EA045762B1 - PRODUCTS BASED ON RECOMBINANT ADENO-ASSOCIATED VIRUSES AND METHODS FOR TREATING PELOVIC-BREAKAL MUSCULAR DYSTROPHY TYPE 2A - Google Patents

Description

This application claims priority under U.S. Provisional Patent Application No.

62/691934, filed June 29, 2018, and US Provisional Patent Application No. 62/865081, filed June 2019, both of which are incorporated herein by reference in their entirety.

Field of technology

Provided herein are products and methods for the treatment of pelvic-brachial muscular dystrophy type 2A. In these methods, recombinant adeno-associated viruses deliver DNA encoding a protein having calpain-3 activity (CAPN3).

Incorporating a sequence listing by reference

This application contains, as a separate part of this disclosure, a sequence listing in machine-readable form (file name: 52684P2_SeqListing.txt; 23755 bytes - ASCII text file created June 26, 2019), which is incorporated herein by reference in its entirety .

BACKGROUND OF THE INVENTION

Forms of muscular dystrophy (MD) are a group of genetic diseases. This group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until adulthood or later in life. These disorders vary in the distribution and extent of muscle weakness (some forms of MD also affect the heart muscle), age at onset, rate of progression, and mode of inheritance.

One MD group is the pelvic-brachial MD (LGMD) group. LGMDs are rare conditions and present differently among individuals with regard to age of onset, areas of muscle weakness, cardiac and respiratory involvement, rate of progression, and severity. LGMD can begin in childhood, adolescence, early adulthood, or even later. Both sexes are affected equally. LGMDs cause weakness in the shoulder and pelvic girdle muscles, and nearby hip and shoulder muscles also sometimes weaken over time. Leg muscle weakness often appears before arm muscle weakness. The facial muscles are usually not affected. As the condition progresses, people may have trouble walking and over time it may become necessary for them to use a wheelchair. Involvement of the shoulder girdle and arm muscles may lead to difficulty raising arms overhead and lifting objects. Some types of LGMD may involve the heart and respiratory muscles.

There are at least nineteen forms of LGMD, and these forms are classified according to the genetic defects associated with them.

Type Type of inheritance Gene or chromosome

LGMD1A Autosomal dominant myotilin gene

LGMD1B Autosomal dominant Lamin A/C gene

LGMDIC 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 dysferlin gene

LGMD2C Autosomal recessive gamma sarcoglycan gene

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 Gene POMT1

LGMD2L Autosomal recessive fukutin gene

Specialized tests to detect LGMD are currently available through the National Commissioning Group's (NCG) national diagnostic program.

Mutations in the calpain-3 (CAPN3) gene lead to the development of LGMD2A, one of the most common forms of pelvic-brachial muscular dystrophy worldwide. There is currently no treatment for this hereditary disease. Previous studies have demonstrated the potential for CAPN3 gene transfer to correct pathological features in CAPN3-deficient mice. However, CAPN3 expression driven by the desmin gene promoter resulted in cardiotoxicity [Bartoli et al., Mol. Ther., 13: 250-259 (2006)]. Prospective studies have examined the expression of this gene in skeletal muscle [Roudaut et al., Circulation, 128: 1094-1104 (2013)].

Adeno-associated virus (AAV) is a replication-defective parvovirus with a single-stranded DNA genome of approximately 4.7 kb in length, including two 145-nucleotide inverted terminal repeats (ITRs). There are several serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete AAV-1 genome is available under GenBank accession number NC_002077; the complete AAV-2 genome is provided under GenBank accession number NC_001401 and in Srivastava et al., J. Virol., 45: 555-564 (1983); The complete AAV-3 genome is available under GenBank accession number NC_1829; The complete AAV-4 genome is available under GenBank accession number NC_001829; The AAV-5 genome is available under GenBank accession number AF085716; The complete AAV-6 genome is available under GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank accession numbers AX753246 and AX753249, respectively; the AAV-9 genome is presented in Gao et al., J. Virol., 78: 6381-6388 (2004); The AAV-10 genome is presented in Mol. Ther., 73(1): 67-76 (2006); and the AAV-11 genome presented in Virology, 330(2): 375-383 (2004). The genome sequence of AAV-rh.74 is presented in US Patent No. 9434928, incorporated herein by reference. Cis-acting sequences that control viral DNA replication (rep), encapsidation/packaging, and integration into the host cell chromosome are contained in the AAV ITR. Three AAV promoters (called p5, p19, and p40 after their relative locations on the map) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) combined with differential splicing of one AAV intron (at nucleotides 2107 and 2227) result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. rep proteins have several enzymatic properties that are ultimately responsible for viral genome replication. The cap gene is expressed under the control of the p40 promoter and encodes three capsid proteins VP1, VP2 and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of three related capsid proteins. One consensus polyadenylation site is located at position 95 on the AAV genome map. A review of the life cycle and genetic features of AAV is presented in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. Infection of cells in culture by AAV is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells, providing the ability to target many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells and can persist essentially throughout the life of these cells as a transcriptionally active nuclear episome (an extrachromosomal element). The AAV proviral genome is inserted as cloned DNA into plasmids, making the construction of recombinant genomes feasible. In addition, because the signals that control AAV replication and genome encapsidation are contained in the ITR of the AAV genome, the approximately 4.3-kb internal portion of the genome. (encoding replication proteins and structural capsid proteins repcap), can be partially or completely replaced by foreign DNA. To produce AAV-based vectors, the rep and cap proteins can be provided in trans. Another important characteristic of AAV is that it is an extremely stable and persistent virus. It easily withstands the conditions used to inactivate adenovirus (56 to 65°C for several hours), making cold storage of AAV less critical. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

There remains a need in the art for treatments for LGMD2A.

Brief description of the invention

Provided herein are methods and products for delivering DNA encoding a protein having calpain-3 (CAPN3) activity. Such methods and products can be used to treat various diseases, such as LGMD2A.

Recombinant adeno-associated viruses (rAAVs) encoding a protein having calpain-3 activity (CAPN3) are provided. Recombinant adeno-associated viruses contain a polynucleotide that contains a nucleotide sequence encoding a protein having CAPN3 activity. The nucleotide sequence encoding a protein having CAPN3 activity, for example, is at least 90% identical to SEQ ID NO: 2 or contains the sequence of SEQ ID NO: 2.

For example, a provided rAAV comprises a polynucleotide that contains a first AAV inverted terminal repeat (ITR), a promoter, a nucleotide sequence encoding a protein having calpain-3 activity (CAPN3), and a second AAV ITR. The nucleotide sequence encoding a protein having CAPN3 activity, for example, is 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

- 2 045762 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, 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. rAAV contains a nucleotide sequence encoding a protein having CAPN3 activity containing the sequence of SEQ ID NO: 2.

In addition, the provided rAAV contains a nucleotide sequence encoding a protein having CAPN3 activity, which contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, at least 91% identical to SEQ ID NO: 7, to 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, 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. rAAV contains a nucleotide sequence encoding a protein having CAPN3 activity, containing the amino acid sequence of SEQ ID NO: 7.

The provided rAAVs contain a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1, 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, at least 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. rAAV contains the polynucleotide sequence of SEQ ID NO: 1.

In one embodiment, the nucleotide sequence is under the transcriptional control of a muscle-specific promoter. For example, the muscle-specific promoter comprises one or more of a human skeletal muscle actin gene element, a cardiac actin gene element, a desmin gene promoter, a skeletal muscle alpha actin gene promoter (ASKA), a troponin I gene promoter (TNNI2), a myocyte binding element -specific enhancer binding factor mef, muscle creatine kinase (MCK) gene promoter, truncated MCK gene promoter (tMCK), myosin heavy chain (MHC) gene promoter, hybrid promoter representing an enhancer of the α-myosin heavy chain gene/tandem enhancer-promoter gene MCK (MHCK7), C5-12 promoter, mouse creatine kinase gene enhancer element, skeletal muscle fast-twitch fiber troponin C gene element, cardiac slow-twitch fiber troponin C gene element, slow-twitch fiber troponin I gene element, nuclear factor response element , hypoxia-inducible element (HIF) (HRE), steroid-inducible element and glucocorticoid response element (gre). In one embodiment, the muscle-specific promoter is a tMCK promoter that contains the sequence of SEQ ID NO: 3.

For example, rAAV comprises a polynucleotide that in one embodiment comprises a first AAV inverted terminal repeat (ITR), a tMCK promoter, a nucleotide sequence encoding a protein having calpain-3 activity, and a second AAV inverted terminal repeat (ITR). The AAV ITR (eg, the first and/or second AAV ITR) is, for example, an AAV2 inverted terminal repeat. rAAV capsid proteins include, for example, the AAV-rh.74 capsid protein or the AAV9 capsid protein.

The provided rAAV contains one or more of the 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 AAVrh.10.

In another embodiment, compositions are provided containing any of the disclosed rAAVs. For example, the compositions are formulated for intramuscular injection or intravenous injection.

Also provided are methods of treating pelvic brachial muscular dystrophy type 2A in a subject, comprising administering to the subject a therapeutically effective amount of any of the disclosed rAAVs or any composition containing the disclosed rAAV. In any of the provided methods, rAAV is administered by intramuscular injection or intravenous injection.

