HK40115222A - Adeno-associated virus vector delivery of muscle specific micro-dystrophin to treat muscular dystrophy - Google Patents

Description

Adeno-associated virus vector delivers muscle-specific microdystrophy proteins to treat muscular dystrophy.

This invention was made with government support under license number NS055958 granted by the National Institutes of Health/National Institute of Neurological Disorders and Stroke. The government holds certain rights to this invention.

This application claims priority to U.S. Provisional Patent Application No. 62/473,148, filed March 17, 2017, which is incorporated herein by reference in its entirety.

By referencing materials submitted electronically

As a separate part of this disclosure, this application contains a sequence list in computer-readable form, which is incorporated in its entirety by reference and is as follows: Filename: 51475_Seqlisting.txt; Size: 29,519 bytes, created on March 13, 2018.

Technical Field

This invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, that express miniaturized human microdystrophin genes, and methods for using these vectors to express microdystrophin in skeletal muscle (including the diaphragm and cardiac muscle) and protect muscle fibers from injury, increase muscle strength, and reduce and/or prevent fibrosis in subjects with muscular dystrophy.

Background Technology

Muscle mass and strength are well-established for daily activities such as movement and breathing, as well as for overall metabolism. Muscle dysfunction results in muscular dystrophy (MD), characterized by muscle weakness and wasting, which severely impacts quality of life. The most fully characterized MD is caused by mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs are caused by membrane fragility associated with the loss of DAPC fascia-cytoskeleton binding. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in 5,000 newborn males.

DMD is caused by mutations in the DMD gene, leading to reduced mRNA and the loss of dystrophin, a 427kD sarcolemma protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51(6):919-28, 1987). DAPC is composed of various proteins on the muscle sarcolemma, forming structural connections between the extracellular matrix (ECM) and cytoskeleton via dystrophin (an actin-binding protein) and α-dystrophin glycan (a laminin-binding protein). These structural connections are used to stabilize the muscle cell membrane during contraction and prevent contraction-induced damage. With the loss of dystrophin, membrane fragility leads to sarcolemma tearing and calcium influx, triggering calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol. 10(2):168-75, 1997). This uncontrolled cycle of muscle degeneration and regeneration eventually depletes the muscle stem cell population (Sacco et al., Cell, 2010.143(7):p.1059-71; Wallace et al., Annu Rev Physiol, 2009.71:p.37-57), leading to progressive muscle weakness, endothelial inflammation, and fibrotic scarring.

Without the membrane-stabilizing effects of dystrophins or microdystrophins, DMD will exhibit an uncontrolled cycle of tissue damage and repair, ultimately replacing lost muscle fibers with fibrotic scar tissue through connective tissue proliferation. Fibrosis is characterized by excessive deposition of ECM matrix proteins, including collagen and elastin. ECM proteins are primarily produced by cytokines such as TGFβ, which are released by activated fibroblasts that respond to stress and inflammation. Although the main pathological feature of DMD is myofibril degeneration and necrosis, fibrosis has the same impact as a pathological consequence. Excessive production of fibrotic tissue limits muscle regeneration and contributes to progressive muscle weakness in patients with DMD. In one study, the presence of fibrosis in initial DMD muscle biopsy tissue was highly correlated with poor exercise outcomes at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol, 2009. 68(7): p. 762-7). These results suggest that fibrosis is a major cause of muscle dysfunction in DMD and highlight the need for early intervention before significant fibrosis develops.

Adeno-associated virus (AAV) is a replication-defective parvovirus with a single-stranded DNA genome approximately 4.7 kb long, consisting of 145 inverted terminal repeats (ITRs). Multiple serotypes of AAV exist. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the genome of AAV serotype 2 (AAV2) was proposed in *Journal of Virology*, 45:555-564 (1983), as corrected by Ruffing et al. in *Journal of General Virology*, 75:3385-3392 (1994). As another example, the complete genome of AAV-1 is available in GenBank accession number NC_002077; the complete genome of AAV-3 is available in GenBank accession number NC_1829; the complete genome of AAV-4 is available in GenBank accession number NC_001829; the genome of AAV-5 is available in GenBank accession number AF085716; the complete genome of AAV-6 is available in GenBank accession number NC_001862; and at least partial genomes of AAV-7 and AAV-8 are available in GenBank. Accessions AX753246 and AX753249 are provided (see also U.S. Patents 7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., Journal of Virology, 78:6381-6388 (2004); the AAV-10 genome is provided in Molecular Therapy, 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004).

Cloning of serotype AAVrh.74 was described in Rodino-Klapac et al., Journal of Translational Medicine 5, 45 (2007). The cis-acting sequences directing viral DNA replication (rep), capsidation/packaging, and host cell chromosome integration are contained in the ITR. Three AAV promoters (named p5, p19, and p40 at their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Differential splicing of the two rep promoters (p5 and p19) with a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227) results in the production of four rep proteins (rep 78, rep 68, rep52, and rep 40) from the rep gene. The rep proteins possess various enzymatic properties ultimately responsible for replicating the viral genome. The cap gene is expressed by the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-shared translation initiation sites are responsible for the production of three related capsid proteins. A single shared polyadenylation site is located at position 95 on the AAV genome map. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

AAV's unique characteristics lie in its attractiveness as a vector for delivering exogenous DNA to cells, for example, in gene therapy. AAV infection of cultured cells is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, allowing for the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells and can essentially prolong the lifespan of these cells as a transcriptionally active nuclear appendage (extrachromosomal element). The AAV proviral genome is infectious as clonal DNA in a plasmid, enabling the construction of recombinant genomes. Furthermore, since the signals guiding AAV replication, genome capsidation, and integration are contained in the ITR of the AAV genome, approximately 4.3 kb of the internal genome (encoding replication and structural capsid proteins, rep-cap) can be partially or entirely replaced with exogenous DNA, such as gene cassettes containing promoters, DNA of interest, and polyadenylation signals. The rep and cap proteins can be provided in trans form. Another significant characteristic of AAV is its extremely stable and robust nature. It readily withstands the conditions used for inactivating adenovirus (56°C to 65°C for several hours), making cold storage of AAV less critical. AAV can even be freeze-dried. Finally, AAV-infected cells are intolerant of repeated infection.

Multiple studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, due to the high vascularization of muscle, recombinant AAV transduction results in the presence of transgenic products in systemic circulation after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). Additionally, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cytokines for proper antibody glycosylation, folding, and secretion, indicating that muscle can stably express secreted protein therapeutic agents.

Functional improvement in patients with DMD and other muscular dystrophys requires genetic restoration in the early stages of the disease. Treatments to increase muscle strength and prevent muscle damage are needed in patients with DMD.

Summary of the Invention

This invention relates to gene therapy vectors, such as AAV, which express microdystrophy protein genes into skeletal muscle, including the diaphragm and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and/or prevent fibrosis.

This invention provides therapies and methods for increasing muscle strength and/or muscle mass by using gene therapy vectors to deliver microdystrophin to address genetic defects observed in DMD. As shown in Example 2, treatment with microdystrophin gene therapy resulted in greater muscle strength in vivo. Furthermore, intramuscular and systemic delivery of microdystrophin gene therapy has demonstrated dystrophin delivery to muscle in in vivo mouse models.

In one embodiment, the present invention provides an rAAV vector comprising a nucleotide sequence of a muscle-specific control element and a nucleotide sequence encoding a microdystrophy protein. For example, the nucleotide sequence encodes a functional microdystrophy protein, wherein the nucleotides have, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1, wherein the protein retains microdystrophy protein activity. Microdystrophy proteins provide stability to the muscle membrane during muscle contraction; for example, microdystrophy proteins act as shock absorbers during muscle contraction.

The present invention also provides an rAAV vector wherein the nucleotide sequence encodes a functional micromuscular dystrophy protein, comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 1 or its complement and encodes a functional micromuscular dystrophy protein.

In one implementation, the rAAV vector is a non-replicating recombinant adeno-associated virus (AAV) called rAAVrh74.MHCK7.micromuscular dystrophy protein. The vector genome contains the minimum elements required for gene expression, including the AAV2 inverted terminal repeat (ITR), micromuscular dystrophy protein, SV40 intron (SD/SA), and a synthetic polyadenylation (Poly A) signal, all under the control of the MHCK7 promoter/enhancer. A schematic diagram of the vector genome and expression cassette is shown in Figure 1. Following IV administration, the AAVrh74 serotype can be used to achieve efficient gene transfer in skeletal and cardiac muscle.

The term "strict" is used to refer to conditions that are generally understood in the art as stringent. The stringency of hybridization is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions used for hybridization and washing are 0.015 M sodium chloride and 0.0015 M sodium citrate at 65 to 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, New York, 1989). Even more stringent conditions (e.g., higher temperatures, lower ionic strengths, higher concentrations of formamide or other denaturing agents) can be used; however, the hybridization rate will be affected. In cases involving deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37°C (for 14-base oligomers), 48°C (for 17-base oligomers), 55°C (for 20-base oligomers), and 60°C (for 23-base oligomers).

To reduce nonspecific and/or background hybridization, additional reagents can be included in the hybridization and wash buffers. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), ficoll, Denhardt solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable reagents may also be used. The concentration and type of these additives can be varied without significantly affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8–7.4; however, under typical ionic strength conditions, the hybridization rate is almost pH-independent. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach , Ch.4, IRL Press Limited (Oxford, UK). Those skilled in the art can adjust hybridization conditions to accommodate these variables and allow DNAs with different sequence correlations to form hybrids.

The term "muscle-specific control element" refers to a nucleotide sequence that regulates the expression of a specific coding sequence in muscle tissue. These control elements include enhancers and promoters. This invention provides a construct comprising the muscle-specific control elements MCKH7 promoter, MCK promoter, and MCK enhancer.

