Combination therapy for the treatment of muscular dystrophy

Abstract

The invention described herein provides gene therapy vectors, e.g., adeno-associated virus (AAV) vectors that co-express a functional protein (e.g., a micro-human micro-dystrophin gene product) and one or more additional coding sequences for RNAi sequences (e.g., siRNA, shRNA, miRNA), antisense sequences, guide sequences for gene-editing enzymes (e.g., sgRNA for CRISPR / Cas9 or crRNA for CRISPR / Cas12a), and / or microRNA, and methods of using such vectors to treat subjects with muscular dystrophy, e.g., DMD / BMD.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 778,646, filed December 12, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Muscular dystrophies (MD) are a group of diseases that cause progressive weakness and loss of muscle mass. In MD, abnormal (mutated) genes do not produce functional wild-type proteins required for healthy muscle formation.

[0003] Muscular dystrophies severely impair the quality of life of affected patients. Duchenne muscular dystrophy (DMD) is one of the most severe muscle diseases, affecting 1 in 5,000 newborn boys. It is the best-characterized muscular dystrophies caused by mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs result from membrane fragility associated with the loss of sarcolemmal sheath-cytoskeleton linkages by DAPC.

[0004] Specifically, DMD is caused by mutations in the DMD gene, leading to a reduction in DMD mRNA and a lack of dystrophin or functional dystrophin, a 427-kDa sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51(6):919-928, 1987). The DAPC is composed of multiple proteins that form structural connections between the extracellular matrix (ECM) and the cytoskeleton in the sarcolemmal sheath via dystrophin, an actin-binding protein, and alpha-dystroglycan, a laminin-binding protein. These structural connections act to stabilize the sarcolemma during contraction and prevent contraction-induced damage.

[0005] Loss of dystrophin as a result of DMD gene mutations leads to disruption of the dystrophin glycoprotein complex and increased muscle membrane fragility. A series of events, including calcium influx into the sarcoplasm, activation of proteases and inflammatory cytokines, and mitochondrial dysfunction, leads to progressive muscle degeneration. Furthermore, neuronal nitric oxide synthase (nNOS) translocation contributes to tissue ischemia, increased oxidative stress, and impaired repair. Disease progression is characterized by increased myonecrosis, fibrosis, and fatty tissue replacement, as well as greater changes in fiber size seen in subsequent muscle biopsies.

[0006] Accumulating evidence suggests that intracellular Ca 2+ (Ca 2+ i These findings suggest that abnormal elevation of sarcoplasmic / endoplasmic reticulum Ca2+ is a critical early pathogenic event that initiates and perpetuates disease progression in DMD. 2+ Normal function of the SERCA pump requires >70% Ca transport from the cytosol. 2+ Reduced SERCA activity is therefore a key factor in Ca sca removal and proper muscle contraction in DMD. 2+ i It is considered a major cause of muscle overload and dysfunction.

[0007] Currently, there is no cure for DMD. Standard treatments include corticosteroids (such as prednisone or deflazacort) to stabilize muscle strength and function, prolong independent ambulation, and slow the progression of scoliosis and cardiomyopathy, bisphosphonates, and denosumab and recombinant parathyroid hormone.

[0008] With the advent of gene therapy, research and clinical trials for the treatment of DMD have focused on gene replacement or other gene therapies aimed at at least partially restoring dystrophin function. These include delivery of a functional copy of the dystrophin gene, e.g., a dystrophin minigene, or repair of the defective dystrophin gene product by exon skipping and nonsense suppression.

[0009] However, due to the wide range of effects caused by dystrophin mutations, there is a need to treat other secondary conditions associated with the primary dystrophin mutation. For example, loss of dystrophin leads to loss of the dystrophin-associated protein complex (DAPC), which in turn leads to the production of nitric oxide (NO) by nNOS and aberrant N-nitrosylation of HDAC2. Such aberrantly N-nitrosylated HDAC2 dissociates from chromatin, releasing the inhibition of specific microRNA cascades, which in turn leads to numerous downstream events, such as increased fibrosis and oxidative stress.

[0010] Specifically, with regard to fibrosis, loss of dystrophin leads to membrane fragility, resulting in disruption of the sarcolemmal sheath and calcium influx, triggering calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr. Neurol. 10(2):168-175, 1997). This uncontrolled cycle of muscle degeneration and regeneration ultimately depletes the muscle stem cell population (Sacco et al., Cell 143(7):1059-1071, 2010; Wallace et al., Annu Rev Physiol 71:37-57, 2009), leading to progressive muscle weakness, endomysial inflammation, and fibrotic scarring.

[0011] In the absence of membrane stabilization by dystrophin or microdystrophin, DMD manifests as an uncontrolled cycle of tissue injury and repair, ultimately resulting in replacement of lost muscle fibers with fibrous scar tissue via connective tissue proliferation.

[0012] Muscle biopsies performed at the minimum age at which DMD is diagnosed (e.g., 4–5 years of age) reveal significant connective tissue proliferation. Muscle fibrosis is detrimental in multiple ways. It reduces the normal passage of endomysial nutrients through the connective tissue barrier, diminishing blood flow and depriving muscles of vascularly derived nutrients. Functionally, it contributes to early loss of ambulation due to limb contractures. Over time, significant muscle fibrosis increases the challenge of treatment. This can be observed in muscle biopsies comparing connective tissue proliferation at successive time points. This process continues to worsen, leading to loss of ambulation, which accelerates and becomes uncontrollable, especially in wheelchair-dependent patients.

[0013] Thus, fibrotic infiltration is severe in DMD and represents a significant obstacle to any potential cure. In this regard, gene replacement therapy alone is usually hampered by the severe fibrosis already present in young children with DMD.

[0014] Fibrosis is characterized by the excessive deposition of ECM matrix proteins, such as collagen and elastin. ECM proteins are primarily produced from cytokines, such as TGF, released by activated fibroblasts in response to stress and inflammation. While the primary pathological hallmark of DMD is muscle fiber degeneration and necrosis, fibrosis as a pathological consequence has an equal impact. The excessive production of fibrous tissue limits muscle regeneration and contributes to the progressive muscle weakness of DMD patients.

[0015] In one study, the presence of fibrosis in the initial DMD muscle biopsy was highly correlated with poor motor outcome at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol 68(7):762-767, 2009). These results indicate that fibrosis is a major cause of muscle dysfunction in DMD and highlight the need to develop therapies that reduce fibrotic tissue.

[0016] Most antifibrotic treatments tested in mdx mice act to block profibrotic cytokine signaling through inhibition of the TGF pathway. MicroRNAs (miRNAs) are single-stranded RNAs of approximately 22 nucleotides that mediate gene repression at the post-transcriptional level by pairing with bases in the 3'UTR of mRNAs, inhibiting translation or promoting mRNA degradation. A 7-bp seed sequence at the 5' end of the miRNA targets the miRNA, and further recognition is provided by the remainder of the target sequence and its secondary structure. miRNAs play an important role in the pathology of muscle diseases and exhibit expression profiles that are uniquely dependent on the type of muscular dystrophy in question (Eisenberg et al., Proc Natl Acad Sci USA 104(43):17016-17021, 2007). Increasing evidence suggests that miRNAs are involved in fibrotic processes in many organs, including the heart, liver, kidneys, and lungs (Jiang et al., Proc Natl Acad Sci USA 104(43):17016-17021, 2007).

[0017] Recently, downregulation of miR-29 has been shown to contribute to myocardial fibrosis (Cacchiarelli et al., Cell Metab 12(4):341-351, 2010). Decreased expression of miR-29 has been genetically associated with muscle in human DMD patients (Eisenberg et al., Proc Natl Acad Sci USA 104(43):17016-17021, 2007).

[0018] The miR-29 family consists of three family members expressed from two bicistronic miRNA clusters. miR-29a is coexpressed with miR-29b (miR-29b-1), and miR-29c is coexpressed with a second copy of miR-29b (miR-29b-2). The miR-29 family shares a conserved seed sequence, with miR-29a and miR-29b each differing by only a single nucleotide from miR-29c. Furthermore, electroporation of miR-29 plasmids (miR-29a and miR-29b-1 clusters) into mdx mouse muscle reduced the expression levels of ECM components, collagen, and elastin, and significantly reduced collagen deposition in muscle within 25 days of treatment (Cacchiarelli et al., Cell Metab 12(4):341-351, 2010).

[0019] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length and contains 145 nucleotide inverted terminal repeats (ITRs).

[0020] AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cultured cells is noncytopathic, and natural infection in humans and other animals is asymptomatic and symptomless. Furthermore, AAV infects many mammalian cell types, making it possible to target many different tissues in vivo. Furthermore, AAV can transduce slow-dividing and non-dividing cells and persist as a transcriptionally active nuclear episome (extrachromosomal element) essentially for the lifetime of those cells. The AAV proviral genome is infectious as DNA cloned into a plasmid, allowing the construction of recombinant genomes. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb region of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is a very stable and abundant virus. It readily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less critical. AAV may also be lyophilized. Finally, cells infected with AAV are not resistant to superinfection.

[0021] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA 93:14082-14087 (1996); and Xiao et al., J Virol 70:8098-8108 (1996). See also Chao et al., Mol Ther 2:619-623 (2000) and Chao et al., Mol Ther 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA 94:13921-13926 (1997). Furthermore, Lewis et al., J Virol 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.

[0022] Although AAV-vector-based gene therapy has encouraged significant investment in the sector, significant challenges remain for commercialization: the production of recombinant viral vectors is considered complex, and scaling up production is a significant technical challenge and is seen as a major barrier to commercialization.

[0023] Specifically, reported clinical doses for AAV-based viral vectors range from 10 to 100 mg / patient, depending on the therapeutic area. 11 ~10 14Thus, from a broader perspective of gene therapy development, current scale-up approaches are insufficient to supply the number of doses required to progress to later phases (e.g., Phase II / III), thereby slowing the development of gene therapy drugs. This is supported by the fact that the majority of clinical trials are very small, involving <100 patients (and in some cases <10), and use adherent cell transfection processes that generate negligible amounts of product. The predicted amount of virus required for later phase progression is estimated based on current productivity (e.g., 5 × 10 cells from a single 10-layer cell factory). 11 vg), there are real concerns that this approach will be inadequate for the material requirements and market needs for later phases, even for ultra-rare diseases with high doses and small patient cohorts, let alone "standard" gene therapy indications.

[0024] As Clement and Grieger note in a recent review (Molecular Therapy-Methods & Clinical Development (2016) 3, 16002, doi:10.1038 / mtm.2016.2), "The use of rAAV in clinical settings highlights the urgent need for production and purification systems capable of generating extremely large quantities of highly pure rAAV particles. A typical FDA-approved investigational drug includes extensive preclinical studies for assessment of toxicology, safety, dosage, and biodistribution, and vector requirements often reach the range of 1E15 to 1E16 vector genomes. Producing such quantities, while technically feasible, still represents an incredible effort using current production systems." This problem is particularly urgent in the case of AAV vectors, which are desirably delivered systemically (as opposed to locally). In a recent paper, Adamson-Small et al. (Molecular Therapy - Methods & Clinical Development (2016) 3, 16031, doi:10.1038 / mtm.2016.31) stated: "Current limitations in vector production and purification hinder widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. ... This is particularly true for the treatment of congenital genetic disorders such as muscular dystrophies, where systemic gene transfer, often dependent on systemic administration of high AAV doses, may be necessary." Indeed, previous studies of rAAV in clinical trials for muscular dystrophies delivered vectors via intramuscular injection, often due to a lack of large-scale production capacity to generate the quantities necessary to support systemic administration. Systemic delivery of two AAV vectors in combination therapy poses an even greater challenge in terms of producing sufficient quantities of the high-quality AAV vectors required for the combination therapy.

[0025] Therefore, functional improvement in patients with DMD and other muscular dystrophies requires both gene restoration and alleviation of symptoms associated with several secondary cascades, such as fibrosis. Alternatively or additionally, muscular dystrophies may benefit from treatments that simultaneously target different disease-causing pathways. There is a need for methods to alleviate such secondary cascade symptoms (e.g., fibrosis) that can be combined with gene restoration methods for more effective treatment of DMD and other muscular dystrophies. Such combination therapies must also overcome significant clinical and commercial challenges, particularly in the systemic delivery of gene therapy vectors: producing sufficient quantities of the gene therapy vector to deliver both therapeutic components to target tissues. Summary of the Invention

[0026] The invention described herein provides viral vectors for gene therapy, the vectors comprising polynucleotide sequences that simultaneously encode a first polypeptide or a first RNA and a second polypeptide or a second RNA.

[0027] For example, the vector can simultaneously encode a first therapeutic protein and a second therapeutic RNA. However, either the first or second RNA, or both, may be non-coding RNAs that do not produce proteins or polypeptides. Such non-coding RNAs may be microRNAs (miRs), shRNAs (short hairpin RNAs), piRNAs, snoRNAs, snRNAs, exRNAs, scaRNAs, long ncRNAs such as Xist and HOTAIR, antisense RNAs, or precursors thereof, and preferably have therapeutic effects, such as those associated with diseases such as cancer, autism, Alzheimer's disease, cartilage-hair hypoplasia, hearing loss, and various forms of muscular dystrophy (MD), including Prader-Willi syndrome, particularly DMD / BMD.

[0028] Such non-coding RNAs may also be the guide RNA(s) of the CRISPR / Cas9 protein or the CRISPR RNA (crRNA) of the CRISPR / Cas12a (formerly Cpf1) protein.

[0029] Accordingly, in one aspect, the present invention provides a recombinant viral vector comprising: a) a polynucleotide encoding a functional gene or protein of interest (GOI), e.g., one effective for treating muscular dystrophy, wherein the polynucleotide comprises a 3'-UTR coding region and is immediately 3' to a heterologous intron sequence that enhances expression of the functional protein encoded by the polynucleotide; b) a regulatory element (e.g., a muscle-specific regulatory element) operably linked to the polynucleotide and driving its expression; and c) one or more coding sequences inserted into the intron sequence or the 3'-UTR coding region, wherein the one or more coding sequences independently encode an RNAi sequence (e.g., siRNA, shRNA, miRNA), an antisense sequence, a guide sequence for a gene-editing enzyme (e.g., a single-stranded guide RNA (sgRNA) for CRISPR / Cas9 or an acrRNA for CRISPR / Cas12a), a microRNA (miRNA), and / or a miRNA inhibitor.

[0030] In certain embodiments, the recombinant viral vector is a recombinant AAV (adeno-associated virus) vector or a recombinant lentiviral vector. In a related aspect, the invention provides a recombinant AAV (rAAV) vector comprising: a) a polynucleotide encoding a functional protein effective in treating muscular dystrophy, wherein the polynucleotide comprises a 3'-UTR coding region and is immediately 3' to a heterologous intron sequence that enhances expression of the functional protein encoded by the polynucleotide; b) a muscle-specific regulatory element operably linked to the polynucleotide and driving its expression; and c) one or more coding sequences inserted into the intron sequence or the 3'-UTR coding region, wherein the one or more coding sequences independently encode an RNAi sequence (siRNA, shRNA, miRNA), an antisense sequence, a microRNA (miRNA), and / or a miRNA inhibitor.

[0031] In certain embodiments, the invention described herein provides viral vectors, e.g., recombinant AAV vectors, that include: a) a dystrophin microgene or minigene encoding a functional micro-dystrophin protein (e.g., microD5), wherein the dystrophin microgene or minigene comprises a 3'-UTR coding region and is immediately 3' to a heterologous intron sequence that enhances expression of the dystrophin microgene or minigene; b) a muscle-specific regulatory element operably linked to and driving expression of the dystrophin microgene or minigene; and c) one or more (e.g., 1, 2, 3, 4, or 5) coding sequence(s) inserted into the intron sequence or the 3'-UTR coding region, wherein the one or more coding sequence(s) independently encode an RNAi sequence (e.g., siRNA, shRNA, miRNA), an antisense sequence, a microRNA (miRNA), and / or a miRNA inhibitor.

[0032] In certain embodiments, the functional dystrophin protein is microD5 and / or the muscle-specific regulatory element / promoter is a CK promoter. The present invention is based, in part, on the surprising discovery that the one or more coding sequence(s) can be inserted into a particular location, e.g., a heterologous intron, and simultaneously, both the functional protein (such as a dystrophin microgene or minigene product) and the one or more coding sequences can be expressed within an infected target cell (e.g., a muscle cell) without significantly reducing expression compared to a similar vector construct containing only the functional protein (e.g., the dystrophin minigene product) or only the one or more coding sequences.