For example, in these methods, treatment results in one or more of (a) an increase in muscle fiber diameter, (b) a decrease in the number of small lobular muscle fibers, (c) a decrease in the number of internal core fibers, (d) a decrease in endomysial connective tissue content, ( e) correcting muscle atrophy and (f) increasing the formation of muscle strength. The muscle fiber affected by the treatment includes one or more of slow-twitch oxidative (STO) muscle fiber, fast-twitch oxidative (FTO) muscle fiber, and fast-twitch glycolytic (FTG) fiber.

Moreover, in any of the provided methods, the treatment results in one or more of (a) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% reduction in the total number of muscle fibers per mm ² 4 weeks after administration; (b) at least 5%, 10%, 15%, 20%, or 25% increase in muscle fiber diameter 4 weeks after administration; (c) at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% reduction in STO muscle fiber number per mm ² at 4 weeks post-administration; (d) at least 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter 4 weeks after administration; (f) at least 5%, 10%, 15% or 20% reduction in FTO muscle fiber number per mm ² 4 weeks after administration; (f) at least 5%, 10%, 15%, or 20% increase in FTO muscle fiber diameter 4 weeks after administration; (g) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% reduction in FTG muscle fiber number per mm ² 4 weeks after administration and (h) at least 5%, 10 %, 15%, 20%, or 25% increase in FTG muscle fiber diameter 4 weeks after administration.

In any of the provided methods, the cardiac muscle of the subject exhibits minimal or low levels of calpain-3 protein expressed from any of the provided rAAVs or a composition containing any of the provided rAAVs. The muscle fiber that is treated with the composition includes one or more of slow twitch oxidative (STO) muscle fiber, fast twitch oxidative (FTO) muscle fiber, and fast twitch glycolytic (FTG) fiber.

Compositions are provided for the treatment of pelvic-brachial muscular dystrophy type 2A containing a therapeutically effective amount of any of the disclosed rAAVs or a composition containing any of the disclosed rAAVs. These compositions for the treatment of pelvic brachial muscular dystrophy type 2A are formulated for administration by intramuscular injection or intravenous injection. In addition, treatment with any of the disclosed compositions for the treatment of pelvic-brachial muscular dystrophy type 2A results in one or more of: (a) an increase in muscle fiber diameter, (b) a decrease in the number of small lobular muscle fibers, (c) a decrease in the number of fibers with the internal nuclei, (d) reducing the content of endomysial connective tissue, (e) correcting muscle atrophy and (f) increasing the formation of muscle strength. The muscle fiber that is treated with the composition includes one or more of slow twitch oxidative (STO) muscle fiber, fast twitch oxidative (FTO) muscle fiber, and fast twitch glycolytic (FTG) fiber.

In addition, treatment with any of the disclosed compositions for the treatment of pelvic-brachial muscular dystrophy type 2A results in one or more of: (a) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% or 40% reduction in total muscle fiber number per mm ² 4 weeks after administration; (b) at least 5%, 10%, 15%, 20%, or 25% increase in muscle fiber diameter 4 weeks after administration; (c) at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% reduction in STO muscle fiber number per mm ² 4 weeks after administration; (d) at least 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter 4 weeks after administration; (f) at least 5%, 10%, 15% or 20% reduction in FTO muscle fiber number per mm ² 4 weeks after administration; (f) at least 5%, 10%, 15%, or 20% increase in FTO muscle fiber diameter 4 weeks after administration; (g) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% reduction in FTG muscle fiber number per mm ² 4 weeks after administration and (h) at least 5%, 10 %, 15%, 20%, or 25% increase in FTG muscle fiber diameter 4 weeks after administration.

Moreover, treatment with any of the provided compositions for the treatment of pelvic brachial muscular dystrophy type 2A results in the subject's cardiac muscle exhibiting minimal or low levels of calpain-3 protein expressed from any of the provided rAAVs or a composition containing any of the provided rAAVs. Following administration of rAAV, cardiac muscle exhibits little or no toxic effect, such as inflammation, necrosis and/or regeneration.

The present invention also provides the use of a therapeutically effective amount of any of the disclosed rAAVs or a composition containing any of the disclosed rAAVs for the preparation of a medicament for the treatment of pelvic brachial muscular dystrophy type 2A. For example, the drug is formulated for administration by intramuscular injection or intravenous injection.

In any of the routes of administration, treatment with the drug results in one or more of: (a) an increase in muscle fiber diameter, (b) a decrease in the number of small lobular muscle fibers, (c) a decrease in the number of fibers with internal nuclei, (d) a decrease in the content endomysial connective tissue, (f) correcting muscle atrophy and (f) increasing muscle strength production. The muscle fiber affected by drug treatment is one or more of slow twitch oxidative (STO) muscle fiber, fast twitch oxidative (FTO) muscle fiber, and fast twitch glycolytic (FTG) fiber.

In addition, in any of the routes of administration of a therapeutically effective amount of any of the disclosed rAAVs or a contemplated composition, treatment with the drug results in one or more of: (a) at least 5%, 10%, 15%, 20%, 25% , 30%, or 35%, or 40% reduction in the total number of muscle fibers per mm ² 4 weeks after administration; (b) at least 5%, 10%, 15%, 20%, or 25% increase in muscle fiber diameter 4 weeks after

- 4 045762 introductions; (c) at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% reduction in STO muscle fiber number per mm ² 4 weeks after administration; (d) at least 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter 4 weeks after administration; (f) at least 5%, 10%, 15% or 20% reduction in FTO muscle fiber number per mm ² 4 weeks after administration; (f) at least 5%, 10%, 15%, or 20% increase in FTO muscle fiber diameter 4 weeks after administration; (g) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% reduction in FTG muscle fiber number per mm ² 4 weeks after administration and (h) at least 5%, 10 %, 15%, 20%, or 25% increase in FTG muscle fiber diameter 4 weeks after administration.

In any of the routes of administration of a therapeutically effective amount of any of the disclosed rAAVs or a contemplated composition, after treatment with a drug, the cardiac muscle of the subject exhibits no or minimal or low levels of calpain-3 protein expressed from the disclosed products or the disclosed composition.

Brief description of graphic materials

In fig. 1A-1F show that gene therapy resulted in restoration of impaired regeneration in CAPN3-KO muscle. In fig. Figure 1A shows a schematic representation of the single-stranded rAAV AAV9.CAPN3. Between the single-stranded 5' and 3' ITRs (inverted terminal repeats) is the muscle creatine kinase (MCK) gene promoter (563 bp), which drives the expression of the CAPN3 open reading frame (2466 bp). The polyadenylation site (poly(A), 53 bp) is also indicated. The tibialis anterior (TA) muscles of CAPN3-KO mice were first injected with CTX, and after 2 weeks, 1x1011 i. d. AAV.CAPN3 to the left TA (Fig. 1B) or PBS to the right TA (Fig. 1C). Four weeks after rAAV injection, muscle diameter increased and lobulated fibers were less common compared to untreated CAPN3-KO muscle. In fig. 1D, showing lobulated fibers with a pattern of subsarcolemmal organelles, the distribution of mitochondria (shown by arrows) suggests partial myotube fusion in untreated CAPN3-KO muscle at higher magnification. Scale bar = 20 µm for BD. In fig. 1E histograms of muscle fiber size distribution (mean ± SEM/ ^mm2 area; data obtained from 3 mice in each group) in treated and untreated TA muscles of CAPN3-KO mice show a shift toward larger diameter fibers with treatment and an increase in small-diameter subpopulation present in the untreated group. In fig. 1F histograms of the size distribution of slow twitch oxidative (STO) fibers demonstrate a greater number of fine fibers (eg, fibers with a diameter equal to 30 μm or less) in untreated CAPN3-KO muscle compared to treated CAPN3-KO muscle.

In fig. 2 shows a schematic representation of an rAAV of the present invention, called AAVrh.74.tMCK.CAPN3.

In fig. 3A-3B show Western blot (panel A) and RT-PCR (panel B) data after administration of AAVrh.74.tMCK.CAPN3 via intramuscular injection (1E11 i.g.) and systemic injection (3E12 i.g. and 6E12 v. g.). These data were compared with normal human muscle lysate (gel loading 60% total protein compared to lysates from mice) and naïve CAPN3-KO mice.

In fig. Figure 4 shows illustrative images of tissue sections from CAPN3-KO (AAV.hCAPN3 gene-injected and untreated) and wild-type (WT) TA muscle tissues stained with SDH. Average fiber size for slow twitch oxidative (STO, dark), fast twitch oxidative (FTO, intermediate), and fast twitch glycolytic (FTG, light) fibers in the TA muscle of mice treated with AAVrh.74.tMCK.CAPN3 appeared normalized to the values for WT. The sizes of fibers depending on the type with and without treatment are illustrated in Table. 4.

In fig. Figure 5 shows the relative levels of CAPN3 protein expression shown in WT (Z18-14) and TA muscles for the low-dose cohort (3E12 wg, Z18-13, Z18-15, Z18-16, Z18-17, Z18- 18), and gastrocnemius (gastrocnemius), cardiac muscle, quadriceps muscle, tibialis anterior (TA) and triceps muscle for the high dose cohort (6E12 i.g., Z1820, Z18-21, Z18-23, Z18- 24, Z18-22) (UT: untreated).