The term "operably linked" refers to the positioning of a regulatory element nucleotide sequence (e.g., a promoter nucleotide sequence) to confer expression of the nucleotide sequence through the regulatory element.

On one hand, the present invention provides an rAAV vector in which the muscle-specific control element is a human skeletal muscle actin gene element, a cardiac muscle actin gene element, a muscle cell-specific enhancer-binding factor (MEF), muscle creatine kinase (MCK), a truncated MCK (tMCK), myosin heavy chain (MHC), a hybrid α-myosin heavy chain enhancer/MCK enhancer promoter (MHCK7), C5-12, a mouse creatine kinase enhancer element, a skeletal fast-twitch muscle fiber troponin c gene element, a slow-twitch muscle fiber cardiac troponin c gene element, a slow-twitch muscle fiber troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

For example, the muscle-specific control element is the MHCK7 promoter nucleotide sequence SEQ ID NO: 2 or the muscle-specific control element is the MCK nucleotide sequence SEQ ID NO: 4. Additionally, in any rAAV vector of the present invention, the muscle-specific control element nucleotide sequence, such as the MHCK7 or MCK nucleotide sequence, is operatively linked to a nucleotide sequence encoding micromuscular dystrophy. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO: 2) is operatively linked to the human micromuscular dystrophy coding sequence (SEQ ID NO: 1), as shown in the constructs provided in FIG1 or FIG10 (SEQ ID NO: 4).

As shown in 3). In another example, the MCK promoter (SEQ ID NO: 4) is operatively linked to the human micromuscular dystrophy protein coding sequence (SEQ ID NO: 1), as shown in the construct (SEQ ID NO: 5) provided in Figure 7 or Figure 11. Alternatively, the present invention provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2. The present invention also provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 4.

In another aspect, the present invention provides an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5. For example, the rAAVrh74.MHCK7.micromuscular dystrophy vector comprises the nucleotide sequence of SEQ ID NO: 3 and is shown in Figure 10. This rAAV vector comprises the MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human micromuscular dystrophy gene, polyA, ampicillin resistance, and a pGEX plasmid backbone having a pBR322 origin or replication.

This invention provides a recombinant AAV vector comprising the human micromuscular dystrophy protein nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 3. This rAAV vector is the AAV serotype AAVrh.74.

The present invention also provides a recombinant AAV vector comprising the nucleotide sequence of the pAAV.MHCK7. micromyotrophic dystrophin construct of SEQ ID NO: 3. This rAAV vector is the AAV serotype AAVrh.74.

The rAAV vector of the present invention can be any AAV serotype, such as serotype AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13.

The present invention also provides pharmaceutical compositions comprising any of the rAAV carriers of the present invention (or sometimes simply referred to herein as "compositions").

In another embodiment, the present invention provides a method for generating rAAV vector particles, comprising culturing cells transfected with any rAAV vector of the present invention and recovering rAAV particles from the supernatant of the transfected cells. The present invention also provides viral particles comprising any recombinant AAV vector of the present invention.

The present invention provides a method for treating muscular dystrophy, comprising administering a therapeutically effective amount of any of the recombinant AAV vectors of the present invention expressing human micromuscular dystrophy protein.

The present invention provides a method for treating muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector comprising the human micromuscular dystrophy protein nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2.

The present invention also provides a method for treating muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector comprising the nucleotide sequence of pAAV.MHCK7. micromuscular dystrophy construct of SEQ ID NO: 3.

"Fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and the abnormal repair process in tissues following injury, including skeletal muscle, cardiac muscle, liver, lungs, kidneys, and pancreas. The deposited ECM components include fibronectin and collagen, such as collagen 1, collagen 2, or collagen 3.

The present invention also provides a method for reducing or preventing fibrosis in subjects suffering from muscular dystrophy, including administering a therapeutically effective amount of any recombinant AAV vector of the present invention.

In another embodiment, the present invention provides a method for preventing fibrosis in subjects with this need, comprising administering a therapeutically effective amount of the recombinant AAV vector of the present invention. For example, any rAAV of the present invention can be administered to subjects with muscular dystrophy to prevent fibrosis, such as the rAAV of the present invention expressing human micromyotrophic protein administered prior to the observation of fibrosis in the subject. Furthermore, the rAAV of the present invention expressing the human micromyotrophic protein gene can be administered to subjects at risk of developing fibrosis, such as those with or diagnosed with muscular dystrophy such as DMD. The rAAV of the present invention can be administered to subjects with muscular dystrophy to prevent new fibrosis in these subjects.

This invention is intended to administer any AAV carrier of the invention prior to the observation of fibrosis in subjects. Furthermore, the rAAV of the invention can be administered to subjects at risk of developing fibrosis, such as those who have or have been diagnosed with muscular dystrophy, such as DMD. The rAAV of the invention can be administered to subjects with muscular dystrophy who already have fibrosis in order to prevent new fibrosis in these subjects.

The present invention also provides methods for increasing muscle strength and/or muscle mass in subjects suffering from muscular dystrophy, comprising administering a therapeutically effective amount of the rAAV vector of the present invention expressing the human micromyotrophic dystrophin gene. These methods may further include the step of administering rAAV expressing micromyotrophic dystrophin.

The present invention is intended to administer any of the AAV carriers of the present invention to patients diagnosed with DMD before fibrosis is observed in subjects or before muscle weakness or muscle mass is reduced.

The present invention also contemplates administering the AAV of the present invention to subjects with muscular dystrophy who already have fibrosis in order to prevent new fibrosis in these subjects or reduce fibrosis in these patients. The present invention also provides administration of any rAAV of the present invention to patients with muscular dystrophy who already have reduced muscle strength or reduced muscle mass to protect muscles from further damage.

In any of the methods of this invention, the subject may have muscular dystrophy such as DMD or any other muscular dystrophy associated with dystrophin.

On the other hand, the rAAV vector expressing microdystrophy contains the coding sequence of the microdystrophy gene, which is operatively linked to muscle-specific control elements other than MHCK7 or MCK. For example, these muscle-specific control elements include human skeletal muscle actin gene elements, cardiac muscle actin gene elements, muscle cell-specific enhancer-binding factor MEF, tMCK (truncated MCK), myosin heavy chain (MHC), C5-12, mouse creatine kinase enhancer elements, troponin C gene elements in fast-twitch skeletal muscle fibers, troponin C gene elements in slow-twitch skeletal muscle fibers, troponin I gene elements in slow-twitch skeletal muscle fibers, hypoxia-inducible nuclear factor, steroid-inducible elements, or glucocorticoid response elements (GRE).

In any of the methods of this invention, the rAAV carrier or composition may be administered by intramuscular or intravenous injection.

Furthermore, in any method of the present invention, the rAAV carrier or composition can be administered systemically. For example, the rAAV carrier or composition can be administered parenterally by injection, infusion, or implantation.

In another embodiment, the present invention provides a composition comprising any rAAV carrier of the present invention for reducing fibrosis in subjects with this need.

Furthermore, the present invention provides a composition comprising any of the recombinant AAV carriers of the present invention for the prevention of fibrosis in patients with muscular dystrophy.

This invention provides compositions comprising any recombinant AAV vector of this invention for the treatment of muscular dystrophy.

The present invention provides a composition comprising a recombinant AAV vector comprising the human micromuscular dystrophy protein nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 2, for the treatment of muscular dystrophy.

The present invention provides a composition comprising a recombinant AAV vector comprising a pAAV.MHCK7 micromuscular dystrophy protein construct containing the nucleotide sequence of SEQ ID NO: 3, for the treatment of muscular dystrophy.

The present invention also provides compositions comprising any rAAV carrier of the present invention for increasing muscle strength and/or muscle mass in subjects suffering from muscular dystrophy. In another embodiment, the present invention provides compositions comprising any rAAV carrier of the present invention for treating muscular dystrophy.

The compositions of the present invention can be formulated for intramuscular or intravenous injection. The compositions of the present invention are also formulated for systemic administration, such as parenteral administration via injection, infusion, or implantation.

In addition, any composition can be formulated for administration to subjects suffering from muscular dystrophy such as DMD or any other muscular dystrophy associated with dystrophin.

In another embodiment, the present invention provides the use of any rAAV carrier of the present invention in the preparation of a medicament for reducing fibrosis in a subject with this need. For example, the subject with this need may suffer from muscular dystrophy, such as DMD or any other muscular dystrophy associated with dystrophin.

In another embodiment, the present invention provides the use of the rAAV carrier of the present invention in the preparation of a medicament for the prevention of fibrosis in subjects suffering from muscular dystrophy.

Furthermore, this invention provides the use of the recombinant AAV vector of this invention in the preparation of a medicament for increasing muscle strength and/or muscle mass in subjects suffering from muscular dystrophy.

The present invention also provides the use of the rAAV carrier of the present invention in the preparation of a medicament for the treatment of muscular dystrophy.

This invention provides the use of a recombinant AAV vector comprising the human micromuscular dystrophy protein nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 in the preparation of a medicament for the treatment of muscular dystrophy.

This invention provides the use of a recombinant AAV vector containing the nucleotide sequence of pAAV.MHCK7. micro dystrophy protein construct of SEQ ID NO: 3 for the treatment of muscular dystrophy.

In any use of the present invention, the medicine may be formulated for intramuscular or intravenous injection. Furthermore, in any use of the present invention, the medicine may be formulated for systemic administration, such as parenteral administration via injection, infusion, or implantation.

Any drug can be prepared for use in subjects suffering from muscular dystrophy such as DMD or any other muscular dystrophy associated with dystrophin.

