, Day 30–3 Years): Examination outcomes for Cohort B included the Timed Up and Go (TUG) Modified for Children.

[0342] Physiotherapy assessment 4-step stair climbing (time frame: screening, day 30-3 years): Exploratory outcomes for Cohort B will include 4-step stair climbing.

[0343] Physical Therapy Assessment Handheld Dynamometry (HHD) (Time Frame: Screening, Day 30-3 Years): Exploratory outcomes for Cohort B included handheld dynamometry (HHD) of the knee extensors and flexors, and elbow flexors and extensors.

[0344] Quantification of micro-dystrophin gene expression by immunofluorescence (Time frame: screening, day 90): Micro-dystrophin gene expression levels were quantified by immunofluorescence and compared before and after muscle biopsy.

[0345] Quantification of micro-dystrophin gene expression by Western blot (Time frame: screening, day 90): Micro-dystrophin gene expression levels were quantified by Western blot analysis and compared before and after muscle biopsy.

[0346] Reduction in CK after gene therapy (time frame: 3 years): Reduction in circulating CK levels.

[0347] Cardiac Magnetic Resonance Imaging (Year 1). Micro-dystrophin gene expression

[0348] Changes from baseline in micro-dystrophin expression via immunofluorescence (IF) fiber intensity were analyzed and quantified. As shown in Table 2, subject 1 (5 years old) demonstrated 78% micro-dystrophin protein expression in muscle fibers of gastrocnemius biopsy after administration of rAAVrh74.MHCK7.micro-dystrophin; subject 2 (4 years old) demonstrated 73.5% micro-dystrophin protein expression in muscle fibers of gastrocnemius biopsy after administration of rAAVrh74.MHCK7.micro-dystrophin; subject 3 (6 years old) demonstrated 77.0% micro-dystrophin protein expression in muscle fibers of gastrocnemius biopsy after administration of rAAVrh74.MHCK7.micro-dystrophin. Subject 4 (age 4) demonstrated 96.2% micro-dystrophin expression in muscle fibers of gastrocnemius biopsies following administration of rAAVrh74.MHCK7.micro-dystrophin. All patients showed robust expression of transduced micro-dystrophin, which was appropriately localized to the sarcolemma as measured by immunohistochemistry (Figure 7). [Table 2]

[0349] Changes in micro-dystrophin gene expression from baseline to day 60 were also assessed by quantification of micro-dystrophin protein expression measured by Western blot of biopsied muscle tissue. As shown in Figures 8A and 8B, Western blot analysis detected micro-dystrophin protein expression in subject 1 (5 years old), subject 2 (4 years old), and subject 3 (6 years old). Figure 8C provides a Western blot analysis detecting micro-dystrophin protein expression in subject 4 (4 years old). All post-treatment biopsies showed healthy levels of micro-dystrophin as measured by Western blot, with subjects 1-4 averaging 74.3% compared to normal utilizing method 1 and 95.8% compared to normal according to method 2 adjusting for fatty and fibrous tissue.

[0350] For each subject, the vector genome copy number per myofiber nucleus was measured. As shown in Table 3, the vector genome copy number per nucleus was greater than 1 for each of the subjects following administration of rAAVrh74.MHCK7.micro-dystrophin. One copy of vector represents approximately 50% expression of the micro-dystrophin gene. An average of 1.6 vector copies per cell nucleus was measured in subjects 1-3, consistent with the high micro-dystrophin expression levels observed. When the value for subject 4 was included, the average vector copy number / μg DNA was 10 5 with an average vector copy number of 3.3 per cell nucleus. [Table 3]

[0351] The protein levels of alpha-sarcoglycan and beta-sarcoglycan in muscle biopsy tissue were measured by immunohistochemistry before and after administration of rAAVrh74.MHCK7.micro-dystrophin. Administration of rAAVrh74.MHCK7 also upregulated DAPC protein in subjects. As shown in Figure 9, the expression of alpha-sarcoglycan and beta-sarcoglycan in muscle biopsy tissue was increased in subject 1 (Figure 9A), subject 2 (Figure 9B), and subject 3 (Figure 9C) compared with the levels of these proteins in muscle biopsy before administration of rAAVrh74.MHCK7. Circulating serum CK levels

[0352] Blood samples were cultured in 10 mL / kg of rAAVrh74.MHCK7.micro-dystrophin vector (2 × 10 14CK levels were measured at each visit and compared to baseline levels obtained prior to administration of rAAVrh74.MHCK7.micro-dystrophin (visit day 0). Baseline serum CK levels (units / liter) are provided in Table 4 below. As shown in FIG. 10, circulating serum CK levels were reduced by approximately 87% two months after administration of rAAVrh74.MHCK7.micro-dystrophin. All subjects showed a significant reduction in serum creatine kinase (CK) levels, with a mean CK reduction of more than 87% after two months of treatment (n=3). CK is an enzyme associated with muscle damage, and patients with DMD uniformly exhibit elevated levels of CK. Indeed, significantly elevated CK is often used as a preliminary diagnostic tool for DMD, followed by genetic testing for confirmation.

[0353] Table 4 and Figure 10 provide the CK levels for each subject. Figure 11 provides the average CK levels over time and demonstrates that the average CK levels significantly decrease over time following administration of rAAVrh74.MHCK7.micro-dystrophin. The average baseline CK level of 27,064 U / L (average, Table 4) decreases by approximately 63% to an average of 9,982 U / L (average, day 270, Table 5). [Table 4] [Table 5] Assessment of effectiveness

[0354] In addition to micro-dystrophin and CK levels, efficacy was measured by the following functional tests: floor rise time, four-step climb, North Star Ambulatory Assessment (NSAA), rise time test, four-step climb test, 10-meter timed test (10m), and 100-meter timed test (100m). The data are provided below in Tables 6 and 7, and demonstrate consistent and durable improvement at 9 months following administration of rAAVrh74.MHCK7.micro-dystrophin. Improvement over time in NSAA is also provided in Figure 12. [Table 6] [Table 7] Safety Assessment

[0355] No serious adverse events (SAEs) were observed in the study. Three subjects had elevations in gamma-glutamyltransferase (GGT), which resolved with increasing steroids within 1 week and returned to baseline. No other clinically significant laboratory findings were observed. Patients generally experienced transient nausea during the first week of treatment, which coincided with the increase in steroid dosage. This did not correlate with elevations in liver enzymes or any other abnormalities. Example 3 Randomized, double-blind, placebo-controlled, whole-body genetic Delivery Phase I / IIa Clinical Trial

[0356] This was a randomized, double-blind, single-dose study using rAAVrh74.MHCK7.micro-dystrophin in DMD subjects. The study included 24 subjects aged 4-7 years. Subjects were randomized to treatment or placebo groups upon enrollment. Twelve subjects received 12 doses of the rAAVrh74.MHCK7.micro-dystrophin vector (approximately 2 x 10 in 10 mL / kg). 14 100 subjects will receive 10 mL / kg of rAAV bearing micro-dystrophin intravenously, and 13 subjects will receive 10 mL / kg of placebo (lactated Ringer's solution). The placebo subjects will receive treatment administered in the same manner as the 12 previously treated subjects one year after the last treatment subject. Subjects will be infused with micro-dystrophin bearing rAAV or lactated Ringer's solution over approximately 1 hour. Pre- and post-treatment (after 90 days) needle muscle biopsies will be taken of the gastrocnemius muscle.

