EA042315B1 - DELIVERY OF MICRODYSTROFIN WITH A VECTOR BASED ON ADENO-ASSOCIATED VIRUS FOR THE TREATMENT OF MUSCULAR DYSTROPHY - Google Patents

Description

This application claims priority over U.S. Provisional Application No. 62/323163, filed April 15, 2016, and U.S. Provisional Application No. 62/473253, filed March 17, 2017, both of which are hereby incorporated by reference in their entirety.

Field of invention

The invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors expressing a miniaturized human microdystrophin gene, and methods of using these vectors to reduce and prevent fibrosis in subjects suffering from muscular dystrophy. According to the invention, combined methods of gene therapy are also proposed to protect muscle fibers from injury, increase muscle strength.

State of the art

The importance of muscle mass and strength in daily physical activities such as moving and breathing, as well as in the metabolism of the whole body, is undeniable. Impaired muscle function causes muscular dystrophies (MD), which are characterized by muscle weakness and wasting, and have a serious impact on quality of life. The most well-characterized MDs are the result of mutations in genes encoding members of the dystrophin-associated protein complex (DABP). These MDs are the result of weak membrane strength associated with loss of the sarcolemmal cytoskeletal attachment of DABA. Duchenne muscular dystrophy (DMD) is one of the most devastating muscular diseases, affecting 1 in 5,000 male births.

This application includes two translational approaches to developing treatments for DMD. In DMD, the fibrous infiltration is deep and a major barrier to any potential therapy. It is also important to consider that gene replacement itself is hampered by the severity of the fibrosis already present in very young children with DMD. In fact, muscle biopsies at the typical age of diagnosis, between 4-5 years, show very significant levels of fibrosis.

DMD is caused by mutations in the DMD gene, resulting in a decrease in the amount of mRNA and the absence of dystrophin, i.e. a 427 kD sarcolemmal protein associated with a dystrophin-associated protein complex (DABA) (Hoffman et al., Cell 51(6):919-28, 1987). DABA is composed of several proteins in the muscle sarcolemma that form a structural link between the extracellular matrix (ECM) and the cytoskeleton through dystrophies, an actin-binding protein, and alpha-dystroglycan, a lamin-binding protein. The function of these structural units is to stabilize the membranes of muscle cells during contraction and to protect against damage caused by contraction. With loss of dystrophin, membrane fragility leads to rupture of the sarcollem 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 breakdown and repair 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, endomysial inflammation, and fibrous scarring.

Without membrane stabilization by dystrophin or microdystrophin, DMD will manifest as uncontrolled cycles of tissue damage and repair, and eventually replace the lost muscle fibers with fibrous scar tissue through connective tissue proliferation. Fibrosis is characterized by excessive deposition of ECM matrix proteins, including collagen and elastin. ECM proteins are mainly produced by cytokines such as TGFe, which is released by activated fibroblasts that respond to stress and inflammation. Although the primary pathological feature of DMD is the destruction and necrosis of muscle fibers, fibrosis as a pathological consequence has the same effect. Excessive fibrous tissue production limits muscle recovery and contributes to progressive muscle weakness in the DMD patient. In one study, the presence of fibrosis in initial muscle biopsies with DMD was strongly correlated with poor motor outcome at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol, 2009. 68(7): p. 762-7). These results point to fibrosis as a major contributor to muscle dysfunction in DMD and highlight the need for the development of treatments that reduce the amount of fibrous tissue. Most of the antifibrotic therapies that have been tested in mice with X-linked muscular dystrophy (mdx) act by blocking fibrotic cytokine signaling through inhibition of the TGFe pathway. MicroRNAs (miRNAs) are single-stranded RNAs of about 22 nucleotides that mediate the repression of gene expression at the post-transcriptional level by base pairing within the 3'-UTR of the mRNA, inhibiting translation or promoting mRNA degradation. Key (seed) sequence of 7 bp. at the 5' end, the siRNA targets the siRNA; additional recognition is provided by the rest of the target sequence as well as its secondary structure. MiRNAs play an important role in the pathology of muscle diseases and have expression profiles that are uniquely dependent on the specific type of muscular dystrophy (Eisenberg et al. Proc Natl Acad Sci USA, 2007. 104(43): p. 17016-21). More evidence suggests that miRNAs are involved in the fibrotic process in many organs, including the heart, liver, kidneys, and lungs (Jiang et al., Proc Natl Acad Sci USA, 2007. 104(43): p.

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17016-21). Recently, downregulation of miR-29 has been shown to promote cardiac fibrosis (Cacchiarelli et al., Cell Metab, 2010. 12(4): p. 341-51), and downregulation of miR-29 has been genetically linked to the muscles of human patients. with DMD (Eisenberg et al. Proc Natl Acad Sci USA, 2007. 104(43): p. 17016-2). The miR-29 family consists of three family members expressed from two bicistronic miRNA clusters.

MiR-29a is co-expressed with miR-29b (miR-29b-1); miR-29c is co-expressed with a second copy of miR-29b (miR-29b-2). The miR-29 family shares a conserved key sequence, while miR-29a and miR-29b differ in only one base from miR-29c. In addition, electroporation of miR-29 plasmid (miR-29a and miR-29b-1 cluster) into mdx mouse muscle reduced expression levels of ECM components, collagen and elastin, and greatly reduced collagen deposition in muscle sections up to 25 days after treatment (Cacchiarelli et al., Cell Metab, 2010. 12(4): pp. 341-51).

Adeno-associated virus (AAV) is a replication-defective parvovirus whose single-stranded DNA genome is about 4.7 kb long, including 145 nucleotide inverted terminal repeats (ITRs). There are several serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983), and in corrected form in Ruffing et al., J Gen Virol, 15: 3385-3392 ( 1994). As other examples, the complete AAV-1 genome is provided under GenBank accession number NC_002077; the complete AAV-3 genome is available under GenBank accession number NC 1829; the complete genome of AAV-4 is available under GenBank accession number NC_001829; the AAV-5 genome is available under GenBank accession number AF085716; the complete AAV-6 genome is available under GenBank accession number NC_00 1862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank accession numbers AX753246 and AX753249, respectively (see also US Pat. Nos. 7,282,199 and 7,790,449 related to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 63816388 (2004); The AAV-10 genome is provided by Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The AAVrh74 serotype is described in Rodino-Klapac et al. J. Trans. Med. 5:45 (2007). The cis-functioning sequences controlling viral DNA replication (rep), encapsulation/packaging, and integration into the host cell chromosome are contained in the ITR. Three AAV promoters (labeled p5, p19, and p40 by their relative positions on the genomic map) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19) combined with differential splicing of one AAV intron (e.g., at AAV2 nucleotides 2107 and 2227) result in the production of four rep proteins (rep 78, rep 68, rep 52 and rep 40) from the rep gene. Rep proteins have several enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation sites are responsible for the production of three related capsid proteins. The only polyadenylation consensus site is at position 95 of 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 has unique features that make it attractive as a vector for the delivery of foreign DNA into cells, for example, in gene therapy. Infection of AAV cells in culture is non-cytopathic, while natural infection in humans and other animals is latent and asymptomatic. In addition, AAV infects many mammalian cells, which allows the use of various tissue types in vivo. In addition, AAV transduces slowly dividing and non-dividing cells, and can essentially persist as a transcriptionally active nuclear episome (extrachromosomal element) during the lifetime of these cells. The proviral AAV genome remains infectious when DNA is cloned into plasmids, which makes it possible to create recombinant genomes. In addition, since the signals governing AAV replication, genome encapsulation, and integration are contained in the ITR of the AAV genome, replacement of some or all of the approximately 4.3 kb content is possible. genome (coding for replication proteins and structural capsid proteins, rep-cap) to foreign DNA, such as a gene cassette containing a promoter, the desired DNA, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It readily withstands the conditions used to inactivate adenovirus (56 to 65°C for several hours), making cold storage of AAV less difficult. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

Numerous studies have demonstrated long-term (greater than 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). Moreover, since the muscle is highly vascularized, transduction with recombinant AAV results in the appearance of transgenic products in the circulatory system 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:

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13921-13926 (1997). In addition, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for proper glycosylation, coagulation, and antibody secretion, indicating that muscle is capable of sustained expression of secreted protein therapeutic agents.

Functional improvement in patients suffering from DMD and other muscular dystrophies requires both gene repair and reduction of fibrosis. There is a need for methods to reduce fibrosis that can be combined with gene repair methods to more effectively treat DMD and other muscular dystrophies. The miR29 molecule is a potential gene regulator and an ideal candidate for reducing muscle fibrosis.

The essence of the invention

The present invention relates to gene therapy methods that directly reduce the three major components of connective tissue (collagen 1, collagen 3 and fibronectin) by delivering miR29 microRNA. In this system, miR29 binds to the 3'UTR of the collagen and fibronectin gene in order to downregulate expression. SUBSTANCE: invention relates to gene therapy vectors, for example based on AAV, expressing miR29 miRNA guide strand, and method of delivering miR29 into muscle to reduce and/or prevent fibrosis.

In addition, the present invention provides combination therapies and approaches to repair and prevent fibrosis using gene therapy vectors delivering miR-29 to suppress fibrosis along with microdystrophin to correct the gene defect seen in DMD. As shown in Examples 5-7, the combined treatment results in greater reduction in fibrosis, increase in muscle size and increase in muscle strength.

In one embodiment, the invention provides an rAAV vector expressing miR-29. For example, the rAAV vector contains a polynucleotide sequence expressing miR29c, such as a nucleotide sequence containing the miR-29c target guide strand SEQ ID NO: 3, the miR-29c guide strand SEQ ID NO: 4, and the native miR scaffold and stem loop -30 (SEQ ID NO: 5). An exemplary polynucleotide sequence containing the miR-29c cDNA in the miR-30 framework is shown as SEQ ID NO: 2 (FIG. 1).