[0033] In certain embodiments, the one or more coding sequences are inserted into the 3'-UTR coding region or after a polyadenylation (polyA) signal sequence (eg, AATAAA).

[0034] In certain embodiments, expression of the functional GOI is substantially unaffected in the presence of the one or more coding sequences (e.g., compared to an otherwise identical control construct in which the one or more coding sequences are not inserted).

[0035] In certain embodiments, in the recombinant AAV (rAAV) vector: a) the polynucleotide is a dystrophin minigene encoding a functional 5-spectrin-like repeat dystrophin protein (e.g., microD5, as described in U.S. Pat. No. 10,479,821, incorporated herein by reference), and / or b) the muscle-specific regulatory element is a CK promoter operably linked to and driving expression of the dystrophin minigene.

[0036] In certain embodiments, the one or more coding sequences comprise an exon-skipping antisense sequence that induces skipping of a defective dystrophin exon, e.g., skipping of any one of dystrophin exons 45-55, or dystrophin exons 44, 45, 51, and / or 53.

[0037] In certain embodiments, the microRNA is miR-1, miR-133a, miR-29c, miR-30c, and / or miR-206. For example, when the microRNA is miR-29c, the miR-29c optionally has a modified flanking backbone sequence that facilitates processing of the guide strand of miR-29c designed for the target sequence. The modified flanking backbone sequence may be derived from or based on other miR sequences, such as miR-30, -101, -155, or -451.

[0038] In certain embodiments, expression of the microRNA in the host cell is upregulated by at least about 1.5-15 fold (e.g., about 2-10 fold, about 1.4-2.8 fold, about 2-5 fold, about 5-10 fold, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 fold) compared to endogenous expression of the microRNA in the host cell.

[0039] In certain embodiments, the RNAi sequence is an shRNA against sarcolipin (shSLN). In certain embodiments, the one or more coding sequences encode one or more of the same or different shRNAs (shSLNs) against sarcolipin.

[0040] In certain embodiments, the shRNA reduces sarcolipin mRNA and / or sarcolipin protein expression by at least about 50%. In certain embodiments, the GOI is CRISPR / Cas9 and the guide sequence is an sgRNA, or the GOI is CRISPR / Cas12a and the guide sequence is a crRNA.

[0041] In certain embodiments, the RNAi sequence (siRNA, shRNA, miRNA), the antisense sequence, the CRISPR / Cas9 sgRNA, the CRISPR / Cas12a crRNA, and / or the microRNA are capable of targeting one or more target genes, such as inflammatory genes, activators of the NF-κB signaling pathway (e.g., TNF-α, IL-1, IL-1β, IL-6, receptor activator of NF-κB (RANK), and Toll-like receptors (TLRs)), NF-κB, NF-κB It antagonizes the function of downstream inflammatory cytokines induced by IL-1, histone deacetylases (e.g., HDAC2), TGF-β, connective tissue growth factor (CTGF), collagen, elastin, components of the extracellular matrix, glucose-6-phosphate dehydrogenase (G6PD), myostatin, phosphodiesterase-5 (PED-5) or ACE, VEGF decoy receptor type 1 (VEGFR-1 or Flt-1), and hematopoietic prostaglandin D synthase (HPGDS).

[0042] In certain embodiments, in patients with Fukuyama congenital muscular dystrophy (FCMD), the vector, e.g., the recombinant AAV (rAAV) vector, wherein a) the polynucleotide encodes a functional Fukutin (FKTN) protein, and / or b) the one or more coding sequences encode an exon-skipping antisense sequence that restores correct splicing of exon 10 of the defective FKTN gene.

[0043] In certain embodiments, in patients with merosin-deficient congenital muscular dystrophy type 1A (MDC1A), the vector, e.g., the recombinant AAV (rAAV) vector, a) the polynucleotide encodes a functional LAMA2 protein, and / or b) the one or more coding sequences encode exon-skipping antisense sequences that restore expression of the C-terminal G domain (exons 45-64) of the defective LAMA2 gene, particularly G4 and G5.

[0044] In certain embodiments, in a DM1 patient, the vector, e.g., the recombinant AAV (rAAV) vector, wherein a) the polynucleotide encodes a functional DMPK protein or a CLCN1 gene, and / or b) the RNAi sequence (siRNA, shRNA, miRNA), the antisense sequence, or the microRNA (miRNA) targets an expanded repeat of a mutant transcript of a defective DMPK gene or encodes an exon skipping antisense sequence that leads to skipping of exon 7A of the CLCN1 gene.

[0045] In certain embodiments, in a dysferlinopathy (LGMD2B or MM) patient, the vector, e.g., the recombinant AAV (rAAV) vector, wherein a) the polynucleotide encodes a functional DYSF protein, and / or b) one or more coding sequences encode an exon skipping antisense sequence that leads to skipping of exon 32 of the defective DYSF gene.

[0046] In certain embodiments, in an LGMD2C patient, the vector, e.g., the recombinant AAV (rAAV) vector, a) the polynucleotide encodes a functional SGCG protein, and / or b) one or more coding sequences encode an exon skipping antisense sequence that leads to skipping of exons 4-7 of a defective LGMD2C gene (e.g., one having a Δ-521T SGCG mutation).

[0047] In certain embodiments, the heterologous intron coding sequence is SEQ ID NO:1. In certain embodiments, the one or more coding sequences are inserted into the intron sequence.

[0048] In certain embodiments, expression of the functional protein is not adversely affected by the insertion of the one or more coding sequences. In certain embodiments, the vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. In certain embodiments, the vector is a derivative of a known serotype. In certain embodiments, the derivative may exhibit desired tissue specificity or tropism, a desired immunogenicity profile (e.g., not susceptible to attack by the immune system of a subject patient), or other desired properties for pharmaceutical compositions or gene therapy for various conditions.

[0049] In certain embodiments, the muscle-specific regulatory element is human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer-binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), C5-12, mouse creatine kinase enhancer element, fast skeletal troponin c gene element, slow cardiac troponin c gene element, slow troponin i gene element, hypoxia-inducible nuclear factor, steroid-inducible factor, or glucocorticoid response element (gre).

[0050] In certain embodiments, the muscle-specific regulatory element comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 of WO2017 / 181015 (hereby incorporated by reference).

[0051] Another aspect of the invention provides compositions comprising any of the vectors, eg, recombinant viral (AAV) vectors of the invention. In certain embodiments, the composition is a pharmaceutical composition further comprising a therapeutically compatible carrier, diluent, or excipient.

[0052] In certain embodiments, the therapeutically acceptable carrier, diluent, or excipient is a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride.

[0053] In certain embodiments, the composition contains at least 1.6×10 13 The dosage form is approximately 10 mL of aqueous solution containing the vector genome. In certain embodiments, the composition contains at least 2×10 per milliliter 12 The vector genome has the potency of

[0054] Another aspect of the invention provides a method of producing the subject composition, comprising producing the vector, e.g., the recombinant AAV vector, in a cell and lysing the cell to obtain the vector.

[0055] In certain embodiments, the vector is an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 vector.

[0056] Another aspect of the present invention provides a method of treating muscular dystrophy or dystrophinopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any one of the recombinant vectors, e.g., a recombinant AAV vector of the present invention, or any one of the compositions of the present invention.

[0057] In certain embodiments, the recombinant vector, e.g., the recombinant AAV vector or the composition, is administered by intramuscular injection, intravenous injection, parenteral administration, or systemic administration.

[0058] In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In certain embodiments, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy, myotonic dystrophy, and merosin-deficient congenital muscular dystrophy type 1A, facioscapulohumeral muscular dystrophy (FSHD), congenital muscular dystrophy (CMD), or limb-girdle muscular dystrophy (LGMDR5 or LGMD2C).

[0059] Another aspect of the invention provides kits for preventing or treating DMD or a related / associated disease in a subject, the kits comprising one or more vectors, e.g., recombinant AAVs described herein, or compositions described herein, instructions for use (written, printed, electronic / optical storage media, or online), and / or packaging. In certain embodiments, the kits also include a known MD (e.g., DMD) therapeutic agent for combination therapy.

[0060] It is understood that any one embodiment described herein, including those set forth solely in the examples or claims, may be combined with any one or more other embodiments of the present invention unless such combination is expressly disclaimed or is otherwise inappropriate. [Brief explanation of the drawings]

[0061] [Figure 1] 1 shows a schematic diagram (not to scale) illustrating a representative recombinant viral (e.g., lentiviral or AAV) vector comprising one or more (i.e., five, as shown) additional coding sequences for a gene of interest (GOI), such as a micro-dystrophin, mini-dystrophin, or dystrophin minigene (e.g., the 5-spectrin-like repeat micro-D5 dystrophin protein described below), and a non-protein-coding RNA (ncRNA), such as an shRNA, between two ITR sequences. The additional ncRNA (e.g., shRNA) coding sequences may be the same or different, and although in this diagram they appear to be within a heterologous intron sequence 5' to the gene of interest (GOI) coding region (e.g., the micro-dystrophin coding sequence), the location of the additional coding sequence is not limited to this. That is, the coding sequence may be located elsewhere within the AAV vector, for example, within the 3'-UTR region, or may be located in both the heterologous intron and the 3'-UTR region. During transcription of the AAV vector genome, a pre-processed mRNA is produced that contains the GOI or dystrophin minigene (e.g., microD5) mRNA and the additional coding sequence as a fusion RNA. After further processing, the GOI, for example, the dystrophin minigene mRNA (as an anti-DMD drug) and the additional coding sequence, for example, the shRNA shown, are separated. [Figure 2A]Figure 12 shows one specific embodiment of a recombinant viral (e.g., lentiviral or AAV) vector in which a single additional microRNA29c coding sequence is inserted into the 3'-UTR region. Transcription and further processing create the mRNA for micro-dystrophin micro-D5 (labeled "SGT-001") and a functional miR29c microRNA. Note that in this illustrative, non-limiting example, the TAG stop codon, the AATAAA polyA signal sequence, and the miR-29c insertion sequence (which happens to be CA) are all underlined. In this illustration, the miR29c coding sequence is depicted as being inserted after the polyA signal sequence, but it can also be inserted elsewhere, for example, in the 3'-UTR region of the mature mRNA before the polyA signal sequence. See also Figure 12. [Figure 2B] Another specific embodiment of a recombinant viral (e.g., lentiviral or AAV) vector is shown, in which a single additional sarcolipin (SLN) shRNA coding sequence (shSLN) is inserted into a heterologous intron. Transcription and further processing creates the mRNA of micro-dystrophin micro-D5 (designated SGT-001) and a functional sarcolipin shRNA. Again, the insertion location of the shSLN is for illustrative purposes only, and it can be inserted in other locations according to the present disclosure, for example, in the 3'-UTR region, or before or after the polyA signal sequence. [Figure 3] Figure 1 shows DAPI staining of nuclei and immunofluorescence staining of dystrophin in cells infected with an AAV vector encoding only microD5 (labeled SGT-001) microdystrophin (left), an AAV vector additionally encoding microRNA 29c with a heterologous intron (center), and an AAV vector additionally encoding sarcolipin shRNA with a heterologous intron (right). Percentage values ​​represent the transfection efficiency, or the percentage of successfully transfected cells. [Figure 4]1 is a schematic diagram showing an AAV vector encoding a sarcolipin-luciferase reporter fusion, with the target location of the shRNA against sarcolipin also shown. [Figure 5] This shows that when cells were co-transfected with an AAV vector expressing both microD5 and shSLN ("SGT001+SLN"), expression of a sarcolipin-luciferase fusion reporter in C2C12 cells was reduced by 86.8% compared to cells co-transfected with an AAV vector expressing only microD5 ("SGT001"). [Figure 6A] This shows that endogenous sarcolipin expression in C2C12 cells (6 days after transfection) was reduced by 55% in C2C12 cells transfected with an AAV vector encoding microD5 and shSLN (labeled "SGT001-shSLN") compared to C2C12 cells transfected with an AAV vector encoding microD5 only (labeled "SGT-001"). [Figure 6B] Immunofluorescence staining images of endogenous SLN expression are shown, and the data in Figure 6A was compiled based on this. [Figure 7] This shows that expression of shSLN in C2C12 cells (by transfection with an AAV vector encoding both microD5 dystrophin and shSLN) reduces the function of endogenous SLN, and calcium reuptake into the sarcoplasmic reticulum is affected over time. Controls include cells transfected with an AAV vector encoding only microD5 dystrophin but no shSLN, and untransfected cells. The relative fluorescence intensity on the y-axis is based on measurements of the fluorescence intensity of the calcium probe Fluo-8. [Figure 8A]The results show that expression of micro-dystrophin in C2C12 cells transfected with SGT-001-shSLN (an AAV vector encoding both micro-D5 dystrophin and shSLN) was delayed 1 day (1d) after transfection, i.e., at approximately 20% of the level, compared to that in C2C12 cells transfected with SGT-001 (an AAV vector encoding micro-D5 dystrophin) alone, but expression of the micro-D5 dystrophin minigene quickly caught up 6 days (6d) after transfection (within error). [Figure 8B] Immunofluorescence staining images of exogenous microD5 dystrophin minigene expression 1 day after transfection are shown, and the data in Figure 8A was compiled based on this. [Figure 8C] Immunofluorescence staining images of exogenous microD5 dystrophin minigene expression 6 days after transfection are shown, from which the data in Figure 8A was compiled. [Figure 9] Several exemplary shRNA designs for mouse SLN are shown. [Figure 10] Nucleotide sequence comparison between mouse (subject) and human (query) sarcolipin sequences and possible shRNA designs for mouse- or human-specific shRNAs, as well as shRNAs common to mouse and human, are shown. [Figure 11] Representative locations within an AAV vector encoding a dystrophin minigene (microD5, designated "SGT-001") are shown that can serve as insertion points for one or more coding sequences, such as the miR-29c coding sequence (as shown) or the coding sequence for an shRNA directed against SLNs. Specifically, while multiple locations within the introns of the SGT-001 minigene can be used, some locations (such as Imir2) may be more preferred due to their lack of adverse effects on dystrophin minigene expression. [Figure 12]1 is a schematic diagram (not to scale) showing one representative and non-limiting embodiment of a recombinant viral (e.g., lentiviral or AAV) vector of the present subject matter. In this particular embodiment shown, the regulatory element is the muscle-specific promoter CK8, and the GOI is a form of a functional DMD gene (microdystrophin or μDys). Coding sequences for RNAi, miRNA, etc. can be inserted into the region of the vector where the "transcript" is indicated, for example, into the intron before the GOI, the 3'-UTR region, or after the polyA signal sequence. Transcription by the promoter results in an initial fusion transcript. [Figure 13] Changes in relative miR-29c expression levels (compared to a control vector expressing μDys only) in human iPS-derived cardiomyocytes are shown for various recombinant viral (e.g., AAV) vectors encoding miR-29c, either as the sole coding sequence of the viral vector ("solo" constructs) or as part of the fusion constructs of the present disclosure ("fusion" constructs). [Figure 14] The relative expression levels of miR-29c in differentiated C2C12 cells or mouse cardiomyocytes are shown for various recombinant AAV vectors encoding miR-29c, either as the sole coding sequence of the viral vector ("solo" construct) or as part of the fusion constructs of the present disclosure ("fusion" construct). [Figure 15] The shSLN-μDys fusion construct of the present disclosure and several solo constructs expressing the same shSLN coding sequence demonstrate approximately 50% knockdown of mouse SLN protein expression levels (bottom panel). The top panel is a loading control. [Figure 16] The relative expression levels of siSLN (processed siRNA product derived from transcribed shSLN) in differentiated C2C12 myotubes or mouse cardiomyocytes are shown for various recombinant AAV vectors encoding shSLN, either as the sole coding sequence of the viral vector ("solo") or as part of the fusion construct of the present disclosure ("fusion"). [Figure 17]1 shows up to 90% knockdown of human SLN mRNA by several subject fusion constructs encoding shSLN in human iPS-derived cardiomyocytes. [Figure 18] Normalized μDys mRNA levels of several Hum-shSLN-μDys fusion constructs in human iPS-derived cardiomyocytes are shown. [Figure 19] 10 is an image of a denaturing agarose gel showing a nearly intact AAV genome in solo and fusion constructs carrying the miR-29c coding sequence. [Figure 20A] 1 shows approximately 1.4- to 2.8-fold upregulation of miR-29c expression in the left gastrocnemius muscle using the miR-29c-μDys fusion construct of the present invention in an AAV9 vector. [Figure 20B] 1 shows approximately 1.4- to 2.8-fold upregulation of miR-29c expression in the diaphragm using the miR-29c-μDys fusion construct of the present invention in an AAV9 vector. [Figure 20C] 1 shows approximately 1.4- to 2.8-fold upregulation of miR-29c expression in the left ventricle using the miR-29c-μDys fusion construct of the present invention in an AAV9 vector. [Figure 21] This shows that miR-29c upregulation fusion AAV9 vector does not reduce μDys expression at the RNA or protein level in gastrocnemius muscle. [Figure 22] Up to 50% downregulation of mSLN mRNA in the diaphragm, left gastrocnemius (left calf muscle), and atrium via AAV9-mediated expression of the shSLN-μDys fusion construct versus μDys-only AAV9 is shown. Up to 50% downregulation of mSLN mRNA was also observed in the tongue (data not shown). [Figure 23] Similar levels of μDys RNA / protein expression in the diaphragm via AAV9 shSLN-μDys fusion constructs were observed in the tongue and atria (data not shown). [Figure 24] 1 shows that AAV9 miR-29c solo and miR-29c-μDys fusion constructs reduce serum CK levels. [Figure 25] 1 shows that AAV9 miR-29c solo and miR-29c-μDys fusion constructs reduce serum TIMP1 levels. [Figure 26] 1 shows almost the same biodistribution of miR-29c or shSLN vector in gastrocnemius muscle derived from several AAV9 miR-29c-μDys fusion vectors or AAV9 shSLN-μDys fusion vectors. [Figure 27] 1 shows similar titers of AAV9 vectors for miR-29c-μDys fusion and shSLN-μDys fusion versus μDys solo constructs in the liver. [Figure 28] The additive effects of the fusion constructs of the present invention over the μDys construct alone in the diaphragm are shown based on their effects on two fibrosis marker genes. [Figure 29] The predicted 2D structure of a representative modified miR-29c construct based on the miR-30E scaffold sequence is shown. [Figure 30] The predicted 2D structure of a representative modified miR-29c construct based on the miR-101 backbone sequence is shown. [Figure 31] 1 shows the predicted 2D structure of a representative modified miR-29c construct based on the miR-451 backbone sequence. DETAILED DESCRIPTION OF THE INVENTION