[0357] The primary objective of this study is to assess the safety of intravenous administration of rAAVrh74.MHCK7.micro-dystrophin for DMD subjects via a peripheral vein in the limb. Safety endpoints will be assessed by changes in hematology, serum chemistry, urinalysis, immune response to rAAVrh74 and micro-dystrophin, and reported medical history and symptom findings. Dystrophin gene expression will serve as the primary outcome measure along with safety. It will be quantified using validated immunofluorescence and immunoblot assays. Reduction in CK following gene therapy will serve as a secondary outcome. Efficacy will be measured by the following functional tests: stand-to-stand time, 4-step climb, North Star Ambulatory Assessment (NSAA), 10-meter timed test (10m), and 100-meter timed test (100m). Exploratory measures will include handheld dynamometry (HHD) for knee extensors and flexors, and elbow flexors and extensors.

[0358] The inclusion criteria for the studies were: Participation age: Between 4 and 7 years old (inclusive). The molecule is characterized by a DMD gene with a frameshift (deletion or duplication) or a premature stop codon mutation between exons 18 and 58. Indicators of symptomatic muscular dystrophy: CK elevation >1000 U / L and <100% of the mean estimated time in the 100 meter walk test Men of any racial group will be eligible. ·Ability to cooperate with physical assessment tests. A stable dose equivalent to oral corticosteroids for at least 12 weeks prior to screening and dosing, expected to remain constant (except for possible adjustments to accommodate weight changes) throughout the study.

[0359] The exclusion criteria for the study were as follows: Active viral infection based on clinical findings. · Signs of cardiomyopathy (including an echocardiogram showing an ejection fraction less than 40%). · Serological evidence of HIV infection or hepatitis B or C infection. Diagnosing (or treating) an autoimmune disease Abnormal laboratory values ​​deemed clinically significant (GGT > 3 × ULN, bilirubin > 3.0 mg / dL, creatinine > 1.8 mg / dL, Hgb < 8 g / Dl or > 18 g / Dl; WBC > 18,500 / cmm, platelets < 50,000). · Concomitant illness or need for chronic drug treatment that, in the opinion of the PI, poses unnecessary risk of 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. The subject has a medical or extenuating medical condition that, in the opinion of the investigator, may impair the subject's ability to comply with the tests or procedures required by the protocol or that may compromise the patient's welfare, safety, or clinical interpretability. Severe infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (participation may be postponed). Have received any investigational medication (other than corticosteroids) or exon skipping agents (including ExonDys51®) for experimental or other purposes in the 6 months prior to the last screening for this study. Have undergone 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 research participation to be disclosed to their doctor and other health care providers. Assessment of effectiveness

[0360] Dystrophin gene expression will serve as the primary outcome measure along with safety. It will be quantified using validated immunofluorescence and immunoblot assays. Reduction of CK following gene therapy will serve as a secondary outcome. Additionally, efficacy will be measured by the following functional tests: floor rise time, 4-step climb, North Star Ambulatory Assessment (NSAA), Timed 10 Meter Test (10m), Timed 100 Meter Test (100m)]. Examination measures will include handheld dynamometry (HHD) for knee extensors and flexors, and elbow flexors and extensors.

[0361] Ultrasound-guided muscle biopsies are used to quantify transgene expression at baseline and day 90. The biopsy is performed on the same muscle as the first biopsy, but on the contralateral leg muscle. One year after all subjects have been dosed, placebo crossover subjects will resume the study timeline at visit day 1. Placebo subjects will not have the following at the second baseline screening: cardiac MRI and muscle biopsy. Placebo subjects will undergo muscle biopsy at day 90 (3 muscle biopsies in total). Frozen sections will be stained for dystrophin using indirect immunofluorescence (IF). Full scanning of slides will be performed and micro-dystrophin intensity and positive fiber percentage will be quantified using a validated image scanning and MuscleMap™ analysis algorithm. Muscle morphometry (including fiber size histograms) will be performed blinded. Blinded frozen muscle biopsy cuttings will be used to perform protein quantification analysis for micro-dystrophin using a validated Western blot method.

[0362] A muscle needle biopsy of the gastrocnemius (unless deemed contraindicated for a particular subject by the PI, in which case the PI will choose an alternative muscle biopsy) will be used to quantify micro-dystrophin expression. Efficacy analysis

[0363] The primary efficacy endpoint is the change from baseline to day 90 in micro-dystrophin protein expression as measured by Western blot of biopsied muscle tissue. Differences in the primary efficacy endpoint between treatment groups are assessed using an analysis of covariance (ANCOVA) model with treatment as fixed factor and baseline value as covariate. Wilcoxon rank sum test is performed as supportive analysis. Changes from baseline in micro-dystrophin expression via immunofluorescence (IF) fiber intensity are similarly analyzed.

[0364] Supportive efficacy endpoints include change from baseline for each scheduled assessment of floor rise time, four-step climb, NSAA, 10-meter timed test (10m), 100-meter timed test (100m), and CK change. Examination measures include HHD for knee extensors and flexors, and elbow flexors and elbow extensors. Treatment group differences are assessed using an ANCOVA model with treatment as fixed factor and baseline value as covariate. Wilcoxon rank sum test is performed as supportive analysis. Example 4

[0365] The tests and studies described above in Examples 2 and 3 are performed utilizing instead the rAAVrh74.MHCK7.micro-dystrophin construct set forth in SEQ ID NO:9; set forth in SEQ ID NO:8 (nucleotides 1-4977); or set forth in SEQ ID NO:6 (nucleotides 56-5022). Example 5 Generation of pAAV.MCK.micro-dystrophin construct

[0366] The pAAV.MCK.micro-dystrophin plasmid was constructed by inserting an MCK expression cassette driving a codon-optimized human micro-dystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski, RJ et al., J. Virol. 61(10):3096-3101 (1987)). A muscle-specific control element was included in the construct to drive muscle-specific gene expression. The control element included the mouse MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). Following the core promoter, the construct contains the 53 bp 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 micro-dystrophin cassette has a consensus Kozak immediately before the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. As previously described by Harper et al., Nat. Med. 8(3):253-61 (2002), the human micro-dystrophin cassette contains the (R4-R23 / Δ71-78) domain.