An exemplary rAAV according to the present invention is pAAV.CMV.Mir29C, which contains the nucleotide sequence of SEQ ID NO: 1; wherein the CMV promoter spans nucleotides 120-526, the EF1a intron spans nucleotides 927-1087 and nucleotides 1380-1854, the miR-29c guide strand spans nucleotides 1257-1284, and the primary key sequence shRNA-miR29-c spans nucleotides 1088-1375, and the poly-A sequence spans nucleotides 1896-2091. In one aspect of the invention, the rAAV vectors of the invention are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

Another illustrative rAAV according to this invention is pAAV.MHC.Mir29C, which contains the nucleotide sequence of SEQ ID NO: 12; moreover, the MCC enhancer spans nucleotides 190-395, the MHC promoter spans nucleotides 396-753, the EF1a intron spans nucleotides 1155-1315 and nucleotides 1609-2083, miR-29c guide strand spans nucleotides 1487-1512, and shRNA-miR29-c with primary key the sequence spans nucleotides 1316-1608, and the poly-A sequence spans nucleotides 2094-2146. In one aspect, the rAAV vectors of this invention are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

In another aspect of the invention, the rAAV vectors of the invention may be operably linked to a muscle-specific regulatory element. For example, a muscle-specific regulatory element is a human skeletal muscle actin gene element, a heart actin gene element, myocyte-specific enhancer binding factor MEF, muscle creatine kinase (MKK), tMKK (truncated MKK), myosin heavy chain (MHC), C5-12 (synthetic promoter), mouse creatine kinase enhancer element, fast twitch skeletal muscle fiber troponin C gene element, slow twitch cardiac muscle fiber troponin C gene element, slow twitch muscle fiber troponin I gene element, hypoxia-induced nuclear factors, steroid-inducible element or glucocorticoid response element (GRE).

For example, any of the rAAV vectors of the invention is operably linked to a muscle-specific regulatory element comprising the MKK enhancer nucleotide sequence of SEQ ID NO: 10 and/or the MKK promoter sequence of SEQ ID NO: 11.

The invention also provides pharmaceutical compositions (sometimes referred to herein simply as compositions) containing any of the rAAV vectors of the invention.

In another embodiment, according to this invention, methods are provided for obtaining an rAAV vector particle, comprising culturing a cell transfected with any rAAV vector according to this invention, and recovering rAAV particles from the supernatant of the transfected

- 3 042315 cells. The invention also provides viral particles containing any of the recombinant AAV vectors of the invention.

In another embodiment, the invention provides methods for reducing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of any rAAV vector of the invention that expresses miR-29. For example, any of the rAAVs of the present invention is administered to subjects suffering from muscular dystrophy to reduce fibrosis and in particular reduce fibrosis in the subject's skeletal muscle or cardiac muscle. These methods may further include the step of administering a microdystrophin-expressing rAAV.

Fibrosis refers to excessive or unregulated deposition of extracellular matrix (ECM) components, and abnormal repair processes in tissues after injury, including skeletal muscle, heart muscle, liver, lungs, kidneys, and pancreas. ECM components that precipitate include fibronectin and collagen, such as collagen 1, collagen 2, or collagen 3.

In another embodiment, the present invention provides methods for preventing fibrosis in a subject in need thereof comprising administering a therapeutically effective amount of any recombinant AAV vector of the present invention expressing miR-29. For example, any of the rAAVs of the invention are administered to subjects suffering from muscular dystrophy to prevent fibrosis, for example, miR-29 expressing rAAVs of the invention are administered before fibrosis is detected in the subject. In addition, an rAAV of the present invention expressing miR-29 is administered to a subject at risk of developing fibrosis, eg, suffering from muscular dystrophy or diagnosed with muscular dystrophy, eg DMD. The rAAV of the present invention is administered to a subject suffering from muscular dystrophy in order to prevent new fibrosis in these subjects. These methods may further include the step of administering a microdystrophin-expressing rAAV.

The invention also provides methods for increasing muscle strength and/or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of any of the miR-29 expressing rAAV vectors of the invention. These methods may further include the step of administering a microdystrophin-expressing rAAV.

The terms combination therapy and combination treatment refer to the administration of a miR-29 expressing rAAV vector according to the invention and a microdystrophin expressing rAAV vector.

In any of the methods of the invention, the subject may be suffering from a muscular dystrophy such as DMD, Becker muscular dystrophy, or any other dystrophin-related dystrophy. In addition, in any of the methods according to this invention, the subject may suffer from dystrophinopathy.

In another embodiment, the present invention provides recombinant AAV vectors containing a nucleotide sequence encoding a microdystrophin protein. The present invention provides an rAAV comprising: a) a nucleotide sequence having at least 85% identity with the nucleotide sequence of SEQ ID NO: 7 and encoding a functional microdystrophin protein, b) a nucleotide sequence of SEQ ID NO: 7, or c) a nucleotide sequence of SEQ ID NO:9.

An exemplary microdystrophin-expressing rAAV according to the present invention is pAAV.mck.-microdystrophin, which contains the nucleotide sequence of SEQ ID NO: 9, and is shown in Fig. 10 and 11. This rAAV vector contains the MCC promoter, a chimeric intron sequence encoding the microdystrophin gene sequence, polyA, ampicillin resistance, and a backbone of the pGEX plasmid with the pBR322 origin of replication. In one aspect of the invention, the recombinant AAV vectors of the invention are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

The present invention provides rAAV vectors encoding a microdystrophin protein that is, 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 common at least 90%, 91%, 92%, 93%, or 94%, and more common at least 95 %, 96%, 97%, 98% or 99% identical in sequence to SEQ ID NO: 8, and the protein retains microdystrophin activity. The microdystrophin protein provides strength to the muscle membrane during muscle contraction, for example, microdystrophin acts as a shock absorber during muscle contraction.

The present invention provides rAAV vectors expressing the microdystrophin protein containing a nucleotide sequence that has 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 more typically at least 95%, 96 %, 97%, 98% or 99% sequence identity with SEQ ID NO: 7,

- 4 042315 and encodes a functional protein microdystrophin.

The present invention provides microdystrophin-expressing rAAV vectors comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 7 or complementary variants thereof and encodes a functional microdystrophin protein.

The term stringent is used to refer to conditions that are commonly understood in the art as stringent. The stringency of hybridization is mainly determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent hybridization conditions and washes are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68°C, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42°C; see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide concentration, or other denaturing agent) may also be used, however the rate of hybridization will be affected. When it comes to hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing 6x SSC with 0.05% sodium pyrophosphate at 37°C (for 14-basic oligonucleotides), 48°C (for 17-basic oligonucleotides), 55°C (for 20-basic oligonucleotides) and 60°C (for 23-basic oligonucleotides).

Other agents may be included in hybridization and wash buffers to reduce non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA) and dextran sulfate, although other suitable agents may be used. The concentration and types of these additives can be changed without significantly affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, under typical ionic strength conditions, the hybridization rate is essentially independent of pH; see Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and allow DNA with varying degrees of sequence relatedness to form hybrids.

In another aspect, microdystrophin-expressing rAAV vectors comprise a microdystrophin gene coding sequence operably linked to a muscle-specific regulatory element. For example, a skeletal muscle specific regulatory element is human skeletal muscle actin gene element, heart actin gene element, myocyte-specific enhancer binding factor MEF, muscle creatine kinase (MKK), tMKK (truncated MKK), myosin heavy chain (MHC), C5 -12 (synthetic promoter), mouse creatine kinase enhancer element, fast twitch skeletal muscle fiber troponin C gene element, slow twitch cardiac muscle fiber troponin C gene element, slow twitch muscle fiber troponin gene element I, hypoxia-induced nuclear factors, steroid-inducible element or glucocorticoid -responding element (GRE).

In addition, according to this invention, rAAV vectors are provided expressing a microdystrophin containing a muscle-specific regulatory element containing the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

The invention also provides pharmaceutical compositions (or sometimes simply referred to herein as compositions) containing any of the rAAV vectors of the invention.

In another embodiment, the invention provides methods for producing an rAAV vector particle, comprising culturing a cell that has been transfected with any rAAV vector of the invention and recovering the rAAV particles from the supernatant of the transfected cells. The invention also provides viral particles containing any of the recombinant AAV vectors of the invention.

The invention also provides methods for producing a functional microdystrophin protein, comprising infecting a host cell with a recombinant AAV vector expressing microdystrophin of the invention and expressing the functional microdystrophin protein in the host cell.

In another embodiment, the present invention provides methods for reducing fibrosis in a subject in need thereof comprising administering a therapeutically effective amount of any microdystrophin expressing rAAV vector of the present invention. For example, any of the rAAVs of the present invention is administered to subjects suffering from muscular dystrophy or dystrophinopathy to reduce fibrosis, and in particular, reduce fibrosis in the subject's skeletal muscle or cardiac muscle.

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In another embodiment, the invention provides methods for preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of any recombinant AAV vector of the invention that expresses microdystrophin. For example, any of the rAAVs of the invention are administered to subjects suffering from muscular dystrophy or dystrophinopathy to prevent fibrosis, for example, a microdystrophin-expressing rAAV of the invention is administered before fibrosis is detected in the subject. In addition, a microdystrophin-expressing rAAV of the present invention is administered to a subject at risk of developing fibrosis, eg, suffering from muscular dystrophy or diagnosed with muscular dystrophy, eg, DMD or Becker muscular dystrophy. The rAAV of the present invention is administered to a subject suffering from dystrophinopathy or dystrophinopathic muscular dystrophy in order to prevent new fibrosis in these subjects.

The invention also provides methods for increasing muscle strength and/or muscle mass in a subject suffering from muscular dystrophy or dystrophinopathy, comprising administering a therapeutically effective amount of any of the miR-29 expressing rAAV vectors of the invention.

Any of the above methods, including the step of administering an rAAV expressing miR-29c according to the invention, may include the additional step of administering any of the rAAVs expressing the microdystrophin described herein. The terms combination therapy and combination treatment refer to the administration of the miR-29 expressing rAAV vector according to the invention and the microdystrophin expressing rAAV vector.

In the methods of administering miR-29 expressing rAAV vector and microdystrophin expressing rAAV vector, these rAAV vectors may be administered simultaneously or sequentially, wherein the miR29 expressing rAAV vector is administered immediately prior to the microdystrophin expressing rAAV, or administered sequentially, with the miR29-expressing rAAV vector administered immediately after the microdystrophin-expressing rAAV. Alternatively, the methods of this invention are carried out such that an AAV vector expressing the microdystrophin protein is administered within about 1-5 hours, or 5-12 hours, or 12-15 hours, or 15-24 hours after administration of miR-expressing rAAV. -29, or the methods of this invention are carried out such that an AAV vector expressing microdystrophin protein is administered for about 1-5 hours, or 5-12 hours, or 1215 hours, or 15-24 hours prior to the introduction of miR-expressing rAAV -29. Alternatively, the methods of this invention are carried out such that an AAV vector expressing microdystrophin protein is administered within about 1 or 6 or 12 or 24 hours after administration of a miR-29-expressing rAAV, or the methods of this invention are carried out as follows: that an AAV vector expressing microdystrophin protein is administered within about 1 or 6 or 12 or 24 hours prior to administration of miR-29 expressing rAAV.

The invention provides for the administration of any of the AAV vectors of the invention to patients diagnosed with dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy, before the subject is found to have fibrosis, or before the subject has had decreased muscle strength, or before how the subject had reduced muscle mass.

