Methods and compositions for treating muscular dystrophy

Abstract

Methods of treating, inhibiting or ameliorating skeletal muscular dystrophy in a subject are provided.SOLUTION: Some embodiments of the methods and compositions provided relate to the treatment, inhibition or amelioration of skeletal muscular dystrophies using a polynucleotide encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) 2a polypeptide. In some embodiments, the muscular dystrophy comprises Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). In some embodiments, the polynucleotide comprises a viral vector, such as an adeno-associated viral (AAV) vector. More embodiments include methods and compositions for screening for therapeutic agents that treat, inhibit or ameliorate skeletal muscular dystrophy, wherein the screening comprises an invitro ventricular muscle tissue model.SELECTED DRAWING: None

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with U.S. government support under grant numbers AR-70517 and AR-69107 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0002] Some embodiments of the methods and compositions provided herein relate to treating, suppressing (preventing), or ameliorating skeletal muscular dystrophy using a polynucleotide encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) 2a polypeptide. In some embodiments, the muscular dystrophy comprises Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). In some embodiments, the polynucleotide comprises a viral vector, such as an adeno-associated virus (AAV) vector. Many embodiments include methods and compositions for screening for therapeutic agents that treat, suppress (prevent), or ameliorate skeletal muscular dystrophy, including in vitro ventricular muscle tissue models. [Background technology]

[0003] Duchenne muscular dystrophy (DMD) is a chronic disease characterized by cardiac and skeletal muscle degeneration, necrosis, and lipid and fibrosis caused by a deficiency of dystrophin, a cytoskeletal protein present in the subsarcolemma. Numerous studies using gene replacement and gene repair to restore dystrophin expression have been conducted. While preclinical data are promising, concerns remain about the potential immunogenicity of newly expressed dystrophin. Furthermore, the complexity of the dystrophin gene—with thousands of pathogenic mutations—makes dystrophin-repair gene therapy challenging. The development of a dystrophin-independent disease-modifying gene therapy could potentially treat all DMD patients without the complications of dystrophin immunity. Summary of the Invention [Means for solving the problem]

[0004] Some embodiments of the methods and compositions provided herein include a method of treating, inhibiting, or ameliorating skeletal muscular dystrophy in a subject, the method comprising administering to the subject a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide.

[0005] In some embodiments, the muscular dystrophy comprises a generalized dystrophin deficiency in the subject. In some embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In some embodiments, the muscular dystrophy comprises DMD.

[0006] In some embodiments, the skeletal muscular dystrophy comprises myocardial remodeling and / or fibrosis.

[0007] In some embodiments, said treating, said inhibiting or said ameliorating results in less myocardial remodeling and / or fibrosis in said subject compared to a subject not administered said polynucleotide.

[0008] In some embodiments, the SERCA polypeptide comprises a SERCA2a polypeptide.

[0009] In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an adeno-associated virus (AAV) vector, a lentiviral vector, and a retroviral vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, the AAV vector encodes an AAV having a serotype selected from AAV serotypes 1 to 12, or a fragment thereof. In some embodiments, the AAV vector encodes an AAV having a serotype selected from AAV serotype 1 (AAV1) and AAV serotype 9 (AAV9), or a fragment thereof.

[0010] In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a cytomegalovirus (CMV) promoter.

[0011] In some embodiments, the polynucleotide is contained in a viral capsid. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid.

[0012] Some embodiments further comprise determining the presence or absence of antibodies to an AAV serotype in said subject.

[0013] In some embodiments, administering the polynucleotide comprises systemic administration, in some embodiments, administering the polynucleotide comprises intravenous administration, in some embodiments, administering the polynucleotide comprises intracoronary infusion, in some embodiments, administering the polynucleotide comprises intrauterine administration.

[0014] In some embodiments, administration of the polynucleotide consists of a single administration of the polynucleotide.

[0015] In some embodiments, the polynucleotide comprises a viral vector and the administration is about 1 x 10 8 Viral genome size: approximately 1 x 10 15 In some embodiments, the polynucleotide is administered in an amount equivalent to about 1 x 10 viral genomes. 13 Viral genome size: approximately 9 x 10 13 The number of viral genomes.

[0016] Some embodiments further comprise administering a vasodilator. In some embodiments, the vasodilator is administered prior to administration of the polynucleotide. In some embodiments, the vasodilator is administered simultaneously with administration of the polynucleotide. In some embodiments, the vasodilator comprises nitroglycerin.

[0017] In some embodiments, the polynucleotide is administered before the onset of muscle tissue damage. In some embodiments, the onset of muscle tissue damage due to the skeletal muscular dystrophy is predicted. In some embodiments, the muscle tissue damage can be measured by muscle histology.

[0018] In some embodiments, the subject is in utero.

[0019] In some embodiments, the subject is a newborn.

[0020] In some embodiments, the subject is at least 3 years old. In some embodiments, the subject is at least 5 years old. In some embodiments, the subject is at least 10 years old.

[0021] In some embodiments, the subject is 20 years of age or younger, or 15 years of age or younger.

[0022] In some embodiments, the subject is between 10 and 20 years old.

[0023] In some embodiments, the subject is non-ambulatory.

[0024] In some embodiments, the subject's cardiac function is reduced compared to cardiac function in a subject without muscular dystrophy.

[0025] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is male.

[0026] In some embodiments, the treatment results in an improvement in the subject's symptoms or indicators of skeletal muscular dystrophy for a period of at least 1 month, 2 months, 3 months, 6 months, 9 months, or 12 months after administration of the polynucleotide, compared to the symptoms or indicators of skeletal muscular dystrophy in an untreated subject. In some embodiments, the period is at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[0027] In some embodiments, the treatment results in an improvement in cardiac tissue or function in the subject compared to cardiac tissue or function in an untreated subject.

[0028] In some embodiments, the treatment results in an improvement in skeletal muscle tissue or function in the subject compared to skeletal muscle tissue or function in an untreated subject.

[0029] In some embodiments, the treatment improves the subject's ventricular function compared to that of an untreated subject. In some embodiments, the improvement in ventricular function comprises an improvement in parameters selected from the group consisting of changes from baseline in left ventricular structure and function assessed by delayed gadolinium enhancement (LGE) cardiac MRI, such as left ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume and / or circumferential strain, regional wall thickness, percentage of left ventricular LGE relative to left ventricular mass, left ventricular viable mass, number of left ventricular segments with LGE; and a composite outcome of changes from baseline in left ventricular function (LVESV).

[0030] In some embodiments, the improvement is Changes from baseline in left ventricular structure and function assessed by delayed gadolinium enhancement (LGE) cardiac MRI, including left ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume and / or circumferential strain, regional wall thickness, percentage of left ventricular LGE relative to left ventricular myocardial mass, left ventricular viable myocardial mass, and number of left ventricular segments with LGE; Composite outcomes of change from baseline in left ventricular function (LVESV), Pulmonary Function Assessment Scale version 2.0 (PUL2.0), selected pulmonary functions, quality of life, and terminal events; and The following items: (a) Skeletal muscle function assessed by PUL2.0, grip strength, key pinch and fingertip pinch strength, elbow flexion strength, 10-meter walk / run time (10MWRT) if ambulatory, incidence of ambulatory failure defined as 10MWRT >30 seconds, and North Star Ambulatory Ability Assessment (NSAA); (b) pulmonary function as assessed by resting vital capacity (SVC), forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), peak expiratory flow (PEF), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak cough flow (PCF), and inspiratory reserve (IFR); or (c) QOL assessed by the DMD Upper Limb Patient-Reported Outcome Scale (DMD UL-PROM) and the Pediatric Outcome Data Collection Questionnaire (PODCI). Change from baseline in The improvement includes improving a parameter selected from the group consisting of:

[0031] Some embodiments further comprise measuring the extent of improvement in the subject after said period of time.

[0032] Some embodiments of the methods and compositions provided herein include the use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to treat, inhibit, or ameliorate skeletal muscular dystrophy in a subject.

[0033] Some embodiments of the methods and compositions provided herein include the use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide in the manufacture of a medicament for treating, inhibiting, or ameliorating skeletal muscular dystrophy in a subject.

[0034] In some embodiments, the muscular dystrophy comprises a generalized dystrophin deficiency in the subject. In some embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In some embodiments, the muscular dystrophy comprises DMD. In some embodiments, the skeletal muscular dystrophy comprises myocardial remodeling and / or fibrosis.

[0035] In some embodiments, the SERCA polypeptide comprises a SERCA2a polypeptide.

[0036] In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an adeno-associated virus (AAV) vector, a lentiviral vector, and a retroviral vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, the AAV vector encodes an AAV having a serotype selected from AAV serotypes 1 to 12, or a fragment thereof. In some embodiments, the AAV vector encodes an AAV having a serotype selected from AAV serotype 1 (AAV1) and AAV serotype 9 (AAV9), or a fragment thereof.

[0037] In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a cytomegalovirus (CMV) promoter.

[0038] In some embodiments, the polynucleotide is contained in a viral capsid. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid.

[0039] In some embodiments, the polynucleotide is formulated for systemic administration, in some embodiments, the polynucleotide is formulated for intravenous administration, in some embodiments, the polynucleotide is formulated for administration by intracoronary infusion, or in some embodiments, the polynucleotide is formulated for intrauterine administration.

[0040] Some embodiments include any of the above uses in combination with a vasodilator, hi some embodiments, the vasodilator comprises nitroglycerin.

[0041] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0042] In some embodiments, the subject is in utero.

[0043] In some embodiments, the subject is an infant.

[0044] In some embodiments, the subject is a newborn.

[0045] In some embodiments, the subject is at least 3 years old. In some embodiments, the subject is at least 5 years old. In some embodiments, the subject is at least 10 years old.

[0046] In some embodiments, the subject is 20 years of age or younger, or 15 years of age or younger.

[0047] In some embodiments, the subject is male.

[0048] Some embodiments of the methods and compositions provided herein include a method of screening for a therapeutic agent that treats, inhibits, or ameliorates skeletal muscular dystrophy in a patient, comprising: (a) contacting a test agent with a ventricular muscle tissue strip; (b) measuring the contraction amplitude of the ventricular muscle tissue strip contacted with the test agent; (c) comparing the contraction amplitude of the ventricular muscle tissue strip contacted with the test agent with the contraction amplitude of the ventricular muscle tissue strip not contacted with the test agent; and (d) determining, based on said comparison, that said test agent contains said therapeutic agent. The method includes:

[0049] In some embodiments, to obtain the ventricular muscle tissue slice, (i) inducing differentiation of a population of induced pluripotent stem cells to obtain a plurality of cardiomyospheric cells comprising a plurality of ventricular myocytes; (ii) dispersing the plurality of cardiomyocytes into single cells to obtain a plurality of cardiomyocytes; and (iii) contacting the plurality of cardiomyocytes with a population of fibroblasts in the presence of collagen under conditions that result in ventricular muscle tissue explants. Do the following.

[0050] Some embodiments further comprise the step of obtaining said ventricular muscle tissue slice, said step of obtaining said ventricular muscle tissue slice comprising: (i) inducing differentiation of a population of induced pluripotent stem cells to obtain a plurality of cardiomyospheric cells comprising a plurality of ventricular myocytes; (ii) dispersing the plurality of cardiomyocytes into single cells to obtain a plurality of cardiomyocytes; and (iii) contacting the plurality of cardiomyocytes with a population of fibroblasts in the presence of collagen under conditions that result in ventricular muscle tissue explants. Includes.

[0051] In some embodiments, the population of induced pluripotent stem cells is obtained from a subject with Duchenne muscular dystrophy (DMD).

[0052] In some embodiments, step (a) is carried out for at least 1 hour. In some embodiments, step (a) is carried out for at least 1 day.

[0053] In some embodiments, the ventricular muscle tissue strip is subjected to an electrical field stimulus at a constant frequency. In some embodiments, step (b) is performed using multiple frequencies.

[0054] In some embodiments, step (b) comprises measuring a parameter selected from force development, normalized force development, velocity variability, force variability, force-frequency relationship, and beta adrenergic response.

[0055] In some embodiments, the muscular dystrophy comprises a generalized dystrophin deficiency in the patient. In some embodiments, the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In some embodiments, the muscular dystrophy comprises DMD.

[0056] In some embodiments, the test agent comprises a polynucleotide. In some embodiments, the polynucleotide encodes a SERCA polypeptide. In some embodiments, the SERCA polypeptide comprises a SERCA2a polypeptide.

[0057] In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an adeno-associated virus (AAV) vector, a lentiviral vector, and a retroviral vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, the AAV vector encodes an AAV having a serotype selected from AAV serotypes 1 to 12, or a fragment thereof. In some embodiments, the AAV vector encodes an AAV having a serotype selected from AAV serotype 1 (AAV1) and AAV serotype 9 (AAV9), or a fragment thereof.

[0058] In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a cytomegalovirus (CMV) promoter. In some embodiments, the promoter comprises an inducible promoter.

[0059] In some embodiments, the polynucleotide is packaged in a viral capsid. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid. [Brief explanation of the drawings]

[0060] [Figure 1A] Schematic diagram of human SERCA2a encoded by an AAV vector with a CMV (cytomegalovirus) promoter. i: intron.

[0061] [Figure 1B] Experimental design: AAV9 (AAV serotype 9) vector encoding SERCA2a was administered via the tail vein of 3-month-old mice at 6 × 10 vg per mouse. Grip strength and treadmill performance were assessed at 11 months of age. When mice reached 21 months of age, serum CK levels, grip strength, treadmill running distance, electrocardiogram, and left ventricular hemodynamics were assessed.

[0062] [Figure 1C] These are immunomicroscopic images of laminin and Flag staining in the hearts of wild-type, mdx, and AAV9.SERCA2a-treated mdx mice. Laminin staining indicates the presence of basement membrane. Flag staining indicates the presence or absence of human SERCA2a expression.

[0063] [Figure 1D] Western blot (left panel) and densitometric analysis (right panel) of lysates prepared from whole hearts of wild-type, mdx, and AAV9.SERCA2a-treated mdx mice. Flag signal indicates the presence of human SERCA2a expression. Vinculin is a loading control. *p<0.05.

[0064] [Figure 1E]Western blot results of sarcoplasmic reticulum / endoplasmic reticulum (SR) preparations from wild-type mice, mdx mice, and AAV9.SERCA2a-treated mdx mice.

[0065] [Figure 1F] The graphs show the change in calcium uptake into the myocardial SR (*p<0.05: comparison between wild-type mice and AAV9.SERCA2a-treated mice) (left panel) and the maximum calcium uptake velocity (Vmax) (*p<0.05) (right panel).