[0367] The pAAV.MCK.micro-dystrophin plasmid contained a human micro-dystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see FIG. 5). This sequence was encapsidated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a rhesus monkey lymph node, as described in Rodino-Klapac et al., J Transl.Med.5:45 (2007). Example 6 rAAV production using hybrid seed train expansion

[0368] The following process can be used to produce the rAAV constructs described herein.

[0369] HEK-293 cells were passaged four times in adherent conditions. Prior to entering the penultimate expansion culture, cells were harvested, centrifuged to wash out serum (300g for 5 min), and resuspended in serum-free growth medium (EXPI293) in suspension shake flasks at a seeding density of 0.5+E6 cells / mL. Cells were then grown for 48-72 hours to expand cell numbers. Suspension cells were then harvested and inoculated into shake flasks or WAVE bags depending on the number of viable cells required to inoculate the bioreactor. After 72 hours, the concentration of viable cells was determined using a cell counting device. The volume required to contain the desired total viable cell number was then added to an adherent bioreactor containing DMEM and 10% FBS. Additional FBS was added appropriately to account for the addition of serum-free suspension culture volume so that the final FBS concentration was maintained at 10%.

[0370] As shown in Figure 18, cell viability in both the seed train and adherent systems was similar. With regard to viable cell density (VCD), in the hybrid seed train system, the VCD after the 6th passage was higher than that after the 1st passage (Figure 19A), which is comparable to the adherent system (Figure 19B).

[0371] Following inoculation into adherent bioreactors (iCELLis®), adherent cultures are transiently transfected with a transgene plasmid carrying a micro-dystrophin construct as described herein, including, for example, the construct set forth 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 (AAV2rep / rh74cap) and a helper plasmid are included. After the desired growth period, rAAV particles are harvested by cell lysis and column chromatography.

[0372] In some embodiments, the rAAV is produced by a suspension seed process comprising: (a) culturing cells in an N-2 container with a first growth medium containing serum; (b) removing said cells from said first medium; (c) inoculating the cells from step (b) in an N-1 container into a second medium that is serum-free or contains a lower concentration of serum than the first medium; (d) culturing said cells under suspension conditions in said N-1 container; and (e) inoculating the third medium in a bioreactor with the cells from step (d) above.

[0373] In some embodiments, the suspension seed process further comprises: (f) transfecting the cells with a transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAVrep and AAVcap genes, and an adenovirus helper plasmid.

[0374] 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 AAVrep gene and the AAVcap gene comprises the AAV2rep gene and the rAAVrh74cap gene.

[0375] In some embodiments, the adenovirus helper plasmid contains the adenovirus type 5 E2A gene, the E4ORF6 gene, and the VA RNA gene.

[0376] In some embodiments, the suspension seed process further comprises the step of (g) lysing the cells. In some embodiments, the cells are lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, or a combination thereof.

[0377] In some embodiments, the suspension seed process further comprises (h) purifying the rAAV by at least one column chromatography step. In some embodiments, 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 using commercially available representative material to evaluate the safety and expansion of rAAVrh74.MHCK7.microdystrophin-derived constructs in subjects with Duchenne muscular dystrophy

[0378] This is a Phase 1b open-label study using commercially available representative material of the rAAVrh74.MHCK7.microdystrophin construct conducted in boys with Duchenne muscular dystrophy. Initially, 20 patients were enrolled, and this Example 7 presents data from the first 11 patients under the age of 8 (e.g., 2 patients are 4-5 years old; 9 patients are 6-7 years old) (Cohort 1; up to 20 male DMD ambulatory subjects aged 4 years or older to less than 8 years old) (Table 7). As further described in Example 8, the study was subsequently expanded to include Cohort 2 (approximately 6 male DMD ambulatory subjects aged 8 years or older and less than 18 years old) and Cohort 3 (approximately 6 DMD non-ambulatory subjects). [Table 8-1] [Table 8-2]

[0379] The primary objective was to evaluate micro-dystrophin expression from rAAVrh74.MHCK7.micro-dystrophin constructs (e.g., commercially available representative material) at 12 weeks post-injection (Part 1) as measured by Western blot in biopsied muscle tissue, with a corresponding endpoint of change in micro-dystrophin protein expression from baseline to Week 12 (Part 1) as measured by Western blot. Secondary objectives were to assess: (1) micro-dystrophin protein expression by immunofluorescence (IF) fiber intensity at Week 12; (2) micro-dystrophin expression by IF percentage of dystrophin positive fibers (PDPF) at Week 12; and (3) safety. The inclusion criteria applicable to Examples 7 and 8 for the study are as follows:

[0380] Subjects must meet all of the following criteria to be eligible to participate in this study: 1. Cohort 1 only (<8 years and ambulatory): Male at birth, ambulatory, aged ≥4 years and <8 years at screening, and NSAA score >17 and ≤26 at the screening visit. 2. Cohort 2 only (ages 8 years and older and ambulatory): Male at birth, ambulatory, aged 8 years or older but less than 18 years at screening, and have an NSAA score of 15 or higher and 26 or lower at the screening visit. 3. Cohort 3 only (non-ambulatory): Male at birth, non-ambulatory for a minimum of 9 months, NSAA ambulation score of "0", unable to perform 10MWR at screening visit, and PUL enrollment item score ≥ 2. Incident loss of ambulation is defined as the age (to nearest month) at which the participant or caregiver reports continuous wheelchair use. 4. Have a confirmed diagnosis of DMD using clinical diagnostic genetic testing prior to screening based on clinical documentation and prior to confirmatory genetic testing. 5. Have the following indicators of symptomatic muscular dystrophy: CK evaluation >1000U / L and Cohorts 1 and 2 only (ambulatory): <95 percent of the time predicted for 100 MWR. 6. Ability to cooperate with physical assessment tests. 7. Stable weekly dose equivalent to oral corticosteroids for at least 12 weeks prior to screening and dosing, expected to remain constant (except for adjustments to accommodate weight changes) through the first year of the study. 8. rAAVrh74 antibody titers below 1:400 (i.e., no increase) as determined by ELISA. 9. Sexually active subjects must agree to use condoms for the entire study period and female sexual partners must also use a medically acceptable form of birth control (e.g., oral contraceptives). 10. Subject 18 years of age or older who has parent(s) or legal guardian(s) who are able to understand and comply with the study visit schedule and all other protocol requirements. 11. Subject aged 18 years or older who is willing to provide informed assent or informed consent (if applicable) and has parent(s) or legal guardian(s) who are willing to provide written informed consent for the subject to participate in the study. The exclusion criteria for the study were as follows:

