Recombinant adeno-associated virus products and methods for treating limb-girdle muscular dystrophy 2A

Abstract

This invention provides products and methods for treating limb-girdle muscular dystrophy 2A. [Solution] A recombinant adeno-associated virus (rAAV) encoding a protein having calpain 3 (CAPN3) activity is provided. The recombinant adeno-associated virus comprises a polynucleotide including a nucleotide sequence encoding a protein having CAPN3 activity. The nucleotide sequence encoding the CAPN3 activity protein is, for example, at least 90% identical to a specific sequence, or includes the specific sequence.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 691,934 filed June 29, 2018, and U.S. Provisional Patent Application No. 62 / 865,081 filed June 21, 2019, both of which are incorporated herein by reference in their entirety.

[0002] This specification provides products and methods for treating limb-girdle muscular dystrophy 2A. In this method, recombinant adeno-associated virus delivers DNA encoding a protein having calpain 3 (CAPN3) activity.

[0003] Inclusion by referencing the sequence list This application includes, as another part of the disclosure, a computer-readable sequence listing (filename: 52684P2_SeqListing.txt; 23,755 bytes - ASCII text file created on June 26, 2019), which is incorporated in its entirety by reference herein. [Background technology]

[0004] Muscular dystrophy (MD) is a group of genetic disorders characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some MDs develop in infancy or childhood, while others may not appear until middle age or later. The disease varies in terms of the distribution and degree of muscle weakness (some MDs also affect the myocardium), age of onset, rate of progression, and genetic pattern.

[0005] One group of MDs is limb-girdle MD (LGMD). LGMD is a rare condition that presents with varying symptoms in different individuals in terms of age of onset, location of muscle weakness, involvement of the heart and respiratory system, rate of progression, and severity. LGMD can develop in childhood, adolescence, young adulthood, or later. Both sexes are equally affected. LGMD causes weakness in the shoulders and hip girdle, and sometimes the surrounding muscles of the upper legs and upper arms also weaken over time. Leg weakness often precedes arm weakness. Facial muscles are usually unaffected. As the condition progresses, walking problems may arise, and wheelchair use may become necessary over time. When shoulder and arm muscles are involved, it may become difficult to raise the arms above the head or lift objects. In some types of LGMD, the heart and respiratory muscles may be involved.

[0006] LGMD has at least 19 subtypes, which are classified by the associated genetic defect. Type: Mode of inheritance (gene or chromosome) LGMD1A autosomal dominant myotirin gene LGMD1B autosomal dominant lamin A / C gene LGMD1C autosomal dominant caveolin gene LGMD1D Autosomal dominant chromosome 7 LGMD1E Autosomal dominant desmin gene LGMD1F Autosomal dominant chromosome 7 LGMD1G Autosomal dominant chromosome 4 LGMD2A autosomal recessive calpain-3 gene LGMD2B autosomal recessive dysferin gene LGMD2C is an autosomal recessive gene for gamma-sarcoglycan. LGMD2D autosomal recessive alpha-sarcoglycan gene LGMD2E autosomal recessive beta-sarcoglycan gene LGMD2F: Autosomal recessive delta-sarcoglycan gene LGMD2G autosomal recessive teletonin gene LGMD2H autosomal recessive TRIM32 LGMD2I autosomal recessive FKRP gene LGMD2J autosomal recessive Titin gene LGMD2K autosomal recessive POMT1 gene LGMD2L autosomal recessive Fukutin gene

[0007] Special tests for LGMD are currently carried out through the National Commissioning Group (NCG), a national diagnostic program.

[0008] Mutations in the calpain 3 gene (CAPN3) cause LGMD2A, one of the most common forms of limb-girdle muscular dystrophy worldwide. At present, there is no treatment for this genetic disease. Previous studies have shown the potential for CAPN3 gene transfer to correct the pathological signs in CAPN3-deficient mice. However, the expression of CAPN3 driven by the desmin promoter resulted in cardiotoxicity [Bartoli et al., Mol. Ther., 13:250-259 (2006)]. Follow-up studies have examined the skeletal muscle expression of the gene [Roudaut et al., Circulation, 128:1094-1104 (2013)].

[0009] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two 145-nucleotide inverted end sequences (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the AAV serotype genomes are known. For example, the whole genome of AAV-1 is available at GenBank deposit number NC_002077; the whole genome of AAV-2 is available at GenBank deposit number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983); the whole genome of AAV-3 is available at GenBank deposit number NC_1829; the whole genome of AAV-4 is available at GenBank deposit number NC_001829; the AAV-5 genome is available at GenBank deposit number AF085716; and the whole genome of AAV-6 is available at GenBank deposit number NC_00 The AAV-7 and AAV-8 genomes were provided in 1862, and at least portions of them are provided to GenBank deposit numbers AX753246 and AX753249, respectively; the AAV-9 genome is provided to Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided to Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided to Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is provided to U.S. Patent No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences that direct viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 in relation to their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) conjugate to the splicing of a single AAV intron (at nucleotides 2107 and 2227), resulting in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. These rep proteins possess multiple enzymatic properties that ultimately drive the replication of the viral genome.The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0010] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cellular, and spontaneous infection in humans and other animals is asymptomatic. Furthermore, because AAV infects many mammalian cells, it can target many different tissues in vivo. Additionally, AAV transducers slow-dividing and non-dividing cells and can persist essentially for the lifespan of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted into a plasmid as cloned DNA, enabling the construction of a recombinant genome. Furthermore, since the signals directing AAV replication and genomic capsid formation are contained within the ITR of the AAV genome, some or all of the approximately 4.3kb of internal genome (coding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. To generate an AAV vector, the rep and cap proteins may be provided in trans. Another important characteristic of AAV is its extreme stability and its heart-like nature. Because AAV can easily withstand the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), the importance of chilling AAV is low. AAV can also be freeze-dried. Finally, AAV-infected cells are not resistant to co-infection.

[0011] There is still a need in this field for the treatment of LGMD2A. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Bartoli et al., Mol. Ther., 13:250-259 (2006) [Overview of the project] [Means for solving the problem]

[0013] Methods and products for delivering DNA encoding a protein having calpain 3 (CAPN3) activity are provided herein. Such methods and products may be used to treat various diseases, such as LGMD2A.

[0014] A recombinant adeno-associated virus (rAAV) encoding a protein having calpain 3 (CAPN3) activity is provided. The recombinant adeno-associated virus contains a polynucleotide comprising a nucleotide sequence encoding the CAPN3 activity protein. The nucleotide sequence encoding the CAPN3 activity protein is, for example, at least 90% identical to SEQ ID NO: 2, or contains the sequence of SEQ ID NO: 2.

[0015] For example, the provided rAAV comprises a polynucleotide including a first AAV inverted terminal sequence (ITR), a promoter, a nucleotide sequence encoding a protein having calpain 3 (CAPN3) activity, and a second AAV ITR. The nucleotide sequence encoding the protein having CAPN3 activity is, for example, at least 90% identical to SEQ ID NO: 2, or at least 91% identical to SEQ ID NO: 2, at least 92% identical to SEQ ID NO: 2, at least 93% identical to SEQ ID NO: 2, at least 94% identical to SEQ ID NO: 2, at least 95% identical to SEQ ID NO: 2, at least 96% identical to SEQ ID NO: 2, or at least 97% identical to SEQ ID NO: 2, at least 98% identical to SEQ ID NO: 2, or at least 99% identical to SEQ ID NO: 2. The nucleotide sequence encoding the protein having CAPN3 activity includes the sequence of SEQ ID NO: 2.

[0016] Furthermore, the provided rAAV includes a nucleotide sequence encoding a CAPN3-active protein, the nucleotide sequence including an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, or at least 91% identical to SEQ ID NO: 7, at least 92% identical to SEQ ID NO: 7, at least 93% identical to SEQ ID NO: 7, at least 94% identical to SEQ ID NO: 7, at least 95% identical to SEQ ID NO: 7, at least 96% identical to SEQ ID NO: 7, or at least 97% identical to SEQ ID NO: 7, at least 98% identical to SEQ ID NO: 7, or at least 99% identical to SEQ ID NO: 7. The rAAV includes a nucleotide sequence encoding a CAPN3-active protein including the amino acid sequence of SEQ ID NO: 7.

[0017] The provided rAAV contains a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1, or at least 91% identical to SEQ ID NO: 1, at least 92% identical to SEQ ID NO: 1, at least 93% identical to SEQ ID NO: 1, at least 94% identical to SEQ ID NO: 1, at least 95% identical to SEQ ID NO: 1, at least 96% identical to SEQ ID NO: 1, or at least 97% identical to SEQ ID NO: 1, at least 98% identical to SEQ ID NO: 1, or at least 99% identical to SEQ ID NO: 1. The rAAV contains the polynucleotide sequence of SEQ ID NO: 1.

[0018] In one embodiment, the nucleotide sequence is under the transcriptional control of a muscle-specific promoter. For example, the muscle-specific promoter includes one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, cleaved MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast contractile troponin c gene element, slow contractile cardiac troponin c gene element, slow contractile troponin i gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid-induced element, and glucocorticoid response element (gre). In one embodiment, the muscle-specific promoter is the tMCK promoter and includes the sequence of Sequence ID No. 3.

[0019] For example, rAAV comprises a polynucleotide including a first AAV inverted terminal sequence (ITR), a tMCK promoter, a nucleotide sequence encoding a protein having calpain 3 activity, and a second AAV inverted terminal sequence (ITR). The AAV ITR (e.g., the first and / or second AAV ITR) is, for example, the AAV2 inverted terminal sequence. The rAAV capsid protein includes, for example, the AAV rh.74 capsid protein or the AAV9 capsid protein.