Attached Figure Description

Figure 1 shows the pAAV.MHCK7. micromyotrophic dystrophin construct. In this construct, the flanking structures of the cDNA expression cassette are AAV2 inverted terminal repeats (ITRs). This construct is characterized by the deletion of in-frame rods (R4-R23), while hinges 1, 2, and 4 ( _H1 , _H2 , and _H4 ) and cysteine-rich domains still produce a 138 kDa protein. Expression of the micromyotrophic dystrophin (3579 bp) is directed by the MHCK7 promoter (795 bp). Introns and 5'UTRs are derived from plasmid pCMVβ (Clontech). The micromyotrophic dystrophin cassette has a consensus Kozak immediately before ATG initiation, as well as a small 53 bp synthetic polyA signal for mRNA termination. The human micromyotrophic dystrophin cassette contains the (R4-R23/Δ71-78) domains as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)).

Figure 2 shows the expression of dystrophin after intramuscular delivery of the AAVrh74.MHCK7 construct. MDX mice were injected with 1 × ^10¹¹ vg into the anterior tibialis muscle (n = 5 per group). Muscle was harvested six weeks later and stained with N-terminal antibodies against dystrophin and hematoxylin and eosin for dystrophin expression.

Figures 3A-3C provide quantification of skeletal muscle strength measurements and microdystrophin expression after intramuscular injection of the AAVrh74.MHCK7 construct. (A) Tibialis anterior muscle of MDX mice was injected with 1 × ^10¹¹ vg of the AAVrh74.MHCK7 construct (n = 5). After six weeks, the tibialis anterior muscle was harvested and in vivo force measurements were performed. The treated group had significantly greater force production than the untreated MDX control.

Figures 4A-4C show extensive transduction of skeletal, diaphragmatic, and cardiac fibers after systemic administration of the AAVrh.74.MHCK7. micromyotrophic protein construct. (A) Mdx mice were systemically treated with 6 × ^10¹² vg (2 × ^10¹⁴ vg/kg) of AAVrh.74.MHCK7. micromyotrophic protein via tail vein at 6 weeks of age. (B) Staining of micromyotrophic protein shows quantification of the percentage of myofibrosis expressing micromyotrophic protein in each tissue. (C) Specific strength measured in the diaphragm at low and high (planned clinical) doses. No significant difference was observed at the low dose; however, a significant improvement was observed at the high dose.

Figure 5 shows the expression of dystrophin after systemic delivery of the AAVrh.74.MHCK7. microdystrophin construct. Mdx mice (n=5) were systemically treated with 6 × ^10¹² vg of AAVrh.74.MHCK7. microdystrophin via tail vein at 6 weeks of age. After 12 weeks of treatment, all muscle tissue was collected and stained for dystrophin and restored DAPC components (showing β-caryogamin).

Figures 6A-6D show the toxicology/safety of AAVrh.74.MHCK7. Hematoxylin and eosin (H&E) staining was performed on the following muscle tissues to analyze toxicity: tibialis anterior (TA), gastrocnemius (GAS), quadriceps femoris (QD), psoas major (PSO), triceps brachii (TRI), and diaphragm (DIA) (Figure 6A). No toxicity was found. The number of muscle fibers with centrally located nuclei (CN) was quantified as an indicator of efficacy (Figure 6B). CN indicates the cycle of muscle degeneration and regeneration, therefore a reduction in CN indicates therapeutic efficacy. (Figure 6C) shows the change in total fiber count with treatment. (D) provides the amount of creatine kinase, showing improvement at high doses. Independent t-tests were used to locate differences (p < .05); data are reported as mean ± SEM.

Figure 7 shows the pAAV.MCK. micromyotrophic protein particle construct.

Figure 8 shows the results of the rAAVrh74.MCK micromyotrophic dystrophin (human) potency assay. Tibial anterior muscle of mdx mice was injected with 3 × ^10⁹ , 3 × ^10¹⁰ , or 1 × ^10¹¹ vg (n = 3 per group). Muscle was harvested after four weeks and stained with N-terminal Dys3 antibody to show dystrophin expression. A linear correlation was found between expression and dose; expression was very low (no effect level) at 3 × ^10⁹ vg and reached 89% at 1 × ^10¹¹ vg.

Figures 9A-9C demonstrate that human micromyotrophic dystrophin improves force generation and protection against damage caused by eccentric contraction. (A) Immunostaining of micromyotrophic dystrophin in extensor digitorum longus (EDL) and TA shows expression in MDX muscle fibers after injection of rAAVrh.74-MCK-micromyotrophic dystrophin (human) via the femoral artery. Muscles with simulated infection were stained in the same manner, and exposure was time-matched. (B) rAAVrh.74-MCK-micromyotrophic dystrophin significantly increased normalized specific force compared to MDX muscle treated with simulated infection (P<0.05). (C) Comparison of mDX muscle infected with rAAVrh.74-MCK-micromyotrophic dystrophin (human) with contralateral MDX EDL muscle infected with simulated infection and WT (WT C57Bl/10) EDL muscle showed a decrease in force during repetitive eccentric contraction at 12 weeks post-gene transfer. Compared with the mdx muscle treated with simulated therapy, rAAVrh.74-MCK-microdystrophy protein (Micro-dys) treatment significantly prevented the loss of force (vs. mdx, P<0.001). Error is SEM.

Figure 10 provides the nucleic acid sequence (SEQ ID NO: 3rAAVrh74.MHCK7. Micromuscular dystrophy protein).

Figure 11 shows the nucleic acid sequence (SEQ ID NO:5) rAAVrh74.MCK. micromyotrophic dystrophy protein.

Figures 12A-12B present the immune responses to systemic delivery of AAVrh74.MHCK7. microdystrophy protein in non-human primates. (A) ELISA-positive responses to AAV capsid and microdystrophy protein peptide libraries. ConA is a positive control, and DMSO is a negative control. There are three libraries of AAVrh74 and four peptide libraries specific to microdystrophy. (B) ELISA-positive titers of circulating neutralizing antibodies against the carrier capsid. Serum was isolated from primates every two weeks and antibody titers were analyzed. The reported titers correspond to the final dilution with a response ratio ≥2.

Figures 13A-B show the systemic delivery of AAVrh74.MHCK7 microdystrophy protein in rhesus monkeys. Anti-FLAG immunofluorescence staining in the left muscle shows strong microdystrophy protein expression.

Figure 14 shows the effect of rAAVrh74.MHCK7. micromyotrophic dystrophin on systemic treatment with transgenic expression. Immunofluorescence staining of micromyotrophic dystrophin with antibodies against N-terminal dystrophin in the heart, diaphragm, psoas, and tibialis anterior (TA) muscles revealed strong expression in animals treated with medium (6e12 vg; 2e14 vg/kg) and high (1.2e13 vg; 6e14 vg/kg) doses at 3 months post-injection. 20x images are shown.

Figure 15 shows the effect of systemic treatment with rAAVrh74.MHCK7. micromyotrophic dystrophin on transgene expression. Immunofluorescence staining of micromyotrophic dystrophin with antibodies against N-terminal dystrophin in the gastrocnemius, quadriceps, triceps, and gluteal muscles showed strong expression in animals treated with medium (6e12 vg; 2e14 vg/kg) and highest doses (1.2e13 vg; 6e14 vg/kg) at 3 months post-injection. 20x images are shown.

Figure 16 shows the effects of systemic treatment with rAAVrh74.MHCK7 micromyotrophic dystrophin on muscle pathology. (A) H&E staining of diaphragm, tibialis anterior, gastrocnemius, and quadriceps muscles from C57BL/6WT, mdx, and rAAVrh74.MHCK7 micromyotrophic dystrophin-treated mice (medium dose -2e14vg/kg; high dose -6e14vg/kg), (B) Quantification of mean fiber size indicating normalized fiber size in all tissues. ****p<0.001, one-way ANOVA; data reported as mean ± SEM. 20x images are shown.

Figure 17 shows the effects of systemic treatment with rAAVrh74.MHCK7. micromyotrophic dystrophin on muscle pathology. (A) H&E staining of triceps, gluteal, and psoas muscles from C57BL/6WT, mdx, and rAAVrh74.MHCK7. micromyotrophic dystrophin-treated mice (medium dose -2e14vg/kg; high dose -6e14vg/kg), (B) quantitative mean fiber size showing larger fibers in a dose-dependent manner. ****p<0.001, one-way ANOVA; data reported as mean ± SEM. 20x images are shown.

Figure 18 shows the effect of systemic treatment with rAAVrh74.MHCK7 micromyotrophic dystrophin on centrifugation. Increased dose indicated reduced centrifugation in all skeletal muscles and the diaphragm. Two-way ANOVA was used to determine the location of differences (p<0.05). Data are reported as mean ± SEM.

Figure 19 shows the effect of systemic treatment with rAAVrh74.MHCK7 micromyotrophic dystrophin on collagen deposition. Increased dose indicates a decrease in collagen accumulation (%) in the diaphragm. *p<0.05, univariate ANOVA; data reported as mean ± SEM. 20x images are shown.

Figure 20 shows the correction for insufficient force in the diaphragm. Diaphragmatic strips were harvested 3 or 6 months after treatment to measure specific force (normalized to cross-sectional area). Treatment restored force to WT level. *p<0.05. Univariate ANOVA was used to determine differences compared to mdx-LR mice.

Figure 21 shows the correction for insufficient force in TA. (A) TA muscle (left and right) was harvested 3–6 months after treatment to measure specific force (normalized to TA weight). Treatment restored force to WT level. (B) Treatment protected TA muscle from fatigue through a strict eccentric contraction protocol. *p<0.05. Univariate ANOVA was used to determine differences compared to mdx-LR mice.