The invention also provides for administering any of the rAAVs of the invention to a subject suffering from a dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy, who has already developed fibrosis, to prevent new fibrosis in these subjects. The invention also contemplates administering any of the rAAVs of the invention to a patient suffering from muscular dystrophy who already has decreased muscle strength or reduced muscle mass to protect the muscle from further damage.

In any of the methods according to this invention, the rAAV vector is administered by intramuscular injection or intravenous injection.

In addition, in any of the methods according to this invention, the rAAV vector or composition is administered systemically. As examples, the rAAV vector or composition is administered parenterally by injection, infusion or implantation.

In another embodiment, the present invention provides a composition comprising any of the rAAV vectors expressing miR29 or any of the rAAV vectors expressing microdystrophin, or comprising both an rAAV vector expressing miR-29 and an rAAV vector expressing microdystrophin, to reduce fibrosis in a subject in need thereof. In addition, the present invention provides compositions comprising any of the recombinant miR29-expressing rAAV vectors or any of the microdystrophin-expressing rAAV vectors, or comprising both a miR-29-expressing rAAV vector and a microdystrophin-expressing rAAV vector. , to reduce fibrosis in a subject suffering from dystrophinopathy or muscular dystrophy such as DMD or Becker muscular dystrophy.

The present invention provides compositions comprising any of the rAAV vectors expressing miR29, or any of the rAAV vectors expressing the microdystrophin protein, or

- 6 042315 containing both an rAAV vector expressing miR-29 and a rAAV vector expressing microdystrophin protein to increase muscle strength and/or muscle mass in a subject suffering from dystrophinopathy or muscular dystrophy such as DMD or Becker muscular dystrophy .

In a further embodiment, the present invention provides compositions comprising any of the miR29-expressing rAAV vectors or any of the microdystrophin protein-expressing rAAV vectors, or comprising both a miR-29-expressing rAAV vector and a rAAV vector, expressing microdystrophin protein, for the treatment of dystrophinopathy or muscular dystrophy such as DMD or Becker muscular dystrophy.

Compositions according to this invention are formulated for intramuscular injection or intravenous injection. The composition according to this invention is also formulated for systemic administration, such as parenteral administration by injection, infusion or implantation. In addition, any of the compositions is formulated for administration to a subject suffering from a dystrophinopathy or muscular dystrophy such as DMD, Becker muscular dystrophy, or any other dystrophin associated dystrophy.

In a further embodiment, the present invention provides the use of any of the rAAV vectors of the present invention expressing miR29, or any of the rAAV vectors expressing microdystrophin, or comprising both a rAAV vector expressing miR-29 and a rAAV vector, expressing microdystrophin for the preparation of a medicament for reducing fibrosis in a subject in need thereof. For example, the patient is in need of treatment suffering from a dystrophinopathy or a muscular dystrophy such as DMD, Becker muscular dystrophy, or any other dystrophin-related dystrophy.

In a further embodiment, the present invention provides the use of any of the rAAV vectors of the present invention expressing miR29, or any of the rAAV vectors expressing microdystrophin, or comprising both a rAAV vector expressing miR-29 and a rAAV vector, expressing microdystrophin for the preparation of a medicament for the prevention of fibrosis in a subject suffering from muscular dystrophy. In a further embodiment, the present invention provides the use of recombinant AAV vectors of the present invention expressing miR29, or any of the rAAV vectors expressing microdystrophin, or comprising both a rAAV vector expressing miR-29 and a rAAV vector expressing microdystrophin, for the preparation of a medicament for increasing muscle strength and/or muscle mass in a subject suffering from a dystrophinopathy or muscular dystrophy such as DMD or Becker muscular dystrophy.

The invention provides for the use of any of the AAV vectors of the invention for the preparation of a medicament for administration to a patient diagnosed with DMD, before the subject is diagnosed with fibrosis, or before the subject has had reduced muscle strength, or before the subject has muscle mass was reduced.

The invention also contemplates the use of any of the AAV vectors of the invention for preparing an administration medicament to administer any of the rAAVs of the invention to a subject suffering from muscular dystrophy who has already developed fibrosis to prevent new fibrosis in those subjects. The invention also contemplates administering any of the rAAVs of the invention to a patient suffering from muscular dystrophy who already has decreased muscle strength or reduced muscle mass to protect the muscle from further damage.

The present invention provides the use of miR296-expressing rAAV vectors of the present invention, or any of the microdystrophin-expressing rAAV vectors, or comprising both a miR-29-expressing rAAV vector and a microdystrophin-expressing rAAV vector, for the preparation of a medicament. for the treatment of muscular dystrophy.

In any of the uses of this invention, the drug is formulated for intramuscular injection. In addition, any drug may be formulated for administration to a subject suffering from a muscular dystrophy such as DMD or any other dystrophin-associated muscular dystrophy.

In addition, any drug according to the present invention may be a combination therapy in which rAAV vectors expressing miR-29 and rAAV vectors expressing microdystrophin are administered simultaneously, or administered sequentially, and the rAAV vector expressing miR29 is administered immediately before rAAV expressing microdystrophin, or administered sequentially, with the rAAV vector expressing miR29 being administered immediately after the rAAV expressing microdystrophin. Alternatively, the drug comprises administering a microdystrophin-expressing AAV vector that is administered within about 1-5 hours after miR-29 expressing rAAV is administered, or the drug contains a microdystrophin-expressing AAV vector that is administered for about 1-5 hours. h before administration of rAAV expressing miR-29.

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Brief description of the drawings

In FIG. 1 shows a schematic of the rAAV vector scAAVCrh.74.CMV.miR29c and the nucleotide sequence of miR-29c in the native miR-30 framework, and the nucleotide sequence of the predicted hairpin structure.

In FIG. 2A-2C show that injection of miR-29c into muscle reduces collagen throughout the muscle and restores miR-29c expression.

In FIG. 3A-3C show that injection of miR-29c improves absolute muscle strength (panel A) and specific muscle strength (panel B), but does not protect against contraction-induced injury (panel C).

In FIG. 4A-4C show the number of microdystrophin-expressing muscle fibers to measure the efficiency of transgene delivery.

In FIG. 5A-5C show that co-delivery of miR-29c with microdystrophin reduces collagen expression (panel A) and fibrosis-induced dystrophin expression.

In FIG. 6A-6D show that intramuscular injection of miR-29c/microdystrophin inhibits extracellular matrix (ECM) in mdx/utrrT mice as measured by collagen 1 alpha (panel A), collagen 3 alpha (panel B), fibronectin (panel C) and TGF-β (panel D).

In FIG. 7A-7C show that intramuscular injection of miR-29c increased absolute force (panel A), normalized specific force (panel B), and provided protection against contraction-induced injury in the muscle (panel C).

In FIG. 8 shows that miR-29c/μ-dys combination increases muscle size in mice treated at 3 months of age. Shown are sections of treated and untreated mdx/utm ^+/ gastrocnemius muscles stained with picrosirius red for collagen staining. Fibrous areas are pink, while intact muscles are green. At a macroscopic level, the miR-29c/μ-dys combination reduces fibrosis and increases total cross-sectional area.

In FIG. 9A-F show that treatment with miR-29c co-delivered with microdystrophin increases muscle hypertrophy and hyperplasia, as shown by an increase in the total weight of the injected gastrocnemius muscle compared with the case when one was administered alone (panel A), an increase in the mean fibers (panel B), increase in muscle cross-sectional area (panel D; not injected: 24.6 vs. miR-29c: 26.3 vs. miR-29c: 26.6 vs. microdis/miR-29c: 33, 1) and an increase in the number of muscle fibers (panel E), but the number of muscle fibers per unit area was not affected (panel F). Panel C compares control mdx/utrn ^+/ with treated miR-29c/μ, -dys mdx/utrn ^+- , mean diameter increased from 25.96 to 30.97 µm.

In FIG. 10A-G show that early treatment with AAV.miR29c/microdystrophin combination therapy is more effective in reducing fibrosis and ECM expression. Panel A shows picrosirius red staining of mice: wild-type, uninjected, AAV.miR-29c, AAV.microdystrophin and AAV.miR-29c/AAV.microdystrophin, injected at 4-5 weeks of age, withdrawn from study twelve weeks post-injection . Panel B shows the quantification of picrosirius red staining, showing that the co-treated muscle had a 51.1% reduction in collagen compared to the uninjected gastrocnemius muscle. Panel C demonstrates that RT-PCR confirms an increase in miR-29c transcription levels in the treated groups. Semi-quantitative RT-PCR shows significant reductions in collagen I and III (panels d, e), fbn (panel f), and TGF-βΙ (panel g) in AAV.miR-29c/AAV.microdystrophin-treated muscle compared to the contralateral limb and each of the single therapies. Error bars, SOS (standard error of the mean) for n=5 (scAAVrh.74.CMV.miR-29c), n=5 (scAAVrh.74.CMV.siR-29c/ssAAVrh.74.MKC.microdystrophin), n= 6 (ssAAVrh. 74.MKC.microdystrophin), n=9 (mdx/utm ^+/ mice). One-way analysis of variance (*p<0.05, **p<0.01, ***p<0.001).

In FIG. 11 shows that early combination therapy restores strength and protects against damage caused by contraction. The magnitude of absolute (panel A) and normalized specific force (panel B) after tetanic contraction in all three treated injected gastrocnemius muscles was significantly increased compared to the untreated muscle mdx/utrn ^+/ (panel C). The muscles were then assessed for loss of strength after repeated eccentric contractions. Only mice co-treated with miR-29c/microdystrophin and microdystrophin alone showed protection against loss of strength compared to untreated mdx/utrn ^+/- muscles (blue). Two-tailed analysis of variance shows significance in the descending curves. Error bars, SOS for n=5 (rAAVrh.74.CMV.miR-29c), n=6 (rAAVrh.74.CMV.miR-29c/rAAVrh.74.MKC.micro-dystrophin), n=5 (rAAVrh .7 4.MCC.microdystrophin), n=15 (mdx/utrn ^+/ mice). One-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

In FIG. 12 shows that combined treatment with miR-29c/microdystrophin increases muscle size in mice treated at 1 month of age. Treated and untreated mdx/utrn ^+/ gastrocnemius muscles were sliced and stained with picrosirius red for collagen staining. Fibrous areas are pink, while intact muscles are green. At the macroscopic level, the miR combination

- 8 042315

29c/microdystrophin reduces fibrosis and increases total cross-sectional area.