[0381] Subjects who meet any of the following criteria will be excluded from this study: 1. Left ventricular ejection fraction <40% on ECHO at screening or clinical signs and / or symptoms of cardiomyopathy. 2. Cohorts 2 and 3 only (ambulatory and non-ambulatory aged 8 years and older): FVC <50% of predicted at screening and / or requirement of nocturnal ventilatory support. 3. Major surgery within 3 months prior to Day 1 or scheduled for any time during the study. 4. Presence of any other significant genetic disease other than DMD. 5. Have current, chronic, or active serologic evidence of human immunodeficiency virus, hepatitis C, or hepatitis B infection. 6. Have been diagnosed with an autoimmune disease. 7. Have any concomitant illness or need for chronic drug treatment that, in the opinion of the investigator, poses unnecessary risk for gene transfer. 8. Has a medical or extenuating medical condition that, in the opinion of the investigator, may impair the subject's ability to comply with tests or procedures required by the protocol or that may compromise patient welfare, safety, or clinical interpretability. 9. Have a symptomatic infection (e.g., upper respiratory tract infection, pneumonia, pyelonephritis, meningitis) within the 4 weeks prior to Day 1. 10. Demonstrate cognitive decline or impairment that, in the opinion of the Investigator, could be confounded with motor development. 11. Treatment with any of the following therapies according to the time frames specified below: At any time: -Gene therapy - Cell-based therapies (e.g., stem cell transplants) -CRISPR / Cas9, or any other form of gene editing Within 12 weeks of Day 1: Use of human growth factors or vamorolone Within 6 months of Day 1: - Any on-study medication - Cohort 1 only: Any treatment designed to increase dystrophin expression (e.g., Translarna™, EXONDYS51, VYONDYS53, VILTEPSO™). Note: Subjects in Cohorts 2 and 3 receiving these treatments are expected to discontinue treatment prior to Day 1. Treatments designed to increase dystrophin expression may be resumed and / or initiated after 72 weeks. 12. Received a live viral vaccine within 4 weeks of the Day 1 visit, or an inactivated vaccine within 2 weeks, or is anticipated to receive a vaccination between Day 1 and the first 3 months after. 13.Has abnormal laboratory values ​​that are considered clinically significant, including but not limited to: Gamma-glutamyltransferase (GGT) >2x the upper limit of normal (ULN) Total bilirubin above the ULN. Note that this does not exclude cases where the elevated total bilirubin is considered to be due to Gilbert syndrome. ·White blood cell count over 18,500 / μl ·Platelet count 150,000 / μL or less 14. If the subject or family member does not want the subject's study participation to be disclosed to their general practitioner / family doctor and other health care providers.

[0382] In the opinion of the investigator, subjects are unlikely to comply with the study protocol. All subjects received the rAAVrh74.MHCK7.micro-dystrophin construct (1.33 × 10 14 vg / kg) (eg, a commercially available representative material). Efficacy assessment:

[0383] Muscle biopsies for assessment of micro-dystrophin expression were collected from all subjects at baseline and week 12. Muscle biopsies were collected using open biopsy or VACORA core biopsy. Biopsies required collection of muscle tissue from the medial gastrocnemius. If not possible to perform on the medial gastrocnemius, prior approval from the sponsor was required for the use of an alternative muscle.

[0384] Biopsy samples were used to quantitate transgene expression by Western blot, IF intensity, and PDPF.

[0385] The mean vector genome copy number per nucleus and change from baseline was determined to be 3.87 (± 2.4). The mean percentage of normal micro-dystrophin expression and change from baseline was determined to be 55.4% (± 43.4) as determined by Western blot. The mean percentage of dystrophin positive fibers was determined to be 70.5% (change from baseline of 57.7% (± 22.2)) and the mean intensity was determined to be 116.9% (change from baseline of 75.9% ± 46.4) as determined by immunofluorescence. These results are consistent with the placebo group patients of Example 3 who received the material described in Example 4 (e.g., mean vector copy number per nucleus - 2.62; percentage of normal expression - 51.7%; percentage of dystrophin positive fibers - 79.2%; percentage intensity 100.6%).

[0386] Figure 20 shows a graph of the mean NSAA scores from Cohort 1 (first 11 patients treated with rAAVrh74.MHCK7.microdystrophin). The first 11 patients improved 3 points from baseline. The 6-7 year old (n=9) improved 2.9 points from baseline. Each time point represents 11 patients.

[0387] Safety of the commercially available representative material was consistent with prior experience with the rAAVrh74.MHCK7.microdystrophin construct. 79 treatment-emergent adverse events were observed in 11 patients. The most common adverse event was vomiting, which typically developed within the first week, was mild, and was treated with standard antiemetics. Increases in liver enzymes were transient and in response to steroids, with no signs of liver dysfunction in any patient. Two patients had serious adverse events that resolved completely. One patient had elevated transaminases and was treated with intravenous steroids. One patient had nausea and vomiting. No adverse events were observed suggestive of complement-mediated events.

[0388] Overall, the rAAVrh74.MHCK7.microdystrophin construct was characterized using commercially available representative material. Robust transduction was observed (e.g., average vector genome copy number per nucleus of 3.87). Average robust expression was observed, appropriately localized to the sarcolemma (e.g., Western blot-55.4%; positive fibers-70.5%; intensity-116.9%). A safe, well tolerated, and consistent safety profile was observed with administration of commercially available representative material. No clinical complement emergence was observed. Taken together, these results are sufficient to support the manufacturing process and analysis and are sufficient to supply the Duchenne population. Example 8

[0389] The test and study described in Example 7 will be expanded to approximately 32 subjects across three cohorts: Cohort 1 will consist of 20 male DMD ambulatory subjects aged 4 years or older to less than 8 years old; Cohort 2 will consist of approximately 6 male DMD ambulatory subjects aged 8 years or older to less than 18 years old; Cohort 3 will consist of approximately 6 male DMD non-ambulatory subjects.