[0066] [Figure 1G] Western blot images of SERCA2a, PLN, and CSQ. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a loading control.

[0067] [Figure 1H] 1 shows a Western blot image of nNOS and a graph showing the quantification results.

[0068] [Figure 2A] Western blot analysis of SERCA2a in four different skeletal muscles from untreated and AAV9.SERCA2a-treated mdx mice. Flag-tag antibody demonstrates the presence of human SERCA2a. Vinculin is a loading control.

[0069] [Figure 2B] These are immunomicrographs of laminin and Flag staining in four different types of skeletal muscle from AAV9.SERCA2a-treated mdx mice. Laminin staining indicates the presence of basement membrane. Flag signal indicates the presence of human SERCA2a expression.

[0070] [Figure 2C]The graphs show the change in calcium uptake into skeletal muscle SR (* p<0.05: comparison between wild-type mice and AAV9.SERCA2a-treated mice, $ p<0.05: comparison between wild-type mice and untreated mdx mice, # p<0.05: comparison between wild-type mice and all mdx mice (regardless of AAV administration)) (left panel) and the maximum velocity of calcium uptake (Vmax) (* p<0.05) (right panel).

[0071] [Figure 3A] These are micrographs of hematoxylin and eosin (H&E) staining and Masson's trichrome staining of the hearts of wild-type mice, mdx mice, and AAV9.SERCA2a-treated mdx mice (fibrotic tissue is stained blue with Masson's trichrome staining) (left panel), and the measurement results of the cardiac fibrotic area (right panel).

[0072] [Figure 3B] ECG evaluation results showing heart rate, PR interval, QRS duration, QTc interval, Q amplitude and cardiomyopathy index.

[0073] [Figure 3C] Cardiac catheter evaluation results showing left ventricular end-systolic volume, dP / dt max, systolic blood pressure, end-diastolic volume, dP / dt min, and ejection fraction.

[0074] [Figure 3D] Pressure-volume loops of wild-type mice, mdx mice, and AAV9.SERCA2a-treated mdx mice. *p<0.05.

[0075] [Figure 3E] These are Masson's trichrome stained micrographs of whole heart cross sections from 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-administered mdx mice.

[0076] [Figure 4A] This is the measurement result of forelimb grip strength.

[0077] [Figure 4B] Graphs showing absolute treadmill distance (left panel) and distance normalized by body weight (right panel). *p<0.05. Data are presented as mean±standard error of the mean.

[0078] [Figure 5A] 1 is a graph showing forelimb grip strength in 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-treated mdx mice.

[0079] [Figure 5B] This graph shows the treadmill running distance of 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-treated mdx mice. *p<0.05. Data are shown as mean±standard error of the mean.

[0080] [Figure 5C] These are the results of measuring serum creatine kinase (CK) levels in 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-administered mdx mice.

[0081] [Figure 6A] These are Masson's trichrome stained micrographs of whole sections of 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-administered mdx mice.

[0082] [Figure 6B] These are high-magnification micrographs of hematoxylin and eosin (HE) staining and Masson's trichrome staining of 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-administered mdx mice.

[0083] [Figure 6C] The results are for the measurement of the fibrotic area. The sample size represents the number of animals used in the test.

[0084] [Figure 6D]The number of muscle fibers with central nuclei was measured. Sample size represents the number of animals used in the study.

[0085] [Figure 6E] 1 is a graph measuring muscle fiber size distribution. Sample size represents the number of muscle fibers measured.

[0086] [Figure 7A] These are Masson's trichrome stained micrographs of whole sections of 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-administered mdx mice.

[0087] [Figure 7B] These are high-magnification micrographs of hematoxylin and eosin (HE) staining and Masson's trichrome staining of 21-month-old wild-type mice, mdx mice, and AAV9.SERCA2a-administered mdx mice.

[0088] [Figure 7C] The results are for the measurement of the fibrotic area. The sample size represents the number of animals used in the test.

[0089] [Figure 7D] The number of muscle fibers with central nuclei was measured. Sample size represents the number of animals used in the study.

[0090] [Figure 7E] 1 is a graph measuring muscle fiber size distribution. Sample size represents the number of muscle fibers measured.

[0091] [Figure 8A] Immunostained micrographs (upper panel) and quantification results (lower panel) of the tibialis anterior muscle from wild-type mice, mdx mice, and AAV.SERCA2a-treated mdx mice. * p<0.05.

[0092] [Figure 8B]Immunostained micrographs (upper panel) and quantification results (lower panel) of the quadriceps muscles of wild-type mice, mdx mice, and AAV.SERCA2a-administered mdx mice. * p<0.05.

[0093] [Figure 8C] Immunostained micrographs (upper panel) and quantification results (lower panel) of gastrocnemius muscles from wild-type mice, mdx mice, and AAV.SERCA2a-administered mdx mice. * p<0.05.

[0094] [Figure 9] Schematic diagram of one embodiment of a Phase 2 trial design. Abbreviations: Pre-Scr: pre-screening, Scr: screening, Rand: randomization, vg: viral genome count, D: day, W: week, M: month. DETAILED DESCRIPTION OF THE INVENTION

[0095] Some embodiments of the methods and compositions provided herein relate to the treatment, suppression (prevention), or amelioration of skeletal muscular dystrophy and cardiac dystrophy using polynucleotides encoding SERCA2a polypeptides. In some embodiments, the muscular dystrophy comprises Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). In some embodiments, the polynucleotide comprises a viral vector, such as an adeno-associated virus (AAV) vector. Many embodiments include methods and compositions for screening for therapeutic agents that treat, suppress, or ameliorate skeletal muscular dystrophy, including in vitro ventricular muscle tissue models.

[0096] DMD is caused by a deficiency of dystrophin. One of the pathogenic features of DMD is a marked increase in cytosolic calcium. Supraphysiological levels of intracellular calcium lead to protein degradation, membrane damage, and ultimately muscle necrosis and dysfunction. Sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) is a calcium pump that transports cytosolic calcium into the sarcoplasmic reticulum during excitation-contraction coupling.

[0097] Some embodiments of the methods and compositions provided herein involve systemic delivery of SERCA2a by AAV to improve calcium recycling and provide long-lasting benefits to DMD. In some embodiments, the delivery is a single administration. As disclosed herein, 3-month-old mdx mice, a model of DMD, are injected with an AAV9 human SERCA2a vector (6×10 12 Intravenous administration of AAV9 SERCA2a (1000 viral genomes / mouse) resulted in robust expression of human SERCA2a in heart and skeletal muscle, sustained for 18 months, as confirmed by immunohistochemistry and Western blot analysis. Concomitantly, significant improvements in sarcoplasmic / endoplasmic reticulum calcium uptake were observed in both heart and skeletal muscle. SERCA2a therapy significantly improved grip strength and treadmill performance, completely prevented myocardial fibrosis, and normalized electrocardiogram (ECG). Cardiac catheterization confirmed normalization of multiple systolic and diastolic hemodynamic parameters in treated mice. Importantly, ventricular dilation was completely prevented, and ejection fraction was restored to wild-type levels. These results demonstrate that a single systemic administration of AAV9 SERCA2a has long-lasting effects in an art-recognized DMD animal model.

[0098] Mounting evidence suggests that excessive calcium influx into the cytoplasm plays a crucial role in the development of DMD. 1-4Specifically, high levels of intracellular calcium activate calcium-sensitive calpain proteases and phospholipase A2, which cause proteolysis and membrane damage. Calcium dysregulation also induces free radical production and impaired mitochondrial function. Ultimately, this elevated calcium leads to muscle fiber necrosis and muscle dysfunction. Therefore, without being bound by theory, it is expected that restoration of calcium homeostasis, as demonstrated by the present disclosure, will alleviate muscle pathology in DMD.

[0099] SERCA is a calcium pump that transports calcium from the cytoplasm into the SR lumen against its concentration gradient. 5,6 In cardiac myocytes, SERCA is responsible for over 70% of calcium export from the cytoplasm. Among the various SERCA isoforms, SERCA1a and SERCA2a are the isoforms naturally expressed in adult muscle. 5 SERCA1a is selectively expressed in skeletal muscle, whereas SERC2a is expressed in both skeletal and cardiac muscle. Some embodiments provided herein include increasing SERCA2a expression to correct excessive calcium influx and alleviate both skeletal muscle disease and cardiomyopathy in DMD.

[0100] Adeno-associated virus serotype 9 (AAV9) is a vector used for systemic gene transfer to skeletal muscle and the heart. 7 Systemic AAV9 therapy has been successfully used to treat neuromuscular diseases in human patients. 8 Additionally, two clinical trials of systemic AAV9 gene therapy for DMD patients have begun. 9,10 Some embodiments disclosed herein involve a single intravenous administration of a human SERCA2a AAV9 vector to achieve lifelong disease freedom in mdx mice, a model of DMD. Mice were treated at 3 months of age and followed for the rest of their lives. 11,12Administration of AAV9 resulted in the expression of human SERCA2a in muscles throughout the body and significantly promoted calcium uptake into the SR. Furthermore, administration significantly improved overall muscle strength and ameliorated fatal dilated cardiomyopathy, which are important results. Some embodiments disclosed herein involve the development of dystrophin-independent gene therapy for DMD based on the administration of a SERCA2a AAV vector (e.g., AAV9).

[0101] Some embodiments of the methods and compositions provided herein include aspects disclosed in U.S. Patent Publication Nos. 2008 / 0076730, 8221738, and 2017 / 0296790, each of which is incorporated herein by reference in its entirety. Some embodiments of the methods and compositions provided herein include aspects disclosed in Wasala NB et al. (2019) Molecular Therapy 28: 845-854, which is incorporated herein by reference in its entirety.

[0102] definition As used herein, the terms "polynucleotide," "nucleic acid," or "nucleic acid molecule" have their common and ordinary meanings as interpreted throughout the present specification, and may refer to, for example, polymers composed of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments obtained by polymerase chain reaction (PCR), and fragments obtained by ligation, cleavage, endonuclease action, or exonuclease action. Nucleic acid molecules may be composed of natural nucleotide monomers (e.g., DNA or RNA), analogs of natural nucleotides (e.g., enantiomers of natural nucleotides), or combinations thereof. Modified nucleotides may have modifications in the sugar moiety and / or pyrimidine or purine base moieties. Modifications in the sugar moiety include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, or azide groups, and the sugar moiety may be etherified or esterified. Furthermore, the entire sugar moiety may be replaced with sterically or electronically similar structures, such as azasugars and carbocyclic sugar analogs. Modified base moieties include alkylated purines, alkylated pyrimidines, acylated purines, acylated pyrimidines, and other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs thereof. Phosphodiester-like linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, and phosphoramidate linkages. The term "nucleic acid molecule" also encompasses so-called "peptide nucleic acids." Peptide nucleic acids contain natural or modified nucleobases attached to a polyamide backbone. Nucleic acids can be single-stranded or double-stranded. In some embodiments, a nucleic acid sequence encoding a fusion protein is provided. In some embodiments, the nucleic acid is RNA or DNA.

[0103] As used herein, the term "encode" has its ordinary and usual meaning as interpreted throughout this specification, and may refer, for example, to the property of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of another macromolecule, such as a predetermined amino acid sequence. Thus, when transcription and translation of mRNA corresponding to a particular gene produces a protein in a cell or other biological system, the gene is said to encode the protein.

[0104] As used herein, the terms "vector," "expression vector," or "construct" have their common and ordinary meanings as interpreted throughout this specification, and refer to a nucleic acid used to introduce heterologous nucleic acid into a cell, which contains various regulatory elements and allows the heterologous nucleic acid to be expressed in the cell. Vectors include, but are not limited to, plasmids, minicircles, yeast, and viral genomes. In some embodiments, the vector is an AAV vector, foamy virus vector, adenovirus vector, retrovirus vector, or lentivirus vector. In some embodiments, the vector is a vector for protein expression in mammalian systems, such as humans.

[0105] As used herein, the term "promoter" has its common and ordinary meaning as interpreted throughout this specification and refers to a region of DNA that initiates transcription of a specific gene. The promoter may be located upstream of the same DNA strand (5' region of the sense strand) near the transcription start site of the gene. The promoter may be a conditional and inducible promoter, or a constitutive promoter. The promoter may be specific for protein expression in bacterial cells, mammalian cells, or insect cells.

[0106] As used herein, the terms "conditional" or "inducible" have their common and ordinary meaning as interpreted throughout this specification and refer to a nucleic acid construct, such as a promoter, that functions to cause gene expression in the presence of an inducer but not substantially cause gene expression in the absence of the inducer. Inducible promoters for mammalian expression constructs include, but are not limited to, promoters inducible by tetracycline, ecdysone, streptogramin antibiotics, macrolide antibiotics, or doxycycline.

[0107] As used herein, the term "constitutive" has its ordinary and usual meaning as interpreted throughout this specification, and refers to a nucleic acid construct, such as a promoter, that functions constitutively to express a polypeptide that is continuously produced.

[0108] As used herein, the terms "infusion," "infused," and "infuse" have their common and ordinary meanings as interpreted throughout this specification, and refer to administration over a period of time substantially longer (usually one minute or longer) than the art-recognized terms "injection" or "bolus injection" (usually less than one minute). The flow rate of an infusion is at least partially affected by the dose, but the flow rate of an "infusion" is slower than the flow rate of an "injection" for the same volume.

[0109] As used herein, the terms "in conjunction with," "in combination with," "simultaneously," or "concurrently" have their common and ordinary meanings as interpreted throughout the present specification, and include the administration of one therapeutic method in addition to the administration of another therapeutic method. For example, it is contemplated that a polynucleotide of the present invention may be injected into a subject, and an art-recognized pharmaceutical composition may be administered to the same individual. As used herein, the terms include simultaneous and sequential administration of multiple therapeutic methods.

[0110] As used herein, "treating" a disease or "treatment" of a disease has its ordinary and ordinary meaning as interpreted in the context of the entire specification and includes stabilizing, curing, or less than curing the disease, and includes arresting or delaying the progression of the disease or signs or symptoms of the disease. The term "prevention" has its ordinary and ordinary meaning as interpreted in the context of the entire specification and includes complete or incomplete prevention of a disease or signs or symptoms of a disease, or delaying the onset or appearance of signs or symptoms of a disease, or altering the course of a disease. The terms "therapeutic," "therapeutic effect," or "clinical effect" include both treatment and prevention.

[0111] As used herein, the term "adeno-associated virus" or "AAV" encompasses all subtypes, serotypes, and pseudotypes, as well as all naturally occurring and recombinant forms. Various AAV serotypes and strains are known in the art and are publicly available from academic or commercial sources, such as the ATCC. Additionally, published AAV serotype and strain sequences and / or sequences available from various databases can be synthesized using known techniques.