[0020] The rAAV provided contains one or more of the following capsid proteins: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, and AAV rh.10.

[0021] In another embodiment, a composition comprising any of the disclosed rAAVs is provided. For example, the composition is formulated for intramuscular or intravenous injection.

[0022] Also provided is a method for treating limb-girdle muscular dystrophy 2A in a subject, comprising administering to the subject a therapeutically effective dose of any of the disclosed rAAVs, or any composition containing the disclosed rAAVs. In any of the methods provided, the rAAV is administered by intramuscular or intravenous injection.

[0023] For example, treatment using these methods results in one or more of the following: (a) an increase in muscle fiber diameter, (b) a decrease in the number of lobular muscle fibers, (c) a decrease in the number of fibers with an internal nucleus, (d) a decrease in endomysial connective tissue content, (e) correction of muscle atrophy, and (f) an increase in muscle strength. The muscle fibers affected by the treatment include one or more of the following: slow contraction oxidative (STO) muscle fibers, fast contraction oxidative (FTO) muscle fibers, and fast contraction glycolysis (FTG) fibers.

[0024] Furthermore, in any of the provided methods, the treatment results in (a) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% of 1 mm by 4 weeks after administration. 2 (b) a decrease in the total number of muscle fibers per unit area, (c) an increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% by 4 weeks after administration, and (d) an increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% by 1 mm by 4 weeks after administration. 2 (d) a decrease in the number of STO muscle fibers per unit area, (e) an increase of at least 5%, 10%, 15%, 20%, or 25% in STO muscle fiber diameter by 4 weeks post-administration, and (f) an increase of at least 5%, 10%, 15%, or 20% in 1 mm by 4 weeks post-administration. 2 (f) a decrease in the number of FTO muscle fibers per unit area, (g) an increase of at least 5%, 10%, 15%, or 20% in FTO muscle fiber diameter by 4 weeks after administration, and (g) an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% in 1 mm by 4 weeks after administration. 2 A decrease in the number of FTG muscle fibers per unit area occurs, and (h) within 4 weeks after administration, one or more of the following occurs: at least 5%, 10%, 15%, 20%, or 25% increase in FTG muscle fiber diameter.

[0025] In any of the methods provided, the cardiomyocyte of interest represents a composition comprising a minimal or low calpain 3 protein expressed from any of the provided rAAVs, or any of the provided rAAVs. The muscle fibers affected by treatment with the composition include one or more of the following: slow contractile oxidative (STO) muscle fibers, fast contractile oxidative (FTO) muscle fibers, and fast contractile glycolytic (FTG) fibers.

[0026] A composition for treating limb-girdle muscular dystrophy 2A is provided, comprising any of the disclosed rAAVs in a therapeutically effective amount, or a composition comprising any of the disclosed rAAVs. These compositions for treating limb-girdle muscular dystrophy 2A are formulated for administration by intramuscular or intravenous injection. Furthermore, treatment of limb-girdle muscular dystrophy 2A with any of the disclosed compositions results in one or more of the following: (a) an increase in muscle fiber diameter, (b) a decrease in the number of small lobe muscle fibers, (c) a decrease in the number of fibers with an internal nucleus, (d) a decrease in endomysial connective tissue content, (e) correction of muscle atrophy, and (f) an increase in muscle strength. The muscle fibers affected by treatment with the composition include one or more of the following: slow contraction oxidative (STO) muscle fibers, fast contraction oxidative (FTO) muscle fibers, and fast contraction glycolysis (FTG) fibers.

[0027] Furthermore, treatment with any of the disclosed compositions for treating limb-girdle muscular dystrophy 2A is (a) within 4 weeks after administration, at least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% of 1 mm 2 (b) a decrease in the total number of muscle fibers per unit area, (c) an increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% by 4 weeks after administration, and (d) an increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% by 1 mm by 4 weeks after administration. 2 (d) a decrease in the number of STO muscle fibers per unit area, (e) an increase of at least 5%, 10%, 15%, 20%, or 25% in STO muscle fiber diameter by 4 weeks post-administration, and (f) an increase of at least 5%, 10%, 15%, or 20% in 1 mm by 4 weeks post-administration. 2 (f) a decrease in the number of FTO muscle fibers per unit area, (g) an increase of at least 5%, 10%, 15%, or 20% in FTO muscle fiber diameter by 4 weeks after administration, and (g) an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% in 1 mm by 4 weeks after administration. 2 A decrease in the number of FTG muscle fibers per unit area occurs, and (h) within 4 weeks after administration, one or more of the following occurs: at least 5%, 10%, 15%, 20%, or 25% increase in FTG muscle fiber diameter.

[0028] Treatment with any of the provided compositions for treating limb-girdle muscular dystrophy 2A shows minimal or low calpain 3 protein expressed from any of the provided rAAVs or from a composition comprising any of the provided rAAVs in the myocardium of a subject. The myocardium after rAAV administration shows no or very little toxic effects such as inflammation, necrosis, and / or regeneration.

[0029] The present disclosure also provides the use of a therapeutically effective amount of the disclosed rAAV or a composition comprising any of the disclosed rAAVs for the preparation of a medicament for the treatment of limb-girdle muscular dystrophy 2A. For example, the medicament is formulated for administration by intramuscular injection or intravenous injection.

[0030] In any of these uses, treatment with this medicament results in one or more of (a) an increase in muscle fiber diameter, (b) a decrease in the number of small-lobed muscle fibers, (c) a decrease in the number of fibers with internal nuclei, (d) a decrease in the endomysial connective tissue content, (e) correction of muscle atrophy, and (f) an increase in muscle force generation. The muscle fibers affected by treatment with this medicament are one or more of slow-twitch oxidative (STO) muscle fibers, fast-twitch oxidative (FTO) muscle fibers, and fast-twitch glycolytic (FTG) fibers.

[0031] Furthermore, in any of the uses of a therapeutically effective amount of any of the disclosed rAAVs or the provided compositions, treatment with this medicament results in (a) a decrease in the total number of muscle fibers per 1 mm of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% by 4 weeks after administration, (b) an increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% by 4 weeks after administration, (c) a decrease in the number of STO muscle fibers per 1 mm of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% by 4 weeks after administration, (d) an increase in STO muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% by 4 weeks after administration, (e) a decrease in the number of muscle fibers per 1 mm of at least 5%, 10%, 15%, or 20% by 4 weeks after administration. 2 per 1 mm of at least 5%, 10%, 15%, 20%, or 25% increase in muscle fiber diameter, (c) a decrease in the number of STO muscle fibers per 1 mm of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% by 4 weeks after administration, (d) an increase in STO muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% by 4 weeks after administration, (e) a decrease in the number of muscle fibers per 1 mm of at least 5%, 10%, 15%, or 20% by 4 weeks after administration. 2 per 1 mm of at least 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter, (e) a decrease in the number of muscle fibers per 1 mm of at least 5%, 10%, 15%, or 20% by 4 weeks after administration. 2(f) a decrease in the number of FTO muscle fibers per unit area, (g) an increase of at least 5%, 10%, 15%, or 20% in FTO muscle fiber diameter by 4 weeks after administration, and (g) an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% in 1 mm by 4 weeks after administration. 2 A decrease in the number of FTG muscle fibers per unit area occurs, and (h) within 4 weeks after administration, one or more of the following occurs: at least 5%, 10%, 15%, 20%, or 25% increase in FTG muscle fiber diameter.