In FIG. 13A-13G show that early treatment (weeks 4-5) with AAV.MKK.miR-29c/microdystrophin combination therapy is more effective in reducing fibrosis and ECM expression. Panel A shows picrosirius red staining of uninjected and AAV.MKK.miR-29c/AAV.MKK.microdystrophin injected mice at 4-5 weeks of age withdrawn from the study 12 weeks post-injection. Initial magnification, x20 Panel B shows a quantification of picrosirius red staining demonstrating that co-treated muscle has a 50.9% reduction in collagen compared to untreated gastrocnemius muscle. Panel C shows RT-PCR confirming an increase in miR-29c transcription levels in the treated group. Semi-quantitative RT-PCR demonstrates significant reductions in collagen 1A (CoIIA, panel D) and collagen 3A (Co13A, panel E), fibronectin (Fbn; panel F), and Tgfp 1 (panel G) in treated AAV.MKK.miR-29c/ AAV. microdystrophin in muscles, compared with treatments of contralateral limbs. (*p<0.05, ****p<0.0001).

In FIG. 14A-14G show that late treatment (treatment at week 12) with AAV.MKK.miR-29c/microdystrophin combination therapy is effective in reducing fibrosis and ECM expression. Panel A shows picrosirius red staining of untreated AAV.MKK.miR-29c and AAV.MKK.miR-29c/AAV.microdystrophin 12 weeks after injection. Original magnification, x20. Panel B shows the quantification of picrosirius red staining, demonstrating that the co-treated muscle had a 30.3% reduction in collagen compared to the untreated gastrocnemius muscle. Panel C shows RT-PCR confirming an increase in miR-29c transcript levels in the treated groups. Semi-quantitative RT-PCR demonstrated significant reductions in collagen 1A (Col1A, panel D), collagen 3A (Col3A, panel E), fibronectin (Fbn; panel F), and TGF01 (panel G) in treated AAV.miR-29c/AAV.microdystrophin muscles compared to the opposite limb. One-way analysis of variance. All data are mean ± SOS (**p<0.01, ****p<0.0001).

In FIG. 15A-15C show that early combination therapy (treatment at weeks 4-5) restores strength and protects against damage caused by contraction. The magnitude of the absolute (panel A) and normalized specific force (panel B) after tetanic contraction of the MKK.miR29c/microdystrophin-treated gastrocnemius muscles was significantly increased compared to the untreated mdx/utm ^+/ muscle (panel C). (C) Muscles were then assessed for loss of strength after repeated eccentric contractions. Mice co-treated with miR-29c/microdystrophin and microdystrophin alone showed protection against loss of strength compared to untreated mdx/utrn ^{+ −} muscles (red). Two-sided analysis of variance. All data are mean ± SOS (****p<0.0001).

In FIG. 16A-16C show that late combination therapy restored strength and protected against contraction-induced damage. Absolute (panel A) and normalized specific force (panel B) after tetanic contraction of injected rAAV.MKK.miR-29c and microdystrophin-expressing rAAV gastrocnemius muscles were significantly increased compared to untreated mdx/utrn ^+/- muscle. Panel C shows that the muscles were then evaluated for loss of strength after repeated eccentric contractions. Mice co-treated with rAAV.MKK.miR-29c/microdystrophin-expressing rAAV showed protection against loss of strength compared to untreated mdx/utrn ^+/- muscles (red). Two-sided analysis of variance. All data are mean ± SOS (**p<0.01, ****p<0.0001).

In FIG. 17A-17D show that combination treatment increases muscle hypertrophy 3 months after injection. Panel A shows that rAAV.MKK.miR.-29c co-delivered with microdystrophin-expressing rAAV failed to increase the total weight of the injected gastrocnemius muscle. Panel B shows that combined treatment with rAAV.MKK.miR29c/microdystrophin-expressing rAAV caused an increase in mean fiber size. Comparing control mdx/utm ^+/ with treated miR-29c/microdystrophin mdx/utm ^+/ , the mean diameter increased from 28.96 to 36.03 µm. Panel C shows that co-delivery resulted in a shift towards wild-type fiber size distribution. Panel D shows that the number of muscle fibers per mm ² in miR-29c/microdystrophin combination therapy is significantly lower than in untreated and wild-type mice (***p<0.01, ****p<0.0001) .

In FIG. 18A, 18B show the nucleotide sequence (SEQ ID NO: 1 pAAV.CMV.Mir29C) of an exemplary rAAV vector containing a mature miR-29c guide strand (nucleotides 12571284) and a native mi-30 backbone (nucleotides 1088-1375). The construct also contains the CMV promoter (nucleotides 120-526), two EF1a introns at nucleotides 927-1087 and 1380-1854, and polA at nucleotides 1896-2091.

In FIG. 19 is a diagram of the rAAV vector pAAV.MKK.microdystrophin.

In FIG. 20A-20D show the nucleotide sequence (SEQ ID NO: 9, pAAV.MKK.microdystrophin) of an exemplary rAAV vector expressing microdystrophin.

- 9 042315

In FIG. 21A-21C show the nucleotide sequence of the human microdystrophin nucleotide sequence (SEQ ID NO: 7)

In FIG. 22A-22C show the nucleotide sequence (SEQ ID NO: 12 pAAV.MKK.miR29C) of an exemplary rAAV vector containing a mature miR-29c guide strand (nucleotides 14871512) and a native mi-30 backbone (nucleotides 1088-1375). The construct also contains an MCC enhancer (nucleotides 190-395), a MCC promoter (nucleotides 396-753), two EF1a introns at nucleotides 1155-1315 and 1609-2083, and polA at nucleotides 2094-2148.

Detailed description of the invention

The present invention provides gene therapy vectors, such as rAAV vectors, overexpressing miR-29 miRNA, and methods for reducing and preventing fibrosis in patients with muscular dystrophy. The invention also provides methods of combination gene therapy which include administering a gene therapy vector expressing miR-29 in combination with a gene therapy vector expressing microdystrophin that has been removed from DMD patients.

Muscle biopsies taken at the earliest age of diagnosis of DMD show marked proliferation of connective tissue. Muscular fibrosis is harmful in many ways. It reduces the normal transport of endomysial nutrients across connective tissue barriers, reduces blood flow and deprives muscles of vascularized nutritional components, and functionally contributes to early loss of ambulation due to limb contracture. Over time, the difficulties of treatment increase as a result of severe fibrosis in the muscles. This can be observed in muscle biopsies comparing connective tissue proliferation at subsequent time points. The process continues to worsen, leading to loss of ambulation and accelerated out of control, especially in patients dependent on wheelchairs.

Without a parallel approach to reducing fibrosis, it is unlikely that the benefits of exon skipping, stop codon stop, or gene replacement therapies will ever be fully realized. Even small molecule or protein replacement strategies are likely to fail without an approach that reduces muscle fibrosis. Previous work on mid-aged mdx mice with existing fibrosis treated with AAV. microdystrophin showed that we were unable to achieve full functional recovery (Human molecular genetics 22, 4929-4937 (2013)). It is also known that the progression of cardiomyopathy in DMD is accompanied by scarring and fibrosis in the ventricular wall. The delivery of miRNAs is particularly innovative due to the lack of immune barriers and the relative ease of delivery. MicroRNAs are small (approximately 200 bp) and therefore can be packaged in AAV along with a therapeutic cassette to correct or bypass a genetic defect.

As used herein, the term AAV is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that replicates only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen AAV serotypes that have been characterized. General information and reviews on AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, p. 169-228, and Berns, 1990, Virology, p. 1743-1764, Raven Press, (New York). However, there is no doubt that these same principles will apply to additional AAV serotypes, since it is well known that the various serotypes are quite closely related both structurally and functionally even at the genetic level (see, for example, Blacklowe, 1988, p. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes appear to have very similar replication properties mediated by homologous rep genes; and all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further predicted by heteroduplex analysis, which shows extensive cross-hybridization between serotypes throughout the genome; and the presence of similar self-annealing segments at the ends that correspond to inverted terminal repeat (ITR) sequences. Similar infectivity patterns also suggest that replication functions in each serotype are under the same regulatory control.

AAV vector, as used herein, refers to a vector containing one or more polynucleotides (or transgenes) of interest that are flanked by AAV terminal repeat (ITR) sequences. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

AAV virion or AAV virus particle or AAV vector particle refers to a viral particle consisting of at least one AAV capsid protein and an encapsulated AAV polynucleotide vector. If the particle contains a heterologous polynucleotide (ie, a polynucleotide other than the wild-type AAV genome, such as a transgene that is to be delivered into a mammalian cell), it is commonly referred to as an AAV vector particle or simply an AAV vector. So

- 10 042315 Thus, the production of an AAV vector particle necessarily involves the production of an AAV vector, since such a vector is contained in an AAV vector particle.

AAV.

Recombinant AAV genomes of the invention comprise a nucleic acid molecule of the invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in rAAV genomes can be from any AAV serotype for which a recombinant virus can be generated, including but not limited to these AAV serotypes: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV12 and AAV-13. The production of pseudo-typed rAAV is described, for example, in WO 01/83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations; see, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of the various AAV serotypes are known in the art. AAV1, AAV6, AAV8 or AAVrh can be used to promote skeletal muscle specific expression.74.

The DNA plasmids of the invention contain the rAAV genomes of the invention. The DNA plasmids are transferred into cells suitable for infection with an AAV helper virus (eg, adenovirus, E1-deleted adenovirus, or herpes virus) to package the rAAV genome into infectious viral particles. Methods for producing rAAV particles, in which the AAV genome to be packaged, the rep and cap genes, and accessory viral functions, are introduced into the cell are standard in the art. Production of rAAV requires that the following components be present in a single cell (herein referred to as a packaging cell): the rAAV genome, separated from (i.e., not in) the rAAV genome the AAV rep and cap genes, and helper virus functions. The rep and cap AAV genes can be from any AAV serotype for which a recombinant virus can be generated and can be from an AAV serotype that is different from the ITR serotype of the rAAV genome, including but not limited to these 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 production of pseudotyped rAAV is described, for example, in WO 01/83692, which is incorporated herein by reference in its entirety.

The way to obtain a packaging cell is to create a cell line that stably expresses all the necessary components for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing the rAAV genome without the rep and cap AAV genes, the rep and cap AAV genes separated from the rAAV genome, and a selection marker such as a neomycin resistance gene are integrated into the genome of the cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC attachment (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites ( Laughlin et al., 1983, Gene, 23:65-73) or direct blunt end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as an adenovirus. The advantages of this method are that cells can be selected and are suitable for large scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells.

General principles for the preparation of rAAV are discussed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbials. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. sci. USA 81:6466 (1984); Tratschin et al., Mol. cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. cell. Biol. 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); US Pat. No. 5,173,414; WO 95/13365 and the corresponding US patent No. 5658776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); W099/11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:11241132; US patent No. 5786211; US patent No. 5871982; and US Pat. No. 6,258,595. The aforementioned documents are hereby incorporated herein by reference in their entirety, with particular reference to those sections of the documents relating to obtaining rAAV.

Thus, the 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 (293 related lineage). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low passage number 293 cells (fetal kidney cells transformed with Adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey fetal lung cells).