[0062] Fibrosis and intracellular Ca 2+Without parallel approaches to treat various secondary cascade symptoms, such as abnormally elevated levels of ATP, the benefits of exon skipping, stop codon readthrough, or gene replacement therapy are unlikely to be fully realized. Even small molecule or protein replacement strategies may fail without approaches to alleviate the symptoms of such secondary cascade events, including myofibrosis. For example, a previous study in aged mdx mice with pre-existing fibrosis treated with AAV microdystrophin showed that complete functional recovery could not be achieved (Human Molecular Genetics 22:4929-4937, 2013). It is also known that the progression of DMD cardiomyopathy is accompanied by scarring and fibrosis of the ventricular wall.

[0063] The present invention relates, in part, to a gene therapy approach for treating patients that not only compensates for defects in dystrophin and its function by providing an alternative functional dystrophin minigene, but also uses one or more additional coding sequences in the same gene therapy vector to directly target one or more secondary cascade genes, thus achieving a combination therapy in one compact vector for systemic delivery.

[0064] In fact, the present invention, and in particular the recombinant AAV (rAAV) vectors of the present invention, are not limited to the treatment of DMD. The present invention can be applied to the treatment of other muscular dystrophies in which genes are defective. For example, the recombinant AAV (rAAV) vectors of the present invention can provide a functional protein and / or one or more coding sequences (non-coding RNA, e.g., RNAi sequences, antisense RNA, miRNA, etc.) for treating muscular dystrophy. The functional protein can provide a wild-type substitute for the defective gene product of muscular dystrophy, or a non-wild-type but effective substitute for treating muscular dystrophy (e.g., the 5-spectrin-like microD5 dystrophin minigene product).

[0065] Accordingly, in one aspect, the present invention provides a recombinant viral vector, e.g., a recombinant lentivirus or AAV (rAAV) vector, comprising: a) a polynucleotide encoding a functional protein effective for treating muscular dystrophy in a patient / subject / individual in need thereof, the polynucleotide comprising a 3'-UTR coding region and immediately 3' to a heterologous intron sequence that enhances expression of the functional protein encoded by the polynucleotide, wherein the wild-type counterpart of the functional protein is defective in muscular dystrophy or the functional protein is not wild-type but is nonetheless effective for treating muscular dystrophy; b) a regulatory element (e.g., a muscle-specific regulatory element) operably linked to the polynucleotide and driving its expression; and c) one or more coding sequences inserted into the intron sequence or the 3'-UTR coding region or elsewhere in the expression cassette for the functional protein, wherein the one or more coding sequences independently encode an RNAi sequence (siRNA, shRNA, miRNA), an antisense sequence, a microRNA (miRNA), and / or a miRNA inhibitor.

[0066] In a related aspect, the invention described herein can also be used as a viral vector to simultaneously deliver / express two or more components of an enzyme-based gene editing system, such as a target sequence-specific (modified) nuclease capable of creating a DNA double-strand break (DSB) at a target genomic site / sequence, and a donor or template sequence matching the (wild-type or desired) target genomic sequence. Such a system allows for the exploitation of endogenous homologous recombination (HR) processes within the target cell to delete a defective / undesired target genomic sequence and replace it with a wild-type or other desired sequence at the desired target genomic location.

[0067] For example, the target sequence-specific (engineered) nucleases may include meganucleases (such as those of the LAGLIDADG family) and variants thereof that recognize unique target genomic sequences, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas gene editing enzymes.

[0068] For example, in the case of CRISPR / Cas, the subject vectors can simultaneously deliver one or more gene editing guide sequence(s) carrying desired sequence(s) for targeting one or more target sequence(s) other than or in addition to the donor sequence, and a compatible editing enzyme that can be encoded as a GOI by the viral vector. Such viral delivery systems can be used to replace undesired sequences occurring in cells, tissues, or organisms with the desired sequences. An example of a CRISPR / Cas enzyme system is CRISPR / Cas9 or CRISPR / Cas12a (formerly Cpf1) and one or more guide sequences (e.g., single-stranded guide RNA or sgRNA in the case of Cas9, or crRNA in the case of Cas12a) required for targeting cells. Cas9 includes wild-type Cas9 and its functional variants. Some Cas9 variants are approximately the same size as the microdystrophin gene and can be functional GOIs encoded by the viral vectors of the present invention. Cas12a is even smaller than Cas9 and can similarly be encoded as a GOI. In certain embodiments, the Cas gene encoded by the viral construct may or may not have UTR and / or intron elements.

[0069] In a related aspect, the present invention provides recombinant lentiviral vectors for use in ex vivo or in vivo gene therapy. In ex vivo gene therapy, cultured host cells are transfected in vitro with the subject viral vectors to express a gene of interest, and then implanted into the body. In vivo gene therapy is a direct method of inserting genetic material into target tissue, with transduction occurring within the patient's own cells. Thus, a lentiviral vector of the invention can comprise: a) a polynucleotide encoding a functional protein effective in treating muscular dystrophy in a patient / subject / individual in need thereof, wherein the polynucleotide comprises a 3'-UTR coding region and is immediately 3' to a heterologous intron sequence that enhances expression of the functional protein encoded by the polynucleotide, wherein the wild-type counterpart of the functional protein is defective in muscular dystrophy, or wherein the functional protein is not wild-type but is nonetheless effective in treating muscular dystrophy; b) a regulatory element (e.g., a muscle-specific regulatory element) operably linked to the polynucleotide and drives its expression; and c) one or more coding sequences inserted into the intron sequence or the 3'-UTR coding region or elsewhere in the expression cassette, wherein the one or more coding sequences independently encode an RNAi sequence (siRNA, shRNA, miRNA), an antisense sequence, a microRNA (miRNA), and / or a miRNA inhibitor.

[0070] As used herein, depending on the context, the term "fusion" may have different meanings and includes fusion proteins, fusion RNA transcripts in which there may be two or more coding sequences (e.g., a coding sequence for a GOI and a coding sequence for one or more RNAi agents inserted / embedded in the 3-UTR region or intron sequence of the GOI), and fusion constructs in which the viral vector comprises the coding sequences for the GOI and the one or more RNAi agents.

[0071] In certain embodiments, the one or more coding sequences are inserted into the 3'-UTR coding region or after a polyadenylation (polyA) signal sequence (eg, AATAAA).

[0072] In certain embodiments, expression of the functional GOI is upregulated or downregulated due to the presence of the one or more coding sequences (e.g., compared to an otherwise identical control construct in which the one or more coding sequences are not inserted).

[0073] In certain embodiments, expression of the functional GOI is substantially unaffected in the presence of the one or more coding sequences (e.g., compared to an otherwise identical control construct in which the one or more coding sequences are not inserted).

[0074] As used herein, "muscular dystrophy (MD)" includes a group of diseases characterized by progressive weakness and loss of muscle mass due to abnormal genes or gene mutations that prevent the production of wild-type proteins necessary for healthy muscle formation. MD includes Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), congenital muscular dystrophies (CMD), particularly those with specific gene mutations, such as those described below, including Fukuyama congenital muscular dystrophy (FCMD) and merosin-deficient congenital muscular dystrophy type 1A (MDC1A), dysferlinopathies (LGMD2B and Miyoshi myopathy), myotonic dystrophy, limb-girdle muscular dystrophies (LGMD), such as LGMD2C, and facioscapulohumeral type (FSHD).

[0075] As used herein, the terms "patient," "subject," and "individual" are used interchangeably and include a mammalian (e.g., human) subject treated, diagnosed, and / or from whom a biological sample is obtained using the subject methods. Typically, the subject is afflicted with or likely to be afflicted with DMD and other related disorders described herein, and in some embodiments, DMD and related cardiomyopathies and dystrophic cardiomyopathies. In certain embodiments, the subject is a human child or adolescent (e.g., 18 years old, 15 years old, 12 years old, 10 years old, 8 years old, 5 years old, 3 years old, 1 year old, 6 months old, 3 months old, less than 1 month old, etc.). In certain embodiments, the child or adolescent is a male. In another particular embodiment, the subject is an adult (e.g., >18 years old), e.g., an adult male.

[0076] The full-length dystrophin gene is 2.6 mb and encodes 79 exons. The 11.5-kb coding sequence results in a 427-kD protein. Dystrophin can be divided into four major domains, including the N-terminal domain, the rod domain, the cysteine-rich domain, and the C-terminal domain. The rod domain can be further divided into 24 spectrin-like repeats and four hinges.

[0077] Functional "dystrophin minigenes" or "dystrophin microgenes" have fewer than 24 spectrin-like repeats and one or more hinge regions compatible with gene therapy delivery vectors (adenovirus and lentivirus) and are described in US7001761, US6869777, US8501920, US7892824, US10479821, and US10166272 (all incorporated herein by reference).

[0078] In one embodiment, the muscular dystrophy is DMD or BMD, and in the recombinant AAV (rAAV) vector: a) the polynucleotide is a dystrophin minigene encoding a functional 5-spectrin-like repeat dystrophin protein (such as the microD5 dystrophin protein described in US 10,479,821, incorporated herein by reference), and / or b) the muscle-specific regulatory element is a CK promoter operably linked to and driving expression of the dystrophin minigene.

[0079] As used herein, "microD5," "microdystrophin minigene encoded by SGT-001," or simply "SGT-001" refers to a specific modified five-repeat microdystrophin protein that contains, from N- to C-terminus, the N-terminal actin-binding domain of the human full-length dystrophin protein, hinge region 1 (H1), spectrin-like repeats R1, R16, R17, R23, and R24, hinge region 4 (H4), and a C-terminal dystroglycan-binding domain. The protein sequences of this five-repeat microdystrophin and related dystrophin minigenes are described in US 10,479,821 and WO 2016 / 115543, which are incorporated herein by reference.

[0080] In certain embodiments, the dystrophin minigene encodes a functional dystrophin protein that differs from microD5, for example, in terms of the specific spectrin-like repeats and / or the number of spectrin-like repeats (e.g., contains at least 4, 5, or 6 spectrin-like repeats of human dystrophin, preferably 1, 2, or 3 extreme N- and / or C-terminal repeats). One or more spectrin-like repeats of human dystrophin can also be replaced with spectrin-like repeats from utrophin or spectrin. In certain embodiments, the dystrophin minigene is smaller than the 5 kb packaging limit of AAV viral vectors, preferably 4.9 kb, 4.8 kb, 4.6 kb, 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, or 4 kb or less.

[0081] In certain embodiments, the dystrophin minigene encodes a microdystrophin protein that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identical to, for example, microD5, and the protein retains microdystrophin activity.

[0082] In certain embodiments, the micro-dystrophin is encoded by the 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 even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity with the polynucleotide sequence that encodes the micro-D micro-dystrophin.The polynucleotide is optionally codon-optimized for expression in mammals, for example, humans.

[0083] In certain embodiments, the nucleotide sequence hybridizes under stringent conditions to a nucleic acid sequence encoding micro-D5 micro-dystrophin, or its complement, and encodes a functional micro-dystrophin protein.

[0084] The term "stringent" refers to conditions commonly understood to be stringent in the art. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989).

[0085] More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agents) can also be used, although the rate of hybridization will be affected. When deoxyoligonucleotide hybridization is involved, further exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

[0086] Other agents may be included in the hybridization and wash buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodS04 (SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents can be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4, although the rate of hybridization is largely independent of pH under typical ionic strength conditions. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art to accommodate these variables and allow DNAs of different sequence relatedness to form hybrids.

[0087] Additional dystrophin minigene sequences can be found, for example, in US2017 / 0368198 (incorporated herein by reference), and SEQ ID NO: 7 of WO2017 / 181015 (incorporated herein by reference).

[0088] In certain embodiments, the nucleotide sequence encoding any dystrophin minigene, such as microD5, can be any based on the protein sequence of the present disclosure. Preferably, the nucleotide sequence is codon-optimized for human expression.

[0089] The micro-dystrophin protein stabilizes muscle membranes during muscle contraction, e.g., micro-dystrophin acts as a shock absorber during muscle contraction. In certain embodiments, at least one of the one or more coding sequences targets one of the secondary cascade genes in DMD.

[0090] For example, in certain embodiments, at least one of the one or more coding sequences encodes a microRNA, such as miR-1, miR-133a, miR-29, particularly miR29c, miR-30c, and / or miR-206. For example, miR-29c directly reduces three major components of connective tissue (e.g., collagen 1, collagen 3, and fibronectin), reducing fibrosis.

[0091] As used herein, "fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in injured tissues, such as skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas. Deposited ECM components include fibronectin and collagen, e.g., collagen 1, collagen 2, or collagen 3.

[0092] As used herein, "miR-29" refers to one of miR-29a, -29b, or -29c. In certain embodiments, miR-29 refers to miR-29c.

[0093] Without wishing to be bound by any particular theory, it is believed that expressed miR29 (such as miR-29a, miR-29b, or miR-29c) binds to the 3'UTR of collagen and fibronectin genes and downregulates the expression of these target genes.

[0094] In another embodiment, at least one of the one or more coding sequences encodes an RNAi sequence, such as an shRNA (shSLN) against sarcolipin. The one or more coding sequences may encode the same or different shRNAs (shSLN) against sarcolipin. In certain embodiments, the shRNA reduces the expression of sarcolipin mRNA and / or sarcolipin protein by at least about 50%.

[0095] As used herein, "sarcolipin (SLN)," "sarcolipin protein," "SLN protein," "sarcolipin polypeptide," and "SLN polypeptide" are used interchangeably and include the expression product of the SLN gene, e.g., the native human SLN protein having the amino acid sequence (MGINTRELFLNFTIVLITVILMWLLVRSYGY) (SEQ ID NO: 1), accession number NP_003054.1. The term preferably refers to the human SLN. The term may also be used to refer to variant SLN proteins, which differ from SEQ ID NO: 1 by 1, 2, 3, 4, 5, 6, 7, or 8 amino acids, optionally at residues 2-5, 10, 14, 17, 20, and 30, preferably 2-5 and 30. The term may also be used to refer to a variant SLN protein that is identical to SEQ ID NO: 1 at residues 6-29 or that differs by up to one, two, or three conservative substitutions at residues 6-29, e.g., L→I and / or I→V. Optionally, the variant SLN has a G30Q substitution. The variant exhibits the functional activity of a native SLN protein, including phosphorylation, dephosphorylation, nitrosylation, and / or ubiquitination of SLN, or binding to SERCA, and / or Ca2+, e.g., derived from ATP hydrolysis. 2+ Uncoupling of transport may involve slowing the rate of calcium uptake into the sarcoplasmic reticulum by SERCA or its role in regulating energy metabolism and weight gain.