[0032] Any therapeutically effective use of the disclosed rAAV or the provided composition will result in the target myocardium showing no, minimal, or low levels of calpain 3 protein expressed from the disclosed or disclosed composition after treatment with the drug. The present invention provides, for example, the following items: (Item 1) Recombinant adeno-associated virus (rAAV) containing a polynucleotide comprising a first AAV inverted terminal sequence (ITR), a promoter, a nucleotide sequence encoding a protein with calpain 3 (CAPN3) activity, and a second AAV ITR. (Item 2) The rAAV according to item 1, wherein the nucleotide sequence encoding the CAPN3-active protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. (Item 3) The rAAV according to item 1 or 2, wherein the nucleotide sequence encoding the CAPN3-active protein is at least 95% identical to SEQ ID NO: 2. (Item 4) The rAAV according to any one of items 1 to 3, wherein the nucleotide sequence encoding the protein having CAPN3 activity includes the sequence of SEQ ID NO: 2. (Item 5) The rAAV according to any one of items 1 to 4, wherein the protein having CAPN3 activity contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. (Item 6) The rAAV according to any one of items 1 to 4, wherein the protein having CAPN3 activity contains an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. (Item 7) The rAAV described in any one of items 1 to 4, wherein the protein having CAPN3 activity includes the amino acid sequence of SEQ ID NO: 7. (Item 8) The rAAV according to any one of items 1 to 7, wherein the polynucleotide comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. (Item 9) The rAAV according to any one of items 1 to 7, wherein the polynucleotide comprises a sequence that is at least 95% identical to SEQ ID NO: 1. (Item 10) The polynucleotide is an rAAV according to any one of items 1 to 7, wherein the polynucleotide includes the sequence of SEQ ID NO: 1. (Item 11) The rAAV described in any one of items 1 to 10, wherein the promoter is a muscle-specific promoter. (Item 12) The muscle-specific promoter is rAAV as described in item 11, comprising one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, cleaved MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast contraction troponin c gene element, slow contraction cardiac troponin c gene element, slow contraction troponin i gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid-induced element, and glucocorticoid response element (gre). (Item 13) The rAAV according to item 11, wherein the muscle-specific promoter is the MCK promoter, the tMCK promoter, or the MHCK7 promoter. (Item 14) The rAAV according to item 11, wherein the muscle-specific promoter is a cleaved MCK promoter containing the nucleotide sequence of SEQ ID NO: 3. (Item 15) The rAAV described in any one of items 1 to 14, wherein the first and second AAV inverted terminal sequences are AAV2 inverted terminal sequences. (Item 16) The rAAV described in any one of items 1 to 15, wherein the rAAV contains one or more of the AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, and AAV rh.10 capsid proteins. (Item 17) The rAAV described in any one of item 16, wherein the rAAV comprises rh.74 capsid protein or AAV9 capsid protein. (Item 18) A composition comprising rAAV as described in any one of items 1 to 17. (Item 19) A method for treating limb-girdle muscular dystrophy 2A in a subject, comprising administering a therapeutically effective amount of rAAV as described in any one of items 1 to 17 or a composition as described in item 18 to the subject. (Item 20) Through the aforementioned treatment, (a) Increase in muscle fiber diameter, (b) Decrease in the number of muscle fibers in the small lobes, (c) Decrease in the number of fibers with a nucleus, (d) Decrease in endomysial connective tissue content, (e) Correction of muscle atrophy, and (f) The method described in item 19, which results in one or more of the increases in muscle strength generation. (Item 21) The method according to item 20, wherein the muscle fibers include one or more of the following: slow contraction oxidative (STO) muscle fibers, fast contraction oxidative (FTO) muscle fibers, and fast contraction glycolysis (FTG) fibers. (Item 22) Through the aforementioned treatment, (a) At least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% of 1 mm within 4 weeks after administration. 2 Decrease in the total number of muscle fibers per unit area (b) An increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% within 4 weeks after administration. (c) At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% of 1 mm within 4 weeks after administration. 2 Decrease in the number of STO muscle fibers per unit (d) At least a 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter within 4 weeks of administration. (e) At least 5%, 10%, 15%, or 20% of 1 mm within 4 weeks after administration 2 Decrease in the number of muscle fibers per FTO (f) An increase of at least 5%, 10%, 15%, or 20% in FTO muscle fiber diameter within 4 weeks of administration. (g) At least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of 1 mm within 4 weeks after administration. 2 The decrease in the number of FTG muscle fibers per unit and (h) The method according to any one of items 19-21, wherein by 4 weeks after administration, one or more of the following increases in FTG muscle fiber diameter occur: at least 5%, 10%, 15%, 20%, or 25%. (Item 23) The method according to any one of items 19 to 22, wherein the administration is by intramuscular injection or intravenous injection. (Item 24) The method according to any one of items 19 to 23, wherein the target myocardium exhibits minimal or low calpain 3 protein expressed from any one of items 1 to 17 or the composition described in item 18. (Item 25) A composition for treating limb-girdle muscular dystrophy 2A, comprising a therapeutically effective amount of rAAV as described in any one of items 1 to 17 or the composition as described in item 18. (Item 26) By the treatment using the above composition, (a) Increase in muscle fiber diameter, (b) Decrease in the number of muscle fibers in the small lobes, (c) Decrease in the number of fibers with a nucleus, (d) Decrease in endomysial connective tissue content, (e) Correction of muscle atrophy, and (f) The composition described in item 25, which results in one or more of the increases in muscle strength generation. (Item 27) The composition according to item 26, wherein the muscle fibers include one or more of the following: slow contraction oxidative (STO) muscle fibers, fast contraction oxidative (FTO) muscle fibers, and fast contraction glycolysis (FTG) fibers. (Item 28) By the treatment using the above composition, (a) At least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% of 1 mm within 4 weeks after administration. 2 Decrease in the total number of muscle fibers per unit area (b) An increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% within 4 weeks after administration. (c) At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% of 1 mm within 4 weeks after administration. 2 Decrease in the number of STO muscle fibers per unit (d) At least a 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter within 4 weeks of administration. (e) At least 5%, 10%, 15%, or 20% of 1 mm within 4 weeks after administration 2 Decrease in the number of muscle fibers per FTO (f) An increase of at least 5%, 10%, 15%, or 20% in FTO muscle fiber diameter within 4 weeks of administration. (g) At least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of 1 mm within 4 weeks after administration. 2 The decrease in the number of FTG muscle fibers per unit and (h) The composition according to any one of items 25 to 27, which results in at least one of the following increases in FTG muscle fiber diameter: 5%, 10%, 15%, 20%, or 25% within four weeks after administration. (Item 29) The composition according to any one of items 25 to 28, wherein the composition is formulated for administration by intramuscular or intravenous injection. (Item 30) The composition according to any one of items 25 to 28, wherein, after treatment with the composition, the target myocardium exhibits minimal or low calpain 3 protein expressed from any one of items 1 to 17 or the composition according to item 18. (Item 31) Use of a therapeutically effective amount of rAAV as described in any one of items 1 to 17 or the composition as described in item 18 for the preparation of a drug for the treatment of limb-girdle muscular dystrophy 2A. (Item 32) As a result of the treatment with the aforementioned drug, (a) Increase in muscle fiber diameter, (b) Decrease in the number of muscle fibers in the small lobes, (c) Decrease in the number of fibers with a nucleus, (d) Decrease in endomysial connective tissue content, (e) Correction of muscle atrophy, and (f) Use as described in item 31, which results in one or more of the increases in muscle strength generation. (Item 33) The use described in item 32, wherein the muscle fibers include one or more of the following: slow contraction oxidative (STO) muscle fibers, fast contraction oxidative (FTO) muscle fibers, and fast contraction glycolysis (FTG) fibers. (Item 34) As a result of the treatment with the aforementioned drug, (a) At least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% of 1 mm within 4 weeks after administration. 2 Decrease in the total number of muscle fibers per unit area (b) An increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% within 4 weeks after administration. (c) At least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% of 1 mm within 4 weeks after administration. 2 Decrease in the number of STO muscle fibers per unit (d) At least a 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter within 4 weeks of administration. (e) At least 5%, 10%, 15%, or 20% of 1 mm within 4 weeks after administration 2 Decrease in the number of muscle fibers per FTO (f) An increase of at least 5%, 10%, 15%, or 20% in FTO muscle fiber diameter within 4 weeks of administration. (g) At least 5%, 10%, 15%, 20%, 25%, 30%, or 35% of 1 mm within 4 weeks after administration. 2 The decrease in the number of FTG muscle fibers per unit and (h) Use as described in any one of items 31-33, in which at least one of the following increases in FTG muscle fiber diameter occurs within 4 weeks of administration: 5%, 10%, 15%, 20%, or 25%. (Item 35) The use described in any one of items 31 to 34, wherein the drug is formulated for administration by intramuscular or intravenous injection. (Item 36) The use according to any one of items 31 to 35, wherein, after treatment with the aforementioned drug, the subject myocardium exhibits minimal or low calpain 3 protein expressed from any one of items 1 to 17 or the composition described in item 18. [Brief explanation of the drawing]

[0033] [Figure 1]Figures 1A–1F show that gene therapy restored regenerative impairment in CAPN3-KO muscle. A schematic diagram of single-stranded AAV9.CAPN3 rAAV is shown in Figure 1A. Between the 5' and 3' single-stranded ITRs (inverted terminal sequences), the muscle creatine kinase (MCK) promoter (563 bp) drives the expression of the CAPN3 open reading frame (2466 bp). The polyadenylation site (polyA, 53 bp) is also labeled. First, tibialis anterior muscle (TA) from CAPN3-KO mice was injected with CTX, and two weeks later, 1 × 10¹¹ vg of AAV.CAPN3 was injected into the left TA (Figure 1B), or PBS was injected into the right TA (Figure 1C). Four weeks after rAAV injection, muscle diameter increased, and lobulated fibers were fewer compared to untreated CAPN3-KO muscle. In Figure 1D, the lobulated fiber and mitochondrial distribution (arrows) with submembrane organelle patterns suggest partial myotubular fusion in untreated CAPN3-KO muscle at higher magnification. For B-D, the scale bar was 20 μm. In Figure 1E, the histogram of muscle fiber size distribution from treated and untreated TA muscle from CAPN3-KO mice (mean ± SEM / mm2 area derived from 3 mice in each group) shows that treatment leads to a shift to larger diameter fibers, and the small-diameter subpopulation present in the untreated group increases. In Figure 1F, the histogram of slow contractile oxidative (STO) fiber size distribution shows a large number of small-diameter fibers (e.g., fiber diameter less than 30 μm) in untreated CAPN3-KO muscle compared to treated CAPN3-KO muscle.

[0034] [Figure 2] Figure 2 shows a schematic diagram of the rAAV of this disclosure, named "AAVrh.74.tMCK.CAPN3".

[0035] [Figure 3]Figures 3A and 3B provide Western blot (Panel A) and RT-PCR (Panel B) data after administration of AAVrh.74.tMCK.CAPN3 by intramuscular injection (1E11 vg) and systemic injection (3E12 vg and 6E12 vg). These data were compared with normal human myolysis (gel loading of 60% of total protein compared to mouse lysates) and untreated CAPN3-KO mice.