[0390] The first two subjects enrolled in each cohort will be sentinel subjects dosed at least one week apart. Cohorts 2 and 3 combined will enroll at least three subjects weighing less than 50 kg and at least three subjects weighing 50 kg or greater. The study will consist of four periods as follows: 1. A screening period (pre-infusion) of approximately 3 weeks during which disease characteristics and baseline treatments will be assessed and pre-infusion evaluations will be completed. 2. Baseline Period (Pre-infusion). This period begins upon confirmation of eligibility and ends the day prior to infusion on Day 1, during which baseline assessments will be completed. 3. Infusion Period. During this period, all subjects will receive a single intravenous (IV) infusion of open-label rAAVrh74.MHCK7.micro-dystrophin within 31 days of rAAVrh74 enzyme-linked immunosorbent assay (ELISA) sample acquisition. On Day 1, subjects weighing less than 70 kg will receive IV rAAVrh74.MHCK7.micro-dystrophin (1.33 x 10 14 vg / kg; for subjects weighing 70 kg or more on day 1, a total fixed dose of 9.31 × 10 15 vg (1.33 × 10 for a 70 kg subject) 14 Subjects will receive 100 mg / kg of rAAVrh74.MHCK7.micro-dystrophin (equivalent to a dose of 100 mg / kg vg / kg). Beginning the day before infusion, subjects will receive at least 1 mg / kg of glucocorticoid (prednisone equivalent) daily in addition to the subject's baseline stable oral corticosteroid dose for at least 60 days after infusion; this will be followed by the addition of 1 mg / kg / day of steroids to a total daily dose of 60 mg / day (excluding the addition of steroids in the event of associated GGT elevation and / or other clinically significant abnormal liver function). If GGT levels are confirmed to be 150 U / L or higher after infusion or other clinically significant abnormal liver function is noted, the addition of glucocorticoids for post-infusion immunosuppression should be increased to 2 mg / kg daily (i.e., if the subject's fixed dose is 60 mg / day, this dose should be increased to 120 mg / day). The investigator may adjust subsequent immunosuppressive therapy for the course of subsequent acute liver trauma or other AEs. A hepatologist should be consulted for severe or very severe elevations in liver biochemistry (including GGT, bilirubin, and ALT vs. baseline) or unresponsive elevations to 2 mg / kg / day or 120 mg / day, respectively. In this situation, IV bolus steroids may be considered. The dosages shown in this Example 8, and in Example 7, are determined using linearized DNA qPCR standards. Otherwise, the dosages shown herein are determined utilizing supercoiled DNA qPCR standards. For example, the dosages shown in Examples 7 and 8 of 1.33×10 14vg / kg is the dosage 2×10 14 vg / kg. The tests and studies described in Examples 7 and 8 above are currently and have been conducted utilizing the rAAVrh74.MHCK7.micro-dystrophin construct set forth in SEQ ID NO:9; set forth in SEQ ID NO:8 (nucleotides 1-4977); or set forth in SEQ ID NO:6 (nucleotides 56-5022). 4. A 260-week follow-up period (post-infusion). Safety, efficacy, and manifestation parameters will be evaluated during this period. Subjects will be expected to attend both remote and in-person visits to complete required procedures / assessments. Part 1 of the follow-up period will begin post-infusion (day 1) and continue through week 12. Part 2 of the follow-up period will begin after week 12 and continue through week 260. Frequent visits (approximately weekly) will be required during the first 12 weeks post-infusion (part 1). Further unscheduled visits will be permitted according to the investigator's clinical judgment. For subjects who complete the study, there will be a final study visit at week 260. For subjects who discontinue post-infusion follow-up early, an early termination visit will be required; however, each subject should be strongly encouraged to continue study follow-up through 260 weeks post-infusion.

[0391] The study will be further expanded to include Cohort 4, consisting of approximately 6 male DMD ambulatory subjects aged 3 years or older but younger than 4 years. Cohort 4 subjects (not receiving oral corticosteroids for their DMD at screening) will begin receiving 1.5 mg / kg / day prednisone / prednisolone one week prior to infusion and continue for at least 60 days after infusion.

[0392] The following exclusion criteria are specified for cohorts 1 and 4 only: Any treatment designed to increase dystrophin expression (e.g., Translarna™, EXONDYS51, VYONDYS53, VILTEPSO™). Treatment designed to increase dystrophin expression may be resumed and / or initiated after 72 weeks. Example 9 Genetic testing

[0393] In additional studies, or in the studies listed above, where applicable, subjects must have a confirmed diagnosis of DMD prior to screening based on documentation of clinical findings and prior to genetic testing for confirmation using a genetic test for clinical diagnosis.

[0394] Genetic testing is used to genotype patients for at least one mutation in human dystrophin (DMD) gene.As used herein, "genotyping" refers to the process of determining the specific allele composition of a cell and / or subject at one or more positions in genome, for example, by determining the nucleic acid sequence of the position.Genotyping refers to nucleic acid analysis and / or analysis at nucleic acid level.

[0395] In one embodiment, the method of treating DMD described herein further comprises genotyping the human dystrophin (DMD) gene of the human subject prior to administering the composition to said human subject.

[0396] In some embodiments, the subject's human dystrophin gene (DMD) is genotyped to characterize mutations in the gene that may be suitable for treatment with the compositions disclosed herein.

[0397] In some embodiments, the subject is genotyped for at least one mutation in exons 18-79 of the DMD gene. Specifically, the subject is genotyped for mutations that are expected to lead to the absence of dystrophin protein in the patient. For example, in some embodiments, the patient is genotyped for frameshift deletions, frameshift duplications, premature stops, or other pathogenic variants in the DMD gene that completely encompass exons 18-79. Identification of at least one of these mutations indicates that the patient is eligible for the treatment of the present disclosure.

[0398] In some embodiments, a patient may be genotyped for the DMD gene to obtain a result indicating that the subject is ineligible for the treatment of the present disclosure. For example, a genotyping result demonstrating a mutation between exons 1-17 or between the inclusive values, an in-frame deletion, an in-frame duplication, a mutation of unknown significance ("VUS"), or a mutation entirely contained within exon 45, indicates that the patient is ineligible for the treatment of the present disclosure.