In fig. Figure 6 shows the number of AAVrh74.tMCK.hCAPN3 vector copies/μg genomic DNA in a cohort receiving a high dose of 6E12 vector genomes via systemic administration in the following muscles: quadriceps (quadriceps), cardiac, tibialis anterior (TA), gastrocnemius (gastrocnemius), triceps , as well as in the liver.

In fig. Figure 7 shows the average fiber diameter values for slow-twitch oxidative (STO, dark), fast-twitch oxidative (FTO, intermediate), and fast-twitch glycolytic (FTG, light) fibers of the left TA muscle after systemic administration of 3E12 and 6E12. g. AAVrh.74.tMCK.CAPN3. Data from naïve CAPN3-KO and WT mice were included.

In fig. Figure 8 shows the data from the run-to-exhaustion test. In fig. 8A presents data for

- 5,045,762 low-dose cohorts who received 3E12v. g. AAVrh.74.tMCK.CAPN3, and the high-dose cohort that received 6E12 v. AAVrh.74.tMCK.CAPN3, 4 weeks after systemic administration. Treated CAPN3-KO mice performed better in the run to exhaustion test compared to matched untreated animals. In fig. 8B shows data from the high dose cohort in which mice were tested 20-24 weeks after systemic administration of 6E12v. g. AAVrh.74.tMCK.CAPN3 (n=5), and corresponding animals not subjected to treatment (n=16).

In fig. Figure 9 shows fresh frozen left ventricular sections stained with hematoxylin and eosin (H&E) from illustrative heart samples from CAPN3-KO mice 4 weeks after systemic injection of the AAVrh7.4.tMCK.hCAPN3 vector at 3E12v doses. and 6E12th century. and corresponding untreated control animals.

In fig. Figure 10 shows the results of Western blot analysis of heart tissue samples from the high-dose cohort (which received 6E12 i.g. AAVrh7.4.tMCK.hCAPN3). This analysis showed no or minimal detectable amounts of calpain-3 protein in the heart of the treated animal. Animal ID numbers Z18-19 and 22 represent lysates from untreated CAPN3-KO mice.

Detailed description

Recombinant AAVs (rAAVs) provided herein comprise a polynucleotide that contains a first AAV inverted terminal repeat (ITR), a promoter, a nucleotide sequence encoding a protein having calpain-3 activity (CAPN3), and a second AAV ITR. In one embodiment, the nucleotide encodes CAPN3. Embodiments include, but are not limited to, an rAAV 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 nucleotide sequence of SEQ ID NO: 2. Additional embodiments include, but are not limited to, rAAV comprising a nucleotide sequence being at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence set forth in SEQ ID NO: 2 and encoding a polypeptide, possessing CAPN3 proteolytic activity. The proteolytic activity of CAPN3 is understood in the art as the activity of proteolysis of potential substrates such as fodrin and HSP60, and/or the activity of autolytic self-cleavage. Thus, as used herein, the term calpain-3 activity protein (CAPN3) refers to a protein having CAPN3 proteolytic activity, which includes, without limitation, proteolysis activity of potential substrates such as fodrin and HSP60, and/or autolytic self-cleavage activity. A protein having CAPN3 activity may have all or part of the activity of the full-length calpain-3 protein. In one embodiment, the 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.

In some embodiments, the nucleotide sequence encoding a protein having CAPN3 activity comprises the sequence of SEQ ID NO: 2. In another embodiment, the protein having CAPN3 activity comprises 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 another embodiment, the protein having CAPN3 activity contains the amino acid sequence of SEQ ID NO: 7. In another embodiment in an embodiment, the rAAV polynucleotide contains a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In another in an embodiment, the polynucleotide contains a sequence that is at least 95% identical to SEQ ID NO: 1. In one embodiment, the polynucleotide contains the sequence of SEQ ID NO: 1.

In another aspect, this document describes a recombinant AAV comprising a nucleotide sequence that encodes a protein having CAPN3 activity and/or that contains a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 2 or its complementary sequence. The term stringent is used to refer to conditions that are often understood in the art to be stringent. The stringency of hybridization conditions is determined primarily by temperature, ionic strength and the concentration of denaturing agents such as formamide. Examples of stringent hybridization and washing conditions are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y. 1989).

In the recombinant genomes described herein, the CAPN3 polynucleotide is functional

- 6 045762 but associated with transcription control elements (including, but not limited to, promoters, enhancers and/or introns), in particular transcription control elements that are functional in the target cells of interest. For example, in various embodiments, methods are provided for transducing muscle cells using muscle-specific transcriptional control elements, including, but not limited to, elements derived from the actin and myosin gene families, such as the myoD gene family [see Weintraub et al., Science, 251: 761-766 (1991)], myocyte-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol, 11: 4854-4862 (1991)], control elements derived from human skeletal muscle actin gene [Muscat et al, Mol Cell Biol, 7: 4089-4099 (1987)], muscle creatine kinase gene sequence elements [see Johnson et al, Mol Cell Biol, 9:3393-3399 (1989)] and the mouse creatine kinase (mCK) gene enhancer element, control elements derived from the skeletal muscle fast-twitch fiber troponin C gene, the cardiac slow-twitch fiber troponin C gene, and slow twitch fiber troponin I gene; hypoxia-inducible nuclear factors [Semenza et al., Proc Natl Acad Sci USA, 88: 5680-5684 (1991)], steroid-inducible elements, and glucocorticoid response element (GRE)-containing promoters [see Mader and White, Proc. Natl. Acad. Sci. USA, 90: 5603-5607 (1993)], tMCK promoter [cm. Wang et al., Gene Therapy, 15: 1489-1499 (2008)], CK6 promoter [see Wang et al., supra] and other control elements. In one embodiment, the nucleotide sequence encoding a protein having calpain-3 activity (CAPN3) is operably linked to a muscle-specific promoter. In one embodiment, the muscle-specific promoter comprises one or more of a human skeletal muscle actin gene element, a cardiac actin gene element, a desmin gene promoter, a skeletal muscle alpha actin gene promoter (ASKA), a troponin I gene promoter (TNNI2), a binding element for myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK) gene promoter, truncated MCK gene promoter (tMCK), myosin heavy chain (MHC) gene promoter, hybrid promoter representing an α-myosin heavy chain gene enhancer/tandem enhancer promoter of the MCK gene (MHCK7), promoter C5-12, enhancer element of the mouse creatine kinase gene, element of the troponin C gene of fast-twitch fibers of skeletal muscle, element of the troponin C gene of slow-twitch fibers of the cardiac muscle, element of the troponin I gene of slow-twitch fibers, element responding to nuclear hypoxia-inducible factor (HIF) (HRE), steroid-inducible element, glucocorticoid response element (gre). In another embodiment, the muscle-specific promoter is an MCK promoter, a tMCK promoter, or an MHCK7 promoter. In some embodiments, the muscle-specific promoter is tMCK, which contains the nucleotide sequence of SEQ ID NO: 3.

Previous studies demonstrated that CAPN3 expression driven by the desmin gene promoter resulted in cardiotoxicity. Prospective studies showed that selective expression of the gene in skeletal muscle led to the elimination of heart defects. The AAV genomes disclosed herein contain the muscle-specific tMCK promoter to restrict CAPN3 expression to skeletal muscle and demonstrated no cardiotoxicity following systemic delivery of the virus at a dose of 6E12v. g. (twice the suggested initial high dose) 4 weeks after gene injection.

The rAAV genomes described herein lack rep and cap AAV DNA. Provided rAAV genomes contain the CAPN3 polynucleotide described above and one or more AAV ITRs flanking the polynucleotide. The AAV DNA in rAAV genomes can be derived from any AAV serotype for which a recombinant virus can be produced, including, 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, AAV12, AAV-13, AAV-rh.74 and AAV-rh.10. Other types of rAAV variants are also being considered, such as rAAVs with capsid protein mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900–1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. AAV1, AAV5, AAV6, AAV8 or AAV9 can be used to promote skeletal muscle-specific expression.

The provided DNA plasmids contain rAAV genomes. DNA plasmids are transferred into cells capable of infection by an AAV helper virus (including, but not limited to, adenovirus, E1 deletion adenovirus, or herpes virus) to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV particles in which the AAV genome to be packaged, the rep and cap genes, and functional elements of the helper virus are provided to the cell are standard in the art. Production of rAAV requires that the following components be present in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (i.e., not contained within) the rAAV genome, and functional viral elements assistant The AAV ITR and rep and cap genes may be derived from any AAV serotype for which a recombinant virus can be produced, and may be derived from an AAV serotype other than the serotype for the rAAV ITR genome, including but not limited to AAV serotypes AAV-1 ,AAV-2,

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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 WO 01/83692, which is incorporated herein by reference in its entirety. Thus, in one embodiment, rAAV comprises one or more of the capsid proteins AAV-1, AAV-2, AAV-3, AAV4, 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. In another embodiment, rAAV comprises an AAV-rh.74 capsid protein or an AAV9 capsid protein.