[0036] [Figure 4] Figure 4 provides representative images of SDH-stained tissue sections of CAPN3 KO (injected AAV.hCAPN3 gene and untreated) and wild-type (WT) TA muscle. In TA muscle of mice treated with AAVrh.74.tMCK.CAPN3, the mean fiber sizes of slow contractile oxidative (STO, dark), fast contractile oxidative (FTO, intermediate), and fast contractile glycolytic (FTG, light) fibers appeared to be normalized to WT values. Fibromas size with and without treatment is shown in Table 4.

[0037] [Figure 5] Figure 5 provides CAPN3 protein expression levels in WT (Z18-14) and TA muscles from the low-dose cohort (3E12 vg, Z18-13, Z18-15, Z18-16, Z18-17, Z18-18), as well as gastrocnemius, cardiac, quadriceps, tibialis anterior (TA), and triceps muscles from the high-dose cohort (6E12 vg, Z18-20, Z18-21, Z18-23, Z18-24, Z18-22) (UT: untreated).

[0038] [Figure 6] Figure 6 shows AAVrh74.tMCK.hCAPN3 vector copy / μg genomic DNA in a whole-body high-dose cohort of 6E12 vector genomes in the following muscles: quadriceps (quad), heart, tibialis anterior (TA), gastrocnemius (gastrocnemius), triceps, and liver.

[0039] [Figure 7]Figure 7 shows the mean fiber diameters of slow contractile oxidative (STO, dark), fast contractile oxidative (FTO, intermediate), and fast contractile glycolytic (FTG, light) fibers from left TA muscle after systemic administration of AAVrh.74.tMCK.CAPN3 at 3E12 and 6E12 vg. Data from untreated CAPN3 KO and WT mice are included.

[0040] [Figure 8A] Figure 8 provides data from the run-to-exhaustion study. Figure 8A provides data from a low-dose cohort administered 3E12 vg of AAVrh.74.tMCK.CAPN3 and a high-dose cohort administered 6E12 vg of AAVrh.74.tMCK.CAPN3 4 weeks after systemic administration. Treated CAPN3 KO mice performed better in the run-to-exhaustion study compared to untreated mice. Figure 8B provides data from a high-dose cohort and untreated mice (n-16) tested 20-24 weeks after systemic administration of 6E12 vg of AAVrh.74.tMCK.CAPN3 (n=5). [Figure 8B] Same as above.

[0041] [Figure 9] Figure 9 provides fresh frozen sections of the left ventricle, hematoxylin and eosin (H&E) stained, from representative cardiac specimens of CAPN3 KO mice 4 weeks after systemic injection of 3E12 vg and 6E12 vg doses of the AAVrh7.4.tMCK.hCAPN3 vector, along with a fitted untreated control.

[0042] [Figure 10] Figure 10 provides Western blot analysis of cardiac tissue from a high-dose cohort (administered 6E12 vg of AAVrh7.4.tMCK.hCAPN3). This analysis showed that the hearts of the treated animals had no or minimal detectable calpain 3 protein. Animal identification numbers Z18-19 and 22 represent lysates from untreated CAPN3 KO mice. [Modes for carrying out the invention]

[0043] The recombinant AAVs (rAAVs) provided herein comprise a polynucleotide comprising a first AAV inverted terminal sequence (ITR), a promoter, a nucleotide sequence encoding a protein having calpain 3 (CAPN3) activity, and a second AAV ITR. In one embodiment, the nucleotide encodes CAPN3. Embodiments, but are not limited, include rAAVs comprising a nucleotide sequence encoding CAPN3 or a protein having CAPN3 activity, wherein the nucleotide sequence is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% identical to the sequence of SEQ ID NO: 2. Additional embodiments include, but are not limited to, rAAVs comprising a nucleotide sequence encoding a polypeptide having CAPN3 proteolytic activity and being at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence described in SEQ ID NO: 2. CAPN3 proteolytic activity is understood in the art as the activity of proteolytically cleaving potential substrates such as fodrin and HSP60, and / or autolytic cleavage activity. Therefore, as used herein, the term “protein having calpain 3 (CAPN3) activity” refers to a protein having CAPN3 proteolytic activity, which includes, but is not limited to, activity of proteolytic substrates such as fodrin and HSP60, and / or autolytic cleavage activity. A protein having CAPN3 activity may have the full or partial activity of the full-length calpain 3 protein. In one embodiment, a protein having CAPN3 activity has at least 60%, 70%, 80%, 90%, 95%, or 99% of the activity of the full-length CAPN3 protein. In another embodiment, a protein having CAPN3 activity contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.

[0044] In some embodiments, the nucleotide sequence encoding the CAPN3-active protein includes the sequence of SEQ ID NO: 2. In another embodiment, the CAPN3-active protein includes an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In yet another embodiment, the CAPN3-active protein includes the amino acid sequence of SEQ ID NO: 7. In yet another embodiment, the polynucleotide of rAAV includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In yet another embodiment, the polynucleotide includes a sequence that is at least 95% identical to SEQ ID NO: 1. In one embodiment, the polynucleotide includes the sequence of SEQ ID NO: 1.

[0045] In another embodiment, recombinant AAVs comprising a nucleotide sequence encoding a protein having CAPN3 activity and / or a nucleotide sequence hybridizing under stringent conditions to the nucleic acid sequence of SEQ ID NO: 2 or its complement are described herein. The term “stringent” is used to refer to conditions that are generally understood to be stringent in the art. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of a denaturing agent such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate (65°C to 68°C) or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide (42°C). Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989).

[0046] In the recombinant genomes described herein, CAPN3 polynucleotides are operably bound to transcriptional regulatory elements (including, but not limited to, promoters, enhancers, and / or introns), particularly transcriptional regulatory elements that function in the target cell of interest. For example, various embodiments provide methods for transducing muscle cells using muscle-specific transcriptional regulatory elements, including, but not limited to, those derived from myosin gene families such as actin and myoD gene families, e.g., from [see Weintraub et al., Science, 251:761-766 (1991)], muscle cell-specific enhancer-binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol, 11:4854-4862 (1991)], regulatory elements derived from human skeletal actin genes [Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)], and muscle creatine kinase sequence elements [Johnson et al., Mol Cell Examples include regulatory elements derived from the mouse creatine kinase enhancer (mCK) element, the fast contractile troponin C gene, the slow contractile cardiac troponin C gene, and the slow contractile troponin I gene of the skeleton: hypoxia-induced nuclear factor [Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)], steroid-induced elements and promoters including the glucocorticoid response element (GRE) [See Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)], the tMCK promoter [See Wang et al., Gene Therapy, 15:1489-1499 (2008)], the CK6 promoter [Wang et al., see above], and other regulatory elements. In one embodiment, a nucleotide sequence encoding a protein having calpain 3 (CAPN3) activity is operably ligated to a muscle-specific promoter.In one embodiment, the muscle-specific promoter includes one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer binding factor mef binding element, muscle creatine kinase (MCK) promoter, cleaved MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) promoter, C5-12 promoter, mouse creatine kinase enhancer element, skeletal fast contraction troponin c gene element, slow contraction cardiac troponin c gene element, slow contraction troponin i gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid-induced element, and glucocorticoid response element (gre). In another embodiment, the muscle-specific promoter is the MCK promoter, tMCK promoter, or MHCK7 promoter. In some embodiments, the muscle-specific promoter is a tMCK containing the nucleotide sequence of sequence number 3.

[0047] Previous studies have shown that CAPN3 expression mediated by the desmin promoter leads to cardiotoxicity. In follow-up studies, selective skeletal muscle expression of the gene resulted in the disappearance of cardiac defects. The AAV genome disclosed herein, containing a muscle-specific promoter and a tMCK that restricts CAPN3 expression in skeletal muscle, showed no cardiotoxicity after systemic delivery of 6E12 vg (twice the proposed initial high dose) of the virus four weeks after gene injection.

[0048] The rAAV genome described herein lacks AAV rep and cap DNA. The provided rAAV genome contains the CAPN3 polynucleotide described above, and one or more AAV ITRs adjacent to the polynucleotide. The AAV DNA of the rAAV genome may be an AAV serotype that may result from recombinant viruses containing, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, and AAV rh.10. Other types of rAAV variants with capsid mutations, such as rAAV, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background technology section above, the nucleotide sequences of various AAV serotype genomes are known in the art. AAV1, AAV5, AAV6, AAV8, or AAV9 may be used to promote skeletal muscle-specific expression.

[0049] The provided DNA plasmid contains the rAAV genome. The DNA plasmid is transferred to a cell tolerant of infection with an AAV helper virus (including, but not limited to, adenovirus, E1 deletion adenovirus, or herpesvirus), which assembles the rAAV genome into infectious viral particles. Techniques for producing rAAV particles that provide the packaged AAV genome, rep and cap genes, and helper virus function to the cell are standard in the art. The production of rAAV requires the presence of the following components within a single cell (indicated herein as a packaging cell): the rAAV genome, the AAVrep and cap genes isolated from the rAAV genome (i.e., in the absence of the rAAV genome), and helper virus function. The AAV ITR and rep and cap genes may originate from any AAV serotype from which the recombinant virus may arise or derive from, but is not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.10, and AAV rh.74. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated herein by reference in its entirety. Therefore, in one embodiment, rAAV comprises one or more of the AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, or AAV rh.10 capsid proteins. In another embodiment, rAAV comprises the AAV rh.74 capsid protein or the AAV9 capsid protein.