Recombinant AAVs (i.e. infectious encapsulated rAAV particles) according to

- 11 042315 to the present invention contain the rAAV genome. In exemplary embodiments, the genomes of both rAAVs do not contain rep and cap AAV DNA, i.e. there is no rep or cap AAV DNA between the ITR genomes. Examples of rAAVs that can be engineered to contain the nucleic acid molecules of this invention are shown in International Patent Application No. PCT/US2012/047999 (WO 2013/016352), incorporated herein by reference in its entirety.

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

In another embodiment, the invention contemplates compositions containing rAAV according to this invention. Compositions according to this invention include rAAV and a pharmaceutically acceptable carrier. The compositions may also contain other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are non-toxic to recipients and are preferably inert at the doses and concentrations used and include: buffers such as phosphate, citrate or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

The titers of rAAV to be administered in the methods of this invention will vary depending on, for example, the particular rAAV, route of administration, goal of treatment, individual and cell type(s) to be targeted, and can be determined by methods in the art. technology. rAAV titers can range from about 1x106, about 1x10 ⁷ , about 1x108, about 1x109, about 1x10 ¹⁰ , about 1x1011, about 1x10 ¹² , about 1x10 ¹³ to about 1x10 ¹⁴ or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (gw).

The invention contemplates methods for transducing a target cell with rAAV, either in vivo or in vitro. In vivo methods include the step of administering an effective dose or multiple effective doses of a composition comprising an rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered prior to development of the disorder/disease, the administration is prophylactic. If the dose is administered after the development of the disorder/disease, the administration is therapeutic. In embodiments, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease condition being treated, that slows or prevents progression to a disorder/disease condition that slows or prevents progression a disorder/disease condition that reduces the severity of the disease, resulting in remission (partial or complete) of the disease and/or prolonging survival. FSHD is an example of a disease that is considered suitable for prevention or treatment by the methods of this invention.

This invention also contemplates combination therapies. The combination used herein includes both simultaneous treatment and sequential treatment. Combinations of the methods of this invention with standard drug therapies (eg, corticosteroids) are specifically contemplated, as are combinations with novel therapies.

Administration of an effective dose of the compositions may be by standard routes, which include, but are not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route(s) of administration and serotype(s) of the rAAV AAV components (particularly the AAV ITR and capsid protein) of the present invention may be selected and/or matched by those skilled in the art, taking into account the infection and/or disease state to be determined. treatment, and target cells/tissue(s) to express miR-29 microRNA and/or microdystrophin.

The present invention provides topical and systemic administration of an effective dose of rAAV and compositions of the present invention, including combination therapy of the present invention. For example, systemic administration is administration to the circulatory system to affect the entire body. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration by injection, infusion, or implantation.

In particular, the actual administration of rAAV according to this invention can be performed with

- 12 042315 using any physical method that will transfer the recombinant rAAV vector into the target tissue of the animal. Administration according to the invention includes, but is not limited to, injection into muscle, bloodstream and/or directly into the liver. It has been demonstrated that simply resuspension of rAAV in phosphate buffered saline is sufficient to obtain a vehicle usable for expression in muscle tissue, and there are no known limitations on vehicles or other components that can be co-administered with rAAV (although rAAV should be avoid compositions that destroy DNA). rAAV capsid proteins can be modified so that rAAV is targeted to a specific target tissue of interest, such as muscle; see, for example, WO 02/053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectables or as topical formulations for delivery to muscles by transdermal transport. Numerous formulations for intramuscular injection and transdermal transport have been developed in the past and may be used in the practice of the invention. The rAAVs may be used with any pharmaceutically acceptable carrier for ease of administration and handling.

The dose of rAAV administered in the methods disclosed herein will vary depending on, for example, the particular rAAV, route of administration, goal of treatment, individual and cell type(s), and may be determined by methods standard in the art. The titers of each rAAV administered can range from about 1x10, about 1x10 ⁷ , about 1x10 ⁸ , about 1x10 ⁹ , about 1x101°, about 1x1011, about 1x10 ¹² , about 1x10 ¹³ , about 1x10 ¹⁴ , to about 1x10 ¹⁵ or more stable to DNase particles (DRP) in ml. Doses may also be expressed in units of viral genomes (gv) (i.e. 1x10 ⁷ hrv, 1x10 ⁸ hrv, 1x10 ⁹ hrv, 1x10 ¹⁰ hrv, 1x1011 hrv, 1x10 ¹² hrv, 1x10 ¹³ hrv, 1x10 ¹⁴ hrv, 1x10 ¹⁵ respectively ). Doses may also be expressed in units of virus genomes (gw) per kilogram (kg) of body weight (i.e. 1 x ^{10 10} gw/kg, 1 x 10 ¹¹ gw/kg, 1 x 10 ¹² gw/kg, 1 x 10 ¹³ gw/kg, 1 x 10 ¹⁴ gw / kg, 1x10 ¹⁵ gw / kg, respectively). AAV titration methods are described in Clark et al., Hum. Gene Ther., 10: 1031-1039 (1999).

In particular, the actual introduction of rAAV according to this invention can be performed using any physical method that will transfer the recombinant rAAV vector into the target tissue of the animal. Administration according to the invention includes, but is not limited to, injection into muscle, bloodstream and/or directly into the liver. It has been demonstrated that simply resuspension of rAAV in phosphate buffered saline is sufficient to obtain a vehicle usable for expression in muscle tissue, and there are no known limitations on vehicles or other components that can be co-administered with rAAV (although rAAV should be avoid compositions that destroy DNA). rAAV capsid proteins can be modified so that rAAV is targeted to a specific target tissue of interest, such as muscle; see, for example, WO 02/053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions may be prepared as injectables or as topical formulations for delivery to muscles by transdermal transport. Numerous formulations for intramuscular injection and transdermal transport have been developed in the past and may be used in the practice of the invention. The rAAVs may be used with any pharmaceutically acceptable carrier for ease of administration and handling.

For intramuscular injection, solutions in an adjuvant such as sesame or peanut oil, or aqueous propylene glycol, as well as sterile aqueous solutions, may be used. If desired, such aqueous solutions may be buffered and the liquid diluent first exposed to isotonic saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. The rAAV dispersion can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, the sterile aqueous media used are readily available for preparation by standard methods well known to those skilled in the art.

Pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile, and must be fluid to the extent that it can be injected with a syringe. It must be stable under the conditions of manufacture and storage, and must be protected from the contaminating effects of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The desired fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. Prevention of exposure to microorganisms can be achieved

- 13 042315 then various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it is preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved through the use of agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the required amount of rAAV into the appropriate solvent with various other ingredients listed above, if necessary, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of injectable sterile solutions, the preferred methods of preparation are vacuum drying and the freeze-drying method, which yield a powder of the active ingredient plus any additional desired ingredient from its pre-sterile filtered solution.

Transduction with rAAV can also be done in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, isogenic or xenogeneic muscle cells may be used, as long as these cells will not elicit an inappropriate immune response in the subject.

Suitable methods for transduction and reintroduction of 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 an appropriate medium, and searching for cells bearing DNA of interest using conventional methods such as Southern blot and/or PCR, or with using selectable markers. The transduced cells can then be formulated into pharmaceutical compositions and the composition administered to the subject by various means such as intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle using, for example, a catheter.

Transduction of cells with the rAAV of the invention results in sustained miR-29 or microdystrophin expression. Thus, the present invention relates to methods for administering/delivering a rAAV that expresses miR-29 and/or microdystrophin to an animal, preferably a human. These methods include transducing tissues (including but not limited to tissues such as muscles, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of the invention. Transduction can be carried out using gene cassettes containing tissue-specific regulatory elements. For example, in one embodiment, the present invention provides methods for transducing muscle cells and muscle tissues driven by muscle-specific regulatory elements, including, but not limited to: those derived from the actin and myosin gene families, e.g. , from the myoD gene family [see Weintraub et al., Science, 251: 761-766 (1991)], myocyte-specific enhancer-binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol 11: 4854-4862 (1991)], regulatory elements derived from human skeletal muscle actin gene [Muscat et al., Mol Cell Biol, 7: 4089-4099 (1987)], cardiac actin gene, muscle creatine kinase element sequences [cf. Johnson et al., Mol Cell Biol, 9:3393-3399 (1989)] and the mouse creatine kinase (mCK) enhancer element, regulatory elements derived from the troponin C gene of fast twitch skeletal muscle fibers, the troponin C gene of slow twitch cardiac muscle fibers and troponin I gene of slow twitch muscle fibers: hypoxia-induced nuclear factors (Semenza et al., Proc Natl Acad Sci USA, 88: 5680-5684 (1991)), steroid-inducible elements and promoters including the glucocorticoid response element (GRE) ( see Mader and White, Proc Natl Acad Sci USA 90: 5603-5607 (1993)) and other regulatory elements.

Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is readily available. The invention considers stable expression of microRNA from transduced myofibrils.

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

The term transduction is used to refer to the introduction/delivery of the miiR29 guide chain or the microdystrophin coding region into a recipient cell, either in vivo or in vitro, by a replication-defective rAAV of the invention, resulting in expression of miR29 or microdystrophin by the recipient cell.

Thus, the present invention provides methods for administering to a patient in need thereof an effective dose (or doses administered substantially simultaneously or at predetermined intervals).

- 14 042315 intervals) rAAV that encodes miR29 and/or microdystrophin.

Examples

Example 1 Validation of models of Duchenne muscular dystrophy.

The mdx mouse provides a convenient but incomplete animal model for studying the pathogenesis of DMD. This model is a hybrid of the mdx mouse with a heterozygous utrophin knockout line (mdx:utrn ^+/- ), which exists with increased fibrosis and more closely mimics the pathology of human DMD. Mdx mice have a nonsense mutation in DMD exon 23, resulting in a relatively mild phenotype and near normal lifespan. By 3 weeks of age, the diaphragm and limb muscles of md mice acquire signs of endomysial inflammation. These symptoms decrease in the muscles of the extremities after the mice reach maturity, and the inflammation in the muscles of the diaphragm continues to gradually worsen. In mdx mice lacking telomerase, muscular dystrophy progressively worsens with age; mdx mice that do not have atrophy (DKO) have a phenotype more characteristic of human DMD with early onset of muscle weakness, severe fibrosis, and premature death. Utrophin, an autosomal paralog of dystrophin, has a high degree of sequence homology that can compensate for the lack of dystrophin in the mdx mouse with double KO (dystrophin plus utrophin); there is a severe phenotype with early death. Premature death of the DKO mouse impedes the progression of inflammation and fibrosis, but the mdx:utm ^+/ mouse is a model similar to human disease, exhibiting an impressive degree of fibrosis and longer survival than DKO, providing a better model for our proposed clinical transfer studies. . A recent report confirms the use of the mdx:utm ^+/ mouse as an ideal model for studying fibrosis in the context of DMD. In this study, an increase in fibrosis, as measured by Sirius red staining, was accompanied by an increase in collagen transcript levels and a decrease in miR29c levels.