[0096] As used herein, "SLN gene," "SLN polynucleotide," and "SLN nucleic acid" are used interchangeably and include naturally occurring human SLN-encoding nucleic acid sequences, e.g., the naturally occurring human SLN gene (RefSeq Accession: NM_003063.2), nucleic acids having sequences from which SLN cDNA can be transcribed, and / or polynucleotides encoding allelic variants and homologs of the foregoing, e.g., any of the variant SLNs described herein. The terms include double-stranded DNA, single-stranded DNA, and RNA.

[0097] In another embodiment, the one or more additional coding sequences of the subject vectors may target any other gene associated with one of the secondary cascade events resulting from a defect in the dystrophin gene, such as inflammatory genes, activators of the NF-κB signaling pathway (e.g., TNF-α, IL-1, IL-1β, IL-6, receptor activator of NF-κB (RANK), and Toll-like receptors (TLRs)), NF-κB, downstream inflammatory cytokines induced by NF-κB, histone deacetylases (e.g., HDAC2), TGF-β, connective tissue growth factor (CTGF), collagen, elastin, components of the extracellular matrix, glucose-6-phosphate dehydrogenase (G6PD), myostatin, phosphodiesterase-5 (PED-5) or ACE, VEGF decoy receptor type 1 (VEGFR-1 or Flt-1), and hematopoietic prostaglandin D synthase (HPGDS). The one or more further coding sequences may be RNAi sequences (siRNA, shRNA, miRNA), antisense sequences, and / or microRNAs that antagonize the function of said target gene.

[0098] The subject recombinant vector designs can simultaneously target one or more (eg, 1, 2, 3, 4, 5) such secondary cascade genes or pathways, eg, SLN, microRNA, etc.

[0099] For example, in certain embodiments, one of the additional coding sequences of the subject vectors downregulates expression of SLN, thus preventing intracellular Ca2+ deficiency, a secondary defect in dystrophic muscle, by increasing calcium reuptake by SERCA. 2+ The nucleic acid sequence may be an RNAi sequence (siRNA, shRNA, miRNA) or an antisense sequence designed to at least partially alleviate the abnormal elevation of ribonucleotides.

[0100] In certain alternative embodiments, instead of or in addition to targeting one of the secondary cascade genes, at least one of the one or more coding sequences may be an exon-skipping antisense sequence that induces skipping of a defective endogenous dystrophin exon, e.g., any one of dystrophin exons 45-55, or dystrophin exons 44, 45, 51, and / or 53, thereby further enhancing the therapeutic effect of the dystrophin minigene (e.g., microD5).

[0101] As used herein, "exon skipping" or "splice switching" antisense oligonucleotide (AON) is a type of antisense sequence that is RNase-H resistant, and acts to regulate pre-mRNA splicing and correct the splicing defect of the pre-mRNA. In antisense-mediated exon skipping therapy, AON is usually used to block specific splicing signals and induce the specific skipping of a specific exon. This results in the correction of the reading frame of the mutant transcript, which can be translated into a partially functional protein with internal defects.

[0102] In certain aspects, the present invention provides recombinant AAV (rAAV) vectors encoding both a dystrophin minigene coding sequence (such as microD5 / SGT-001) and one or more additional sequences for targeting one or more additional target genes involved in the secondary cascade resulting from loss of dystrophin function. Such constructs include both a dystrophin minigene and one or more additional coding sequences inserted into a heterologous intron 5' of the dystrophin minigene and / or the 3'-UTR region of the dystrophin minigene.

[0103] Specifically, in one aspect, the present invention provides a recombinant AAV (rAAV) vector comprising: a) a dystrophin minigene encoding a functional micro-dystrophin protein, wherein the dystrophin minigene comprises a 3'-UTR coding region and is immediately 3' to a heterologous intron sequence that enhances expression of the dystrophin minigene; b) a muscle-specific regulatory element operably linked to and driving expression of the dystrophin minigene; and c) one or more (e.g., 1, 2, 3, 4, or 5) coding sequence(s) inserted into the intron sequence or the 3'-UTR coding region, wherein the one or more coding sequence(s) independently encode an RNAi sequence (siRNA, shRNA, miRNA), an antisense sequence, a microRNA (miRNA), and / or a miRNA inhibitor.

[0104] For example, the rAAV vector can include a polynucleotide sequence that expresses miR-29 (e.g., miR-29c), such as a nucleotide sequence comprising a miR-29c target guide strand (ACCGATTTCAAATGGTGCTAGA, SEQ ID NO: 3 of WO2017 / 181015, which is incorporated herein by reference), a miR-29c guide strand (TCTAGCACCATTTGAAATCGGTTA, SEQ ID NO: 4 of WO2017 / 181015, which is incorporated herein by reference), and a native miR-30 backbone and stem loop (GTGAAGCCACAGATG, SEQ ID NO: 5 of WO2017 / 181015, which is incorporated herein by reference).

[0105] An exemplary polynucleotide sequence comprising the cDNA of miR-29c in a miR-30 backbone is shown in SEQ ID NO: 2 and FIG. 1 of WO2017 / 181015 (hereby incorporated by reference).

[0106] In certain embodiments, the microRNA-29 coding sequence encodes miR-29c. In certain embodiments, miR-29c optionally has a modified flanking backbone sequence that facilitates the processing of the guide strand of miR-29c designed for the target sequence. For example, the modified flanking backbone sequence may be derived from or based on miR-30 (miR-30E), -101, -155, or -451.

[0107] In certain embodiments, the microRNA is miR-1, miR-133a, miR-30c, and / or miR-206. In certain embodiments, expression of the microRNA in the host cell is upregulated by at least about 1.5-15 fold (e.g., about 2-10 fold, about 1.4-2.8 fold, about 2-5 fold, about 5-10 fold, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 fold) compared to endogenous expression of the microRNA in the host cell.

[0108] In certain embodiments, vectors of the present invention encode antisense or RNAi sequences (e.g., siRNA, shRNA, miRNA) that antagonize the function of sarcolipin (SLN). In certain embodiments, vectors of the present invention encode shRNA (shSLN) that antagonize the function of sarcolipin. Exemplary shSLN sequences include those disclosed in Figures 9 and 10 (e.g., the underlined sequences in Figure 9 and the highlighted sequences in Figure 10). Further exemplary shSLN sequences include SEQ ID NOs: 7-11, as disclosed in WO2018 / 136880 (incorporated herein by reference).

[0109] The invention also relates, in part, to gene therapy vectors, e.g., lentiviruses or AAVs, that express the one or more coding sequence(s) and the dystrophin minigene, and methods for delivering same to muscle to alleviate and / or prevent secondary cascade symptoms while restoring dystrophin function.

[0110] In one embodiment, the muscular dystrophy is a congenital muscular dystrophy (CMD) associated with a known genetic defect, such as the fukutin gene or the FKRP (fukutin-related protein) gene. Thus, in certain embodiments, the congenital muscular dystrophy is Fukuyama congenital muscular dystrophy (FCMD).

[0111] Congenital muscular dystrophies (CMDs) are a group of muscular dystrophies that are evident at or near birth. In certain embodiments, the methods and rAAVs of the present invention target CMDs, particularly titin (CMD with cardiomyopathy), SEPN1 (CMD with desmin inclusion or (early) CMD with spinal rigidity), integrin-alpha7 (CMD with integrin alpha7 mutations), integrin-alpha9 (CMD with joint hyperlaxity), plectin (CMD with familial junctional epidermolysis bullosa), fukutin (Fukuyama CMD or MDDGA4), fukutin-related protein (FKRP) (CMD with muscle hypertrophy), and rAAVs of the present invention target CMDs, particularly titin (CMD with cardiomyopathy), SEPN1 (CMD with desmin inclusion or (early) CMD with spinal rigidity), integrin-alpha7 (CMD with integrin alpha7 mutations), integrin-alpha9 (CMD with joint hyperlaxity), plectin (CMD with familial junctional epidermolysis bullosa), fukutin (Fukuyama CMD or MDDGA4), fukutin-related protein (FKRP) (CMD with muscle hypertrophy). MD or MDC1C), LARGE (MDC1D), DOK7 (CMD with myasthenic syndrome), lamin A / C (CMD with spinal rigidity and lamin A / C abnormalities), SBP2 (CMD with spinal rigidity and selenoprotein deficiency), choline kinase beta (CMD with structural mitochondrial abnormalities), laminin alpha 2 (merosin-deficient CMD or MDC1A), POMGnT1 (Santavuori muscle-eye-brain disease), COLGA1, COL6A2, or COL6A3 (Ullrich It can be used to treat CMD with known genetic defects in genes such as B3GNT1 (Walker-Warburg syndrome: MDDGA type), B3GNT1 (Walker-Warburg syndrome: MDDGA type), POMT1 (Walker-Warburg syndrome: MDDGA type 1), POMT2 (Walker-Warburg syndrome: MDDGA type 2), ISPD (MDDGA3, MDDGA4, MDDGB5, MDDGA6, and MDDGA7), GTDC2 (MDDGA8), TMEM5 (MDDGA10), B3GALNT2 (MDDGA11), or SGK196 (MDDGA12).

[0112] Thus, the lentiviral or rAAV vectors of the invention can include a polynucleotide encoding any of the wild-type genes (such as those listed above) defective in a CMD, or a functional equivalent thereof, to treat a CMD in a subject in need thereof. The one or more additional coding sequences can encode RNAi sequences (siRNA, shRNA, miRNA), antisense sequences, or microRNAs (miRNAs) that eliminate or modify the mutant CMD gene, or secondary cascade genes that are upregulated by the loss of the wild-type gene function.

[0113] For example, Fukuyama congenital muscular dystrophy (FCMD) is caused by a mutated FKTN gene, and the one or more additional coding sequences encode an exon-skipping antisense oligonucleotide to restore correct exon 10 splicing in the defective FKTN gene in the patient.

[0114] In another example, the congenital muscular dystrophy is merosin-deficient congenital muscular dystrophy type 1A (MDC1A), which is caused by a mutation in exon 65 of the LAMA2 gene.

[0115] Thus, the lentiviral or rAAV vectors of the invention can comprise a polynucleotide encoding a functional LAMA2 protein. The one or more additional coding sequences can encode exon-skipping antisense sequences that restore expression of the C-terminal G domain (exons 45-64) of LAMA2, particularly G4 and G5, which are most important for mediating interaction with α-dystroglycan. For example, exon 4 of the mutant LAMA2 gene can be skipped to treat MDC1A.

[0116] In one embodiment, the muscular dystrophy is myotonic dystrophy (DM), eg, DM1 or DM2. Therefore, the lentivirus or rAAV vector of the present invention may contain a polynucleotide encoding a defective functional myotonic dystrophy protein kinase (DMPK) protein in DM1 or a functional CCHC-type zinc finger nucleic acid binding protein gene (CNBP) protein in DM2. The one or more additional coding sequences may encode an RNAi sequence (siRNA, shRNA, miRNA), an antisense sequence, or a microRNA (miRNA) that can target the expanded repeat of the mutant transcript of the DMPK gene or CNBP gene and be used for degradation by RNase. The one or more additional coding sequences may also encode an exon skipping antisense sequence that causes exon 7A skipping of the CLCN1 gene in DM1 patients.

[0117] In one embodiment, the muscular dystrophy is a dysferlinopathy caused by mutations in the dysferlin (DYSF) gene, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM).

[0118] Thus, the lentiviral or rAAV vectors of the invention can comprise a polynucleotide encoding a functional DYSF protein defective in LGMD2B or MM, and the one or more additional coding sequences can also encode an exon-skipping antisense sequence that results in skipping of exon 32 of the DYSF gene defective in dysferlinopathy patients.

[0119] In one embodiment, the muscular dystrophy is limb-girdle muscular dystrophy (LGMD) and is caused by a mutation in any of the four sarcoglycan genes, namely, alpha (LGMD2D), beta (LGMD2E), gamma (LGMD2C), and delta (LGMD2F), particularly gamma sarcoglycan (LGMD2C), which is encoded by the SGCG gene.

[0120] Thus, a lentiviral or rAAV vector of the invention can include a polynucleotide encoding a functional sarcoglycan protein defective in LGMD, e.g., an SGCG gene defective in LGMD2C. The one or more additional coding sequences can also encode an exon skipping antisense sequence that results in skipping of exons 4-7 of a defective LGMD2C gene, e.g., one with a Δ-521T SGCG mutation.

[0121] In one embodiment, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD), which is caused by a mutation in the DUX4 gene. Thus, the one or more additional coding sequences may encode an RNAi sequence (siRNA, shRNA, miRNA), an antisense sequence, or a microRNA (miRNA) that reduces the expression of DUX4 or a downstream target, such as PITX1.

[0122] In certain embodiments, the one or more additional coding sequences encode exon-skipping antisense sequences that target the 3'-UTR of DUX4 and reduce its expression, because the DUX4 coding sequence is located entirely in the first exon of the gene, and exon skipping that targets elements of the 3' UTR of the mRNA can disrupt permissive polyadenylation or disrupt splicing of intron 1 or 2, thus disrupting functional DUX4 mRNA.

[0123] Facioscapulohumeral muscular dystrophy (FSHD) is an inherited autosomal dominant disorder clinically characterized by progressive muscle degeneration. It is the third most common muscular dystrophy after Duchenne muscular dystrophy (DMD) and myotonic dystrophy. FSHD is genetically characterized by pathogenic truncation of a subset of macrosatellite repeats on chromosome 4, resulting in abnormal expression of the double homeobox protein 4 (DUX4) gene.

[0124] There are two types of FSHD: FSHD1 and FSHD2. FSHD1 is the most common form, affecting more than 95% of all FSHD patients. Genetic analysis has linked FSHD1 to a genetic truncation of the macrosatellite D4Z4 repeat sequence on chromosome 4. FSHD2, on the other hand, has a normal number of D4Z4 repeats but instead contains a heterozygous mutation in the SMCHD1 gene, a chromatin modifier, on chromosome 18p. Patients with FSHD1 and FSHD2 share similar clinical features.

[0125] Current drug therapies do not cure FSHD but focus on managing FSHD symptoms, including the myostatin inhibitor raspatercept and anti-inflammatory biologics (ATYR1940). The principle of anti-inflammatory biologics is to suppress inflammation, which is commonly seen in muscle pathology in FSHD patients, and slow phenotypic progression. Therefore, one or more coding sequences of the present subject matter may encode RNAi reagents or antisense RNAs directed against genes in the myostatin or inflammatory pathways. At the same time, the RNAi reagents, e.g., small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), or microRNAs (miRNAs), or antisense oligonucleotides, can be used to knock down the expression of the myopathic DUX4 gene and its downstream molecules, such as paired-like homeodomain transcription factor 1 (PITX1). Indeed, in vitro studies have shown that DUX4 mRNA expression was successfully suppressed by administering antisense oligonucleotides to primary skeletal muscle cells from FSHD patients and by using miRNA against DUX4 delivered using AAV vectors in a DUX4 mouse model. Furthermore, successful systemic suppression of PITX1 expression has already been demonstrated in vivo.

[0126] In certain embodiments, the one or more additional coding sequences can encode the same sequence (e.g., siRNA, shRNA, miRNA, or antisense), and thus the copy number of the additional coding sequences can be adjusted or fine-tuned based on administration considerations.

[0127] In certain embodiments, the one or more additional coding sequences can encode different sequences and target different targets, or can target the same target.For example, in certain embodiments, one additional coding sequence is the antisense of target, and another additional coding sequence is the shRNA of the same target.Alternatively, two additional coding sequences are both shRNA, but they target different regions of the same target.