[0050] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from the rAAV genome, and a selection marker such as a neomycin resistance gene is incorporated into the cell genome. The AAV genome is introduced into bacterial plasmids by procedures such as GC tailing [Samulski et al., Proc. Natl. Acad. S6. USA, 79:2077-2081 (1982)], by adding a synthetic linker containing restriction endonuclease cleavage sites [Laughlin et al., Gene, 23:65-73 (1983)], or by direct blunt-end linking [Senapathy & Carter, J. Biol. Chem., 259:4661-4666 (1984)]. Subsequently, the packaging cell line is infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus instead of plasmid to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0051] The general principles of rAAV production are reviewed, for example, in Carter, *Current Opinions in Biotechnology*, 1533-1539 (1992); and Muzyczka, *Curr. Topics in Microbial. and Immunol.*, 158:97-129 (1992). Various approaches include: Ratschin et al., Mol. Cell. Biol., 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol., 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); Lebkowski et al., Mol. Cell. Biol., 7:349 (1988); Samulski et al., J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO95 / 13365 and the corresponding U.S. Patent No. 5,658,776; WO 95 / 13392;WO96 / 17947;PCT / US98 / 18600;WO97 / 09441(PCT / US96 / 14423);WO97 / 08298(PCT / US96 / 13872);WO97 / 21825(PCT / US96 / 20777);WO 97 / 06243(PCT / FR96 / 01064);WO99 / 11764;Perrin et al., Vaccine, 13:1244-1250(1995);Paul et al., Human Gene Therapy, 4:609-615(1993);Clark et al., Gene Therapy This is described in 3:1124–1132 (1996); U.S. Patent Nos. 5,786,211, 5,871,982, 6,258,595, and McCarty, Mol. Ther., 16(10):1648–1656 (2008). The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on these sections of the documents relating to the production of rAAV.

[0052] Therefore, packaging cells that produce infectious rAAV are provided. In one embodiment, the packaging cells may be stable transformed cancer cells such as HeLa cells and PerC.6 cells (congener 293 strain). In another embodiment, the packaging cells are non-transformed cancer cells such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey lung cells).

[0053] Therefore, the recombinant AAVs provided herein are replication-deficient, infectious, capsidized viral particles containing a recombinant genome. Examples include, but are not limited to, a genome containing the sequence described in SEQ ID NO: 1 encoding CAPN3, an rAAV containing a genome essentially consisting of the sequence described in SEQ ID NO: 1 encoding CAPN3, and an rAAV containing a genome consisting of the sequence described in SEQ ID NO: 1 encoding CAPN3 (named "AAVrh.74.tMCK.CAPN3"). The genome of an rAAV lacks AAVrep and capDNA; that is, there is no AAVrep or capDNA between the ITRs of the rAAV genome.

[0054] The sequence of AAVrh.74.tMCK.CAPN3 is described in Sequence ID No. 1, with the AAV2 ITR extending from nucleotides 1 to 128, the tMCK promoter from nucleotides 165 to 884, the chimeric intron from nucleotides 937 to 1069, the Kozak sequence from nucleotides 1101 to 1106, the CAPN3 polynucleotide from nucleotides 1107 to 3572, the polyA signal from nucleotides 3581 to 3780, and the second AAV2 ITR from nucleotides 3850 to 3977.

[0055] rAAV may be purified by methods known in the art, such as column chromatography or a cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0056] In another embodiment, a composition comprising rAAV as described herein is provided. The provided composition comprises rAAV on a pharmaceutically acceptable carrier. The composition may also include other components such as diluents and adjuvants. The acceptable carrier, diluent and adjuvant are preferably nontoxic to the recipient and inactive at the dose and concentration used, and include phosphates, citrates or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

[0057] The potency of rAAV administered in the methods described herein may vary depending, for example, on the specific rAAV, dosage form, therapeutic target, individual, and targeted cell type(s), and may be determined by standard methods in the art. The potency of rAAV is approximately 1 × 10⁶ per ml. 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14、Or it may be in the range of DNase-resistant particles (DRPs) or higher. Doses may also be expressed in units of viral genome (vg). Exemplary doses disclosed include 1E11 vg, 3E12 vg, and 6E12 vg.

[0058] A method for transducing target cells, such as muscle cells containing rAAV, in vivo or in vitro is contemplated herein. The in vivo method comprises the step of administering an effective dose or effective repeated dose of the rAAV-containing composition provided herein to a subject in need (e.g., animals, including, but not limited to, human patients). When administered before the onset of disease / disease, the administration is prophylactic. When administered after the onset of disease / disease, the administration is therapeutic. An effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease, reduces the severity of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival. Compared to the subject before treatment, the treatment herein results in one or more of the following: increased muscle fiber diameter, decreased number of lobular muscle fibers, decreased number of fibers with an internal nucleus, decreased endomysial connective tissue content, correction of muscle atrophy, and increased muscle strength. In one embodiment, the muscle fibers include one or more of the following: slow contraction oxidative (STO) muscle fibers, fast contraction oxidative (FTO) muscle fibers, and fast contraction glycolytic (FTG) fibers. In one embodiment, by treatment, (a) by 4 weeks after administration, at least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% of 1 mm 2 (b) a decrease in the total number of muscle fibers per unit area, (c) an increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, or 25% by 4 weeks after administration, and (d) an increase in muscle fiber diameter of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% by 1 mm by 4 weeks after administration. 2 (d) a decrease in the number of STO muscle fibers per unit area, (e) an increase of at least 5%, 10%, 15%, 20%, or 25% in STO muscle fiber diameter by 4 weeks post-administration, and (f) an increase of at least 5%, 10%, 15%, or 20% in 1 mm by 4 weeks post-administration. 2(f) a decrease in the number of FTO muscle fibers per unit area, (g) an increase of at least 5%, 10%, 15%, or 20% in FTO muscle fiber diameter by 4 weeks after administration, and (g) an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% in 1 mm by 4 weeks after administration. 2 A decrease in the number of FTG muscle fibers per unit area occurs, and (h) within 4 weeks after administration, one or more of the following occurs: at least 5%, 10%, 15%, 20%, or 25% increase in FTG muscle fiber diameter. In one embodiment, the method of the present disclosure reduces the expression of calpain 3 protein from rAAV to none, minimal, or low in the cardiac muscle of a subject administered with rAAV.

[0059] Assays for investigating these results are understood in the art and / or described in the examples herein. The use of the methods herein is intended for the prevention or treatment of disorders / diseases (e.g., muscular dystrophy) caused by defects in CAPN3 activity or CAPN3 expression. LGMD2A is an example of a disease intended for prevention or treatment by the method.

[0060] Combination therapy is also being considered. The combinations used herein include both concurrent and sequential treatments. Combining the methods described herein with standard medical treatments (e.g., corticosteroids) is particularly intended, as is combining them with novel therapies.

[0061] The effective dose of the composition may be administered by standard routes in the art, including, but not limited to, intramuscular, parenteral, intravenous, subarachnoid, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route(s) and serotype(s) of the AAV components of rAAV (particularly AAV ITR and capsid protein) may be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease condition being treated, as well as the target cells / tissues(s) that will express CAPN3. In one embodiment, rAAV is administered by intramuscular, intravenous, intraperitoneal, subcutaneous, epicutaneous, vaginal, intradermal, or nasal injection. In another embodiment, rAAV is administered by intramuscular or intravenous injection.

[0062] In particular, the practical administration of rAAV as described herein can be achieved using any physical method for delivering the rAAV recombinant vector to the target tissue of an animal. Administration includes, but is not limited to, direct injection into muscle, bloodstream, and / or liver. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carrier or other components that can be co-administered with rAAV. The capsid protein of rAAV may be modified so that rAAV targets specific target tissues such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in practice by this method. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0063] For intramuscular injection, solutions in adjuvants such as sesame or peanut oil, aqueous solutions of propylene glycol, and sterile aqueous solutions can be used. Such aqueous solutions can be buffered if desired, and the liquid diluent can be isotonic first with physiological saline or glucose. Solutions of rAAV as free acid (DNA contains acidic phosphate groups) or pharmacokinetically acceptable salts can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0064] Pharmaceutical forms suitable for systemic (e.g., intravenous) injection include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and liquid to the extent that it can be easily injected. It must be stable under manufacturing and storage conditions and protected against microbial contamination such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action is provided by various antimicrobial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Often, it is preferable to include isotonic agents such as sugars or sodium chloride. Sustained absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0065] Sterile injectable solutions are prepared by mixing the required amount of rAAV in a suitable solvent with the various other components listed above, as needed, and then sterilizing by filter. Generally, dispersants are prepared by mixing the sterilized active ingredient with a sterile medium containing a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, in some embodiments, the preparation method includes vacuum drying and / or freeze-drying techniques, each of which can produce a powder of the active ingredient and any additional desired components from its previously sterilized by filter.

[0066] Transduction using rAAV may also be performed in vitro. In one embodiment, desired target muscle cells are removed from the subject, transduced with rAAV, and then reintroduced into the subject. Alternatively, syngeneic or heterogeneic muscle cells can be used if these cells do not produce an inappropriate immune response in the subject.

[0067] Appropriate methods for transducing and retransducing transduced cells into a target are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells in a suitable culture medium, for example, and screening those cells for the target DNA using conventional techniques such as Southern blotting and / or PCR, or using a selection marker. The transduced cells are then formulated into a pharmaceutical composition, which can be introduced into a target by various techniques, such as intramuscular, intravenous, subcutaneous and intraperitoneal injection, or injection into smooth muscle and cardiac muscle using a catheter, for example.

