Obtaining recombinant vectors based on adeno-associated virus (AAV) for treatment of muscular dystrophy

Abstract

FIELD: biotechnology.SUBSTANCE: method for producing a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by propagating cells in a hybrid seed bioreactor system is described, comprising: (a) culturing adherent cells under adherent conditions with a first growth medium containing serum in container N-2; (b) removing adherent cells from the first medium; (c) inoculating adherent cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than in the first medium in container N-1; (d) culturing adherent cells in container N-1 under suspension conditions; (e) inoculating adherent cells from step (d) into a third medium in a bioreactor for growing adherent cultures; and (f) transfecting the adherent cells with a transgenic plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenoviral helper plasmid, to produce rAAV containing the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO: 7.EFFECT: expanding the scope of treatments for muscular dystrophy.18 cl, 8 tbl, 11 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 253,998, filed October 8, 2021, U.S. Provisional Patent Application No. 63 / 243,944, filed September 14, 2021, U.S. Provisional Patent Application No. 63 / 209,733, filed June 11, 2021, and U.S. Provisional Patent Application No. 63 / 189,676, filed May 17, 2021, all of which are incorporated herein by reference in their entireties.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The contents of the sequence listing provided in electronic form in an ASCII text file (Name: 4140_052PC04_Seqlisting_ST25.txt; Size: 60,194 bytes; and Creation date: May 13, 2022) filed with the application are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0003] The present invention relates to the field of gene therapy. In particular, the present invention provides gene therapy vectors, such as adeno-associated virus (AAV)-based vectors, for expressing a miniaturized human microdystrophin gene, wherein said AAVs are obtained from adherent cells cultured in suspension conditions. The present invention also provides methods of using such vectors to express microdystrophin in skeletal muscles, including diaphragm and cardiac muscle, and to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy.BACKGROUND

[0004] The importance of muscle mass and strength for daily activities such as locomotion and breathing, as well as for whole-body metabolism, is undeniable. Deficits in muscle function cause muscular dystrophies (MDs), which are characterized by muscle weakness and wasting and have a significant impact on quality of life. The best-characterized MDs result from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs arise from membrane fragility associated with loss of sarcolemmal cytoskeletal binding to DAPC. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting one in 5,000 male births.

[0005] DMD results from mutations in the DMD gene, resulting in reduced mRNA and absence of dystrophin, a 427-kDa sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51: 919–28, 1987). DAPC consists of several proteins in the muscle sarcolemma that form a structural link between the extracellular matrix (ECM) and the cytoskeleton via dystrophin, an actin-binding protein, and alpha-dystroglycan, a laminin-binding protein. These structural links stabilize the muscle cell membrane during contraction and protect against contraction-induced damage. With loss of dystrophin, membrane fragility leads to sarcolemmal ruptures and a calcium influx that triggers calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol. 10: 168-75 (1997)). This uncontrolled cycle of muscle destruction and regeneration eventually depletes the muscle stem cell population (Sacco et al., Cell 143: 1059–1071 (2010); Wallace et al., Annu Rev Physiol 71: 37–57 (2009)), resulting in progressive muscle weakness, endomysial inflammation, and fibrous scarring.

[0006] Without membrane stabilization by dystrophin or microdystrophin, DMD will manifest as uncontrolled cycles of tissue damage and repair, ultimately replacing lost muscle fibers with fibrous scar tissue through connective tissue proliferation. Fibrosis is characterized by excessive deposition of ECM matrix proteins, including collagen and elastin. ECM proteins are primarily produced from cytokines such as TGFβ, which are released by activated fibroblasts responding to stress and inflammation. Although the primary pathological feature of DMD is muscle fiber degeneration and necrosis, fibrosis as a pathological consequence leads to the same results. Overproduction of fibrous tissue limits muscle regeneration and contributes to progressive muscle weakness in patients with DMD.In one study, the presence of fibrosis in baseline DMD muscle biopsies was strongly correlated with poor motor outcome at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol 68: 762–767 (2009)). These findings indicate that fibrosis is a major factor contributing to the development of muscle dysfunction in DMD and highlight the need for early intervention before overt fibrosis occurs.

[0007] International Publication No. WO 2019 / 245973 A1, incorporated herein by reference in its entirety, describes AAV vector delivery of the microdystrophin gene for the treatment of muscular dystrophy (e.g., DMD) in a human subject. However, there is a continuing need in the art for improved methods for producing such AAV vectors, particularly methods suitable for large-scale production of such vectors for gene therapy. SUMMARY OF THE INVENTION

[0008] The present invention relates to gene therapy vectors, such as AAV vectors produced by the suspension seeding method described herein, that express the human microdystrophin gene in skeletal muscles, including diaphragm and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis.

[0009] The present invention relates to a method for producing a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by a suspension seeding method, comprising: (a) culturing cells with a first growth medium containing serum in container N-2; (b) removing cells from the first medium; (c) inoculating the cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than in the first medium, in container N-1; (d) culturing the cells in container N-1 under suspension conditions; and (e) inoculating a third medium into a bioreactor with the cells from step (d).

[0010] According to some aspects, the rAAV used in the method described in the present application comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1. According to some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 7. According to some aspects, the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 7.

[0011] According to some aspects, the suspension seeding method further comprises: (f) transfecting adherent cells with a transgenic plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

[0012] In some aspects, a transgenic plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct comprises: a nucleic acid sequence as set forth in SEQ ID NO: 9; nucleotides 55-5021 of SEQ ID NO: 3; or nucleotides 1-4977 of SEQ ID NO: 8. In some aspects, a plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and a rAAVrh74 cap gene. In some aspects, an adenovirus helper plasmid comprises an adenovirus 5 E2A gene, E40RF6, and VA-RNA.

[0013] According to some aspects, the method for seeding a suspension further comprises: (g) lysing the adherent cells. According to some aspects, the adherent cells are lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, ultrasonic disruption, high-pressure extrusion, detergent lysis, or combinations thereof.

[0014] In some aspects, the method for seeding a suspension further comprises (h) purifying the rAAV using at least one column chromatography step. In some aspects, said at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0015] According to some aspects, the method for seeding the suspension further comprises culturing the cells with the first growth medium in container N-3.According to some aspects, the method for seeding the suspension further comprises culturing the cells with the first growth medium in container N-4.

[0016] In some aspects, the bioreactor is a bioreactor for growing adherent cultures. In some aspects, rAAV is purified from a culture obtained in the bioreactor for growing adherent cultures.

[0017] According to some aspects, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. According to some aspects, 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. According to some aspects, the third medium in the bioreactor comprises DMEM and 10% FBS.

[0018] According to some aspects, the adherent cells are cultured under suspension conditions for approximately 48-72 hours.

[0019] According to some aspects, container N-1 is a suspension shaking flask.

[0020] According to some aspects, 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. According to some aspects, the adherent cells are HeLa cells or HEK-293 cells. According to some aspects, the adherent cells are HEK-293 cells. According to some aspects, the adherent cells are not adapted to suspension. According to some aspects, culturing the cells under suspension conditions does not alter the dependence of the cells on the substrate. According to some aspects, culturing does not contribute to altering the cells to create a new cell line.

[0021] According to the present invention, there is also provided a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is produced by any of the methods described herein. In some aspects, the composition comprises: a) rAAV particles comprising a nucleic acid sequence presented in SEQ ID NO: 9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO: 3; and / or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO: 8.

[0022] According to some aspects of the present invention, there is provided a composition for treating muscular dystrophy in a subject in need thereof, comprising a recombinant adeno-associated virus (rAAV) rAAV.rh74MHCK7.microdystrophin, wherein the rAAV is produced in adherent cells, and wherein said adherent cells are cultured in container N-1 under suspension conditions. According to some aspects, rAAV comprises the nucleotide sequence of human microdystrophin presented in SEQ ID NO: 1. According to some aspects, rAAV comprises the MHCK7 promoter sequence presented in SEQ ID NO: 7. According to some aspects, rAAV comprises the MHCK7 promoter sequence presented in SEQ ID NO: 7 and the nucleotide sequence of human microdystrophin presented in SEQ ID NO: 1.

[0023] According to some aspects, the composition comprises: (a) rAAV comprising a nucleic acid sequence as presented in SEQ ID NO: 9; (b) rAAV particles comprising a nucleic acid sequence as presented in SEQ ID NO: 9; (c) rAAV comprising nucleotides 55-5021 of SEQ ID NO: 3; (d) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO: 3; (e) rAAV comprising nucleotides 1-4977 of SEQ ID NO: 8; and / or (1) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO: 8.

[0024] The present invention also provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering a composition comprising rAAV described herein to said human subject. In some aspects, the rAAV is administered via a systemic route and at a dose of from about 5.0×10 12vg / kg to approximately 1.0×10 15 vg / kg. In some aspects, the systemic route of administration is the intravenous route, and the dose of rAAV administered is approximately 2×10 14 vg / kg.

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

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

[0027] In some aspects, the level of microdystrophin gene expression in a subject's cell is increased after rAAV administration compared to the level of microdystrophin gene expression before rAAV administration. In some aspects, microdystrophin gene expression in a cell is detected by measuring the microdystrophin protein level using Western blotting in a muscle biopsy before and after rAAV administration. In some aspects, the expression is at least 55.4% after rAAV administration compared to the expression before administration.

[0028] According to some aspects, the average percentage of microdystrophin-positive fibers in the subject's muscle tissue increases after rAAV administration compared to the number of microdystrophin-positive fibers before rAAV administration. According to some aspects, the average percentage of microdystrophin-positive fibers is at least 70.5%, and the average intensity is at least 116.9%, as determined by immunofluorescence (IF) in muscle biopsies before and after rAAV administration. According to some aspects, microdystrophin transduction by the number of vector genomes averages at least 3.87 vector genome copies per nucleus.

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

[0030] In some aspects, the method for treating muscular dystrophy further comprises genotyping the DMD gene of a human subject prior to administering the composition to said human subject. In some aspects, the genotyping detects at least one mutation in exons 18-79 of the DMD gene. In some aspects, the at least one mutation is a frameshift deletion, a frameshift duplication, a premature stop, or another pathogenic variant resulting in the absence of expression of the human dystrophin protein.

[0031] The present invention also provides the use of the composition described herein for the treatment of muscular dystrophy in a human subject in need thereof. According to some aspects, the present invention also provides the use of the composition described herein for the preparation of a medicament for the treatment of muscular dystrophy.

[0032] According to some aspects, muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. According to some aspects, muscular dystrophy is Duchenne muscular dystrophy. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 shows the rAAV.MHCK7.micro-dystrophin construct. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeat (ITR) sequences. The construct is characterized by an in-frame deletion of the stem (R4-R23), while hinges 1, 2, and 4 (H-1, H2, and H4) and the cysteine-rich domain remain to produce a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) is regulated by the MHCK7 promoter (795 bp). The intron and 5'-UTR are obtained from the pCMVB plasmid (Clontech). The microdystrophin cassette had a Kozak consensus sequence immediately upstream of the ATG start codon and a small synthetic polyA signal of 53 bp for mRNA termination. The human microdystrophin cassette contained domains (R4-R23 / Δ71-78) as described previously by Harper et al. (Nature Medicine 8: 253-261 (2002)).

[0034] Fig. 2 shows the nucleic acid sequence (SEQ ID NO: 3) of AAVrh74.MHCK7.micro-dystrophin.

[0035] Fig. 3 shows the map of the AAV helper plasmid pNLREP2-Caprh74.

[0036] Fig. 4 shows the Ad pHELP helper plasmid.

[0037] Fig. 5 shows the construction of the rAAV.MCK.micro-dystrophin plasmid.

[0038] Fig. 6 shows the nucleic acid sequence (SEQ ID NO: 5) of rAAVrh74.MCK.micro-dystrophin.

[0039] Fig. 7 shows the expression of the microdystrophin gene in muscle fibers in a gastrocnemius muscle biopsy measured by immunocytochemistry.

[0040] Figures 8A-8C show Western blots demonstrating microdystrophin protein expression at the correct molecular weight. Referring to Figures 8A and 8B, Western blot analysis revealed microdystrophin protein expression in Subject 1 (5 years old), Subject 2 (4 years old), and Subject 3 (6 years old). Referring to Figure 8C, samples from Subject 4 (*) were diluted 1:4 (to the linear range) when exceeding the ULDQ (>80%) in the original assay, and the mean values ​​were multiplied by the dilution correction factor to determine the final value compared to normal. The average microdystrophin expression compared to normal was 182.7% in Method 1 and 222.0% in Method 2.

[0041] Fig. 9A-9C show that administration of rAAVrh74.MHCK7.micro-dystrophin increased the expression of DAPC, alpha-sarcoglycan, and beta-sarcoglycan proteins in subject 1 (Fig. 9A), subject 2 (Fig. 9B), and subject 3 (Fig. 9C).

[0042] Fig. 10 is a graph showing the sustained sharp decrease in creatine kinase (CK) values ​​upon administration of rAAVrh74.MHCK7.micro-dystrophin.

[0043] Figure 11 is a graph showing the mean change in creatine kinase (CK) from baseline to day 270. These data demonstrate that the CK level was significantly decreased over time after rAAVrh74.MHCK7.micro-dystrophin administration.

[0044] Fig. 12 is a graph showing the mean change in NSAA and the mean change in CK from baseline to day 270. These data demonstrate that the NSAA level increased significantly over time after rAAVrh74.MHCK7.micro-dystrophin administration.

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

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

[0047] Fig. 15 shows the nucleic acid sequence (SEQ ID NO: 8) of the AAVrh74.MHCK7.micro-dystrophin plasmid construct, which contains the kanamycin resistance gene.

[0048] Fig. 16 is a visualization of a method for cell propagation in a hybrid seed bioreactor system as described in the present application.

[0049] Fig. 17 is a visualization of the production of an AAV particle using the cell expansion method in a hybrid seed bioreactor system as described in this application.

[0050] Figs. 18A-18B are graphs showing the viability of HEK-293 cells cultured according to the cell propagation method in the hybrid seed bioreactor system described in the present application (Fig. 18A) and only under adherent conditions (Fig. 18B).

[0051] Figs. 19A-19B are graphs showing the density of viable HEK-293 cells cultured according to the cell expansion method in the hybrid seed bioreactor system described in the present application (Fig. 19A) and only under adherent conditions (Fig. 19B).

[0052] Figure 20 is a graph showing the mean NSAA score in Cohort 1 (the first 11 patients treated with rAAVrh74.MHCK7.microdystrophin), as described in Example 7. The first 11 patients demonstrated an improvement of 3 points compared to baseline. Patients aged 6 to 7 years (n=9) demonstrated an improvement of 2.9 points compared to baseline. Eleven patients are represented at each time point.

[0053] Fig. 21A-21C show the expression of microdystrophin in skeletal and cardiac muscles (immunofluorescence) of DMD rats mdx at 12 weeks (Fig. 21B) and 24 weeks (Fig. 21C) after treatment with the delanistrogens moxeparvovec compared to saline (Fig. 21A), as described in Example 11. Definitions: LTA=left tibialis anterior; HRT=heart.

[0054] Fig. 22A-22B are histograms depicting the quantification of microdystrophin expression (immunofluorescence) (Fig. 22A) and vector transduction (vector genome copies) (Fig. 22B) in muscle tissues of DMD rats mdx at 12 weeks and 24 weeks after treatment with delanistrogens moxiparvovec as described in Example 11. Definitions: TA=tibia; HRT=heart; MG=medial gastrocnemius; LG=lateral gastrocnemius; DIA=diaphragm; TRI=triceps; PSO=psoas major.

[0055] Fig. 23A-23B are histograms illustrating the increase in locomotion (Fig. 23A) and upright activity (Fig. 23B) in DMD rats mdxafter 12 weeks and 24 weeks of treatment with the delanistrogens moxeparvovec compared to saline as described in Example 11. Rats' locomotion (ambulation and upright activity) in activity cages was measured by the number of laser beam interruptions per hour. Each point represents a value for an individual animal. Data are presented as mean ± SD; ***=p<0.001; **=p<0.01. SD=standard deviation.

[0056] Figures 24A-24B show a significant reduction in muscle degeneration as determined by central nucleation analysis in skeletal muscle at 12 weeks and 24 weeks after delanistrogene moxeparvovec gene transfer in DMD rats mdxcompared to saline as described in Example 11. Figure 24A shows hematoxylin and eosin (H & E) staining of the gastrocnemius muscle. Figure 24B shows histograms showing the percentage of fibers with central nuclei. Bars are represented as mean ± SD; ****=p<0.0001. SD=standard deviation.

[0057] Fig. 25A-25B show the analysis of collagen deposition in skeletal and cardiac muscles demonstrating the reduction of fibrosis after treatment with the delanistrogenstogen moxeparvovec in DMD rats mdxafter 12 weeks and 24 weeks compared to saline as described in Example 11. Figure 25A shows Masson's trichome staining 12 weeks after treatment. Figure 25B presents histograms quantifying collagen deposition in skeletal and cardiac muscle at 12 and 24 weeks after treatment. Key: HRT=heart; MG=medial gastrocnemius; DIA=diaphragm. Data are presented as mean ± SD; ****=p<0.0001; *=p<0.05. SD=standard deviation.

[0058] Fig. 26 is a histogram showing that serum troponin I levels do not change significantly after 1 week and 12 weeks of treatment with delanistrogens moxeparvovec in DMD rats mdx compared to saline as described in Example 11. Bars represent mean + SD. Each point represents the value for an individual animal.

[0059] Fig. 27A-27C are histograms analyzing the cardiac function determined by echocardiography in DMD rats mdx after 24 weeks of treatment with delanistrogens moxeparvovec, compared to saline, as described in Example 11. Fig. 27A illustrates data for left ventricular end-systolic diameter (LVESD). Fig. 27B illustrates data for ejection fraction (%) (EF). Fig. 27C illustrates data for shortening fraction (%) (FS). DETAILED DESCRIPTION OF THE INVENTION

[0060] According to the present invention, there are provided vectors for gene therapy, for example, rAAV, which express human microdystrophin, wherein rAAV is produced in adherent mammalian cells, and wherein said adherent cells are cultured in an N-1 container under suspension conditions.

[0061] The present invention relates to a method for producing a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by a suspension seeding method, comprising: (a) culturing cells with a first growth medium containing serum in container N-2; (b) removing cells from the first medium; (c) inoculating the cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than in the first medium, in container N-1; (d) culturing the cells in container N-1 under suspension conditions; and (e) inoculating a third medium into a bioreactor with the cells from step (d).

[0062] The present invention also provides compositions (e.g., pharmaceutical compositions) comprising the rAAV described herein, as well as methods for treating muscular dystrophy (e.g., DMD) using the compositions described herein.

[0063] Muscle biopsies taken at the earliest age at diagnosis of DMD reveal marked connective tissue proliferation. Muscle fibrosis has numerous deleterious consequences. It reduces the normal transport of endomysial nutrients across connective tissue barriers, reduces blood flow, and deprives muscles of vascular nutrients. It also contributes to early loss of ambulation due to limb contractures. Over time, treatment challenges increase as a result of severe muscle fibrosis. This can be observed in muscle biopsies by comparing connective tissue proliferation at successive time points. This process continues to worsen, leading to loss of ambulation and accelerated decline, especially in wheelchair-dependent patients.

[0064] Without early treatment that includes a parallel approach to reducing fibrosis, it is unlikely that the benefits of exon skipping, stop codon frameshifting, or gene replacement therapies will ever be fully realized. Even small molecule or protein replacement strategies are likely to fail without an approach that reduces muscle fibrosis. Previous work in aged mdx mice with fibrosis treated with AAV.micro-dystrophin demonstrated that the authors were unable to achieve full functional recovery (Liu, M. et al., Mol Ther 11: 245-256 (2005)). It is also known that the progression of cardiomyopathy in DMD is accompanied by scarring and fibrosis in the ventricular wall. Definitions

[0065] Unless otherwise defined, all technical and scientific terms used in this application have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, this application, including definitions, shall prevail. Unless the context otherwise requires, singular terms include the plural, and plural terms include the singular.

[0066] In the present description, a term describing an object in the singular refers to one or more such objects; for example, "polynucleotide" should be understood as one or more polynucleotides. Therefore, the terms in the singular, the terms "one or more," and "at least one" can be used interchangeably in this application.

[0067] Furthermore, as used in this application, “and / or” should be understood as a specific description of each of two specific features or components, together with or separately from the other. Thus, the term “and / or” used in an expression such as “A and / or B” in the context of this application includes “A and B,” “A or B,” (only) “A,” and (only) “B.” Similarly, the term “and / or” used in an expression such as “A, B and / or C” is intended to include each of the following aspects: 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; (only) A; (only) B; and (only) C.

[0068] As used in this application, the term "approximately" is used to denote concepts such as "approximately," "roughly," "about," or "in the vicinity." When used in conjunction with a numerical range, the term "approximately" modifies the range by extending the boundaries above and below the specified numerical values. In general, the term "approximately" is used in this application to vary a numerical value above and below the stated value by a deviation of 10 percent in either direction (above or below), unless otherwise specified.

[0069] It should be understood that the term "at least" before a number or series of numbers includes the number following the term "at least" and all subsequent numbers or integers that can logically be included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be represented by 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 specified property. When "at least" appears before a series of numbers or a range of numbers, it should be understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, without regard to the number of significant digits).

[0070] Nucleotide sequences are represented in the context of this application as single strands only, in the 5'-3' direction, from left to right, unless otherwise specifically indicated. Nucleotides and amino acids are represented in the context of this application in accordance with the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature, or (for amino acids) either by the one-letter code or the three-letter code in accordance with the requirements of 37 CFR §1.822, as applicable.

[0071] As used in this application, the term "polynucleotide" or "nucleic acid" means a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides in this application are presented in the 5' to 3' direction. A polynucleotide according to the present invention may be a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. Nucleotide bases are designated in this application by a single-letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0072] As used in this application, the term "polypeptide" encompasses both peptides and proteins unless otherwise specified.

[0073] The term "coding sequence" or "coding" sequence is used in this application to refer to a region of DNA or RNA (the transcribed region) that "encodes" a particular protein, such as insulin or glucokinase. The coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide, in vitro or in vivo, when under the control of an appropriate regulatory region, such as a promoter. The boundaries of the coding sequence are defined by a start codon at the 5' end (amino) and a translation stop codon at the 3' end (carboxy). The coding sequence may include, but is not limited to, prokaryotic or eukaryotic cDNA, prokaryotic or eukaryotic genomic DNA, and synthetic DNA sequences. A transcription termination sequence may be located in the 3' direction to the coding sequence.

[0074] A gene may contain several functionally linked fragments, such as a promoter, a 5' leader sequence, an intron, a coding sequence, and a 3' untranslated sequence, such as one containing a polyadenylation site or a signal sequence. As used herein, the term "gene expression" refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0075] As used in this application, the term "promoter" refers to a nucleic acid sequence or fragment whose function is to control the transcription of one or more genes (or coding sequence) located upstream of the transcription initiation site of a gene, and is structurally defined by the presence of a DNA-dependent RNA polymerase binding site, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences that are known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions.An "inducible" promoter is one that is regulated depending on physiological or developmental conditions. A "tissue-specific" promoter is preferentially active in certain types of differentiated cells / tissues.

[0076] As used in this application, the term "enhancer" is a cis-acting element that stimulates or inhibits the transcription of adjacent genes. An enhancer that inhibits transcription is also called a "silencer." Enhancers can function (e.g., be linked to the coding sequence) in any orientation, at distances of up to several thousand base pairs (kb) from the coding sequence, and downstream of the transcribed region.

[0077] The term "operably linked" refers to the positioning of a nucleotide sequence of a regulatory element, such as a promoter nucleotide sequence, to ensure expression of said nucleotide sequence by said regulatory element.

[0078] As used in this application, the term "transgene" refers to a gene (e.g., microdystrophin) or a nucleic acid molecule introduced into a cell. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide. In some aspects, a 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 although the gene is normally expressed in the cell, the condition and / or disease may still develop. In some aspects, a transgene provides increased expression or overexpression of the gene. A transgene may contain sequences that are native to the cell, contain sequences that are not naturally found in the cell, or it may contain combinations of these sequences.In some aspects, the transgene may comprise a sequence that can be operably linked to appropriate regulatory sequences for gene expression. In some aspects, the transgene is not integrated into the host cell genome.

[0079] As used in this application, 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 co-infection with a helper virus provides specific functions. Currently, there are thirteen characterized serotypes of AAV. 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 expected that the same principles will apply to additional AAV serotypes, since it is well known that the various serotypes are quite closely related, both structurally and functionally, and even genetically. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes appear to exhibit very similar replication properties mediated by homologous rep genes; and they all carry three related capsid proteins, like the proteins expressed in AAV2. The degree of relatedness is further supported by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes throughout the genome; and by the presence of similar self-annealing segments at the ends, corresponding to "inverted terminal repeat" (ITR) sequences. Similar patterns of infectivity also suggest that 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 containing one or more polynucleotides of interest (or transgenes, such as microdystrophin) flanked by AAV terminal repeat (ITR) sequences. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0081] As used in this application, the terms "AAV virion," "AAV viral particle," or "AAV vector particle" refer to a viral particle consisting of at least one AAV capsid protein and an encapsidated AAV vector polynucleotide. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered into a mammalian cell), it is commonly referred to as an "AAV vector particle" or simply an "AAV vector." Thus, producing an AAV vector particle necessarily includes producing an AAV vector. Thus, the vector is contained in an AAV vector particle.