[0128] In certain embodiments, expression of the functional protein, eg, a dystrophin minigene product, is not adversely affected by the insertion of the one or more coding sequence(s). By the early 1990s, it was discovered that many intronless transgenes, while fully expressed in tissue culture cells in vitro, failed to express the same transgene in vivo (e.g., in transgenic mice carrying the transgene), and that insertion of certain heterologous intron sequences between the promoter and the (intronless) coding sequence of the transgene greatly enhanced transgene expression in vivo.

[0129] In particular, Palmiter et al. (Proc. Natl. Acad. Sci. USA 88:478-482, 1991, incorporated herein by reference) showed that several heterologous introns inserted between the metallothionein promoter and the growth hormone transgene improved transgene expression, and proposed the addition of certain heterologous introns as a general strategy for improving transgene expression. These include heterologous introns selected from the native rGH first intron, intron A of the rat insulin II (rIns-II) gene, intron B of the hβG gene, and the SV40 small t intron.

[0130] Similar findings were confirmed by Choi et al. (Mol. Cell. Biol. 11(6):3070-3074, 1991, incorporated herein by reference). They reported that in transgenic mice carrying the human histone H4 promoter linked to the bacterial gene for chloramphenicol acetyltransferase (CAT), the presence of a 230-bp heterologous hybrid intron in the transcription unit significantly enhanced CAT activity (5- to 300-fold compared to a similar transgene with the intervening sequence precisely deleted). This hybrid intron, consisting of an adenovirus splice donor and an immunoglobulin G splice acceptor, stimulated expression in a wide range of tissues in the animals. Because the hybrid intron stimulated the expression of tissue plasminogen activator and factor VIII in tissue culture, Choi concluded that the enhancement seen in these mice was unlikely to be specific to CAT but instead was generally applicable to the expression of any cDNA in transgenic mice.

[0131] Thus, in certain embodiments, the heterologous intron in the lentiviral or rAAV vector of the present subject matter is selected from the group consisting of the first intron of native rGH, intron A of the rat insulin II (rIns-II) gene, intron B of the hβG gene, the small t intron of SV40, and the hybrid intron of Choi.

[0132] In one particular embodiment, the heterologous intron sequence is SEQ ID NO:1: GTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG.

[0133] In certain embodiments, the one or more additional coding sequences are inserted entirely into the heterologous intron sequence (SEQ ID NO: 1), or entirely into the 3'-UTR region, or into both regions. For example, the microRNA-29c coding sequence can be inserted into the intron coding sequence as shown in SEQ ID NO: 2 below: GTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGATCTCTTACACAGGCTGACCGATTTCTCCTGGTGTTCAGAGTTCTGTTTTTGTCTAGCACCATTTGAAATCGGTTATGATGTAGGGGGAAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAG.

[0134] The miR-29c sequence in SEQ ID NO: 2 is ATCTCTTACACAGGCTGACCGATTTCTCCTGGTGTTCAGAGTCTGTTTTTGTCTAGCACCATTTGAAATCGGTTATGATGTAGGGGGA (SEQ ID NO: 3).

[0135] In certain embodiments, the lentivirus or rAAV further comprises two lentivirus or AAV LTR / ITR sequences flanking the polynucleotide (such as the dystrophin minigene) and the additional coding sequence(s).

[0136] In certain embodiments, the lentivirus or rAAV vector of the present invention can be operably linked to a muscle-specific regulatory element. For example, the muscle-specific regulatory element can be human skeletal actin gene element, cardiac actin gene element, muscle cell-specific enhancer-binding factor MEF, muscle creatine kinase (MCK), tMCK (truncated MCK), myosin heavy chain (MHC), C5-12 (synthetic promoter), mouse creatine kinase enhancer element, fast skeletal troponin C gene element, slow cardiac troponin C gene element, slow troponin I gene element, hypoxia-inducible nuclear factor, steroid-inducible factor, or glucocorticoid response element (GRE).

[0137] In certain embodiments, the muscle-specific regulatory element is 5' to the heterologous intron sequence, which is 5' to the dystrophin minigene, which comprises a 3'-UTR region containing a translation stop codon (e.g., TAG), a polyA adenylation signal (e.g., AATAAA), and an mRNA cleavage site (e.g., CA).

[0138] In certain embodiments, the muscle-specific regulatory element comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 of WO2017 / 181015. SEQ ID NO: 10 of WO2017 / 181015:

[0139] [ka]

[0140] SEQ ID NO: 11 of WO2017 / 181015:

[0141] [ka]

[0142] In certain embodiments, the rAAV vectors of the invention can be operably linked to muscle-specific regulatory elements comprising the nucleotide sequence of the MCK enhancer (see SEQ ID NO: 10 of WO2017 / 181015, which is incorporated herein by reference) and / or the sequence of the MCK promoter (see SEQ ID NO: 11 of WO2017 / 181015, which is incorporated herein by reference).

[0143] In certain embodiments, the rAAV further comprises a promoter operably linked to and capable of driving the transcription of the dystrophin minigene and the additional coding sequence.

[0144] An exemplary promoter is the CMV promoter. In certain embodiments, the rAAV further comprises a polyA adenylation sequence for inserting a polyA sequence into the transcribed mRNA.

[0145] In certain embodiments, the rAAV vectors of the invention are of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0146] Another aspect of the invention provides a method for producing a viral vector, e.g., an rAAV vector of the invention, comprising culturing cells transfected with any viral vector, e.g., an rAAV vector of the invention, and recovering the virus, e.g., rAAV particles, from the supernatant of the transfected cells.

[0147] Another aspect of the invention provides a viral particle comprising any of the viral vectors, eg, a recombinant AAV vector of the invention. Another aspect of the invention provides a method for producing a functional protein (such as a microdystrophin protein) defective in or effective in treating a muscular dystrophy, and one or more additional coding sequence(s), comprising infecting a host cell with a subject recombinant AAV vector that co-expresses the functional protein of the invention (e.g., microdystrophin) and the coding sequence product (e.g., RNAi, siRNA, shRNA, miRNA, antisense, microRNA or inhibitor thereof) in the host cell.

[0148] Another aspect of the present invention provides a method of treating a muscular dystrophy (such as DMD or BMD) or a dystrophinopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a viral vector, e.g., a recombinant AAV vector of the present invention, or a composition of the present invention.

[0149] The present invention contemplates administering any of the viral vectors, e.g., the AAV vectors of the invention, to a patient diagnosed with a dystrophinopathy or muscular dystrophy, e.g., DMD or BMD or any other MD, particularly a defective dystrophin-associated muscular dystrophy, preferably before the subject exhibits one or more secondary cascade symptoms, such as fibrosis, or before the subject experiences a loss of muscle strength, or before the subject experiences a loss of muscle mass.

[0150] The present invention also contemplates administering any of the viral vectors, e.g., the rAAV of the present invention, to subjects suffering from a dystrophinopathy or muscular dystrophy, e.g., DMD or BMD or any other MD, particularly a dystrophin-associated muscular dystrophy, and who have already developed one or more secondary cascade symptoms, such as fibrosis, to prevent or slow further disease progression in these subjects.

[0151] Another aspect of the present invention provides a recombinant viral vector, e.g., an AAV vector, comprising a nucleotide sequence encoding a functional protein deficient in or effective in treating a muscular dystrophy (e.g., a microdystrophin protein) and one or more additional coding sequences.

[0152] In certain embodiments, the present invention provides an rAAV comprising a nucleotide sequence having at least 85%, 90%, 95%, 97%, or 99% identity to a nucleotide sequence encoding a functional micro-dystrophin protein, e.g., micro-D5.

[0153] The viral vector, e.g., rAAV vector, may contain a muscle-specific promoter, e.g., the MCK promoter, a heterologous intron sequence effective for enhancing expression of the dystrophin gene, the coding sequence of the microdystrophin gene, a poly(A) adenylation signal sequence, and ITR / LTR repeats flanking these sequences. The viral vector, e.g., rAAV vector, may optionally further contain ampicillin resistance and a plasmid backbone sequence or pBR322 origin of replication for amplification in a bacterial host.

[0154] In one embodiment, the recombinant AAV vector of the invention is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13.

[0155] In any of the methods of the invention, the rAAV vector can be administered by intramuscular or intravenous injection. In any of the methods of the invention, the viral vector, e.g., an rAAV vector, or composition is administered systemically, e.g., the viral vector, e.g., an rAAV vector, or composition is administered parenterally by injection, infusion, or implantation.

[0156] Another aspect of the invention provides compositions, e.g., pharmaceutical compositions, comprising any of the viral vectors, e.g., the rAAV vectors of the invention. In certain embodiments, the composition is a pharmaceutical composition which may further comprise a therapeutically compatible carrier or excipient.

[0157] In another embodiment, the present invention provides a composition comprising any of the subject functional proteins (e.g., microdystrophin) and a viral vector, e.g., an rAAV vector, co-expressing the one or more additional coding sequences for treating a subject afflicted with a dystrophinopathy or muscular dystrophy, e.g., DMD or Becker muscular dystrophy.

[0158] The compositions (e.g., pharmaceutical compositions) of the present invention can be formulated for intramuscular or intravenous injection.The compositions of the present invention can also be formulated for systemic administration, for example, parenteral administration by injection, infusion or implantation.Furthermore, any of the compositions is formulated for administration to subjects suffering from dystrophinopathy or muscular dystrophy, such as DMD, Becker muscular dystrophy or any other dystrophin-related muscular dystrophy.

[0159] In a further embodiment, the invention provides the use of any of the viral vectors, e.g., the rAAV vectors of the invention, co-expressing a subject functional protein (e.g., microdystrophin) and the one or more additional coding sequences, for the preparation of a medicament for ameliorating a subject afflicted with a dystrophinopathy or muscular dystrophy, e.g., DMD, Becker muscular dystrophy, or any other dystrophin-associated muscular dystrophy.

[0160] The present invention contemplates the use of any of the viral vectors, e.g., the AAV vectors of the invention, for the preparation of a medicament for administration to a patient diagnosed with DMD before one or more secondary cascade symptoms, e.g., fibrosis, are observed in the subject.

[0161] The present invention also contemplates the use of any of the viral vectors, e.g., the AAV vectors of the present invention, for the preparation of a medicament for preventing or slowing the progression of disease in subjects suffering from muscular dystrophy and who have already developed secondary cascade symptoms, such as fibrosis, by administering any of the viral vectors, e.g., the rAAV of the present invention.

[0162] The present invention also provides the use of a viral vector, e.g., an rAAV vector of the present invention, that co-expresses a subject functional protein, e.g., microdystrophin, and said one or more additional coding sequences, for the preparation of a medicament for the treatment of muscular dystrophy, e.g., DMD / BMD.

[0163] In any of the uses of the present invention, the medicament can be formulated for intramuscular injection.Furthermore, any of the medicaments can be formulated for administration to a subject suffering from muscular dystrophy, such as DMD or any other dystrophin-related muscular dystrophy.

[0164] The present invention also provides gene therapy vectors, e.g., rAAV vectors, that co-express a subject functional protein (e.g., microdystrophin) and the one or more additional coding sequences in patients with muscular dystrophy.

[0165] It is to be understood that any one embodiment of the invention described herein may be combined with any one or more additional embodiments of the invention, including embodiments described only in the examples or only in one of the sections above or below, or in one aspect of the invention.

[0166] AAV As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are at least 13 characterized serotypes of AAV. General descriptions and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York, incorporated herein by reference. However, since it is well known that the various serotypes are very closely related structurally and functionally, even at the genetic level, it is fully expected that these same principles will apply to additional AAV serotypes. See, e.g., Blacklowe, 1988, in Parvoviruses and Human Disease, J.R.P.Tattison, ed., pp. 165-174, and Rose, Comprehensive Virology 3:1-61 (1974). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome, and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions of each serotype are under similar regulatory control.

[0167] As used herein, "AAV vector" refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeat sequences (ITRs), which can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0168] An "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 encapsidated polynucleotide AAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, because such a vector is contained within an AAV vector particle, the production of an AAV vector particle necessarily includes the production of an AAV vector.

[0169] A recombinant AAV genome of the invention comprises a nucleic acid molecule of the invention and one or more AAV ITRs flanking the nucleic acid molecule. AAV has multiple serotypes, and the nucleotide sequences of the genomes of these AAV serotypes are known.For example, the nucleotide sequence of AAV serotype 2 (AAV2) genome is shown in Srivastava et al., J Virol 45:555-564 (1983), and revised by Ruffing et al., J Gen Virol 75:3385-3392 (1994).Both are incorporated herein by reference. As other examples, the complete genome of AAV-1 is set forth in GenBank Accession No. NC_002077 (incorporated herein by reference), the complete genome of AAV-3 is set forth in GenBank Accession No. NC_001829 (incorporated herein by reference), the complete genome of AAV-4 is set forth in GenBank Accession No. NC_001829 (incorporated herein by reference), the genome of AAV-5 is set forth in GenBank Accession No. AF085716 (incorporated herein by reference), and the genome of AAV The entire genome of AAV-6 is set forth in GenBank Accession No. NC_001862 (incorporated herein by reference), at least portions of the genomes of AAV-7 and AAV-8 are set forth in GenBank Accession Nos. AX753246 (incorporated herein by reference) and AX753249 (incorporated herein by reference), respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the genome of AAV-9 ... Gao et al. (2004), which is incorporated herein by reference. The genome of AAV-10 is set forth in Mol. Ther. 13(1):67-76 (2006), which is incorporated herein by reference, and the genome of AAV-11 is set forth in Virology 330(2):375-383 (2004), which is incorporated herein by reference. The AAVrh74 serotype is described in Rodino-Klapac et al., J. Trans. Med. 5:45 (2007), which is incorporated herein by reference.

[0170] The AAV DNA of the rAAV genome may be from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, Rh10, Rh74, and AAV-2i8.

[0171] The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0172] In certain embodiments, AAV1, AAV6, AAV8, or AAVrh.74 may be used to promote skeletal muscle-specific expression. In certain embodiments, the AAV serotype of the subject AAV vector is AAV9.

[0173] Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 based on their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.

[0174] These two rep promoters (p5 and p19), along with alternative splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.

[0175] The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are involved in the production of the three related capsid proteins.

[0176] A single consensus polyadenylation site is located in the AAV genome at map position 95. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology 158:97-129 (1992).

[0177] The DNA plasmids of the present invention comprise the rAAV genome of the present invention. The DNA plasmids are transferred into cells that can be infected with an AAV helper virus (e.g., adenovirus, El-deleted adenovirus, or herpesvirus) to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided to the cell, are standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from the rAAV genome (i.e., not within the rAAV), and helper virus functions. The AAV rep and cap genes may be from any AAV serotype from which a recombinant virus can be derived, including but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13, which are different from the ITRs of the rAAV genome.

[0178] The method for generating packaging cells involves creating a cell line that stably expresses all components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., Proc. Natl. Acad. Sci. USA 79:2077-2081, 1982), the addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., Gene 23:65-73, 1983), or direct blunt-end ligation (Senapathy & Carter, J. Biol. Chem. 259:4661-4666, 1984). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that these cells are selectable and are suitable for large-scale production of rAAV.

[0179] In another example of a suitable method, the rAAV genome and / or the rep and cap genes are introduced into packaging cells using adenovirus or baculovirus rather than a plasmid.

[0180] The general principles of rAAV production are reviewed, for example, in Carter, Current Opinions in Biotechnology 1533-1539, 1992, and Muzyczka, Curr. Topics in Microbial and Immunol. 158:97-129, 1992. Various approaches have been described in, for example, 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., Mol. Cell. Biol. 7:349, 1988; Samulski et al. al., J. Virol. 63:3822-3828, 1989, U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al., Vaccine 13:1244-1250, 1995, Paul et al. al., Human Gene Therapy 4:609-615, 1993, Clark et al., Gene Therapy 3:1124-1132, 1996, U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis on the sections thereof related to the production of rAAV.