Figure 22 shows the distribution of mean VG copies in various tissues from three MDX mice after IV delivery of rAAVrh74.MHCK7 microdystrophy protein.

Figure 23. Serum chemistry of mice systemically injected with ssAAVrh74.MHCK7. micromuscular dystrophy protein and serum chemistry of age-matched controls were analyzed by an independent CRO (Charles River Laboratories), showing normal values for all analyzed chemistry. The only outliers were elevated AST and ALT observed in MDX-carrier-treated animals [MDX-LR (lactated Ringer's solution)], which were normalized by treatment. Elevated AST and ALT are known in DMD. ALT = alanine aminotransferase, ALP/K = alkaline phosphatase, AST = aspartate aminotransferase, BUN = blood urea nitrogen, B/C = blood to creatinine ratio, CREAT = creatine, GLU = glucose, TP = total protein, TBIL = total bilirubin, DBIL = direct bilirubin

Figure 24 shows the biodistribution protein blots of muscles and organs from mdx mice that received systemic injections of rAAVrh74.MHCK7 microdystrophy protein.

Figure 25 shows the map of the pNLREP2-Caprh74 AAV helper plasmid.

Figure 26 shows the Ad helper plasmid pHELP.

Detailed Implementation

This invention provides gene therapy vectors, such as rAAV vectors overexpressing human microdystrophy protein, and methods for reducing and preventing fibrosis in patients with muscular dystrophy. Muscle biopsies taken at the earliest age of DMD diagnosis reveal prominent connective tissue hyperplasia. Muscle fibrosis is detrimental in several ways. It reduces the normal transport of nutrients within the muscle through the connective tissue barrier, reduces blood flow and deprives muscles of vascularized nutrients, and functionally promotes early loss of walking through limb contractures. Over time, the treatment challenge increases exponentially due to the significant fibrosis in the muscles. This can be observed in muscle biopsies comparing connective tissue proliferation at consecutive time points. This process continues to worsen, leading to loss of walking and accelerated loss of control, particularly in wheelchair-dependent patients.

Without early intervention, including parallel approaches that reduce fibrosis, the full benefits of exon skipping, stop codon readout, or gene replacement therapy cannot be realized. Even small molecule or protein replacement strategies may fail without methods to reduce muscle fibrosis. Previous work in aged MDX mice with existing fibrosis treated with AAV. micromuscular dystrophy demonstrated that full functional recovery cannot be achieved (Liu, M. et al., Mol Ther 11, 245-256 (2005)). The progression of DMD cardiomyopathy is also known to be accompanied by scarring and fibrosis in the ventricular wall.

As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that is generated only in cells, with some functions provided by co-infected helper viruses. Thirteen AAV serotypes have been characterized. Basic information and reviews of AAVs can be found, for example, Carter, 1989, *Handbook of Parvoviruses*, Vol. 1, pp. 169–228, and Berns, 1990, *Virology*, pp. 1743–1764, Raven Press, (New York). However, it is entirely reasonable to expect that the same principles will be applicable to additional AAV serotypes, given that it is well known that the various serotypes are closely related in both structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165–174, edited by J.R. Pattison; and Rose, Comprehensive Virology 3:1–61 (1974)). For instance, all AAV serotypes exhibit remarkably similar replication characteristics mediated by homologous rep genes; and all possess three associated capsid proteins, such as those expressed in AAV2. The degree of correlation is further demonstrated by heteroduplex analysis, which reveals extensive cross-hybridization along the genome length between serotypes; and the presence of similar self-annealing fragments at the ends, corresponding to “inverted terminal repeats” (ITRs). Similar patterns of infectivity also suggest that replication function is under similar regulatory control for each serotype.

As used herein, “AAV vector” refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeat sequences (ITRs). When present in host cells transfected with a vector that encodes and expresses the rep and cap gene products, such an AAV vector can be replicated and packaged into infectious viral particles.

"AAV virion," "AAV virus particle," or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and a capsidated polynucleotide AAV vector. If the particle includes heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is generally referred to as an "AAV vector particle" or simply an "AAV vector." Therefore, the production of AAV vector particles necessarily involves the production of AAV vectors, thus the AAV vector particle contains a vector.

AAV

The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs of the side-linked nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which recombinant viruses can be derived, including but not limited to AAV serotypes AAVrh.74, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAV is disclosed, for example, in WO 01/83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To initiate skeletal muscle-specific expression, AAV1, AAV6, AAV8, or AAVrh.74 can be used.

The DNA plasmid of this invention contains the rAAV genome of this invention. The DNA plasmid is transferred into cells that allow infection with AAV helper viruses (e.g., adenovirus, E1-deficient adenovirus, or herpesvirus) to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV particles are standard in the art, wherein the AAV genome to be packaged, the rep and cap genes, and helper viral functions are provided to the cell. The production of rAAV requires the presence of the following components within a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e., not contained within) the rAAV genome, and the helper viral functions. The AAV rep and cap genes can be derived from any AAV serotype from which recombinant viruses can be derived, and can be derived from AAV serotypes that differ from the rAAV genomic ITR, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAVs is disclosed, for example, in WO 01/83692, which is incorporated herein by reference in its entirety.

The method for generating packaging cells involves creating cell lines that stably express all the essential components for AAV particle production. This includes, for example, plasmids (or multiple plasmids) containing the rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separated from the rAAV genome, and optional markers such as neomycin resistance genes integrated into the cell's genome. The AAV genome has been introduced into bacterial plasmids via procedures such as GC tailing (Samulski et al., 1982, Proceedings of the National Academy of Sciences of the United States of America, 79:2077-2081), by adding synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or by direct blunt-end ligation (Senapathy and Carter, 1984, Journal of Biochemistry, 259:4661-4666). The packaging cell lines are then infected with helper viruses such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and/or rep and cap genes into packaging cells.

The general principles of rAAV generation are commented on, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial and Immunol., 158:97-129. Various methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proceedings of the National Academy of Sciences, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., Journal of Virology, 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol. 7:349 (1988). Samulski et al., *Journal of Virology*, 63:3822-3828 (1989); US Patent No. 5,173,414; WO 95/13365 and corresponding US Patent No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/062 43 (PCT/FR96/01064); WO 99/11764; Perrin et al., Vaccine, 13:1244-1250 (1995); Paul et al., Human Gene Therapy, 4:609-615 (1993); Clark et al., Gene Therapy, 3:1124-1132 (1996); U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. The aforementioned references are incorporated herein by reference in their entirety, with particular emphasis on those relating to the generation of rAAV.

Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (homogeneous 293 lineage). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passaged 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey embryonic lung cells).

The recombinant AAV (i.e., infectious capsidated rAAV particles) of the present invention comprises an rAAV genome. In an exemplary embodiment, the genomes of both rAAVs lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAVs that can be constructed to comprise the nucleic acid molecules of the present invention are set forth in International Patent Application No. PCT/US2012/047999 (WO 2013/016352), which is incorporated herein by reference in its entirety.

In one exemplary embodiment, the recombinant AAV vector of the present invention is generated via a triple transfection method (Xiao et al., J Virol 72, 2224-2232 (1998)), which uses the AAV vector plasmid pAAV.MHCK7.micromuscular dystrophy, pNLRep2-Caprh74, and pHelp, where pAAV contains a micromuscular dystrophy gene expression cassette flanking an AAV2 inverted terminal repeat (ITR) sequence. It is this sequence that is capsidred into the AAVrh74 viral particle. The plasmid contains the micromuscular dystrophy sequence and the MHCK7 enhancer and core promoter elements of the muscle-specific promoter to drive gene expression. The expression cassette also contains an SV40 intron (SD/SA) to promote high levels of gene expression, and bovine growth hormone polyadenylation signaling for efficient transcription termination.

pNLREP2-Caprh74 is an AAV helper plasmid that encodes four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins derived from serotype rh74. A schematic diagram of the pNLREP2-Caprh74 plasmid is shown in Figure 25.

The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. The plasmid contains adenovirus genomic regions important for AAV replication, namely E2A, E4ORF6, and VA RNA (adenovirus E1 function was provided by 293 cells). The adenovirus sequence present in this plasmid represents only about 40% of the adenovirus genome and does not contain cis-elements crucial for replication, such as adenovirus terminal repeat sequences. Therefore, infectious adenovirus is not expected to be generated from this production system. A schematic diagram of the pHELP plasmid is shown in Figure 26.

rAAV can be purified using standard methods in the art, such as column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art and are included, for example, Clark et al., Human Gene Therapy, 10(6):1031-1039 (1999); Schenpp and Clark, Methods in Molecular Medicine, 69:427-443 (2002); U.S. Patent Nos. 6,566,118 and WO 98/09657.

In another embodiment, the present invention relates to compositions comprising the rAAV of the present invention. The compositions of the present invention comprise rAAV and a pharmaceutically acceptable carrier. The compositions may also comprise other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to the recipient and are preferably inert at the dosage and concentration used, including buffers and surfactants such as pluronics.

In the method of this invention, the titer of the rAAV to be administered will vary depending on, for example, the specific rAAV, the route of administration, the therapeutic target, the individual, and one or more targeted cell types, and can be determined by methods standard in the art. The rAAV titer range can be from about 1× ^10⁶ , about 1× ^10⁷ , about 1× ^10⁸ , about 1× ^10⁹ , about 1× ^10¹⁰ , about 1× ^10¹¹ , about 1× ^10¹² , about 1× ^10¹³ to about 1× ^10¹⁴ or more DNase-resistant particles (DRP) per milliliter. The dosage can also be expressed in units of viral genome (vg).