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

[0083] By "muscle cell" or "muscle tissue" is meant a cell or group of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, such as that found in the gastrointestinal tract, bladder, blood vessels, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0084] As used herein, the term "transduction" refers to the introduction / delivery of a microdystrophin coding region into a recipient cell either in vivo or in vitro using a replication-deficient rAAV of the present invention, which results in the expression of microdystrophin by the recipient cell.

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

[0086] As used in this application, the term "vector" refers to a recombinant plasmid or virus that contains a polynucleotide to be delivered into a host cell in vitro or in vivo. "Recombinant" means different from that typically found in nature.

[0087] The "serotype" of a virus vector or capsid is determined by a distinct immunological profile based on capsid protein sequences and capsid structure.

[0088] "AAV Cap" means AAV Cap, VP1, VP2 and VP3 proteins and their analogs.

[0089] "AAV Rep" refers to AAV Rep proteins and their analogs.

[0090] As used in this application, the term "flanked" in relation to a sequence that is flanked by other elements indicates the presence of one or more flanking elements upstream and / or downstream, i.e., 5' and / or 3', relative to the sequence. The term "flanked" does not necessarily mean that the sequences are contiguous. For example, there may be inserted sequences between the nucleic acid encoding the transgene and the flanking element. A sequence (e.g., a transgene) that is "flanked" by 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, inserted sequences may be present between them.

[0091] As used in this application, the term "gene therapy" refers to the insertion of nucleic acid sequences (e.g., a nucleic acid containing a promoter operably linked to a polynucleotide encoding a transgene, such as microdystrophin) into the cells and / or tissues of an individual to treat a disease or pathological condition. Such transgenes may be exogenous. An exogenous molecule or sequence is a molecule or sequence not normally found in the cell, tissue, and / or organism of the individual being treated.

[0092] As used herein, the term "genotyping" refers to the process of determining the specific allelic composition of a cell and / or subject at one or more positions in the genome, for example, by determining the nucleic acid sequence at that position. Genotyping refers to nucleic acid analysis and / or analysis at the nucleic acid level. In some aspects, the human dystrophin gene (DMD) in a subject is genotyped to characterize a mutation in the gene that is particularly amenable to treatment with the compositions disclosed herein. Numerous genotyping techniques are known to those skilled in the art.

[0093] The term "stringent" is used to refer to conditions that are commonly understood in the art as stringent. The stringency of hybridization conditions is mainly determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual. 2 ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). More stringent conditions (such as higher temperature, lower ionic strength, higher concentration of formamide or other denaturing agent) can also be used, but this will affect the rate of hybridization. When hybridizing deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6x SSC 0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides). Other agents may be included in the hybridization and wash buffers to reduce nonspecific and / or background hybridization.Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4 (SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentrations and types of these additives can be varied without significantly affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8–7.4; however, under ordinary conditions of ionic strength, the hybridization rate is nearly independent of pH. See Anderson, M. L. M., et al., Nucleic Acid Hybridization: A Practical Approach. Ch. 4, IRL Press Limited (Oxford, England) (1998). A person skilled in the art will be able to adjust the hybridization conditions to account for these variables and to allow DNA with different sequence relationships to form hybrids.

[0094] As used in this application, 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, said solutions provide essential and nonessential amino acids, vitamins, energy sources, lipids, and micronutrients required by the cell for minimal growth and / or survival. Said solution may also contain components that enhance growth and / or survival beyond the minimal rate, including hormones and growth factors. Said solution is prepared with a pH and salt concentration optimal for cell survival and proliferation.The medium may also be a "defined medium" or "chemically defined medium"—a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. Defined media do not contain components of animal origin, and all components have a known chemical structure. Those skilled in the art will recognize that a defined medium may contain recombinant glycoproteins or proteins, such as, but not limited to, hormones, cytokines, interleukins, and other signaling molecules.

[0095] As used in this application, the term "basal medium composition" or "basal medium" refers to any cell culture medium used for culturing cells that has not been modified by either the addition or the selective removal of a particular component.

[0096] As used in this application, the terms "culture," "cell culture," and "eukaryotic cell culture" refer to a eukaryotic cell population, either attached to a surface (i.e., adherent) or in suspension, that is maintained or grown in a medium under conditions suitable for the survival and / or growth of the cell population. Those skilled in the art will understand that these terms, in this context, can refer to a combination comprising a population of mammalian cells and a medium in which the population is suspended.

[0097] As used in this application, the term "batch culture" refers to a cell culture method in which all components that will ultimately be used in cell culture, including the medium and the cells themselves, are provided at the beginning of the culture process. Batch culture is typically stopped at some point, and the cells and / or components in the medium are collected and optionally purified.

[0098] As used in this application, the term "fed-batch culture" refers to a cell culturing method in which additional components are introduced into the culture at some time after the start of the culturing process. Fed-batch culture may be initiated using a basal medium. The culture medium, with which additional components are introduced into the culture at some time after the start of the culturing process, is a nutrient medium. The proposed components typically contain additional nutrition for cells that have been depleted during the culturing process. Fed-batch culture is typically stopped at a certain point, and the cells and / or components in the medium are collected and optionally purified.

[0099] As used in this application, the term "perfusion culture" refers to a cell culture method in which additional components are continuously or semi-continuously introduced into the culture after the culture process has begun. These components typically provide additional nutrients for cells that have been depleted during the culture process. A portion of the cells and / or components in the medium is typically collected on a continuous or semi-continuous basis and optionally purified.

[0100] The "growth phase" of cell culture refers to the period of exponential cell growth (logarithmic phase), during which cells typically divide rapidly. During this phase, cells are cultured for a specific period of time, typically 1 to 4 days, and under conditions that ensure maximum cell growth. The growth cycle for a host cell can be determined for the specific host cell provided without undue experimentation. "The period of time and conditions that ensure maximum cell growth" and the like refer to those culture conditions that are determined to be optimal for cell growth and division for a specific cell line. In some aspects, during the growth phase, cells are cultured in a nutrient medium containing the necessary supplements, typically at a temperature of approximately 25-40°C, in a humidified, controlled atmosphere that achieves optimal growth for the specific cell line.

[0101] In some aspects, the cells are maintained in the growth phase for a period of approximately one to seven days, such as two to six days, such as six days. The duration of the growth phase for specific cells can be determined without undue experimentation. For example, the duration of the growth phase will be a period of time sufficient for the specific cells to proliferate to a viable cell density in the range of approximately 20-80% of the maximum possible viable cell density if the culture were maintained under growth conditions.In some aspects, "maximal growth rate" refers to the growth rate of a particular cell line / clone measured during its exponential growth phase when the cells are in fresh culture medium (e.g., measured during culture with sufficient nutrients and in the absence of significant growth inhibition from any culture components).

[0102] As used in this application, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. This term, as used in this application, also refers to the proportion of cells that are alive at a given time and to the sum of the number of live and dead cells in a culture at a given time.

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

[0104] As used in this application, the term "bioreactor" or "culture vessel" refers to any vessel used for growing mammalian cell cultures. The bioreactor may be of any size as long as it is suitable for culturing mammalian cells.

[0105] As used in this application, the term "bioreactor cycle" may include one or more of the lag phase, log phase, or plateau phase growth periods during a cell culture cycle.

[0106] As used in this application, the terms "N-1 culture vessel," "N-1 seed culture vessel system," "N-1 vessel," "N1 culture," or "N1 container" refer to a culture vessel that is located immediately before the N culture vessel (production culture vessel) and is used to grow a cell culture to a high density of viable cells for subsequent inoculation into the N (production) culture vessel. The cell culture grown in the N-1 culture vessel can be obtained after culturing the cells in several vessels before the N-1 culture vessel, for example, in N-4, N-3, and N-2 vessels.

[0107] As used in this application, the terms "N culture vessel," "production culture vessel," "N vessel," "N bioreactor," or "production bioreactor" refer to the cell culture in the bioreactor downstream of the N-1 bioreactor. The N culture is used to produce AAV.

[0108] As used in this application, the terms "seeding" or "inoculation" refer to the process of introducing a cell culture into a bioreactor or other vessel. In one aspect, the cells were previously replicated in another bioreactor or vessel. In another aspect, the cells were frozen and thawed immediately before introducing them into the bioreactor or vessel. This term refers to any number of cells, including a single cell. rAAV and methods for producing rAAV (or a composition containing rAAV) (e.g., rAAVrh74.MHCK7.microdystrophin)

[0109] According to the present invention, there is provided a composition comprising a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.micro-dystrophin, wherein said rAAV is produced in adherent mammalian cells, and wherein said adherent cells are cultured in a container N-1 under suspension conditions. In some aspects, the rAAV is of the AAVrh.74 serotype (e.g., rAAV.MHCK7.micro-dystrophin).

[0110] The present invention also provides a method for producing a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by a suspension seeding method, comprising: (a) culturing the cells with a first growth medium containing serum in a container N-2; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than in the first medium in container N-1; (d) culturing the cells in container N-1 under suspension conditions; and (e) inoculating a third medium into the bioreactor with the cells from step (d).

[0111] According to some aspects, the rAAV used in the method described in the present application comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1. According to some aspects, the rAAV comprises the MHCK7 promoter sequence presented in SEQ ID NO: 7. According to some aspects, the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 7.

[0112] According to some aspects, the suspension seeding method further comprises: (f) transfecting adherent cells with a transgenic plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

[0113] In some aspects, a transgenic plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct comprises: a nucleic acid sequence as set forth in SEQ ID NO: 9; nucleotides 55-5021 of SEQ ID NO: 3; or nucleotides 1-4977 of SEQ ID NO: 8. In some aspects, a plasmid comprising an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and a rAAVrh74 cap gene. In some aspects, an adenovirus helper plasmid comprises an adenovirus 5 E2A gene, E4ORF6, and VA-RNA.

[0114] According to some aspects, the method for seeding a suspension further comprises: (g) lysing the adherent cells. According to some aspects, the adherent cells are lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, ultrasonic disruption, high-pressure extrusion, detergent lysis, or combinations thereof.

[0115] In some aspects, the method of seeding a suspension further comprises (h) purifying the rAAV using at least one column chromatography step. In some aspects, said at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0116] According to some aspects, the method for seeding the suspension further comprises culturing the cells with the first growth medium in container N-3.According to some aspects, the method for seeding the suspension further comprises culturing the cells with the first growth medium in container N-4.

[0117] In some aspects, the bioreactor is a bioreactor for growing adherent cultures. In some aspects, rAAV is purified from a culture obtained in the bioreactor for growing adherent cultures.

[0118] According to some aspects, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. According to some aspects, 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. According to some aspects, the third medium in the bioreactor comprises DMEM and 10% FBS.

[0119] According to some aspects, the adherent cells are cultured under suspension conditions for approximately 48-72 hours.

[0120] According to some aspects, container N-1 is a suspension shaking flask.

[0121] According to some aspects, 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. According to some aspects, the adherent cells are HeLa cells or HEK-293 cells. According to some aspects, the adherent cells are HEK-293 cells. According to some aspects, the adherent cells are not adapted to suspension. According to some aspects, culturing the cells under suspension conditions does not alter the dependence of the cells on the substrate. According to some aspects, culturing does not contribute to altering the cells to create a new cell line.

[0122] According to some aspects, a suspension seeding method used to produce rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells comprises: (a) culturing the cells with a first growth medium containing serum in container N-2; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than that in the first medium in container N-1; (d) culturing the cells in container N-1 under suspension conditions; (e) inoculating a third medium in a bioreactor with the cells from step (d); (f) transfecting the cells with a transgene plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid, (g) lysis of the cells, and (h) purification of the rAAV by at least one column chromatography step.

[0123] In some aspects, rAAVs are described in International Publication WO 2019 / 245973 A1, which is expressly incorporated by reference in its entirety into this application.

[0124] Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of approximately 4.7 kb, including a 145-nucleotide inverted terminal repeat (ITR). There are several serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the genome of AAV serotype 2 (AAV2) is presented in the article by Srivastava et al., J Virol. 45: 555–564 (1983) and in Ruffing et al., J Gen Virol. 75: 3385–3392 (1994).As other examples, the complete genome of AAV-1 is submitted to GenBank under the accession number NC_002077; the complete genome of AAV-3 is submitted to GenBank under the accession number NC_1829; the complete genome of AAV-4 is submitted to GenBank under the accession number NC_001829; the genome of AAV-5 is submitted to GenBank under the accession number AF085716; the complete genome of AAV-6 is submitted to GenBank under the accession number NC_00 1862; at least fragments of the genomes of AAV-7 and AAV-8 are submitted to GenBank under the accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 relating to AAV-8); The AAV-9 genome is presented in Gao et al., J. Virol., 78: 6381–6388 (2004); the AAV-10 genome is presented in Mo / . Ther., 13(1): 67–76 (2006); and the AAV-11 genome is presented in Virology, 330(2):375–383 (2004). Cloning of serotype AAVrh.74 is described in Rodino-Klapac et al., Journal of Translational Medicine 5: 45 (2007).Cis-functioning sequences directing viral DNA replication (rep), encapsidation / packaging, and integration into the host cell chromosome are contained within the ITR. Three AAV promoters (named p5, p19, and p40 based on their relative positions on the genomic map) drive the expression of two internal AAV open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), combined with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 in AAV2), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins possess multiple enzymatic properties that are ultimately responsible for viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of three related capsid proteins.The single consensus polyadenylation site is located at position 95 of the AAV genome map. The life cycle and genetics of AAV are described in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97–129 (1992).

[0125] AAV possesses unique features that make it promising as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is latent and asymptomatic. Furthermore, AAV infects a wide range of mammalian cells, allowing for targeting of many different tissues in vivo. Furthermore, AAV transduces slowly dividing and non-dividing cells and can persist essentially for the life of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious when the DNA is cloned into plasmids, enabling the construction of recombinant genomes. Furthermore, since signals directing AAV replication, encapsidation, and genome integration are contained within the ITR of the AAV genome, some or all of the internal approximately 4.3 kbThe genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, the DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another significant feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenoviruses (from 56°C to 65°C for several hours), facilitating cold storage of AAV. AAV can even be lyophilized. Finally, cells infected with AAV are not resistant to superinfection.

[0126] Numerous 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: 211–222 (2001). Furthermore, because muscle is highly vascularized, transduction with recombinant AAV results in the appearance of transgene products in the systemic circulation following intramuscular injection, 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). Furthermore, Lewis et al., J Virol.76: 8769–8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for proper glycosylation, folding, and antibody secretion, indicating that muscle is capable of stable expression of secreted protein therapeutic agents.

[0127] The recombinant AAV genomes of the present invention comprise a nucleic acid molecule of the present invention and one or more AAV ITRs flanking said nucleic acid molecule. The AAV DNA in the rAAV genomes can be obtained from any AAV serotype for which a recombinant virus can be produced, including, but not limited to, 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 described, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. 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 stimulate specific expression in skeletal muscle.

[0128] The DNA plasmids of the present invention comprise the rAAV genomes of the present invention. The DNA plasmids are transformed into cells that can be infected with an AAV helper virus (e.g., an adenovirus, an E1-deleted adenovirus, or a herpes virus) to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV particles in which the AAV genome to be packaged, the rep and cap genes, and the helper virus functions are introduced into a cell are standard in the art. Production of rAAV requires the presence in a single cell (referred to herein as a "packaging cell") of the following components: the rAAV genome, the AAV rep and cap genes separated from (i.e., not included in) the rAAV genome, and the helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be produced, and may be from an AAV serotype other than the ITR of the rAAV genome, including 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, but not limited to those mentioned. The production of pseudotyped rAAV is described, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0129] The method for producing a packaging cell involves creating a cell line that stably expresses all the necessary components for producing AAV particles. For example, a plasmid (or several plasmids) containing the rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene are integrated into the cell's genome. AAV genomes are introduced into bacterial plasmids using 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 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 advantages of this method are that the cells are selective and suitable for industrial rAAV production. Other examples of suitable methods for introducing rAAV genomes and / or the rep and cap genes into packaging cells use adenovirus or baculovirus, rather than plasmids.

[0130] General principles for rAAV production are reviewed, for example, by Carter, Current Opinions in Biotechnology 1533–539 (1992); and Muzyczka, N., Curr. Topics Microbial. Immunol. 158: 97–129 (1992). Various approaches are described by 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., 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); US Patent No. 5,786,211; US Patent No. 5,871,982 and US Patent No. 6,258,595.The above documents are incorporated by reference in their entirety into this application, with particular emphasis on those sections of the documents that relate to the production of rAAV.

[0131] Thus, the present invention provides packaging cells that produce infectious rAAV. According to one aspect, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (a related line of 293). According to another aspect, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with E1 adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus macaque fetal lung cells).

[0132] The recombinant AAV (i.e., infectiously encapsulated rAAV particles) of the present invention comprises an rAAV genome. According to exemplary aspects, the genomes of both rAAVs do not contain AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genomes. Examples of rAAVs that can be engineered to contain nucleic acid molecules of the present invention are disclosed in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352), which is incorporated herein by reference in its entirety.

[0133] According to an exemplary aspect, 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. RAAV contains a microdystrophin gene expression cassette flanked by AAV2 inverted terminal repeat (ITR) sequences. It is this sequence that is encapsidated into AAVrh74 virions. The plasmid contains the microdystrophin sequence and the MHCK7 enhancer and the core promoter elements of the muscle-specific promoter to drive gene expression. The expression cassette also contains the SV40 (SD / SA) intron to promote highly efficient gene expression, and the bovine growth hormone gene polyadenylation signal is used for efficient transcription termination.

[0134] pNLREP2-Caprh74 is an AAV helper plasmid that encodes 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 Fig. 3.

[0135] The adenovirus helper plasmid pHELP is 11,635 bp and was obtained from Applied Viromics. The plasmid contained regions of the adenovirus genome important for AAV replication, namely E2A, E40RF6, and VA-RNA (adenovirus E1 functions were provided by 293 cells). The adenovirus sequences present in this plasmid constituted only ~40% of the adenovirus genome and lacked cis-elements critical for replication, such as adenovirus terminal repeats. Thus, this production system is presumably incapable of generating infectious adenovirus. A schematic map of the pHELP plasmid is shown in Fig. 4.

[0136] RAAV as described herein can be purified by methods routinely used in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include those described, for example, by Clark et al., Hum. Gene Ther. 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med. 69 421-443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0137] According to one aspect, the present invention relates to an rAAV comprising a nucleotide sequence of a muscle specific control element and a nucleotide sequence encoding a microdystrophin protein. For example, the nucleotide sequence encodes a functional microdystrophin protein, wherein the nucleotide is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more preferably at least 90%, 91%, 92%, 93% or 94% and even more preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, wherein said protein retains microdystrophin activity. Microdystrophin protein provides stability to the muscle membrane during muscle contraction, for example, microdystrophin acts as a shock absorber during muscle contraction.

[0138] In one aspect, the rAAV is rAAVrh74.MHCK7.microdystrophin, i.e., a viral form of rAAV serotype rh74 capsid particles encapsidating a genome or nucleic acid expression cassette comprising a microdystrophin transgene driven by the MHCK7 promoter / enhancer, and also referred to by the nonproprietary name delanistrogene moxeparvovec in the context of administration to a subject. In some aspects, when describing subjects administered delanistrogene moxeparvovec in the Examples section, the data (e.g., histograms) may be referred to as "Treated" for simplicity.

[0139] According to one aspect, rAAVrh74.MHCK7.microdystrophin is rAAVrh74.MHCK7.microdystrophin with the sequence of SEQ ID NO: 9 or comprising 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 wherein the adherent cells are cultured in container N-1 under suspension conditions. According to one aspect, rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, rAAVrh74.MCK.microdystrophin is rAAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0140] The present invention also relates to rAAV, wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or its complements, and encodes a functional microdystrophin protein.

[0141] According to one aspect, rAAV is a non-replicating recombinant adeno-associated virus (AAV) called rAAVrh74.MHCK7.micro-dystrophin, comprising the sequence of SEQ ID NO: 9, nucleotides 55-5021 of the sequence of SEQ ID NO: 3, nucleotides 1-4977 of the sequence of SEQ ID NO: 8, or nucleotides 56-5022 of the sequence of SEQ ID NO: 6, wherein rAAV is produced in adherent cells, and wherein the adherent cells are cultured in container N-1 under suspension conditions. This vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeats (ITR), microdystrophin, the SV40 intron (SD / SA), and the synthetic polyadenylation signal (PolyA), all under the control of the MHCK7 promoter / enhancer. A schematic of the vector genome and expression cassette is shown in Fig. 1. The AAVrh74 serotype can be used to achieve efficient gene transfer into skeletal and cardiac muscle following intravenous administration.

[0142] According to one aspect, the present invention relates to rAAV, wherein the muscle-specific control element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer binding factor (MEF), muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), a hybrid α-myosin heavy chain enhancer / MCK enhancer-promoter (MHCK7), C5-12, a mouse creatine kinase enhancer element, a skeletal fast-twitch troponin c gene element, a slow-twitch cardiac troponin c gene element, a slow-twitch troponin i gene element, hypoxia-inducible nuclear factors, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0143] For example, the muscle-specific control element is the nucleotide sequence of the MHCK7 promoter of SEQ ID NO: 2 or SEQ ID NO: 7, or the muscle-specific control element is the nucleotide sequence of MCK of SEQ ID NO: 4. Furthermore, in any of the rAAV vectors of the present invention, the nucleotide sequence of the muscle-specific control element, for example, the nucleotide sequence of MHCK7 or MCK, is operably linked to a nucleotide sequence encoding a microdystrophin protein. For example, the nucleotide sequence of the MHCK7 promoter (SEQ ID NO: 2 or SEQ ID NO: 7) is operably linked to a sequence encoding human microdystrophin (SEQ ID NO: 1), as shown in the construct shown 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 sequence encoding human microdystrophin (SEQ ID NO: 1), as shown in the construct shown in Fig. 5 or Fig. 6 (SEQ ID NO: 5). In another aspect, the present invention relates to a rAAV vector comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 1 and SEQ ID NO: 7. The present invention also provides a rAAV vector comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 4.

[0144] According to a further aspect, the present invention relates to an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8. For example, the AAVrh74.MHCK7.microdystrophin vector comprises a nucleotide sequence within the ITR of the sequence of SEQ ID NO: 3 inclusive, shown in Fig. 2. According to another aspect, the rAAV vector comprises a 5' ITR, a MHCK7 promoter, a chimeric intron sequence, a sequence encoding the human microdystrophin gene, polyA and a 3' ITR. According to one aspect of the present invention, the vector comprises nucleotides 55-5021 of the sequence of SEQ ID NO: 3. The plasmid shown in SEQ ID NO:3 additionally contains the ampicillin resistance gene and the pGEX plasmid backbone with the pBR322 origin of replication.

[0145] According to another aspect, the present invention relates to a rAAV comprising the nucleotide sequence of SEQ ID NO: 9, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in container N-1 under suspension conditions. For example, the AAVrh74.MHCK7.microdystrophin vector construct comprises the nucleotide sequence presented in SEQ ID NO: 9 and shown in Fig. 13. Said rAAV vector construct comprises the MHCK7 promoter, a chimeric intron sequence, a sequence encoding the human microdystrophin gene and polyA. According to one aspect, the rAAV vector construct further comprises an ITR in the 5' direction to the promoter and an ITR in the 3' direction to polyA. According to one aspect, the rAAV is AAVrh74.

[0146] According to another aspect, the rAAVrh74.MHCK7.microdystrophin vector (i.e., the viral vector) comprises a nucleotide sequence within and including the ITR of SEQ ID NO: 8 and is shown in Fig. 15. The rAAV vector comprises a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, a sequence encoding the human microdystrophin gene, polyA, and a 3' ITR. According to one aspect, the vector comprises nucleotides 1-4977 of SEQ ID NO: 9. The plasmid shown in SEQ ID NO: 3 further comprises a kanamycin resistance gene and a pGEX plasmid backbone with a pBR322 origin of replication.

[0147] According to another aspect of the present invention, there is provided a plasmid comprising the AAVrh74.MHCK7.microdystrophin construct. In one aspect, the plasmid comprises a 5'-ITR, an MHCK7 promoter, a chimeric intron sequence, a sequence encoding the human microdystrophin gene, polyA and a 3'-ITR. In one aspect, the plasmid comprises a kanamycin resistance gene and optionally comprises a pGEX plasmid backbone with a pBR322 origin of replication. In a specific aspect, the plasmid is presented in SEQ ID NO: 8 and is shown in Figs. 14 and 15.

[0148] The present invention relates to a recombinant AAV vector comprising the nucleotide sequence of human microdystrophin presented in SEQ ID NO: 1 and the nucleotide sequence of the MHCK7 promoter presented in SEQ ID NO: 2 or SEQ ID NO: 7, wherein said rAAV is produced in adherent cells and wherein the adherent cells are cultured in container N-1 under suspension conditions. Said rAAV vector is AAV serotype AAVrh.74.

[0149] The present invention also relates to a rAAV comprising the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct within the ITR sequence of SEQ ID NO: 3 inclusive, the nucleotide sequence within the ITR sequence of SEQ ID NO: 8 inclusive, or the nucleotide sequence presented in SEQ ID NO: 9, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured in container N-1 under suspension conditions. Said rAAV vector is AAV serotype AAVrh.74.

[0150] The rAAV vectors of the present invention may have any AAV serotype, such as serotype AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13.

[0151] The present invention also provides pharmaceutical compositions (or sometimes referred to herein simply as “compositions”) comprising any of the rAAV vectors of the present invention.