Example 2 Delivery of miR29 to DMD mice reduces fibrosis.

Preliminary studies have shown that there is a significant increase in Sirius red staining of collagen and a decrease in miR-29c levels in human DMD patients and mdx/utm ^+/ mice. Delivery of the miR-29 gene using muscle-specific AAV vectors is potentially safe and effective. To obtain the rAAV vector, herein referred to as rAAVrh.74.CMV.miR29c, the 22 bp miR29c sequence (guide strand SEQ ID NO: 3 and guide strand SEQ ID NO: 4) was cloned into the miR-30 framework, under control of the CMV promoter. The expression cassette (SEQ ID NO: 2) was cloned into a self-complementary AAV plasmid and packaged using AAVrh.74, a serotype known to be well expressed in muscle. miR-29c cDNA was synthesized using a custom primer containing miR-29c target (sense), miR-30 stem loop, and miR-29c guide (antisense) strand in miR-30 scaffold. Three bases of the miR-29c sequence were modified. This sequence was then cloned into a self-complementary AAV containing ITR plasmid under the control of the CMV promoter and the polyA sequence.

As shown in FIG. 1, plasmid pAAV.CMV.miR29C contains the mir29c cDNA in a miR-30 stem loop framework flanked by AAV2 inverted terminal repeat (ITR) sequences. It is this sequence that was encapsidated into AAVrh.74 virions. In addition, several nucleotides of the miR-29c target sequence were altered to mimic Watson-Crick pairing at this site, as in shRNA-miR(luc). According to the shRNA-luc design, the hairpin must be perfectly complementary throughout its length. In addition, the more changes in the escort strand, the more likely it is to eliminate any endogenous mechanism that regulates the processing of miR-29, which could recognize microRNA through the stem. The 19th base of the guide strand was modified to cytosine to mimic the nucleotide that precedes the cleavage site in the natural mi-29c sequence, and the corresponding base on the other strand was changed to preserve mating.

The gene therapy vector scAAVrh.74.CMV.miR29c (1x1011 gv) was injected into the quadriceps muscle of a 3-month-old mdx/utm ^+/ ' mouse. The quadriceps muscle was analyzed 3 months post-injection by Sirius Red staining and analyzed using NIH ImageJ software as described in Nevo et al. (PloS One, 6: e18049 (2011). miR29c, collagen and elastin levels were determined by RT-PCR. Delivery of miR-29c to young mdx/utrn ^+/- mice significantly increased miR29c levels and significantly reduced Sirius red staining in the quadriceps muscle 6-month-old mdx/utm ^+/ mice (3 months post-injection) Decrease in collagen and elastin levels in treated muscles was observed when assessed by quantitative PCR.

The demonstration of increased fibrosis and reduced expression of miR29 in mdx/utrn ^+/ ' mice and dystrophin-deficient patients confirms that the mouse model is a replacement for human disease. Initial results using AAV-delivered miR29 as an antifibrotic therapy suggest that there is a significant beneficial effect in reducing Sirius red staining and levels of collagen and elastin, which are key factors in fibrosis.

- 15 042315

Example 3 Injection of MiR-29c reduces collagen and restores miR-29c.

To determine if rAAVrh.74.CMV.MiR-29c could reduce fibrosis, 12-week-old mdx/utrn ^+/- mice received an intramuscular injection of rAAVrh.74.CMV.MiR-29c with 5x1011 gv into the left gastrocnemius muscle (GAS). Mice were analyzed 12 weeks after injection. Picrosirius red staining showed a significant reduction in collagen staining throughout the gastrocnemius muscles (FIG. 2A) compared to untreated contralateral gastrocnemius muscle mdx/utrn +/-. Quantification of picrosirius red staining showed that the treated muscle had an 18.3% reduction in collagen compared to the untreated muscle (treated, 23.3% ± 1.3 vs. untreated, 29.5% ± 0.7) (Fig. 2B). To confirm overexpression of miR-29c in the treated muscle, total RNA was isolated from gastrocnemius muscle from 24-week-old DT mice treated with miR-29c and mdx/utrn ^+/ and subjected to quantitative reverse transcription PCR (RTPCR) to analyze miR expression. -29c. The results showed that the amount of miR-29 was significantly increased in the gastrocnemius muscle of treated mice compared to untreated mice (Fig. 2D).

Example 4 MiR-29c improves absolute and specific muscle strength but does not protect against contraction-induced damage.

Knowing that fibrosis can affect muscle function, we wanted to test whether reducing fibrosis by increasing miR-29c expression could protect the mdx/utm ^+/ muscle from contraction-induced injury and increase overall strength. The functional properties of the gastrocnemius muscle from mdx/utm ^+/ mice treated with rAAVrh.74.CMV.MiR-29c were evaluated. Twelve weeks after injection, calf muscles were obtained for in vivo strength measurements.

The calf procedure follows the protocol outlined in Hakim et al. (Methods Mol Biol. 709: 75-89, 2011) to analyze the physiology of the transversus abdominis but adapted to the gastrocnemius. Briefly, mice were anesthetized using a mixture of ketamine/xylazine. The skin of the hind limb was removed to expose the calf muscle and Achilles tendon. The distal tendon was opened and a double straight knot was tied around the tendon with a 4-0 thread as close to the muscle as possible, another double straight knot was tied right next to the first knot, and then the tendon was cut. The exposed muscle was constantly moistened with saline. Mice were then transferred to a temperature-controlled platform and maintained at 37°C. The knee was attached to the platform with a needle through the tendon of the patella, a tendon suture to the shoulder level of a force transducer (Aurora Scientific, Aurora, ON, Canada), and the leg was secured with tape. Muscle contractions of the gastrocnemius muscle were induced by stimulation of the sciatic nerve through bipolar platinum electrodes. Once the muscle was stabilized, the optimal length was determined by gradually stretching the muscle until the maximum contraction force was reached. After a 3-minute rest period, the gastrocnemius muscle was stimulated at 50, 100, 150, and 200 Hz, with a 1-minute rest period between each stimulus to determine maximum tetanic force. The length of the muscle was measured. After a 5-minute rest, the susceptibility of the gastrocnemius muscle to damage caused by the contraction was assessed. After 500 ms of stimulation, the muscle was lengthened by 10% of the optimal length. This was achieved by stimulating the muscle at 150 Hz for 700 ms. After stimulation, the muscle was returned to the optimal length. The cycle was repeated every minute for a total of 5 cycles. The specific force was calculated by dividing the maximum tetanic force by the cross-sectional area of the gastrocnemius muscle. After eccentric contractions, mice were euthanized and the gastrocnemius muscle was dissected, weighed and frozen for analysis.

Each gastrocnemius muscle was subjected to a series of repeated eccentric contractions. Comparing the ratio of the strength of each contraction compared to the first contraction, it turned out that after the fifth contraction, the untreated muscle was weakened to 0.56 ± 0.05 versus 0.50 ± 0.04 (p < 0.0001). The injected group showed a slight decrease in protection compared to the DT control, attenuated to 0.92 ± 0.02 (Fig. 3C). These data indicate that a decrease in fibrosis due to an increase in miR-29c expression leads to an increase in both absolute and specific strength, but does not provide significant muscle protection from contraction-induced injury.

The rAAVrh.74.MiR-29c treated gastrocnemius muscle showed a significant improvement in absolute strength compared to the untreated mdx/utrn gastrocnemius muscle (rAAV.miR-29c- 2277 ± 161.7 vs. untreated mdx/utrn ^+/ - - 1722 ± 145.7 , Fig. 3a), as well as a specific improvement in normalized specific force in the rAAVrh.74.miR-29c treated gastrocnemius muscle compared to the untreated gastrocnemius muscle (rAAV.miR-29c - 204.7 ± 11.7 vs. untreated mdx/utrn - - 151.6 ± 14.5, Fig. 3b). Strength was still significantly reduced compared to wild-type control (rAAV.miR-29c - 204.7 ± 11.7 vs. wild-type -312.0 ± 34.1).

Example 5 Co-delivery with microdystrophin further reduces fibrosis.

To determine whether a miR-29c/microdystrophin combination gene therapy approach was preferable for reducing fibrosis, 12-week-old mdx/utrn ^+/- mice received an intramuscular injection of rAAVrh.74.CMV.MiR-29c at 5x1011 gv in the left gastrocnemius muscle . Next

- 16 ⁰⁴²³¹⁵ gene therapy vectors were administered by intramuscular injection (IM) into the left gastrocnemius muscle (GAS) of 3-month-old mdx/utrn ^+/- mice, DMD mouse model: scAAVrh.74.CMV.miR-29c only, coded with rAAVrh.74. MKK.microdystrophin, and only rAAVrh.74.MKK.microdystrophin.

The pAAV.MKK.microdystrophin plasmid contains the human microdystrophin cDNA expression cassette flanked by AAV2 terminal inverted repeat (ITR) sequences as shown in FIG. 10. This sequence was encapsulated in AAV rh.74 virions. The pAAV.MKK.microdystrophin plasmid was constructed by inserting an MCK expression cassette driving a codon-optimized human microdystrophin cDNA sequence into an AAV cloning vector as described in Rodino-Klapac et al. (Mol Ther. 2010 Jan; 18(1): 109-17). The MCK promoter/enhancer sequence was used to drive the expression of muscle specific genes and consisted of a mouse MCK enhancer core (206 bp) fused to a 351 bp MCC promoter core. (proximal). The promoter core is followed by 53 bp. mouse MCC exon1 (untranslated) for efficient transcription initiation, followed by SV40 late 16S/19S splice signals (97 bp) and a small 5'UTR (61 bp). The intron and 5'-UTR are obtained from the plasmid rCMBβ (Clontech). The microdystrophin cassette has a Kozak consensus just before the ATG startcodon and a small 53 bp polyA synthetic signal. for mRNA termination. The human microdystrophin cassette contains (R4-R23/A71-78) domains. Complementary DNA was codon-optimized for human use and synthesized using GenScript (Piscataway, NJ).

Mice were analyzed 12 and 24 weeks after injection. First, the number of microdystrophin-expressing muscle fibers was used to evaluate the efficiency of transgene delivery and to make sure we had the same amount of microdystrophin expressed in each group. We found that microdystrophin did not differ between groups treated with microdystrophin alone (71.85 ± 2.25%) compared to miR-2 9c/microdystrophin combination therapy (75.03 ± 1.91%) (Fig. 4) .