This invention contemplates methods for transducing target cells with rAAV in vivo or in vitro. In vivo methods comprise the step of administering an effective dose or multiple effective doses of a composition comprising the rAAV of this invention to an animal (including a human) in need. If the dose is administered before the onset of a symptom/disease, the administration is prophylactic. If the dose is administered after the onset of a symptom/disease, the administration is therapeutic. In embodiments of this invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with a treatable symptom/disease state, slows or prevents progression to a symptom/disease state, slows or prevents the progression of a symptom/disease state, reduces the severity of the disease, causes remission of the disease (partial or complete), and/or prolongs survival. An example of a disease contemplated for prevention or treatment using the methods of this invention is DMD.

This invention also contemplates combination therapies. As used herein, combinations include both simultaneous and sequential treatment. Particular consideration is given to combinations of the methods of this invention with standard medical treatments (e.g., corticosteroids), as well as with novel therapies.

The composition can be administered at effective doses via standard routes in the art, including but not limited to intramuscular, parenteral, intravenous, oral, oral, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal routes. Considering the infection and/or ongoing disease state and one or more target cells/tissues expressing micromuscular dystrophy proteins, those skilled in the art can select and/or match one or more routes of administration and one or more serotypes of the AAV components (particularly the AAV ITR and capsid protein) of the rAAV of the present invention.

This invention provides effective doses of rAAV and topical and systemic administration of the compositions of this invention. For example, systemic administration involves delivery to the circulatory system, thereby affecting the entire body. Systemic administration includes enteral administration, such as through absorption via the gastrointestinal tract, and parenteral administration via injection, infusion, or implantation.

In particular, the actual administration of the rAAV of the present invention can be achieved by any physical method of delivering the recombinant rAAV vector to the target tissue of an animal. Administration according to the invention includes, but is not limited to, injection into muscle and injection into the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has proven sufficient to provide a vector that can be used for expression in muscle tissue, and there are no known limitations on the vectors or other components that can be co-administered with rAAV (although DNA-degrading compositions should be avoided in the normal manner when using rAAV). The capsid protein of rAAV can be modified such that rAAV targets 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 formulated as injectable or topical formulations that can be delivered to muscle via transdermal transport. Many formulations for both intramuscular injection and transdermal transport have been previously developed and are available for use in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and treatment.

In the methods disclosed herein, the dose of rAAV to be administered will vary depending on, for example, the specific rAAV, the route of administration, the therapeutic target, the individual, and one or more targeted cell types, and can be determined using methods standard in the art. The titer of each rAAV administered may range from about 1 × ^10⁶ , about 1 × ^10⁷ , about 1 × ^10⁸ , about 1 × ^10⁹ , about 1 × ^10¹⁰ , about 1 × ^10¹¹ , about 1 × ^10¹² , about 1 × ^10¹³ , about 1 × ^10¹⁴ , or about 1 × ^10¹⁵ or more of DNase resistance particles (DRP) per milliliter. Dosage can also be expressed in units of viral genome (vg) (i.e., 1× ^10⁷ vg, 1× ^10⁸ vg, 1× ^10⁹ vg, 1× ^10¹⁰ vg, 1×10¹¹ vg, 1× ^10¹² vg, 1× ^10¹³ vg, 1× ^10¹⁴ vg, 1× ^{10¹⁵ vg} , respectively). Dosage can also be expressed in units of viral genome (vg) per kilogram (kg) of body weight (i.e., 1× ^10¹⁰ vg/kg, 1× ^10¹¹ vg/kg, 1×10¹² vg/kg, 1× ^10¹³ vg/kg, 1× ^10¹⁴ vg/kg, 1× ^{10¹⁵ vg} /kg, respectively). The method for titrating AAV is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

In particular, the actual administration of the rAAV of the present invention can be achieved by any physical method of delivering the recombinant rAAV vector to the target tissue of an animal. Administration according to the invention includes, but is not limited to, injection into muscle and injection into the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has proven sufficient to provide a vector that can be used for expression in muscle tissue, and there are no known limitations on the vectors or other components that can be co-administered with rAAV (although DNA-degrading compositions should be avoided in the normal manner when using rAAV). The capsid protein of rAAV can be modified such that rAAV targets 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 formulated as injectable or topical formulations that can be delivered to muscle via transdermal transport. Many formulations for both intramuscular injection and transdermal transport have been previously developed and are available for use in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and treatment.

For intramuscular injection, adjuvants such as sesame oil or peanut oil or aqueous propylene glycol, as well as sterile aqueous solutions, can be used. If desired, such aqueous solutions can be buffered, and the liquid diluent should first be isotonic with saline or glucose. rAAV solutions as free acids (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared in water appropriately mixed with surfactants such as hydroxypropyl cellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oils. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, the sterile aqueous media used are readily available using standard techniques well known to those skilled in the art.

Suitable drug carriers, diluents, or excipients for injectable use include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and its flowability must be sufficient to allow for smooth injection. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, suitable flowability can be maintained by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, will be preferred. Extended absorption of the injectable composition can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

As needed, a sterile injectable solution is prepared by incorporating rAAV in the desired amount into a suitable solvent containing the various other components listed above, followed by filtration sterilization. Typically, a dispersion is prepared by incorporating the sterilized active ingredient into a sterile carrier containing a base dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient plus any additional desired components from their previously sterile filtered solution.

rAAV transduction can also be performed in vitro. In one embodiment, desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or allogeneic muscle cells can be used, where these cells will not elicit an inappropriate immune response in the subject.

Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells (e.g., in a suitable culture medium) and those cells with DNA of interest can be screened using conventional techniques such as Southern blotting and/or PCR, or by using selection markers. The transduced cells can then be formulated into a pharmaceutical composition and the composition can be introduced into a subject using various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and myocardium using, for example, a catheter.

Transduction of cells with rAAV of the present invention results in sustained expression of microdystrophy protein. Therefore, the present invention provides methods for administering/delivering rAAV expressing microdystrophy to animals, preferably humans. These methods include transduction of tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of the present invention. Transduction can be performed using a gene cassette containing tissue-specific control elements. For example, one embodiment of the present invention provides a method for transducing muscle cells and muscle tissue guided by muscle-specific control elements, said control elements including, but not limited to, those derived from actin and myosin gene families, such as control elements derived from the myoD gene family (see Weintraub et al., Science, 251:761-766 (1991)); muscle cell-specific enhancer-binding factor MEF-2 (Cserjesi and Olson, Molecular Cell Biology 11:4854-4862 (1991)); control elements derived from human skeletal muscle actin gene (Muscat et al., Molecular Cell Biology 7:4089-4099 (1987)) and cardiac actin gene; muscle creatine kinase sequence elements. Elements (see Johnson et al., Molecular Cell Biology, 9:3393-3399 (1989)) and mouse creatine kinase enhancer (mCK) elements; control elements derived from the skeletal fast twitching troponin C gene, slow twitching cardiac troponin C gene, and slow twitching troponin I gene: hypoxia-induced nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)); steroid-induced elements and promoters including glucocorticoid response elements (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)) and other control elements.

Muscle tissue is an attractive target for DNA delivery in vivo because it is not a vital organ and is easily accessible. This invention contemplates the sustained expression of microdystrophy proteins from transduced myofibrils.

"Muscle cells" or "muscle tissue" refers to cells or groups of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, such as tissue from the digestive tract, bladder, blood vessels, or heart). These muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardioblasts.

The term "transduction" is used to refer to the administration or delivery in vivo or in vitro of the coding region of micromyotrophic dystrophin to recipient cells via the replication-defective rAAV of the present invention, resulting in the expression of micromyotrophic dystrophin in the recipient cells.

Therefore, the present invention provides a method for administering an effective dose (or multiple doses administered substantially simultaneously or multiple doses administered at intervals) of rAAV encoding micromuscular dystrophy to a patient in need.

Example

Example 1

Generate pAAV.MHCK7 micromyodystrophy protein construct

The pAAV.MHCK7.micromyotrophic dystrophin plasmid contains a human micromyotrophic dystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 1). The micromyotrophic dystrophin construct is characterized by the deletion of in-frame rods (R4-R23), while hinges 1, 2, and 4 and cysteine-rich domains still produce a 138 kDa protein. Expression of micromyotrophic dystrophin (3579 bp) is initiated by the MHCK7 promoter (795 bp). Plasmids were constructed from the pAAV.MCK.micromyotrophic dystrophin plasmid by removing the MCK promoter and inserting the MHCK7 promoter. Following the core promoter, a 53 bp endogenous mouse MCK exon 1 (untranslated) is present for efficient transcription initiation, followed by the SV40 late 16S/19S splicing signal (97 bp) and a small 5'UTR (61 bp). The introns and 5'UTR are derived from plasmid pCMVβ (Clontech). The micromyotrophic cassette possesses a consensus Kozak immediately prior to ATG initiation, along with a small 53 bp synthetic polyA signal for mRNA termination. The human micromyotrophic cassette contains the (R4-R23/Δ71-78) domain as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)). Complementary DNA for human use was codon-optimized and synthesized by GenScript (Piscataway, NJ) (Mol Ther 18, 109-117 (2010)). The only viral sequence contained in this vector is the inverted terminal repeat of AAV2, which is essential for viral DNA replication and packaging. The micromyotrophic cassette possesses a small 53 bp synthetic polyA signal for mRNA termination.

Previous studies have confirmed the use of the MHCK7 promoter for myocardial expression (Salva et al., Mol Ther 15, 320-329 (2007)) and AAVrh74 for expression in skeletal, diaphragmatic, and myocardial regions (Sondergaard et al., Annals of Clinical and Transl Neurology 2, 256-270 (2015)). The sequence of the construct in Figure 1 was capsidred into the AAVrh.74 virion. Molecular clones of the AAVrh.74 serotype were cloned from rhesus macaque lymph nodes and described in Rodino-Klapac et al., Journal of Translational Medicine 5, 45 (2007).