[0068] Transduction of cells using rAAV by the methods described herein results in the sustained expression of CAPN3 or a CAPN3-active protein. Thus, methods are provided for administering rAAV expressing CAPN3 or a CAPN3-active protein to a subject, preferably a human. The subjects of this disclosure include, but are not limited to, humans, dogs, cats, horses, cattle, pigs, sheep, goats, chickens, rodents (e.g., rats and mice), and primates. These methods include transduction of tissues using one or more rAAVs described herein (including, but not limited to, organs such as muscles, livers and brains, and glands such as salivary glands).

[0069] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The method described herein provides sustained expression of CAPN3 from transduced muscle cells.

[0070] "Muscle cells," "muscle fibers," or "muscle tissue" means cells or groups of cells derived from any type of muscle [e.g., skeletal muscle and smooth muscle (e.g., gastrointestinal tract, bladder, blood vessels, or cardiac tissue)]. Such muscle cells may be differentiated or undifferentiated, including myoblasts, muscle cells, myotubes, cardiomyocytes, and cardiomyocytes.

[0071] The term "transduction" is used to refer to the administration / delivery of CAPN3 to recipient cells via the described rAAV, either in vivo or in vitro, resulting in the expression of CAPN3 by the recipient cells.

[0072] Therefore, a method is provided for administering an effective dose of rAAV (or a dose administered essentially simultaneously, or a dose administered at regular intervals) that codes for CAPN3 to a subject in need of it.

[0073] Compared to subjects before treatment, the method described herein results in one or more of the following in subjects: an increase in muscle fiber diameter, a decrease in the number of small lobe slow contraction oxidative (STO) muscle fibers, a decrease in the number of fibers with an internal nucleus, a decrease in endomysial connective tissue content, correction of muscle atrophy, and an increase in muscle strength. [Examples]

[0074] The aspects and embodiments are illustrated by the following examples. Example 1 describes the production of AAV9.MCK.CAPN3. Example 2 describes the intramuscular administration of AAV9.MCK.CAPN3. Example 3 describes the production of AAVrh.74.tMCK.CAPN3. Example 4 describes the intramuscular administration of AAVrh.74.tMCK.CAPN3. Example 5 describes the intravenous administration of AAVrh.74.tMCK.CAPN3. Example 6 shows an endpoint study. Example 7 describes toxicity and in vivo distribution studies. Example 8 describes in vivo bioactivity studies after intramuscular injection. Example 9 describes in vivo bioactivity studies after systemic injection. Example 10 describes the evaluation of systemic AAVrh.74.tMCK.CAPN3 gene delivery. Example 11 describes the evaluation of cardiotoxicity after systemic injection of the AAVrh.74.tMCK.CAPN3 vector. Example 12 describes the in vivo physiological analysis.

[0075] Example 1 Production of AAV9.MCK.CAPN3 An AAV vector (named AAV.CAPN3) carrying the CAPN3 gene under a muscle-specific MCK promoter was produced (Figure 1A). DNA containing an open reading frame of mouse CAPN3 (NM_007601.3) between two Not1 restriction sites was synthesized by Eurofin Genomics, USA, and then subcloned into an AAV.MCK (muscle creatine kinase) vector described earlier by Rodino-Klapac et al., Journal of Translational Medicine, 5:45-55 (2007). The rAAV vector was produced by a modified cross-packaging approach, which allows the AAV2 vector genome to be packaged into an AAV capsid serotype [Rabinowitz et al., J Virol. 76(2):791-801 (2002)]. Production was achieved using three standard plasmid DNA / CaPO4 precipitation methods with HEK293 cells. 293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. The produced plasmids were (i) pAAV.MCK.microdys, (ii) rep2-capX modified AAV helper plasmid encoding cap serotype 1, 6, or 8-like isolates, and (iii) adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I / II RNA genes. To enable comparison between serotypes, capsidized vector genome (vg) titers were determined using a Prism 7500 Taqman detector system (PE Applied Biosystems) with a quantitative PCR-based titration method. [Clark et al., Hum Gene Ther. 10(6):1031-1039 (1999)]. The primers and fluorescent probes targeted the MCK promoter and were as follows: MCK forward primer, 5-CCCGAGATGCCTGGTTATAATT-3 (SEQ ID NO: 4); MCK reverse primer, 5-GCTCAGGCAGCAGCAGGTGTTG-3 (SEQ ID NO: 5); MCK probe, 5-FAM-CCAGACATGGGCTGCTCCCC-TAMRA-3 (SEQ ID NO: 6). Final titer (vgml).-1 ) is the Prism 7500 real-time detector system (PE Applied The results were determined by quantitative reverse transcriptase PCR using specific primers and probes for the MCK promoter from Biosystems (Grand Island, New York, USA). Ali-coated viruses were kept at -80°C until use.

[0076] Example 2 Intramuscular administration of AAV9, MCK, and CAPN3 To demonstrate whether WT CAPN3 can restore the regenerative failure process in CAPN3 knockout (CAPN3-KO) mice, 30 μl of CTX was first injected into the TA muscle (n=4) of anesthetized CAPN3-KO mice [Kramerova et al., Hum Mol Genet 13(13):1373-1388 (2004)], and two weeks later, 20 μl of 1x10¹⁶ CTX was administered via intramuscular injection. 11 We transduced wild-type CAPN3 using vg AAV9.MCK.CAPN3. TA muscle from another CAPN3-KO cohort (n=4), which served as a control, received the same amount of PBS two weeks after CTX injection.

[0077] Mice were killed six weeks after CTX injection, the TA muscle was removed, and the tissue was processed for cryostat sections. For routine histopathological evaluation, 12 μm thick sections were first stained with H&E, and muscle fiber type-specific diameter measurements were obtained from SDH-stained sections of TA from three mice in each group. Three random images of TA (per section per animal) were taken at 20x magnification to generate fiber diameter measurements and fiber type-specific histograms.

[0078] Succinate dehydrogenase (SDH) enzyme histochemistry was used to evaluate metabolic fiber type differentiation [slow contractile oxidation (STO), fast contractile oxidation (FTO), and fast contractile glycolysis (FTG)]. Fiber type-specific diameter measurements were obtained using 12 μm thick SDH-stained sections 4 and 12 weeks after final cardiotoxin injection. Three images were taken at 20x magnification using an Olympus BX41 microscope and SPOT camera (Olympus BX61, Japan) along the midline axis (per section per animal) of the gastrocnemius muscle (deep zone mainly consisting of STO, intermediate zone showing a checkerboard appearance of STO, FTO, or FTG, and superficial zone mainly consisting of FTG fibers). This approach was chosen to capture changes in the oxidative state of fibers in each zone in response to metabolic changes during regeneration. The diameters of dark (STO), intermediate (FTO), and bright (FTG) muscle fibers were determined by measuring the shortest distance between muscle fibers using Zeiss Axiovision LE4 software (v.4.8). Fiber diameter histograms were created individually for STO, and combined for FTG and FTO, obtained from three animals, with endomysial area (mean ± SEM) in mm². 2 The total fast-contracting fiber population (FTG / O), expressed as the number of fibers per unit area, is shown. The average fiber diameter was derived from combining all three fiber types. An average of 900 to 1700 fibers were measured per group. TA muscle was used to evaluate fiber formation (see below).

[0079] Four weeks after AAV9.MCK.CAPN3 injection, a significant increase in muscle diameter was observed, with a clear decrease in the nucleus and a much smaller number of small-diameter fibers exhibiting a lobulated pattern (Figure 1B). Untreated CAPN3-KO muscle was mm 2 The tissues have more than 31.6% fibers per unit area, are mainly composed of small and lobulated STO fibers, and treatment improves myotubular fusion, thus reducing the number of individual small fibers per unit area (Figure 1, C and D; Table 1). [Table 1]

[0080] The fiber size distribution histograms of treated TA muscle showed a shift to larger diameter fibers with treatment, and the excess number of small-diameter fibers in untreated CAPN3-KO control muscle was of the STO histochemical fiber type (Figures 1E and 1F). In summary, these findings indicate that CAPN3 replacement via gene therapy in CAPN3-KO muscle rescued regeneration defects, as evidenced by normalization of fiber diameter and a reduction in the number of STO fiber populations.

[0081] Example 3 Production of AAVrh.74.tMCK.CAPN3 An AAV vector (named AAVrh74.tMCK.CAPN3) carrying the CAPN3 gene under a cleaved muscle-specific MCK promoter (tMCK promoter) was produced. DNA containing an open reading frame of mouse CAPN3 (NM_007601.3) between two Not1 restriction sites was synthesized by Eurofin Genomics, USA, and subsequently inserted into the AAV-producing plasmid. A plasmid map is shown in Figure 2.

[0082] Subsequently, the rAAV vector was prepared using the approach described in Example 1.

[0083] Example 4 Intravenous administration of AAVrh.74.tMCK.CAPN3 CAPN3-KO mice at 6 months of age were administered a low dose (3 x 10) via injection into the tail vein. 12 vg) and high dose (6x10 12 The mice were administered AAVrh.74.tMCK.CAPN3 (vg). For the endpoint study, the mice were killed 20 weeks after gene injection. Age-matched, vehicle-treated CAPN3-KO mice were provided as controls. [Table 2]

[0084] The endpoint studies performed as described in Example 7 below include muscle physiology (TA force generation or in vivo muscle contractility assay, and protection from eccentric contraction), muscle histopathology, hCAPN3 detection using qPCR, and Western blot analysis.