[0152] In a further aspect, the present invention provides methods for producing an rAAV vector particle, comprising culturing a cell transfected with any rAAV vector of the present invention and isolating the rAAV particles from the supernatant of the transfected cells. The present invention also provides viral particles comprising any of the recombinant AAV vectors of the present invention. Compositions comprising rAAV and administration of said compositions

[0153] According to another aspect, the present invention provides compositions comprising rAAV according to the present invention. The compositions according to the present invention comprise rAAV and a pharmaceutically acceptable carrier. Said compositions may also contain other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients and preferably inert at the dosages and concentrations used and include buffers and surfactants, such as Pluronics.

[0154] ​​According to the present invention, there is provided a composition for the treatment of muscular dystrophy (e.g., DMD) in a subject in need thereof, comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin, wherein said rAAV is produced in adherent cells, and wherein the adherent cells are cultured in a container N-1 under suspension conditions.

[0155] According to some aspects of the present invention, there is provided a composition comprising a recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin, wherein the rAAV is produced in adherent mammalian cells by the suspension seeding method described herein. According to some aspects, the composition comprises: a) rAAV particles comprising the nucleic acid sequence presented in SEQ ID NO: 9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO: 3; and / or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO: 8.

[0156] The titers of rAAV administered in the methods of the present invention depend, for example, on the particular rAAV, the route of administration, the purpose of treatment, the individual, and the target cell type(s), and can be determined by methods routinely used in the art. rAAV titers can range from approximately 1×10 6 , approximately 1×10 7 , approximately 1×108 , approximately 1×10 9 , approximately 1×10 10 , approximately 1×10 11 , approximately 1×10 12 , approximately 1×10 13 up to approximately 1×10 14 or more DNase-resistant particles (DRP) per ml. Doses can also be expressed in viral genome units (vg). One typical method for determining the titer of encapsidated vector genome is quantitative PCR, such as in the methods described in the source (Pozsgai et al., Mol. Ther. 25: 855–869 (2017)).

[0157] The present invention provides methods for transducing a target cell with rAAV in vivo or in vitro. The in vivo methods comprise the step of administering an effective dose or multiple effective doses of a composition comprising rAAV according to the present invention to an animal (including a human) in need thereof. If the dose is administered before the development of a disorder / disease, the administration is prophylactic. If the dose is administered after the development of a disorder / disease, the administration is therapeutic.According to aspects of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease being treated, wherein said dose slows or prevents progression to the disorder / pathological condition, slows or prevents progression of the disorder / pathological condition, reduces the severity of the disease, leads to remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease that can be prevented or treated by the methods of the present invention is DMD.

[0158] The present invention also provides combination therapy. In this application, combination includes both simultaneous treatment and sequential treatment. In particular, combinations of the methods of the present invention with standard medical treatments (e.g., corticosteroids), as well as combinations thereof with new treatments, are contemplated.

[0159] Administration of an effective dose of the compositions can be carried out by standard methods in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal or vaginal administration. The route(s) of administration and the AAV serotype(s) of the rAAV components (in particular, the AAV ITR and capsid protein) according to the present invention can be selected and / or adjusted by those skilled in the art taking into account the infection and / or disease condition being treated and the target cells / tissue(s) that are to express the microdystrophin protein.

[0160] The present invention provides local and systemic administration of an effective dose of rAAV and the compositions of the present invention. For example, systemic administration is administration into the circulatory system, thereby affecting the entire body. Systemic administration includes enteral administration, for example, by absorption through the gastrointestinal tract, and parenteral administration via injection, infusion, or implantation.

[0161] In particular, the actual administration of rAAV according to the present invention can be carried out by any physical method that allows the transport of the recombinant rAAV vector into the target tissue of the animal. Administration according to the present invention includes, but is not limited to, injection into muscle and injection into the bloodstream. It has been demonstrated that simple resuspension of rAAV in phosphate-buffered saline is sufficient to produce a carrier suitable for expression in muscle tissue, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although for rAAV, DNA-damaging compositions should generally be avoided). The capsid proteins of rAAV can be modified such that rAAV targets a specific tissue of interest, such as muscle. See, for example, WO 02 / 053703, the contents of which are incorporated herein by reference.Pharmaceutical compositions can be formulated as injectables or topical formulations for delivery to muscle via 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 invention. RAAV can be administered with any pharmaceutically acceptable carrier for ease of administration and handling.

[0162] According to one aspect of the present invention, the AAVrh74.MHCK7.microdystrophin described herein is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl2), 200 mM sodium chloride (NaCl) and 0.001% poloxamer 188.

[0163] The dose of rAAV to be administered in the methods described herein varies depending on, for example, the particular rAAV, the route of administration, the purpose of treatment, the individual, and the cell type(s) being targeted, and can be determined by methods routinely used in the art. Titers of each rAAV administered can range from approximately 1×10 6 , approximately 1×10 7 , approximately 1×10 8 , approximately 1×10 9 , approximately 1×10 10 , approximately 1×10 11 , approximately 1x1012, approximately 1×10 13 , approximately 1×10 14 , approximately 2×10 14 up to approximately 1×10 15 or more DNase-resistant particles (DRP) per ml. Dosages can 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, 1×1014 vg, 2×10 14 vg, 1×10 15 vg, respectively). Dosages can 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 (vg / kg, respectively). AAV titration methods are described in Clark et al., Hum. Gene Ther., 10: 1031–1039 (1999).

[0164] In particular, the actual administration of rAAV according to the present invention can be carried out by any physical method that allows the transport of the recombinant rAAV vector into the target tissue of the animal. Administration according to the present invention includes, but is not limited to, injection into muscle and injection into the bloodstream. It has been demonstrated that simple resuspension of rAAV in phosphate-buffered saline is sufficient to produce a carrier suitable for expression in muscle tissue, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although for rAAV, DNA-damaging compositions should generally be avoided). The capsid proteins of rAAV can be modified such that rAAV targets a specific tissue of interest, such as muscle. See, for example, WO 02 / 053703, the contents of which are incorporated herein by reference.Pharmaceutical compositions can be formulated as injectables or topical formulations for delivery to muscle via 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 invention. RAAV can be administered with any pharmaceutically acceptable carrier for ease of administration and handling.

[0165] For intramuscular injection, solutions in an excipient such as sesame or peanut oil, or in an aqueous propylene glycol solution, as well as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered if necessary, and the liquid diluent is first rendered isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. rAAV dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal conditions of storage and use, such preparations contain a preservative to prevent the growth of microorganisms.In this regard, all sterile aqueous media used are easily obtained using standard techniques well known to those skilled in the art.

[0166] Pharmaceutical carriers, diluents, or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the immediate reconstitution of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be protected against contamination by microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures of these components, and vegetable oils.Proper fluidity can be maintained, for example, by using a coating, such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. The action of microorganisms can be prevented by the use of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents in the composition, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be ensured by the use of agents that delay absorption, such as aluminum monostearate and gelatin.

[0167] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in an appropriate solvent with various other ingredients listed above, as required, followed by filter sterilization. Typically, dispersions are prepared by incorporating the sterilized active ingredient into a sterile carrier medium containing a basic dispersion medium and the required other ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield the active ingredient in powder form in combination with any additional desired ingredient from a previously filter-sterilized solution.

[0168] rAAV transduction can also be performed in vitro. According to one aspect, the desired target muscle cells are isolated from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, isogenic or xenogenic muscle cells can be used in cases where these cells will not elicit an inappropriate immune response in the subject.

[0169] Suitable methods for transducing and re-administering transduced cells to a subject are known in the art. According to one aspect, cells can be transduced in vitro by combining rAAV with muscle cells, for example, in an appropriate medium, and screening to detect cells that contain the DNA of interest using traditional methods such as Southern blotting and / or PCR, or using selectable markers. The transduced cells can then be incorporated into pharmaceutical compositions, and said composition is administered to the subject by various routes, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth and cardiac muscle, using, for example, a catheter.

[0170] Transduction of cells with rAAV according to the present invention results in stable expression of microdystrophin protein. Accordingly, the present invention provides methods of administering / delivering rAAV that expresses microdystrophin protein to an animal, preferably a human. These methods comprise transducing tissues (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more rAAVs according to the present invention. Transduction can be carried out using gene cassettes containing tissue-specific control elements. For example, according to one aspect of the present invention, there are provided methods of transducing muscle cells and muscle tissues driven by muscle-specific control elements, including, but not limited to, those derived from the actin and myosin gene families, for example, from the myoD gene family (see Weintraub et al., Science, 251: 761–766 (1991)), myocyte-specific enhancer-binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11: 4854–4862 (1991)), control elements derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7: 4089–4099 (1987)), the cardiac actin gene, muscle creatine kinase sequence elements (see Johnson et al., Mol Cell Biol 9:3393–3399 (1989)) and the mouse creatine kinase (mCK) enhancer element, control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene, and the slow-twitch troponin I gene: hypoxia-inducible nuclear factors (Semenza et al., Proc Nail Acad Sci USA 88: 5680–5684 (1991)), steroid-inducible elements and promoters, including the glucocorticoid response element (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA 90: 5603–5607 (1993)) and other control elements.

[0171] Muscle tissue is a suitable target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present invention provides for the sustained expression of microdystrophin from transduced muscle fibers.

[0172] Thus, the present invention provides methods for administering an effective dose (or doses administered substantially simultaneously, or doses administered at intervals) of rAAV that encode microdystrophin to a subject in need thereof (e.g., a subject suffering from muscular dystrophy).

[0173] The present invention provides nucleic acid molecules comprising a nucleotide sequence as set forth in SEQ ID NO: 3, 8 or 9. The present invention also provides rAAVs comprising a nucleic acid sequence as set forth in SEQ ID NO: 9, or nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 55-5021 of SEQ ID NO: 3, and rAAV particles comprising a nucleic acid sequence as set forth in SEQ ID NO: 9, or nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 55-5021 of SEQ ID NO: 3, wherein said rAAVs are produced in adherent cells, and wherein the adherent cells are cultured in container N-1 under suspension conditions.

[0174] According to another aspect of the present invention, there are provided compositions comprising a nucleic acid molecule comprising a nucleotide sequence presented in SEQ ID NO: 3, 8 or 9, an rAAV comprising a nucleotide sequence presented in SEQ ID NO: 9, or nucleotides 1-4977 of the sequence SEQ ID NO: 8 or nucleotides 55-5021 of the sequence SEQ ID NO: 3, and rAAV particles comprising a nucleotide sequence presented in SEQ ID NO: 9, or nucleotides 1-4977 of the sequence SEQ ID NO: 8 or nucleotides 55-5021 of the sequence SEQ ID NO: 3, wherein said rAAVs are produced in adherent cells, and wherein the adherent cells are cultured in container N-1 under suspension conditions. Any of the methods described in this application can be carried out using the said compositions. Propagation of adherent cells in a hybrid seed reactor system

[0175] Some aspects of the present invention relate to a method for cell propagation, comprising: (a) culturing cells using a first medium containing serum; in a container N-2; (b) removing cells from the first medium; (c) inoculating the cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than that in the first medium, in container N-1; (d) culturing the cells in container N-1 under suspension conditions; and (e) inoculating a third medium into a bioreactor with the cells from step (d). According to one aspect, the second medium is a serum-free medium. According to another aspect, the second medium contains serum at a concentration lower than the serum concentration in the first medium.

[0176] Some aspects of the present invention relate to cell propagation in a seed bioreactor system comprising (a) culturing cells with a first medium containing serum in a container N-3; (b) removing cells from the first medium; (c) inoculating the cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than that in the first medium in a container N-2; (d) culturing the cells in container N-2 under suspension conditions; (e) and inoculating the cells from step (d) into the second medium in vessel N-1; and (f) inoculating a third medium into the bioreactor with the cells from step (d). According to one aspect, the second medium is a serum-free medium. According to another aspect, the second medium contains serum at a concentration lower than the serum concentration in the first medium.

[0177] Some aspects of the present invention relate to a method for expanding adherent cells, comprising (a) culturing adherent cells under adherent conditions in a first medium containing serum; (b) removing adherent cells from the first medium; (c) suspending the adherent cells in a second medium that does not contain serum or contains serum at a concentration lower than in the first medium; (d) culturing the adherent cells under suspension conditions; and (e) inoculating a third medium into a bioreactor with the adherent cells from step (d). According to some aspects, the method may further comprise transferring the adherent cells from step (a) at least once under adherent conditions. According to some aspects, the method may further comprise transferring the adherent cells from step (d) at least once under suspension conditions. According to one aspect, the second medium is a serum-free medium.According to another aspect, the second medium contains serum at a concentration lower than in the first medium in container N-1.

[0178] The first medium, second medium and third medium may be any medium suitable for the specific cells being cultured. In some aspects, 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 containing albumins, 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 derived from plant or animal sources), and combinations thereof.The growth medium may be commercially available media such as 5x concentrated DMEM / F12 (Invitrogen), CD OptiCHO feed (Invitrogen), CD EfficientFeed (Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (Irvine Scientific), BD Recharge (Becton Dickinson), Cellvento Feed (EMD Millipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria), ActiCHO (PAA / GE Healthcare), Ham's F10 (Sigma), Minimum Essential Medium (MEM), Sigma, RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM), Sigma.

[0179] According to some aspects, a serum-free second growth medium that does not contain serum or contains serum at a concentration lower than the concentration of the first serum, is substantially free (contains no more than trace levels) of calcium ions, fetal bovine serum (FBS), fibronectin, collagen, laminin, or proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix that promote cell attachment. According to one aspect, the second medium is a serum-free medium. According to another aspect, the second medium contains serum at a concentration lower than the serum concentration in the first medium.

[0180] According to some aspects, the growth medium may have a pH of about 6.5 to about 7.5, about 6.5 to about 7.4, about 6.5 to about 7.3, about 6.5 to about 7.2, about 6.5 to about 7.1, about 6.5 to about 7.0, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.5 to about 6.7, about 6.6 to about 7.5, about 6.6 to about 7.4, about 6.6 to about 7.3, about 6.6 to about 7.2, about 6.6 to about 7.1, about 6.6 to about 7.0, about 6.6 to about 6.9, about 6.6 to about 6.8, from about 6.7 to about 7.5, from about 6.7 to about 7.4, from about 6.7 to about 7.3, from about 6,7 to about 7.2, from about 6.7 to about 7.1, from about 6.7 to about 7.0, from about 6.7 to about 6.9, from about 6.8 to about 7.5, from about 6.8 to about 7.4, from about 6.8 to about 7.3, from about 6.8 to about 7.2, from about 6.8 to about 7.1, from about 6.8 to about 7.0, from about 6.9 to about 7.5, from about 6.9 to about 7.4, from about 6.9 to about 7.3, from about 6.9 to about 7.2, from about 6.9 to about 7.1, from about 7.0 to about 7.5, from about 7.0 to about 7.4, from about 7.0 to about 7.3, from about 7.0 to about 7.2, from about 7.1 to about 7.5, from about 7.1 to about 7.4, from about 7.1 to about 7.3, from about 7,2 to about 7.5, from about 7.2 to about 7.4, or from about 7.3 to about 7.5.,

[0181] According to some aspects, the cells can be cultured at a temperature of from 32°C to about 39°C, from about 32°C to about 37°C, from about 32°C to about 37.5°C, from about 34°C to about 37°C, from about 35°C to about 37°C, from about 35.5°C to about 37.5°C, from about 36°C to about 37°C, or about 36.5°C. According to some aspects, the cells can be incubated at a temperature of about 37°C from the beginning to the end of the culture period. According to some aspects, the temperature can be changed or can vary slightly during the culture period, for example, hourly or daily.In some aspects, the temperature may change or shift (e.g., increase or decrease) approximately one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, fourteen days, or fifteen days after the start of the culturing period or at any time during the culturing period. In some aspects, the temperature may shift upward by approximately 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°C. In some aspects, the temperature may be shifted downward by approximately 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°C.

[0182] According to some aspects, cell culturing can be performed using an atmosphere containing from about 1% to about 15% CO2. According to some aspects, 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] According to some aspects, culturing the cells can be carried out while maintaining a dissolved oxygen (dO2) level in the cell culture of from about 3% to about 55%, from about 3% to about 50%, from about 3% to about 45%, from about 3% to about 40%, from about 3% to about 35%, from about 3% to about 30%, from about 3% to about 25%, from about 3% to about 20%, from about 3% to about 15%, from about 5% to about 55%, from about 5% to about 50%, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10%,from about 10% to about 55%, from about 10% to about 50%, from about 10% to about 45%, from about 10% to about 40%, from about 10% to about 35%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 55%, from about 15% to about 50%, from about 15% to about 45%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 15% to about 25%, from about 15% to about 20%, from about 20% to about 55%, from about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%,from about 20% to about 25%, from about 25% to about 55%, from about 25% to about 50%, from about 25% to about 45%, from about 25% to about 40%, from about 25% to about 35%, from about 25% to about 30%, from about 30% to about 55%, from about 30% to about 50%, from about 30% to about 45%, from about 30% to about 40%, from about 30% to about 35%, from about 35% to about 55%, from about 35% to about 50%, from about 35% to about 45%, from about 35% to about 40%, from about 40% to about 55%, from about 40% to about 50%, from about 40% to about 45%, from about 45% to about 55%, from about 45% to about 50%, or from about 50% to about 55%.

[0184] According to some aspects, the pH of cell culture can be maintained at a certain pH value by adding a basic solution such as an alkaline base solution. The pH of the cell culture can be maintained at a pH of from about 6.5 to about 7.5, from about 6.5 to about 7.4, from about 6.5 to about 7.3, from about 6.5 to about 7.2, from about 6.5 to about 7.1, from about 6.5 to about 7.0, from about 6.5 to about 6.9, from about 6.5 to about 6.8, from about 6.5 to about 6.7, from about 6.6 to about 7.5, from about 6.6 to about 7.4, from about 6.6 to about 7.3, from about 6.6 to about 7.2, from about 6.6 to about 7.1, from about 6.6 to about 7.0, from about 6.6 to about 6.9, from about 6,6 to about 6.8, from about 6.7 to about 7.5, from about 6.7 to about 7.4, from about 6.7 to about 7.3, from about 6.7 to about 7.2, from about 6.7 to about 7.1, from about 6.7 to about 7.0, from about 6.7 to about 6.9, from about 6.8 to about 7.5, from about 6.8 to about 7.4, from about 6.8 to about 7.3, from about 6.8 to about 7.2, from about 6.8 to about 7.1, from about 6.8 to about 7.0, from about 6.9 to about 7.5, from about 6.9 to about 7.4, from about 6.9 to about 7.3, from about 6.9 to about 7.2, from about 6.9 to about 7.1, from about 7.0 to about 7.5, from about 7.0 to about 7.4, from about 7.0 to about 7.3, from about 7,0 to about 7.2, from about 7.1 to about 7.5, from about 7.1 to about 7.4, from about 7.1 to about 7.3, from about 7.2 to about 7.5, from about 7.2 to about 7.4, or from about 7.3 to about 7.5.,

[0185] In some aspects, the cell culture in suspension conditions can be carried out in any cell culture vessel suitable for steady-state or agitated / shaken cell propagation in suspension conditions, using, for example, a T-flask, a spinner flask, a shake flask, or a combination thereof. In some aspects, container N-1 is a shake flask.

[0186] In some aspects, the suspension conditions may include some form of mixing. In some aspects, the mixing may comprise rotational mixing. According to some aspects, the stirring can be carried out at a frequency of from about 25 rpm to about 500 rpm, from about 25 rpm to about 480 rpm, from about 25 rpm to about 460 rpm, from about 25 rpm to about 440 rpm, from about 25 rpm to about 420 rpm, from about 25 rpm to about 400 rpm, from about 25 rpm to about 380 rpm, from about 25 rpm to about 360 rpm, from about 25 rpm to about 340 rpm, from about 25 rpm to about 320 rpm, from about 25 rpm to about 300 rpm, from about 25 rpm to about 280 rpm,from about 25 rpm to about 260 rpm, from about 25 rpm to about 240 rpm, from about 25 rpm to about 220 rpm, from about 25 rpm to about 200 rpm, from about 25 rpm to about 180 rpm, from about 25 rpm to about 160 rpm, from about 25 rpm to about 140 rpm, from about 25 rpm to about 120 rpm, from about 25 rpm to about 100 rpm, from about 25 rpm to about 80 rpm, from about 25 rpm to about 60 rpm, from about 25 rpm to about 40 rpm, from about 25 rpm to about 35 rpm, from about 25 rpm to about 30 rpm, from about 50 rpm to about 500 rpm, from about 50 rpm to about 480 rpm, from about 50 rpm to about 460 rpm,from about 50 rpm to about 440 rpm, from about 50 rpm to about 420 rpm, from about 50 rpm to about 400 rpm, from about 50 rpm to about 380 rpm, from about 50 rpm to about 360 rpm, from about 50 rpm to about 340 rpm, from about 50 rpm to about 320 rpm, from about 50 rpm to about 300 rpm, from about 50 rpm to about 280 rpm, from about 50 rpm to about 260 rpm, from about 50 rpm to about 240 rpm, from about 50 rpm to about 220 rpm, from about 50 rpm to about 200 rpm, from about 50 rpm to approximately 180 rpm, from approximately 50 rpm to approximately 160 rpm, from approximately 50 rpm to approximately 140 rpm, from approximately 50 rpm to approximately 120 rpm,from about 50 rpm to about 100 rpm, from about 50 rpm to about 80 rpm, from about 50 rpm to about 60 rpm, from about 75 rpm to about 500 rpm, from about 75 rpm to about 480 rpm, from about 75 rpm to about 460 rpm, from about 75 rpm to about 440 rpm, from about 75 rpm to about 420 rpm, from about 75 rpm to about 400 rpm, from about 75 rpm to about 380 rpm, from about 75 rpm to about 360 rpm, from about 75 rpm to about 340 rpm, from about 75 rpm to about 320 rpm, from about 75 rpm to about 300 rpm, from about 75 rpm to about 280 rpm, from about 75 rpm to about 260 rpm, from about 75 rpm to about 240 rpm,from about 75 rpm to about 220 rpm, from about 75 rpm to about 200 rpm, from about 75 rpm to about 180 rpm, from about 75 rpm to about 160 rpm, from about 75 rpm to about 140 rpm, from about 75 rpm to about 120 rpm, from about 75 rpm to about 100 rpm, from about 75 rpm to about 80 rpm, from about 100 rpm to about 500 rpm, from about 100 rpm to about 480 rpm, from about 100 rpm to about 460 rpm, from about 100 rpm to about 440 rpm, from about 100 rpm to about 420 rpm, from about 100 rpm to about 400 rpm, from about 100 rpm to about 380 rpm, from about 100 rpm to about 360 rpm, from about 100 rpm to about 340 rpm,from about 100 rpm to about 320 rpm, from about 100 rpm to about 300 rpm, from about 100 rpm to about 280 rpm, from about 100 rpm to about 260 rpm, from about 100 rpm to about 240 rpm, from about 100 rpm to about 220 rpm, from about 100 rpm to about 200 rpm, from about 100 rpm to about 180 rpm, from about 100 rpm to about 160 rpm, from about 100 rpm to about 140 rpm, from about 100 rpm to about 120 rpm, from about 150 rpm to about 500 rpm, from about 150 rpm to about 480 rpm, from about 150 rpm to about 460 rpm, from about 150 rpm to about 440 rpm, from about 150 rpm to about 420 rpm,from about 150 rpm to about 400 rpm, from about 150 rpm to about 380 rpm, from about 150 rpm to about 360 rpm, from about 150 rpm to about 340 rpm, from about 150 rpm to about 320 rpm, from about 150 rpm to about 300 rpm, from about 150 rpm to about 280 rpm, from about 150 rpm to about 260 rpm, from about 150 rpm to about 240 rpm, from about 150 rpm to about 220 rpm, from about 150 rpm to about 200 rpm, from about 150 rpm to about 180 rpm, from about 150 rpm to about 160 rpm, from about 200 rpm to about 500 rpm, from about 200 rpm to 480 rpm, from about 200 rpm to about 460 rpm, from about 200 rpm to about 440 rpm,from about 200 rpm to about 420 rpm, from about 200 rpm to about 400 rpm, from about 200 rpm to about 380 rpm, from about 200 rpm to about 360 rpm, from about 200 rpm to about 340 rpm, from about 200 rpm to about 320 rpm, from about 200 rpm to about 300 rpm, from about 200 rpm to about 280 rpm, from about 200 rpm to about 260 rpm, from about 200 rpm to about 240 rpm, from about 200 rpm to about 220 rpm, from about 240 rpm to about 500 rpm, from about 240 rpm to about 480 rpm, from about 240 rpm to about 460 rpm, from about 240 rpm to about 440 rpm, from about 240 rpm to about 420 rpm,from about 240 rpm to about 400 rpm, from about 240 rpm to about 380 rpm, from about 240 rpm to about 360 rpm, from about 240 rpm to about 340 rpm, from about 240 rpm to about 320 rpm, from about 240 rpm to about 300 rpm, from about 240 rpm to about 280 rpm, from about 240 rpm to about 260 rpm, from about 260 rpm to about 500 rpm, from about 260 rpm to about 480 rpm, from about 260 rpm to about 460 rpm, from about 260 rpm to about 440 rpm, from about 260 rpm to about 420 rpm, from about 260 rpm to about 400 rpm, from about 260 rpm to about 380 rpm, from about 260 rpm to about 360 rpm,from about 260 rpm to about 340 rpm, from about 260 rpm to about 320 rpm, from about 260 rpm to about 300 rpm, from about 260 rpm to about 280 rpm, from about 280 rpm to about 500 rpm, from about 280 rpm to about 480 rpm, from about 280 rpm to about 460 rpm, from about 280 rpm to about 440 rpm, from about 280 rpm to about 420 rpm, from about 280 rpm to about 400 rpm, from about 280 rpm to about 380 rpm, from about 280 rpm to about 360 rpm, from about 280 rpm to about 340 rpm, from about 280 rpm to about 320 rpm, from about 280 rpm to about 280 rpm, from about 300 rpm to about 500 rpm,from about 380 rpm to about 480 rpm, from about 380 rpm to about 460 rpm, from about 380 rpm to about 440 rpm, from about 380 rpm to about 420 rpm, from about 380 rpm to about 400 rpm, from about 400 rpm to about 500 rpm, from about 400 rpm to about 480 rpm, from about 400 rpm to about 460 rpm, from about 400 rpm to about 440 rpm, or from about 400 rpm to about 420 rpm. The stirring can be carried out continuously or intermittently.