[0112] As used herein, the term "serotype" refers to a characteristic that distinguishes one AAV from another, defined by the reactivity of the capsid protein with a given antiserum. At least 12 serotypes of human AAV, including AAV1 to AAV12, are known, but additional serotypes continue to be discovered, and the use of newly discovered serotypes is also anticipated. For example, "AAV2 serotype" refers to an AAV containing the capsid protein encoded by the AAV2 cap gene and a genome including the 5' inverted terminal repeat (ITR) sequence and 3' inverted ITR sequence of the same AAV2 serotype.

[0113] The term "pseudotyped" AAV refers to an AAV containing capsid proteins from one serotype and a viral genome containing 5' inverted terminal repeat (ITR) sequences and 3' inverted ITR sequences from a different or heterologous serotype. Pseudotyped rAAVs are considered to have the cell surface binding characteristics of the capsid serotype and the genetic characteristics consistent with the ITR serotype. Pseudotyped rAAVs may contain AAV capsid proteins and ITRs, including VP1, VP2, and VP3, from any AAV serotype, including primate AAV serotypes AAV1 through AAV12, as long as the capsid protein and ITR serotypes are heterologous. In pseudotyped rAAVs, the 5' and 3' ITRs may be from the same serotype or from heterologous serotypes. Pseudotyped rAAVs are produced using standard techniques described in the art.

[0114] "Chimeric" rAAV vectors encompass AAV vectors containing heterologous capsid proteins. These AAV vectors are rAAV vectors in which the capsid proteins VP1, VP2, and VP3 are chimeric, i.e., rAAV vectors in which the capsid proteins VP1, VP2, and VP3 are not necessarily all of the same serotype. The term "chimeric AAV" as used herein encompasses AAVs in which the capsid proteins VP1, VP2, and VP3 are of different serotypes. For example, but not limited to, the capsid proteins VP1, VP2, and VP3 may be the capsid proteins of AAV1 and AAV2, or a mixture of other parvovirus capsid proteins. Chimeric AAVs may also contain other viral or other proteins, such as proteins that target AAV to desired cells or tissues. The term "chimeric rAAV" as used herein also encompasses rAAVs in which the 5'ITR and 3'ITR are chimeric. The present invention encompasses chimeric rAAV vectors containing ITRs of different AAV serotypes, such as the ITRs of AAV1 and AAV2. The chimeric rAAV may also contain synthetic sequences.

[0115] rAAV viral vectors can be produced by many methods known in the art, such as the transient transfection method described in U.S. Patent No. 6,001,650 and U.S. Patent No. 6,258,595. These documents are incorporated herein by reference. Generally, the production of rAAV vectors requires four common elements: 1) Permissive host cells for replication, including standard host cells known in the art, such as 293-A cell line, 293-S cell line (obtained from BioReliance), VERO cell line, HeLa cell line, etc., that are applicable to the vector production system described herein; 2) Helper virus function: When used in the transduction production system, it is supplied as the plasmid pAd Helper 4.1, which expresses the E2a gene, E4-orf6 gene, and VA gene of adenovirus type 5 (Ad5); 3) a rep-cap construct with trans-packaging functions; and 4) The gene of interest flanked by AAV ITR sequences. Production by transfection may be carried out similarly to the method described in Sandalon et al., J. Virology, 2004; 78(22):12355-12365, which is incorporated herein by reference.

[0116] Therapeutic methods and compositions Some embodiments of the methods and compositions provided herein include therapeutic methods and agents for treating, inhibiting, or ameliorating muscular dystrophy in a subject. Aspects useful for certain embodiments provided herein, such as therapeutic compositions and methods, including kits, and delivery routes and methods, are disclosed in U.S. Patent No. 8,221,738 and U.S. Patent Publication No. 2017 / 0252462, each of which is incorporated herein by reference in its entirety. In some embodiments, the muscular dystrophy is present in skeletal muscle. For example, the muscular dystrophy is present in a muscle other than cardiac muscle. In some embodiments, the muscular dystrophy is present in Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, or myotonic dystrophy. In some embodiments, the muscular dystrophy is present in a mutation in the dystrophin gene. In some embodiments, the muscular dystrophy is present in a subject with generalized dystrophin deficiency. For example, the level or function of dystrophin expressed in the skeletal muscle of a subject with muscular dystrophy is reduced compared to the level or function of dystrophin expressed in the skeletal muscle of a subject without muscular dystrophy. In some embodiments, the muscular dystrophy is selected from DMD and BMD. In some embodiments, the muscular dystrophy includes DMD. In some embodiments, the muscular dystrophy includes myocardial remodeling and / or fibrosis.

[0117] Some embodiments include administering to a subject a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide. In some embodiments, the SERCA polypeptide comprises a SERCA2 polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2 isoform selected from a SERCA2a polypeptide and a SERCA2c polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2a polypeptide.

[0118] In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a cytomegalovirus (CMV) promoter.

[0119] In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an adeno-associated virus (AAV) vector, a lentiviral vector, and a retroviral vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, the polynucleotide is encapsulated in a viral capsid. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid. In some embodiments, the AAV vector encodes an AAV having a specific serotype or a fragment thereof. In some embodiments, the AAV or fragment thereof comprises a viral capsid protein or a fragment thereof. In some embodiments, the AAV or fragment thereof has a serotype selected from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAVrhlO), AAV serotype 11 (AAV11), and AAV serotype 12 (AAV12). In some embodiments, the AAV or fragment thereof has a serotype selected from AAV1 and AAV9. In some embodiments, the AAV or fragment thereof has the AAV1 serotype. In some embodiments, the AAV or fragment thereof has the AAV9 serotype. In some embodiments, the AAV or fragment thereof may comprise a hybrid serotype consisting of one or more of the above serotypes.

[0120] Some embodiments further comprise determining the presence or absence of antibodies to an AAV serotype in the subject. In some embodiments, the AAV or fragment thereof encoded by the polynucleotide may be selected to have an AAV serotype that is different from the AAV serotype recognized by the antibodies identified in the subject.

[0121] In some embodiments, administration of the polynucleotide comprises systemic administration. In some embodiments, administration of the polynucleotide comprises injection, infusion, or implantation. In some embodiments, administration of the polynucleotide comprises intravenous administration. In some embodiments, administration of the polynucleotide comprises intrauterine administration, for example, intrauterine administration to a subject in utero who is predicted to develop the muscular dystrophy.

[0122] In some embodiments, administering the polynucleotide comprises one or more administrations of the polynucleotide to the subject.

[0123] In some embodiments, administering the polynucleotide comprises a single administration of the polynucleotide to the subject. In some embodiments, administering the polynucleotide comprises two administrations of the polynucleotide to the subject. In some embodiments, administering the polynucleotide comprises three administrations of the polynucleotide to the subject. In some embodiments, administering the polynucleotide comprises four administrations of the polynucleotide to the subject. In some embodiments, administering the polynucleotide comprises five administrations of the polynucleotide to the subject.

[0124] In some embodiments, the polynucleotide comprises a viral vector. In some embodiments, the polynucleotide is encapsulated in a viral capsid or a functional fragment thereof. In some such embodiments, the dosage of the polynucleotide can be measured as viral genome counts (vg) or DNase-resistant particle counts (DRP).

[0125] In some embodiments, the dose of the polynucleotide is at least 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12vg, 1×10 13 vg, 1×10 14 vg, 1×10 15 vg, 1×10 16 vg, 1×10 17 vg, 1×10 18 vg, 1×10 19 vg, 1×10 20 In some embodiments, the polynucleotide is administered in an amount of at least 1×10 vg, or in an amount ranging between any two of these amounts. 8 DRP, 1×10 9 DRP, 1×10 10 DRP, 1×10 11 DRP, 1×10 12 DRP, 1×10 13 DRP, 1×10 14 DRP, 1×10 15 DRP, 1×10 16 DRP, 1×10 17 DRP, 1×10 18 DRP, 1×10 19 DRP, 1×10 20 In some embodiments, the polynucleotide is administered in an amount of about 1 x 10 DRP, or in an amount ranging between any two of these amounts. 8 vg~approx. 1×10 20 vg, approx. 1×10 9 vg~approx. 1×10 17 vg, approx. 1×10 10 vg~approx. 1×10 16 vg, approx. 1×10 11 vg~approx. 1×10 15 vg or approximately 1×10 12 vg~approx. 1×10 14 In some embodiments, the dose of the polynucleotide is about 1 x 10 13 vg~approx. 9×10 13 In some embodiments, the dose of the polynucleotide is about 1 x 10 8 DRP ~ approx. 1×10 20 DRP, approx. 1×10 9 DRP ~ approx. 1×10 17 DRP, approx. 1×10 10 DRP ~ approx. 1×1016 DRP, approx. 1×10 11 DRP ~ approx. 1×10 15 DRP or approximately 1 × 10 12 DRP ~ approx. 1×10 14 In some embodiments, the dose of the polynucleotide is about 1 x 10 13 DRP ~ approx. 9×10 13 It is DRP.

[0126] Some embodiments further comprise administering a vasodilator. In some embodiments, the vasodilator is administered prior to administration of the polynucleotide. In some embodiments, the vasodilator is administered simultaneously with administration of the polynucleotide. In some embodiments, the vasodilator comprises nitroglycerin.

[0127] In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is male. In some embodiments, the subject is an infant. As used herein, "infant" includes a human child under the age of four. In some embodiments, the subject is a newborn. As used herein, "newborn" includes a human infant under one month of age. In some embodiments, the subject is in utero. In some embodiments, the subject does not exhibit substantial physical symptoms of muscular dystrophy. For example, the subject may be a subject predicted to develop muscular dystrophy, such as a subject with a mutation in a gene such as the dystrophin gene. In some embodiments, the subject does not exhibit physical symptoms of muscular dystrophy, such as elevated creatine kinase, pseudohypertrophy of the gastrocnemius muscle, or Gowers' sign, which indicates weakness in proximal muscles. In some embodiments, the polynucleotide is administered before the onset of muscle tissue damage. In some embodiments, the onset of muscle tissue damage due to skeletal muscular dystrophy is predicted. In some embodiments, the muscle tissue damage may or may not be measurable by muscle histology.In some embodiments, the subject is at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old, 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old, 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 1 year ... The age range is 1 month or younger, 1 year or younger, 2 years or younger, 3 years or younger, 4 years or younger, 5 years or younger, 6 years or younger, 7 years or younger, 8 years or younger, 9 years or younger, 10 years or younger, 11 years or younger, 12 years or younger, 13 years or younger, 14 years or younger, 15 years or younger, 16 years or younger, 17 years or younger, 18 years or younger, 19 years or younger, or 20 years or younger, or an age range with any two of these age ranges as the upper and lower limits, for example, 1 month to 20 years old, 1 month to 15 years old, 1 month to 12 years old, 1 month to 10 years old, 3 years to 20 years old, 3 years to 15 years old, 3 years to 10 years old, 8 years to 20 years old, or 8 years to 15 years old.

[0128] In some embodiments, the treatment results in a long-term improvement in one or more symptoms or indicators of skeletal muscular dystrophy, such as DMD, in a treated subject compared to one or more such symptoms in an untreated subject with skeletal muscular dystrophy. In some embodiments, the improvement is observed for a period of at least 3 months, 6 months, 9 months, or 12 months after administration of the treatment. In some embodiments, the improvement is observed for a period of at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years after administration of the treatment. In some embodiments, the improvement is an improvement in one or more indicators of cardiac function or cardiac tissue. In some embodiments, the improvement is an improvement in one or more indicators of skeletal muscle function or skeletal muscle tissue. In some embodiments, the improvement is an improvement in one or more indicators of cardiac and skeletal muscle function or cardiac and skeletal muscle tissue. The improvement includes those described herein, for example, improvements in the indicators described in the Examples below. For example, improvement in cardiac and / or skeletal muscle function or cardiac and / or skeletal muscle tissue includes one or more of the following: Left ventricular structure and function assessed by delayed gadolinium enhancement (LGE) cardiac MRI included changes from baseline in left ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume and / or circumferential strain, regional wall thickness, left ventricular LGE expressed as a percentage of left ventricular myocardial mass and in grams, left ventricular viable myocardial mass expressed in grams, and the number of left ventricular segments with LGE; Composite outcome of change from baseline in left ventricular function (LVESV), PUL2.0, pulmonary function (select one parameter), quality of life, and terminal events; and The following items: (a) Skeletal muscle function assessed by PUL2.0, grip strength, key pinch and fingertip pinch strength, elbow flexion strength, 10-meter walk / run time (10MWRT) if ambulatory, incidence of ambulatory failure defined as 10MWRT >30 seconds, and North Star Ambulatory Ability Assessment (NSAA); (b) pulmonary function assessed by resting vital capacity (SVC), forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), peak expiratory flow (PEF), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak cough flow (PCF), and inspiratory reserve (IFR); and (c) QOL assessed by DMD UL-PROM and PODCI Change from baseline in

[0129] Some embodiments include a method of treating, inhibiting, or ameliorating skeletal muscular dystrophy, such as DMD, in a subject, such as a human male, prior to the onset of muscle tissue damage predicted to result from the skeletal muscular dystrophy, the method comprising administering to a subject a human subject, the subject, a human male, a human subject, or a human subject, the ... 13 vg as a single intravenous dose. Some embodiments further include administering in combination with a vasodilator, such as nitroglycerin.

[0130] Some embodiments include a method of treating, inhibiting, or ameliorating skeletal muscular dystrophy, such as DMD, in a subject, such as a human male, prior to the onset of muscle tissue damage predicted to result from the skeletal muscular dystrophy, the method comprising administering to a subject a human subject, the subject, a human male, a human subject, or a human subject, the ... 13 vg via intracoronary infusion. Some embodiments further include administering in combination with a vasodilator, such as nitroglycerin.

[0131] Some embodiments include a method of treating, inhibiting, or ameliorating skeletal muscular dystrophy, such as DMD, in a subject, such as a human male, prior to the onset of muscle tissue damage predicted to result from the skeletal muscular dystrophy, the method comprising administering to a subject a human subject, the subject, a human male, a human subject, or a human subject, the ... 13 vg via intracoronary infusion, wherein the administration is via antegrade epicardial intracoronary infusion into the left and / or right coronary artery using a commercially available guide catheter or diagnostic cardiac catheter and a B. Braun Perfusor® Space syringe pump at a flow rate of 300 mL / hr over 10 minutes. In some embodiments, intravenous infusion of nitroglycerin is administered at the maximum tolerated dose for at least 10 minutes prior to and concurrently with the AAV1 vector infusion.