Calf muscles were analyzed 12 months post-injection to evaluate collagen accumulation by Sirius Red staining and subsequent quantification with ImageJ. Additional results included miR-29c and collagen transcript levels, calf strength measurements, fiber diameter measurements, and Western blot analysis for proteins involved in muscle regeneration (MyoD, Myogenin). The amount of fibrosis was analyzed by picrosirius red staining, which revealed a significant reduction in collagen staining in all gastrocnemius muscles in all treated groups (Fig. 5A) compared to untreated contralateral gastrocnemius mdx/utm ^+/ or treated with microdystrophin alone. Quantification by picrosirius red staining shows that co-treated muscle had a 40.8% percent reduction in collagen compared to untreated muscle (treated - 17.47% ± 0.75 vs. untreated - 29.5 ± 0.7) ( Fig. 5B). To confirm miR-29c expression, RT-PCR was performed on gastrocnemius muscle and all treatment groups had an increase in miR-29c compared to untreated muscle (Fig. 5C).

Similarly to DMD tissue, there was a significant decrease in miR-29c levels in mdx/utm ^+/ muscle, which correlated with an increase in fibrosis as measured by picrosirius red staining. After 3 months of treatment with scAAV.miR.-29c alone, there was a significant reduction in fibrosis (treated 23.5 ± 1.3 versus untreated 27.8 ± 0.6) in the gastrocnemius muscle. When co-delivered with microdystrophin, there was an additional decrease in collagen (41%) when stained with picrosirius red (combined treatment: 17.47 ± 0.75 vs. untreated: 29.5 ± 0.7) (p<0.0001) (Fig. 5B). To confirm miR-29c expression, RT-PCR was performed on gastrocnemius muscle and all treatment groups had an increase in miR-29c compared to untreated muscle (Fig. 5B).

At 24 weeks post-injection, the results were similar to those seen 12 weeks post-injection. The decrease in collagen was 47%, as determined by picrosirius red staining, compared with untreated muscle (combination therapy: 16.5 ± 1.23 vs. untreated: 31.07 ± 0.93, p<0.0001) and a concomitant increase in the level miR-29c transcription.

To further confirm the reduction in collagen observed with picrosirius red staining, muscle RT-PCR was performed to quantify levels of Col1A, Col3A transcriptotes, as well as another ECM component, fibronectin (Fbn). RT-PCR analysis showed a decrease in Col1A and Col3A after each treatment, however, only the microdystrophin and miR-29c treated group showed a significant decrease (FIGS. 6A and 6B). The analysis showed that Fbn was significantly reduced only in the co-treated group (FIG. 6C).

It has previously been shown that TGF-e1 levels are elevated in dystrophic muscle, which likely plays a role in initiating the fibrotic cascade. TGF-e1 is a known pro-fibrotic cytokine that reduces miR-29c levels and is responsible for the conversion of myoblasts to myofibroblasts with increased collagen and muscle fibrogenesis. RT-PCR analysis shows that co-processed

- 17 042315 tan muscle had significantly lower levels of TGF-β 1 compared with untreated muscle and with treatments alone (FIG. 6D). At 6 months post-injection, the co-treated muscle continued to show reductions in Col1A, Col3A, Fbn, and TGF-βΤ levels, while there was only a slight decrease in Col1A mRNA levels in the miR-29 and microdystrophin alone treated groups.

Increases in specific and absolute strength were observed in muscle treated with miR-29c alone compared to an untreated limb, which, when combined with microdystrophin, resulted in absolute and specific strengths that were indistinguishable from wild-type. We also observed a significant increase in the mass of those calf muscles that were co-treated.

Initial results with rAAV.miR.-29c as an antifibrotic therapy indicate a beneficial effect in reducing collagen levels, a key factor in fibrosis. In addition, when combined with microdystrophin to improve membrane stability, increasing miR29 levels normalizes muscle strength.

Example 6: Additional increase in absolute strength and added protection from damage caused by contraction.

Knowing that miR-29-treated muscle had a modest but significant increase in absolute and specific strength, the effect of combination therapy of miR-29c overexpression and microdystrophin gene replacement on muscle function was investigated. At 12 weeks post-injection, we isolated the gastrocnemius muscle, for which we performed in vivo strength measurements. The rAAVrh.74.MiR-29c vector described in Example 2 above, and rAAV.

Co-treated rAAVrh.74.MiR-29c and rAAV expressing mirodystrophin, gastrocnemius muscle showed a significant improvement in absolute strength compared to untreated mdx/utrn gastrocnemius muscle (co-treated - 3582.4 ± 79.4 nM vs. untreated mdx/utrn ^+/ - 1722 ± 145.7 nM versus wild-type - 3005 ± 167.3 nM) (Fig. 7), as well as a specific improvement in the normalized specific force in the treated rAAVrh.74.miR-29c/microdis gastrocnemius muscle, compared with the untreated gastrocnemius muscle (co-treated mice 244.2 ± 6.6 nM/mm ² vs untreated mdx/utm ^+/- 151.6 ± 14.5 nM/mm ² vs 312.0 ± 34.1 nM/mm ² ) (Fig. .7). Both absolute and specific strengths did not differ significantly from the control wild type.

Each gastrocnemius muscle was subjected to a series of repeated eccentric contractions. Comparing the ratio of the strength of each contraction with the first contraction, it was found that after the fifth contraction, the untreated muscle was weakened to 0.54 ± 0.06 compared to the jointly treated 0.66 ± 0.04 (p < 0.0001), which can be explained the contribution of microdystrophin, since only microdystrophin treated was also attenuated to 0.66 ± 0.04. The treated group was still significantly lower than the wild type, which attenuated to 0.92 ± 0.02 (Fig. 7C). Similar results were found 24 weeks after injection. These data show that the reduction in fibrosis and gene replacement leads to an increase in both absolute and specific strength, and significantly protects muscles from damage caused by contraction.

Example 7 Combination treatment increases muscle hypertrophy and hyperplasia.

MiR-29c co-delivered with microdystrophin increased the total mass of the injected gastrocnemius muscle compared to those injected individually for three months (Fig. 8, Fig. 9A). To investigate the source of the increased muscle mass, the diameters of the myofibrils were measured. miR-29c/μ-dys combination treatment demonstrated an increase in mean fiber size. Comparing control mdx/utrn ^+/- to treated miR-29c/μ-dys mdx/utrn ^+/- , the mean diameter increased from 25.96 to 30.97 µm (FIG. 9B). Co-delivery resulted in a shift in fiber size distribution towards that of the wild type (FIG. 9C). Although the average fiber size was increased, this does not explain the 30% increase in muscle mass. The total cross-sectional area of the muscle was also measured. The calf muscles from all groups were completely scanned and the total area measured. Muscles co-treated with microdystrophin/miR-29c had a significant increase in cross-sectional area compared to untreated and treated with either alone (uninjected: 24.6 vs. miR-29c: 26.3 vs. microdystrophin: 26.6 compared to microdystrophin/ miR-29c: 33.1) (Fig. 8, 9D).

miR-29 has been reported to be involved in the yoD/Pax7/myogenin pathway, and it has been suggested that miR-29c may influence the regeneration and activation of satellite cells (muscle stem cells), leading to their differentiation into a myogenic lineage. To test this, the total number of muscle fibers from the complete scans was counted. Increased number of muscle fibers after miR-29c/μ-dys combination therapy (Fig. 9E). Finally, given that muscle fiber diameter in mdx/utrn+/- mice is heterogeneous with many small fibers and some hypertrophic fibers, it was determined whether treatment affected the number of fibers per unit area (cells/mm ² ). Treatment with the miR-29c/μ-dys combination did not differ from wild type (FIG. 9F).

- 18 042315

Example 8 Early treatment with the combination prevents fibrosis.

Given the potential importance of combining miR-29c and microdystrophin as a preventive therapy for DMD, a group of young mdx/utrn ^+/- mice were treated at 4 weeks of age. Using the same paradigm as for the other groups described herein, the following treatments were compared for fibrosis prevention efficacy administered by intramuscular injection into the gastrocnemius muscle: scAAVrh.74.CMV.miR-29c, ssAAVrh74.MKK.microdystrophin+scAAVrh. 74.CMV.miR-29c or only ssAAVrh74.MKK.microdystrophin, in equal doses. Mice were sacrificed 12 weeks after injection. There was a significant reduction in collagen staining in the gastrocnemius muscles in all treated groups compared to the untreated contralateral gastrocnemius muscle mdx/utrn ^+/- (FIG. 10A). Quantification by picrosirius red staining showed that microdystrophin/miR-29c co-treated muscle had a 51% reduction in collagen compared to untreated muscle (treated 11.32 ± 1.18 versus untreated 23.15 ± 0, 90) (p<0.0001) (FIG. 10) and RT-PCR confirmed a decrease in Col1A, Col3A, Fbn and TGF-e1 after combination therapy (FIGS. 10D and E).

Example 9 Early combination therapy restores strength and protects against contraction-induced damage better than late treatment.

In vivo strength measurements were performed on the gastrocnemius muscles of mice treated at an early stage with combination therapy as described in Example 8 ^. compared to untreated mdx/utm ^+/ mice, and there was no difference compared to wild type (co-treated: 2908 ± 129.5 mN versus untreated: 1639.4 ± 116.9 mN versus wild type: 3369 .73 ± 154.1 mN). Specific force was also normalized to wild-type levels after combination therapy (co-treated 338.9 ± 22.34 mN/ ^mm2 versus untreated 184.3 ± 13.42 mN/ ^mm2 versus DT3 64.3 ± 7.79 mN/mm ² ) (FIGS. 11A, B and 12).

Each calf muscle was then subjected to a series of repeated eccentric contractions. Comparing the strength ratio of each contraction with the fifth contraction, the untreated muscle was attenuated to 0.53 ± 0.04 compared to the co-treated 0.82 ± 0.04 (p < 0.0001). The combination treatment group was somewhat lower than wild-type but not significantly different, attenuated to 0.93 ± 0.01 (Fig. 11C). These data show that the reduction in fibrosis and gene replacement leads to an increase in both absolute and specific strength and significantly protects muscles from contraction-induced injury.