[0187] According to some aspects, the cells are subcultured no more than two times under suspension conditions.

[0188] According to some aspects, the cells are cultured in suspension culture for about 24 to about 96 hours. According to some aspects, the cells are cultured in suspension culture for about 36 to about 84 hours. According to some aspects, the cells are cultured in suspension culture for about 48 to about 72 hours. According to some aspects, the cells are cultured in suspension culture for about 54 to about 66 hours. According to some aspects, 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] According to some aspects of the present invention, the cells are adherent cells. In some aspects, the adherent cells are HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, or COS cells. In some aspects, the adherent cell is a human cell. In some aspects, the adherent cell is a HeLa cell or a HEK-293 cell. In some aspects, the adherent cell is a HEK-293 cell.

[0190] In some aspects, the adherent cells are not adapted to suspension. In some aspects, culturing the cells in suspension does not alter the cell's substrate dependence. In some aspects, the method does not alter the cells to produce a new cell line. The methods disclosed herein do not alter the genomic or transcriptomic profile of the cells. The methods disclosed herein do not alter the phenotype of the cell.

[0191] According to some aspects, the cells are passaged several times under adherent conditions in a growth medium supplemented with serum before inoculating container N-1. According to some aspects, the cells are cultured in containers N-2, N-3, N-4, N-5, N-6, N-7, N-8, N-9, or N-10 before inoculating container N-1. According to some aspects, the cells are cultured in a container with N-3 and N-2. According to some aspects, the cells are cultured in containers N-4, N-3, and N-2.

[0192] In some aspects, the bioreactor is a bioreactor for growing adherent cultures. In some aspects, adherent cells are purified from the culture obtained in the bioreactor for growing adherent cultures.

[0193] According to some aspects, the bioreactor comprises at least one, more preferably a plurality of carriers, on which the expanded cells are attached, which can be either floating or fixed in the bioreactor. Preferably, said carriers can be obtained, for example, using polyethylene terephthalate, polystyrene, polyester, polypropylene, DEAE-dextran, collagen, glass, alginate or acrylamide. According to some aspects, the bioreactor can be a bioreactor containing granular microcarriers (e.g., beads under the trademark Cytodex®, commercially available from GE Healthcare Inc., a division of General Electric Corp.) or matrix-type carriers (e.g., discs under the trademark Fibra-Cell™, commercially available from Eppendorf Corp.).In some aspects, the bioreactor utilizes a polyester fibrous support, such as the support used in the iCELLis® nano or iCELLis® 500 bioreactors (commercially available from Advanced Technology Materials Inc. (Brussels, Belgium) and Pall corporation (Fall River, Mass)).

[0194] According to some aspects, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. According to some aspects, the at least one factor; promoting cell adhesion is selected from the group consisting of FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix; and combinations thereof. According to some aspects, said at least one factor promoting cell adhesion can be added to the third medium immediately before, during, or after inoculation of suspension cells into the bioreactor.

[0195] According to some aspects, the growth medium comprises DMEM and about 10% by weight of FBS. According to some aspects, the growth medium comprises from about 2% to about 20% by weight of FBS. According to some aspects, the growth medium comprises from about 3% to about 19% by weight of FBS. According to some aspects, the growth medium comprises from about 4% to about 18% by weight of FBS. According to some aspects, the growth medium comprises from about 5% to about 17% by weight of FBS. According to some aspects, the growth medium comprises from about 6% to about 16% by weight of FBS. According to some aspects, the growth medium comprises from about 7% to about 15% by weight of FBS. According to some aspects, the growth medium comprises from about 8% to about 14% by weight of FBS.According to some aspects, the growth medium comprises from about 9% to about 13% by weight of FBS. According to some aspects, the growth medium comprises from about 10% to about 12% by weight of FBS. According to some aspects, 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% by weight of FBS.

[0196] In some aspects, the suspension-propagated cells from step (d) can be inoculated directly into the bioreactor. In some aspects, the amount of cells inoculated into the bioreactor varies depending on the size of the bioreactor. In some aspects, a 4 m bioreactor is used. 2 (e.g. iCELLis® nano bioreactor). According to some aspects, a 4 m2 bioreactor 2 inoculate from approximately 1×10 8 up to 1×10 9 cells. According to some aspects, a 4 m2 bioreactor 2 inoculate from approximately 3×10 8 up to 7×10 8 cells. According to some aspects, a 4 m2 bioreactor 2 inoculate from approximately 4×10 8 up to 6×10 8 cells. According to some aspects, a 4 m2 bioreactor 2 inoculate approximately 5×10 8cells. In some respects, equivalent cell densities are used for bioreactors of other sizes.

[0197] According to some aspects, the method of the present invention may further comprise culturing cells in a bioreactor. According to some aspects, culturing the cells comprises batch culturing. According to some aspects, culturing the cells comprises fed-batch culturing. According to some aspects, culturing the cells comprises perfusion culturing.

[0198] Fed-batch culturing involves gradually (batch) or continuously adding a nutrient culture medium to a starting cell culture without substantially or significantly removing the growth medium from the cell culture. The cell culture in fed-batch culturing may be placed in a bioreactor (e.g., a production bioreactor, such as a 10,000-L production bioreactor). In some aspects, the nutrient culture medium may be the same as the growth medium. The nutrient culture medium may be in liquid form or as a dry powder. In some aspects, the nutrient culture medium is a concentrated form of the growth medium and / or is added as a dry powder.In some aspects, a first liquid nutrient culture medium and a different second liquid nutrient culture medium can be added (e.g., continuously added) to the growth medium. In some aspects, the addition of the first liquid nutrient culture medium and the addition of the second liquid nutrient culture medium to the culture can begin approximately simultaneously. In some aspects, the total volume of the first liquid nutrient culture medium and the second liquid nutrient culture medium added to the culture over the entire culturing period can be approximately the same.

[0199] When continuously adding the nutrient culture medium, the rate of addition of the nutrient culture medium may be maintained constant or may increase (e.g., uniformly increase) during the culturing period. Continuous addition of the nutrient culture medium may be started at a specific time during the culturing period (e.g., when the cells reach the target viable cell density, e.g., a viable cell density of approximately 1×10 6 cells / ml, approximately 1.1×10 6 cells / ml, approximately 1.2×10 6 cells / ml, approximately 1.3×10 6 cells / ml, approximately 1.4×10 6 cells / ml, approximately 1.5×10 6 cells / ml, approximately 1.6×10 6 cells / ml, approximately 1.7×10 6 cells / ml, approximately 1.8×10 6 cells / ml, approximately 1.9×10 6 cells / ml or approximately 2.0×10 6cells / ml). In some aspects, continuous addition of nutrient culture medium may be initiated on day 2, day 3, day 4, or day 5 of the culture period.

[0200] In some aspects, the gradual (periodic) addition of nutrient culture medium may begin when the cells reach a target cell density (e.g., approximately 1×10 6 cells / ml, approximately 1.1×10 6 cells / ml, approximately 1.2×10 6 cells / ml, approximately 1.3×10 6 cells / ml, approximately 1.4×10 6 cells / ml, approximately 1.5×10 6 cells / ml, approximately 1.6×10 6 cells / ml, approximately 1.7×10 6 cells / ml, approximately 1.8×10 6 cells / ml, approximately 1.9×10 6 or approximately 2.0×10 6cells / ml). In some aspects, the gradual addition of the fed-batch culture medium may be performed at regular intervals (e.g., every day, every second day, or every third day) or may be performed upon reaching a certain target cell density (e.g., a target cell density that increases over the culturing period). In some aspects, the amount of added nutrient culture medium may be gradually increased between the first gradual addition of nutrient culture medium and subsequent additions of nutrient culture medium. In some aspects, the volume of liquid nutrient culture medium added to the initial cell culture during any 24-hour culturing period may be a certain fraction of the initial volume of the bioreactor containing the culture or a certain fraction of the volume of the initial culture.

[0201] According to some aspects, the addition of the liquid nutrient culture medium (continuously or intermittently) may occur at a time that is from 6 hours to about 7 days, from about 6 hours to about 6 days, from about 6 hours to about 5 days, from about 6 hours to about 4 days, from about 6 hours to about 3 days, from about 6 hours to about 2 days, from about 6 hours to about 1 day, from about 12 hours to about 7 days, from about 12 hours to about 6 days, from about 12 hours to about 5 days, from about 12 hours to about 4 days, from about 12 hours to about 3 days, from about 12 hours to about 2 days, from about 1 day to about 7 days, from about 1 day to about 6 days, from about 1 day to about 5 days,from about 1 day to about 4 days, from about 1 day to about 3 days, from about 1 day to about 2 days, from about 2 days to about 7 days, from about 2 days to about 6 days, from about 2 days to about 5 days, from about 2 days to about 4 days, from about 2 days to about 3 days, from about 3 days to about 7 days, from about 3 days to about 6 days, from about 3 days to about 5 days, from about 3 days to about 4 days, from about 4 days to about 7 days, from about 4 days to about 6 days, from about 4 days to about 5 days, from about 5 days to about 7 days, or from about 5 days to about 6 days after the start of the culturing period.

[0202] In some aspects, the volume of liquid nutrient culture medium added (continuously or intermittently) to the initial cell culture during any 24-hour period may be from 0.01× to about 0.3× the capacity of the bioreactor. The specified portion may be from about 0.01× to about 0.28×, from about 0.01× to about 0.26×, from about 0.01× to about 0.24×, from about 0.01× to about 0.22×, from about 0.01× to about 0.20×, from about 0.01× to about 0.18×, from about 0.01× to about 0.16×, from about 0.01× to about 0.14×, from about 0.01× to about 0.12×, from about 0.01× to about 0.10×, from about 0.01× to about 0.08×, from about 0.01× to about 0.06×, from about 0.01× to approximately 0.04×, from approximately 0.02× to approximately 0.3×, from approximately 0,02× to about 0.28×, from about 0.02× to about 0.26×, from about 0.02× to about 0.24×, from about 0.02× to about 0.22×, from about 0.02× to about 0.20×, from about 0.02× to about 0.18×, from about 0.02× to about 0.16×, from about 0.02× to about 0.14×, from about 0.02× to about 0.12×, from about 0.02× to about 0.10×, from about 0.02× to about 0.08×, from about 0.02× to about 0.06×, from about 0.02× to about 0.05×, from about 0.02× to about 0.04×, from about 0.02× to about 0.03×, from about 0.025× to about 0.3×, from about 0.025× to about 0.28×, from about 0.025× to about 0.26×, from about 0.025× to about 0.24×, from about 0.025× to about 0.22×, from about 0,025× to about 0.20×, from about 0.025× to about 0.18×, from about 0.025× to about 0.16×, from about 0.025× to about 0.14×, from about 0.025× to about 0.12×, from about 0.025× to about 0.10×, from about 0.025× to about 0.08×, from about 0.025× to about 0.06×, from about 0.025× to about 0.04×, from about 0.05× to about 0.3×, from about 0.05× to about 0.28×, from about 0.05× to about 0.26×, from about 0.05× to about 0.24×, from about 0.05× to about 0.22×, from about 0.05× to about 0.20×, from about 0.05× to about 0.18×, from about 0.05× to about 0.16×, from about 0.05× to about 0.14×, from about 0.05× to about 0.12×, from about 0.05× to about 0.10×, from about 0,1× to about 0.3×, from about 0.1× to about 0.28×, from about 0.1× to about 0.26×, from about 0.1× to about 0.24×, from about 0.1× to about 0.22×, from about 0.1× to about 0.20×, from about 0.1× to about 0.18×, from about 0.1× to about 0.16×, from about 0.1× to about 0.14×, from about 0.1×, from about 0.15× to about 0.3×, from about 0.15× to about 0.2×, from about 0.2× to about 0.3×, or from about 0.25× to about 0.3× of the capacity bioreactor,

[0203] According to some aspects, the volume of liquid nutrient culture medium added (continuously or intermittently) to the original cell culture during any 24-hour period during the culturing period may be from 0.02× to about 1.0×, from about 0.02× to about 0.9×, from about 0.02× to about 0.8×, from about 0.02× to about 0.7×, from about 0.02× to about 0.6×, from about 0.02× to about 0.5×, from about 0.02× to about 0.4×, from about 0.02× to about 0.3×, from about 0.02× to about 0.2×, from about 0.02× to about 0.1×, from about 0.02× to about 0.08×, from about 0.02× to about 0.06×, from about 0.02× to about 0.05×, from about 0.02× to about 0.04×, from about 0.02× to about 0.03×, from about 0.05× to about 1.0×,from about 0.05× to about 0.8×, from about 0.05× to about 0.7×, from about 0.05× to about 0.6×, from about 0.05× to about 0.5×, from about 0.05× to about 0.4×, from about 0.05× to about 0.3×, from about 0.05× to about 0.2×, from about 0.05× to about 0.1×, from about 0.1× to about 1.0×, from about 0.1× to about 0.9×, from about 0.1× to about 0.8×, from about 0.1 to about 0.7×, from about 0.1× to about 0.6×, from about 0.1× to about 0.5×, from about 0.1× to about 0.4×, from about 0.1× to about 0.3×, from about 0.1× to about 0.2×, from about 0.2× to about 1.0×, from about 0.2× to about 0.9×, from about 0.2× to about 0.8×, from about 0.2× to about 0.7×,from about 0.2× to about 0.6×, from about 0.2× to about 0.5×, or from about 0.2× to about 0.4× the volume of the original cell culture.

[0204] According to some aspects, the total amount of culture medium added (continuously or intermittently) during the entire culturing period may be from about 1% to about 40% (e.g., from about 1% to about 35%, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, from about 1% to about 5%, from about 1% to about 4%, from about 2% to about 40%, from about 2% to about 35%, from about 2% to about 30%, from about 2% to about 25%, from about 2% to about 20%, from about 2% to about 15%, from about 2% to about 10%, from about 2% to about 5%, from approximately 3% to approximately 40%,from about 3% to about 35%, from about 3% to about 30%, from about 3% to about 25%, from about 3% to about 20%, from about 3% to about 15%, from about 3% to about 10%, from about 3% to about 5%, from about 4% to about 40%, from about 4% to about 35%, from about 4% to about 30%, from about 4% to about 25%, from about 4% to about 20%, from about 4% to about 15%, from about 4% to about 10%, from about 4% to about 8%, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% up to approximately 15%, from approximately 5% to approximately 10%,from about 10% to about 40%, from about 10% to about 35%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 10% to about 15%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 15% to about 25%, from about 15% to about 20%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, from about 20% to about 25%, from about 25% to about 40%, from about 25% to about 35%, from about 25% to about 30%, from about 30% to about 40%, from about 30% to about 35%, or from about 35% to about 40% of the volume of the original culture.

[0205] According to some aspects, two different nutrient culture media are added (continuously or gradually) during fed-batch culturing. According to some aspects, the amount or volume of the first nutrient culture medium and the second nutrient culture medium may be substantially the same or may be different. According to some aspects, the first nutrient culture medium may be in liquid form, and the second nutrient culture medium may be in solid form. According to some aspects, the first nutrient culture medium and the second nutrient culture medium may be liquid culture media.

[0206] Perfusion culturing comprises removing a first volume of growth medium from a bioreactor and adding a second volume of a second growth medium to a production bioreactor, wherein said first volume and said second volume are approximately equal. Cells are retained in the bioreactor using a cell retention device or by techniques such as settling the cells in a settling cone. In some aspects, the removal and addition of growth medium may be performed simultaneously or sequentially, or in some combination thereof. In some aspects, the removal and addition can be performed continuously, such as at a rate that allows for the removal and replacement of 0.1% to 800%, 1% to 700%, 1% to 600%, 1% to 500%, 1% to 400%, 1% to 350%, 1% to 300%, 1% to 250%, 1% to 100%, 100% to 200%, 5% to 150%, 10% to 50%, 15% to 40%, 8% to 80%, or 4% to 30% of the bioreactor capacity.

[0207] According to some aspects, the first volume of the removed first growth medium and the second volume of the added second growth medium can be maintained at approximately the same level during each 24-hour period. According to some aspects, 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 vary and depend on the conditions of a specific cell culture system. According to some aspects, 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] According to some aspects, the removed and added volume may be changed by gradually increasing during each 24-hour period. According to some aspects, the volume of the first growth medium removed and the volume of the second growth medium added during each 24-hour period may increase during the culturing period. According to some aspects, the volume added may be from 0.5% to about 20% of the bioreactor capacity during a 24-hour period for the increase. According to some aspects, the volume may be increased during the culturing period to a volume of from about 25% to about 150% of the bioreactor capacity or the volume of the first liquid culture medium during a 24-hour period.

[0209] According to some aspects, after the first 48-96 hours of the culturing period, in each 24-hour period, the first volume of the first growth medium is removed and a second volume of the second growth medium is added, which is from about 10% to about 95%, from about 10% to about 20%, from about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, from about 85% to about 95%, from about 60% to about 80% or about 70% of the volume of the first growth medium.

[0210] In some aspects, the first growth medium and the second growth medium may be the same type of medium. In some aspects, the first growth medium and the second growth medium may be different. In some aspects, the second liquid culture medium may be more concentrated with respect to one or more components of the medium.

[0211] According to some aspects, the first volume of the first growth medium can be removed using any automated system. According to some aspects, alternating tangential flow filtration can be used. According to some aspects, the first volume of the first growth medium can be removed by filtering or gravity flowing the first volume of the first growth medium through a sterile cell-screening molecular weight cutoff membrane. According to some aspects, the first volume of the first growth medium can be removed by stopping or significantly reducing the stirring speed for a period of at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour, and removing or aspirating the first volume of the growth medium from the upper part of the production bioreactor.

[0212] According to some aspects, a second volume of the second liquid culture medium can be added to the first liquid culture medium using a pump. According to some aspects, the second liquid culture medium can be added to the first liquid culture medium manually, for example, by pipetting or introducing the second volume of the second liquid culture medium directly into the first liquid culture medium, or by an automated method.

[0213] According to some aspects, the method further comprises contacting the cells with a first polynucleotide sequence. According to some aspects, the method further comprises transfecting the cells with the polynucleotide sequence. According to some aspects, the polynucleotide sequence is a plasmid. According to some aspects, the plasmid encodes a capsid of a recombinant viral particle selected from the group consisting of AAV, lentivirus, herpes virus, polyoma virus, and vaccinia virus. According to some aspects, the cells are transfected before inoculation into the bioreactor. According to some aspects, the cells are transfected after inoculation into the bioreactor. According to some aspects, the cells are contacted or transfected with a second polynucleotide and comprise a nucleic acid encoding a transgene. According to some aspects, the cells are cultured under conditions allowing production of the viral vector.According to some aspects, the method further includes isolating the resulting viral vector.

[0214] In some aspects, the polynucleotide is a viral vector. In some aspects, the viral vector is an adenovirus or adeno-associated virus (AAV) vector. These vectors infect a wide variety of dividing and non-dividing cell types, including synovial cells and liver cells. The episomal nature of adenovirus and AAV vectors after entry into the cell makes these vectors suitable for therapeutic use (Russell, J. Gen. Virol. 81: 2573-2604 (2000)); Goncalves, Virol J. 2(1):43 2005)), as noted above. AAV vectors can result in very stable long-term transgene expression (up to 9 years in dogs (Niemeyer et al., Blood 113(4):797–806 (2009)) and up to 2 years in humans (Nathwani et al., N Engl J Med. 365(25): 2357–2365 (2011); Simonelli et al., Mol Ther. l8(3):643–50 (2010), Epub 2009 Dec. 1)).In some aspects, adenoviral vectors are modified to reduce the host response, as reviewed by Russell (2000, see above). Gene therapy approaches using AAV vectors are described by Wang et al., 2005, J Gene Med. 9 Mar (Epub ahead of print); Mandel et al., Curr Opin Mol Ther. 6:482–90 (2004); Martin et al., Eye 78:1049–55 (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] According to some aspects, the first polynucleotide sequence comprises one or more of a reverse terminal repeat, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one protein involved in AAV packaging, 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 that allow the production of a recombinant viral particle. In some aspects, the method further comprises isolating the resulting recombinant viral particle.

[0217] In some aspects, the viral vector comprises a transgene operably linked to appropriate regulatory sequences. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control protein transcription or translation. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). In some aspects, the regulatory sequence may comprise a promoter sequence. In some aspects, the promoter sequence may be the cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat (LTR) promoters such as the Moloney murine leukemia virus (MMLV), Rous sarcoma virus, or HTLV-1 promoters, the simian virus 40 (SV 40) early promoter, or the herpes simplex virus thymidine kinase promoter.

[0218] According to some aspects, the viral vector includes an additional nucleotide sequence encoding an additional polypeptide. According to some aspects, the additional polypeptide may be a (selectable) marker polypeptide that enables identification, selection, and / or screening of cells containing the viral vector. According to some aspects, the marker polypeptide may be a GFP fluorescent protein and selectable marker genes of HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), aminoglycoside phosphotransferase Tn5 (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, low-affinity nerve growth factor gene. Sources for obtaining the indicated marker genes and methods for their use are suggested in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3 еedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. Method for producing viral vectors (e.g., AAV)

[0219] Certain aspects of the present invention relate to a method for producing a viral vector (e.g., rAAV, as described herein) comprising propagating cells according to any of the propagation methods in a seed bioreactor system described herein, inoculating a growth medium into a bioreactor containing cells, transfecting the cells with a polynucleotide sequence encoding a viral particle, and culturing the cells in the bioreactor under conditions permitting production of the viral particle. In certain aspects, the production of viral vectors is disclosed in U.S. Patent Application Ser. No. 63 / 123,602, which is expressly incorporated herein by reference.

[0220] Methods for introducing exogenous nucleic acid into host cells are well known in the art and vary depending on the host cell used. Techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polyethyleneimine-mediated transfection, polybrene-mediated transfection, protoplast fusion, electroporation, viral or phage infection, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into nuclei. Transfection can be transient or stable.

[0221] In some aspects, the polynucleotide sequence is a plasmid. In some aspects, the plasmid encodes a viral particle from AAV.

[0222] In some aspects, the polynucleotide sequence is a viral vector. In some aspects, the viral vector encodes a viral particle. In preferred aspects, the viral particle is derived from AAV. In some aspects, the rAAV comprises the nucleic acid sequence of SEQ ID NO: 9. In some aspects, the present invention provides rAAV particles comprising the nucleic acid sequence of SEQ ID NO: 9. In some aspects, the present invention relates to rAAV comprising nucleotides 55-5021 of SEQ ID NO: 3. In some aspects, the present invention relates to rAAV particles comprising nucleotides 55-5021 of SEQ ID NO: 3. In some aspects, the rAAV comprises nucleotides 1-4988 of SEQ ID NO: 8. In some aspects, the present invention relates to rAAV particles comprising nucleotides 1-4977 of SEQ ID NO: 8.

[0223] In some aspects, the viral vector is an AAV vector. In some aspects, the AAV vector may comprise a recombinant AAV vector (rAAV). As used herein, an "rAAV vector" refers to a recombinant vector comprising a portion of an AAV genome encapsidated within a protein shell of a capsid protein derived from an AAV serotype described herein. In some aspects, the AAV vector may comprise inverted terminal repeats (ITRs) derived from an adeno-associated virus serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVRH10, AAV11, AAV12, and others.

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

[0225] The complete genome of several AAV serotypes and the corresponding ITRs have been previously sequenced (Chiorini et al. J. of Virology 73: 1309-1319 (1999)). They can be cloned or produced by chemical synthesis as known in the art, using, for example, an oligonucleotide synthesizer available from, for example, Applied Biosystems Inc. (Fosters, Calif., USA) or using standard molecular biology techniques. ITRs can be cloned from the AAV virus genome or excised from a vector containing AAV ITRs. The ITR nucleotide sequences can either be ligated at either end of a nucleotide sequence encoding one or more therapeutic proteins using standard molecular biology techniques, or the wild-type AAV sequence between the ITR repeats can be replaced with the desired nucleotide sequence.

[0226] According to some aspects, the viral capsid component of the packaged viral vectors may be a parvovirus capsid, such as AAV Cap and / or chimeric capsids. Examples of suitable parvovirus capsid components include capsid components from the Parvoviridae family, such as autonomous parvovirus or dependovirus. For example, the viral capsid may be an AAV capsid (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8 AAV 9, AAV10, AAVRH10, AAV11 or AAV12 capsid; one skilled in the art will appreciate that there are likely other variants, not yet identified, that perform the same or similar function), or may include components from two or more AAV capsids. The full complement of AAV Cap proteins includes VP1, VP2 and VP3.An ORF containing nucleotide sequences encoding AAV VP capsid proteins may contain less than the full complement of AAV Cap proteins or may provide the full complement of AAV Cap proteins.