[0399] A number of genotyping techniques are known to those of skill in the art. Example 10 A Phase 3 Multinational, Randomized, Double-Blind, Placebo-Controlled Systemic Gene Delivery Study to Evaluate the Safety and Efficacy of Derandistrogen Moxeparvovec in Subjects With Duchenne Muscular Dystrophy

[0400] This is a randomized, double-blind, placebo-controlled, two-part study of systemic gene delivery of the investigational drug rAAVrh74.MHCK7.microdystrophin (also described in Examples 6-8 above) in approximately 120 ambulatory male DMD subjects aged 4 years or older to <8 years. Randomization will be stratified by age group at randomization (4 years or older to <6 years vs. 6 years or older to <8 years) and NSAA total score at screening (22 years or younger vs. >22 years); at least 50% of subjects must be randomized into the 4 years or older to <6 years age group at randomization. All patients will receive intravenous (IV) investigational drug (1.33 x 10 14 In the treatment arm, participants will receive a single intravenous (IV) infusion of the study drug on day 1. Participants will then receive a single IV infusion of a matching placebo in year 2. In the placebo arm, participants will receive an IV infusion of a matching placebo on day 1. Participants will then have the opportunity to receive a single IV infusion of the study drug in year 2. The study will consist of four periods as follows: Screening period (pre-infusion). Beginning up to 31 days prior to the Day 1 infusion, during this period disease characteristics and baseline treatments will be assessed and pre-infusion evaluations will be completed. Baseline period (pre-infusion). This period begins upon confirmation of eligibility and ends the day prior to infusion on Day 1, during which baseline assessments will be completed. Infusion period. During this period, subjects will receive a single intravenous (IV) infusion of study drug or placebo in a double-blind fashion within 31 days of rAAVrh74 enzyme-linked immunosorbent assay (ELISA) sample acquisition. During the infusion period in part 1, approximately 60 subjects will receive IV study drug (1.33 × 10 14 vg / kg; as determined using linear DNA qPCR standards) and approximately 60 subjects will receive a placebo (saline, 0.9% sodium chloride solution). During the infusion period of part 2, subjects who received placebo in part 1 will receive IV study drug, and subjects who received study drug in part 1 will receive a placebo. All subjects, parents / caregivers, investigators, and site staff, except for the unblinded site pharmacist, will be blinded to the subject's treatment (study drug or placebo). All subjects will receive additional glucocorticoids (prednisone equivalents) for at least 60 days, beginning the day before the infusion. A 104-week follow-up period (post-Part 1 infusion), during which safety and efficacy parameters in Part 1 and Part 2 will be evaluated. Subjects will be expected to attend both remote and in-person visits to complete necessary procedures / assessments. Additionally, unscheduled visits will be permitted according to the investigator's clinical judgment. For subjects who complete the study, there will be a final study visit at Week 52 in Part 2. For subjects who discontinue follow-up post-infusion early, an end-of-study / early termination visit will be required; however, each subject should be strongly encouraged to continue study follow-up through 52 weeks after each infusion. Inclusion / Exclusion Criteria: Selection criteria:

[0401] Subjects must meet all of the following criteria to be eligible to participate in this study: 1. Be male at birth, ambulatory, and aged ≥4 years but <8 years at the time of randomization. 2. Have a confirmed diagnosis of DMD using clinical diagnostic genetic testing prior to screening based on documentation of clinical findings and prior to confirmatory genetic testing. The genetic report must document a frameshift deletion, frameshift duplication, premature stop ("nonsense"), canonical splice site mutation, or other pathogenic variant in the DMD gene entirely within exons 18-79 (inclusive) that is predicted to result in loss of dystrophin protein. a. Mutations between exons 1 and 17, inclusive, are ineligible. b. In-frame deletions, in-frame duplications, and variants of unknown significance ("VUS") are ineligible. c. Mutations entirely contained within exon 45 (inclusive) are not eligible. 3. Ability to cooperate with physical assessment tests. 4.NSAA score >16 and <29 at the screening visit. 5. Time required to rise from the floor at the time of the screening visit is less than 5 seconds. 6. Stable daily dose of oral corticosteroids for at least 12 weeks prior to screening and dosing and regimen expected to remain constant (except for adjustments to accommodate weight changes) throughout the study. 7. rAAVrh74 antibody titers less than 1:400 (i.e., not elevated) as determined by ELISA. 8. Sexually active subjects must agree to use condoms for the entire study period and female sexual partners must also use a medically acceptable form of birth control (e.g., oral contraceptives). 9. Have parent(s) or legal guardian(s) who are able to understand and comply with the study visit schedule and all other protocol requirements. 10. Willing to provide informed consent (if applicable) and have parent(s) or legal guardian(s) who are willing to provide informed consent for the subject to participate in the study. Exclusion criteria

[0402] Subjects who meet any of the following criteria will be excluded from this study: 1. Left ventricular ejection fraction <40% on ECHO at screening or clinical signs and / or symptoms of cardiomyopathy. 2. Having major surgery within 3 months prior to Day 1 or any surgery or procedure scheduled at any time during the study that would interfere with the conduct of the study. 3. The presence of any other clinically significant disease (including cardiac, pulmonary, hepatic, renal, hematological, immunological, or behavioral disease, or infectious or malignant disease), or a concurrent illness or requirement for chronic drug treatment that, in the opinion of the investigator, poses unnecessary risk of gene transfer, or a medical or extenuating medical condition that, in the opinion of the investigator, may impair the subject's ability to comply with the tests or procedures required by the protocol or may impair the subject's welfare, safety, or clinical interpretability. 4. Have current, chronic, or active serologic evidence of human immunodeficiency virus, hepatitis C, or hepatitis B infection. 5. Have a symptomatic infection (e.g., upper respiratory tract infection, pneumonia, pyelonephritis, meningitis) within the 4 weeks prior to Day 1. 6. Demonstrate cognitive decline or impairment that, in the opinion of the Investigator, could be confounded with motor development. 7. Treatment with any of the following therapies according to the time frames specified below: At any time: -Gene therapy - Cell-based therapies (e.g., stem cell transplants) -CRISPR / Cas9, or any other form of gene editing Within 12 weeks of Day 1 and any time during the study: Use of human growth factors or vamorolone Within 6 months of Day 1 and any time during the study: - Any on-study medication - any treatment designed to increase dystrophin expression (e.g., Translarna™, EXONDYS51™, VILTEPSO™) 8. Received a live viral vaccine within 4 weeks of the Day 1 visit, an inactivated vaccine within 2 weeks, or anticipated to receive a vaccination between Day 1 and the first 3 months after. 9. Have abnormal laboratory values ​​that are considered clinically significant, including but not limited to: Gamma-glutamyltransferase >2x the upper limit of normal (ULN) Glutamate dehydrogenase (GLDH) > 15U / L Total bilirubin above ULN. Note: This is not excluded if the elevated total bilirubin is confirmed to be due to Gilbert syndrome. ·White blood cell count over 18,500 / μl ·Platelet count 150,000 / μL or less 10. If the family member does not want the subject's study participation to be disclosed to their general practitioner / family doctor and other health care providers. 11. In the Investigator's opinion, the subject is unlikely to comply with the study protocol. Genetic testing

[0403] Subjects must have a confirmed diagnosis of DMD using a clinically diagnostic genetic test prior to screening based on documentation of clinical findings and prior to confirmatory genetic testing. The genetic report must document a frameshift deletion, frameshift duplication, premature stop, or other pathogenic variant in the DMD gene that completely encompasses exons 18 to 79 (inclusive) that would be expected to result in the absence of dystrophin protein. a. Mutations between exons 1 and 17, inclusive, are ineligible. b. In-frame deletions, in-frame duplications, and variants of unknown significance ("VUS") are ineligible. c. Mutations entirely contained within exon 45 (inclusive) are not eligible. Statistical methods: Sample size:

[0404] The sample size for this study was based on power to detect the primary efficacy endpoint (change in NSAA total score) from baseline to 52 (part 1).