These experiments suggest that gene replacement should begin in the neonatal period. Efforts are clearly being made towards the identification of DMD and other muscular dystrophies in the neonatal period. The Ohio Newborn Screening Study illustrates the potential for identifying DMD in newborns using Neurol SK 7. as a biomarker (>2000 U/s) with DNA confirmation on the same dried blood spot (Mendell et al., Ann. Neurol. 71: 304-313, 2012). This methodology is now expanding to other states in the US (PPMD May 16, 2016: Next steps in newborn screening) and other countries, especially in the UK (British National Screening Committee) and China (Perkin Elmer™ launches screening in China).

miR-29 has also shown promise as a treatment for cardiac, pulmonary and hepatic fibrosis. Myocardial infarction in mice and humans is associated with a decrease in miR-29 levels. Roij et al. (Proc. Natl. Acad. Sci, USA 105:13027-13032, 2008) demonstrated that exposure of miR29b mimetic to fibroblasts reduces collagen transcripts, providing a pathway to clinical applications in cardiac fibrosis. Subsequent studies have shown that in a mouse bleomycin-induced pulmonary fibrosis model, attenuation of fibrosis can be achieved with miR.-2 9b.14. delivered by a transposon-based Sleeping Beauty (SB) system. The miR-29b mimetic is currently in a clinical Phase 1 safety-tolerance local intradermal trial in healthy volunteers (miRagen Therapeutics™ MRG-201). Compared to miR-29 oligonucleotide delivery, which would require repeated administration associated with the half-life of the oligonucleotides, AAV gene therapy could potentially provide a route for single gene transfer-delivery.

Example 10 Treatment with muscle-specific expression of miR-29 and microdystrophin reduces fibrosis and ECM expression.

AAV vectors containing the miR29c sequence and a muscle-specific MCC promoter were also generated and tested as combination therapy with AAV vectors expressing microdystrophin. To obtain the rAAV vector, herein designated rAAV.MKK.miR.29c, the 22-nucleotide miR29c sequence (target strand SEQ ID NO: 3 and guide strand SEQ ID NO: 4) was cloned into the miR-30 framework. , under the control of the MCC promoter (SEQ ID NO: 11). The expression cassette (SEQ ID NO: 12) was cloned into a single chain AAV plasmid and packaged using AAVrh74, a serotype known to be well expressed in muscle. cDNA miR-29c was synthesized using a special primer containing the strand

- 19 042315 miR-29c (sense) target, miR-30 loop-on-stem, and miR-29c guide (antisense) strand in a miR-30 scaffold. Three bases of the miR-29c sequence were modified. This sequence was then cloned into a single-stranded AAV ITR-containing plasmid under the control of the MCC promoter and the polyA sequence. Plasmid pAAV.MKK.miR29C contains the mir29c cDNA in miR-30 stalk-loop flanked by AAV2 terminal inverted repeat (ITR) sequences. It is this sequence that was encapsidated into AAVrh74 virions. In addition, several nucleotides of the miR-29c target sequence were altered to mimic Watson-Crick pairing at this site, as in shRNA-miR(luc). According to the shRNA-luc design, the hairpin must be perfectly complementary throughout its length. In addition, the more changes in the escort strand, the more likely it is to eliminate any endogenous mechanism that regulates the processing of miR-29, which could recognize microRNA through the stem. The 19th base of the guide strand was modified to cytosine to mimic the nucleotide that precedes the cleavage site in the natural mi-29c sequence, and the corresponding base on the other strand was changed to preserve mating. Early treatment with AAV.MKK.miR-29c/microdystrophin combination therapy was more effective in reducing fibrosis and ECM expression. 4-5 week old mdx/utm ^+/ ' mice received an intramuscular injection of rAAVrh.74.MKK.MiR-29c and rAAVrh74.MKK.microdystrophin at 5x1011 gw into the left gastrocnemius muscle as described in Example 5. Muscles were harvested after twelve weeks after injection. Picrosirius red staining of muscles harvested from uninjected mice and mice injected with rAAV.MKK.miR-29c/rAAV.MKK.-microdystrophin combination therapy showed that co-treated muscle had a 50.9% reduction in collagen compared to untreated gastrocnemius muscle (see Fig. 13A and 13B). RT-PCR confirmed an increase in miR-29c transcription levels in the treated group (Fig. 13C). Semi-quantitative RT-PCR showed a significant decrease in the levels of collagen A1 and collagen 3A (Fig. 13D, E), fibronectin (Fig. 13F) and TGFe1 (Fig. 13G) in AAV.MKK.miR29c/AAV.microdystrophin-treated muscle, compared with contralateral limb therapy. (*p<0.05, ****p<0.0001). Late treatment with AAV.MKK.miR-29c/microdystrophin combination therapy is effective in reducing fibrosis and ECM expression. Three-month-old mdx/utm ^+/ mice received an intramuscular injection of rAAVrh.74.MKK.MiR-29c and rAAVrh.74.MKK.microdystrophin at 5x1011 gw into the left gastrocnemius muscle as described in Example 5. Muscles were harvested twelve weeks after injection. Picrosirius red staining of untreated muscle treated with AAV.MKK.miR-29c and AAV.MKK.miR-29c/AAV.microdystrophin muscle showed that the co-treated muscle had a 30.3% reduction in collagen compared to untreated gastrocnemius muscle (see Fig. 14A and 14B). RT-PCR confirmed an increase in miR-29c transcription levels in the treated groups (Fig. 14C). Semi-quantitative RT-PCR showed a significant decrease in the levels of collagen 1A and collagen 3A (Fig. 14D, E), fibronectin (Fig. 14F) and TGFel (Fig. 14G) in AAV.miR29c/AAV.microdystrophin-treated muscle, compared with contralateral therapy. limbs. One-way analysis of variance. All data are mean ± SOS (**p<0.01, ****p<0.0001).

Example 11 Early combination therapy restores strength and protects against contraction-induced damage better than late treatment.

In vivo strength measurements were performed on the gastrocnemius muscles of mice that were early treated with miR-29 and muscle-specific microdystrophin as described in Examples 8 ^and 9. .MKK.miR-29c/ and microdystrophin-expressing rAAV showed a significant improvement in absolute strength compared to untreated mdx/utm ^+/ mice, and there was no difference compared to wild type (Fig. 15a). Specific strength was also normalized to wild-type levels after combination therapy (Fig. 15b).

Muscles were then assessed for loss of strength following repeated eccentric contractions as described in Example 9. Only mice co-treated with rAAV.MKK.miR-29c/rAAV.MKK.microdystrophin and rAAV.MKK.microdystrophin alone showed protection against loss of strength by compared with untreated muscles mdx/utrn ^+/- (Fig. 15c).

In 12-week-old mdx/utm ^+/ mice, co-treatment with rAAV.MKK.miR-29c/ and microdystrophin-expressing rAAV restored strength and protected against damage caused by contraction. The magnitude of absolute (FIG. 16A) and normalized specific force (FIG. 16B) after tetanic contraction of injected rAAV.MKK.miR-29c and microdystrophin-expressing rAAV in gastrocnemius muscles was significantly increased compared to untreated mdx/utm ^+/ muscle. Muscles were then assessed for loss of strength following repeated eccentric contractions as described in Example 9. Mice co-treated with MKK.miR-29c/microdystrophin showed protection against loss of strength compared to untreated muscles with mdx/utrn ^+/- (Fig. 16C ). These data show that the reduction in fibrosis and gene replacement leads to an increase in both absolute and specific strength and significantly protects muscles from contraction-induced injury.

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Example 12 Early combination treatment increases muscle hypertrophy and hyperplasia.

Co-delivery of rAAV.MKK.miR-29 with microdystrophin-expressing rAAV did not increase the total weight of the injected gastrocnemius muscle compared to those injected individually for three months (FIG. 17A). The diameters of myofibrils were also measured. miR-29c/microdystrophin combination treatment demonstrated an increase in mean fiber size. Comparing control mdx/utrn ^+/- with treated miR-29c/microdystrophin mdx/utrn ^+/- , the mean diameter increased from 28.96 to 36.03 µm (FIG. 17B). Co-delivery resulted in a shift in fiber size distribution towards that of the wild type (FIG. 17C). The number of muscle fibers per mm ² in miR-29c/microdystrophin combination therapy is significantly lower than in untreated and wild-type mice (Fig. 17d; ***p<0.01, ****p<0.0001).

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Claims (10)

Suitability as a Murine Model of Duchenne Muscular Dystrophy. PloS one 10, e0117306 (2015). 31. Rodino-Klapac, L.R., et al. Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model. Human molecular genetics 22, 4929-4937 (2013). 32. Cushing, L., et al. MIR-29 is a Major Regulator of Genes Associated with Pulmonary Fibrosis. Am J Respir Cell Mol Biol (2010). 33. Roderburg, C., et al. Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis. Hepatology 53, 209-218 (2011). 34. Nevo, Y., et al. The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy. PloS one 6, el8049 (2011). 35. Rodino-Klapac, L.R., et al. A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy. J Transl Med 5, 45 (2007) . 36. Mulieri, L.A., Hasenfuss, G., Ittleman, F., Blanchard, E.M. & Alpert, N.R. Protection of human left ventricular myocardium from cutting injury with 2,3-butanedione monoxime. Circ Res 65, 1441-1449 (1989). 37. Rodino-Klapac, L.R., et al. Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery. Molecular therapy: the journal of the American Society of Gene Therapy 18, 109-117 (2010). 38. Grose, W.E., et al. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer. PloS one 7, e39233 (2012) . 39. Liu, M., et al. Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury. Mol Ther 11, 245-256 (2005). CLAIM 1. Recombinant AAVrh74 vector containing 5'-3' inverted terminal repeat (ITR), muscle-specific regulatory element, chimeric intron sequence, nucleotide sequence of SEQ ID NO: 7, poly-A tail and ITR. 2. The AAVrh74 recombinant vector according to claim 1, wherein the muscle-specific regulatory element contains the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11. 3. The AAVrh74 recombinant vector according to claim 1 or 2, further comprising a chimeric intron sequence between said nucleotide sequence and said muscle-specific regulatory element, wherein said chimeric intron sequence is shown at nucleotides 844-993 of SEQ ID NO: 9. 4. The recombinant AAVrh74 vector according to claim 1, additionally containing a poly-A tail on the 3' side of the specified nucleotide sequence, where the sequence of the specified poly-A tail is given in nucleotides 4585-4640 of SEQ ID NO: 9. 5. A composition for use in a method of treating muscular dystrophy in a subject, comprising a recombinant AAVrh74 according to any one of claims 1-4 and a pharmaceutically acceptable carrier. 6. A method of treating muscular dystrophy in a subject, comprising administering a composition according to claim 5. 7. A method of reducing or preventing fibrosis in a subject suffering from muscular dystrophy, comprising administering a composition according to claim 5. 8. A method for increasing muscle strength or muscle mass in a subject suffering from muscular dystrophy, comprising administering a composition according to claim 5. 9. The method according to any one of claims 6 to 8, wherein the recombinant AAVrh74 vector is administered by intramuscular injection, intravenous injection, parenteral administration, or systemic administration. 10. The method of claim 7 or 9, wherein the recombinant AAVrh74 vector is administered before fibrosis is detected in the subject, or before muscle strength decreases in the subject, or before muscle mass decreases in the subject. -