The method for producing a packaging cell is to create a cell line that stably expresses all the necessary components for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing the rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene are integrated into the genome of the cell. AAV genomes were introduced into bacterial plasmids using procedures such as GC tail formation [Samulski et al., Proc. Natl. Acad. S6. USA, 79:2077-2081 (1982)], the addition of synthetic linkers containing restriction endonuclease cleavage sites [Laughlin et al., Gene, 23:65-73 (1983)], or by direct blunt end ligation [Senapathy & Carter, J. Bioh Chem., 259:4661-4666 (1984)]. The packaging cell line is then infected with a helper virus, such as an adenovirus. The advantages of this method are that the cells can be selected and are suitable for large-scale rAAV production. Other examples of suitable methods use adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells.

A review of the general principles for the preparation of rAAV is presented, 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 are described in Ratschin et al., Mol. Cell. Biol., 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol., 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); Lebkowski et al., Mol. Cell. Biol. 7:349 (1988); Samulski et al., J. Virol., 63:3822-3828 (1989); US Patent No. 5173414; WO 95/13365 and related US Patent No. 5658776; WO 95/13392; WO 96/17947; PCT/US 98/18600; WO 97/09441 (PCT/US 96/14423); WO 97/08298 (PCT/US 96/13872); WO 97/21825 (PCT/US 96/20777); WO 97/06243 (PCT/FR 96/01064); WO 99/11764; Perrin et al., Vaccine, 13:1244-1250 (1995); Paul et al., Human Gene Therapy, 4:609-615 (1993); Clark et al., Gene Therapy 3:11241132 (1996); US Patent No. 5786211; US Patent No. 5871982; US Patent No. 6258595 and McCarty, Mol. Ther., 76(10): 1648-1656 (2008). The foregoing documents are hereby incorporated herein by reference in their entirety, with particular emphasis on those portions of the documents that relate to the acquisition of rAAV.

Thus, packaging cells are provided that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells, such as HeLa cells and PerC.6 cells (293 cognate lineage). In another embodiment, the packaging cells are cells that are not 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 (fetal lung cells of rhesus monkey).

Recombinant AAVs provided herein are thus replication-defective, infectious, encapsidated viral particles that contain a recombinant genome. Examples include, but are not limited to, rAAV containing a genome containing the sequence set forth in SEQ ID NO: 1 encoding CAPN3, rAAV containing a genome substantially consisting of the sequence set forth in SEQ ID NO: 1 encoding CAPN3, and rAAV (referred to as AAVrh .74.tMCK.CAPN3) containing a genome consisting of the sequence presented under SEQ ID NO: 1, encoding CAPN3. The rAAV genomes lack rep and cap AAV DNA, meaning that the rAAV genome lacks rep or cap AAV DNA between the ITRs.

The sequence of AAVrh.74.tMCK.CAPN3 is presented under SEQ ID NO: 1, in which the AAV2 ITR spans nucleotides 1-128, the tMCK promoter spans nucleotides 165-884, the chimeric intron spans nucleotides 937-1069, the Kozak sequence spans nucleotides 1101-1106, the CAPN3 polynucleotide spans nucleotides 1107-3572, the poly(A) signal spans nucleotides 3581-3780, and the second AAV2 ITR spans nucleotides 3850-3977.

rAAV can be purified using methods known in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include those disclosed, for example, in Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schnepp and Clark, Methods Mol. Med., 69: 427-443 (2002); US patent No. 6566118 and WO 98/09657.

In another embodiment, compositions are provided containing rAAV described herein. The provided compositions contain rAAV in a pharmaceutically acceptable carrier. The compositions may also contain other ingredients such as diluents and auxiliary

- 8 045762 new funds. Acceptable carriers, diluents and auxiliaries are non-toxic to the patients receiving them and are preferably inert in the dosages and concentrations used, and they include buffers such as phosphate, citrate or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or non-ionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

Titers of rAAV to be administered in the methods described herein may vary depending on, for example, the specific rAAV, route of administration, purpose of treatment, individual, and cell type(s) being targeted, and can be determined using the methods , standard in the art. rAAV titers can range from about 1x101°, about 1x1011, about 1x10 ¹² , about 1x10 ¹³ to about 1x10 ¹⁴ or more DNase resistant particles (DRP) per ml. Dosages can also be expressed in units of viral genomes (vg). Exemplary doses disclosed include 1E11v. g., 3E12 century. and 6E12th century. G.

Disclosed herein are methods for transducing a target cell, such as a muscle cell, with rAAV in vivo or in vitro. The in vivo methods include the step of administering an effective dose or multiple effective doses of a composition containing rAAV provided herein to a subject in need thereof (eg, an animal, including but not limited to a human patient). If the dose is administered before the development of the disorder/disease, then the administration is prophylactic. If the dose is administered after the disorder/disease has developed, the administration is therapeutic. An effective dose is a dose that results in a reduction in intensity (elimination or amelioration) of at least one symptom associated with the disorder/disease condition being treated, which results in slowing or preventing progression to the disorder/disease condition that results in slowing or preventing the progression of a disorder/disease condition that results in a reduction in the severity of the disease, that results in remission (partial or complete) of the disease, and/or that results in prolongation of survival. Compared to a subject before treatment, the methods described herein result in one or more of an increase in muscle fiber diameter, a decrease in the number of small lobular muscle fibers, a decrease in the number of fibers with internal nuclei, a decrease in endomysial connective tissue content, correction of muscle atrophy, and increased formation muscle strength. In one embodiment, the muscle fiber includes one or more of a slow twitch oxidative (STO) muscle fiber, a fast twitch oxidative (FTO) muscle fiber, and a fast twitch glycolytic (FTG) fiber. In one embodiment, treatment results in one or more of: (a) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35%, or 40% reduction in total muscle fiber number per mm ² 4 weeks after administration; (b) at least 5%, 10%, 15%, 20%, or 25% increase in muscle fiber diameter 4 weeks after administration; (c) at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 42% reduction in STO muscle fiber number per mm ² 4 weeks after administration; (d) at least 5%, 10%, 15%, 20%, or 25% increase in STO muscle fiber diameter 4 weeks after administration; (f) at least 5%, 10%, 15% or 20% reduction in FTO muscle fiber number per mm ² 4 weeks after administration; (f) at least 5%, 10%, 15%, or 20% increase in FTO muscle fiber diameter 4 weeks after administration; (g) at least 5%, 10%, 15%, 20%, 25%, 30%, or 35% reduction in FTG muscle fiber number per mm ² 4 weeks after administration and (h) at least 5%, 10 %, 15%, 20%, or 25% increase in FTG muscle fiber diameter 4 weeks after administration. In one embodiment, the method of the present invention results in no or minimal or low levels of rAAV-expressed calpain-3 protein in the cardiac muscle of a subject receiving rAAV.

Assays to examine these results are understood in the art and/or described in the examples herein. Disclosed herein is the use of the methods described herein for the prevention or treatment of disorders/diseases (eg, forms of muscular dystrophy) caused by defects in CAPN3 activity or defects in CAPN3 expression. LGMD2A is an example of a disease for which prevention or treatment is considered in the methods.

Types of combination therapy are also considered. The combination used herein includes both simultaneous treatment and sequential treatment. In particular, combinations of the methods described herein with standard medical treatments (for example, the use of corticosteroids), as well as combinations with new types of

- 9 045762 therapy.

Administration of an effective dose of the compositions can be accomplished by routes standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, intrathecal, oral, buccal, intranasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal routes. The route(s) of administration and serotype(s) of the AAV components in rAAV (particularly the ITR and AAV capsid protein) can be selected and/or tailored by one of ordinary skill in the art based on the infection and/or disease state being treated and the cells /target tissue(s) that will express CAPN3. In one embodiment, rAAV is administered by intramuscular injection, intravenous injection, intraperitoneal injection, subcutaneous injection, cutaneous injection, intravaginal injection, intradermal injection, or intranasal injection. In another embodiment, rAAV is administered by intramuscular injection or intravenous injection.

In particular, the actual administration of the rAAV described herein can be accomplished by any physical method that will transport the recombinant rAAV vector into the target tissue of the animal. Administration includes, but is not limited to, injection into the muscle, bloodstream, and/or directly into the liver. It has been demonstrated that simple resuspension of rAAV in phosphate buffered saline is sufficient to obtain a carrier medium useful for expression in muscle tissue, and there are no known restrictions on the vehicles or other components that can be co-administered with rAAV. The rAAV capsid proteins can be modified so that the rAAV is targeted to a specific target tissue of interest, such as muscle. See, for example, WO 02/053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations that are to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practical implementation of the methods. rAAV can be used in conjunction with any pharmaceutically acceptable carrier for ease of administration and handling.

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

Pharmaceutical forms suitable for systemic (eg, intravenous) injection use include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions for immediate use. In all cases, the form must be sterile and must be fluid to the extent that it can be easily administered through a syringe. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating effects of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohol (eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of exposure to microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases it will be preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved through the use of absorption retarding agents, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating rAAV in the required amount into an appropriate diluent, optionally with various other ingredients listed above, followed by sterilizing filtration. Typically, dispersions are prepared by incorporating the sterilized active ingredient into a sterile carrier medium that contains the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, in some embodiments, the preparation methods include vacuum drying and/or freeze drying techniques, each of which can provide a powder based on the active ingredient and any additional required ingredient from a solution thereof, first subjected to sterilizing filtration.