[0227] According to some aspects, one or more of the AAV Cap proteins may be a chimeric protein, including AAV Cap amino acid sequences from two or more viruses, preferably two or more AAVs. For example, the capsid of a chimeric virus may include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. According to some aspects, the rAAV genome present in the rAAV vector does not contain any nucleotide sequences encoding viral proteins, such as the AAV rep (replication) or cap (capsid) genes. According to some aspects, the rAAV genome may further comprise a marker or reporter gene, such as a gene encoding, for example, an antibiotic resistance gene, a fluorescent protein (e.g., gfp), or a gene encoding a chemically, enzymatically, or otherwise detectable and / or selectable product (e.g., lacZ, aph, etc.), known in the art.

[0228] According to some aspects, the rAAV genome present in said rAAV vector may further comprise a promoter sequence operably linked to a nucleotide sequence encoding a transgene.

[0229] According to some aspects, a suitable 3'-untranslated sequence can also be operably linked to modified nucleic acid sequences encoding a transgene. Suitable 3'-untranslated regions can be regions naturally associated with the nucleotide sequence or can be obtained from various genes, such as, for example, the 3'-untranslated region of bovine growth hormone (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal, and enhancer sequence).

[0230] Unless otherwise indicated, methods known to those skilled in the art can be used to produce recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvovirus Rep and / or Cap sequences, and transiently and stably transactivated packaging cells. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed. (Cold Spring Harbor, NY, 1989); AUSUBEL et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

[0231] According to some aspects, a suitable 3'-untranslated sequence can also be operably linked to nucleic acid sequences encoding the transgene. Suitable 3'-untranslated regions can be regions naturally associated with the nucleotide sequence or can be obtained from various genes, such as, for example, the 3'-untranslated region of bovine growth hormone (e.g., the bGH polyadenylation signal, the SV40 polyadenylation signal, the SV40 polyadenylation signal, and the enhancer sequence).

[0232] According to some ampects, additional nucleotide sequences may 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 to produce lentiviral constructs, vectors, and transiently and stably transactivated packaging cells. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd 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] According to some aspects, the methods of the present invention include transfecting cells with a transgenic plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct, a plasmid comprising the AAV rep gene and the AAV cap gene, and an adenoviral helper plasmid. According to some aspects, the transgenic plasmid comprising the rAAVrh74.MHCK7.microdystrophin construct comprises: the nucleic acid sequence presented in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, or nucleotides 1-4977 of SEQ ID NO: 8. According to some aspects, the plasmid comprising the AAV rep gene and the AAV cap gene comprises the AAV2 rep gene and the rAAVrh74 cap gene. According to some aspects, the adenovirus helper plasmid contains the adenovirus 5 E2A gene, E4ORF6 and VA-RNA.

[0235] According to some aspects, the method further comprises isolating the resulting viral particle. The viral vector replicates within the cell, thereby amplifying and producing viral particles. Viral infection results in lysis of the transfected cells. Thus, the lytic characteristics of viral vectors, such as AAV, allow for two distinct pathways for the production and isolation of viral particles. The first pathway involves collecting viral particles prior to cell lysis using external factors to lyse the cells. The second pathway involves collecting viral particles from the supernatant after nearly complete cell lysis by the resulting virus.

[0236] Methods that can be used for active cell lysis are known to those skilled in the art. In some aspects, cells can be lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, ultrasonic disruption, high-pressure extrusion, detergent lysis, combinations of the above methods, and the like.

[0237] In some aspects, cells can be lysed using at least one detergent. In some aspects, the detergent can include anionic, cationic, zwitterionic, and nonionic detergents. In some aspects, the concentration of the detergent can be approximately 0.1%-5% (w / w). In some aspects, the detergent can be Triton X-100.

[0238] In some aspects, a nuclease can be used to remove contaminating nucleic acids, i.e., native nucleic acids, from transfected cells. In some aspects, the nuclease can be BENZONASE®, PULMOZYME®, or any other DNase and / or RNase commonly used in the art.

[0239] Methods for collecting or isolating viral vectors from transfected cells are described in detail in document WO 2005 / 080556, which is incorporated herein by reference in its entirety.

[0240] According to some aspects, the time of collection or isolation of the viral vector is from about 24 to 120 hours after transfection, from about 36 to 108 hours, from about 48 to about 96 hours after transfection, from about 60 to about 84 hours after transfection. According to some aspects, the time of collection or isolation of the vector is about 72 hours after transfection.

[0241] In some aspects, the isolated viral particle may be further purified. In some aspects, purification of viral particles may be carried out in multiple steps, including clarification, ultrafiltration, diafiltration, or separation by chromatography. Such methods have been described in WO 2005 / 080556, the entire contents of which are incorporated herein by reference. In some aspects, purification may be accomplished by a filtration step, removing cellular debris and other impurities from the cell lysate. In some aspects, ultrafiltration is used to concentrate the virus solution. In some aspects, diafiltration, buffer exchange, or ultrafiltration may be used to remove and exchange salts, sugars, etc. One skilled in the art will know how to find optimal conditions for each purification step.

[0242] In some aspects, purification can be achieved by density gradient centrifugation. In some aspects, the purification process employs at least one chromatography step. In some aspects, the viral vector can be purified using anion exchange chromatography, size exclusion chromatography, or a combination thereof. Methods for treating muscular dystrophy (e.g., DMD)

[0243] The present invention provides methods for treating muscular dystrophy in a human subject in need thereof, comprising the step of administering a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the rAAV is administered systemically at a dose of about 5.0×10 12 vg / kg to approximately 1.0×10 15vg / kg, and wherein rAAV is produced in adherent mammalian cells, and wherein the adherent cells are cultured in an N-1 container under suspension conditions. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0244] The present invention also provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering a composition comprising rAAV described herein to said human subject. In some aspects, the rAAV is administered via a systemic route and at a dose of from about 5.0×10 12 vg / kg to approximately 1.0×10 15 vg / kg. In some aspects, the systemic route of administration is the intravenous route, and the dose of rAAV administered is approximately 2×1014 vg / kg.

[0245] In some aspects, the rAAV dose is administered at a concentration of approximately 10 mL / kg. In some aspects, the rAAV is administered by injection, infusion, or implantation. In some aspects, the rAAV is administered by infusion over approximately one hour. In some aspects, the rAAV is administered intravenously through a peripheral vein of the extremity.

[0246] In some aspects, muscular dystrophy is either Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, muscular dystrophy is Duchenne muscular dystrophy.

[0247] In some aspects, the level of expression of the microdystrophin gene in a cell of the subject is increased after administration of rAAV compared to the level of expression of the microdystrophin gene before administration of rAAV. In some aspects, expression of the microdystrophin gene in the cell is detected by measuring the level of microdystrophin protein using Western blotting in a muscle biopsy before and after administration of rAAV. In some aspects, the expression is at least 55.4% after administration of rAAV compared to the expression before administration.

[0248] According to some aspects, the average percentage of microdystrophin-positive fibers in the subject's muscle tissue increases after rAAV administration compared to the number of microdystrophin-positive fibers before rAAV administration. According to some aspects, the average percentage of microdystrophin-positive fibers is at least 70.5%, and the average intensity is at least 116.9%, as determined by immunofluorescence (IF) in muscle biopsies before and after rAAV administration. According to some aspects, microdystrophin transduction by the number of vector genomes averages at least 3.87 vector genome copies per nucleus.

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

[0250] In some aspects, the method of treating muscular dystrophy further comprises genotyping the DMD gene of a human subject prior to administering the composition to said human subject. In some aspects, the genotyping detects at least one mutation in exons 18-79 of the DMD gene. In some aspects, the at least one mutation is a frameshift deletion, a frameshift duplication, a premature stop, or another pathogenic variant resulting in the absence of expression of the human dystrophin protein.

[0251] In some aspects, detection of a frameshift deletion, frameshift duplication, premature stop, or other pathogenic variant that results in lack of expression of human dystrophin protein means that the subject can be administered the compositions described herein.

[0252] According to one aspect, at least one mutation in the DMD gene is a mutation in exons 1-17, an in-frame deletion, an in-frame duplication, a variant of undetermined significance (VUS), or a mutation contained entirely in exon 45, which means that the subject is not suitable for administration of the compositions described herein.

[0253] The present invention also provides the use of the composition described herein for the treatment of muscular dystrophy in a human subject in need thereof. According to some aspects of the present invention, the present disclosure also provides the use of the composition described herein for the preparation of a medicament for the treatment of muscular dystrophy.

[0254] In some aspects, muscular dystrophy is either Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, muscular dystrophy is Duchenne muscular dystrophy.

[0255] For example, the administered dose of rAAV is approximately 5.0×10 12 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg up to 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 5.0×1012 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 5.0×10 13 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 2.0×10 13 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 1.0×10 13 vg / kg, or from 1.0×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg up to 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 5.0×10 13 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 5.0×1014 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or from 1.0×10 13 vg / kg to approximately 1.0×10 15 vg / kg, or from 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg up to 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 5.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or from 5.0×10 13 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 14 vg / kg up to approximately 6.0x10 14 vg / kg, or from 1.0x10 14 vg / kg to approximately 5.0×1014 vg / kg, or from 1.0×10 14 vg / kg to approximately 4.0×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.0×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 5.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 4.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 1.0×10 15 , or from approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.0×10 14vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from 1.5×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from approximately 1.5×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.5×10 14 vg / kg up to 5.0×10 14 , or from approximately 1.5×10 14 vg / kg up to 4.0×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from approximately 1.5×10 14vg / kg to approximately 3.25×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from 1.75×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or approximately 1.75×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.75×10 14 vg / kg up to 5.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg up to 4.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.25×10 14vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.25×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg up to 1.0×10 15 vg / kg, or from approximately 2.0×10 14 vg / kg up to 6.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg up to 5.0×10 14 , or from approximately 2.0×10 14 vg / kg to approximately 4.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from approximately 2.0×1014 vg / kg to approximately 3.25×10 14 vg / kg.

[0256] According to some aspects, the age of the human subject is from about 4 to less than 8 years old. According to some aspects, the age of the human subject is about 4, about 4.25, about 4.5, about 4.75, about 5, about 5.25, about 5.5, about 5.75, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, about 7.5, or about 7.75 years old.

[0257] According to some aspects, the age of the subject representing a human is from approximately 8 to less than 18 years old.In some aspects, the human subject is about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.5, about 9.75, about 10, about 10.25, about 10.5, about 10.75, about 11, about 11.25, about 11.5, about 11.75, about 12, about 12.25, about 12.5, about 12.75, about 13, about 13.25, about 13.5, about 13.75, about 14, about 14.5, about 14.75, about 15, about 15.25, about 15.5, about 15.75, about 16, about 16.25, about 16.5, about 16.75, approximately 17, approximately 17.25, approximately 17.5, or approximately 17.75 years.

[0258] According to one aspect, the methods of the present invention comprise systemic administration of rAAV, wherein the systemic route of administration is the intravenous route, and the dose of rAAV administered is approximately 2.0×10 14 vg / kg. According to another aspect, the methods of the present invention comprise systemic administration of rAAV, wherein said systemic route of administration is intravenous, and the dose of rAAV administered is approximately 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 13vg / 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 14vg / kg, or approximately 5.0×10 14 vg / kg, or approximately 6.0×10 14 vg / kg, or approximately 1×10 15 vg / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin of the sequence SEQ ID NO: 9, comprises nucleotides 55-5021 of the sequence SEQ ID NO: 3, nucleotides 1-4977 of the sequence SEQ ID NO: 8, or nucleotides 56-5022 of the sequence SEQ ID NO: 6. According to one aspect, the rAAV is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of the sequence SEQ ID NO: 5.

[0259] In any of the methods according to the present invention, the dose of rAAV can be administered in an amount of from about 5 ml / kg to about 15 ml / kg, or from about 8 ml / kg to about 12 ml / kg, or from 8 ml / kg to about 10 ml / kg, or from 5 ml / kg to about 10 ml / kg, or from about 10 ml / kg to 12 ml / kg, or from about 10 ml / kg to 15 ml / kg, or from 10 ml / kg to about 20 ml / kg. According to a particular aspect, the dose is or the rAAV is administered at a dose of about 10 ml / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin with the sequence of SEQ ID NO: 9, comprises nucleotides 55-5021 of the sequence of SEQ ID NO: 3, nucleotides 1-4977 of the sequence of SEQ ID NO: 8, or nucleotides 56-5022 of the sequence of SEQ ID NO: 6. According to one aspect, the rAAV is AAVrh74.MCK.microdystrophin, comprising nucleotides 56-4820 of the sequence SEQ ID NO: 5.

[0260] In any of the methods according to the present invention, the rAAV dose can be administered by injection, infusion, or implantation. For example, the rAAV dose is administered by infusion over approximately one hour. In addition, the rAAV dose is administered intravenously through a peripheral vein of an extremity, such as a peripheral vein of the arm or a peripheral vein of the leg. Alternatively, the infusion can be administered over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours, or approximately 3 hours. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin with the sequence of SEQ ID NO: 9, comprises nucleotides 55-5021 of the sequence of SEQ ID NO: 3, nucleotides 1-4977 of the sequence of SEQ ID NO: 8, or nucleotides 56-5022 of the sequence of SEQ ID NO: 6. In one aspect, rAAV is AAVrh74.MCK.microdystrophin. In one aspect, AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0261] The rAAV administered by any of the methods of the present invention may comprise the human microdystrophin nucleotide sequence presented in SEQ ID NO: 1, the MHCK7 promoter sequence presented in SEQ ID NO: 2 or SEQ ID NO: 7. In addition, the rAAV administered by any of the methods of the present invention comprises the human microdystrophin nucleotide sequence presented in SEQ ID NO: 1 and the MHCK7 promoter sequence presented in SEQ ID NO: 2 or SEQ ID NO: 7. For example, the rAAV may comprise the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct as set forth in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin with the sequence SEQ ID NO: 9, contains nucleotides 55-5021 of the sequence SEQ ID NO: 3, nucleotides 1-4977 of the sequence SEQ ID NO: 8 or nucleotides 56-5022 of the sequence SEQ ID NO: 6.

[0262] In one aspect, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0263] In any of the methods according to the present invention, the rAAV administered has the serotype AAVrh7.4.

[0264] According to some aspects, the methods of the present invention are for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. An exemplary aspect is a method of treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, comprising the step of administering a dose of a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the mode of administration is intravenous infusion, and the dose of rAAV administered is approximately 2×10 14vg / kg for about one hour, and wherein the rAAV vector comprises the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct with the sequence of SEQ ID NO: 9 or nucleotides 55-5021 of the sequence of SEQ ID NO: 3, nucleotides 1-4977 of the sequence of SEQ ID NO: 8 or nucleotides 56-5022 of the sequence of SEQ ID NO: 6. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin with the sequence of SEQ ID NO: 9 or comprises nucleotides 55-5021 of the sequence of SEQ ID NO: 3, nucleotides 1-4977 of the sequence of SEQ ID NO: 8, or nucleotides 56-5022 of the sequence of SEQ ID NO: 6. According to one aspect, rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0265] In any of the methods for treating muscular dystrophy, the level of microdystrophin gene expression in a subject's cell is increased after administration of rAAV. The expression of the microdystrophin gene in the cell is detected by measuring the level of microdystrophin protein using Western blotting in a muscle biopsy before and after administration of rAAV. In particular, the level of microdystrophin protein increases by at least about 70% to at least about 80%, or by at least about 70% to at least about 90%, or by at least about 80% to at least about 90% after administration of rAAV compared to the level of microdystrophin before administration of rAAV.For example, the microdystrophin protein level increases 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 microdystrophin level before administration of rAAV.

[0266] In addition, microdystrophin gene expression in a cell is detected by measuring the microdystrophin protein level using immunohistochemistry in muscle biopsies before and after rAAV administration. The microdystrophin protein level increases by at least about 70% to at least about 80%, or by at least about 70% to at least about 90%, or by at least about 80% to at least about 90% after rAAV administration compared to the microdystrophin level before rAAV administration.For example, the microdystrophin protein level increases 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 microdystrophin level before administration of rAAV.

[0267] In any of the methods for treating muscular dystrophy, the serum CK level of the subject is reduced after administration of rAAV compared to the serum CK level before administration of rAAV. For example, the serum CK level of the subject is reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, 60 days after administration of rAAV compared to the serum CK level before administration of rAAV. In particular, in any of the methods for treating muscular dystrophy according to the present invention, the serum CK level of the subject is reduced by about 87% 60 days after administration of rAAV compared to the serum CK level before administration of rAAV,or in any of the methods for treating muscular dystrophy according to the present invention, the serum CK level of the subject is reduced by about 72% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy according to the present invention, the serum CK level of the subject is reduced by about 73% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy according to the present invention, the serum CK level of the subject is reduced by about 78% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy according to the present invention,The subject's serum CK level is reduced by approximately 95% 60 days after rAAV administration compared to the serum CK level before rAAV administration. In any of the muscular dystrophy treatments, the number of microdystrophin-positive fibers in the subject's muscle tissue increases after rAAV administration compared to the number of microdystrophin-positive fibers before rAAV administration. For example, the number of microdystrophin-positive fibers is detected by measuring microdystrophin protein levels using Western blotting or immunohistochemistry in muscle biopsies before and after rAAV administration.

[0268] In any of the methods for treating muscular dystrophy described herein, administration of rAAV increases the expression of DAPC proteins, such as alpha-sarcoglycan or beta-sarcoglycan. For example, the level of alpha-sarcoglycan in a subject is increased after administration of rAAV compared to the level of alpha-sarcoglycan before rAAV administration. In addition, the level of beta-sarcoglycan in a subject is increased after administration of rAAV compared to the level of beta-sarcoglycan before rAAV administration. The level of alpha-sarcoglycan or beta-sarcoglycan is determined by measuring the level of alpha-sarcoglycan or beta-sarcoglycan protein using Western blotting or immunohistochemistry in muscle biopsies before and after rAAV administration.

[0269] In any of the treatments for muscular dystrophy, the subject's disease progression is slowed after administration of rAAV, as determined by any of the following: six-minute walk test, floor rise time, four-step climb, four-step ascent and descent, North Star Ambulatory Assessment (NSAA), timed 10-meter walk test, timed 100-meter walk test, handgrip dynamometry (HHD), timed stand-up walk test, and / or Gross Motor Subtest Scaled (Bayley-III) score.

[0270] For example, in any of the methods, the subject has an improvement 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 in the NSAA score at least 270 days after rAAV administration compared to the NSAA score before rAAV administration. Additionally, in any of the methods, the subject shows an improvement of at least about 0.8 seconds in the time required to rise from the floor at least 270 days after rAAV administration compared to the time to rise from the floor before rAAV administration. Additionally, in any of the methods, the subject shows an improvement of at least about 1.2 seconds in the time required to climb 4 steps at least 270 days after rAAV administration compared to the time to climb 4 steps before rAAV administration.Additionally, in any of the methods, the subject shows an improvement of at least about 7 seconds in the 100-meter timed run test at least 270 days after rAAV administration compared to the 100-meter timed run test result before rAAV administration.

[0271] According to another aspect, the present invention relates to methods for expressing a microdystrophin gene in a cell of a patient, comprising administering to the patient a nucleotide sequence of an AAVrh74.MHCK7.micro-dystrophin construct comprising the 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 microdystrophin gene in a cell of the patient is detected by measuring the level of microdystrophin protein using Western blotting or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. In addition, the patient's microdystrophin gene expression is measured by detecting more vector genomes per nucleus, with 1 vector genome per nucleus accounting for approximately 50% of microdystrophin expression, and more than 1 copy per nucleus indicating the level of microdystrophin expression.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.

[0272] According to some aspects, the present invention relates to methods for reducing serum CK levels in a patient in need thereof, the method comprising administering to the patient a nucleotide sequence of an AAVrh74.MHCK7.micro-dystrophin construct comprising the 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 serum CK level of the patient is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, through 60 days after rAAV administration compared to serum CK levels before rAAV administration. In particular,the subject's serum CK level is reduced by approximately 87% 60 days after rAAV administration compared to the serum CK level before rAAV administration or in any of the methods for treating muscular dystrophy according to the present invention, the subject's serum CK level is reduced by approximately 72% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy according to the present invention, the subject's serum CK level is reduced by approximately 73% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy according to the present invention, the subject's serum CK level is reduced by approximately 78% 60 days after rAAV administration compared to the serum CK level before rAAV administration or in any of the methods for treating muscular dystrophy according to the present invention,The subject's serum CK level is reduced by approximately 95% 60 days after rAAV administration compared to the serum CK level prior to rAAV administration.

[0273] The present invention also provides methods for increasing the number of microdystrophin-positive fibers in muscle tissue of a patient, comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. For example, the number of microdystrophin-positive fibers is detected by measuring the level of dystrophin protein using Western blotting or immunohistochemistry on muscle biopsies before and after rAAV administration. In addition, the patient's microdystrophin gene expression is measured by detecting more vector genomes per nucleus, with 1 vector genome per nucleus accounting for approximately 50% of microdystrophin expression, and more than 1 copy per nucleus indicating the level of microdystrophin expression.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.

[0274] According to another aspect, the present invention relates to methods for increasing the expression of alpha-sarcoglycan in a patient in need thereof, comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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 level of alpha-sarcoglycan is determined by measuring the level of alpha-sarcoglycan protein by Western blotting or immunohistochemistry on muscle biopsies before and after rAAV administration.

[0275] Further, the present invention provides methods for increasing the expression of beta-sarcoglycan in a patient in need thereof, comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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 level of beta-sarcoglycan is determined by measuring the level of beta-sarcoglycan protein by Western blotting or immunohistochemistry on muscle biopsies before and after rAAV administration.

[0276] The present invention also relates to methods of treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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 the progression of the disease in the patient is slowed, as determined by any of the following criteria: a six-minute walk test, a floor rise time, a 4-step ascent, a 4-step ascent and descent, a North Star Ambulatory Assessment (NSAA), a timed 10-meter walk test, a timed 100-meter walk test, a handgrip dynamometry (HHD), a stand-up test and timed walking and / or Gross Motor Subtest Scaled (Bayley-III) score.

[0277] For example, in any of the methods, the subject has an improvement 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 in the NSAA score at least 270 days after rAAV administration compared to the NSAA score before rAAV administration. Furthermore, in any of the methods, the subject shows an improvement of at least about 0.8 seconds in the time required to rise from the floor at least 270 days after rAAV administration compared to the time to rise from the floor before rAAV administration. Additionally, in any of the methods, the subject shows an improvement of at least about 1.2 seconds in the time required to climb 4 steps at least 270 days after rAAV administration compared to the time to climb 4 steps before rAAV administration.Additionally, in any of the methods, the subject shows an improvement of at least about 7 seconds in the 100-meter timed run test at least 270 days after rAAV administration compared to the 100-meter timed run test performance before rAAV administration.

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

[0279] The present invention also relates to methods for reducing or preventing fibrosis in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of a rAAV comprising the nucleotide sequence of human microdystrophin presented in SEQ ID NO: 1 and the nucleotide sequence of the MHCK7 promoter presented in SEQ ID NO: 2 or SEQ ID NO: 7; or an rAAV vector comprising the nucleotide sequence of the microdystrophin construct AAVrh74.MHCK7.micro-dystrophin presented in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. In one aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin comprising nucleotides 55-5021 of SEQ ID NO: 3. In another aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of the sequence SEQ ID NO: 9. According to another aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin comprising nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 56-5066 of SEQ ID NO: 6. According to a further aspect, rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0280] According to another aspect, the present invention relates to methods for preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7; or an rAAV vector comprising the nucleotide sequence of the AAV74.MHCK7.micro-dystrophin construct of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. For example, any of the rAAVs of the present invention can be administered to subjects suffering from muscular dystrophy to prevent fibrosis, such as an rAAV of the present invention expressing the human microdystrophin protein administered before fibrosis is observed in the subject.Furthermore, the rAAV of the present disclosure expressing the human microdystrophin gene can be administered to a subject at risk of developing fibrosis, such as subjects suffering from or diagnosed with muscular dystrophy, such as DMD. The rAAV of the present invention can be administered to a subject suffering from muscular dystrophy to prevent new fibrosis in such subjects.

[0281] The present invention provides for the administration of rAAV before fibrosis is detected in a subject. Furthermore, rAAV can be administered to a subject at risk of developing fibrosis, such as subjects suffering from muscular dystrophy or diagnosed with muscular dystrophy, such as DMD. RAAV can be administered to a subject suffering from muscular dystrophy who has already developed fibrosis to prevent new fibrosis in such subjects.

[0282] The present invention also provides methods for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of the nucleotide sequence of human microdystrophin SEQ ID NO: 1 and the nucleotide sequence of the MHCK7 promoter SEQ ID NO: 2 or SEQ ID NO: 7; or an rAAV comprising the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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 invention provides for administering rAAV vectors to subjects diagnosed with DMD before the subject is diagnosed with fibrosis, or before muscle strength is reduced, or before muscle mass is reduced.

[0284] The present invention also provides for the administration of a nucleotide sequence of human microdystrophin having the sequence of SEQ ID NO: 1 and a nucleotide sequence of the MHCK7 promoter having the sequence of SEQ ID NO: 2 or SEQ ID NO: 7; or rAAV comprising the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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, for the purpose of preventing new fibrosis in such subjects or reducing fibrosis in such subjects. The present invention also provides for the administration of the nucleotide sequence of human microdystrophin SEQ ID NO: 1 and the nucleotide sequence of the MHCK7 promoter SEQ ID NO: 2 or SEQ ID NO: 7; or an rAAV vector containing the nucleotide sequence of the AAVrh74.MHCK7 construct.micro-dystrophin 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 decreased muscle strength or decreased muscle mass, in order to protect the muscle from further damage.