Table 1 shows the molecular characteristics of plasmid pAAV.MHCK7.micro dystrophin (SEQ ID NO: 3).

Example 2

Intramuscular expression study using rAAV.MHCK7 microdystrophy protein.

Expression studies were conducted using a human micromyotrophic dystrophin construct (rAAVrh74.MHCK7. micromyotrophic dystrophin; described in Example 1) via intramuscular injection. The anterior tibialis muscle of mdx mice (spontaneous Dmd ^mdx mutant mice that do not express dystrophin) was injected with a cassette containing 1 × ^10¹¹ vg (n = 5 per group). Muscle was harvested after six weeks and stained with N-terminal antibodies against dystrophin and hematoxylin and eosin (HE) for dystrophin (Dys3) expression. Figure 2 shows the reduction and centrally localized diffuse gene expression in the nucleus compared to untreated muscle with the 1 × ^10¹¹ vg dose. Furthermore, a decrease in central nucleation and an increase in the mean fiber/box ratio were observed after treatment with the micromyotrophic dystrophin construct.

The expression level of the rAAVrh74.MHCK7 micromyotrophic dystrophy protein construct was approximately 73%.

In addition to measuring the localization and expression levels of microdystrophy proteins, skeletal muscle strength was measured after intramuscular injection. Intramuscular expression of the pAAV.MHCK7. microdystrophy construct resulted in significantly greater absolute and specific force production compared to the untreated control (maps 3A and 3B, respectively).

Example 3

rAAVrh.74.MHCK7 microdystrophy protein was systemically delivered to mdx mice.

Micromyotrophic dystrophin (MMD) was administered to MDX mice at 6 weeks of age via tail vein at doses of 2 x ^10¹² vg (8 x ^10¹³ vg/kg) or a high dose (planned clinical dose) of 6 x ^10¹² vg (2 x ^10¹⁴ vg/kg). After 12 weeks of treatment, all muscles were collected and stained for MMD and the recovery of DAPC components. Mice receiving systemic injection (tail vein) showed high levels of MMD staining in all muscles. Figure 4A shows extensive transduction of skeletal, diaphragmatic, and cardiac fibers after a systemic dose of 6 x ^10¹² vg (2 x ^10¹⁴ vg/kg). Figure 4B shows the quantification of the percentage of muscle fibers expressing MMD in each tissue. Finally, improvement in diaphragmatic function was tested (Figure 4C). No significant difference was observed at low doses; however, significant improvement was observed at high doses. Importantly, Figure 5 shows that the other components of DAPC were fully restored after MMD delivery. The protein shown is β-sarcosin (B-SG).

The toxicology/safety of AAVrh.74.MHCK7. micromuscular dystrophy protein was assessed by administering the carrier via intravenous (iv) injection into the tail vein of mdx mice according to Table 2. No evidence of toxicity was found in any of the muscle tissues analyzed, including: tibialis anterior (TA), gastrocnemius (GAS), quadriceps femoris (QD), psoas major (PSO), triceps brachii (TRI), and diaphragm (DIA) (Figs. 6A and 6B). With high doses of 6 × ^10¹² vg (2 × ^10¹⁴ vg/kg), the number of centrally placed nuclei decreased. Historically, central nucleation in skeletal muscle of untreated age-matched mdx mice averaged ~80%. Finally, preliminary data from a small sample size (n = 3) indicated a decrease in serum CK release levels (U/L) in mice (D) treated with high doses. Independent t-tests were used to locate differences (p <.05); data are reported as mean ± SEM.

Table 2. Overview of toxicological/safety studies of rAAVrh.74.MHCK7. micromuscular dystrophy protein in mice.

Example 4

Generation of pAAV.MCK. Micromuscular Dystrophy Protein Construct

The pAAV.MCK.micromyotrophic protein cassette was constructed by inserting the MCK expression cassette of the driver codon-optimized human micromyotrophic dystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski et al., J.Virol. 61(10):3096-3101). A muscle-specific regulatory element was included in the construct to drive muscle-specific gene expression. This regulatory element contained a mouse MCK core enhancer (206 bp) fused to a 351 bp MCK core promoter (proximal). Following the core promoter, the construct contained a 53 bp endogenous mouse MCK exon 1 (untranslated) for efficient transcription initiation, followed by an SV40 late 16S/19S splicing signal (97 bp) and a small 5'UTR (61 bp). The introns and 5'UTR were derived from plasmid pCMVβ (Clontech). The micromyotrophic cassette contained a consensus Kozak immediately prior to ATG initiation, along with a small 53 bp synthetic polyA signal for mRNA termination. The human micromuscular dystrophy cassette contains the (R4-R23/Δ71-78) domains as previously described by Harper et al., Nat. Med. 8(3):253-61, 2002.

pAAV.MCK. micromuscular dystrophy protein particles contain a human micromuscular dystrophy protein cDNA expression cassette flanked by AAV2 inverted terminal repeat (ITR) sequences (see Figure 7). This sequence was capsidred into AAVrh.74 viral particles. Molecular clones of the AAVrh.74 serotype were cloned from rhesus macaque lymph nodes and described in Rodino-Klapac et al., Journal of Tran. Med. 45 (2007).

Example 5

Potency and dosage analysis using rAAV.MCK microdystrophy protein

Expression studies were conducted using a human micromyotrophic dystrophin construct (rAAV.MCK. micromyotrophic dystrophin; described in Example 1) via intramuscular injection. MDX mice (spontaneous Dmd ^mdx mutant mice that do not express dystrophin) were injected intramuscularly with 3 × ^10⁹ , 3 × ^10¹⁰ , or 1 × ^10¹¹ vg (n = 3 per group). After four weeks, muscle was harvested and stained for dystrophin expression using antibodies specific to N-terminal Dys3 and hematoxylin and eosin (HE). Figure 8 shows the linear correlation between expression and dose, where expression was very low (no effect level) at 3 × ^10⁹ vg and 89% at 1 × ^10¹¹ vg.

Example 6

Vascular delivery of rAAV.MCK microdystrophy protein to MDX mice

Using an isolated limb perfusion model (Rodino-Klapac et al., J. Trans. Med. 5(45): 1-11, 2007), mdx mice (n = 10) were injected with 1 × ^10¹¹ vg of rAAVrh.74.MCK. micromuscular dystrophy protein via the femoral artery, and the results were analyzed. Three months after gene transfer, lower limb muscles were harvested, and efficacy studies showed significant improvements in both force and resistance to eccentric contraction-induced damage (Figure 9).

Immunostaining of rAAVrh.74-MCK-micro dystrophin in extensor digitorum longus (EDL) and TA muscles after treatment showed expression of dystrophin in MDX muscle fibers (Fig. 9A). Muscles with simulated infection were stained in the same manner, and exposure was time-matched. Fig. 9B demonstrates that rAAVrh.74-MCK-micro dystrophin significantly increased normalized specific force compared to treated MDX muscle (contralateral MDX, P < 0.05). Furthermore, the decrease in force during repetitive eccentric contractions was compared between mDX muscle infected with rAAVrh.74-MCK-micro dystrophin (human) and contralateral MDX EDL muscle with simulated infection (blue) and wild-type (WTC57Bl/10) EDL muscle at 12 weeks post-gene transfer (Fig. 9C). Compared with the mdx muscle treated with simulated therapy, rAAVrh.74-MCK-micro dystrophin (Micro-dys) treatment significantly prevented the loss of force (compared to mdx, P<0.001).

Example 7

Primate research

To apply preclinical findings in mice to a clinical paradigm, systemic administration was performed to non-human primates (NHPs) to evaluate the safety and efficacy of future clinical trials. The effects of a total dose of 2 × ^10¹⁴ vg of AAVrh74.MHCK7.micromuscular dystrophy protein.FLAG, delivered intravenously via the cephalic vein, were investigated in NHPs. This dose (based on animal weight) was proportional to the systemic dose administered to mice and corresponded to the intermediate dose (6.0 × ^10¹² vg total dose) administered to mice.

Baseline chemical and immunological studies were performed, including enzyme-linked immunosorbent assay (ELISpot) analysis, to measure T cell activity against AAVrh.74 capsid and micromyotrophic dystrophin, as well as anti-AAV antibody titers. Three peptide libraries were used for AAVrh.74 capsid protein (Genemed Synthesis, San Antonio, TX), each containing 34–36 peptides, 18 amino acids long with 11 overlapping residues. Four peptide libraries included micromyotrophic dystrophin-FLAG protein (Genemed Synthesis), each 18 amino acids long with 11 overlapping residues. Concanavalin A (ConA) (Sigma, 1 μg/mL) was used as a positive control, and 0.25% dimethyl sulfoxide (DMSO) was used as a negative control. These studies were repeated every two weeks throughout the study. Animals were euthanized three months post-treatment for complete histological necropsy. Immunological assays showed no unexpected response of ELISpot to the capsid or transgene (Fig. 12A), and no unexpected antibody response to the AAVrh74 capsid was observed by ELISA (Fig. 12B).

In addition, a complete blood count and chemobiopsy showed a slight elevation in liver enzymes, which were normalized to baseline without intervention or treatment, as shown in Table 3 below.

Table 3

No other unexpected chemical values were observed throughout the study. Finally, a comprehensive analysis of all skeletal muscles, using immunofluorescence staining with a FLAG-specific antibody and Western blot analysis with a mouse monoclonal antibody against dystrophin, showed widespread expression in muscle fibers (Figure 14).