Transduction by rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced to the subject. Alternatively, syngeneic or xenogeneic muscle cells may be used as long as the cells will not induce an unacceptable immune response in the subject.

Suitable methods for performing transduction and introducing the transduced cells back into the subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells, for example, in a suitable medium, and screening for those cells that contain DNA of interest using conventional techniques such as Southern blotting and/or or PCR, or using selectable markers. The transduced cells can then be formulated into pharmaceutical compositions, and the composition can be administered to the subject using various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle using, for example, a catheter.

Transduction of cells with rAAV using the methods described herein results in sustained expression of CAPN3 or a protein having CAPN3 activity. Thus, methods are provided for administering rAAV that expresses CAPN3 or a protein having CAPN3 activity to a subject, preferably a human. Subjects of the present invention include, but are not limited to, human, dog, cat, horse, cow, pig, sheep, goat, chicken, rodent (eg, rats and mice), and primate. These methods include transduction of tissues (including, but not limited to, tissues such as muscle tissue, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs described herein.

Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The methods described herein provide sustained expression of CAPN3 in transduced muscle cells.

By muscle cell, muscle fiber, or muscle tissue is meant a cell or group of cells derived from any type of muscle [eg, skeletal muscle and smooth muscle (eg, from the digestive tract, bladder, blood vessels, or cardiac tissue)]. Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts.

The term transduction is used to refer to the introduction/delivery of CAPN3 into a recipient cell in vivo or in vitro via the described rAAV, resulting in expression of CAPN3 by the recipient cell.

Thus, methods are provided for administering an effective dose (or doses administered substantially simultaneously or doses administered at intervals) of rAAV that encodes CAPN3 to a subject in need thereof.

As noted above, the methods described herein result in the subject, compared to the pre-treatment subject, developing one or more of an increase in muscle fiber diameter, a decrease in the number of small lobulated slow twitch oxidative (STO) muscle fibers, a decrease in the number of internal core fibers , reducing the content of endomysial connective tissue, correcting muscle atrophy and increasing the formation of muscle strength.

Examples

Aspects and embodiments are illustrated using the following examples. Example 1 describes the production of AAV9.MCK.CAPN3. Example 2 describes 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 intravenous administration of AAVrh.74.tMCK.CAPN3. Example 6 describes endpoint studies. Example 7 describes toxicology and biodistribution studies. Example 8 describes in vivo testing of biological activity following intramuscular administration. Example 9 describes in vivo testing of biological activity following systemic injection. Example 10 describes an evaluation of systemic delivery of the AAVrh.74.tMCK.CAPN3 gene. Example 11 describes the assessment of cardiotoxicity following systemic injection of the AAVrh.74.tMCK.CAPN3 vector. Example 12 describes an in vivo physiological assay.

Example 1: Obtaining AAV9.MCK.CAPN3

An AAV vector (termed AAV.CAPN3) carrying the CAPN3 gene under the control of the muscle-specific MCK promoter was generated (Fig. 1A). DNA containing the mouse CAPN3 open reading frame (NM_007601.3) between two restriction sites for Not1 was synthesized at Eurofins Genomics, USA, and then subcloned into the single-stranded AAV.MCK (muscle creatine kinase) vector previously described in Rodino-Klapac et al. , Journal of Translational Medicine, 5:45-55 (2007). rAAV vectors were produced using a modified cross-packing approach, whereby the AAV vector genome

- 11 045762 type 2 can be packaged into AAV capsids of several serotypes. [Rabinowitz et al., J Virol. 76 (2):791801 (2002)]. Production was carried out using a standard three-plasmid method with DNA precipitation using CaPO4 and using HEK293 cells. 293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. The production plasmids were: (i) pAAV.MCK.microdys, (ii) rep2/capX-modified AAV helper plasmids encoding the cap of serotypes 1, 6 or a serotype 8-like isolate, and (iii) a helper plasmid based on adenovirus type 5 (pAdhelper), expressing the genes of adenovirus E2A, ORF6 E4 and RNA VA I/II. To allow comparisons between serotypes, a qPCR titration method was used to determine the titer of encapsidated vector genomes (vg) using a Prism 7500 TaqMan real-time detection system (PE Applied Biosystems). [Clark et al., Hum Gene Ther. 10 (6): 1031-1039 (1999)]. The primer and fluorescent probe for targeting the MCK promoter were as follows: forward primer for MCK - 5CCCGAGATGCCTGGTTATAATT-3 (SEQ ID NO: 4); the reverse primer for MCK is 5GCTCAGGCAGCAGGTGTTG-3 (SEQ ID NO: 5) and the probe for MCK is 5-FAMCCAGACATGTGGCTGCTCCCCC-TAMRA-3 (SEQ ID NO: 6). The final titer (vg- ^ml'1 ) was determined by quantitative reverse transcriptase PCR using MCK promoter-specific primers and probes using a Prism 7500 real-time detection system (PE Applied Biosystems, Grand Island, New York, USA). Aliquoted viruses were kept at -80°C until use.

Example 2. Intramuscular administration of AAV9.MCK.CAPN3

To demonstrate whether CAPN3 WT can restore the impaired regeneration process in CAPN3 knockout (CAPN3-KO) mice, into the TA muscles of CAPN3-KO mice (n=4) [Kramerova et al., HumMolGenet 13(13): 1373-1388 (2004)]] were first injected with 30 μl of CTX under anesthesia, and 2 weeks later they were transduced to express wild-type CAPN3 with AAV9.MCK.CAPN3 at 1x1011 in. in a volume of 20 µl by intramuscular injection. TA muscles from another CAPN3-KO cohort (n=4), serving as controls, were injected with the same volume of PBS 2 weeks after CTX injection.

Mice were sacrificed 6 weeks after CTX injection, and TA muscles were removed and processed for cryosectioning. Twelve-μm-thick transverse sections were first stained with H&E for standard pathological evaluation; diameter measurements of specific muscle fiber types were obtained using SDH-stained TA transverse sections from 3 mice from each group. Three random photographs of TA were taken (for each section from each animal) at a magnification of 20X, and fiber diameter measurements were made and histograms were constructed for specific fiber types.

Histochemical analysis using the enzyme succinate dehydrogenase (SDH) was used to assess the metabolic differentiation of fiber types [slow twitch oxidative (STO), fast twitch oxidative (FTO) and fast twitch glycolytic (FTG)]. Measurements of the diameter of specific muscle fiber types were obtained using 12-μm-thick transverse sections stained with SDH at 4 and 12 weeks after the final cardiotoxin injection. Three photographs were taken, each showing three distinct zones of the gastrocnemius muscle (deep zone consisting predominantly of STO, intermediate zone showing STO and FTO or FTG in a checkerboard pattern, and superficial zone consisting predominantly of FTG fibers). along the midline axis (for each section from each animal) at 20X magnification using an Olympus BX41 microscope and a SPOT camera (Olympus BX61, Japan). This approach was chosen to capture changes in the oxidative state of fibers in each zone in response to metabolic changes during regeneration. Dark (STO), intermediate (FTO), and light (FTG) fiber diameters were determined by measuring the shortest distance within a muscle fiber using Zeiss AxioVision LE4 software (version 4.8). Fiber diameter histograms were plotted separately for STO; and for FTG and FTO were combined to represent the total population of fast twitch fibers (FTG/O) obtained from 3 animals and expressed as numbers per ^mm2 endomysial area (mean ± SEM). The average fiber diameter was obtained by combining the values for all 3 fiber types. On average, 900-1700 fibers were measured per group. TA muscles were used to assess fibrosis (see below).

Four weeks after injection of AAV9.MCK.CAPN3, there was a significant increase in muscle diameter, as well as a marked decrease in the number of internal nuclei and a much smaller number of thin fibers with a lobulated pattern (Fig. 1B). In untreated CAPN3-KO muscle, there were 31.6% more fibers per ^mm2 area and these were mainly composed of thin and lobulated STO fibers, indicating that the treatment resulted in improved myotube fusion and therefore a decrease in the number of individual fine fibers per unit area (Fig. 1C and D; Table 1).

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Table 1. Fiber size of the tibialis anterior muscle

Untreated, quantity per mm ² Untreated, diameter Treated with AAV.CAPN3, quantity per mm ² Treated with using AAV.CAPN3, diameter STO 355 32.72 ± 0.4 233 39.81 ±0.6* FTG/O 116 44.26 ± 0.9 99 50.40 ± 1.2* All fiber types 471 35.55 ±0.4 322 43.08 ±0.6*

*p < 0.0001 compared with the same parameter for the wild type

Histograms of fiber size distribution in treated TA muscle showed a shift toward larger diameter fibers with treatment, and an excess number of fine fibers in untreated control CAPN3-KO muscle were assigned to the histochemical STO fiber type (Fig. 1E and 1F). Taken together, these findings demonstrate that replacement of CAPN3 by gene therapy in CAPN3-KO muscle alleviated defective regeneration, as evidenced by a trend toward normalization of fiber size and a decrease in fiber number in the STO population.

Example 3. Obtaining AAVrh.74.tMCK.CAPN3

An AAV vector (termed AAVrh74.tMCK.CAPN3) carrying the CAPN3 gene under the control of a truncated muscle-specific MCK promoter (tMCK promoter) was generated. DNA containing the mouse CAPN3 open reading frame (NM_007601.3) between two restriction sites for Not1 was synthesized at Eurofin Genomics, USA, and then inserted into an AAV-based production plasmid. The plasmid map is shown in Fig. 2.

rAAV vectors were then prepared using the approach described in Example 1.