[0132] Therapeutic agent screening Some embodiments of the methods and compositions provided herein include screening for therapeutic agents that treat, inhibit, or ameliorate skeletal muscular dystrophy in a patient. In some embodiments, the skeletal muscular dystrophy comprises a generalized dystrophin deficiency in the patient. In some embodiments, the skeletal muscular dystrophy is selected from DMD and BMD. In some embodiments, the muscular dystrophy comprises DMD. In some embodiments, the muscular dystrophy comprises myocardial remodeling and / or fibrosis.

[0133] Some such embodiments include: (a) contacting a test agent with a ventricular muscle tissue strip; (b) measuring the contraction amplitude of the ventricular muscle tissue strip contacted with the test agent; (c) comparing the contraction amplitude of the ventricular muscle tissue strip contacted with the test agent with the contraction amplitude of the ventricular muscle tissue strip not contacted with the test agent; and (d) determining, based on said comparison, that said test agent contains said therapeutic agent. Includes.

[0134] In some embodiments, to obtain the ventricular muscle tissue slice, (i) inducing differentiation of a population of induced pluripotent stem cells to obtain a plurality of cardiomyospheric cells comprising a plurality of ventricular myocytes; (ii) dispersing the plurality of cardiomyocytes into single cells to obtain a plurality of cardiomyocytes; and (iii) contacting the plurality of cardiomyocytes with a population of fibroblasts in the presence of collagen under conditions that result in ventricular muscle tissue explants. Do the following. Some embodiments further comprise the step of obtaining said ventricular muscle tissue slice, said step of obtaining said ventricular muscle tissue slice comprising: (i) inducing differentiation of a population of induced pluripotent stem cells to obtain a plurality of cardiomyospheric cells comprising a plurality of ventricular myocytes; (ii) dispersing the plurality of cardiomyocytes into single cells to obtain a plurality of cardiomyocytes; and (iii) contacting the plurality of cardiomyocytes with a population of fibroblasts in the presence of collagen under conditions that result in ventricular muscle tissue explants. Includes. In some embodiments, the population of induced pluripotent stem cells is obtained from a subject with a muscular dystrophy, such as DMD or BMD.

[0135] In some embodiments, the test agent is contacted with the ventricular muscle tissue strip for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 18 hours, 24 hours, or any two of these values. In some embodiments, the test agent is contacted with the ventricular muscle tissue strip for at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, or any two of these values.

[0136] In some embodiments, an electric field stimulus is applied to the ventricular muscle tissue strip contacted with the test agent during measurement of the contraction amplitude of the ventricular muscle tissue strip. In some embodiments, the frequency of the electric field may be constant during measurement. In some embodiments, the frequency of the electric field may be modulated during measurement. In some embodiments, a parameter is determined during measurement. In some embodiments, the parameter is selected from force development, normalized force development, velocity variability, force variability, force-frequency relationship, and beta-adrenergic response.

[0137] In some embodiments, the test agent comprises a polynucleotide. In some embodiments, the polynucleotide encodes a SERCA polypeptide. In some embodiments, the SERCA polypeptide comprises a SERCA2 polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2 isoform selected from a SERCA2a polypeptide and a SERCA2c polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2a polypeptide.

[0138] In some embodiments, the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a CMV promoter. In some embodiments, the promoter comprises an inducible promoter.

[0139] In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an AAV vector, a lentiviral vector, and a retroviral vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, the polynucleotide is encapsulated in a viral capsid. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid. In some embodiments, the AAV vector encodes an AAV or a fragment thereof having a specific serotype. In some embodiments, the AAV or fragment thereof comprises a viral capsid protein or a fragment thereof. In some embodiments, the AAV or fragment thereof has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, the AAV or fragment thereof has a serotype selected from AAV1 and AAV9. In some embodiments, the AAV or fragment thereof has the AAV1 serotype. In some embodiments, the AAV or fragment thereof has the AAV9 serotype. In some embodiments, the AAV or fragment thereof may comprise a hybrid serotype consisting of one or more of the above serotypes.

[0140] Systems and Kits Some embodiments of the methods and compositions provided herein include systems and kits for treating, inhibiting, or ameliorating skeletal muscular dystrophy in a subject. Some such embodiments include a polynucleotide encoding a SERCA2 polypeptide. Some embodiments include a pharmaceutical composition comprising the polynucleotide and a pharmaceutically acceptable excipient. In some embodiments, the SERCA polypeptide comprises a SERCA2 polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2 isoform selected from a SERCA2a polypeptide and a SERCA2c polypeptide. In some embodiments, the SERCA2 polypeptide comprises a SERCA2a polypeptide. In some embodiments, the polynucleotide comprises a promoter operably linked to a nucleic acid encoding the SERCA polypeptide. In some embodiments, the promoter comprises a constitutive promoter. In some embodiments, the promoter comprises a CMV promoter. In some embodiments, the polynucleotide comprises a vector. In some embodiments, the vector is selected from an AAV vector, a lentiviral vector, and a retroviral vector. In some embodiments, the vector comprises an AAV vector. In some embodiments, the polynucleotide is encapsulated in a viral capsid. In some embodiments, the polynucleotide comprises a nucleic acid encoding the viral capsid. In some embodiments, the AAV vector encodes an AAV or a fragment thereof having a specific serotype. In some embodiments, the AAV or a fragment thereof comprises a viral capsid protein or a fragment thereof. In some embodiments, the AAV or a fragment thereof has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, the AAV or a fragment thereof has a serotype selected from AAV1 and AAV9.In some embodiments, the AAV or fragment thereof has the AAV1 serotype. In some embodiments, the AAV or fragment thereof has the AAV9 serotype.

[0141] More embodiments include a container, such as a sterile vial, containing a single dose of the polynucleotide for treating, inhibiting, or ameliorating skeletal muscular dystrophy in a subject. In some embodiments, the polynucleotide comprises a viral vector. In some embodiments, the polynucleotide is encapsulated in a viral capsid or a functional fragment thereof. In some such embodiments, the dose of the polynucleotide can be measured as viral genome count (vg) or DRP.

[0142] In some embodiments, the dose of the polynucleotide is at least 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 1×10 15 vg, 1×10 16 vg, 1×10 17 vg, 1×10 18 vg, 1×10 19 vg, 1×10 20 In some embodiments, the polynucleotide is administered in an amount of at least 1×10 vg, or in an amount ranging between any two of these amounts. 8 DRP, 1×10 9 DRP, 1×10 10 DRP, 1×10 11 DRP, 1×10 12 DRP, 1×10 13 DRP, 1×10 14 DRP, 1×10 15 DRP, 1×10 16 DRP, 1×10 17 DRP, 1×10 18 DRP, 1×1019 DRP, 1×10 20 In some embodiments, the polynucleotide is administered in an amount of about 1 x 10 DRP, or in an amount ranging between any two of these amounts. 8 vg~approx. 1×10 20 vg, approx. 1×10 9 vg~approx. 1×10 17 vg, approx. 1×10 10 vg~approx. 1×10 16 vg, approx. 1×10 11 vg~approx. 1×10 15 vg or approximately 1×10 12 vg~approx. 1×10 14 In some embodiments, the dose of the polynucleotide is about 1 x 10 13 vg~approx. 9×10 13 In some embodiments, the dose of the polynucleotide is about 1 x 10 8 DRP ~ approx. 1×10 20 DRP, approx. 1×10 9 DRP ~ approx. 1×10 17 DRP, approx. 1×10 10 DRP ~ approx. 1×10 16 DRP, approx. 1×10 11 DRP ~ approx. 1×10 15 DRP or approximately 1 × 10 12 DRP ~ approx. 1×10 14 In some embodiments, the dose of the polynucleotide is about 1 x 10 13 DRP ~ approx. 9×10 13 It is DRP.

[0143] Some embodiments further include a vasodilator, such as nitroglycerin.

[0144] Specific Embodiments Some embodiments of the methods and compositions provided herein include the use of a vasodilator. Aspects useful for certain embodiments provided herein are disclosed in U.S. Patent No. 8,221,738, which relates to compositions, kits, and delivery routes / methods, and is incorporated herein by reference in its entirety. In some embodiments, the polynucleotide is administered in combination with a vasodilator. In some embodiments, the vasodilator may be administered to a subject prior to administration of the polynucleotide. In some embodiments, the vasodilator may be administered to a subject prior to or simultaneously with administration of the polynucleotide. Examples of vasodilators include adenosine, histamine (or histamine inducers), alpha-blockers, theobromine, papaverine, ethanol, tetrahydrocannabinol (THC), minoxidil, nitric oxide (including nitric oxide enhancers), nitroglycerin, and the like. In some embodiments, the vasodilator is administered systemically, for example, orally, transdermally, intravenously, or by injection or infusion. In some embodiments, the infusion comprises intracoronary infusion. [Example]

[0145] Example 1: In vivo administration of SERCA2a to a DMD model A single intravenous injection of AAV9 SERCA2a vector into young mdx mice improved calcium uptake into the SR at 21 months of age To test SERCA2a as a disease-modifying gene therapy for DMD, we packaged the Flag-tagged human SERCA2a gene into AAV9 and inoculated 6 × 10 mice at 3 months of age with SERCA2a. 12 The virus was administered via the tail vein at a dose of 10 ... 11,12 Therefore, when treated mice reached 21 months of age, terminal functional measurements were performed and hearts and skeletal muscles were removed.

[0146] Immunofluorescence staining using Flag antibodies confirmed robust expression of human SERCA2a in the hearts of AAV-injected mice (Figure 1C). Western blot analysis of whole-cell lysates confirmed a significant increase in the total amount of SERCA2a in the heart (Figure 1D, 1G, and 1H). Western blot analysis of cardiac SR preparations confirmed that Flag-tagged human SERCA2a was correctly localized and enriched in the SR of AAV-injected mice (Figure 1E). No substantial changes were observed in the expression of other calcium-regulatory proteins, such as phospholamban and calsequestrin (Figure 1G). Calcium uptake into the SR in mdx hearts was significantly reduced (Figure 1F). This calcium uptake defect was completely normalized in the hearts of AAV9-injected mdx mice (Figure 1F).

[0147] We also examined AAV9-mediated SERCA2a expression and calcium uptake in skeletal muscle (Fig. 2). Western blot and immunostaining using the Flag tag confirmed widespread expression of human SERCA2a in the forelimb, upper hindlimb (quadriceps), and lower hindlimb (tibialis anterior and gastrocnemius) muscles (Fig. 2A, 2B). Similar to the results observed in the heart, SERCA2a administration significantly enhanced calcium uptake into the SR in skeletal muscle. The maximum calcium uptake rate in the treated mice was comparable to that in normal mice (Fig. 2C).

[0148] In addition to SERCA2a and other calcium-regulating proteins, we also examined the expression of neuronal nitric oxide synthase (nNOS) (Figure 1H). No significant difference was observed in nNOS levels between normal and mdx hearts. Furthermore, no significant change was observed in nNOS levels in the hearts of mice administered AAV.SERCA2a (Figure 1H). The nNOS levels in the skeletal muscle of mdx mice were significantly lower than those of BL10 mice, consistent with previous research findings. 14,15 The nNOS levels in skeletal muscle of AAV.SERCA2a-injected mice were not significantly different from those of uninjected mdx mice (Fig. 1H).

[0149] Systemic administration of SERCA2a to young mdx mice prevented the development of dilated cardiomyopathy in life-long mdx mice. Untreated mdx mice showed features characteristic of dilated cardiomyopathy at 21 months of age (Figure 3A-E). 16,17 Characteristics characteristic of dilated cardiomyopathy include myocardial fibrosis, ventricular dilation, ECG abnormalities, and hemodynamic disturbances (Figures 3A-3E). Cardiac catheterization in mdx mice confirmed significant increases in ventricular volumes at end-systole and end-diastole, significant decreases in cardiac contractility, and a rightward / downward shift in the pressure-volume loop (Figures 3C-3E; Table 1). Systemic administration of SERCA2a completely prevented myocardial fibrosis and normalized the PR interval, QRS duration, corrected QT (QTc) interval, Q amplitude, cardiomyopathy index, end-systolic volume, end-diastolic volume, systolic blood pressure, end-systolic pressure, maximum and minimum rates of change in ventricular pressure (dP / dt max and dP / dt min), blood pressure at dP / dt max, volume at dP / dt max and dP / dt min, preload-adjusted maximum power, and ejection fraction (Figures 3A-3E; Table 1). Treatment resulted in significant improvements in stroke work (Table 1). Heart rate, left ventricular relaxation time constant (Tau), stroke volume index, cardiac index, end-diastolic pressure, and maximum power also tended to improve (Fig. 3A-D; Table 1). Cardiac anatomical parameters (weight and weight ratio) of treated mice were unremarkable (Table 2). [Table 1] [Table 2]

[0150] SERCA2a administration to 3-month-old mdx mice significantly improved forelimb grip strength and treadmill running performance at 11 and 21 months of age. Skeletal muscle function and whole-body performance were assessed at 11 and 21 months of age using noninvasive grip strength and treadmill running tests (Figures 4A-4B, 5A-5C). Forelimb grip strength was significantly higher in SERCA2a-treated mdx mice compared with untreated mdx mice (Figures 4A and 5A). Treadmill running showed that both absolute running distance and weight-normalized running distance recovered to wild-type levels at 11 months of age (Figure 4B). The running distance of treated mdx mice significantly exceeded that of untreated mdx mice up to 21 months of age (Figure 5B). There was also a trend toward decreased serum creatine kinase (CK) levels in treated mice at 21 months of age (p = 0.09) (Figure 5C). Histological examination of skeletal muscle was performed using hematoxylin-eosin staining, Masson's trichrome staining, and muscle fiber immunostaining (Figures 6A-6E, 7A-7E, 8A-8C). Although grip strength and running ability improved, the improvement observed in histological examination of skeletal muscle was minimal (Figures 6A-6E, 7A-7E).

[0151] In embodiments disclosed herein, a single systemic administration of human SERCA2a improved muscle and cardiac function throughout life in mdx mice, a model of DMD. Three-month-old mdx mice were intravenously administered an AAV9 vector expressing Flag-tagged human SERCA2a. At 21 months of age, sustained and widespread expression of human SERCA2a was observed in striated muscles throughout the body. Administration normalized impaired calcium uptake into the SR in cardiac and skeletal muscles (Figures 1A-1H, 2A-2C). In physiological assays, SERCA2a administration significantly increased forelimb grip strength and treadmill running distance (Figures 4A-4B, 5A-5C). Surprisingly, systemic administration of SERCA2a completely prevented myocardial fibrosis and normalized cardiac electrophysiology (Figures 3A-3E). Key systolic and diastolic hemodynamic parameters were restored to wild-type levels (Figures 3A-3E; Table 1). These results demonstrate that mechanism-based gene therapy using disease-modifying factors (such as SERCA2a, which restores cytosolic calcium homeostasis in DMD) is an important approach to effectively treat at least some muscular dystrophies.