[0405] Assuming a standard deviation of 3.5 across all subjects and a decline rate of 10% at week 52 (part 1) with a type I error of 0.05 (2-sided), a sample size of 120 with a 1:1 randomization ratio would have approximately 90% power to detect a difference in the mean change from baseline to week 52 (part 1) of 2.2 between the investigational drug and placebo groups.

[0406] The study will also be powered in the age group 4 years and older to less than 6 years. At least 60 subjects aged 4 years and older to less than 6 years will participate in the study. Assuming a standard deviation of 3.2 for the primary endpoint in the age group 4 years and older to less than 6 years and a decline rate of 10% at week 52 (part 1) with a type I error of 0.05 (two-sided), a sample size of 60 with a randomization ratio of 1:1 will have at least 80% power to detect a difference in the mean change in NSAA total score from baseline to week 52 (part 1) of 2.5 between the investigational drug and placebo groups.

[0407] A test procedure to adjust for multiplicity will be used to adjust the overall type I error at the two-sided level of 0.05. Details will be specified in the SAP. Randomization

[0408] Subjects will be randomized in a 1:1 ratio to receive either the study drug or a placebo by a single IV infusion. Subjects who received the study drug in part 1 of the study will receive a placebo in part 2. Subjects who received a placebo in part 1 of the study will have the opportunity to receive the study drug in part 2.

[0409] Randomization will be stratified by age group at randomization (≥4 to <6 years or ≥6 to <8 years) and NSAA total score at screening (≤22 years or >22 years); at least 50% of subjects must be randomized into the ≥4 to <6 years age group at randomization. All patients will receive intravenous (IV) investigational drug (1.33 × 10 14 Each patient will have the opportunity to receive 100 mg / kg of 1000 mg ... Corticosteroids

[0410] Subjects must be on a stable daily dose of oral corticosteroids for at least 12 weeks prior to the first screening visit, and this dose will remain constant (except for modifications to accommodate weight changes) throughout the study. Type of corticosteroid, frequency of administration, start and end dates of corticosteroid dosing, and any changes in dosage will be recorded in the subject's source paperwork or on the eCRF. Pre-infusion immunosuppressants

[0411] The day before infusion (study drug or placebo), subjects will begin additional glucocorticoids (prednisone equivalents) for immunosuppression in addition to the subject's baseline stable oral corticosteroid for DMD. Subjects receiving a baseline daily corticosteroid dose for their DMD will receive the subject's usual DMD corticosteroid in addition to an additional 1 mg / kg / day immunosuppressant dose. The 1 mg / kg / day dose will be continued up to a total daily dose of 60 mg / day. Post-infusion immunosuppressants

[0412] For the first 60 days after infusion, subjects will be maintained on a stable baseline daily oral corticosteroid dose for the subject's DMD, plus 1 mg / kg / day glucocorticoid (prednisone equivalent) for immunosuppression. Early taper may be permitted to manage adverse events (AEs) with medical monitor approval. A total daily dose of 1 mg / kg / day will be continued (except for the addition of steroids in the event of GGT elevation and / or other clinically significant liver dysfunction).

[0413] If the subject is unable to tolerate oral immunosuppressive glucocorticoids due to vomiting, glucocorticoids should be administered intravenously.

[0414] If GGT levels are confirmed to be ≥150 U / L after infusion or other clinically significant liver function abnormalities are present, the addition of glucocorticoids for post-infusion immunosuppression should be increased. The investigator may adjust subsequent immunosuppressive therapy for the course of subsequent acute liver trauma or other AEs. A hepatologist should be consulted for severe or non-serious elevations in liver biochemistry (including GGT, bilirubin, GLDH, and ALT vs. baseline) or unresponsive elevations to 2 mg / kg / day or 120 mg / day, respectively. IV bolus steroids may be considered in this situation. · If the subject is taking 1mg / kg of steroids for immune suppression in addition to the subject's DMD steroids, this dose should be increased to 2mg / kg in addition to the subject's DMD steroids for immune suppression that they are taking. If the subject is on a fixed dose of 60 mg / day, this dose should be increased to 120 mg / day.

[0415] Subjects with normal GGT values ​​on Day 60 and no signs of acute liver injury should have their immunosuppressive glucocorticoids tapered over a 2-week period. The tapering period may be adjusted to manage AEs according to the investigator's judgment, but continuation of steroids at doses above the baseline daily regimen beyond 90 days should be discussed with the medical monitor. Immunosuppressive glucocorticoids in subjects with elevated GGT values ​​and / or signs of acute liver injury on Day 60 should be managed as above until GGT values ​​are normal (or clearly trending toward normal) and all signs of acute liver injury have resolved, at which point the subject's immunosuppressive glucocorticoids should be tapered over a 2-week period. Once additional steroids for immunosuppression have been tapered, the subject's baseline daily steroid regimen for DMD should be maintained with any necessary adjustments based on body weight. Efficacy Analysis: North Star Ambulatory Assessment (NSSA)

[0416] The primary endpoint and several secondary endpoints will be tested in a hierarchical fashion using appropriate multiple testing approaches that robustly adjust for family-wise type I error rates at the two-sided 0.05 level.

[0417] For the primary endpoint of change in NSAA total score from baseline to week 52 (part 1), summary statistics will be provided by treatment group for NSAA total score at baseline, each post-baseline visit during part 1, and change from baseline to each post-baseline visit during part 1. For the baseline and week 52 (part 1) visits where 2 NSAA scores are collected, the mean NSAA score will be used in the analysis.

[0418] The primary analysis will compare the two treatment groups for change in NSAA total score from baseline to week 52 (part 1) using a restricted maximum likelihood based mixed model repeated measures analysis. In this model, the response vector will consist of the change from baseline in NSAA total score at each post-baseline visit during part 1. The model will include covariates for treatment group (categorical), visit (categorical), treatment group by visit interaction, and age group at randomization.