[0085] Example 5 Intramuscular administration of AAVrh.74.tMCK.CAPN3 In aged and juvenile CAPN3-KO muscles, cardiotoxin (CTX)-induced synchronous necrosis followed by regenerative responses to CAPN3 introduction into regenerating muscle via rAAV treatment are measured.

[0086] In cohorts of young (2 months old) and older (6 months old) mice, CTX was injected into both TA muscles to induce synchronous necrosis two weeks before rAAV injection into the left TA muscle. AAVrh.74.tMCK.CAPN3 was administered in a volume of 20 μl at a rate of 1 × 10⁶ 11 It is administered via intramuscular injection in vg. The endpoint trial was conducted at 8 weeks after gene transfer (1 x 10⁻¹⁴). 11 The correction of regenerative defects was evaluated by comparing quantitative histological and physiological outcomes from the left TA to the untreated right TA (with the efficacy established in our previous trials) at the VG dose. [Table 3]

[0087] Eight weeks after rAAV injection, endpoint studies performed as described in Example 6 below included muscle physiology (generation of TA force and protection from eccentric contraction), quantitative muscle histopathology, and hCAPN3 detection using qPCR and Western blot analysis.

[0088] Example 6 Endpoint testing Generation of TA force and protection from eccentric contraction A protocol for evaluating the functional outcomes of the TA muscle is performed on muscle tissue extracted from mice [Wein et al., Nature Medicine, 20(9):992-1000 (2014)]. Mice are anesthetized with a ketamine / xylazine mixture. The skin of the hind limb is excised using a dissecting microscope to expose the TA muscle and patella. The distal TA tendon is incised, and a double square knot is tied with 4-0 sutures to the muscle as close as possible to the tendon, thereby cutting the tendon. The exposed muscle is kept constantly moist with saline. The mouse is then transferred to a heat-controlled platform and maintained at 37°C. The knee is fixed to the platform and the foot is secured with tape, with a needle passed through the patellar ligament, which is the distal TA tendon suture, to the horizontal arm of a force transducer (Aurora Scientific, Aurora, ON, Canada). TA muscle contraction is induced by stimulating the sciatic nerve via bipolar platinum electrodes. Once the muscle stabilized, the optimal length was determined by gradually stretching the muscle until maximum contractile force was achieved. After a 3-minute resting period, the TA was stimulated at 50, 100, 150, and 200 Hz, with a 1-minute resting period between each stimulation to determine the maximum tetanic force. Muscle length was measured. After a 5-minute rest, the sensitivity of the TA muscle to contraction-induced injury was assessed. After 500 ms of stimulation, the muscle was stretched to 10% of its optimal length. This included 700 ms of muscle stimulation at 150 Hz. After stimulation, the muscle returned to its optimal length. The cycle was repeated for a total of 10 cycles at 1-minute intervals. The specific force was calculated by dividing the maximum tetanic force by the cross-sectional area of ​​the TA muscle. After eccentric contraction, the mice were euthanized, the TA muscle was dissected, weighed, and frozen for analysis. Data analysis was performed blinded but not randomly.

[0089] In vivo muscle contraction assay This assay measures the total torque generated by either the plantar or dorsiflexor muscles of the lower limb and is performed using a muscle physiology apparatus (Aurora Scientific, ON, Canada). The animals are anesthetized with isoflurane. Once anesthetized, the hind limb hair is removed with clippers as needed. If hair removal with clippers is insufficient, a thin layer of hair removal cream (Nair) is applied and thoroughly washed with lukewarm water to prevent discomfort. The hind limb to be measured is attached to a footplate with adhesive tape. The limb is firmly secured with a blunt clamp. Either the tibial or fibular element of the sciatic nerve is stimulated with two sterile, disposable 28-gauge unipolar electrodes inserted subcutaneously into the skin near the nerve. The mouse's body temperature is maintained by a conductive temperature-controlled heating pad (set to 37°C) or a radiant heat source and monitored with a temperature probe.

[0090] Histopathological examination For histological analysis, all muscle and organ tissues are embedded in 7% tragacanth gum and rapidly frozen in liquid nitrogen-cooled isopentane. Frozen sections (12 μm) are collected for immunohistochemical and Western blot analysis.

[0091] Western blot analysis for human CAPN3 detection CAPN3 protein quantification in mouse muscle tissue is evaluated using Western blotting. The CAPN3 enzyme is lysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and migrates as a 94 kDa band with an approximately 60 kDa autolysated product using Novocastra's clinical-grade antibody that recognizes the N-terminus of NCL-CALP-12A2. Furthermore, the NCL-CALP-2C4 antibody recognizes the same CAPN3 molecule (94 kD) and an additional fragment (30 kD) in skeletal muscle; both antibodies are suitable for protein detection. Semi-quantitative measurement of CAPN3 protein expression levels in samples from calpain knockout mice after therapeutic rAAV vector delivery is performed and compared with untreated controls.

[0092] quantitative muscle histology Cross-sectional images of TA and quadriceps femoris treated with AAVrh.74.tMCK.CAPN3 and untreated subjects were stained with hematoxylin and eosin and imaged using Zeiss Axiovision L4 software (20 random images, 4 per section per animal). Fiber diameter was compared between treated and control subjects.

[0093] statistical analysis Student's t-tests or one-way ANOVA multiple comparison tests should be performed as appropriate.

[0094] Example 7 Toxicity testing / In vivo distribution testing Toxicity / distribution studies will be conducted using established effective doses and one-log high doses. Toxicity studies will be performed by systemic (tail vein) delivery of rAAV to 6-8 week old CAPN3-KO mice, including comparison with normal C57Bl6 normal mice. A cohort of 6-10 mice will be included, and complete necropsies will be performed using GLP-like methods.

[0095] Serum collected from blood samples is used for blood biochemical tests: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin (total and direct), blood urea nitrogen, creatinine, creatine kinase, glucose, and total protein.

[0096] A complete autopsy is performed by a complete and systematic examination and dissection of the animal's internal organs and carcass. Tissues / organs are collected, including the gonads, brain, spleen, kidneys, jejunum, colon, pancreas, heart, lungs, stomach, liver, inguinal lymph nodes, spinal gastrocnemius muscle, and quadriceps muscle. Tissues / organs for histopathological study are collected and fixed in 10% neutral buffered formalin (10% NBF), with the exception of all skeletal muscle specimens placed on blocks with OCT, which are rapidly frozen in liquid nitrogen-cooled methylbutane for frozen sections.

[0097] Example 8 In vivo bioefficacy studies after intramuscular injection The bioactivity test was performed after intramuscular (IM) injection of AAVrh.74.tMCK.CAPN3(1E11 vg) into the tibialis anterior muscle (TA) of CAPN3 KO mice (n=3), as described above in Example 5.

[0098] Four weeks after administration, gene delivery was analyzed by reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis. For Western blot analysis, samples equivalent to the entire 50 μg of muscle protein extract were separated on a 3-8% acrylamide, tris-acetate SDS gel and transferred to a PVDF membrane. Immunodetection was performed using monoclonal antibodies produced against s-synthetic peptides containing AA1-AA19 of the human calpain 3 sequence (Leica), and muscle-specific actin antibody (Leica) as a loading control. Figure 3A shows the presence of 94 kD calpain 3 protein in TA muscle after intramuscular injection. RT-qPCR analysis demonstrated that the relative expression level of the human calpain 3 gene returned to normal levels four weeks after gene transfer compared to WT mice (see Figure 3B). RT-qPCR data were calibrated using WT C57BL / 6 with mouse GAPDH as the reference gene.

[0099] Furthermore, quantitative histopathological analysis was performed after intramuscular administration. As shown, the diameter of TA muscle fibers in treated CAPN3 KO mice was compared to the diameter of untreated control (TA injected with lactate Ringer's solution) muscle. In TA muscle injected with AAV.hCAPN3, the mean fiber sizes of slow contractile oxidative (STO, dark), fast contractile oxidative (FTO, intermediate), and fast contractile glycolytic (FTG, light) fibers appeared to be normalized to the WT value. Quantification of fiber type size is shown in Table 4, showing an increase with treatment. [Table 4]

[0100] In summary, in in vivo bioactivity studies of CAPN3 KO mice (n=2) after IM injection of the vector (1E11 vg) into the tibialis anterior muscle (TA), 4 weeks after gene delivery, 1) RT-qPCR and Western blot analysis showed expression of CAPN3 transcript and full-length 94kDa calpain 3 protein, and 2) histological analysis showed an increase in TA muscle fiber diameter compared to the control (Ringer).

[0101] Example 9 In vivo bioefficacy studies after systemic injection AAVrh.74.tMCK.CAPN3 (3E12 vg or 6E12 vg) was systemically injected into the tail vein of CAPN3-KO mice, followed by in vivo bioactivity studies. The low-dose CAPN3 KO cohort (n=5; mice labeled Z18-13, Z18-15, Z18-16, Z18-17, and Z18-18) was administered 3E12 vg in 300 μl of Ringer's lactate solution. Four weeks after gene injection, the mice were evaluated for running fatigue using a run-to-exhaustion treadmill test and then euthanized for tissue collection. The muscles of the upper and lower extremities (TA, gastrocnemius (GAS), quadriceps, triceps), heart, liver, lungs, spleen, and pulmonary testes were excised, and the tissue samples were frozen with isopentane and cooled to liquid nitrogen.