Example 4. Intravenous administration of AAVrh.74.tMCK.CAPN3

CAPN3-KO mice aged 6 months received AAVrh.74.tMCK.CAPN3 at low (3x10 ¹² i.g.) and high doses (6x10 ¹² i.g.) via tail vein injection. Mice were sacrificed 20 weeks after gene injection for endpoint studies. Age-matched vehicle-treated CAPN3-KO mice served as controls.

Table 2. Treatment cohorts

Cohorts Treatment Total number of mice Age at start of treatment Dose used during treatment (AAVrh.74.tMCK.CAPN3) Processing duration Age at end point CAPN3- KO 40

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Low dose AAV. CAPN3 8 24 weeks Ze12 century in 300 µl saline, i.v. 20 weeks 44 weeks Processing using saline solution 8 24 weeks 20 weeks 44 weeks High dose AAV. CAPN3 8 24 weeks 6th-12th century in 300 µl saline, i.v. 20 weeks 44 weeks Processing using saline solution 8 24 weeks 20 weeks 44 weeks Wild type control animals Processing using saline solution 8 24 weeks 20 weeks 44 weeks

Endpoint studies conducted as described in Example 7 below included muscle physiological analysis (in vivo TA force generation or muscle contractility analysis and eccentric contraction protection analysis), muscle pathology analysis, hCAPN3 detection by qPCR and Western blot analysis.

Example 5. Intramuscular administration of AAVrh.74.tMCK.CAPN3

Regenerative responses to synchronous cardiotoxin (CTX)-induced necrosis were measured in aged and young CAPN3-KO muscles after introducing CAPN3 into the regenerating muscle via rAAV treatment.

In cohorts consisting of young (2 months old) and old mice (6 months old), CTX was injected into both TA muscles to induce synchronous necrosis 2 weeks before rAAV injection into the left TA muscle. AAVrh.74.tMCK.CAPN3 in the amount of 1x1011 v. g in a volume of 20 μl was administered via intramuscular injection. Endpoint studies were performed 8 weeks after gene transfer (at a dose of 1x1011 i.g. with efficacy established in previous studies by the present inventors) to evaluate correction of the regeneration defect by comparing quantitative histological and physiological outcomes for left TA and untreated right TA .

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Table 3. Treatment cohorts

Cohorts Treatment Total number of mice Bilateral injection of STX into the TA muscle; age/delivery route/vine Age at start of gene therapy Dose used during treatment (AAVrh.74.tMCK. CAPN3), left TA Processing duration Age at end point CAPN3- KO CTX+ AAV. CAPN 3 16 Young 8 6 weeks/i.m./ 30 µl 8 weeks lell in. in 30 µl PBS, i.m. 8 weeks 16 weeks Old 8 22 weeks/i.m./ 30 µl 24 weeks lell in. in 30 µl PBS, i.m. 8 weeks 32 weeks Wild type animals Only oCTX 18 Young 8 6 8 30 µl 8 weeks 16 weeks/i.m./ 30 µl weeks PBS weeks Old 8 22 weeks/i.m./ 30 µl 24 weeks 30 µl PBS 8 weeks 32 weeks

Eight weeks after rAAV injection, the endpoint studies described in Example 6 below were performed, including muscle physiological analysis (TA strength generation and eccentric contraction protection), quantitative muscle pathology analysis, hCAPN3 detection by qPCR, and Western blot analysis. .

Example 6: Endpoint Studies

Formation of strength in the TA and protection against eccentric contractions

A protocol for assessing functional outcomes for the TA muscle was performed on muscles extracted from mice [Wein et al., Nature Medicine, 20(9):992-1000 (2014)]. Mice were anesthetized with ketamine/xylazine. Using a dissecting microscope, the skin of the hind limb was removed to expose the TA muscle and patella. The distal TA tendon was excised and a double straight knot was tied using a 4-0 surgical suture around the tendon as close to the muscle as possible and the tendon was cut. The exposed muscle was constantly moistened with saline. The mice were then transferred to a temperature-controlled heating platform and kept at 37 degrees. The knee was attached to the platform using a needle passed through the patellar tendon, the distal TA tendon suture was attached to the arm of a force transducer lever (Aurora Scientific, Opopa, Ontario, Canada), and the foot was secured using adhesive tape. Contractions of the TA muscle were induced by stimulation of the sciatic nerve using bipolar platinum electrodes. After stabilization of the muscle, the optimal length was determined by gradually stretching the muscle until maximum contraction force was achieved. After a 3-minute rest period, TA stimulation was performed at 50, 100, 150, and 200 Hz, allowing a 1-minute rest period between stimulations, to determine the maximum force of tetanic contraction. The length of the muscle was measured. After a 5-minute rest, the susceptibility of the TA muscle to contraction-induced damage was assessed. After 500 ms stimulation, the muscle lengthened by 10% of its optimal length. This involved stimulating the muscle at 150 Hz for 700 ms. After stimulation, the muscle returned to its optimal length. The cycle was repeated every minute for a total of 10 cycles. Specific force was calculated by dividing the maximum force of tetanic contraction by the cross-sectional area of the TA muscle. After eccentric contractions, mice were euthanized, and the TA muscle was excised, weighed, and frozen for analysis. Data analysis was performed in a blinded but not randomized fashion.

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In Vivo Muscle Contractility Analysis

This analysis measured the cumulative amount of torque produced by the plantaris or dorsiflexor muscles of the lower extremity and was performed using a muscle physiology instrument (Aurora Scientific, Ontario, Canada). The animal was anesthetized with isoflurane. After the animal was anesthetized, hair from the dorsal and hind limb areas was removed, if necessary, using a hair clipper. If hair removal with a hair clipper was not sufficient, a thin layer of hair removal cream (Nair) was applied and the application area was thoroughly cleaned with warm water to prevent discomfort. The hind limb intended for measurements was attached to the pedal using adhesive tape. The limb was held immobile using a rounded clamp. The tibial or peroneal component of the sciatic nerve was stimulated using two sterile, disposable, 28-gauge monopolar electrodes inserted through the skin and placed subcutaneously near the nerve. The mouse's temperature was maintained using a conductive temperature-controlled heating pad (set at 37°C) or a thermal radiation source and monitored using a temperature sensor.

Pathohistological examination

For histological analysis, all muscles and organs were embedded in 7% gum tragacanth and flash frozen in isopentane cooled with liquid nitrogen. Frozen sections (12 μm) were collected for immunohistochemistry and Western blot analysis.

Western blot analysis for detection of human CAPN3

Quantification of CAPN3 protein in mouse muscle tissue samples was performed using Western blotting. The CAPN3 enzyme was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and migrated as a 94 kDa band along with an approximately 60 kDa autolysis product, as detected using the Novocastra clinical grade antibody NCL-CALP-12A2. recognizing the N-terminus. In addition, the NCLCALP-2C4 antibody recognizes CAPN3 with the same molecular weight (94 kDa), as well as an additional fragment (30 kDa) in skeletal muscle; and both of these antibodies are suitable for protein detection. CAPN3 protein expression levels were measured semiquantitatively in samples from calpain knockout mice following delivery of the rAAV therapeutic vector and compared with untreated control animals.

Quantitative histological analysis of muscles

Transverse sections of TA and quadriceps muscles from animals treated with AAVrh.74.tMCK.CAPN3 compared to uninjected controls were stained with hematoxylin and eosin and photographed using Zeiss AxioVision L4 software. (4 random photographs at 20X magnification for each section from each animal). Diameters, which determine fiber size, were compared between treated and control animals.

Statistical analysis

Multiple comparison tests were performed using Student's t test or one-way ANOVA where appropriate.

Example 7: Toxicology/Biodistribution Studies

Toxicology/biodistribution studies were performed using the established effective dose and the one log higher dose. Toxicology studies were performed by systemic (tail vein) delivery of rAAV to CAPN3-KO mice at 6-8 weeks of age and included comparisons with normal values in normal C57BL6 mice. The study included cohorts of 6-10 mice and performed complete necropsy procedures using GLP-compliant techniques.

Serum collected from the blood samples was used for clinical biochemical analysis measuring the levels of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin (total and direct), blood urea nitrogen, creatinine, creatine kinase, glucose and total protein.

A full autopsy was performed, including a thorough and systematic examination and dissection of the animal's internal organs and musculoskeletal frame. Tissues/organs collected included gonads, brain, spleen, kidneys, jejunum, colon, pancreas, heart, lung, stomach, liver, inguinal lymph nodes, spinal cord, gastrocnemius, and quadriceps muscle. Tissues/organs for pathological studies were collected and fixed in 10% neutral buffered formalin (10% NBF), except for all skeletal muscle samples, which were mounted in OCT blocks and snap frozen in methylbutane cooled with liquid nitrogen for cryosectioning. .

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Example 8 In Vivo Biological Activity Testing After Intramuscular Injection

In vivo biological activity testing was performed after intramuscular (IM) injection.