[0181] In certain embodiments, the AAV vectors of the invention are produced according to the method described in Adamson-Small et al. (Molecular Therapy-Methods & Clinical Development (2016) 3, 16031; doi:10.1038 / mtm.2016.31, incorporated herein by reference), i.e., a scalable method for producing high-titer, high-quality adeno-associated type 9 vectors using an HSV platform. This is an entirely herpes simplex virus (HSV)-based production and purification method, employing 1 x 10 AAV vectors per 10 layers of CellSTACK HEK293 producer cells. 14 More than 1 x 10 rAAV9 vector genomes per cell in the final fully purified product. 5 It is possible to generate vector genomes exceeding 1000 kJ / s. This represents a 5- to 10-fold increase over transfection-based methods. Furthermore, rAAV vectors produced by this method exhibited improved biological properties compared to transfection-based production, including increased infectivity indicated by a high transducing unit-to-vector genome ratio and a reduced amount of total capsid protein, as indicated by a low empty-to-filled ratio. This method can also be easily adapted for large-scale Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP) production of rAAV9 vectors, enabling preclinical and clinical trials and establishing a platform for later phase and commercial production. While AAV9 was used in this study, this method is likely scalable to other serotypes and should bridge the gap between preclinical research, early-phase clinical trials, and the large-scale global development of gene therapy-based drugs for genetic diseases and disorders.

[0182] The present invention therefore provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (a homologous 293 lineage). In another embodiment, the packaging cells are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0183] The recombinant AAV of the present invention (i.e., infectious, encapsidated rAAV particles) comprise an rAAV genome. In an exemplary embodiment, both rAAV genomes lack AAV rep and cap DNA; in other words, there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAVs that can be constructed to contain the nucleic acid molecules of the present invention are described in International Patent Application No. PCT / US2012 / 047999 (WO2013 / 016352), which is incorporated herein by reference in its entirety.

[0184] The rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including those disclosed in, for example, Clark et al., Hum. Gene Ther. 10(6):1031-1039, 1999; Schenpp and Clark, Methods Mol. Med. 69:427-443, 2002; U.S. Patent No. 6,566,118; and WO 98 / 09657.

[0185] Further coding sequences In addition to the coding sequence for a dystrophin protein, such as microD5, the recombinant vector of the present invention also contains one or more additional coding sequences for a target gene(s) in one of the secondary complications / secondary cascades associated with or resulting from the loss of dystrophin.

[0186] In certain embodiments, the vectors of the invention encode exon-skipping antisense sequences capable of correcting specific dystrophin gene mutations. For example, the exon-skipping antisense sequence induces skipping of a specific exon during pre-messenger RNA (pre-mRNA) splicing of a defective dystrophin gene in a subject, resulting in restoration of the reading frame and partial production of an internally truncated protein, similar to the expression of dystrophin protein seen in Becker muscular dystrophy.

[0187] In certain embodiments, the exon-skipping antisense sequence skips or splices out the frame-disrupted exon (mutated exon) and / or adjacent exons, restoring the correct transcriptional reading frame and producing a truncated but functional dystrophin protein.

[0188] In certain embodiments, the exon-skipping antisense sequence induces single exon skipping. In certain embodiments, the exon-skipping antisense sequence induces multiple exon skipping, for example, skipping of one or more or all of exons 45-55 (i.e., native exon 44 is directly linked to exon 56). For example, 11 antisense sequences can be used together to skip all 11 exons, including exons 45-55. A cocktail of 10 AONs was used in the mdx52 mouse model (exon 52 deleted) to induce skipping of exons 45-51 and 53-55, restoring functional dystrophin expression.

[0189] In certain embodiments, the exon skipping antisense sequence induces skipping of exon 51 of dystrophin pre-mRNA. Successful skipping of exon 51 could theoretically treat approximately 14% of all DMD patients.

[0190] In certain embodiments, the exon skipping antisense sequence targets an exon splice enhancer (ESE) site in exon 51 of the dystrophin gene, thus causing exon 51 to be skipped, producing a truncated but partially functional dystrophin protein.

[0191] In certain embodiments, the exon skipping antisense sequence induces skipping of one or more of exons 44, 45, and 53. In certain embodiments, the exon skipping antisense sequence targets the same target sequence as that of casimersen (exon 45), NS-065 / NCNP-01 or golodirsen (exon 53), or eteplirsen or Exondys51 (exon 51).

[0192] In certain embodiments, the exon skipping antisense sequence targets cryptic splicing donor and / or acceptor sites of the mutant FCMD / FKTN gene in Fukuyama congenital muscular dystrophy (FCMD) patients and restores correct exon 10 splicing.

[0193] Fukuyama congenital muscular dystrophy (FCMD) is a rare autosomal recessive disorder and the second most common form of childhood muscular dystrophy in Japan. The gene responsible for FCMD (also known as FKTN) encodes the protein fukutin, a putative glycosyltransferase that glycosylates α-dystroglycan, a member of the dystrophin-associated glycoprotein complex (DAGC). FCMD pathogenesis results from the ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into the 3'-untranslated region (UTR) of the fukutin gene, which activates a new cryptic splice donor in exon 10 and a new cryptic splice acceptor at the SVA insertion site, thereby inducing aberrant mRNA splicing between the cryptic donor and acceptor sites. This results in premature exon 10 exon 10 excision. In FCMD patient cells and in vivo mouse models, a cocktail of three in vivo PMOs targeting the cryptic splice control region was shown to prevent pathogenic SVA exon trapping and restore normal FKTN protein levels and α-dystroglycan O-glycosylation.

[0194] In certain embodiments, the antisense sequence targets a pathological expansion of a 3- or 4-nucleotide repeat, e.g., a CTC triplet repeat in the 3'-UTR region of the DMPK gene in DM1 patients, or a CCTG repeat within the first intron of the CNBP gene in DM2 patients.

[0195] Myotonic dystrophy (DM) is the most common form of adult muscular dystrophy. It is an autosomal dominant disease that can be classified into myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by a pathological expansion of a CTC triplet in the 3'-UTR region of the myotonic dystrophy protein kinase (DMPK) gene, whereas DM2 is caused by a pathological expansion of a CCTG tract in the first intron of the CCHC-type zinc finger nucleic acid binding protein (CNBP) gene. RNA gain-of-function toxicity resulting from repeat-expanded transcript RNA aggregates leads to aberrant splicing (spliceopathy). Toxic RNA aggregates disrupt the function of alternative splicing regulators, such as muscleblind-like (MBNL) protein and CUG-binding protein 1 (CUGBP1), in DM1 by sequestering and depleting the former within nuclear RNA foci and by increasing the expression and phosphorylation of the latter. Altered function of MBNL and CUGBP1 proteins leads to aberrant splicing of the pre-mRNAs of their target genes, namely, insulin receptor (INSR), muscle chloride channel (CLCN1), bridging integrator 1 (BIN1), and dystrophin (DMD), respectively, which are associated with insulin resistance, muscle tone, muscle weakness, and dystrophic muscle processes (all typical symptoms of myotonic dystrophy).

[0196] Therefore, the expansion of the CUG repeat in the DMPK gene sequesters the MBNL1 protein and causes aberrant splicing of several downstream genes, resulting in the DM1 phenotype. Alternatively, antisense oligonucleotides can be used to target such expanded repeats in mutant transcripts for degradation by RNase H, thereby restoring splicing of downstream genes. 2'-O-Methoxyethyl gapmer AONs have been used to target the expanded CUG in mutant RNA transcripts for degradation by RNase H, resulting in a reduction in mutant mRNA transcripts and restoration of protein expression.

[0197] In certain embodiments, the exon skipping antisense sequence results in the skipping of exon 7A of the CLCN1 gene in a DM1 patient. Because chloride channel 1 (CLCN1) contributes to muscle tone in DM1 patients, correcting the aberrant splicing of this gene may also be effective in treating DM1. By using phosphorodiamidate morpholino oligomers (PMOs) with ultrasound-irradiated bubble liposomes to enhance PMO delivery to the muscles of DM1 mice (HSALR), skipping of exon 7A of CLCN1 was achieved in vivo, resulting in improved muscle tone and Clcn1 protein expression in skeletal muscle.

[0198] In certain embodiments, the exon skipping antisense sequence targets exons 17, 32, 35, 36, and / or 42, preferably exons 32 and / or 36, of the DYSF gene for exon skipping in dysferlinopathy (e.g., LGMD2B or MM) patients with DYSF mutations.

[0199] Dysferlinopathy is a general term encompassing muscular dystrophies caused by mutations in the dysferlin (DYSF) gene. The dysferlin gene encodes a sarcolemmal sheath protein required for repair of muscle membrane damage. It consists of a calcium-dependent C2 lipid-binding domain and an integral transmembrane domain. There are two common dysferlinopathies, limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM), both of which have clinically distinct phenotypes and autosomal recessive inheritance. LGMD2B is characterized by proximal muscle weakness, while MM is characterized by distal muscle weakness. The initial clinical phenotypes of LGMD2B and MM differ. However, as the disease progresses, the clinical symptoms of both conditions overlap and become more similar, with patients experiencing muscle weakness in both the proximal and distal limbs. Dysferlin-deficient muscle fibers exhibit defective membrane repair.

[0200] Dysferlinopathy can be treated by exon skipping using antisense oligonucleotides, in part because of the mild phenotype observed in patients with truncated mutant DYSF proteins that express only 10% of wild-type levels. Specifically, in the case of a compound heterozygous female patient with LGMD2B, the patient had a null allele in intron 31 and a DYSF branchpoint mutation in the other allele. Natural in-frame skipping of exon 32 resulted in a truncated dysferlin protein expressed at approximately 10% of wild-type levels, which was sufficient to partially complement the null mutation. The patient exhibited mild symptoms and was ambulatory at age 70. Recently, it has been shown that skipping exon 32 in the patient's cells resulted in pseudo-dysferlin expression levels, which rescued membrane repair in vitro in treated cells exposed to hypoosmotic stress and laser damage localized to the sarcolemmal sheath.

[0201] In certain embodiments, the exon-skipping antisense sequence targets exon 4 of the LAMA2 gene for exon skipping in merosin-deficient congenital muscular dystrophy type 1A (MDC1A) patients with LAMA2 mutations. In certain embodiments, exon skipping restores expression of the C-terminal G domains (exons 45-64), particularly G4 and G5, which are most important for mediating interaction with α-dystroglycan.

[0202] Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the 65-exon LAMA2 gene that result in complete or partial loss of laminin-α2 chain expression. The laminin-α2 chain, along with the beta-1 (β1) and gamma-1 (γ1) chains, is part of a heterotrimeric laminin isoform known as laminin-211 or merosin, and is specifically expressed in the basement membrane of skeletal muscle, including neuromuscular junctions, and Schwann cells (peripheral nerves). Laminin-α2 interacts with the dystrophin-dystroglycan complex (DGC) to mediate cell signaling, adhesion, and tissue integrity in skeletal muscle and peripheral nerves. While not always the case, partial expression of laminin-α2 causes mild MDC1A, while complete absence of laminin-α2 causes severe MDC1A. The C-terminal G domains (exons 45-64), particularly G4 and G5, are most important for mediating the interaction with α-dystroglycan. Mutations that eliminate G4 and G5 are associated with a severe phenotype, even in the presence of truncated laminin-α2 expression.

[0203] Exon skipping has been explored for the treatment of MDC1A, in that PMO-mediated skipping of exon 4 corrects the open reading frame, leading to restoration of the truncated laminin-α2 chain and a slight extension of patient lifespan.

[0204] In certain embodiments, the exon-skipping antisense sequence induces skipping of exons 4-7 of the most common Δ-521T mutation in the LGMD2C / SGCG gene and restoration of the reading frame to produce an internally truncated SGCG protein for the treatment of patients with limb-girdle muscular dystrophy type 2C with the Δ-521T SGCG mutation. In certain embodiments, exon skipping restores expression of an internally truncated SGCG protein that retains the intracellular, transmembrane, and most distal carboxy termini of the wild-type SGCG protein.

[0205] Dystrophin-associated proteins (DAPs) are complexes within the sarcolemma. Their transmembrane components connect the cytoskeleton to the extracellular matrix in mature muscle fibers and are essential for maintaining the integrity of the sarcolemma. The sarcoglycan subcomplex within the DGC consists of four single-pass transmembrane subunits: α-, β-, γ-, and δ-sarcoglycans. The α- to δ-sarcoglycan genes, i.e., α (LGMD2D), β (LGMD2E), γ (LGMD2C), and δ (LGMD2F), are expressed predominantly (β) or exclusively (α, γ, and δ) in striated muscles. Mutations in any of the four sarcoglycan genes can cause secondary deficiencies of other sarcoglycan proteins, possibly by destabilizing the sarcoglycan complex, resulting in sarcoglycanopathies, i.e., autosomal recessive limb-girdle muscular dystrophies (LGMDs). Disease-causing mutations in the α-δ genes cause disruptions within the dystrophin-associated protein (DAP) complex in the muscle cell membrane.

[0206] In humans, gamma-sarcoglycan (LGMD2C) is a protein encoded by the SGCG gene. Severe childhood autosomal recessive muscular dystrophy (SCARMD) is a progressive muscle-wasting disease that segregates with microsatellite markers in the gamma-sarcoglycan gene. Mutations in the gamma-sarcoglycan gene were first reported in the Maghreb countries of North Africa, where gamma-sarcoglycanopathies have a higher than normal incidence. One of the most common mutations in LGMD2C patients, Δ-521T, is a deletion of a thymine from a five-thymine stretch at nucleotide bases 521-525 in exon 6 of the gamma-sarcoglycan gene. This mutation shifts the reading frame, resulting in the absence of gamma-sarcoglycan protein and secondary reduction of beta- and delta-sarcoglycans, resulting in a severe phenotype. This mutation occurs in both Maghreb populations and other countries.

[0207] Exon skipping has been explored for the treatment of LGMD2C, which carries the Δ-521T mutation, in that the resulting internally truncated SGCG protein provides functional and pathological benefits to correct the γ-sarcoglycan defect in a Drosophila model lacking γ-sarcoglycan, in heterologous cell expression studies, and in transgenic mice. A cellular model of the human muscle disease has also been generated, demonstrating that multiple exon skipping can be induced with RNA encoding mutant human γ-sarcoglycan.

[0208] In certain embodiments, the vectors of the present invention encode an antisense sequence or an RNAi sequence (such as an siRNA, shRNA, or miRNA) that antagonizes the function of sarcolipin (SLN). In certain embodiments, the vectors of the present invention encode an shRNA (shSLN) that antagonizes the function of sarcolipin.

[0209] Exemplary shSLN sequences include those disclosed in Figures 9 and 10 (e.g., the underlined sequences in Figure 9 and the highlighted sequences in Figure 10). Further exemplary shSLN sequences include SEQ ID NOs: 7-11, as disclosed in WO2018 / 136880 (incorporated herein by reference).

[0210] Additional shSLN sequences can be designed based on any art-recognized method using the human SLN mRNA sequence shown below.

[0211] [ka]

[0212] In certain embodiments, the vectors of the invention encode antisense or RNAi sequences (such as siRNA, shRNA, miRNA, etc.) that antagonize the function of one or more target genes, eg, inflammatory genes.

[0213] IκB kinase / nuclear factor-kappa B (NF-κB) signaling is persistently elevated in immune cells and regenerating muscle fibers in both animal models and patients with DMD. Furthermore, TNF-α and NF-κB activators, such as IL-1 and IL-6, are upregulated in DMD muscles. Therefore, inhibiting components of the NF-κB signaling cascade, such as NF-κB itself, its upstream activators, and downstream inflammatory cytokines, in conjunction with replacing / repairing the defective dystrophin gene, is beneficial for treating patients.

[0214] Thus, in certain embodiments, the vectors of the invention encode antisense or RNAi sequences (e.g., siRNA, shRNA, miRNA) that antagonize the function of one or more inflammatory genes, such as NF-κB, TNF-α, IL-1 (IL-1β), IL-6, receptor activator of NF-κB (RANK), and Toll-like receptors (TLRs).

[0215] In certain embodiments, the vectors of the present invention encode antisense or RNAi sequences (e.g., siRNA, shRNA, miRNA) that antagonize the function of histone deacetylases, such as HDAC2. In DMD, the absence of dystrophin at the sarcolemmal sheath leads to the delocalization and downregulation of nitric oxide synthase (nNOS), which alters HDAC2 S-nitrosylation and its chromatin binding. HDAC2 activity was specifically increased in dystrophin-deficient mdx mice, which lack the NO pathway. In contrast, rescue of nNOS expression in mdx animals improved the dystrophic phenotype. Furthermore, deacetylase inhibitors had strong morphofunctional effects on dystrophic muscle fibers. Indeed, the histone deacetylase inhibitor gibinostat is currently under evaluation as a potential disease-modifying treatment for DMD. The data show that in both mouse and human dystrophic cells, the absence of dystrophin correlates with HDAC2 binding to a specific subset of miRNAs (see below), and that upon dystrophin rescue, HDAC2 is released from these promoters.