[0102] CAPN3 expression was evaluated in TA muscle using RT-qPCR. At low doses of 3E12 vg, CAPN3 mRNA expression levels were low, as observed with high CT values ​​(>27). Western blot analysis showed corresponding undetectable protein bands. Although low expression data was observed at low doses in this tissue, systemic administration of 3E12 vg demonstrated both functional and histological benefits.

[0103] Next, a high dose (6E12 vg) was administered systemically to investigate whether protein expression could be detected with higher dose vector delivery. High-dose cohort (mice designated Z18-20, Z18-21, Z18-23, and Z18-24) CAPN3-KO mice were administered 6E12 vg AAVrh7.4.tMCK.hCAPN3 vector (twice the dose used in the low-dose cohort) via systemic injection into the tail vein, and euthanized 4 weeks after injection. RT-qPCR showed variable levels of CAPN3 expression in the quadriceps, triceps, GAS, TA, and myocardium.

[0104] To determine the relative expression of CAPN3 mRNA, muscle tissue samples were collected from CAPN3 KO mice treated with the tMCK.hCAPN3 vector at doses of 3E12 vg (low-dose cohort 1) and 6E12 vg (high-dose cohort 2). Total RNA was isolated from both cohorts, and qPCR of CAPN3 against mouse GAPDH was assayed together with previous samples from the vector-administered cohort via IM injection (1E11 vg; see above in Example 8).

[0105] The relative expression of CAPN3 was determined by the following method: CT=CT CAPN3 -CT mGAPDH ΔΔCT = ΔCT - ΔCT Calibrator* CAPN3 relative expression = 2 -ΔΔCT The relative expression of CAPN3 in each tissue and the original CT values ​​are shown in Table 5 and Figure 5 below. Table 5 provides data for IM delivery (mouse numbers Z18-11 and Z18-12) and systemic administration. [Table 5-1] [Table 5-2]

[0106] Overall, CAPN3 mRNA expression in CAPN3 KO muscle after systemic delivery exhibited animal and tissue-specific variability, being relatively low compared to IM delivery at 1E11 vg (<1% of IM delivery), particularly in the 3E12 low-dose cohort. Consequently, the full-length 94kDa protein was below the detection limit by Western blotting. However, robust gene expression and significant amounts of full-length calpain 3 protein were shown after systemic injection of a 6E12 vg systemic dose in the high-dose cohort.

[0107] Example 10 Evaluation of systemic AAVrh74.tMCK.hCAPN3 gene delivery Gene transfer efficiency was evaluated by qPCR to calculate vector genome copies in CAPN3 KO mouse tissue samples after systemic delivery of AAVrh74.tMCK.hCAPN3 in 6E12 vg. Vector genome loading was measured in skeletal muscles of the lower and upper limbs (quadriceps, TA muscle, gastrocnemius, triceps), heart, and liver. Genomic DNA was isolated from frozen tissue samples. The qPCR assay was performed on an ABI 7500 (Applied Biosystems) using the following primer set: “5'-CGGAGAGCAACTGCATAAG-3' (forward direction; SEQ ID NO: 8); The primer pair “5'-GGCTGATGATGGCTGAATAG-3' (reverse direction; SEQ ID NO: 9)” exclusively amplifies the product from the 5' region of the hCAPN3 ORF and amplifies the downstream region specific to the expression vector, including the intronic element portion. The final result is reported as the mean copy number of the AAVrh74 vector per microgram of genomic DNA.

[0108] As shown in Figure 6, the liver had the highest vector genome copy number after systemic vector delivery. The vector genome distribution varied among muscle groups. Overall, quadriceps and cardiac tissue showed higher values ​​compared to other muscle groups. Experimental variability was also noted. Mice No. Z18-21 showed relatively low copy numbers in all muscle groups compared to the other three mice.

[0109] In 3E12 vg whole-body treated CAPN3 KO mice, improvements in both functional and histological features were observed. However, RT-qPCR detected only low levels of muscle calpain 3 expression in total RNA isolates, and in certain muscle tissues, the full-length 94kDa protein was not detectable by Western blotting (see Figure 3A). However, after systemic administration of 6E12 vg, robust gene expression and significant amounts of full-length calpain 3 protein were shown (see Figure 3B). The data demonstrate that calpain 3 gene expression returned to normal levels compared to WT mice 4 weeks after gene transduction with AAVrh74.tMCK.hCAPN3 particles. RT-qPCR data were calibrated using mouse GAPDH as the reference gene and WT C57BL / 6.

[0110] Histopathological examination As described above, a trend in efficacy was observed at 4 weeks post-injection. In both cohorts (3E12 and 6E12), a significant increase in fiber size was observed in the TA muscle from CAPN3 KO mice after systemic delivery of AAVrh.74.tMCK.hCAPN3 4 weeks after administration. As shown in Figure 7, total fiber diameter was significantly increased in both treatment cohorts compared to the untreated KO group (p<0.00001). Treatment resulted in normalization of fiber size, and there was no dose-dependent difference between the treatment cohorts (p=0.78058). Table 6 shows the results for wild-type and CAPN3 after systemic AAV.hCAPN3 gene therapy in 3E12 and 6E12 vg mice. This provides muscle fiber size in KO mice. [Table 6]

[0111] In all cohorts, there was no histopathological evidence of cardiotoxicity following systemic administration of the AAVrh7.4.tMCK.hCAPN3 vector at week 4. Although varying levels of virus were found in cardiac tissue, no protein bands were detected in cardiac tissue by Western blotting in any of the cohorts.

[0112] Functionality study: Run-to-Exhaust test Prior to data collection for the Run-to-Exhaustion trial, mice were acclimatized to a treadmill (Columbus Instruments) for 3 days, running for 15 minutes once daily at a speed of 10 m / min. The protocol used required placing the mice on a treadmill with a 15-degree incline. The treadmill was turned on at a speed of 1 m / min, and the speed was increased by 1 m / min until the mouse became exhausted. Fatigue was assessed when the mouse sat on a resting pad for at least 15 seconds. Time, speed, and distance to exhaustion were recorded.

[0113] Figure 8A provides data from the Run-to-Exhaustion test for a low-dose cohort administered 3E12 vg of AAVrh.7.4.tMCK.hCAPN3 and a low-dose cohort administered 6E12 vg of AAVrh.7.4.tMCK.hCAPN3 4 weeks after systemic administration. CAPN3 KO mice treated in both cohorts performed better in the Run-to-Exhaustion test compared to untreated mice. There was no clear dose-dependent difference in Run-to-Exhaustion performance between the low-dose and high-dose cohorts, nor were there any statistically significant differences in muscle fiber diameter.

[0114] Mice from high-dose cohort 2 (n=16) were further analyzed 20–24 weeks after administration of 6E12 vg of AAVrh7.4.tMCK.hCAPN3. As shown in Figure 8B, treated CAPN3 KO mice continued to perform better in the Run-to-Exhastion test compared to untreated mice (p<0.00001).

[0115] Example 11 Evaluation of cardiotoxicity after systemic injection of the AAVrh7.4.tMCK.hCAPN3 vector. After euthanasia of the cohort mice four weeks post-administration, serum and organ samples were collected. 300 μl of 3E12 vg of AAVrh.74.tMCK.hCAPN3 vector in Ringer's lactate solution was administered via tail vein injection to CAPN3-KO mice in low-dose cohort 1. 6E12 vg of AAVrh7.4.tMCK.hCAPN3 vector was administered via tail vein injection to CAPN3-KO mice in high-dose cohort 2. Both cohorts were euthanized four weeks after injection. Two sections of the ventricle—apical, superficial, and deep—were examined. No inflammation, necrosis, or regeneration was found in the tissue sections, indicating that no myocardial toxicity was observed from systemic delivery of AAVrh7.4.tMCK.hCAPN3 vector at two different doses four weeks after injection. Mouse numbers Z18-19 and Z18-22 (lactated Ringer's solution / untreated) were used as control knockout animals. Figure 9 provides fresh frozen sections from H&E-stained hearts. No muscle fiber necrosis, regeneration, or inflammation was observed. Although varying amounts of virus were present in the cardiac tissue, no protein bands were detected by Western blotting in any cohort. Figure 10 provides Western blotting analysis showing that full-length calpain 3 protein was below the detection limit in transduced cardiac tissue.

[0116] Example 12 In Vivo Physiological Analysis Physiological evaluation is performed after IM or systemic administration of the AAVrh7.4.tMCK.hCAPN3 vector. During in vivo physiological evaluation, mice are anesthetized with inhaled isoflurane. Once the animals are anesthetized, hair is removed from the back and hind limbs with clippers as needed. If hair removal with clippers is insufficient, a thin layer of depilatory cream is applied. For in vivo physiological force measurements, hind limb torque was measured using a non-invasive force footplate (Aurora Scientific, Canada) connected to a force-detecting device after supramaximal stimulation of the sciatic nerve. The hind limb to be measured is secured to the footplate with adhesive tape. The limb is firmly fixed with a blunt clamp. Either the tibia or fibula component of the sciatic nerve is stimulated with two sterile, disposable 28-gauge monopolar electrodes inserted subcutaneously into the skin near the nerve. The mouse's body temperature is maintained by a conductive temperature-controlled heating pad (set to 37°C) or a radiant heat source and monitored with an infrared temperature probe.

[0117] While this disclosure provides specific embodiments, it will be understood that modifications and alterations can be made to those skilled in the art. Therefore, only the limitations set forth in the claims should be made to the present invention.

[0118] All documents referenced in this application are incorporated herein by reference in their entirety.

Claims

[Claim 1] The invention described herein.

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