[0152] DMD is a disease caused by a deficiency of dystrophin. Therefore, experimental studies of DMD gene therapy have focused on restoring dystrophin expression. Dystrophin gene replacement therapy and dystrophin gene repair therapy have shown very promising results in mouse and canine DMD models. 18-20 Currently, several clinical trials using systemic AAV micro-dystrophin gene therapy are underway. 10 Despite these advances, dystrophin-based gene therapy has limitations. For example, the newly regenerated dystrophin may be recognized by the immune system as a new antigen, potentially triggering an immune response. 21 Mutation-targeted exon skipping and genome editing approaches require individualized tailoring depending on the mutation. The development of new therapies that utilize genes naturally expressed in DMD patients may overcome these challenges.

[0153] Abnormal elevation of intracellular calcium is one of the major causes of myonecrosis in DMD. In normal muscle, cytosolic calcium levels are maintained at physiological levels by coordinated regulation of calcium influx and recycling between the extracellular space, cytoplasm, and intracellular calcium storage organelles. In dystrophic muscle, calcium homeostasis is disrupted. Increased calcium influx into the cytosol occurs via dysfunctional calcium channels and ryanodine receptors (RyRs), which result in calcium leakage. 22,23 In addition, decreased SERCA activity also reduces the amount of calcium released from the cytoplasm.

[0154] Calcium regulation has been a therapeutic target for DMD since the early 1980s 24 However, the clinical efficacy of calcium channel blocking drugs has not been confirmed in human clinical trials. 25Recently, calcium leaking RyRs have been recognized as a major cause of excess calcium influx into the cytoplasm, and researchers have begun to search for chemicals that stabilize RyRs. 22,26 .

[0155] Another approach to restore calcium homeostasis is to enhance calcium uptake by SERCA. This could be achieved by modulating SERCA activity or by increasing SERCA expression. Suppression of inhibitory SERCA regulators (e.g., phospholamban and sarcolipin) has been shown to prevent heart failure in a hamster model of limb-girdle muscular dystrophy and to ameliorate the dystrophic phenotype in a mouse model of DMD. 27-29 Additionally, SERCA overexpression has been tested using either SERCA1a or SERCA2a. Three separate research groups have investigated increased expression of SERCA1a in mouse models of DMD using transgenic techniques or neonatal AAV gene transfer. 30-32 These studies demonstrated significant improvements in skeletal muscle calcium uptake, histology, and function. Administration of the human SERCA2a vector to 12-month-old mdx mice resulted in significant improvements in several ECG parameters at 20 months of age. 33 Furthermore, when the same human SERCA2a vector was used in the skeletal muscles of newborn mice with limb-girdle muscular dystrophy, a significant suppression of the cycle of muscle degeneration and regeneration was observed. 30 .

[0156] Despite these results, however, it remains unclear whether SERCA treatment provides long-term protection to both skeletal muscle and the heart when treatment is initiated at an age comparable to the ages targeted in ongoing DMD gene therapy trials. 10 With this in mind, the experiments disclosed herein focused on mdx mice, which are the equivalent age of a teenage boy. 34Treated mice were followed for the expected lifespan to assess both skeletal and cardiac muscle outcomes. Long-term follow-up of all striated muscles is important, as lifelong protection of both skeletal and cardiac muscles is necessary for this treatment to be established as an effective DMD treatment. 35 Furthermore, we used a model that perfectly reproduces the dilated cardiomyopathy of DMD patients. 36 With improvements in medical care and respiratory support, many patients are living longer than ever before, and cardiac complications remain the leading cause of death in DMD patients. 37 The embodiments disclosed herein focus on cardiac effects (particularly blood pump function) in phenotypic models. Furthermore, SERCA2a was used instead of SERCA1 because SERCA1 is not expressed in the hearts of wild-type animals. SERCA2a is a better therapeutic target because it can be used to treat both skeletal and cardiac muscle. Furthermore, we used a human SERCA2a vector, which has a proven safety profile in hundreds of human heart failure patients. 38-40 .

[0157] Significant improvements were observed in whole-body muscle function (forearm grip strength and treadmill performance), cardiac electrophysiology (ECG), and cardiac contractility (hemodynamics). Furthermore, SERCA2a administration effectively suppressed myocardial remodeling and fibrosis, although it did not suppress skeletal muscle pathology. Further studies are needed to clarify this discrepancy, but it is likely due to the timing of AAV administration. Three-month-old mdx mice exhibit significant skeletal muscle disease but no cardiac pathology. 41,42 SERCA2a administration may be more effective before rather than after the onset of tissue damage. Supporting this idea, experiments in which SERCA expression was initiated in utero in transgenic mice or AAV SERCA vectors were delivered to neonatal mice showed significant improvement in skeletal muscle histology. 30,31 In addition, administration of SERCA2a to 12-month-old mdx mice did not suppress myocardial fibrosis. 33 .

[0158] Although grip strength and treadmill running performance improved (Figures 4A-4B, 5A-5C), no improvement was observed in skeletal muscle histology (Figures 6A-6E, 7A-7E). However, improvements in muscle function may occur independently of improvements in muscle pathology. 47 .

[0159] Methods and Materials experimental animals All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) and in accordance with NIH guidelines. C57 / BL10 (wild-type control, stock no. 000476) and dystrophin-deficient mdx mice (stock no. 001801) were purchased from The Jackson Laboratory (Bar Harbor, MN, USA) and bred in a barrier facility.

[0160] The classic symptom of DMD cardiac disease is dilated cardiomyopathy. Myocardial damage has been observed in various DMD mouse models, including mdx, mdx4cv, mdx5cv, D2-mdx, utrophin / dystrophin double knockout mice, utrophin heterozygous mdx mice, Cmah / dystrophin double knockout mice, MyoD / dystrophin double knockout mice, and integrin / dystrophin double knockout mice. However, dilated cardiomyopathy is not observed in aged female mdx mice. 17 , aged female mdx4cv mice 48 , MyoD / dystrophin double knockout mice 49 MyoD / dystrophin double knockout mice are genetically distinct from DMD patients. In addition to the null mutation in the dystrophin gene, these mice also have a null mutation in the MyoD gene. Furthermore, MyoD / dystrophin double knockout mice are not available. Because the severity of cardiac disease in aged female mdx mice and aged female mdx4cv mice is similar, we used aged female mdx mice. 16,17We believe that the use of female mice does not diminish the translational relevance of this study. Girls who lose the activity of the dystrophin gene, which resides on both X chromosomes, exhibit a phenotype characteristic of DMD. Therapies developed to treat affected boys may be equally effective in affected girls, and vice versa. Sample sizes for functional measurements are summarized in Table 3. [Table 3-1]

[0161] AAV production and delivery The cis SERCA2a packaging plasmid is a modified version of a previously published construct. 33,39 Specifically, we generated a construct in which a Flag tag was fused in-frame to the C-terminus of human SERCA2a cDNA. SERCA2a expression is controlled by a cytomegalovirus promoter, a hybrid intron, and a bovine growth hormone polyadenylation signal. AAV9 vectors were constructed, purified, and titered according to our published protocol. 50 . Total of 6 x 10 per fish 12 vg of the AAV9 SERCA2a vector was administered via the tail vein of conscious 3-month-old mdx mice.

[0162] Morphological examination Flag-tagged human SERCA2a was confirmed by immunohistochemistry using a monoclonal antibody against the Flag tag (1:500, Sigma-Aldrich, Cat. No. F1804, clone M2). Laminin was detected with a polyclonal antibody (1:200, Sigma-Aldrich, Cat. No. L9393). General histological examination was performed by hematoxylin and eosin staining. Fibrosis was examined by Masson's trichrome staining. 51 Slides were observed using a Nikon E800 fluorescence microscope with identical exposure settings. Photomicrographs were taken with a QImaging Retiga 1300 camera. 51The fibrotic area and myofiber cross-sectional area (CSA) of whole heart sections were quantified from Masson's trichrome stained images using the free selection tool in Photoshop software. 35 The method is briefly described below. The micrometer scale was selected in the measurement scale setting options in Photoshop. Fibrotic areas were marked with the Quick Selection tool. The sum of the areas of all fibrotic areas was expressed as a percentage of the total cardiac cross-sectional area (CSA). For myofiber CSA measurements, the micrometer scale was selected and the circumference of each individual fiber was marked with the Quick Selection tool. CSA was then calculated in Photoshop.

[0163] Calcium uptake into the SR Calcium uptake into the SR was measured by Millipore filtration. 28 The method is briefly described below. Approximately 150 mg of total protein extract was dissolved in 1.5 mL of Ca 2+ The uptake medium (40 mmol / L imidazole [pH 7.0], 100 mmol / L KCl, 5 mmol / L MgCl2, 5 mmol / L NaN3, 5 mmol / L potassium oxalate, 0.5 mmol / L EGTA) was supplemented with various concentrations of CaCl2 (0.03–3 mmol / L free CaCl2). 2+ (1 mCi / mmol 45 Ca 2+ The SR was incubated at 37°C in the presence of Ca. 2+ To obtain maximal stimulation of Ca uptake, ruthenium red was added to a final concentration of 1 mmol before the addition of substrate. 2+ The uptake was initiated by adding ATP to a final concentration of 5 mmol. The reaction was stopped after 1 minute by filtering the reaction solution. The assay was performed in duplicate. 2+ Uptake rate and Ca required for 50% effective concentration 2+ Concentrations (EC50) were determined by nonlinear curve fitting analysis using GraphPad Prism software version 7.0.

[0164] SR fraction SR fractions were prepared at 4°C unless otherwise specified. Briefly, the method is as follows: The excised tissue (approximately 25–40 mg) was homogenized in 1 mL of ice-cold Buffer A (pH 7.0; 10 mM imidazole, 0.3 M sucrose, 0.5 M dithiothreitol [DTT], 40 mM CaCl2, 1× protease inhibitor cocktail; Roche, Indianapolis, IN, USA). The resulting crude lysate was centrifuged at 3,000 × g for 20 min. The homogenization and centrifugation were repeated. The supernatant was centrifuged at 10,000 × g for 20 min. The resulting supernatant was transferred to a 5 mL Beckman tube, and KCl was added to a final concentration of 0.5 mM in Buffer A. The lysate was incubated on ice for 20–30 min with occasional agitation. Each sample was then centrifuged at 245,419 × g for 40 min. The resulting pellet was resuspended in buffer B (pH 7.5; 20 mM Tris, 0.3 M sucrose, 0.6 M KCl, 0.5 mM DTT, 40 mM CaCl) and centrifuged at 245,419 × g for 40 minutes. The pellet was then resuspended in resuspension buffer (pH 7.0; 10 mM imidazole, 0.3 M sucrose, 0.25 mM DTT, 1× protease inhibitor cocktail). Protein concentration was measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA).

[0165] Western blotting Heart and muscle whole lysates were prepared as previously described. 14The method is briefly described below. Each tissue was snap-frozen in liquid nitrogen, then finely pulverized in liquid nitrogen and homogenized in a buffer containing 10% sodium dodecyl sulfate, 5 mM ethylenediaminetetraacetic acid, 62.5 mM Tris-HCl (pH 6.8), and a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The crude lysate was heated at 95°C for 3 min, cooled on ice for 2 min, and centrifuged at 16,000 × g for 2 min. The supernatant was collected as whole muscle lysate. Protein concentration was measured using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). A SERCA2a polyclonal antibody (1:2,500, Badrilla, Leeds, UK, Cat. No. A010-23S) detects both endogenous and human SERCA2a. Human SERCA2a expression was confirmed using an anti-Flag antibody (1:500, Sigma, St. Louis, MO, USA, Cat. No. F1804, clone M2). Western blot quantification was performed using LI-COR Biosciences Image Studio software version 5.0.21 (https: / / www.licor.com). Band intensities of each protein were normalized to the corresponding loading control blotted in the same manner. Furthermore, the relative band intensities were normalized to the wild-type control. Western blots of whole heart lysates were first performed using glyceraldehyde 3-phosphate dehydrogenase (1:3,000, Millipore, Billerica, MA, USA, Cat. No. MAB374, clone 6C5) as a loading control (Figure 1G, Figure 1H). Quantitative data from this experiment are shown in Figure 1D (right panel). Western blots of cardiac SERCA2 were also performed using vinculin (1:2,000, Abcam, Cambridge, MA, USA, Cat. No. Ab155120) as a loading control. Representative data from this experiment are shown in Figure 1D (left panel). Western blots of skeletal muscle total lysates were also performed using vinculin as a loading control (Figure 2A).

[0166] Treadmill running test Treadmill endurance measurements were performed in the same manner as previously reported. 52 The method is briefly described below. Mice were acclimated for 5 days on a 7° incline treadmill (Columbus Instruments, Columbus, OH, USA). Each day, the acclimation protocol consisted of placing the mice on a stationary, flat treadmill for 2 minutes, followed by a 7° incline treadmill for 5 minutes. All running acclimation was performed on the 7° incline treadmill. On the first day, mice were run at 5 m / min for 15 minutes, followed by 10 m / min for 5 minutes. On the second day, mice were run at 5 m / min for 5 minutes, followed by 10 m / min for 15 minutes, followed by 12 m / min for 5 minutes. On the third day, mice were run at 5 m / min for 5 minutes, followed by 10 m / min for 15 minutes, followed by 12 m / min for 10 minutes. On days 4 and 5, mice were run at 5 m / min for 5 min, 10 m / min for 20 min, 12 m / min for 5 min, and 15 m / min for 5 min. Distance traveled was measured on day 6. On the day of distance measurement, mice were placed on a stationary treadmill for 2 min, then run at 5 m / min for 5 min. The treadmill speed was then increased by 1 m / min every 5 min. Total distance traveled was calculated when the mice reached exhaustion. Exhaustion was determined when the animals stopped running, did not attempt to re-enter the treadmill, and remained in contact with the shocker (minimum setting) for typically 1–3 s. Animals that did not run were excluded from analysis.

[0167] Serum CK activity measurement Fresh serum was obtained by tail vein bleeding, and CK activity was measured using a CK liqui-UV test kit from Stanbio Laboratory (Boerne, TX, USA) according to the manufacturer's instructions.