[0285] The present invention also provides methods for treating cardiomyopathy in a human subject suffering from muscular dystrophy (e.g., DMD), comprising administering any of the compositions described herein (e.g., delanistrogene moxeparvovec) to said human subject. In some aspects, the method is used to improve cardiac function in subjects with DMD. In some aspects, cardiac function is improved by, for example, increasing the ejection fraction (EF); increasing the shortening fraction (FS); decreasing the left ventricular diastolic internal diameter (LVIDd); decreasing the left ventricular end-systolic diameter (LVESD); and / or maintaining or decreasing serum troponin levels. In any of the methods of the present invention, the subject may suffer from muscular dystrophy, such as DMD, or any other dystrophin-associated muscular dystrophy.

[0286] According to other aspects of any of the methods of the present invention described herein, the CK level in the serum of the subject is reduced after administration of rAAV compared to the CK level in the serum before administration of rAAV by a percentage level selected from the group consisting of: a) at least 78% at 90, 180, or 270 days after administration; b) at least 46, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 85% at 270 days after administration; c) at least 72, 73, 74, or 95% at 180 days after administration; d) at least 87, 88, 93, or 95% at 90 days after administration; d) at least 70% at 270 days after administration; f) 70 to 95% at 90, 180 or 270 days after administration; g) at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% at 90, 180 or 270 days after administration;andh) 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% at 90, 180 or 270 days after administration.

[0287] According to another aspect of the present invention, there are provided compositions for the treatment of muscular dystrophy in a human subject in need thereof, wherein said composition comprises a dose of a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the composition is for systemic administration, and the dose of rAAV is from about 1×10 14 vg / kg to approximately 4×10 14vg / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of the sequence of SEQ ID NO: 9, comprises 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. According to one aspect, the rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0288] For example, the composition of the present invention comprises a dose of rAAV of from about 5.0×10 12 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg up to 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 5.0×1012 vg / kg to approximately 1.0×10 14 vg / kg, or approximately 5.0×10 12 vg / kg to approximately 5.0×10 13 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 2.0×10 13 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 1.0×10 13 vg / kg, or from 1.0×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg up to 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 5.0×10 13 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 5.0×1014 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or from 1.0×10 13 vg / kg to approximately 1.0×10 15 vg / kg, or from 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg up to 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 5.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or from 5.0×10 13 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 14 vg / kg to approximately 6.0×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 5.0×1014 vg / kg, or from 1.0×10 14 vg / kg to approximately 4.0×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.0×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 5.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 4.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 1.0×10 15 , or from approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.0×10 14vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from 1.5×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from approximately 1.5×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.5×10 14 vg / kg up to 5.0×10 14 , or from approximately 1.5×10 14 vg / kg up to 4.0×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.75×10114 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.25×1014 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from 1.75×10* 14 vg / kg to approximately 1.0×10 15 vg / kg, or approximately 1.75×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.75×10 14 vg / kg up to 5.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg up to 4.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.25×10 14 vg / kg, or approximately 1.75×10 14vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.25×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg up to 1.0×10 15 vg / kg, or from approximately 2.0×10 14 vg / kg up to 6.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg up to 5.0×10 14 , or from approximately 2.0×10 14 vg / kg to approximately 4.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.25×10 14vg / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of the sequence of SEQ ID NO: 9, comprises 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. According to one aspect, the rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0289] According to one aspect of the present invention, the compositions of the present invention are for intravenous administration and comprise a dose of rAAV of approximately 2.0×10 14vg / kg. According to another aspect, the compositions of the present invention are intended for intravenous administration and comprise a dose of rAAV that is approximately 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 6.0×1013 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. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of the sequence of SEQ ID NO: 9, comprises 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. According to another aspect, the rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0290] In any of the compositions of the present invention, the rAAV dose is delivered in an amount of from about 5 ml / kg to about 15 ml / kg, or from about 8 ml / kg to about 12 ml / kg, or from 8 ml / kg to about 10 ml / kg, or from 5 ml / kg to about 10 ml / kg, or from about 10 ml / kg to 12 ml / kg, or from about 10 ml / kg to 15 ml / kg, or from 10 ml / kg to about 20 ml / kg. According to a particular aspect, the composition comprises a dose of rAAV delivered in an amount of about 10 ml / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin with the sequence of SEQ ID NO: 9, comprises nucleotides 55-5021 of the sequence of SEQ ID NO: 3, nucleotides 1-4977 of the sequence of SEQ ID NO: 8, or nucleotides 56-5022 of the sequence of SEQ ID NO: 6. According to another aspect, rAAV is AAVrh74.MCK.microdystrophin.According to one aspect, AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0291] The compositions of the present invention are intended for administration by injection, infusion, or implantation. For example, the compositions are intended for administration by infusion over approximately one hour. In addition, the compositions of the present invention are intended for intravenous administration through a peripheral vein of an extremity, such as a peripheral vein of the arm or a peripheral vein of the leg. Alternatively, the infusion can be administered 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 present invention comprises an rAAV comprising the nucleotide sequence of human microdystrophin presented in SEQ ID NO: 1 and the MHCK7 promoter sequence presented in SEQ ID NO: 2 or SEQ ID NO: 7, or an rAAV vector comprising the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct presented in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6.

[0293] In particular, the compositions of the present invention are used for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present invention provides compositions for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the composition is prepared for administration by intravenous infusion over about one hour, and the dose of rAAV administered is about 2×10 14 vg / kg, and wherein the rAAV comprises the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct presented in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6.

[0294] According to another aspect, the present invention also provides a composition comprising rAAV for reducing fibrosis in a subject in need thereof. Furthermore, the present invention provides a composition comprising rAAV vectors for preventing fibrosis in a subject suffering from muscular dystrophy.

[0295] The present invention also provides compositions containing rAAV for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy. In a further aspect, the present invention provides compositions containing any of the rAAVs of the present invention for the treatment of muscular dystrophy.

[0296] According to other aspects of any of the compositions of the present invention, after administration of said composition to a human subject in need of treatment for muscular dystrophy, the serum CK level of the subject is reduced, compared to the serum CK level before administration of the composition, by a percentage level selected from the group consisting of: a) at least 78% at 90, 180 or 270 days after administration; b) at least 46, 55, 70 or 85% at 270 days after administration; c) at least 72, 73, 74 or 95% at 180 days after administration; d) at least 87, 99, 93 or 95% at 90 days after administration; d) at least 70% at 270 days after administration;f) from 70 to 95% at 90, 180 or 270 days after administration;g) at least 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% at 90, 180 or 270 days after administration;andh) 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% at 90, 180 or 270 days after administration.;

[0297] According to another aspect, the present disclosure provides the use of a dose of a recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin for the production of a medicament for the treatment of muscular dystrophy in a human subject in need thereof, wherein said medicament is for systemic administration, and the dose of rAAV is from about 1×10 14 vg / kg to approximately 4×10 14vg / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of the sequence of SEQ ID NO: 9, comprises 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. According to one aspect, the rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0298] For example, the medicinal product contains a dose of rAAV of approximately 5.0×10 12 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg up to 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 5.0×10 12vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 5.0×10 13 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 2.0×10 13 vg / kg, or from approximately 5.0×10 12 vg / kg to approximately 1.0×10 13 vg / kg, or from 1.0×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg up to 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 5.0×10 13 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 5.0×10 14vg / kg, or from approximately 1.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or from 1.0×10 13 vg / kg to approximately 1.0×10 15 vg / kg, or from 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg up to 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 5.0×10 14 vg / kg, or from approximately 5.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or from 5.0×1013 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 14 vg / kg to approximately 6.0×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 5.0×10 14vg / kg, or from 1.0×10 14 vg / kg to approximately 4.0×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from 1.0×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.0×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or from 1.0×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 5.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 4.0×10 14 , or from approximately 1.25×10 14 vg / kg up to 1.0×10 15 , or from approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.25×1014 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from 1.5×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or from approximately 1.5×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.5×10 14 vg / kg up to 5.0×10 14 , or from approximately 1.5×10 14 vg / kg up to 4.0×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.25×10 14vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or from approximately 1.5×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from 1.75×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or approximately 1.75×10 14 vg / kg up to 6.0×10 14 , or from approximately 1.75×10 14 vg / kg up to 5.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg up to 4.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.25×10 14 vg / kg, or approximately 1.75×10 14vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.25×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg up to 1.0×10 15 vg / kg, or from approximately 2.0×10 14 vg / kg up to 6.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg up to 5.0×10 14 , or from approximately 2.0×10 14 vg / kg to approximately 4.0×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or from approximately 2.0×10 14 vg / kg to approximately 3.25×10 14vg / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of the sequence of SEQ ID NO: 9, comprises 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. According to one aspect, the rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0299] According to one aspect, the medicaments of the present invention are prepared for systemic administration of a dose of rAAV, wherein the systemic route of administration is an intravenous route, and the dose of rAAV administered is approximately 2.0×10 14vg / kg. According to one aspect, the medicament of the present invention is formulated for systemic administration of a dose of rAAV, wherein the systemic route of administration is the intravenous route, and the dose of rAAV is approximately 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 13vg / 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. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of the sequence of SEQ ID NO: 9, comprises 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. According to one aspect, the rAAV is AAVrh74.MCK.microdystrophin. According to one aspect, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0300] In any of the uses according to the present invention, the medicament comprises a dose of rAAV of from about 5 ml / kg to about 15 ml / kg, or from about 8 ml / kg to about 12 ml / kg, or from 8 ml / kg to about 10 ml / kg, or from 5 ml / kg to about 10 ml / kg, or from about 10 ml / kg to 12 ml / kg, or from about 10 ml / kg to 15 ml / kg, or from 10 ml / kg to about 20 ml / kg. According to a particular aspect, the dose of rAAV is about 10 ml / kg. According to one aspect, the rAAV is AAVrh74.MHCK7.microdystrophin. According to one aspect, AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin with the sequence of SEQ ID NO: 9, comprises nucleotides 55-5021 of the sequence of SEQ ID NO: 3, nucleotides 1-4977 of the sequence of SEQ ID NO: 8, or nucleotides 56-5022 of the sequence of SEQ ID NO: 6. According to one aspect, rAAV is AAVrh74.MCK.microdystrophin.According to one aspect, AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin comprising nucleotides 56-4820 of SEQ ID NO: 5.

[0301] In any of the uses according to the present invention, the medicinal product is intended for administration by injection, infusion, or implantation. For example, the medicinal product is intended for administration by infusion over approximately one hour. Additionally, the medicinal product is intended for intravenous administration through a peripheral vein of an extremity, such as a peripheral vein of the arm or a peripheral vein of the leg. Alternatively, the infusion can be administered 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 according to the present invention, the medicament comprises an rAAV comprising the nucleotide sequence of human microdystrophin presented in SEQ ID NO: 1 and the MHCK7 promoter sequence presented in SEQ ID NO: 2 or SEQ ID NO: 7, or the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct presented in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 56-5022 of SEQ ID NO: 6.

[0303] A specific use of the present invention is for the production of a medicament for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. For example, according to the present invention, there is provided the use of a dose of the recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin for the production of a medicament for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy in a subject that is a human in need thereof, wherein the medicament is intended to be administered by intravenous infusion for about one hour, and the dose of rAAV to be administered is approximately 2×10 14 vg / kg, and wherein the rAAV comprises the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct presented in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6.

[0304] According to a further aspect, the present invention relates to the use of rAAV for the production of a medicament for reducing fibrosis in a subject in need thereof. For example, the subject in need thereof may suffer from muscular dystrophy, such as DMD or any other dystrophin-related muscular dystrophy.

[0305] According to another aspect, the present invention provides the use of rAAV for the production of a medicament for preventing fibrosis in a subject suffering from muscular dystrophy.

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

[0307] The present invention also provides the use of rAAV for producing a medicament for treating muscular dystrophy.

[0308] The present invention provides the use of an rAAV vector comprising the nucleotide sequence of human microdystrophin SEQ ID NO: 1 and the nucleotide sequence of the MHCK7 promoter SEQ ID NO: 2 or SEQ ID NO: 7, for producing a medicament for treating muscular dystrophy or an rAAV vector comprising the nucleotide sequence of the AAVrf74.MHCK7.microdystrophin construct 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 treating muscular dystrophy.

[0309] According to other aspects of any of the uses according to the present invention, the CK level in the serum of the subject is reduced after administration of rAAV to the subject, compared to the CK level in the serum before administration of rAAV, by a percentage level selected from the group consisting of: a) at least 78% at 90, 180 or 270 days after administration; b) at least 46, 55, 70 or 95% at 270 days after administration; c) at least 72, 73, 74 or 95% at 180 days after administration; d) at least 87, 88, 93 or 95% at 90 days after administration; d) at least 70% at 270 days after administration;f) from 70 to 95% at 90, 180 or 270 days after administration;g) at least 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% at 90, 180 or 270 days after administration; and h) 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% at 90, 180 or 270 days after administration.

[0310] In any of the compositions for treating muscular dystrophy or the use of a medicament for treating muscular dystrophy, the level of expression of the microdystrophin gene in a cell of a subject increases after administration of said composition or medicament. Expression of the microdystrophin gene in a cell is detected by measuring the level of microdystrophin protein using Western blotting in a muscle biopsy before and after administration of the composition or medicament. In particular, the level of microdystrophin protein increases by at least about 70% to at least about 80%, or by at least about 70% to at least about 90%, or by at least about 80% to at least about 90% after administration of the composition or medicament, compared to the level of microdystrophin before administration of the composition or medicament.For example, the level of microdystrophin protein increases by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition compared to the level of microdystrophin before administration of the composition or drug.

[0311] Furthermore, expression of the microdystrophin gene in the cell is detected by measuring the microdystrophin protein level using immunohistochemistry in muscle biopsies before and after administration of the composition or drug. The microdystrophin protein level increases by at least about 70% to at least about 80%, or from at least about 70% to at least about 90%, or from at least about 80% to at least about 90% after administration of rAAV compared to the microdystrophin level before administration of the composition or drug.For example, the level of microdystrophin protein increases 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 drug, compared to the level of microdystrophin before administration of the composition or drug.

[0312] In any of the compositions for the treatment of muscular dystrophy, the serum CK level of the subject is reduced after administration of rAAV compared to the serum CK level before administration of said composition or drug. For example, the serum CK level of the subject is reduced by from about 65% to about 90%, or from about 65% to about 95%, or from about 75% to about 90%, or from about 80% to about 90%, or from about 85% to about 95%, or from about 87% to about 95%, or from about 87% to about 90%, 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug. In particular,in any of the compositions for the treatment of muscular dystrophy according to the present invention, the serum CK level of a subject is reduced by about 87% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or in any of the compositions for the treatment of muscular dystrophy or about a drug for the treatment of muscular dystrophy according to the present invention, the serum CK level of a subject is reduced by about 72% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or in any of the compositions for the treatment of muscular dystrophy according to the present invention,the serum CK level of the subject is reduced by approximately 73% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or in any of the compositions for the treatment of muscular dystrophy or the use of the drug for the treatment of muscular dystrophy according to the present invention, the serum CK level of the subject is reduced by approximately 78% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or in any of the compositions for the treatment of muscular dystrophy or the use of the drug for the treatment of muscular dystrophy according to the present invention,The serum CK level in the subject is reduced by approximately 95% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug. In any of the compositions for the treatment of muscular dystrophy or the use of a drug for the treatment of muscular dystrophy, the number of microdystrophin-positive fibers in the muscle tissue of the subject increases after administration of the composition or drug compared to the number of microdystrophin-positive fibers before administration of the composition or drug. For example, the number of microdystrophin-positive fibers is detected by measuring the level of microdystrophin protein using Western blotting or immunohistochemistry on muscle biopsies before and after administration of the composition or drug.

[0313] In any of the compositions for treating muscular dystrophy or the use of a medicament for treating muscular dystrophy, administration of the composition or medicament increases the expression of DAPC proteins, such as alpha-sarcoglycan or beta-sarcoglycan. For example, the level of alpha-sarcoglycan in a subject increases after administration of the composition or medicament compared to the level of alpha-sarcoglycan before administration of the composition or medicament. In addition, the level of beta-sarcoglycan in a subject increases after administration of the composition or medicament compared to the level of beta-sarcoglycan before administration of the composition or medicament. The level of alpha-sarcoglycan or beta-sarcoglycan is determined by measuring the level of alpha-sarcoglycan or beta-sarcoglycan protein using Western blotting or immunohistochemistry in a muscle biopsy before and after administration of the composition or medicament.

[0314] In any of the compositions for treating muscular dystrophy or the use of a medicament for treating muscular dystrophy, the progression of the disease in a subject is slowed after administration of the composition or medicament, as determined by any of the following criteria: a six-minute walk test, time to rise from the floor, a four-step ascent, a four-step ascent and descent, a North Star Ambulatory Assessment (NSAA), a timed 10-meter walk test, a timed 100-meter walk test, handgrip dynamometry (HHD), a timed stand-up walk test, and / or a Gross Motor Subtest Scaled (Bayley-III) score.

[0315] For example, after administration of any of the compositions for the treatment of muscular dystrophy or the use of a drug for the treatment of muscular dystrophy, the subject shows an improvement 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 in the NSAA score at least 270 days after administration of the composition or drug compared to the NSAA score before administration of rAAV. Additionally, in any of the methods, the subject shows an improvement of at least about 0.8 seconds in the time required to rise from the floor at least 270 days after administration of the composition or drug compared to the time to rise from the floor before administration of the composition or drug.Furthermore, in any of the methods or uses of the present invention, the subject shows an improvement of at least about 1.2 seconds in the time required to climb 4 steps at least 270 days after administration of the composition or drug compared to the time to climb 4 steps before administration of the composition or drug. Furthermore, in any of the methods or uses according to the present invention, the subject shows an improvement of at least about 7 seconds in the 100-meter timed test at least 270 days after administration of the composition or drug compared to the results in the 100-meter timed test before administration of the composition or drug.

[0316] According to another aspect, the present invention provides compositions for expressing a microdystrophin gene in a cell of a patient, comprising the nucleotide sequence of an AAVrh74.MHCK7.micro-dystrophin construct presented in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. According to a further aspect, the present invention provides the use of a dose of the nucleotide sequence of an AAVrh74.Micro-dystrophin construct comprising the 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 producing a drug for expressing the microdystrophin gene in a patient's cell.For example, microdystrophin gene expression in a patient's cells is detected by measuring microdystrophin protein levels using Western blotting or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.microdystrophin construct. Additionally, microdystrophin gene expression in the patient is measured by detecting a higher number of vector genomes per nucleus, with one vector genome per nucleus representing approximately 50% microdystrophin expression, and more than one copy per nucleus indicating a high level of microdystrophin expression. 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] According to a further aspect, the present invention relates to compositions for reducing serum CK levels in a patient in need thereof, wherein the composition comprises the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct as set forth in SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. Furthermore, the present invention provides the use of a dose of the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct 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 production of a medicinal agents to reduce serum CK levels in a patient who requires it. For example,the serum CK level in the patient is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, 60 days after administration of the composition or drug, compared to the serum CK level before administration of the composition or drug. In particular, the serum CK level in the subject is reduced by about 87% 60 days after administration of the composition or drug, compared to the serum CK level before administration of the composition or drug, or is reduced by about 72% 60 days after administration of the composition or drug, compared to the serum CK level before administration of the composition or drug,or is reduced by approximately 73% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or is reduced by approximately 78% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or is reduced by approximately 95% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug.

[0318] According to the present invention, there are also provided compositions for increasing the number of microdystrophin-positive fibers in the muscle tissue of a patient, comprising the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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 addition, the present invention relates to the use of a dose of the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct 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 production of a medicament for increasing the number microdystrophin-positive fibers in the patient's muscle tissue.For example, the number of microdystrophin-positive fibers is detected by measuring dystrophin protein levels using Western blotting or immunohistochemistry on muscle biopsies before and after administration of a composition or drug. Additionally, the patient's microdystrophin gene expression is measured by detecting a greater number of vector genomes per nucleus, with 1 vector genome per nucleus representing approximately 50% microdystrophin expression, and more than 1 copy per nucleus indicating a microdystrophin expression level. For example, cells have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0319] According to another aspect, the present invention provides compositions for increasing the expression of alpha-sarcoglycan in a patient in need thereof, comprising a nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct having the sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 56-5022 of SEQ ID NO: 6. The present invention also provides the use of a dose of the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct comprising the 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 producing a drug to increase alpha-sarcoglycan expression in a patient in need thereof.For example, alpha-sarcoglycan levels are determined by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or drug.

[0320] Furthermore, the present invention provides compositions for increasing the expression of beta-sarcoglycan in a patient in need thereof, comprising the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. The present invention also provides the use of the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct 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 production of a medicament for increasing the expression beta-sarcoglycan in a patient who needs it.For example, beta-sarcoglycan levels are determined by measuring beta-sarcoglycan protein levels by Western blotting or immunohistochemistry on muscle biopsies before and after administration of the composition or drug.

[0321] The present invention also relates to the use of a dose of the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct 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 the treatment of a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, such that administration of the medicament results in a slowdown in the progression of the disease in the patient, as determined by any of the following criteria: a six-minute walk test, a floor rise time, a 4-step climb, a 4-step ascent and descent, a North Star Ambulatory Assessment (NSAA), a timed 10-meter walk test, a timed 100-meter walk test, a handgrip dynamometry (HHD), a test with timed standing and walking and / or assessment according to the Gross Motor Subtest Scaled (Bayley-III).

[0322] For example, the subject demonstrates an improvement in the NSAA scale score of at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.4, 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 drug compared to the NSAA scale score before administration of the composition or drug. In addition, the subject demonstrates an improvement of at least about 0.8 seconds in the time required to rise from the floor at least 270 days after administration of the composition or drug compared to the time to rise from the floor before administration of the composition or drug.In addition, the subject demonstrates an improvement of at least approximately 1.2 seconds in the time required to climb 4 steps at least 270 days after administration of the composition or drug compared to the time required to climb 4 steps before administration of the composition or drug. Furthermore, the subject demonstrates an improvement of at least approximately 7 seconds in the 100-meter timed walk test at least 270 days after administration of the composition or drug compared to the results in the 100-meter timed walk test before administration of the composition or drug.

[0323] It should be understood that the Detailed Description section, rather than the Summary and Abstract sections, is intended to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, illustrative aspects of the present invention contemplated by the authors and, thus, are in no way intended to limit the present invention and the appended claims.

[0324] The following examples are provided for illustrative purposes and should not be construed as limiting the invention. The numerical ranges described include each integer value within each range and include the lowest and highest specified integer. EXAMPLES Example 1A) Preparation of the AAVrh74.MHCK7.microdystrophin construct

[0325] The AAVrh74.MHCK7.micro-dystrophin plasmid contains the human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeat (ITR) sequences (see Fig. 1). The microdystrophin construct was characterized by an in-frame deletion of the stem (R4-R23), while hinges 1, 2, and 4 and the cysteine-rich domain remain unchanged to produce a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) was driven by the MHCK7 promoter (792 bp). The plasmid was constructed from the rAAV.MCK.micro-dystrophin plasmid by deletion of the MCK promoter and insertion of the MHCK7 promoter. The core promoter is followed by the endogenous exon 1 (untranslated) of mouse MCK 53 bp for efficient transcription initiation, followed by the SV40 late splicing signals 16S / 19S (150 bp) and a short 5'UTR (61 bp). The intron and 5'-UTR were obtained from the pCMVß plasmid (Clontech).The microdystrophin cassette had a Kozak consensus sequence immediately upstream of the ATG start codon and a short 53 bp synthetic polyA signal for mRNA termination. The human microdystrophin cassette contained domains (R4-R23 / Δ71-78) as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)). Complementary DNA was codon-optimized for human use and synthesized by GenScript (Piscataway, NJ) (Mol Ther 18, 109-117 (2010)). The only viral sequences included in this vector were the AAV2 inverted terminal repeats, which are required for both viral DNA replication and packaging. The microdystrophin cassette has a short 53 bp synthetic polyA signal for mRNA termination.

[0326] Previous studies have confirmed expression using the MHCK7 promoter in cardiac muscles (Salva et al., Mol. Ther., 15, 320–329 (2007)) and expression of AAVrh74 in skeletal, cardiac muscles, and diaphragm (Sondergaard et al., Annals of Clinical and Transl Neurology, 2, 256–270 (2015)). The sequence of the construct shown in Fig. 1 was encapsidated into AAVrh.74 virions. The molecular clone of serotype AAVrh.74 was cloned from the lymph node of a rhesus macaque and is described in Rodino-Klapac et al., Journal of Translational medicine, 5, 45 (2007).