Together, the data demonstrate that systemic delivery of AAVrh74.MHCK7.micromalnutrition protein.FLAG establishes safety and efficacy, and that it is widely expressed in all skeletal muscles of non-human primates.

Example 8

Preclinical studies demonstrating efficacy

Preclinical studies were conducted to demonstrate the efficacy of systemic delivery of rAAVrh74.MHCK7. micromyotrophic dystrophin in treating skeletal and cardiac defects in MDX mice. As described in Example 1, an AAVrh74 vector containing a codon-optimized human micromyotrophic dystrophin transgene driven by the muscle- and heart-specific promoter MHCK7 was used in this study.

Systemic injection via tail vein in MDX (dystrophin ineffective) mice

rAAVrh74.MHCK7. Micromuscular dystrophy protein was used in a dose-response study. The results of this study indicate that systemic injection in MDX mice effectively normalized histological and functional outcomes measured in the limbs and diaphragm in a dose-dependent manner. Furthermore, no significant carrier-related toxicity was reported following formal histopathological examination by a board-certified veterinary pathologist.

The vector used in this study was generated under research-grade conditions using a triple transfection method with HEK293 cells at Nationwide Children’s Hospital Viral Vector Core. Characterization of the generated vector included titer determination using supercoiled standards via qPCR, endotoxin level determination (EU/mL), and sterility assessment. SDS-PAGE analysis of the generated vector was used to verify consistency with the expected rAAV banding pattern. The vector was generated using a plasmid containing a micromyotrophic dystrophin construct, a muscle-specific MHCK7 promoter driving expression, a shared Kozak sequence (CCACC), an SV40 chimeric intron, and a synthetic polyadenylation site (53 bp) (Figure 1). The micromyotrophic dystrophin expression cassette was cloned between AAV2 ITRs packaged in the AAVrh74 vector to enhance transduction in bone and heart tissues.

The potency of the rAAVrh74.MHCK7 micromuscular dystrophy protein assay was determined by intramuscular injection of the carrier into MDX mice. Wild-type mice were used as a positive control, and sterile lactated Ringer's solution was injected into MDX mice as a negative control.

Table 4: Overview of the study design for rAAVrh74.MHCK7. Micromuscular dystrophy protein

The animals shown in Table 4 were administered the drug at the specified age (4–5 weeks), with tail vein injection for systemic delivery. For accurate administration via intramuscular injection, the animals were briefly anesthetized by inhalation of isoflurane. The dose was administered by direct injection into the tibialis anterior muscle of the lower limb. For accurate systemic administration, anesthesia is not required. The dose was administered via the vasculature through the tail vein. Care was taken to ensure accurate deposition of the entire carrier dose into the bloodstream. After administration, the animals were placed on a heated pad until spontaneous movement resumed, then returned to their cages. Each animal was observed weekly throughout the study period.

Mice were overdosed on a mixture of ketamine/toluidine (200 mg/kg/20 mg/kg) at the appropriate ages listed in Table 4. Blood was collected by cardiac puncture, and whole blood was sent for complete blood count (CBC) analysis. Serum was stored at -80°C until analysis of serum chemistry was performed at the Charles Rivers Laboratory. Tissue was then collected and sent for independent veterinary histopathology and internal analysis.

Intramuscular delivery of rAAVrh74.MHCK7 microdystrophy protein to dystrophy-deficient mice at a total dose of 1 x ^10¹¹ vg resulted in approximately 70% dystrophy protein expression in the injected TA muscle. Immunofluorescence imaging of the vector-treated mice confirmed the expression of the microdystrophy protein gene.

rAAVrh74.MHCK7. Recovery of dystrophin expression after systemic treatment with micromyotrophic dystrophin

The efficacy assay of the rAAVrh74.MHCK7. micromuscular dystrophy protein test item was performed by systemic injection in mdx mice (genotype: C57BL/10ScSn-Dmd ^mdx /J) at dose escalations of low, medium, and high (2.0 x ^10¹² vg total dose; 6.0 x ^10¹² vg total dose; 1.2 x ^10¹³ vg total dose) to assess transgenic expression and efficacy of the vector at systemic delivery at 3 and 6 months post-injection. Mice were injected at 4–5 weeks of age, and complete necropsy was performed at 3 and 6 months post-injection. Based on the mean animal weight per group, these doses were equivalent to: 8 × ^10¹³ vg/kg, 2 × ^10¹⁴ vg/kg, and 6 × ^10¹⁴ vg/kg. An equal volume of lactated Ringer's solution was injected as a negative control. An equal volume of lactated Ringer's solution was injected into C57BL/6 mice as a positive control. Safety was determined by systemic injection in WT mice at a total dose of 6.0 × ^10¹² vg (referred to as the WT TX-medium dose group). Immunofluorescence staining was performed on skeletal muscles including the tibialis anterior (TA), gastrocnemius (GAS), quadriceps femoris (QUAD), gluteal muscles (GLUT), psoas, triceps brachii (TRI), diaphragm (DIA), and heart to determine the recovery of dystrophin and to ensure the efficacy of the viral vector for rAAVrh74.MHCK7 microdystrophin.

Skeletal muscle (TA, QUAD, GLUT, TRI), as well as the heart and diaphragm, were extracted for analysis. Organs were also removed for toxicological and biodistribution studies. Microdystrophy protein transgene expression remained high 3–6 months after treatment. This was accompanied by improved muscle tissue pathology and function, with no adverse effects on off-target organs.

Reversal of dystrophin in rAAVrh74.MHCK7.mdx mice treated systemically with micromyotrophic dystrophin Phenotype

Hematoxylin and eosin (H&E) staining of skeletal muscle, diaphragm, and heart was performed to determine the reversal and improvement of dystrophin pathology following systemic injection of rAAVrh74.MHCK7 micromyotrophic dystrophin at a total dose of 2 x ^10¹² vg (low dose; n = 1), 6 x ^10¹² vg (medium dose; n = 8), and 1.2 x ^10¹³ vg (high dose; n = 8), with euthanasia performed 12 weeks post-injection for each dose. At 24 weeks post-injection, the reversal and improvement of dystrophin pathology in a second group of animals treated with the medium dose (6 x ^10¹² vg total dose) were assessed (n = 5).

Immunofluorescence staining of human micromyotrophic dystrophin was used to determine the expression of the micromyotrophic transgene in six skeletal muscles (TA, GAS, QUAD, GLUT, psoas, TRI) as well as the left and right sides of the diaphragm and heart in all dystrophin-deficient mice injected with the micromyotrophic dystrophin vector. This was done to determine the recovery of dystrophin and to ensure the efficacy of the rAAVrh74.MHCK7.micromyotrophic dystrophin viral vector at total doses of 2 × ^10¹² vg (low dose; n = 2), 6 × ^10¹² vg (medium dose; n = 8), and 1.2 × ^10¹³ vg (high dose; n = 8), with each dose administered euthanized 12 weeks post-injection.

To assess expression and transduction efficiency, quantification was performed using images from all three dose groups and from the left and right sides of each muscle. Four 20X images were taken for each muscle, and the percentage of microdystrophin-positive fibers in each image was determined to obtain the mean transduction percentage for each muscle. Figures 14 and 15 present representative images of mice treated with intermediate doses ( ⁶ × ^10¹² vg; 2 × 10¹⁴ vg/kg) and high doses (1.2 × ^10¹³ vg; 6 × ^10¹⁴ vg/kg). Age-matched dystrophin-deficient mice injected with lactated Ringer's solution served as negative controls, and wild-type mice injected with lactated Ringer's solution served as positive controls. In all animals analyzed, the heart showed ≥75%.

Muscles from untreated animals exhibited extensive myopathy, including focal areas of fatty infiltration, centronucleation, fibrosis, and necrosis. H&E staining in Figures 16 and 17 shows this dystrophic phenotype in dystrophin-deficient mice compared to normal WT mice, and the improvement in muscle pathology following treatment with medium-dose (6 × ^10¹² vg; 2 × ^10¹⁴ vg/kg) or high-dose (1.2 × ^10¹³ vg; 6 × ^10¹⁴ vg/kg) treatment. Quantification of histological parameters showed a reduction in centronucleation (Figure 18) and normalization of mean fiber diameter in all muscles of treated mice in a dose-dependent manner (Figures 16 and 17). Sirius Red staining compared to the untreated (mdx LR) cohort showed reduced collagen deposition in the diaphragm in both medium- and high-dose groups (Figure 19).

Functional assessment of systemic treatment using rAAVrh74.MHCK7 microdystrophy protein

To determine whether micromyotrophic dystrophin gene transfer provides a functional strength benefit to diseased muscles, the functional properties of the diaphragm and anterior tibialis muscle were evaluated at three dose levels in MDX mice, WT mice, and vector-treated mice. Dose escalation included low (8 × ^10¹³ vg/kg), medium (2 × ^10¹⁴ vg/kg), and high (6 × ^10¹⁴ vg/kg). In animals receiving a total dose of 6 × ^10¹² vg (medium dose) of rAAVrh74.MHCK7. micromyotrophic dystrophin systemically, functional assessments of systemic treatment with rAAVrh74.MHCK7. micromyotrophic dystrophin were performed 24 weeks post-injection using in vitro and in vivo assessments of specific force and reduction in force output following eccentric contraction in the TA. Additionally, specific force output in the diaphragm was assessed in the same animals.

As outlined in the previous figures, histopathology showed a more normalized environment, with improved central nucleation, collagen deposition, and fiber size at both medium and high doses. Tail vein delivery of rAAVrh74.MHCK7. micromuscular dystrophy protein resulted in a progressively improved diaphragmatic force output (176.9 mN/ ^mm² in the medium-dose group vs. 227.78 mN/ ^mm² in the high-dose group). Furthermore, the long-term treatment group, representing mice 6 months post-injection (medium dose 2 × ^10¹⁴ vg/kg), showed no deviation in long-term diaphragmatic force output (176.9 mN/ ^mm² vs. 194.9 mN/ ^mm² ) (Figure 20).