[0216] In certain embodiments, the vectors of the present invention encode antisense sequences, RNAi sequences (e.g., siRNA, shRNA, miRNA), or microRNAs that antagonize the function of TGF-β or connective tissue growth factor (CTGF). Elevated TGF-β levels in muscular dystrophy stimulate fibrosis and impair muscle regeneration by inhibiting satellite cell activation. Antifibrotic agents, including losartan, an angiotensin II type 1 receptor blocker that reduces TGF-β expression, have been tested in mouse models of muscular dystrophy. HT-100 (halofuginone) has also been shown to prevent fibrosis via the TGF-β / Smad3 pathway in muscular dystrophy. Meanwhile, FG-3019, a fully human monoclonal antibody that inhibits the action of connective tissue growth factor (CTGF), a central mediator in the pathogenesis of fibrosis, is being evaluated in an open-label phase 2 trial in patients with idiopathic pulmonary fibrosis (IPF).

[0217] In certain embodiments, the vectors of the present invention encode microRNAs (miRs), such as miR-1, miR-29c, miR-30c, miR-133, and / or miR-206. The distinct HDAC2 nitrosylation status in Duchenne versus wild-type conditions deregulates the expression of specific subsets of microRNA genes. Several identified microRNA-regulated circuits, such as those linking miR-1 to the G6PD enzyme and cellular redox state, or miR-29 to extracellular proteins and fibrotic processes, explain the pathogenesis of some DMDs. The muscle-specific (myomiR) miR-1 and miR-133, as well as the ubiquitous miR-29c and miR-30c, which were downregulated in mdx, were restored to wild-type levels in exon-skipping-treated animals. In the mdx model, when dystrophin synthesis was restored via exon skipping, levels of miR-1, miR-133a, miR-29c, miR-30c, and miR-206 increased, but expression of miR-23a remained unchanged.

[0218] In certain embodiments, the vector of the present invention encodes the microRNA inhibitor that inhibits the function of the microRNA that is upregulated in DMD or its related diseases.For example, the expression level of inflammatory miR-223 is upregulated in the muscle of mdx mice, and is downregulated in exon skipping treated mice.Its reduction is consistent with the observed improvement of muscle inflammation due to the dystrophin rescue by exon skipping.

[0219] The mdx animals suffer from extensive fibrotic degeneration, and miR-29 has been shown to target the mRNAs of key factors involved in fibrotic degeneration, such as collagen, elastin, and structural components of the extracellular matrix. In mdx mice, miR-29 expression is impaired, and collagen (COL1A1) and elastin (ELN) mRNAs are upregulated. Thus, miR-29c expression alleviates fibrotic degeneration in DMD patients, in part, by downregulating collagen and elastin expression and pathological extracellular matrix modifications associated with collagen and elastin expression.

[0220] In certain embodiments, the vectors of the present invention encode antisense or RNAi sequences (e.g., siRNA, shRNA, miRNA) that antagonize the function of G6PD (glucose-6-phosphate dehydrogenase). One important issue in dystrophic muscle is its sensitivity and response to oxidative stress, which has been suggested to be involved in disease progression. G6PD is a cytoplasmic enzyme in the pentose phosphate pathway that provides cells with reduced energy by maintaining NADPH levels. This results in a high ratio of reduced to oxidized glutathione (GSH / GSSG). GSH is a major antioxidant molecule that protects cells from oxidative damage. G6PD mRNA is deregulated in mdx muscle. It contains three putative binding sites for the miR-1 family in its 3'-UTR region, and miR-1 and miR-206 can suppress G6PD expression. Indeed, there was an inverse correlation between G6PD and miR-1 expression, and in vitro differentiation of C2 myoblasts showed that increased miR-1 levels correlated with decreased G6PD protein, mRNA, and GSH / GSSG ratios. In mdx mice, where miR-1 is downregulated, G6PD was detected at higher levels than in WT muscle, whereas the amount of G6PD was reduced in exon-skipping-treated mdx mice, where miR-1 expression was restored. Notably, increased G6PD levels were accompanied by a decreased GSH / GSSG ratio in mdx mice.

[0221] In certain embodiments, the vectors of the present invention encode antisense or RNAi sequences (e.g., siRNA, shRNA, miRNA) that antagonize the function of myostatin. Myostatin is a negative regulator of muscle mass. Inhibition or blockage of endogenous myostatin compensates for the severe muscle wasting common to many types of muscular dystrophies, including DMD. MYO-029, a myostatin-blocking antibody, is currently in clinical trials in adult subjects with BMD and other dystrophies. Other clinical trials are also being conducted using follistatin and myostatin inhibitors such as PF-06252616 (NCT02310764) and BMS-986089.

[0222] In certain embodiments, the vectors of the present invention encode antisense or RNAi sequences (e.g., siRNA, shRNA, miRNA) that antagonize the function of phosphodiesterase-5 (PED-5) or ACE, or VEGF decoy receptor type 1 (VEGFR-1 or Flt-1). Loss of dystrophin leads to the translocation of neuronal nitric oxide synthase and a decrease in muscle-derived nitric oxide in the microvasculature, leading to functional muscle ischemia and further muscle damage. Therefore, several inhibitors of phosphodiesterase-5 or ACE, or VEGF decoy receptor type 1 (VEGFR-1 or Flt-1), have been tested as part of strategies to increase blood flow to muscles, including pharmaceutical inhibition of either phosphodiesterase-5 or ACE.

[0223] In certain embodiments, the vectors of the present invention encode antisense or RNAi sequences (e.g., siRNA, shRNA, miRNA) that antagonize the function of hematopoietic prostaglandin D synthase (HPGDS). Prostaglandin D2 (PGD2) is produced by various inflammatory cells, and hematopoietic PGD synthase (HPGDS) is known to be expressed in necrotic muscle of DMD patients. Administration of an HPGDS inhibitor reduced urinary excretion of tetranor-PGDM, a urinary metabolite of PGD2, and suppressed muscle necrosis in the mdx mouse model of DMD. A novel HPGDS inhibitor, TAS-205, is being evaluated in clinical trials for the treatment of DMD.

[0224] RNAi and antisense design RNA interference (RNAi) involves the creation of small RNA molecules that are complementary to and bind to endogenous target mRNAs, leading to their functional inactivation, including degradation.

[0225] The RNAi pathway is found in many eukaryotic organisms, including plants and animals. It is initiated in the cytoplasm by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) or short hairpin RNA (shRNA) molecules into short, double-stranded fragments (siRNAs) of approximately 21 nucleotides. Each siRNA then unwinds into two single-stranded RNAs (ssRNAs): a passenger strand and a guide strand. The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most commonly studied outcome is post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence within an mRNA molecule and induces cleavage by Argonaute 2 (Ago2), the catalytic component of RISC. In some organisms, this process can spread throughout the body, despite the initial molar concentration of siRNA being limiting.

[0226] Besides siRNAs and shRNAs, another type of small RNA molecule central to RNA interference is microRNA (miRNA). MicroRNAs are genomically encoded non-coding RNAs that help regulate gene expression, particularly during development. Mature miRNAs are structurally similar to siRNAs but must first undergo extensive post-transcriptional modifications before reaching maturity. miRNAs are expressed from very long RNA-coding genes as primary transcripts known as pri-miRNAs, which are then processed in the cell nucleus by the microprocessor complex, consisting of the RNase III enzyme Drosha and the dsRNA-binding protein DGCR8, into 70-nucleotide stem-loop structures called pre-miRNAs. Once the pre-miRNA is transported into the cytosol, its dsRNA portion is anchored and cleaved by Dicer to generate the mature miRNA molecule, and the two strands can be separated into a passenger strand and a guide strand. The miRNA guide strand, like the siRNA guide strand, can be incorporated into the same RISC complex.

[0227] Thus, both dsRNA pathways, miRNA and siRNA / shRNA, require the processing of precursor molecules (pri-miRNA, pre-miRNA, and dsRNA or shRNA) that contain scaffold sequences to generate the mature functional guide strand of the miRNA or siRNA, and both pathways ultimately converge at the RISC complex.

[0228] After incorporation into RISC, siRNAs base-pair with and cleave target mRNAs, preventing them from being used as translation templates. However, unlike siRNAs, miRNA-loaded RISC complexes scan cytoplasmic mRNAs for potential complementarity. Instead of destructive cleavage (by Ago2), miRNAs target the 3'-UTR region of mRNAs, where they usually bind with imperfect complementarity, thus blocking ribosome access for translation.

[0229] siRNAs differ from miRNAs in that miRNAs, especially animal miRNAs, typically exhibit imperfect base-pairing with their targets, inhibiting the translation of many different mRNAs with similar sequences. In contrast, siRNAs typically form perfect base pairs and direct mRNA cleavage at only a single specific target.

[0230] Historically, RNAi applications have used siRNAs and shRNAs. siRNAs are typically double-stranded RNA molecules 20–25 nucleotides in length. siRNAs temporarily inhibit target mRNAs until they are degraded within the cell. shRNAs are typically approximately 80 base pairs in length and contain a region of internal hybridization that creates a hairpin structure. As previously mentioned, shRNA molecules are processed within the cell to form siRNAs, which then knock down gene expression. One advantage of shRNAs is that they can be incorporated into plasmid vectors and integrated into genomic DNA for longer-term or stable expression and thus longer knockdown of target mRNAs.

[0231] shRNA designs are commercially available. For example, Cellecta offers RNAi screening services for any target gene (e.g., all 19,276 protein-coding human genes) using its human whole genome shRNA library or its mouse DECIPHER shRNA library (targeting approximately 10,000 mouse genes). ThermoFisher Scientific offers Ambion's Silencer Select siRNA (classic 21-mers), which, according to the manufacturer, incorporate the latest improvements in siRNA design, off-target effect prediction algorithms, and chemistry.

[0232] ThermoFisher Scientific also offers Ambion® Pre-miR™ miRNA precursor molecules, which are chemically modified small, double-stranded RNA molecules designed to mimic endogenous mature miRNAs. These Pre-miR miRNA precursors enable functional analysis of miRNAs by upregulating miRNA activity and can be used to identify and validate miRNA target sites, screen for miRNAs that regulate target gene expression, and screen for miRNAs that affect the function of target genes (e.g., SLNs) or cellular processes.

[0233] ThermoFisher Scientific also offers Ambion® Anti-miR™ miRNA inhibitors, which are chemically modified single-stranded nucleic acids designed to specifically bind to and inhibit endogenous microRNA (miRNA) molecules.

[0234] Antisense sequence designs are also commercially available from many commercial and public sources, such as IDT (Integrated DNA Technologies) and GenLink. Design considerations may include oligo length, secondary / tertiary structure of the target mRNA, protein binding sites on the target mRNA, the presence of CG motifs in either the target mRNA or the antisense oligo, tetraplex formation in the antisense oligo, and the presence of motifs that enhance or decrease antisense activity.

[0235] The design of exon skipping antisense oligonucleotide is known in the art.For example, see Shimo et al., Camilla Bernardini (ed.), Duchenne Muscular Dystrophy: Methods and Protocols, Methods in Molecular Biology, vol.1687, DOI 10.1007 / 978-1-4939-7374-3_10, Chapter 10 (published by Springer Science+Business Media LLC), 2018), which discusses in detail the design of effective exon skipping oligonucleotide, taking into account factors such as target site selection, oligo length, oligo chemistry, and melting temperature relative to RNA strand.The use of a cocktail of antisense oligonucleotides to skip multiple exons is also discussed. Specific genes and muscular dystrophies addressed include DMD (Duchenne muscular dystrophy), LAMA2 (merosin-deficient CMD), DYSF (dysferlinopathy), FKTN (Fukuyama CMD), DMPK (myotonic dystrophy), and SGCG (LGMD2C), the entire contents of which are incorporated herein by reference.

[0236] For example, protein / gene sequences and their mutations in affected disease genes are publicly available online at NCBI and the Leiden Muscular Dystrophy pages. Potential target sites for efficient exon skipping can be obtained using the Human Splicing Finder website at www.umd.be / HSF. The secondary structure of the target mRNA can be evaluated, for example, using the mfod web server on the Albany.edu website. Oligo lengths are typically 8-30 mers. Calculation of the GC content of oligos can be performed using the OligoCalc website on the Northwestern University server. Searches for any off-target sequences can be performed using the GGGenome website. Oligo melting temperatures can be estimated using the LNA Oligo Prediction Tool at sg.idtdna.com or OligoAnalyzer 3.1 software.

[0237] Improved guide strand generation for RNAi (miR, siRNA, shRNA) In certain embodiments, the coding sequence encodes an RNAi reagent, eg, an miR, siRNA, or shRNA.

[0238] In certain embodiments, with regard to the design of miRs and / or shRNAs / siRNAs, the wild-type backbone sequence from which the mature miR or mature siRNA is generated can be modified to improve guide strand generation and minimize / eliminate passenger strand generation. Since both strands of the mature miR / siRNA / shRNA (after cleavage) can theoretically be incorporated into the RISC complex and serve as guide strands for RNAi, it is advantageous to selectively enhance the utilization of the designed guide strand and minimize the utilization of the mostly complementary passenger strand in the RISC complex, for example, to reduce or minimize off-target effects (e.g., due to unintended cleavage of the target sequence when the passenger strand is loaded into RISC).

[0239] One approach that can be used to achieve this goal (enhancing leading strand production and minimizing / eliminating passenger strand production) is by using hybrid constructs in which a designed mature miR / siRNA / shRNA sequence containing the desired guide strand is embedded within the scaffold sequence of another miR sequence that favors guide strand production and disfavors passenger strand production.

[0240] This principle has been demonstrated in the design of several modified miR-29c and shSLN constructs, but the same principle can be easily adopted for other RNAi reagents targeting any other sequence.

[0241] For all designs shown below, the design strategy employed involves modifying the nucleotide sequences of the adjacent scaffold sequences, loop sequences, and passenger strands to maintain the 2D and 3D structures of the native scaffold sequences. In this regard, for miRNA / shRNA design, the 2D / 3D structure of the native scaffold sequence mainly refers to the distance between the stem-loop and the adjacent scaffold polynucleotide sequence, the structure of the central stem, the position and / or size of the bulge, the presence and location of any internal loops and mismatches within the stem, etc. A specific exemplary 2D structure map of a miR-29c hybrid construct based on the selected scaffold sequences of miR-30E, miR-101, and miR-451 is provided below as an example.

[0242] A. Hybrid miR-29c with miR-30 backbone sequence (29c-M30E) Fellmann et al. (Cell Rep. 5(6):1704-1713, 2013, incorporated herein by reference) described a systematic approach to optimizing experimental miR-30 scaffolds by identifying conserved elements 3' of the basic stem that are critical for optimal processing of so-called "shRNAmirs," i.e., synthetic shRNAs embedded in endogenous microRNA contexts. The resulting optimized scaffolds, termed "miR-Es," significantly increased the levels and knockdown efficacy of mature shRNAs. This approach can easily convert existing miR and shRNA reagents into miR-Es, generating more effective miRs and shRNAs.

[0243] Applying this technology, a 29c-M30E hybrid sequence was generated based on the desired mature miR-29c sequence and the modified / optimized miR-30 backbone sequence described in Fellmann et al. This 29c-M30E sequence (see Figure 29 for its predicted 2D structure) was incorporated into the following subject viral vectors used in the following examples: μDys-29c-M30E-i2, EF1A-29c-M30E, and U6-29c-M30E. The 5' to 3' sequences of the following 29c-M30E sequences are contiguous and are artificially separated into different lines to represent different segments of the contiguous sequence.

[0244] [ka]

[0245] Specifically, in the above contiguous sequence, the middle line represents the passenger strand sequence, the double-underlined loop sequence, and the mature miR-29c guide sequence. Note that the passenger and guide sequences are reverse-complementary to each other and can snap back to form a stem-loop structure with the intervening loop sequence. Note, however, that a perfect reverse-complementary sequence is not required. Due to possible internal bulges, etc., the two strands may not necessarily be 100% complementary to each other in some cases (see the guide and passenger strands in the last sequence of this subsection). The top and bottom lines represent the flanking scaffold sequences of M30E, which have been optimized to enhance guide sequence production and minimize passenger strand production.