[0327] Table 1 shows the molecular features of the plasmid AAVrh74.MHCK7.micro-dystrophin (SEQ ID NO: 3) B) Generation of the AAVrh74.MHCK7.micro-dystrophin construct from a plasmid encoding kanamycin resistance (KAN)

[0328] Cloning of MHCK7.μDys.KAN was accomplished by isolating the MHCK7.uDys fragment from the MHCK7.μDys.AMP plasmid and the kanamycin backbone and annealing them using the NEBuilder cloning process. The MHCK7.μDys fragment was isolated by restriction enzyme digestion with SnaBI. Digestion was performed in a total reaction of 50 μl in 1x CutSmart Buffer (NEB) and 1 μl SnaBI at 37°C for 1 hour. The resulting fragment was isolated by electrophoresis using 1% agarose gel 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 digestion with the restriction enzyme XbaI in a 50 μl reaction with 1x CutSmart buffer (NEB) and 1 μl XbaI at 37°C for 1 hour. The resulting fragment was isolated by electrophoresis using a 1% agarose gel at 105 volts for 1.5 hours.The band corresponding to the KAN backbone was excised and purified using a gel purification kit (Macherey-Nagel). The resulting fragment had a DNA concentration of 8.1 ng / μl. The two fragments were annealed using the NEB Builder cloning process, which has the ability to join two fragments with overlapping sequences. The NEBuilder cloning reaction was performed according to the manufacturer's protocol at 50°C for 15 min, using a 1:1 ratio of MHCK7.μDys to the kanamycin backbone in 1x NEBuilder HiFi DNA Assembly Master Mix for a total reaction volume of 20 μl. The resulting clone was transformed into NEB® Stable Competent E. coli (C3040) by adding 2.5 μl of the cloning product to the cells, followed by 30 min on ice, 30 s at 42°C, and an additional 5 min on ice. After transformation, 950 µl of growth medium were added to the cells and they were left to grow at 30°C for 1.5 hours, shaking at 225 rpm.After expansion, 450 μl of the cells were plated on an LB agar plate supplemented with 50 μg / ml kanamycin and incubated overnight at 30°C in a dry incubator. A colony was picked from this plate and grown overnight in LB containing 50 μg / ml kanamycin. DNA was isolated from 3 ml of this culture using the 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 further confirmed by sequencing. The resulting plasmid is presented as SEQ ID NO: 8 and is shown in Figs. 14 and 15. The sequence of the construct shown in Fig. 13, which corresponds to the sequence of SEQ ID NO: 9 and nucleotides 1-4977 of the sequence of SEQ ID NO: 8, was encapsidated into AAVrh.74 virions as described above.Example 2: Clinical trial of systemic gene delivery in Duchenne muscular dystrophy.

[0329] The study was a single-dose, controlled trial using rAAVrh74.MHCK7.micro-dystrophin, containing nucleotides 55-5021 of SEQ ID NO: 3, in subjects with DMD. 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 (2×10 14 (vg / kg in 10 ml / kg). The rAAVrh74.MHCK7.micro-dystrophin composition was prepared in a buffer solution containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl2), 200 mM sodium chloride (NaCl) and 0.001% poloxamer 188.

[0330] In the rAAVrh74.MHCK7.micro-dystrophin study, the micro-dystrophin vector was administered via a peripheral vein in the arm, allowing delivery to all muscles in the body. Cohort A included six subjects with DMD aged 3 months to 3 years, and Cohort B included six subjects with DMD aged 4 years to 7 years. All subjects received the micro-dystrophin vector (2×10) intravenously. 14 (vg / kg in 10 ml / kg). The titer of the encapsulated vector genome for the administered dose was determined by quantitative PCR using the Prism 7500 Taqman Detection System (PE Applied Biosystems) with primers targeting the MHCK7 promoter, compared to a supercoiled DNA plasmid standard (Pozsgai et al., Mol. Ther. 25(4): 855–869 (2017)).

[0331] Patients received 1-hour infusions in the pediatric intensive care unit (PICU) at Nationwide Children's Hospital. Before gene therapy, a muscle biopsy was performed during the screening visit. Subjects underwent a second muscle biopsy to determine whether the gene provided a replacement for the missing dystrophin protein 90 days after administration. After gene transfer, patients were closely monitored for any adverse effects of treatment. This monitoring included blood and urine tests, as well as a physical examination at the screening visits and on days 0, 1, 7, 14, 30, 60, 90, and 180, as well as at months 9, 12, 18, 24, 30, and 36 to ensure there were no adverse effects from the gene injection.

[0332] Cohort A subjects (n=6) were patients aged 3 months to 3 years and received intravenous rAAVrh74.MHCK7.micro-dystrophin vector (2×10 14(vg / kg in 10 mL / kg). One day before gene transfer, subjects in cohort A started receiving prednisone or deflazacort at a dose of 1 mg / kg and continued taking these drugs for 30 days while monitoring the immune response. If the result was negative on day 30, steroids were gradually tapered over 1 week. If the T-cell response to AAV or microdystrophies was >125 SFC / 106 PBMCs, steroids were maintained until levels fell below this threshold.

[0333] Cohort B subjects (n=6) were 4 to 7 years old and received intravenous rAAVrh74.MHCK7.micro-dystrophin vector (2×10 14 (vg / kg in 10 ml / kg). For these subjects, a stable dose of corticosteroids was maintained throughout the study, but it could be increased for a short time if the T-cell response to AAV or microdystrophies was >125 SFC / 106 PBMC. Selection criteria

[0334] The following inclusion criteria were used in the study:• Age of participation: Cohort A: 3 months to 7 years and Cohort B: 4 years to and including 7 years.• Molecular characterization of the DMD gene with a frameshift (deletion or duplication) or premature stop codon mutation between exons 18-58.• Increased CK>1000 U / L.• Cohort A subjects: below average in the Bayley-III gross motor assessment, defined as a score ≤9.• Cohort B: below average in the 100-meter walk test, defined as <80% of predicted.• Boys of any ethnicity.• Eligibility for motor testing.• Cohort A subjects: naive to corticosteroid treatment.• Cohort B subjects: were taking a stable oral corticosteroid equivalent dose for at least 12 weeks prior to screening and the dose was expected to remain constant (except for modifications to account for weight changes) throughout the study.

[0335] The exclusion criteria for the study were as follows:• Active viral infection based on clinical observations.• Evidence of cardiomyopathy, including echocardiogram with an ejection fraction below 40%.• Serologic evidence of HIV infection or hepatitis B or C infection.• Diagnosis (or ongoing treatment) of an autoimmune disease.• Abnormal laboratory test results considered clinically significant.• Concomitant disease or requirement for chronic drug therapy that, in the opinion of the Clinical Trial Director, poses unnecessary risks to gene transfer.• Subjects with AAVrh74 or AAV8 antibody titers>1:400 as determined by ELISA.• The presence of a medical condition or mitigating circumstance that, in the opinion of the investigator, may compromise the subject's ability to comply with protocol-required trial or procedures or compromise the subject's welfare, safety, or clinical interpretability.• Severe infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (study enrollment may be delayed).• The patient has received any investigational medications (except corticosteroids) or exon skipping medications (including ExonDys 51®), experimental or otherwise, within the last 6 months prior to screening for this study.• The patient has received any type of gene therapy, cell therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy.• The family does not wish to disclose information about the patient's participation in the study to the primary care physician and other health care professionals. Performance criteria.

[0336] The primary efficacy outcome was safety, defined as the number of participants experiencing adverse events (time frame: 3 years). Adverse events were observed and assessed for severity and relationship to the study drug.

[0337] The secondary outcome measures were as follows:

[0338] Gross Motor Subtest Scaled (Bayley-III) score (time frame: screening, day 30-3 years): The total Gross Motor Scaled Score was a measure of motor development. Cohort A was assessed using the Bayley-III gross motor scale at each follow-up visit from day 30 until age 3. Any subject aged 43-47 months inclusive at screening had a score calculated relative to the normative data for children aged 42 months. Normative data for children aged 1-42 months were obtained using the Bayley-III.

[0339] Physical therapy evaluation of the 100-meter timed walk test (100m) (time frame: screening, day 30-3 years): The 100m timed walk test was the primary outcome measure of motor performance for cohort B. The 100m timed walk test was the study outcome measure initiated for cohort A when the child reached 3 years of age.

[0340] North Star Ambulatory Assessment (NSAA) Physical Therapy Assessment (Time Frame: Screening, Day 30-3 years): The North Star Ambulatory Assessment (NSAA) was a study outcome measure initiated for the cohort of Cohort A when the child reached four years of age and for Cohort B. The NSAA measures the quality of ambulation in boys with Duchenne muscular dystrophy.

[0341] Physical therapy evaluations using the Timed Up and Walk Test modified for children (TUG) (Time frame: screening, day 30-3 years): The effectiveness assessment for cohort B included the Timed Up and Walk Test modified for children (TUG).

[0342] Evaluation of physical therapy with 4-step ascent and descent (Time frame: Screening, Day 30-3 years): The effectiveness assessment for cohort B included the 4-step ascent and descent test.

[0343] Physical therapy evaluation with handgrip dynamometry (HHD) (time frame: screening, day 30-3 years): The effectiveness assessment for cohort B included handgrip dynamometry (HHD) for knee extensors and knee flexors, and elbow flexors and elbow extensors.

[0344] Quantification of microdystrophin gene expression by immunofluorescence (Time frame: Screening, Day 90): Microdystrophin gene expression levels were quantified by immunofluorescence and compared in muscle biopsies before and after treatment.

[0345] Quantification of microdystrophin gene expression by Western blot (Time frame: Screening, Day 90): Microdystrophin gene expression levels were quantified by Western blot and compared in muscle biopsies before and after treatment.

[0346] Decrease in CK after gene therapy (time frame: 3 years):Decrease in the level of CK in circulating blood.

[0347] Cardiac magnetic resonance imaging (after 1 year). Microdystrophin gene expression

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

[0349] The change in microdystrophin gene expression from baseline to day 60 was also assessed by quantifying microdystrophin protein expression measured by Western blotting of biopsied muscle tissue. As shown in Figs. 8A and 8B, Western blotting detected microdystrophin protein expression in Subject 1 (at age 5 years), Subject 2 (at age 4 years), and Subject 3 (at age 6 years). Fig. 8C shows the Western blot analysis detecting microdystrophin protein expression in Subject 4 (at age 4 years). All post-treatment biopsies showed robust microdystrophin levels measured by Western blot, with the mean for subjects 1-4 being 74.3% of normal using method 1 and 95.8% of normal using method 2, adjusted for adipose and fibrous tissue.

[0350] The vector genome copy per muscle fiber nucleus was measured for each subject. As shown in Table 3, the vector genome copy per nuclease was greater than 1 for each subject after rAAVrh74.MHCK7.micro-dystrophin administration. One vector copy indicates approximately 50% microdystrophin gene expression. A mean value of 1.6 vector copies per cell nucleus was measured in subjects 1–3, consistent with the observed high levels of microdystrophin expression. When including values ​​for subject 4, the mean vector copy number / μg DNA was >105, with a mean value of 3.3 vector copies per cell nucleus.

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

[0352] Blood samples were obtained every 30 days after intravenous infusion of rAAVrh74.MHCK7.micro-dystrophin vector (2×10 14(vg / kg in 10 mL / kg). CK levels were measured at each visit and compared with the baseline obtained before rAAVrh74.MHCK7.micro-dystrophin administration (visit day 0). Baseline serum CK levels (units / liter) are presented in Table 4 below. As shown in Fig. 10, circulating serum CK levels decreased by approximately 87% 2 months after rAAVrh74.MHCK7.micro-dystrophin administration. All subjects exhibited a significant reduction in serum creatine kinase (CK) levels, with a mean CK reduction of more than 87% 2 months after treatment (n=3). CK is an enzyme associated with muscle damage, and patients with DMD uniformly exhibit high CK levels. Indeed, a significantly elevated CK level is often used as a preliminary diagnostic tool for DMD, followed by confirmatory genetic testing.

[0353] Table 4 and Fig. 10 show the CK levels for each subject. Fig. 11 shows the mean CK levels over time and also demonstrates that the mean CK levels significantly decreased over time after rAAVrh74.MHCK7.micro-dystrophin administration. The mean CK level at baseline of 27,064 U / L (mean for Table 4) decreased by approximately 63% to a mean of 9,982 U / L (mean, day 270, Table 5). Evaluation of effectiveness

[0354] In addition to microdystrophin and CK levels, efficacy was measured using the following functional tests: floor rise time, 4-step climb, North Star Ambulatory Assessment (NSAA), timed rise test, 4-step climb, timed 10-m walk test (10m), and timed 100-m walk test (100m). The data are presented in Tables 6 and 7 below, and these data demonstrate a stable, sustained improvement 9 months after rAAVrh74.MHCK7.micro-dystrophin administration. The improvement in NSAA over time is also shown in Fig. 12. Safety assessment

[0355] No serious adverse events (SAEs) were observed in the study. Three patients experienced elevated gamma-glutamyl transferase (GGT) levels, which resolved with steroid titration within a week and returned to baseline levels. No other clinically significant laboratory findings were observed. Patients experienced transient nausea, typically during the first week of therapy, coinciding with the steroid dose increase. This did not correlate with elevated liver enzymes or any other abnormalities. Case 3: Randomized, double-blind, placebo-controlled, systemic gene delivery clinical trial. Phase I / IIa systemic gene delivery.

[0356] The study was a randomized, double-blind, single-dose study using rAAVrh74.MHCK7.micro-dystrophin in subjects with DMD. Twenty-four subjects aged 4 to 7 years were enrolled in the study. Subjects were randomized to receive drug or placebo at the time of study entry. Twelve subjects received intravenous rAAVrh74.MHCK7.microdystrophin vector (2×10 14 Thirteen subjects received 10 ml / kg placebo (lactated Ringer's solution) and 12 subjects received 10 ml / kg placebo (lactated Ringer's solution). Placebo subjects crossed over to treatment, which was administered in the same manner as the 12 previously treated subjects, one year after the last treated subject's dose. Subjects received infusions of rAAV carrying microdystrophin or lactated Ringer's solution over approximately 1 hour. Pre- and post-treatment (90 days) needle muscle biopsies were performed on the gastrocnemius muscles.

[0357] The primary objective of this study was to evaluate the safety of intravenous administration of rAAVrh74.MHCK7.micro-dystrophin to subjects with MDC via a peripheral vein in the extremities. Safety endpoints were assessed by changes in hematology, serum biochemistry, urinalysis, and the immunological response to rAAVrh74 and microdystrophin, as well as by history and symptom observations. Dystrophin gene expression served as the primary outcome measure along with safety. Quantification was performed using validated immunofluorescence and immunoblot assays. A decrease in CK after gene therapy served as a secondary efficacy criterion. Performance was measured using the following functional tests: time taken to rise from the floor, 4-step climb, North Star Ambulatory Assessment (NSAA), 10-meter walk test (10m), 100-meter walk test (100m).The study included handgrip dynamometry (HHD) for the knee extensors and knee flexors, as well as the elbow flexors and elbow extensors.

[0358] The inclusion criteria for the study were as follows:• Study age: 4 to 7 years inclusive.• Molecular characterization of the DMD gene with a frameshift (deletion or duplication) or premature stop codon mutation between exons 18-58.• Evidence of symptomatic muscular dystrophy: CK level increased >1000 U / L and 100-meter walking time below the mean percentage of predicted time.• Boys of any ethnicity are eligible to participate.• Eligibility for motor function testing.• Patients are receiving a stable oral corticosteroid equivalent dose for at least 12 weeks prior to screening and the dose is expected to remain constant (except for modifications to account for weight changes) throughout the study.

[0359] The exclusion criteria for the study were as follows:• Active viral infection based on clinical observations.• Evidence of cardiomyopathy, including echocardiogram with an ejection fraction below 40%.• Serologic evidence of HIV infection or hepatitis B or C infection.• Diagnosis (or ongoing treatment) of an autoimmune disease.• Abnormal laboratory values ​​considered clinically significant (GGT >3x ULN, bilirubin ≥3.0 mg / dL, creatinine ≥1.8 mg / dL, Hgb <8 or >18 g / dL; white blood cell count >18,500 / cm), platelets ≤50,000.• Concomitant disease or need for chronic drug therapy that, in the opinion of the Clinical Trial Director, poses unnecessary risks to the gene transfer.• Subjects with AAVrh74 antibody titers or AAV8 >1:400 as determined by ELISA. If the endpoint titer is positive at screening, testing may be repeated before exclusion.• The presence of a medical condition or mitigating circumstance that, in the opinion of the investigator, may compromise the subject's ability to comply with protocol-required trial or procedures or compromise the subject's health, safety, or clinical interpretability.• Severe infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (study enrollment may be delayed).• The patient has received any investigational drugs (except corticosteroids) or exon skipping drugs (including ExonDys 51®), experimental or otherwise, within the last 6 months prior to screening for this study.• The patient has received any type of gene therapy, cell therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy.• The family does not wish to disclose information about the patient's participation in the study to the primary care physician and other health care professionals. Evaluation of effectiveness.

[0360] Dystrophin gene expression serves as the primary outcome measure along with safety. Quantification was performed using validated immunofluorescence and immunoblot assays. The reduction in CK after gene therapy serves as a secondary efficacy criterion. In addition, efficacy was measured using the following functional tests: time to rise from the floor, four-step climb, North Star Ambulatory Assessment (NSAA), 10-meter walk test (10m), and 100-meter walk test (100m). Handgrip dynamometry (HHD) for knee extensors and knee flexors, as well as elbow flexors and elbow extensors, was included in the study.

[0361] Ultrasound-guided muscle biopsies were used to quantify transgene expression from baseline to day 90. Biopsies were performed on the same muscle as the baseline biopsy but on the contralateral leg. One year after dosing, all patients; those crossing over to placebo resumed the study schedule at Visit 1. At the second baseline screening, placebo-treated patients did not undergo the following tests: cardiac MRI and muscle biopsy. Placebo-treated subjects underwent muscle biopsy on day 90 (a total of 3 muscle biopsies). Frozen sections were stained for dystrophies using indirect immunofluorescence (IF). Whole-slide scanning was performed, and microdystrophin intensity and the percentage of positive fibers were quantified using a validated image scan and the MuscleMap™ analysis algorithm.Muscle morphometry was performed blinded, including fiber size histograms. Blinded frozen muscle biopsy sections were used for quantitative microdystrophin protein analysis using a validated Western blot method.

[0362] Puncture biopsy of the gastrocnemius muscle was used to quantify microdystrophin expression (unless there were contraindications for a particular subject, in the investigator's judgment, otherwise the investigator chose an alternative to biopsy). Efficacy analysis

[0363] The primary efficacy endpoint is the change from baseline to day 90 in microdystrophin protein expression, measured by Western blot analysis of biopsied muscle tissue. Differences between treatment groups for the primary efficacy endpoint were assessed using an analysis of covariance (ANCOVA) model with treatment as a fixed factor and baseline as a covariate. The Wilcoxon rank-sum test was used as a secondary analysis. Changes in microdystrophin expression from baseline were similarly analyzed using immunofluorescence (IF) fiber intensity.

[0364] Secondary efficacy endpoints included change from baseline to each scheduled assessment in floor rise time, four-step climb, NSAA, 10-meter walk test (10m), 100-meter walk test (100m), and change in CK. The study included handgrip dynamometry (HHD) for knee extensors and knee flexors, as well as elbow flexors and elbow extensors. Differences between treatment groups for the primary efficacy endpoint were assessed using an analysis of covariance (ANCOVA) model with treatment as a fixed factor and baseline value as a covariate. The Wilcoxon rank-sum test was used as a secondary analysis. Example 4

[0365] The tests and studies described in Examples 2 and 3 above were alternatively performed using the rAAVrh74.MHCK7.microdystrophin construct shown in SEQ ID NO: 9; shown in SEQ ID NO: 8, nucleotides 1-4977; or shown in SEQ ID NO: 6, nucleotides 56-5022. Example 5 Preparation of the pAAV.MCK.micro-dystrophin construct

[0366] Plasmid pAAV.MCK.microdystrophin was constructed by inserting the MCK expression cassette driving a codon-optimized human microdystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski et al., J. Virol., 61(10):3096–3101 (1987)). A muscle-specific regulatory element was included in the construct to drive muscle-specific gene expression. This regulatory element comprised the mouse MCK core enhancer (206 bp) fused to the MCK core promoter 351 bp (proximal). Following the core promoter, the construct included the endogenous mouse MCK exon 1 (untranslated) 53 bp. for efficient transcription initiation, followed by the SV40 late 16S / 19S splicing signals (97 bp) and a short 5'UTR (61 bp). The intron and 5'-UTR were obtained from the pCMVβ plasmid (Clontech). The microdystrophin cassette had a Kozak consensus sequence immediately upstream of the ATG start codon and a small synthetic polyA signal of 53 bp.for mRNA termination. The human microdystrophin cassette contains domains (R4-R23 / Δ71-78) as described previously in Harper et al., Nat. Med. S(3):253-61 (2002).

[0367] The pAAV.MCK.micro-dystrophin plasmid contained the human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeat (ITR) sequences (see Fig. 5). This sequence was encapsidated into AAVrh.74 virions. The molecular clone of serotype AAVrh.74 was cloned from the lymph node of a rhesus macaque and is described in Rodino-Klapac et al., J Transl. Med. 5: 45 (2007). Example 6 Production of rAAV using a hybrid seed reactor system

[0368] The following method can be used to obtain the rAAV constructs described in this application.

[0369] HEK-293 cells were passaged four times under adherent conditions. Before introduction into the penultimate expansion culture, cells were collected and centrifuged to wash out serum (at 300 g for 5 min) and resuspended in serum-free growth medium (EXPI293) in shake flasks under suspension conditions at a seeding density of 0.5+E6 cells / mL. The cells were then allowed to grow and expand for 48-72 hours. The suspension cells were then collected and inoculated into shake flasks or disposable bioreactors, depending on how many viable cells were required for inoculation in the bioreactor. After 72 hours, viable cell concentrations were determined using cell counting equipment. The required volume, preferably containing total viable cells, was then added to the adherent bioreactor containing DMEM and 10% FBS.Additionally, FBS was added appropriately to account for the addition of the serum-free suspension culture volume, such that the final FBS concentration was maintained at 10%.

[0370] As shown in Fig. 18, the cell viability in both the seeding reactor system and the adhesive system was similar. Regarding the viable cell density (VCD), in the hybrid seeding reactor system, the VCD after the 6th transfer was higher than that after the 1st transfer (Fig. 19A), which was comparable to the adhesive system (Fig. 19B).

[0371] After inoculation into the adherent bioreactor (iCELLis®), the adherent culture was briefly transfected with a transgene plasmid carrying the microdystrophin construct described in this application, including, for example, the construct shown in SEQ ID NO: 9, contained in the transgene plasmid in the sequence SEQ ID NO: 8. In addition to the transgene plasmid, a rep / cap plasmid (AAV2 rep / rh74 cap) and a helper plasmid were included. After the required growth period, rAAV particles were collected by cell lysis and column chromatography.

[0372] According to some aspects, rAAV is produced by a suspension seeding method comprising: (a) culturing cells with a first growth medium containing serum in container N-2; (b) removing cells from the first medium; (c) inoculating the cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than that in the first medium in container N-1; (d) culturing the cells in container N-1 under suspension conditions; and (e) inoculating a third medium in a bioreactor with the cells from step (d).

[0373] In some aspects, the suspension seeding method further comprises: (f) transfecting cells with a transgenic plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

[0374] According to some aspects, a transgenic 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. According to some aspects, a plasmid containing an AAV rep gene and an AAV cap gene comprises an AAV2 rep gene and a rAAVrh74 cap gene.

[0375] According to some aspects, the adenovirus helper plasmid comprises the adenovirus 5 E2A gene, E4ORF6, and VA-RNA.

[0376] According to some aspects, the method for seeding a suspension further comprises: (g) lysing the cells. According to some aspects, the cells can be lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, ultrasonic disruption, high-pressure extrusion, detergent lysis, and combinations thereof.

[0377] In some aspects, the method for seeding a suspension further comprises: (h) purifying the rAAV using at least one column chromatography step. In some aspects, said at least one column chromatography step comprises anion exchange chromatography or size exclusion chromatography, or a combination thereof. Example 7. Open-label systemic gene delivery study using commercially representative material to evaluate the safety and expression of the rAAVrh74.MHCK7.microdystrophm construct in subjects suffering from Duchenne muscular dystrophy.

[0378] The study was an open-label, phase 1b study using a commercially representative rAAVrh74.MHCK7.microdystrophin construct in boys with Duchenne muscular dystrophy. Twenty patients were initially enrolled, and Table 7 presents data from the first 11 patients aged <8 years (e.g., 2 patients aged 4-5 years; 9 patients aged 6-7 years) (Cohort 1; up to 20 ambulating male DMD patients aged ≥4 to <8 years) (Table 7). The study was later expanded to include cohort 2 (approximately 6 ambulatory male DMD patients aged ≥8 to <18 years) and cohort 3 (approximately 6 non-ambulatory male DMD patients), as further described in Example 8.