[0419] The NSAA is a clinician-administered scale that rates the performance of various functional activities (Mazzone, E et.al., Neuromuscul Disord. 20(11):712-716(2010)). It was designed for use in boys with DMD who are able to stand and has been used in boys with DMD in the study age range (4 years and older and younger than 8 years) (Connolly AM et al., ``Motor and cognitive assessment of infants and young boys with Duchenne Muscular Dystrophy: results from the Muscular Dystrophy Association DMD Clinical Research Network,'' Neuromuscul Disord. 23(7):529-539(2013));Mercuri E et al., ``Revised North Star Ambulatory Assessment for Young Boys with Duchenne Muscular Dystrophy,'' PLoS One 11(8):e0160195(2016)).

[0420] During this assessment, subjects perform 17 different functional activities including 10 MWR, sit to rise, stand on one leg, climb a staircase, descend a staircase, sit to lie, rise from floor, lift head off floor, stand on heels, and jump.

[0421] Subjects will be rated as follows: 2=normal, no obvious alterations in activity; 1=performs task without physical assistance from another person, although the method is modified; and 0=unable to perform task independently.

[0422] Details of NSAA administration are provided in the Clinical Evaluator Manual.

[0423] Two NSAA scores will be collected on two separate days at baseline, on two separate days at week 52 during part 1, and on two separate days at week 52 during part 2. Time required to get up from the floor

[0424] The f...

Claims

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

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

1.

3. The method of claim 2, wherein the rAAV comprises an 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 micro-dystrophin nucleotide sequence of SEQ ID NO: 1 and an MHCK7 promoter sequence of SEQ ID NO:

7.

5. The suspension seed process comprises: The method of any one of claims 1 to 3, further comprising the step of (f) transfecting the adherent cells with a transgene plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct, a plasmid comprising AAVrep and AAVcap genes, and an adenovirus helper plasmid.

6. The transgene plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct, 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. The method described in claim 6, wherein the plasmid containing the AAVrep gene and the AAVcap gene contains the AAV2rep gene and the rAAVrh74cap gene.

8. The method according to any one of claims 6 to 7, wherein the adenovirus helper plasmid comprises the E2A gene, the E4ORF6 gene, and the VA RNA gene of adenovirus type 5.

9. The suspension seed process comprises: (g) lysing the adherent cells The method of any one of claims 1 to 3, further comprising:

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

11. The suspension seed process comprises: (h) purifying the rAAV by at least one column chromatography step. The method of any one of claims 1 to 3, 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. The method of claim 1 , wherein the bioreactor is an adherent bioreactor.

14. 14. The method of claim 13, wherein the third medium in the bioreactor comprises at least one factor that promotes cell adhesion.

15. The method of claim 14, 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.

16. 16. The method of claim 14 or 15, wherein the third medium in the bioreactor comprises DMEM and 10% FBS.

17. The method according to any one of claims 1 to 3, wherein the adherent cells are cultured under suspension conditions for about 48 to 72 hours.

18. The method of any one of claims 1 to 3, wherein the N-1 container is a suspension shake flask.

19. The method of any one of claims 1 to 3, 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.

20. The method of claim 19, wherein the adherent cells are HeLa cells or HEK-293 cells.

21. The method of claim 20, wherein the adherent cells are HEK-293 cells.

22. The method of any one of claims 1 to 3, wherein the adherent cells are not adapted to suspension.

23. The method according to any one of claims 1 to 3, wherein the step of culturing the cells under suspension conditions does not alter the adhesion-dependence of the cells.

24. 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 3.

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

8.

25. The composition of claim 24 comprising:

26. 26. The composition of claim 25 for use in a method of treating muscular dystrophy in a human subject in need thereof, the method comprising administering to the human subject the composition of claim 25.

27. The composition is administered using a systemic route, and the rAAV is administered in an amount of about 5.0×10 １２ vg / kg ~ approx. 1.0×10 １５ 27. The composition of claim 26, administered at a dose of 1000 mg / kg.

28. The systemic administration route is an intravenous route, and the dose of the rAAV administered is about 2×10 １４ 28. The composition of claim 27, wherein the total amount of the composition is 100 mg / kg.

29. The composition described in any one of claims 26 to 28, characterized in that the dose of rAAV is administered at a concentration of about 10 mL / kg.

30. The composition according to any one of claims 26 to 28, characterized in that the composition is administered by injection, infusion or implantation.

31. 31. The composition of claim 30, wherein the composition is administered by infusion over a period of approximately 1 hour.

32. The composition according to any one of claims 26 to 28, characterized in that the composition is administered by the intravenous route via a peripheral vein of a limb.

33. The composition according to any one of claims 26 to 28, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

34. 34. The composition of claim 33, wherein the muscular dystrophy is Duchenne muscular dystrophy.

35. The composition of any one of claims 26 to 28, wherein the expression level of the micro-dystrophin gene in the cells of the subject is increased after administration of the composition compared to the expression level of the micro-dystrophin gene before administration of the composition.

36. 36. The composition of claim 35, wherein expression of the micro-dystrophin gene in the cells is detected by measuring micro-dystrophin protein levels by Western blot in muscle biopsied before and after administration of the composition.

37. 37. The composition of claim 36, wherein the expression is at least 55.4% after administration of the composition compared to before administration.

38. 29. The composition of any one of claims 26-28, wherein the average percentage of micro-dystrophin positive fibers in the muscle tissue of the subject is increased after administration of the composition compared to the number of micro-dystrophin positive fibers before administration of the composition.

39. 39. The composition of claim 38, wherein the average percentage of micro-dystrophin positive fibers is at least 70.5% and the average intensity is at least 116.9% as detected by immunofluorescence (IF) in muscle biopsies before and after administration of the composition.

40. The composition of any one of claims 26 to 28, wherein the micro-dystrophin transduction by vector genome count is an average vector genome copy number of at least 3.87 per nucleus.

41. Composition according to any one of claims 26 to 28, characterized in that said composition is administered to a genotyped patient.

42. 42. The composition of claim 41, wherein the genotyped patient is genotyped for at least one mutation in exons 18-79 of the human dystrophin (DMD) gene.

43. The composition of claim 42, wherein the method further comprises the step of genotyping the DMD gene of the human subject prior to administration of the composition to the human subject.

44. 44. The composition of claim 43, wherein said genotyping detects at least one mutation in exons 18-79 of the DMD gene, wherein said at least one mutation is a frameshift deletion, frameshift duplication, premature stop, or other pathogenic variant that results in the abolition of expression of human dystrophin protein.

45. 26. The composition of claim 25 for treating muscular dystrophy in a human subject in need thereof.

46. 26. Use of the composition of claim 25 in the manufacture of a medicament for the treatment of muscular dystrophy.

47. 47. The composition of claim 45 or the use of claim 46, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

48. 48. The composition or use of claim 47, wherein the muscular dystrophy is Duchenne muscular dystrophy.

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