[0168] Forelimb grip strength measurement Forelimb grip strength was measured using a computerized grip strength meter (Columbus Instruments, Columbus, OH, USA) as previously described. 43,53The grip strength meter is equipped with a pull bar attached to a force transducer and a digital display. Mice were first allowed to habituate to the apparatus for approximately 5 minutes. Then, the mouse was allowed to grasp the pull bar by holding the tip of its tail. The mouse was gently pulled away from the grip bar. When the mouse was no longer able to grasp the bar, a measurement was recorded. This protocol was repeated five times with at least a 30-second rest period between measurements. The three highest readings were averaged to obtain absolute grip strength. Normalized grip strength was calculated by dividing absolute grip strength by body weight.

[0169] ECG and hemodynamic measurements Cardiac function was assessed using published protocols. 54,55 Specifically, 12-lead ECG measurements were performed using a commercially available system from AD Instruments (Colorado Springs, Colorado, USA). 51,56 The amplitude of the Q wave was determined using the lead I tracing. Other ECG parameters were analyzed using the lead II tracing. The QTc interval was calculated by correcting the QT interval for heart rate. 57 The cardiomyopathy index was calculated by dividing the QT interval by the PQ segment. 58 Left ventricular hemodynamics was assessed using a closed-chest approach as previously reported. 51,54 The obtained pressure-volume (PV) loops were analyzed using PVAN software (Millar Instruments, Houston, TX, USA). The cardiac diastolic time constant (Tau) was calculated. 59 Body surface area was also calculated. 60 .

[0170] Assessment of muscle fiber types by immunostaining Primary antibodies for each muscle fiber type were obtained from the Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa. Specifically, type I muscle fibers were detected using BA-D5 (1:5) as the primary antibody and Alexa Flour 350 goat anti-mouse IgG2b (1:50, Invitrogen, Cat. No. A21140) as the secondary antibody. Type IIa muscle fibers were detected using SC-71 (1:10) as the primary antibody and Alexa Flour 594 goat anti-mouse IgG1 (1:100, Invitrogen, Cat. No. A21125) as the secondary antibody. Type IIb muscle fibers were detected using BF-F3 (1:10) as the primary antibody and FITC goat anti-mouse IgM (1:100, Jackson ImmunoResearch, Cat. No. 115-095-075) as the secondary antibody. Laminin was detected using a polyclonal primary antibody (1:200, Sigma-Aldrich, Cat. No. L9393) and goat anti-rabbit IgG Alexa Flour Plus 647 (1:100, Invitrogen, Cat. No. A32733) as the secondary antibody. For quantification, five random images were collected per fluorescent channel from each tissue and overlaid using ImageJ. Counting was performed using the ImageJ multipoint tool. Results were exported to Excel, and percentage calculations were performed.

[0171] statistical analysis Data are presented as mean ± standard error of the mean. For physiological assays, data from individual subjects were presented as scatter plots. One-way analysis of variance and Tukey's multiple comparison analysis were performed using GraphPad Prism software version 7.0 for Mac OSX (GraphPad, La Jolla, CA, USA). p < 0.05 was considered statistically significant.

[0172] Example 2: In vitro human ventricular muscle tissue model Differentiation of DMD-derived iPS cells into cardiomyocytes Human induced pluripotent stem cells (hiPSCs) derived from DMD patients were directly induced to differentiate into human ventricular myocytes (hvCMs), yielding over 90% yield of ventricular subtype hiPSC-derived CMs (Weng, Z., et al. (2014). Stem Cells Dev 23: 1704-1716). Briefly, the method is as follows: hiPSCs were dispersed into single cells and cultured overnight under hypoxic conditions in ultra-low attachment plates containing mTeSR1 containing 1 ng / ml bone morphogenetic protein 4 (BMP4). Cell clusters were then treated with GlutaMAX (Thermo Fisher Scientific) in StemPro-34 medium supplemented with 50 μg / ml ascorbic acid (Sigma-Aldrich), 10 ng / ml activin A, 10 ng / ml BMP4, and 10 μM ROCK inhibitor Y-27632 under hypoxic conditions at 5% O2 from day 1 to day 4. On days 4–8 after differentiation, hypoxic cell clusters were treated with 50 μg / ml ascorbic acid and 5 mM IWR-1 in StemPro-34 medium. From day 8 onward, cell clusters were maintained under normoxic conditions using RPMI 1640 medium containing 1x B27 supplement (Thermo Fisher Scientific) and 50 μg / ml ascorbic acid.

[0173] Contractility evaluation of human ventricular tissue strips (hvCTS) Three-dimensional multicellular myocardial tissue (hvCTS) was prepared and contractile activity was measured using a previously published method to evaluate the contractility and dynamics of hvCM (Turnbull, IC, et al. (2014). FASEB J 28: 644-654; and Cashman, TJ, et al., (2016). J Vis Exp 109: e53447). The method is briefly described below. Cardiospheres obtained 15–16 days after the initiation of hiPSC differentiation were dispersed into single cells and added to RPMI 1640 containing 1× B27, 50 μg / ml ascorbic acid, and 10 μM ROCK inhibitor Y-27632. After recovery in an incubator for 3 days, hvCTS was prepared. To generate hvCTS, 1.0 × 10 cardiomyocytes induced to differentiate from hiPSCs were used. 6 and 1.0 x 10 human foreskin fibroblasts 5 Each cell was added to 100 μl of an ice-cold solution consisting of 2 mg / ml type I collagen (Thermo Fisher Scientific), 0.80–0.95 mg / ml Matrigel, 15 mM NaOH, 0.9x minimum essential medium (Sigma-Aldrich), 25 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and 0.1x hvCTS maintenance medium. This 100 μl cell-collagen mixture was added to a PDMS (polydimethylsiloxane) bioreactor with force-sensing cantilever posts at both ends of a rectangular well and returned to the incubator to allow the formation of hvCTS attached between the posts. The resulting hvCTS were maintained in DMEM medium supplemented with 10% neonatal calf serum (Gibco). The medium was replaced with half a change every 2–3 days until the tissue was ready for testing 7–8 days after tissue preparation.

[0174] On the day of testing, the contraction amplitude of hvCTS is measured using a custom-built post-tracking contractile force measurement system that records the displacement of a cantilever post placed on a temperature-controlled heating plate in hvCTS maintenance medium containing HEPES buffer at 37 °C. During testing, the hvCTS is paced by applying electrical field stimuli of different frequencies. Force generation and contractile kinetics are analyzed using custom-built data processing and analysis software.

[0175] Infection with AAV1.SERCA2a AAV1.CMV.SERCA2a is a recombinant adeno-associated vector serotype 1 containing the CMV promoter driving the cardiac isoform of the sarcoplasmic reticulum calcium ATPase pump (SERCA2a). 1.0 x 10 12 A solution of AAV1.SERCA2a was used at a concentration of viral genome (vg) / ml. DMD-derived human ventricular tissue strips (hvCTS) were transduced with AAV1.CMV.SERCA2a by adding 0.1 μl, 1 μl, 10 μl, or 100 μl of vector solution. Human DMD-derived hvCTS were exposed to the virus solution for 0, 2, 4, 7, or 14 days. At each time point, all parameters described below were measured using human DMD-derived hvCTS and compared with measurements taken before transduction on day 0. Additionally, AAV1.GFP (green fluorescent protein) virus transduced into DMD-hvCTS was used as a control at each time point. Parameters measured included force generation, normalized force generation, velocity fluctuation, force fluctuation, force-frequency relationship, and β-adrenergic response. Abnormalities in all of these parameters were observed in DMD-hvCTS transduced with AAV1.GFP, and these abnormalities are expected to be improved in DMD-hvCTS transduced with AAV1.SERCA2a.

[0176] hvCTS contractility analysis On the day of measurement, each hvCTS was photographed from the side. PDMS post height and mean tissue height were determined using image analysis in ImageJ (NIH). A custom-written MATLAB data processing executable was used to convert the measured hvCTS contraction amplitude into force production. This force data was then analyzed and visualized in a separate MATLAB data analysis software.

[0177] Example 3: Clinical Trial of DMD Treatment the purpose A Phase 2 randomized, double-blind, placebo-controlled study will be conducted to evaluate the safety and efficacy of SRD-001 (AAV1 / SERCA2a) in subjects with secondary cardiomyopathy due to DMD. Primary objective: To evaluate the safety and efficacy of a single intracoronary administration of SRD-001 in subjects with secondary cardiomyopathy due to DMD. Secondary objective: To evaluate the effect of SRD-001 on skeletal muscle function and quality of life.

[0178] Study design This Phase 2, multicenter, randomized, double-blind, placebo-controlled study evaluated the safety and efficacy of a single dose of SRD-001 administered via antegrade epicardial coronary artery infusion in subjects with secondary cardiomyopathy due to DMD who were anti-AAV1 neutralizing antibody (NAb) negative. 2+ This is an adeno-associated virus serotype 1 (AAV1) vector expressing a transgene for ATPase 2a isoform (SERCA2a).

[0179] method After providing informed consent, subjects with a clinical diagnosis of DMD and evidence of cardiomyopathy will undergo a series of tests and procedures during a 30-day screening period prior to randomization to determine subject eligibility and obtain baseline measurements of specific parameters. A total of 50 eligible subjects will then be randomized 1:1 to receive either SRD-001 or placebo.

[0180] On Day 1, subjects will undergo diagnostic angiography without left heart catheterization, followed by a single dose of study drug via antegrade epicardial coronary infusion to deliver to the three major cardiac regions of the left ventricle (anterior, lateral, and inferior / posterior), unless contraindicated. Subjects will be continuously monitored for at least 4 hours before and after the procedure and will be discharged the same day unless a periprocedural complication requires overnight hospitalization.

[0181] Subjects will be followed at pre-specified visits throughout the 24-month study period. A telephone assessment will be conducted at Week 1, followed by in-person follow-ups or assessments as soon as possible if clinically indicated. At Week 2, Months 1, 3, 6, 12, 18, and 24, subjects will undergo a series of safety and efficacy assessments, including physical and laboratory tests, 12-lead electrocardiogram (ECG) measurements, and collection of adverse events (AEs). At Months 12 and 24 only, subjects will undergo delayed gadolinium enhancement (LGE) cardiac MRI. Additionally, at 6, 12, 18, and 24 months, subjects will undergo assessments of skeletal muscle function, as assessed by the Performance of Upper Limb version 2.0 (PUL2.0), grip strength, key and fingertip pinch strength, elbow flexion strength, and, if ambulatory, 10-meter walk / run time and the North Star Ambulatory Assessment; pulmonary function; and quality of life, as assessed by the DMD Upper Limb Patient-Reported Outcome Measures (DMD UL-PROM) and the Pediatric Outcomes Data Collection Instrument (PODCI). At the 24-month visit, each subject will complete the study. Figure 9 outlines the phase 2 study design.

[0182] Evaluation items Primary efficacy endpoint: Changes from baseline to 12 and 24 months in left ventricular structure and function assessed by delayed gadolinium enhancement (LGE) cardiac MRI: left ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume, circumferential strain, regional wall thickness, left ventricular LGE as a percentage of left ventricular myocardial mass and in grams, left ventricular viable myocardial mass in grams, and number of left ventricular segments with LGE. Alternative primary efficacy endpoints: Composite outcomes of change from baseline to 12 and 24 months in left ventricular function (LVESV), PUL2.0, pulmonary function (select one parameter), QOL, and eventual events Secondary efficacy endpoints: Changes from baseline to months 6, 12, 18, and 24 in: (a) Skeletal muscle function assessed by PUL2.0, grip strength, key pinch and fingertip pinch strength, elbow flexion strength, 10-meter walk / run time (10MWRT) if ambulatory, incidence of ambulatory failure defined as 10MWRT >30 seconds, and North Star Ambulatory Ability Assessment (NSAA); (b) pulmonary function assessed by resting vital capacity (SVC), forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), peak expiratory flow (PEF), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak cough flow (PCF), and inspiratory reserve (IFR); and (c) QOL assessed by DMD UL-PROM and PODCI.

[0183] Primary safety endpoint: The incidence of the following from Day 1 to Month 24: all-cause death, serious adverse events, treatment-emergent adverse events related to the study drug or administered procedure, and cell-mediated immune responses. Secondary safety endpoints: Incidence and severity of all adverse events from Day 1 to Month 24.

[0184] Diagnosis and main selection criteria A sample size of 50 subjects is considered appropriate for the primary and secondary objectives of the study. Number of subjects (planned): Maximum N=50.

[0185] Eligibility for study participation for subjects with DMD and evidence of cardiomyopathy will be assessed as follows: 1. Anti-AAV1 neutralizing antibody titer <1:2 or unknown within 60 days of randomization. 2. Male subjects aged 10 years or older at the time of consent. 3. If 18 years of age or older, be willing and able to provide informed consent to participate in the study. If under 18 years of age, have parental or guardian consent. 4. Subjects with a diagnosis of DMD based on clinical symptoms and phenotype consistent with DMD (e.g., family history of DMD, elevated creatine kinase, dystrophin muscle biopsy, gastrocnemius pseudohypertrophy, Gowers sign, and / or failure to gain weight before age 7) and confirmatory genetic testing performed by a certified laboratory. If applicable, subjects have lost the ability to walk independently by their 18th birthday. However, standing without assistance or walking a few steps independently is not considered ambulation. 5. Cardiomyopathy with left ventricular scarring by delayed gadolinium enhancement in at least four segments as assessed by cardiac MRI and a left ventricular ejection fraction of less than 40% at screening. 6. Have a specific score in PUL2.0. 7. If able to walk, walking / running speed of 10m less than 1m / sec. 8. Patient receives standard of care at an established multidisciplinary DMD center, including regular cardiac and pulmonary monitoring, systemic glucocorticoids, and range of motion exercises at home. 9. Receiving systemic glucocorticoid therapy for at least 12 months prior to randomization, with stable dose except for weight-based dose adjustments or ≤10% reduction in steroid dose due to toxicity for at least 6 months prior to randomization. 10. Receiving current and up-to-date vaccinations, including annual influenza; meningococcal and meningococcal B; tetanus-diphtheria-pertussis (Tdap); and pneumococcal polysaccharide, unless contraindicated. 11. Agree to use condoms and spermicide during sexual intercourse to protect partners from viral shedding for 6 months after receiving the study drug. 12. The partner of childbearing potential agrees to use adequate contraception (oral contraceptives, injectable contraceptives, intrauterine devices or surgical sterilization, and / or condoms in combination with spermicide) to prevent pregnancy for 6 months after administration of the study drug. 13. Subject is determined by the investigator to be willing and able to comply with the requirements of the study protocol.