AAVrh.74.tMCK.CAPN3 (1E11 i.g.) into the tibialis anterior (TA) muscle of CAPN3-KO mice (n=3), as described above in example 5.

At 4 weeks post-administration, gene delivery was analyzed using reverse transcription quantitative PCR (RT-qPCR) and Western blot assays. For Western blot analysis, samples corresponding to 50 μg of whole muscle protein extracts were separated on a 3–8% acrylamide gel with Tris-acetate buffer and SDS and transferred to a PVDF membrane. Immunoassays were performed using a monoclonal antibody raised against a synthetic peptide containing amino acids 1–19 of the human calpain-3 sequence (Leica) and an antibody against muscle-specific actin (Leica) as a loading control. In fig. Figure 3A demonstrates the presence of the 94 kDa calpain-3 protein in TA muscle after intramuscular injection. RT-qPCR analysis demonstrated a return of relative levels of human calpain-3 gene expression to normalized levels compared with WT mice 4 weeks after gene transfer (see Fig. 3B). Mouse GAPDH was used as the reference gene, and C57BL/6 WT was used to calibrate the RT-qPCR data.

In addition, quantitative pathological analysis was performed after intramuscular injection. As shown, TA muscle fiber diameter in treated CAPN3-KO mice was compared with that of untreated control muscle (TA injected with lactated Ringer's solution). The mean fiber size for slow-twitch oxidative (STO, dark), fast-twitch oxidative (FTO, intermediate), and fast-twitch glycolytic (FTG, light) fibers in AAV.hCAPN3-injected TA muscle appeared normalized to WT values. Quantitative assessment of fiber size depending on type is presented in table. 4 and shows an increase with processing.

Table 4

WT (zl8-14) Treated (zl8-ll) Not processed (2 exposed :18-22 L) Quantity Diameter (µm) Quantity Diameter (µm) Quantity Diameter (µm) STO 246 28.06 0.27 ± 142 28.89 0.32 ± 240 25.57 ±0.27 FTO 63 36.65 0.53 ± 86 36.71 0.58 ± 110 32.19 ±0.48 FTG 82 42.55 0.53 ± 86 43.68 0.66 ± 128 35.49 ±0.50 All types of fiber 391 32.45 0.38 ± 314 35.08 0.45 ± 478 29.75 ± 0.30

Briefly, in vivo bioactivity testing following IM vector injection (1E11 i.g.) into the tibialis anterior (TA) muscle in CAPN3-KO mice (n=2) demonstrated that 4 weeks after gene delivery 1) RT assays qPCR and Western blotting demonstrated expression of CAPN3 transcripts and the 94 kDa full-length calpain-3 protein and 2) histological analysis demonstrated an increase in TA muscle fiber diameter compared to control muscle (TA injected with lactated Ringer's solution).

Example 9 In Vivo Biological Activity Testing After Systemic Injection

In vivo biological activity testing was performed after systemic injection of AAVrh.74.tMCK.CAPN3 (3E12 i.g. or 6E12 i.g.) into the tail vein of CAPN3-KO mice. The low-dose CAPN3KO cohort (n=5; mice designated Z18-13, Z18-15, Z18-16, Z18-17, Z18-18) received 3E12v. g. in 300 µl of lactated Ringer's solution. At 4 weeks after gene injection, mice were assessed for running fatigue using a treadmill exhaustion test and then euthanized for tissue collection. Muscles of the upper and lower extremities (TA, gastrocnemius (GAS), quadriceps, triceps), heart, liver, spleen, lung, ovaries and testes were removed, and tissue samples were frozen in isopentane and cooled in liquid nitrogen.

CAPN3 expression in TA muscles was assessed using RT-qPCR. At a low dose of 3E12 c. d. CAPN3 mRNA expression levels were low, as observed by high CT values >27. Western blot analysis did not show detectable bands of the corresponding protein. Although data on low expression were obtained in this tissue at a low dose, with systemic administration of 3E12 c. d. demonstrated both functional and histological beneficial effects.

Therefore, a higher dose (6E12 i.g.) was administered systemically to investigate the possibility of detecting protein expression at a higher dose of the delivered vector. A high-dose cohort of CAPN3-KO mice (mice designated Z18-20, Z18-21, Z18-23, and Z18-24) received 6E12 i.v. g of AAVrh7.4.tMCK.hCAPN3 vector (which is twice the dose used for the low-dose cohort via systemic tail vein injection) and was euthanized 4 weeks after injection. RT-qPCR showed variable expression levels

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CAPN3 in quadriceps, triceps, GAS, TA and cardiac muscle.

To determine the relative expression of CAPN3 mRNA, muscle tissue samples were collected from CAPN3-KO mice treated with the tMCK.hCAPN3 vector at a dose of 3E12. g. (cohort 1, receiving a low dose) and 6E12 c. g. (cohort 2, receiving a high dose). Total RNA was isolated from both cohorts and CAPN3 versus mouse GAPDH was analyzed by qPCR along with previously obtained samples from the IM vector-treated cohort (1E11 i.g.; see Example 8 above).

The relative expression of CAPN3 was determined using the method below:

^CT = ^CT CAPN3 ^- CTmGAPDH

ΔΔST=ΔST - ΔSTcalibrator*

Relative expression of CAPN3=2^ ^CT

The relative expression of CAPN3 in each tissue and the baseline CT value are shown in Table. 5 below and in FIG. 5. In table. Figure 5 presents data for IM delivery (mice nos. Z18-11 and Z18-12) and systemic delivery.

Table 5. RT-PCR for CAPTN3

Mouse no. Textile Genoti p Dose for treatments (DRAP per mouse) CT value ACT AACT 2(- DAST) CAPN3 mGAPDH Z18-14* TA W.T. 0 22,437 22,456 15,234 15,274 7,193 0.000 1.0003 Z18-19 TA CAPN3- KO 0 35,259 32,705 15,159 15,176 18,814 11,621 0.0003 Z18-11 TA 1E11 24,338 24.217 15,800 15,835 8,460 1.267 0.4155 Z18-12 TA 21,030 21.104 14,906 15,058 6,085 -1.108 2.1548 Z18-13 TA ZE12 32,376 32,430 15,236 15,203 17,183 9,990 0.0010 Z18-15 TA 27,407 27,443 14,510 14,520 12,910 5,717 0.0190 Z18-16 TA 28,609 28,333 15,229 15,259 13,227 6,034 0.0153 Z18-17 TA 28,675 28,670 14,997 15,005 13,671 6,478 0.0112 Z18-18 TA 27,869 28.128 14,522 14,544 13,466 6,273 0.0129 Z18-20 Caviar foot 6E12 22,271 22,439 15,939 15,974 6,398 -0.795 1.7347 Heart 21,996 22,051 15,267 15,315 6,732 -0.461 1.3762 Four- heading 21,008 21,202 15,203 15,407 5,800 -1.393 2.6262 TA 23,806 24,173 16,169 16,385 7,713 0.520 0.6975 Three-headed I 24,083 24,361 15,978 16,097 8,185 0.992 0.5027 Z18-21 Caviar foot 25,330 25.221 15,461 15,462 9,814 2.621 0.1625 I'm cordial 25,024 24,819 15,032 15,097 9.857 2,664 0.1577 Four- heading 26,278 26.108 15,285 15,370 10,866 3.673 0.0784 TA 26,649 26,697 15,017 15,010 11,659 4,466 0.0452 Three-headed I 27,040 27,134 15,321 15,343 11,755 4,562 0.0423

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Z18-23 Caviar foot 24,150 24,144 16.225 16.117 7,976 0.783 0.5812 I'm cordial 22,799 22,495 14,593 14,502 8,099 0.906 0.5335 Four- heading 24,248 24,076 16,511 16,504 7.655 0.462 0.7262 TA 25,554 25,338 16,124 16,054 9,357 2,164 0.2231 Three-headed I 24,396 24,383 15,363 15,277 9,070 1.877 0.2723 Z18-24 Caviar foot 24,444 24.165 18,083 18,036 6,245 -0.948 1.9297 I'm cordial 22,769 22.425 15,100 15,077 7,508 0.315 0.8037 Quadriceps 22,754 22,521 15,637 15,672 6,983 -0.210 1.1568 TA 23,491 23,555 15,979 15,974 7,547 0.354 0.7826 Three-headed I 24,554 24,403 16,370 16,329 8.128 0.935 0.5229 Z18-22 Caviar foot 0 31,878 31,962 15,433 15,527 16,440 9,247 0.0016 I'm cordial 31,407 32,964 16,006 15,972 16,197 9,004 0.0019 Quadriceps 32,332 33,464 16,528 16,468 16,400 9,207 0.0017 TA 35,584 33,917 16,451 16,372 18,339 11,146 0.0004 Three-headed I 33,615 32,786 15,742 15,628 17,516 10,323 0.0008 Human tissue Muscle 23,547 23,539 37,743 38,262 pAAV.tM CK.hCAP N3 1 pg/µl 16,328 16,413 n. O. 38,302

* Calibrator

Overall, CAPN3 mRNA expression in CAPN3-KO muscle following systemic delivery was characterized by animal- and tissue-type-specific variability and lower relative expression compared to IM delivery at 1E11v dose. g. (<1% of that observed with IM delivery); which was particularly true for the low-dose 3E12 cohort. Accordingly, the level of the full-length 94-kDa protein was below the detection threshold for Western blotting. However, after systemic injection of a systemic dose of 6E12 v. g, the high-dose cohort exhibited robust gene expression and significant amounts of full-length calpain-3 protein.