[0379] The primary objectives were to evaluate microdystrophin expression from the rAAVrh74.MHCK7.microdystrophin construct (e.g., commercially representative material) at 12 weeks (Part 1 of the study) post-infusion, measured by Western blotting of muscle tissue, with a corresponding endpoint of change in microdystrophin protein expression amount from baseline to week 12 (Part 1 of the study), measured by Western blotting. Secondary objectives were to evaluate: (1) microdystrophin protein expression by immunofluorescence (IF) intensity of fibers at week 12; (2) microdystrophin expression by IF percentage of dystrophin-positive fibers (PDPF) at week 12; and (3) safety. The study inclusion criteria applicable to Examples 7 and 8 are as follows:

[0380] A subject must meet all of the following criteria to be eligible to participate in this study: 1. Cohort 1 only (ambulatory patients <8 years): Male patient, ambulatory, aged ≥4 to <8 years at screening and has an NSAA score >17 and <26 at the screening visit. 2. Cohort 2 only (ambulatory patients >8 years): Male patient, ambulatory, aged ≥8 to <18 years at screening and has an NSAA score ≥15 and ≤26 at the screening visit. 3. Cohort 3 only (bedridden patients): Male patient, bedridden for at least 9 months, with an NSAA gait score of 0 and the inability to perform the 10MWR test at the screening visit, and a PUL score ≥2.Onset of loss of ambulation is defined as the participant's age or caregiver-reported age at initiation of continuous wheelchair use, normalized to the nearest month.4. Has a definitive diagnosis of MDC prior to screening based on clinical documentation and preliminary confirmatory genetic testing using a clinical diagnostic genetic test.5. Has features of symptomatic muscular dystrophy:• CK elevation >1000 U / L and• Cohorts 1 and 2 (ambulatory patients) only: below 95 percent of the predicted time to complete the 100MWR test.6. Is able to complete a motor assessment study.7. Patients are receiving a stable weekly dose equivalent of oral corticosteroids for at least 12 weeks prior to screening, and the dose is expected to remain constant (except for modifications to account for weight changes) throughout the study.8.Has rAAVrh74 antibody titers ≤1:400 (i.e., not elevated) as determined by ELISA.9. Subjects who are sexually active agree to use condoms throughout the study, and the female sexual partner must also use a medically acceptable form of contraception (e.g., oral contraceptives).10. Has parent(s) or legal guardian(s) or is a subject>18 years of age who is / are capable of understanding and complying with the study visit schedule and all other protocol requirements.11. Is willing to provide informed consent as a minor or assent (if applicable) and has parents or legal guardians or is a subject ≥18 years of age who is willing to provide written informed consent for the subject to participate in the study.The study exclusion criteria were as follows:

[0381] A patient who met any of the following criteria was excluded from this study: 1. Has a left ventricular ejection fraction <40% on screening echoct or clinical signs and / or symptoms of cardiomyopathy. 2. Cohorts 2 and 3 only (ambulatory >8 years and bedridden patients): FVC <50% of predicted value at screening and / or need for ventilatory support at night. 3. Major surgery within 3 months prior to Day 1 or planned surgery at any time during the study. 4. Presence of any other significant genetic disease other than DMD. 5. Has serologic evidence of current, chronic, or active human immunodeficiency virus, hepatitis C, or hepatitis B. 6. Has a diagnosis of an autoimmune disease. 7. Has a comorbidity or need for chronic drug therapy that, in the opinion of the investigator, creates unnecessary risks for gene transfer. 8.The presence of a medical condition or mitigating circumstance that, in the opinion of the investigator, may compromise the subject's ability to comply with protocol-required test procedures or compromise the subject's health, safety, or clinical interpretability.9. Has had a symptomatic infection (e.g., upper respiratory tract infection, pneumonia, pyelonephritis, meningitis) within 4 weeks prior to Day 1.10. Demonstrates cognitive delay or impairment that, in the opinion of the investigator, may impair motor development.11.Taking any of the following medicinal products within the specified time frames:• At any time:- Gene therapy- Cell therapy (e.g., stem cell transplant)- CRISPR / Cas9 or any other form of gene editing• Within 12 weeks of Day 1:- Human growth factor or vamorolone• Within 6 months of Day 1:- Any investigational medicinal product- For Cohort 1 only: any medicinal product designed to increase dystrophin expression (e.g., Translarna™, EXONDYS 51, VYONDYS 53, VILTEPSO™). NOTE: Patients in Cohorts 2 and 3 receiving such medicinal products are expected to discontinue treatment before Day 1. Medications designed to increase dystrophin expression may be restarted and / or started after Week 7212.Received a live virus vaccine within 4 weeks or an inactive vaccine within 2 weeks of the Day 1 visit or expects to receive a vaccine within the first 3 months after Day 1. 13. Has abnormal laboratory values ​​considered clinically significant, including but not limited to: • Gamma-glutamyl transferase (GGT) >2× upper limit of normal (ULN) • Total bilirubin >ULN. Of note, elevated total bilirubin suspected of being due to Gilbert's syndrome is not an exception. • White blood cell count >18,500 / μL • Platelets ≤150,000 / μL 14. The subject or family does not wish to disclose information about the subject's participation in the study to the general practitioner / primary care physician and other healthcare professionals.

[0382] The investigator judged the patient unlikely to comply with the study protocol. All subjects received the rAAVrh74.MHCK7.microdystrophin construct (1.33×10 14(g / kg) (eg, commercially representative material) by single intravenous infusion. Efficacy assessment:

[0383] Muscle biopsies for microdystrophin expression assessment were collected from all subjects at baseline and week 12. Muscle biopsies were collected using open biopsy or VACORA cutting biopsy. Muscle biopsies required tissue harvesting from the medial gastrocnemius muscle. If the medial gastrocnemius muscle was not viable, prior consent from the Sponsor was required for use of an alternative muscle.

[0384] The biopsy specimen was used to quantify transgene expression by Western blotting, IF intensity, and PDPF.

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

[0386] Fig. 20 graphically shows the mean NSAA score from Cohort 1 (the first 11 patients treated with rAAVrh74.MHCK7.microdystrophin). The first 11 patients demonstrated a 3-point improvement from baseline. Patients aged 6 to 7 years (n=9) demonstrated a 2.9-point improvement from baseline. Eleven patients are represented at each time point.

[0387] The safety of the commercially representative material was consistent with previous experience with the rAAVrh74.MHCK7.microdystrophin construct. Seventy-nine treatment-emergent adverse events were observed in 11 patients. The most common adverse event was vomiting, which typically began within the first week, was mild, and was treated with standard antiemetics. Liver enzyme elevations were transient and responsive to steroids, with no evidence of liver dysfunction in any patient. Serious adverse events were observed in two patients and resolved completely. One patient experienced transaminase elevations and received intravenous steroids. Nausea and vomiting were observed in one patient. No adverse events suggestive of a complement-mediated event were observed.

[0388] Overall, the performance of the rAAVrh74.MHCK7.microdystrophin construct was validated with commercially representative material. Robust transduction was observed (e.g., 3.87 average vector genome copies per nucleus). Moderate, robust expression with proper localization to the sarcolemmal membrane was observed (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 representative material. No clinical manifestations of complement were observed. Collectively, these results validate the manufacturing process and analytics and are sufficient for provision of the Duchenne population. Example 8

[0389] The trials and studies described in Example 7 are expanded to approximately 32 subjects in 3 cohorts: Cohort 1 consists of up to 20 ambulatory male subjects with DMD aged ≥4 to <8 years; Cohort 2 consists of approximately 6 ambulatory male subjects with DMD aged ≥8 to <18 years; and Cohort 3 consists of approximately 6 non-ambulatory male subjects with DMD.

[0390] The first 2 enrolled subjects in each cohort will be marker subjects, at least 1 week apart. Cohorts 2 and 3 will include at least 3 subjects weighing <50 kg and at least 3 subjects weighing ≥50 kg. The study consisted of 4 periods: 1. A screening (pre-infusion) period of up to approximately 3 weeks, during which disease signs and baseline therapy were assessed, and pre-infusion assessments were completed. 2. A run-in (pre-infusion) period, which began after eligibility was confirmed and ended on the day preceding Day 1 of the infusion, during which baseline assessments were completed. 3. An infusion period during which all subjects received a single intravenous (IV) infusion of rAAVrh74.MHCK7.micro-dystrophin without placebo control for 31 days prior to sample collection for rAAVrh74 enzyme-linked immunosorbent assay (ELISA). Subjects weighing <70 kg received intravenous rAAVrh74.MHCK7.micro-dystrophin (1.33×1014 vg / kg); subjects weighing ≥70 kg on day 1 received a total fixed dose of rAAVrh74.MHCK7.micro-dystrophin 9.31×10 15 vg / kg, which is equivalent to a dose of 1.33×10 14g / kg for a 70 kg subject. Beginning on the day prior to infusion, subjects received at least 1 mg / kg glucocorticoid (prednisone equivalent) daily in addition to their initial stable oral corticosteroid dose for at least 60 days post-infusion; the addition of 1 mg / kg / day steroids was maintained up to a total daily dose of 60 mg / day (except for added steroids in case of a corresponding increase in GGT and / or other clinically significant liver dysfunction). After infusion, the added glucocorticoid for immunosuppression should be increased to 2 mg / kg / day (or, if the subject is receiving a fixed dose of 60 mg / day, it should be increased to 120 mg / day) if the GGT level is confirmed to be ≥150 U / L or there are other clinically significant liver dysfunctions after infusion. The investigator may make subsequent adjustments to immunosuppressive therapy in response to subsequent acute liver injury or other AEs.A hepatologist should be consulted for significant or severe elevations in liver biochemistry parameters (including GGT, bilirubin, and ALT compared to baseline), or for elevations that do not respond to 2 mg / kg / day or 120 mg / day, respectively. In this situation, intravenous bolus steroids may be considered. The dosages given in this Example 8 and in Example 7 are determined using a linearized DNA standard for qPCR. Otherwise, the dosages given herein are determined using a supercoiled DNA qPCR standard. For example, a dose of 1.33 x 10. 14 vg / kg, indicated in examples 7 and 8, corresponds to a dose of 2×10 14vg / kg, as otherwise described herein. The tests and studies described in Examples 7 and 8 above were conducted using the rAAVrh74.MHCK7.micro-dystrophin construct shown in SEQ ID NO: 9; as set forth in SEQ ID NO: 8, nucleotides 1-4977; or as set forth in SEQ ID NO: 6; nucleotides 56-5022.4. A 260-week follow-up period (post-infusion) will be included during which safety, efficacy, and expression parameters will be assessed. Subjects were required to attend the study site both remotely and in person to complete required procedures / assessments. Part 1 of the follow-up period began after the infusion (Day 1) and lasted until Week 12. Part 2 of the follow-up period began after Week 12 and lasted until Week 260. The first 12 weeks (Part 1) after the infusion required frequent visits (nearly weekly). Additional unscheduled visits are permitted according to the clinical judgment of the Investigator.For patients who complete the study, the final study visit will be at week 260. Patients who discontinue follow-up early after the infusion will require an early discontinuation visit; however, each patient should be strongly encouraged to continue study follow-up until week 260 after the infusion.

[0391] The study was further expanded to include Cohort 4, which consists of approximately 6 ambulatory male subjects with DMD aged ≥3 to <4 years. Subjects in Cohort 4 who were not taking oral corticosteroids for DMD at the time of screening were started on prednisone / prednisolone at a dose of 1.5 mg / kg / day 1 week before the infusion and continued for at least 60 days after the infusion.

[0392] The following exclusion criteria are specified for cohorts 1 and 4 only: Taking any medication intended to increase dystrophin expression (e.g., Translarna™, EXONDYS 51, VYONDYS 53, VILTEPSO™). Medications intended to increase dystrophin expression may be restarted and / or started after week 72. Example 9Genetic Diagnosis

[0393] In additional studies or, alternatively, in the studies described above, as appropriate, subjects had a definitive diagnosis of DMD prior to screening based on clinical data documentation and preliminary confirmatory genetic testing using a clinical diagnostic genetic test.

[0394] A genetic test is used to genotype a patient for at least one mutation in the human dystrophin gene (DMD). As used in this application, the term "genotyping" refers to the process of determining the specific allelic composition of a cell and / or subject at one or more positions in the genome, for example, by determining the nucleic acid sequence at that position. Genotyping refers to nucleic acid analysis and / or analysis at the nucleic acid level.

[0395] According to one aspect, the method for treating DMD described in the present application further comprises genotyping the human dystrophin gene (DMD) from a human subject prior to administering the composition to said human subject.

[0396] In some aspects, the human dystrophin gene (DMD) in a subject is genotyped to characterize a mutation in the gene that can be treated with the compositions disclosed herein.

[0397] According to some aspects, the subject is genotyped for at least one mutation in exons 18-79 of the DMD gene. In particular, the subject is genotyped for a mutation that is expected to result in the absence of the dystrophin protein in the patient. For example, in some aspects, the subject is genotyped for a frameshift deletion, a frameshift duplication, a premature stop, or another pathogenic variant in the DMD gene, contained entirely between exons 18-79. Identification of at least one of these mutations indicates that the patient is suitable for treatment in accordance with the present invention.

[0398] In some aspects, genotyping the DMD gene in a patient may result in a determination that the subject is not suitable for treatment according to the present invention. For example, a genotyping result that reveals a mutation between exons 1-17 inclusive, an in-frame deletion, an in-frame duplication, a variant of uncertain significance ("VUS"), or a mutation contained entirely within exon 45 indicates that the patient is not suitable for treatment according to the present invention.

[0399] Numerous genotyping techniques are known to those skilled in the art. Example 10: A multinational, randomized, double-blind, placebo-controlled phase 3 systemic gene delivery study to evaluate the safety and efficacy of delanistrogens moxeparvovec in patients with Duchenne muscular dystrophy

[0400] The study is a randomized, double-blind, placebo-controlled, 2-part study of systemic gene delivery of the study drug rAAVrh74.MHCK7.microdystrophin (as also described in Examples 6-8 above) in approximately 120 ambulatory male patients with DMD, aged ≥4 to <8 years. Randomization was stratified by age group at randomization (≥4 to <6 years vs. ≥6 to <8 years) and NSAA total score at screening (≤22 vs. >22); at least 50% of subjects were randomized to the age group ≥4 to <6 years at randomization. All patients had the opportunity to receive intravenous (IV) study drug (1.33×10 14vg / kg) in either Part 1 or Part 2 of the study. In the treatment group, participants received a single intravenous (IV) infusion of study drug on Day 1. Participants then received a single IV infusion of matching placebo at Year 2. In the placebo group, participants received a matching IV infusion of placebo on Day 1. Participants then had the opportunity to receive one IV infusion of study drug at Year 2. The study consisted of 4 periods:• A Screening (pre-infusion) period, which began up to 31 days before the Day 1 infusion and during which disease characteristics and baseline therapy were assessed, and pre-infusion assessments were completed.• A Baseline (pre-infusion) period, which began after study participation was confirmed and ended on the day before the Day 1 infusion, during which baseline assessments were completed.• An infusion period in which a single intravenous (IV) infusion of blinded study drug or placebo was administered for 31 days from the date of rAAVrh74 enzyme-linked immunosorbent assay (ELISA) sample collection. Approximately 60 subjects received study drug IV (1.33 x 10. 14vg / kg; determined using a linear DNA qPCR standard), and approximately 60 subjects received placebo (saline, 0.9% sodium chloride) during the infusion period in Part 1 of the study. During the infusion period in Part 2 of the study, subjects who received placebo in Part 1 of the study received study drug IV, and subjects who received study drug in Part 1 of the study received placebo. All patients, parents / caregivers, investigators, and study site staff, except for the open-label pharmacist, will be blinded to study drug or placebo. Beginning on the day prior to infusion, all subjects received an additional glucocorticoid (prednisone equivalent) for at least 60 days.• A 104-week follow-up period (post-infusion in Part 1 of the study) during which safety and efficacy parameters were assessed in Part 1 and Part 2 of the study. Subjects were required to visit the study site both remotely and in-person to complete required procedures / assessments. Additional unscheduled visits were permitted according to the clinical judgment of the Investigator. For patients who completed the study, the last study visit occurred at week 52 of Part 2 of the study. Patients who discontinued follow-up early after an infusion required a study completion / early discontinuation visit; however, each patient was strongly encouraged to continue follow-up until 52 weeks after each infusion.INCLUSION / EXCLUSION CRITERIA:Inclusion Criteria.

[0401] Subject must meet all of the following criteria to be eligible to participate in this study: 1. Is male, ambulatory, and ≥4 to <8 years of age at the time of randomization. 2. Has a definitive diagnosis of DMD prior to screening based on documentation of clinical data and preliminary confirmatory genetic testing using a clinical diagnostic genetic test. The genetic report must describe a frameshift deletion, frameshift duplication, premature stop (nonsense), canonical splice site mutation, or other pathogenic variant in the DMD gene contained entirely between exons 18-79 (inclusive) that is expected to result in the absence of dystrophin protein. a. Mutations between exons 1-17 (inclusive) are not allowed. b. Deletions, in-frame duplications, and variants of uncertain significance (VUS) are not allowed. c.Mutations contained entirely within exon 45 (inclusive) are not allowed. 3. Is capable of undergoing motor testing. 4. Has an NSAA score >16 and <29 at the screening visit. 5. The patient requires <5 seconds to rise from the floor at the screening visit. 6. A stable daily dose of oral corticosteroids is expected for at least 12 weeks prior to screening, and the dose and regimen are expected to remain constant (except for modifications to account for changes in body weight) throughout the study. 7. The patient has rAAVrh74 antibody titers <1:400 (i.e., not elevated) as determined by ELISA. 8. Subjects who are sexually active agree to use condoms throughout the study, and the female sexual partner must also use a medically acceptable form of contraception (e.g., oral contraceptives). 9.Has parent(s) or legal guardian(s) who are(are) able to understand and comply with the study visit schedule and all other protocol requirements.10. Is willing to provide informed consent (if applicable) and has parent(s) or legal guardian(s) who are(are) willing to provide informed consent for the subject's participation in the study.Exclusion Criteria.

[0402] A patient who met any of the following criteria was excluded from this study: 1. Has a left ventricular ejection fraction <40% based on screening echocardiography or clinical signs and / or symptoms of cardiomyopathy. 2. Major surgery within 3 months prior to Day 1 or planned surgery or procedures that may interfere with the study at any time during this study. 3.The presence of any other clinically significant disease, including cardiac, pulmonary, hepatic, renal, hematological, immunological, or behavioral disease, or infection, or malignancy, or comorbidity, or need for chronic drug therapy, which, in the opinion of the investigator, creates unnecessary risks for gene transfer, or a medical condition or mitigating circumstance that, in the opinion of the investigator, may compromise the subject's ability to comply with protocol-required study conditions or procedures or compromise the subject's health, safety, or clinical interpretability. 4. Has serologic evidence of current, chronic, or active human immunodeficiency virus, hepatitis C, or hepatitis B. 5. Has had a symptomatic infection (e.g., upper respiratory tract infection, pneumonia, pyelonephritis, meningitis) within 4 weeks prior to Day 1. 6.7. Use of any of the following medications within the specified time frames:• At any time:- Gene therapy- Cell therapy (e.g., stem cell transplant)- CRISPR7Cas9 or any other form of gene editing• Within 12 weeks from Day 1 and any time during the study:- Use of human growth factor or vamorolone• Within 6 months from Day 1 and any time during the study:- Any investigational medication- Any treatment designed to increase dystrophin expression (e.g., Translarna™, EXONDYS 51™, VILTEPSO™)8. Received a live virus vaccine within 4 weeks or an inactive vaccine within 2 weeks of the Day 1 visit or anticipates receiving a vaccine within the first 3 months after Day 1.9.Has abnormal laboratory values ​​considered clinically significant, including but not limited to:• Gamma-glutamyl transferase >2× upper limit of normal (ULN)• Glutamate dehydrogenase (GLDH) >15 U / L• Total bilirubin > ULN. Note: elevated total bilirubin due to Gilbert's syndrome is not an exception.• White blood cell count >18,500 / μL• Platelets ≤150,000 / μL10. The family does not wish to disclose the subject's participation in the study to the general practitioner / primary care physician and other healthcare professionals.11. The investigator feels that the patient is unlikely to comply with the study protocol.Genetic Diagnosis

[0403] Subjects must have a definitive diagnosis of DMD prior to screening based on clinical data reports and preliminary confirmatory genetic testing using a clinical diagnostic genetic test. The genetic report must describe a frameshift deletion, frameshift duplication, premature stop, or other pathogenic variant in the DMD gene contained entirely between exons 18-79 (inclusive) that is expected to result in the absence of dystrophin protein.a. Mutations between exons 1-17 (inclusive) are not allowed.b. In-frame deletions, in-frame duplications, and variants of uncertain significance (“VUS”) are not allowed.c. Mutations that are contained entirely within exon 45 (inclusive) are not allowed.Statistical Methods:Sample Size

[0404] The sample size for this study is based on the estimated power for the primary efficacy endpoint of change in NSAA total score from baseline to week 52 (Part 1 of the study).

[0405] Assuming a standard deviation of 3.5 for all subjects and a dropout rate of 10% at week 52 (Part 1) with a type 1 error of 0.05 (two-sided), a sample size of 120 subjects with a 1:1 randomization ratio provides approximately 90% power to detect a mean difference of 2.2 in the change in NSAA total score from baseline to week 52 (Part 1 of the study) between the study drug group and the placebo group.

[0406] The study is also conducted for the age group of ≥4 to <6 years. At least 60 patients aged ≥4 to <6 years are enrolled in the study. Assuming a standard deviation of 3.2 for the primary endpoint in the age group of ≥4 to <6 years and a dropout rate of 10% at week 52 (part 1 of the study), with a type 1 error of 0.05 (two-sided), a sample size of 60 subjects with a 1:1 randomization ratio provides at least 80% power to detect a mean difference of 2.5 in the change in NSAA total score from baseline to week 52 (part 1 of the study) between the study drug group and the placebo group.

[0407] The study procedure for adjusting for multiplicity was used to control the overall type 1 error at the two-sided 0.05 level, details are shown in SAP.Randomization

[0408] Subjects were randomized 1:1 to receive STUDY MEDICINE or placebo via a single intravenous infusion. Subjects who received STUDY MEDICINE in Part 1 of the study received placebo in Part 2. Subjects who received placebo in Part 1 of the study had the option of receiving study medication in Part 2.

[0409] Randomization was stratified by age group at randomization (≥4 to <6 years or ≥6 to <8 years) and NSAA total score (≤22 or >22) at screening; at least 50% of patients were randomized to the age group ≥4 to <6 years at randomization. All patients had the opportunity to receive intravenous (IV) study drug (1.33×10 14 (vg / kg) in either Part 1 or Part 2 of the study. Corticosteroids

[0410] Subjects received a stable daily dose of oral corticosteroids for at least 12 weeks prior to screening, and the dose and treatment regimen remained constant (except for modifications to account for changes in body weight) throughout the study. All changes in corticosteroid type, dosing frequency, corticosteroid start and end dates, and dosage were recorded in the patient's primary records and in the eCRF. Pre-infusion immunosuppressants

[0411] On the day before the infusion (study drug or placebo), subjects began taking an additional glucocorticoid (prednisone equivalent) for immunosuppression in addition to their baseline stable oral corticosteroids for DMD. Subjects receiving daily corticosteroid doses at baseline for DMD treatment took their usual dose of DMD corticosteroids in addition to the added immunosuppressant dose of 1 mg / kg / day. The dose of 1 mg / kg / day was maintained for a total daily dose of 60 mg / day. Post-infusion immunosuppressants

[0412] For the first 60 days after infusion, subjects continued to receive the initial stable daily oral corticosteroid dose for the treatment of DMD and additionally received 1 mg / kg / day of glucocorticoid (prednisone equivalent) for immunosuppression. Earlier dose reduction to address an adverse event (AE) may be permitted with the approval of the medical monitor. The do...

Claims

1. A method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by cell propagation in a hybrid seed bioreactor system, comprising: (a) culturing adherent cells under adherent conditions with a first growth medium containing serum in container N-2; (b) removal of adherent cells from the first medium; (c) inoculating the adherent cells from step (b) into a second medium that does not contain serum or contains serum at a concentration lower than that in the first medium, in container N-1; (d) culturing adherent cells in container N-1 under suspension conditions; (e) inoculating the adherent cells from step (d) into a third medium in a bioreactor for growing adherent cultures; and (f) transfecting adherent cells with a transgenic plasmid containing the rAAVrh74.MHCK7.microdystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenoviral helper plasmid to produce rAAV containing the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO:

7.

2. The method according to claim 1, characterized in that said transgenic plasmid containing the rAAVrh74.MHCK7.microdystrophin construct contains: nucleic acid sequence SEQ ID NO: 9; nucleotides 55-5021 of SEQ ID NO: 3; or nucleotides 1-4977 of SEQ ID NO:

8.

3. The method according to claim 1, characterized in that the plasmid containing the AAV rep gene and the AAV cap gene contains the AAV2 rep gene and the rAAVrh74 cap gene.

4. The method according to claim 1, characterized in that said adenovirus helper plasmid includes the adenovirus 5 gene E2A, E4ORF6 and VA-RNA.

5. The method according to any one of paragraphs 1-4, characterized in that the method for cell propagation in the hybrid seed bioreactor system additionally includes: (g) lysis of adherent cells.

6. The method according to claim 5, characterized in that the adherent cells are lysed by freezing-thawing, shear in a solid medium, hypertonic and / or hypotonic lysis, shear in a liquid medium, destruction by ultrasound, high-pressure extrusion, lysis with a detergent, or combinations thereof.

7. The method according to any one of paragraphs 1-6, characterized in that the method for cell propagation in the hybrid seed bioreactor system additionally includes: (h) purifying the rAAV using at least one column chromatography step.

8. The method according to claim 7, characterized in that said at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

9. The method according to any one of paragraphs 1-8, characterized in that the method of cell propagation in the hybrid seed bioreactor system further comprises culturing the cells with the first growth medium in container N-3 and / or in container N-4.

10. The method according to claim 1, characterized in that rAAV is purified from the culture obtained in the bioreactor for growing adhesive cultures.

11. The method according to claim 9 or 10, characterized in that the third medium in the bioreactor contains at least one factor promoting cell adhesion, wherein the at least one factor promoting cell adhesion is selected from the group consisting of serum, fetal bovine serum (FBS), fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix and combinations thereof.

12. The method according to claim 11, characterized in that said third medium in the bioreactor contains DMEM and 10% FBS.

13. The method according to any one of paragraphs 1-12, characterized in that the adherent cells are cultured under suspension conditions for approximately 48-72 hours.

14. The method according to any one of claims 1-13, characterized in that 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.

15. The method according to claim 14, characterized in that said adhesive cells are HeLa cells or HEK-293 cells.

16. The method according to any one of paragraphs 1-15, characterized in that the adhesive cells are not adapted to the suspension.

17. The method according to any one of paragraphs 1-16, characterized in that culturing cells in suspension conditions does not change the dependence of the cells on the type of substrate.

18. The method according to any one of paragraphs 1-17, characterized in that the culturing does not change the cells to form a new cell line.

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