[0186] Main exclusion criteria Subjects meeting any of the following criteria will be excluded from the study: Subjects with DMD, such as elbow flexion contracture of more than 30° in both limbs, obesity index greater than 45, or a mutation in the dystrophin gene for which exon 44 skipping is indicated, or a dystrophin deletion mutation such as (but not limited to) exon 307. Other exclusion criteria were as follows: 1. Received intravenous administration of vasoactive drugs, vasodilators, or diuretics within 30 days prior to screening or enrollment. 2. Patients with restrictive cardiomyopathy, hypertrophic cardiomyopathy, acute myocarditis, pericardial disease, amyloidosis, infiltrative cardiomyopathy, untreated thyroid disease, or focal left ventricular (LV) aneurysm. 3. Underwent cardiac surgery, percutaneous coronary intervention (PCI), valvuloplasty, or valve replacement within 30 days prior to screening. 4. Myocardial infarction (e.g., ST-segment elevation myocardial infarction [STEMI] or extensive non-ST-segment elevation myocardial infarction) within 90 days prior to screening. Extensive non-ST-segment elevation myocardial infarction is defined as a creatine kinase test (CK-MB) greater than 3 times the upper limit of normal (ULN) or a troponin greater than 5 times the ULN. 5. History of cardiac transplantation, left ventriculoplasty (LVRS), cardiomyoplasty, passive restraint device (e.g., CorCap® cardiac assist device), mechanical circulatory support device (MCSD), or cardiac shunt surgery. 6. Likely to undergo cardiac resynchronization therapy, cardiomyoplasty, LVRS, conventional revascularization, or valve repair within 6 months of treatment. 7. Hemodynamic instability likely requires immediate cardiac transplantation or MCSD implantation. 8. Known hypersensitivity to radiopaque agents used in angiography. High-dose corticosteroid pretreatment has been or is likely to be required prior to angiography. 9. The investigator has determined that there is significant luminal stenosis in the left main coronary artery or the ostium of the right coronary artery. 10. Liver function tests (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase) >3×ULN, total bilirubin >2×ULN, or known underlying liver disease (e.g., cirrhosis, chronic hepatitis B or C virus infection). 11. Currently undergoing hemodialysis, likely to require hemodialysis within 12 months, or an estimated glomerular filtration rate (GFR) of 20 mL / min / 1.73 m as calculated by the Modification of Diet in Renal Disease (MDRD) formula. 2 The following is the result. 12. Have a bleeding diathesis or thrombocytopenia defined as a platelet count <75,000 / μL. 13. Have anemia defined as hemoglobin <9g / dL. 14. Have pulmonary dysfunction as follows: a. FVC <35% predicted. b. In the combined screening test, the FVC and PEF measurements do not fall within ±15% of each other. c. If the investigator determines that the patient is at risk for respiratory failure in the near future or meets criteria for initiation of non-invasive ventilatory support, defined as serum bicarbonate ≥ 29 mmol / L at screening. d. History of chronic respiratory disease unrelated to DMD (such as, but not limited to, asthma, bronchitis, and tuberculosis) requiring continuous or intermittent treatment. e. If you have had an acute respiratory illness within 30 days prior to screening. f. If non-invasive ventilation has been initiated within 30 days prior to screening or if it is anticipated that non-invasive ventilation will need to be initiated within 12 months after screening. 15. Planned or anticipated to undergo thoracic or spinal surgery, or lower limb surgery if ambulatory, within 12 months of randomization. 16. Initiation of metformin or insulin treatment within 3 months prior to randomization. 17. Received human growth hormone (HGH) within 3 months prior to randomization unless the dose was stable for at least 24 months prior to randomization. 18. Within the past five years, you have been diagnosed with or treated for cancer for which surgical removal is considered curative (excluding basal cell carcinoma and in situ carcinoma). (However, even if you have a history of cancer, you are not excluded if you have been disease-free for at least five years since your diagnosis or treatment.) 19. Previous participation in a gene transfer trial is permitted if the trial was unblinded or if there is documentation that the subject was randomized to a placebo control group and did not receive an active gene transfer agent. 20. Has received another investigational drug intervention or participated in another clinical trial within 30 days or 5 half-lives of the drug prior to screening, except if the subject is enrolled in a non-therapeutic observational study (registry) or the observational portion of an intended-to-treat study where the sponsoring institution approves enrollment. 21. In the opinion of the investigator, the subject has a recent history of psychiatric illness (including drug or alcoholism) that is likely to impair their ability to comply with the procedures set out in the clinical trial protocol. 22. Has any other concurrent medical condition not specifically excluded in the protocol that may compromise the subject's safety or the objectives of the study.

[0187] Investigational drug, dosage and administration method Using a commercially available guide catheter or diagnostic cardiac catheter and a B. Braun Perfusor® Space syringe pump, 3 × 10 syringes were infused at a flow rate of 300 mL / hr for 10 minutes. 13 vg of SRD-001 will be administered via antegrade epicardial coronary infusion into the left and / or right coronary artery. An intravenous infusion of nitroglycerin will be administered at the maximum tolerated dose for a minimum of 10 minutes prior to and concurrently with the SRD-001 infusion.

[0188] Treatment duration Single intracoronary injection.

[0189] Reference treatment, dosage, and administration method A placebo drug containing the same excipients as SRD-001 but without the AAV1 / SERCA2a active ingredient will be administered via antegrade epicardial coronary infusion into the left and / or right coronary artery at a flow rate of 300 mL / hr over 10 minutes using a commercially available guide catheter or diagnostic cardiac catheter and a B. Braun Perfusor® Space syringe pump. An intravenous infusion of nitroglycerin will be administered at the maximum tolerated dose for a minimum of 10 minutes before and simultaneously with the placebo infusion.

[0190] Evaluation criteria Efficacy: LGE cardiac MRI, PUL2.0, key pinch and fingertip pinch strength, elbow flexion strength, 10MWRT and NSAA (if ambulatory), pulmonary function, [NT-proBNP], and QOL assessed by DMD UL-PROM and PODCI.

[0191] Safety: Subject disposition (need for hospitalization), adverse events (AEs), concomitant medications, laboratory tests (complete blood count [CBC], white blood cell [WBC] differential, platelet count, serum chemistry for basic metabolic panel and comprehensive liver panel, lactate dehydrogenase [LDH], and uric acid), troponin T, enzyme-linked immunospot (ELISpot), urinalysis, physical examination (including weight) and vital signs, and 12-lead electrocardiogram (ECG).

[0192] statistical methods Analysis population: The intention-to-treat (ITT) population is all subjects randomized in the study, aggregated and analyzed according to their randomized treatment assignment. The modified ITT (mITT) population is all subjects actually treated in the study, aggregated and analyzed according to their randomized treatment assignment. The per-protocol (PP) population is subjects treated in the study without protocol violations that materially affect the completeness, accuracy, and / or reliability of the trial data. The safety population is subjects treated in the study, aggregated and analyzed according to the treatment actually received.

[0193] The analytical data cutoff is performed twice. Month 12: After all randomized subjects have completed the 12-month visit (unless discontinued earlier). Month 24: After all randomized subjects have completed the 24-month visit (unless discontinued earlier).

[0194] Safety and efficacy data will be reported for the ITT population. Safety data will be collected for the safety population at 12 and 24 months. Efficacy analyses will be performed for the mITT population at the analysis data cutoffs of 12 and 24 months. Efficacy analyses will also be performed using the ITT and PP populations.

[0195] method: Data will be tabulated by treatment group and by visit during the study. Categorical variables will be tabulated as frequencies and percentages for each category. Continuous variables will be tabulated as number of subjects, mean, standard deviation, median, and range. Clinical event rates will be tabulated as the number of events occurring during the one-year follow-up period for each patient. Treatment effects on clinical event rates will be tabulated as hazard ratios estimated by a semiparametric joint frailty model that accounts for competing risks of terminal events.

[0196] Composite outcomes are used to examine overall treatment effects at the group and subject levels. For group-level analyses, outcomes are categorized into one of the following domains: 1. Cardiac structural parameters: LGE cardiac MRI 2. Functional parameters: PUL2.0, grip strength, key pinch and fingertip pinch strength, elbow flexion strength, and 10MWRT (ambulatory subjects only) 3. Pulmonary parameters: Pulmonary function parameters 4. Biomarker: NT-proBNP (% and absolute change) 5. QOL parameters: DMD UL-PROM and PODCI 6. Clinical Outcome: Final Event

[0197] Treatment effects on change from baseline will be analyzed by comparing the distributions of change scores using analysis of covariance, controlling for baseline values, or by using a chi-square test to test for association (or Fisher's exact test if fewer than five subjects in the treatment group fall into the change category). Treatment effects on clinical outcomes will be analyzed using a joint frailty model. Hypothesis testing will be two-sided.

[0198] A "success" area in the treatment group is one that meets the following criteria: superiority of SRD-001 is demonstrated at the 0.20 significance level for at least one outcome within that area, and superiority of SRD-001 is descriptively demonstrated for other outcomes within that area.

[0199] If there are at least two "success" domains in the SRD-001 group and no clinically significant deterioration (as predefined) is observed in any of these domains, then improvement due to the study drug will be considered to have been observed at the group level.

[0200] For subject-level analysis, each subject will be scored for each outcome as clinically significantly improved (+1), clinically significantly worsened (-1), or unchanged (0), and a subject-level activity score will be calculated as the sum of all outcomes. Clinically significant change will be predefined. A two-tailed t-test will be used to test for treatment effect based on mean scores. Subject-level improvement due to the investigational drug will be considered to have been achieved if superiority of SRD-001 is observed at a significance level of 0.20.

[0201] SRD-001 activity will be considered "significant" if an improvement in the SRD-001 group (as defined above) is detected at either the group or subject level and a descriptive improvement in the SRD-001 group is evident at both the group and subject levels. Because this efficacy analysis is exploratory, the significance level will not be adjusted for multiplicity.

[0202] Evaluation Schedule The evaluation schedule is shown in Table 3. [Table 3-2]

[0203] Expected Results Improvement in one or more of the above primary and / or secondary endpoints will be observed in the treatment group compared to the placebo group at one or more of the following time points: 6 months, 12 months, 18 months, and 24 months.

[0204] Abbreviations in Table 3: AAV1: adeno-associated virus serotype 1 ALT: alanine aminotransferase AST: aspartate aminotransferase BUN: Blood urea nitrogen CBC: complete blood count CK-MB: Creatine kinase test DMD UL-PROM: Patient-reported outcome measure for the upper limb in DMD d: number of days ECG: electrocardiogram ELISpot: enzyme-linked immunospot LGE: delayed gadolinium enhancement LDH: lactate dehydrogenase MRI: Magnetic Resonance Imaging NAb: Neutralizing antibody NSAA: North Star Ambulatory Assessment NT proBNP: N-terminal prohormone of brain natriuretic peptide PODCI: Pediatric Outcomes Data Collection Questionnaire PUL2.0: Upper Limb Functional Assessment Scale Version 2.0 WBC: white blood cell count a, Before screening b, before randomization c. Before administration of the study drug d, 2 to 4 hours after administration of the study drug e. Within 18 to 30 hours after cardiac catheterization and administration of the investigational drug f. Informed consent for this study will usually be obtained at least 30 days before screening. g, height (screening only), weight, systolic and diastolic blood pressure, pulse and temperature h, AE / SAE reporting begins from the time the subject signs the initial informed consent for the study. Clinical event reporting begins from the time the subject signs the informed consent for this study. i, performed in a central laboratory. On day 1, point-of-care testing was performed as required as a physical examination for cardiac catheterization and angiography, per standard of care. j, ELISpot can be performed at scheduled time points as well as any time clinically indicated. k, CBC (WBC differential), hemoglobin, hematocrit and platelet count l, basic metabolic panel (glucose, sodium, potassium, chloride, bicarbonate, BUN, creatinine, calcium), comprehensive liver panel (albumin, alkaline phosphatase, total protein, ALT, AST, direct bilirubin, total bilirubin), LDH and uric acid m, cardiac MRI performed before functional testing. n, randomization will occur before Day 1 to coincide with the recommended shipping lead time for the investigational drug. If angiography has not been performed within the past 2 months, it should be performed immediately prior to infusion and, if tolerated, should be administered with intravenous nitroglycerin. On Day 2, subjects will be contacted by telephone to assess for possible delayed complications related to cardiac catheterization (e.g., bleeding from the puncture site) and appropriate treatment will be administered if necessary. At Week 1, subjects will be contacted by telephone for a general safety assessment, unless clinically indicated, in-person assessment will be conducted as soon as possible.

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[0206] As used herein, the word "comprising" is synonymous with "including," "containing," or "characterized by," and is an inclusive or open-ended term that does not exclude other elements or method steps not recited.

[0207] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention are susceptible to modification, as are the manufacturing methods and equipment. Such modifications will be readily apparent to those skilled in the art from a consideration of this disclosure or from practice of the invention disclosed herein. Therefore, it is not intended that the present invention be limited to the specific embodiments disclosed herein, but rather that the present invention will embrace all modifications and alternatives falling within the true scope and spirit of the invention.

[0208] All references cited herein, including, but not limited to, published patent applications, unpublished patent applications, patents, and scientific literature, are incorporated herein by reference in their entirety and made a part of this specification. In the event that a publication, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or control with respect to any such conflicting matter.

[0209] The present specification includes the following inventions. [1] A method for treating, suppressing, or ameliorating skeletal muscular dystrophy in a subject, the method comprising administering to the subject a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide. [2] The method according to [1], wherein the muscular dystrophy comprises a generalized dystrophin deficiency in the subject. [3] The method according to [1] or [2] above, wherein the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). [4] The method according to [3], wherein the muscular dystrophy includes DMD. [5] The method according to any one of [1] to [4] above, wherein the skeletal muscular dystrophy includes myocardial remodeling and / or fibrosis. [6] The method according to any one of [1] to [5] above, wherein the treatment, inhibition, or improvement suppresses myocardial remodeling and / or fibrosis in the subject compared to a subject to which the polynucleotide is not administered. [7] The method according to any one of [1] to [6] above, wherein the SERCA polypeptide comprises a SERCA2a polypeptide. [8] The method according to any one of [1] to [7] above, wherein the polynucleotide comprises a vector. [9] The method according to [8] above, wherein the vector is selected from an adeno-associated virus (AAV) vector, a lentivirus vector, and a retrovirus vector.

[10] The method according to [9], wherein the vector comprises an AAV vector.

[11] The method described in

[10] above, wherein the AAV vector encodes an AAV having a serotype selected from AAV serotypes 1 to 12, or a fragment thereof.

[12] The method described in [9] or

[10] above, wherein the AAV vector encodes an AAV or a fragment thereof having a serotype selected from AAV serotype 1 (AAV1) and AAV serotype 9 (AAV9).