Example 10. Evaluation of systemic delivery of the AAVrh74.tMCK.hCAPN3 gene

The efficiency of gene transfer was assessed by qPCR by counting the copy number of vector genomes in tissue samples from CAPN3-KO mice after systemic delivery of AAVrh74.tMCK.hCAPN3 at a dose of 6E12. d. We determined the load of vector genomes in the skeletal muscles of the upper and lower extremities (quadriceps, TA, gastrocnemius, triceps), heart and liver. Genomic DNA was isolated from frozen tissue samples. qPCR analysis was performed on an ABI 7500 (Applied Biosystems) using the following primer set: 5'CGGAGAGCAACTGCATAAG-3' (forward; SEQ ID NO: 8); 5'-GGCTGATGATGGCTGAATAG-3' (reverse; SEQ ID NO: 9). The primer pair exclusively amplified product from the 5'-terminal region of the hCAPN3 ORF and a downstream region unique to the expression vector spanning portions of the intronic element. The final results are given as the average number of copies of the AAVrh74 vector per

- 19,045,762 micrograms of genomic DNA.

As shown in FIG. 6, the highest copy number of vector genomes was present in the liver after systemic delivery of the vector. The distribution of vector genomes was variable depending on the muscle group. Overall, higher values were observed for quadriceps and cardiac tissue compared to other muscles. Variability between experiments was also noted; as in the case of mouse #Z18-21, which showed relatively lower copy number in all muscle groups compared to the other 3 mice.

In CAPN3-KO mice treated with systemic administration of 3E12 b. g., there was an improvement in both functional and histological features, but only low levels of muscle calpain-3 expression were detected in total RNA isolates using RT-qPCR, and the full-length 94 kDa protein for a specific muscle tissue could not be detected using Western blotting (see Fig. 3A). However, after administration of a systemic dose of 6E12 v. g showed robust gene expression and significant amounts of full-length calpain-3 protein (see Fig. 3B). Data demonstrate that calpain-3 gene expression returned to normal levels compared to WT mice 4 weeks after gene transfer in AAVrh74.tMCK.hCAPN3 particles. Mouse GAPDH was used as the reference gene and C57BL/6 WT was used to calibrate the RTqPCR data.

Pathohistological examination

As discussed above, there was a trend toward increased efficacy 4 weeks after injection. A significant increase in fiber size was observed in TA muscle from CAPN3-KO mice following systemic delivery of AAVrh.74.tMCK.hCAPN3 4 weeks post-injection in both cohorts (3E12 and 6E12). As shown in FIG. 7, total fiber diameter was significantly increased in both treated cohorts compared to the corresponding untreated KO animals (p < 0.00001). Treatment resulted in normalization of fiber size, and no dose-dependent differences were observed between treatment cohorts (p=0.78058). In table Figure 6 shows muscle fiber sizes for wild-type and CAPN3-KO mice after systemic gene therapy with AAV.hCAPN3 at doses of 3E12 and 6E12. G.

Table 6

WT (n=3) 6E12 CAPN3 (n=3) ZE 12 CAPN3 (n=4) KO (n=4) If- quality Diameter p (µm) If- quality Diameter p (µm) If- quality Diameter p (µm) If- quality Diameter p (µm) 532 30.0 ± 0.6 441 31.7 ± 0.8 464 30.7 ± 1.2 858 27.2 ± 0.8 278 40.7 ± 1.1 345 38.8 ± 1.1 447 40.8 ± 1.4 364 35.4 ± 1.1 275 46.1 ± 1.3 226 45.8 ± 1.4 403 44.1 ± 1.6 455 39.7 ± 1.2 1085 38.9 ± 1.8 1012 38.8 ± 2.0 1314 38.5 ± 2.4 1677 34.1 ± 1.8

Pathological evidence of cardiotoxicity after systemic injection of the AAVrh7.4.tMCK.hCAPN3 vector after 4 weeks was not observed in any of the cohorts. Variable amounts of virus were detected in cardiac tissue, but protein bands were not detected in cardiac tissue by Western blotting in any of the cohorts.

Functional Properties Study: Run to Exhaustion Test

Mice were habituated to a treadmill (Columbus Instruments) by running for 15 minutes at 10 m/min once daily for 3 days until data for the run to exhaustion test was obtained. The protocol used required the mice to be placed on a treadmill set at an inclination of 15°. The speed of movement of the treadmill when turned on was 1 m/min, and the speed was increased by 1 m every minute until the mouse was exhausted. Exhaustion was defined as the mouse sitting on the resting platform for at least 15 s. The time, speed and distance covered before exhaustion was recorded.

In fig. Figure 8A shows run-to-exhaustion test data for the low-dose cohort that received 3E12v. g. AAVrh7.4.tMCK.hCAPN3, and high-dose cohort 2, which received 6E12 i. d. AAVrh7.4.tMCK.hCAPN3, as assessed 4 weeks after systemic administration. Treated CAPN3-KO mice in both cohorts performed better in the test

- 20,045,762 runs to exhaustion compared to corresponding untreated animals. There were no significant dose-related differences in the run to exhaustion test or statistically significant differences in muscle fiber diameter between the low- and high-dose cohorts.

Mice from high-dose cohort 2 (n=16) were further analyzed 20-24 weeks after 6E12v administration. g. AAVrh7.4.tMCK.hCAPN3. As shown in FIG. 8B, treated CAPN3-KO mice continued to perform better in the run to exhaustion test compared to the corresponding untreated animals (p < 0.00001).

Example 11. Cardiotoxicity assessment after systemic injection of the AAVrh7.4.tMCK.hCAPN3 vector

After cohort mice were euthanized 4 weeks postinjection, serum and organ samples were collected. Low dose CAPN3-KO cohort 1 (n=5) received the AAVrh.74.tMCK.hCAPN3 vector at a dose of 3E12v. g. in 300 µl of lactated Ringer's solution by injection into the tail vein. Cohort 2 of high-dose CAPN3-KO mice received the AAVrh7.4.tMCK.hCAPN3 vector at a dose of 6E12v. g via the tail vein, and both cohorts were euthanized 4 weeks after injection. Two sections were examined through the apex of the heart, the superficial and deep regions of the ventricles. Tissue sections showed no signs of inflammation, necrosis, or regeneration, indicating no observable toxic effects on cardiac muscle after systemic administration of the AAVrh7.4.tMCK.hCAPN3 vector at two different doses 4 weeks after injection. Mice Nos. Z18-19 and Z18-22 (lactated Ringer's solution/naïve) served as KO control animals. In fig. Figure 9 shows fresh frozen heart sections stained with H&E. No signs of necrosis, regeneration or inflammation of muscle fibers were observed. Although variable amounts of virus were present in cardiac tissue, protein bands were not detected by Western blotting in any of the cohorts. In fig. 10 shows the results of Western blot analysis, which demonstrate that the level of full-length calpain-3 protein is below the detection threshold in cardiac tissue after transduction.

Example 12 In Vivo Physiological Analysis

Physiological assessments were performed after IM or systemic administration of the AAVrh7.4.tMCK.hCAPN3 vector. During in vivo physiological assessments, mice were anesthetized by isoflurane inhalation. After the animal was anesthetized, hair from the dorsal and hind limb areas was removed, if necessary, using a hair clipper. If hair removal with a hair clipper was not sufficient, a thin layer of hair removal cream was applied. During in vivo physiological force measurements, hindlimb torque was measured using a noninvasive force pedal coupled to a force detector motor (Aurora Scientific, Canada) following supramaximal sciatic nerve stimulations. The hind limb intended for measurements was attached to the pedal using adhesive tape. The limb was held immobile using a rounded clamp. The tibial or peroneal component of the sciatic nerve was stimulated using two sterile, disposable, 28-gauge monopolar electrodes placed subcutaneously near the nerve. The mouse's temperature was maintained using a conductive temperature-controlled heating plate (set at 37°C) or a thermal radiation source and monitored using an infrared temperature sensor.

Although the present invention provides specific embodiments, it should be understood that variations and modifications thereof will occur to those skilled in the art. Accordingly, the invention should be subject only to those limitations indicated in the claims.

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

Claims (7)

1. Recombinant adeno-associated virus (rAAV) containing the polynucleotide sequence SEQ ID NO: 1. 2. rAAV according to claim 1, wherein rAAV contains one or more of the 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 AAVrh.10. 3. rAAV according to any one of claims 1 or 2, where rAAV contains the capsid protein of 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, AAV rh.10, or its variant. 4. rAAV according to any one of claims 2 or 3, wherein the rAAV contains rh.74 capsid protein or AAV9 capsid protein. - 21 045762 5. A composition for treating pelvic-brachial muscular dystrophy type 2A in a subject, comprising rAAV according to any one of claims 1 to 4. 6. A method of treating pelvic-brachial muscular dystrophy type 2A in a subject, comprising administering to the subject a therapeutically effective amount of rAAV according to any one of claims 1 to 4 or a composition according to clause 5. 7. The method according to claim 6, where the administration is carried out by intramuscular injection or intravenous injection.