Furthermore, functional deficits in the tibialis anterior muscle were observed in MDX mice compared to WT mice. MDX mice showed a 50% reduction in force output (171.3 mN/ ^mm² vs. 291.65 mN/ ^mm² ) and greater force loss after eccentric contraction compared to WT mice (32% loss in MDX; 5% loss in WT). Systemic delivery of a medium dose of rAAVrh74.MHCK7. microdystrophin resulted in a 65.5% recovery of dystrophin and specific force output in the tibialis anterior muscle, improving to 235.4 mN/mm², and protecting the muscle from repetitive eccentric contraction injury with only a 25% reduction in force (Figure 21). The medium-dose WT group represents the wild-type treatment group to demonstrate the absence of toxicity after carrier treatment and to maintain functional outcome measurements.

Summarize

After initial demonstration of bioefficacy via intramuscular injection, similar or increased recovery of microdystrophy proteins was achieved through simultaneous transduction of skeletal muscle, diaphragm, and heart via intravascular delivery. This efficacy demonstrated a dose-dependent reversal of dystrophy characteristics by reducing inflammation, decreasing degenerated fibers, and improving functional recovery by preventing eccentric contractions in the tibialis anterior and diaphragm. Functional benefits of the carrier included progressively improved whey generation in the diaphragm and TA to wild-type levels.

Example 9

Toxicology and biodistribution of rAAVrh74.MHCK7. Micromuscular dystrophy protein for systemic treatment

Organs and tissues from MDX mice that had been systemically injected with rAAVrh74.MHCK7 micromyotrophic dystrophin were collected for real-time quantitative PCR to detect specific sequences of the vector DNA. Proteins extracted from all collected organs and tissues were run on Western blots to detect micromyotrophic dystrophin in off-target organs.

The test substance was administered intravenously at three dose levels: low (2 × ^10¹² vg; 8 × ^10¹³ vg/kg), medium (6 × ^10¹² vg; 8 × ^10¹⁴ vg/kg), and high (1.2 × ^10¹³ vg; 6 × ^10¹⁴ vg/kg) at 4–5 weeks of age. To assess the safety of the vector, frozen sections of muscle tissue and all major organs harvested from the same group of mice previously described were subjected to H&E staining. Organs and muscles from C57BL6 WT mice, which had been systemically treated with the vector at a medium dose, were also included. Lactated Ringer's solution-treated MDX and WT mice were also included for histopathological analysis. These portions were formally reviewed for toxicity by a third-party, board-certified veterinary pathologist, and no adverse effects were detected in any samples from any mice; the results are summarized below.

The group details and research design are shown in Figure 4 below.

Table 5: Study Design for Safety of rAAVrh74.MHCK7 Micromuscular Dystrophin

Histopathological studies of vector-transduced tissues

Intravenous injection of rAAVrh74.MHCK7 micromyotrophic dystrophin did not induce any microscopic changes in the myofibrils of any skeletal muscle examined. Furthermore, no treatment-related lesions were observed in any tissues under histological evaluation. Any changes observed in both treated and control mice were considered incidental findings. In summary, these data indicate that the test substance was well tolerated by the test subjects. Moreover, administration of rAAVrh74.MHCK7 micromyotrophic dystrophin reduced myofibril atrophy in treated MDX mice compared to a reference sample from age-matched untreated MDX mice, thus suggesting that the test substance may improve the degree of myopathy associated with MDX deficiency.

In addition to examining diseased MDX mice treated systemically with the carrier, rAAVrh74.MHCK7 micromyotrophic dystrophin was also systemically delivered to five C57BL/6WT mice at the same minimum effective dose (MED) established in the aforementioned MDX mouse studies: a total dose of 6 x ^10¹² vg (2 x ^10¹⁴ vg/kg). This allowed for intravenous delivery of the test substance in healthy WT mice to more definitively determine whether any side effects were solely due to treatment. Various skeletal muscles, including the diaphragm, heart, and five other organs, were again collected here, and H&E sections of each tissue were formally reviewed by an independent veterinary pathologist.

Vector genome biodistribution

The presence of testant-specific DNA sequences was examined using real-time quantitative PCR (qPCR). Biodistribution analysis was performed on tissue samples collected from three mdx animals administered the vector at each dose level. A positive signal was any detected amount equal to or greater than 100 single-stranded DNA copies/μg of genomic DNA. Tissues were harvested at necropsy and a vector-specific primer-probe set specific to the MHCK7 promoter sequence was used. Figure 22 and Table 6 below depict the vector genomic copies detected in each tissue sample from mice injected with rAAVrh74.MHCK7. micromyotrophic dystrophin.

Table 6: Vector genome copy numbers in organs and muscles from three mdx mice administered the vector at each dose level. Values are shown in vg/μg genomic DNA.

Different levels of rAAVrh74.MHCK7. micromuscular dystrophy protein transcripts were detected in all collected tissues. As expected, the highest levels were observed in skeletal muscle and heart. The lowest levels were detected in gonads, lungs, kidneys, and spleen. These data demonstrate that the test substance was effectively delivered to all study tissues in the vector-administered mice.

As the qPCR results above show, intravenous delivery of rAAVrh74.MHCK7.micromuscular dystrophy resulted in varying levels of the vector transcript in most tissues, with the highest levels observed in the liver, heart, and quadriceps (medium dose) and the liver, heart, and gastrocnemius (high dose). Therefore, the aim of this part of the study was to determine protein expression of the human micromuscular dystrophy transgene in these tissues to ensure the function of the muscle-specific MHCK7 promoter. Western blotting was used to detect micromuscular dystrophy expression in tissue samples.

Protein expression and vector biodistribution were also assessed using qPCR and Western blotting (Figure 23), and these data indicated normal vector levels in ex vivo organs and minimal detection of micromyotrophic dystrophin in high-dose treated liver. These results correlated with the pathologist-determined lack of toxicity in the liver. Additionally, serum chemistry was analyzed by an independent CRO (Charles River Laboratories), indicating normal values for all analyzed chemicals. Three abnormal values were found in the liver enzyme AST, two confirmed in the mdx-LR group and one in the medium-dose group (Figure 23). A subset of animals underwent creatine kinase (CK) analysis; however, samples were analyzed before and after physiological evaluation. Serum analysis confirmed the lack of toxicity after test substance delivery.

Varying levels of microdystrophy protein expression were observed in all skeletal muscle and cardiac samples (Figure 24). However, the protein was detected at the lowest levels in the high-dose group in the liver. This is considered a benign result, and its presence in the liver is likely due to expression in hepatic smooth muscle. Importantly, no adverse histopathological effects were reported in the liver by independent pathologists.

Summarize

Histopathological examination concluded that the mdx-LR group exhibited extensive myopathy, affecting all seven skeletal muscles assessed, as well as the right ventricular wall of the heart. Key findings of histopathological evaluation included marked and widespread myofiber atrophy (30–75% of normal myofiber size), mild to moderate mononuclear cell inflammation, increased interstitial space, and increased cytoplasmic mineral deposition. The diaphragm showed the most significant changes in mononuclear cell infiltration and myofiber atrophy. The heart exhibited small lesions with minimal mononuclear cell accumulation in the ventricular myocardium. The carrier-administered cohort showed a significant reduction in myopathy in all skeletal tissues and the heart. The reductions observed in histopathology were dose-dependent, with the high-dose group showing significantly less degeneration and inflammation. Carrier treatment with rAAVrh74.MHCK7. micromyodystrophy protein did not have the side effects recorded in the WT-treated cohort and the carrier-administered mdx cohort. Incidental findings of mild vacuolation of hepatocyte cytoplasm were observed in the liver and lungs of both mdx and WT mice, regardless of treatment. Therefore, the test substance is safe, effective, and its protective effect is dose-dependent.

Claims (10)

1. A recombinant AAV vector containing nucleotide sequences of muscle-specific control elements and nucleotide sequences encoding micromuscular dystrophy proteins. 2. The recombinant AAV vector according to claim 1, wherein the nucleotide sequence encoding microdystrophy protein includes a) A nucleotide sequence that is at least 85% identical to the nucleotide sequence SEQ ID NO: 1 and encodes a functional micromuscular dystrophy protein, or b) The nucleotide sequence of SEQ ID NO: 1. 3. A recombinant AAV vector comprising the human micromuscular dystrophy protein nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 2. 4. A recombinant AAV vector containing the nucleotide sequence of pAAV.MHCK7.micromyopathy construct of SEQ ID NO: 3. 5. A composition comprising the recombinant AAV carrier of any one of claims 1-4 and a pharmaceutically acceptable carrier. 6. Use of the recombinant AAV carrier of any one of claims 1-4 or the composition of claim 5 in the preparation of a medicament for increasing muscle strength or muscle mass in subjects suffering from muscular dystrophy. 7. Use of the recombinant AAV carrier of any one of claims 1-4 or the composition of claim 5 in the preparation of a medicament for the treatment of muscular dystrophy. 8. Use of the recombinant AAV carrier of any one of claims 1-4 or the composition of claim 5 in the preparation of a medicament for reducing or preventing fibrosis in subjects suffering from muscular dystrophy. 9. The use of a recombinant AAV vector containing the human micromuscular dystrophy protein nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 in the preparation of a medicament for the treatment of muscular dystrophy. 10. Use of a recombinant AAV vector containing the nucleotide sequence of pAAV.MHCK7. micro dystrophin construct of SEQ ID NO: 3 for the treatment of muscular dystrophy.