[13] The method according to any one of [1] to

[12] above, wherein the polynucleotide comprises a promoter operably linked to a nucleic acid encoding the SERCA polypeptide.

[14] The method according to

[13] , wherein the promoter comprises a constitutive promoter.

[15] The method according to

[13] or

[14] , wherein the promoter comprises a cytomegalovirus (CMV) promoter.

[16] The method according to any one of [1] to

[15] above, wherein the polynucleotide is encapsulated in a viral capsid.

[17] The method according to

[16] , wherein the polynucleotide comprises a nucleic acid encoding the viral capsid.

[18] The method according to any one of [1] to

[17] above, further comprising the step of determining the presence or absence of antibodies against an AAV serotype in the subject.

[19] The method according to any one of [1] to

[18] above, wherein the administration of the polynucleotide comprises systemic administration.

[20] The method according to any one of [1] to

[19] above, wherein the administration of the polynucleotide comprises intravenous administration.

[21] The method according to any one of [1] to

[20] above, wherein the administration of the polynucleotide comprises intracoronary injection.

[22] The method according to any one of [1] to

[21] above, wherein the administration of the polynucleotide comprises intrauterine administration.

[23] The method according to any one of [1] to

[22] above, wherein the administration of the polynucleotide consists of a single administration of the polynucleotide.

[24] The polynucleotide comprises a viral vector, and the administration is at least about 1 x 10 8 Viral genome size: approximately 1 x 10 15 The method according to any one of [1] to

[23] above, comprising administering the polynucleotide in an amount equal to the number of viral genomes.

[25] The dose of the polynucleotide is about 1 × 10 13 Viral genome size: approximately 9 x 10 13 The method according to

[24] above, wherein the number of viral genomes is determined.

[26] The method according to any one of [1] to

[25] above, further comprising the step of administering a vasodilator.

[27] The method according to

[26] , wherein the vasodilator is administered before administration of the polynucleotide.

[28] The method according to

[26] or

[27] above, wherein the vasodilator is administered simultaneously with the administration of the polynucleotide.

[29] The method according to any one of

[26] to

[28] above, wherein the vasodilator comprises nitroglycerin.

[30] The method according to any one of [1] to

[29] above, wherein the polynucleotide is administered before the onset of muscle tissue damage.

[31] The method according to

[30] , wherein the onset of muscle tissue damage due to skeletal muscular dystrophy is predicted.

[32] The method according to

[30] or

[31] , wherein the muscle tissue damage is measurable by muscle histological examination.

[33] The method according to any one of [1] to

[32] above, wherein the subject is present in the uterus.

[34] The method according to any one of [1] to

[32] above, wherein the subject is a newborn.

[35] The method according to any one of [1] to

[32] above, wherein the subject is at least 3 years old.

[36] The method according to any one of [1] to

[32] above, wherein the subject is at least 5 years old.

[37] The method according to any one of [1] to

[32] above, wherein the subject is at least 10 years old.

[38] The method according to any one of [1] to

[32] above, wherein the subject is 20 years of age or younger or 15 years of age or younger.

[39] The method according to any one of [1] to

[32] above, wherein the subject is aged between 10 and 20 years.

[40] The method according to any one of [1] to

[39] above, wherein the subject is unable to walk.

[41] The method according to any one of [1] to

[40] above, wherein the cardiac function of the subject is reduced compared to the cardiac function of a subject without muscular dystrophy.

[42] The method according to any one of [1] to

[41] above, wherein the subject is a mammal.

[43] The method according to any one of [1] to

[42] above, wherein the subject is a human.

[44] The method according to any one of [1] to

[43] above, wherein the subject is male.

[45] The method according to any one of [1] to

[44] above, wherein the treatment results in an improvement in the symptoms or indicators of skeletal muscular dystrophy of the subject for a period of at least 1 month, 2 months, 3 months, 6 months, 9 months, or 12 months after administration of the polynucleotide, compared to the symptoms or indicators of skeletal muscular dystrophy of an untreated subject.

[46] The method according to

[45] , wherein the period is at least 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[47] The method according to any one of [1] to

[46] above, wherein the treatment results in an improvement in the cardiac tissue or cardiac function of the subject compared to the cardiac tissue or cardiac function of an untreated subject.

[48] ​​The method according to any one of [1] to

[47] , wherein the treatment results in an improvement in the subject's skeletal muscle tissue or skeletal muscle function compared to the skeletal muscle tissue or skeletal muscle function of an untreated subject.

[49] The method according to any one of [1] to

[48] above, wherein the treatment results in an improvement in the ventricular function of the subject compared to the ventricular function of an untreated subject.

[50] The method described in

[49] , wherein the improvement in ventricular function comprises an improvement in parameters selected from the group consisting of changes from baseline in left ventricular structure and function assessed by delayed gadolinium enhancement (LGE) cardiac MRI, such as left ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume and / or circumferential strain, regional wall thickness, percentage of left ventricular LGE relative to left ventricular myocardial mass, left ventricular viable myocardial mass, and number of left ventricular segments with LGE; and a composite outcome of changes from baseline in left ventricular function (LVESV).

[51] The improvement was assessed by cardiac MRI with delayed gadolinium enhancement (LGE) in terms of left ventricular structure and function, including changes from baseline in left ventricular ejection fraction, end-diastolic volume, end-systolic volume, stroke volume and / or circumferential strain, regional wall thickness, percentage of left ventricular LGE relative to left ventricular mass, left ventricular viable mass, and number of left ventricular segments with LGE; a composite outcome of changes from baseline in left ventricular function (LVESV), Pulmonary Function Assessment Scale Version 2.0 (PUL2.0), selected pulmonary function, quality of life, and terminal events; and the following items: (a) P The method of any one of the above

[45] to

[50] , comprising improvement in parameters selected from the group consisting of: (a) skeletal muscle function assessed by UL2.0, grip strength, key pinch and fingertip pinch strength, elbow flexion strength, 10-meter walk / run time (10MWRT) if ambulatory, incidence of ambulatory inability defined as 10MWRT > 30 seconds, and the North Star Ambulation Assessment (NSAA); (b) pulmonary function assessed by resting vital capacity (SVC), forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), peak expiratory flow (PEF), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak cough flow (PCF), and inspiratory reserve volume (IFR); or (c) change from baseline in quality of life assessed by the DMD Ultimate Patient-Reported Outcome Measure for Upper Limb (DMD UL-PROM) and the Pediatric Outcomes Data Collection Questionnaire (PODCI).

[52] The method according to any one of

[45] to

[51] above, further comprising measuring the degree of improvement of the subject after the period.

[53] Use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide to treat, inhibit, or ameliorate skeletal muscular dystrophy in a subject.

[54] Use of a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide in the manufacture of a medicament for treating, inhibiting, or ameliorating skeletal muscular dystrophy in a subject.

[55] The use according to

[53] or

[54] , wherein the muscular dystrophy comprises a generalized dystrophin deficiency in the subject.

[56] The use according to any one of

[53] to

[55] above, wherein the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[57] The use according to

[56] , wherein the muscular dystrophy includes DMD.

[58] The use according to any one of

[53] to

[57] above, wherein the skeletal muscular dystrophy includes myocardial remodeling and / or fibrosis.

[59] The use according to any one of

[53] to

[58] above, wherein the SERCA polypeptide comprises a SERCA2a polypeptide.

[60] The use according to any one of

[53] to

[59] above, wherein the polynucleotide comprises a vector.

[61] The use according to

[60] , wherein the vector is selected from an adeno-associated virus (AAV) vector, a lentivirus vector, and a retrovirus vector.

[62] The use according to

[61] above, wherein the vector comprises an AAV vector.

[63] The use described in

[62] above, wherein the AAV vector encodes an AAV having a serotype selected from AAV serotypes 1 to 12, or a fragment thereof.

[64] The use according to

[62] or

[63] , wherein the AAV vector encodes an AAV or a fragment thereof having a serotype selected from AAV serotype 1 (AAV1) and AAV serotype 9 (AAV9).

[65] The use according to any one of

[53] to

[64] above, wherein the polynucleotide comprises a promoter operably linked to the nucleic acid encoding the SERCA polypeptide.

[66] The use according to

[65] above, wherein the promoter comprises a constitutive promoter.

[67] The use according to

[65] or

[66] above, wherein the promoter comprises a cytomegalovirus (CMV) promoter.

[68] The use according to any one of

[53] to

[67] above, wherein the polynucleotide is encapsulated in a viral capsid.

[69] The use according to

[68] , wherein the polynucleotide comprises a nucleic acid encoding the viral capsid.

[70] The use according to any one of

[53] to

[69] above, wherein the polynucleotide is prepared for systemic administration.

[71] The use according to any one of

[53] to

[70] above, wherein the polynucleotide is prepared for intravenous administration.

[72] The use according to any one of

[53] to

[71] above, wherein the polynucleotide is prepared for administration by intracoronary injection.

[73] The use according to any one of

[53] to

[72] above, wherein the polynucleotide is prepared for intrauterine administration.

[74] The use according to any one of

[53] to

[73] above, in combination with a vasodilator.

[75] The use according to

[74] , wherein the vasodilator comprises nitroglycerin.

[76] The use according to any one of

[53] to

[75] above, wherein the subject is a mammal.

[77] The use according to any one of

[53] to

[76] above, wherein the subject is a human.

[78] The use according to any one of

[53] to

[77] above, wherein the subject is present in uterus.

[79] The use according to any one of

[53] to

[77] above, wherein the subject is an infant.

[80] The use according to any one of

[53] to

[77] above, wherein the subject is a newborn.

[81] The use according to any one of

[53] to

[77] above, wherein the subject is at least 3 years old.

[82] The use according to any one of

[53] to

[77] above, wherein the subject is at least 5 years old.

[83] The use according to any one of

[53] to

[77] above, wherein the subject is at least 10 years old.

[84] The use according to any one of

[53] to

[77] above, wherein the subject is 20 years of age or younger or 15 years of age or younger.

[85] The use according to any one of

[53] to

[84] above, wherein the subject is male.

[86] A method for screening for a therapeutic agent that treats, inhibits, or ameliorates skeletal muscular dystrophy in a patient, the method comprising the steps of: (a) contacting a test agent with a ventricular muscle tissue strip; (b) measuring the contraction amplitude of the ventricular muscle tissue strip contacted with the test agent; (c) comparing the contraction amplitude of the ventricular muscle tissue strip contacted with the test agent with the contraction amplitude of a ventricular muscle tissue strip not contacted with the test agent; and (d) determining that the test agent contains the therapeutic agent based on the comparison.

[87] The method according to

[86] , wherein the steps of (i) inducing differentiation of a population of induced pluripotent stem cells to obtain a plurality of cardiomyocytes containing a plurality of ventricular muscle cells; (ii) dispersing the plurality of cardiomyocytes into single cells to obtain a plurality of cardiomyocytes; and (iii) contacting the plurality of cardiomyocytes with a population of fibroblasts in the presence of collagen under conditions that allow the production of ventricular muscle tissue fragments.

[88] The method according to

[86] or

[87] , further comprising the step of obtaining the ventricular muscle tissue fragment, the step of obtaining the ventricular muscle tissue fragment comprising: (i) inducing differentiation of a population of induced pluripotent stem cells to obtain a plurality of cardiomyocytes, each of which contains a plurality of ventricular muscle cells; (ii) dispersing the plurality of cardiomyocytes into single cells to obtain a plurality of cardiomyocytes; and (iii) contacting the plurality of cardiomyocytes with a population of fibroblasts in the presence of collagen under conditions that allow the obtaining of the ventricular muscle tissue fragment.

[89] The method according to any one of

[86] to

[88] above, wherein the population of induced pluripotent stem cells is obtained from a subject with Duchenne muscular dystrophy (DMD).

[90] The method according to any one of

[86] to

[89] above, wherein step (a) is carried out for at least 1 hour.

[91] The method according to any one of

[86] to

[90] above, wherein step (a) is carried out over at least one day.

[92] The method according to any one of

[86] to

[91] above, wherein an electric field stimulus of a certain frequency is applied to the ventricular muscle tissue strip.

[93] The method according to

[92] , wherein step (b) is carried out using a plurality of frequencies.

[94] The method according to any one of

[86] to

[93] , wherein step (b) comprises measuring a parameter selected from force production, normalized force production, velocity variability, force variability, force-frequency relationship and beta-adrenergic response.

[95] The method according to any one of

[86] to

[94] above, wherein the muscular dystrophy comprises a systemic dystrophin deficiency in the patient.

[96] The method according to any one of

[86] to

[95] above, wherein the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD).

[97] The method according to

[96] , wherein the muscular dystrophy includes DMD.

[98] The method according to any one of

[86] to

[97] above, wherein the test agent comprises a polynucleotide.

[99] The method according to

[98] , wherein the polynucleotide encodes a SERCA polypeptide.

[0100] The method described in

[99] , wherein the SERCA polypeptide comprises a SERCA2a polypeptide.

[0101] The method according to any one of

[98] to

[0100] above, wherein the polynucleotide comprises a vector.

[0102] The method described in

[0101] above, wherein the vector is selected from an adeno-associated virus (AAV) vector, a lentivirus vector, and a retrovirus vector.

[0103] The method described in

[0102] above, wherein the vector comprises an AAV vector.

[0104] The method described in

[0103] above, wherein the AAV vector encodes an AAV or a fragment thereof having a serotype selected from AAV serotypes 1 to 11.

[0105] The method described in

[0103] or

[0104] , wherein the AAV vector encodes an AAV or a fragment thereof having a serotype selected from AAV serotype 1 (AAV1) and AAV serotype 9 (AAV9).

[0106] A method described in any one of

[98] to

[0105] above, wherein the polynucleotide comprises a promoter operably linked to a nucleic acid encoding the SERCA polypeptide.

[0107] The method described in

[0106] above, wherein the promoter comprises a constitutive promoter.

[0108] The method described in

[0106] or

[0107] above, wherein the promoter comprises a cytomegalovirus (CMV) promoter.

[0109] The method described in

[0106] or

[0107] above, wherein the promoter comprises an inducible promoter.

[0110] A method according to any one of

[98] to

[0109] above, wherein the polynucleotide is packaged in a viral capsid.

[0111] The method described in

[0110] above, wherein the polynucleotide comprises a nucleic acid encoding the viral capsid.

Claims

[Claim 1] 1. A method of treating, inhibiting, or ameliorating skeletal muscular dystrophy in a subject, comprising: administering to the subject a polynucleotide comprising a nucleic acid encoding a sarcoplasmic / endoplasmic reticulum calcium ATPase (SERCA) polypeptide. A method comprising:

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