[0246] In a similar design, siRNA targeting human SLN is embedded in the same M30E backbone sequence in miR-30E-hSLN-c1 (compare the upper and lower lines of the sequences immediately above and below this paragraph, as well as the double-underlined loop sequence). However, the guide and passenger strands are different. This so-called c1-M30E sequence is incorporated into the following subject viral vectors used in the following examples: c1-M30E-i2, c1-M30E-3UTR, and c1-M30E-pa.

[0247] [ka]

[0248] A similarly designed second siRNA that also targets human SLN is embedded in the same M30E backbone sequence in miR-30E-hSLN-c2 (compare the upper and lower lines of the sequences immediately above and below this paragraph, as well as the double-underlined loop sequence). However, the guide and passenger strands are different. This so-called c2-M30E sequence is incorporated into the following subject viral vectors used in the following examples: c2-M30E-i2, c2-M30E-3UTR, and c2-M30E-pa.

[0249] [ka]

[0250] The sequence of modified miR-29c using the native miR-30 scaffold sequence ("M30N") is also provided below for comparison. Note that the guide strand in this case is 5' to the loop sequence. This M30N scaffold sequence also enhanced guide strand production, although to a lesser extent than the M30E scaffold sequence in the experimental system examined (data not shown).

[0251] [ka]

[0252] B. Hybrid miR-29c (29c-101) with miR-101 backbone sequence A different miR-29c hybrid (29c-101, see Figure 30 for its predicted 2D structure) using the miR-101 backbone sequence is shown below using the same naming conventions used herein, where the top and last two lines represent the miR-101 backbone sequence, and the second line is the mature miR-29c with the passenger strand, loop sequence, and guide strand. This 29c-101 sequence has been incorporated into the following subject viral vectors used in the following examples: μDys-29c-101-i2, μDys-29c-3UTR-101.

[0253] [ka]

[0254] C. Hybrid miR-29c (29c-155) with miR-155 backbone sequence A different miR-29c hybrid (29c-155) using the miR-155 scaffold sequence is shown below using the same naming conventions used herein, where the top and bottom lines represent the miR-155 flanking scaffold sequence, and the second line is the mature miR-29c with the guide strand, loop sequence, and passenger strand. This 29c-155 sequence was incorporated into the subject viral vector used in the following examples: EF1A-29c-155.

[0255] [ka]

[0256] Another miR-29c hybrid (29c-19nt) similarly using the miR-155 backbone sequence is shown below using the same naming conventions as used herein. Here, the top and bottom lines represent the miR-155 flanking backbone sequence (identical to that in the sequence immediately above), and the second line is the mature miR-29c with the guide strand, loop sequence, and passenger strand. Note that the loop sequence here is 19 nt instead of the 17 nt loop in the sequence above. This 29c-19nt sequence was incorporated into the following subject viral vectors used in the following examples: EF1A-29c-19nt, 29c-19nt-μDys-pA, and 29c-19nt-μDys-3UTR.

[0257] [ka]

[0258] D. Hybrid shSLNs with miR-155 backbone sequences (shmSLN-v2 and c1 / c2-m155) The miR-155 backbone sequence containing shSLN is shown below using the same naming conventions used herein, where the top and bottom lines represent the miR-155 flanking backbone sequence, and the second line is the mature shRNA (shmSLN) targeting mouse SLN, containing the guide strand, loop sequence (19 nt), and passenger strand. This shmSLN-v2 sequence was incorporated into the following subject viral vectors used in the following examples: EF1A-mSLN, fusion-v1, and μDys-shmSLN-v1.

[0259] [ka]

[0260] The miR-155 backbone sequence containing shSLN is shown below using the same naming conventions used herein. Here, the top and bottom lines represent the miR-155 flanking backbone sequence, and the second line is another mature shRNA (shmSLN) targeting mouse SLN, containing the guide strand, loop sequence (19 nt), and passenger strand. Compared to the similar / related shmSLN sequences described above, the presence of an extra dinucleotide base pair, TT:AA (or strictly speaking, UU at the 3' end of the siRNA), correlates with increased potency of the resulting guide strand siRNA. This shmSLN-v2 sequence was incorporated into the following subject viral vectors used in the following examples: EF1A-mSLN-v2, fusion-v2, and μDys-shmSLN-v2.

[0261] [ka]

[0262] Another miR-155 backbone sequence containing shSLN is shown below using the same naming conventions used herein, where the top and bottom lines represent the miR-155 flanking backbone sequence, and the second line is the mature shRNA targeting human SLN, containing the guide strand, loop sequence (19 nt), and passenger strand. This c1-m155 sequence was incorporated into the following subject viral vectors used in the following examples: c1-m155-pa, c1-m155-i2, and c1-m155-3UTR.

[0263] [ka]

[0264] Another miR-155 backbone sequence containing shSLN is shown below using the same naming conventions used herein, where the top and bottom lines represent the miR-155 flanking backbone sequence, and the second line is the mature shRNA targeting human SLN with a different guide strand, loop sequence (19 nt), and passenger strand. This c2-m155 sequence was incorporated into the following subject viral vectors used in the following examples: c2-m155-pa, c2-m155-i2, and c2-m155-3UTR.

[0265] [ka]

[0266] E. Hybrid miR-29c (29c-451) with miR-451 backbone sequence A miR-29c hybrid (29c-451, see Figure 31 for its predicted 2D structure) using the miR-451 scaffold sequence is shown below using the same naming conventions as herein, where the top two and bottom two lines represent the flanking scaffold sequence of miR-451, and the third line is the mature miR-29c with the guide strand, loop sequence, and passenger strand.

[0267] [ka]

[0268] F. U6-driven miR-29c and shSLN The experimental section below also describes the use of certain "solo" viral vector constructs that express only miR-29c or only shSLN. Such solo expression cassettes are driven by a strong Pol III U6 promoter. Because this strong U6 promoter directly generates pre-miRNA or shSLN without any flanking nucleotide sequences, such sequences do not belong to the modified miR-29c or modified shSLN sequences. However, for comparison purposes, such sequences are also described here using the same nomenclature.

[0269] The miR-29c driven by the U6 promoter is shown below (U6-29c-v1). Here, the second line is the mature miR-29c with the passenger strand, loop sequence, and guide strand. This was used to generate a "solo" control vector in the pGFP-U6-shAAV-GFP vector. The nucleotides in the first line of the following consecutive sequence are the first five nucleotides after the transcription start site of the U6 promoter, and the T6 transcription termination sequence precedes the sequence used to clone the last line of the following consecutive sequence.

[0270] [ka]

[0271] The shSLN driven by the U6 promoter is shown below (U6-shmSLN-v1), where the second line is the mature shSLN containing the passenger strand, loop sequence, and guide strand, which is used in the U6-shmSLN-v1 vector in the examples.

[0272] [ka]

[0273] The shSLN driven by the U6 promoter is shown below (U6-mSLN-v4). Here, the second line is the mature shSLN containing the passenger strand, loop sequence, and guide strand. This is used in the U6-mSLN-v4 vector in the examples (see Figure 15).

[0274] [ka]

[0275] Compositions and pharmaceutical compositions In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The composition of the present invention comprises the rAAV and a pharmaceutically acceptable carrier. The composition may also include other components, such as a diluent and an adjuvant. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients and preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, 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, i.e., glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, pluronic, or polyethylene glycol (PEG).

[0276] Medication and Administration The titer of the rAAV administered in the methods of the invention will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the targeted cell type(s), and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×108 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 From about 1 × 10 14 Doses may range from up to or exceeding DNase resistant particles (DRP). Doses may also be expressed in units of viral genomes (vg).

[0277] Methods for transducing target cells with rAAV in vivo or in vitro are contemplated by the present invention. The in vivo method comprises administering an effective dose or effective multiple doses of a composition comprising an rAAV of the present invention to an animal (including a human) in need thereof. When the dose is administered before the onset of a disorder / disease, the administration is prophylactic. When the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, thereby delaying or preventing progression to the disorder / disease state, delaying or preventing progression of the disorder / disease state, attenuating the extent of the disease, resulting in remission (partial or complete) of the disease, and / or prolonging survival. Examples of diseases contemplated for prevention or treatment by the methods of the present invention are PMD or other diseases characterized by defects in myelin production, degeneration, regeneration, or function.

[0278] For administration, effective and therapeutically effective amounts (also referred to herein as doses) can be estimated initially based on results of in vitro assays and / or animal model studies, e.g., IC determined in cell culture. 50 A dose can be formulated in animal models to achieve a circulating concentration range including . Such information can be used to more accurately determine useful doses in subjects of interest.

[0279] Administration of an effective dose of the composition may be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or intravaginal. The route(s) of administration and serotype(s) of the AAV components of the rAAV of the present invention (particularly the ITRs and capsid proteins of the AAV) can be selected and / or adapted by one skilled in the art taking into account the infection and / or disease state to be treated and the target cell / tissue(s) expressing the one or more coding sequences and / or micro-dystrophin.

[0280] Specifically, the formulations described herein may be administered by, but are not limited to, injection, infusion, perfusion, inhalation, lavage, and / or ingestion. Routes of administration may include, but are not limited to, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, intramuscular, intravesical, intrapericardial, intraumbilical, intraocular, mucosal, oral, subcutaneous, and / or subconjunctival.

[0281] The present invention provides for local or systemic administration of an effective dose of the rAAV and compositions of the present invention, including the combination therapy of the present invention. For example, systemic administration is administration into the circulatory system, affecting the entire body. Systemic administration includes enteral administration, e.g., absorption through the digestive tract, and parenteral administration by injection, infusion, or implantation.

[0282] In particular, the actual administration of the rAAV of the present invention can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal, such as skeletal muscle. Administration according to the present invention includes, but is not limited to, injection into muscle, the bloodstream, and / or directly into the liver. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations on the carrier or other components that can be co-administered with rAAV (although compositions that degrade DNA should generally be avoided with rAAV). The capsid protein of rAAV can be modified to target the rAAV 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.

[0283] The pharmaceutical composition can be prepared as an injection formulation or a local formulation delivered to the muscle by transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in the practice of the present invention. The rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0284] The dose of rAAV administered in the methods disclosed herein will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the targeted cell type(s), and can be determined using standard methods in the art.

[0285] The actual dosage administered to a particular subject may be determined by a physician, veterinarian, or researcher, taking into consideration parameters such as, but not limited to, physical and physiological factors, including body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathic diseases of the subject, and / or route of administration.

[0286] The titer of each rAAV administered was approximately 1 × 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×109 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 to, or about 1 × 10 15 Doses may range from up to or exceeding DNase-resistant particles (DRPs). Doses may also be expressed in units of viral genomes (vg) (i.e., 1 x 10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 1×10 15 Doses are sometimes expressed in units (vg) of viral genomes per kilogram (kg) of body weight (i.e., 1 × 10 10 vg / kg, 1 × 10 11 vg / kg, 1 × 10 12 vg / kg, 1 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1 × 10 15 A method for measuring AAV titer is described in Clark et al., Hum. Gene Ther. 10:1031-1039, 1999.

[0287] An exemplary dose is about 1 x 10 10 ~Approx. 1×10 15 In some embodiments, the dose may range from 1 x 10 vector genomes (vg) per kilogram of body weight. 10 vg / kg body weight, 1×10 11 vg / kg body weight, 1×10 12 vg / kg body weight, 1×10 13 vg / kg body weight, 1×10 14 vg / kg body weight, or 1 × 10 15 The dose may include 1 x 10 vg / kg body weight. 10 vg / kg / day, 1×10 11 vg / kg / day, 1×10 12vg / kg / day, 1×10 13 vg / kg / day, 1×10 14 vg / kg / day, or 1 × 10 15 Doses may range from 0.1 mg / kg / day to 5 mg / kg / day, or from 0.5 mg / kg / day to 1 mg / kg / day, or from 0.1 mg / kg / day to 5 μg / kg / day, or from 0.5 mg / kg / day to 1 μg / kg / day. In other non-limiting examples, doses may include 1 μg / kg / day, 5 μg / kg / day, 10 μg / kg / day, 50 μg / kg / day, 100 μg / kg / day, 200 μg / kg / day, 350 μg / kg / day, 500 μg / kg / day, 1 mg / kg / day, 5 mg / kg / day, 10 mg / kg / day, 50 mg / kg / day, 100 mg / kg / day, 200 mg / kg / day, 350 mg / kg / day, 500 mg / kg / day, or 1000 mg / kg / day. A therapeutically effective amount may be achieved in a single administration or in multiple administrations over the course of a treatment regimen (ie, over the course of days, weeks, months, etc.).

[0288] In some embodiments, the pharmaceutical composition contains at least 1.6×10 13 In some embodiments, the dosage form is a 10 mL aqueous solution having at least 2 x 10 vector genomes per milliliter. 12 In some embodiments, the dosage form comprises a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride. In some embodiments, the pharmaceutical composition is contained in a dosage form of 10 mL of a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride, and contains at least 1.6 x 10 13 It has a vector genome of

[0289] In some embodiments, the pharmaceutical composition contains 1×10 10 ~1×10 15 A 10 mL solution containing 1 x 10 vector genomes 11 ~1×10 14A 10 mL solution containing 1 x 10 vector genomes 12 ~2×10 13 of vector genomes in a 10 mL solution, or approximately 1.6 x 10 13 The dosage form may comprise a 10 mL aqueous solution containing at least 1 x 10 vector genomes. In some embodiments, the aqueous solution is a sterile aqueous solution containing about 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride. In some embodiments, the dosage form contains at least about 1 x 10 vector genomes per milliliter (vg / mL). 11 Super, about 1×10 12 More than vg / mL, approximately 2×10 12 More than vg / mL, approximately 3×10 12 > 4 × 10 vg / mL or approximately 4 × 10 12 It has a potency of > vg / mL.

[0290] In some embodiments, at least one AAV vector is provided as part of a pharmaceutical composition. The pharmaceutical composition may, for example, comprise at least 0.1% w / v of the AAV vector. In some other embodiments, the pharmaceutical composition may comprise 2% to 75% of the compound by weight of the pharmaceutical composition, or 25% to 60% of the compound by weight of the pharmaceutical composition.

[0291] In some embodiments, the dosage form is included in a kit. The kit may further include instructions for use of the dosage form. For intramuscular injection purposes, sterile aqueous solutions, as well as solutions in adjuvants such as sesame or peanut oil, or aqueous propylene glycol solutions can be used. Such aqueous solutions can be buffered, if necessary, and the diluents are first rendered isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed are readily obtainable by standard techniques well known to those skilled in the art.

[0292] In some embodiments, for injection, the formulation may be prepared as an aqueous solution, for example, but not limited to, Hank's solution, Ringer's solution, and / or physiological saline buffer solution. The solution may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the formulation may be in lyophilized and / or powder form, and may be reconstituted with an appropriate vehicle (e.g., sterile pyrogen-free water) before use.

[0293] Any formulation disclosed herein may advantageously include any other pharmaceutically acceptable carrier(s), including those that do not produce significant side, allergic, or other adverse reactions that may outweigh the benefits of administration, whether for research, prophylactic, and / or therapeutic treatment. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, which is incorporated herein by reference for its relevant teachings. Furthermore, formulations may be prepared to meet sterility, pyrogenicity, general safety, and purity standards required by the U.S. FDA's Division of Biological Standards and Quality Control and / or other relevant U.S. and foreign regulatory agencies.

[0294] Exemplary commonly used pharmaceutically acceptable carriers may include, but are not limited to, bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, and vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffers, chelating agents (e.g., EDTA), gels, binders, disintegrating agents, and / or lubricants.

[0295] Exemplary buffers may include, but are not limited to, citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0296] Exemplary preservatives may include, but are not limited to, phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides, hexamethonium chloride, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, and / or 3-pentanol.

[0297] Exemplary isotonicity agents can include polyhydric sugar alcohols, including, but not limited to, trihydric or higher sugar alcohols (eg, glycerin, erythritol, arabitol, xylitol, sorbitol, and / or mannitol).

[0298] Exemplary stabilizers may include, but are not limited to, organic sugars, polyhydric sugar alcohols, polyethylene glycols, sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, and / or polysaccharides.

[0299] The formulation may also be a depot preparation. In some embodiments, such long-acting formulations may be administered by, but not limited to, implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the compound may be formulated with suitable polymeric and / or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as a sparingly soluble derivative (e.g., as a sparingly soluble salt).

[0300] Furthermore, in various embodiments, the AAV vector can be delivered using a sustained-release system, such as a semipermeable matrix of solid polymers containing the AAV vector. Various sustained-release materials have been established and are well known to those skilled in the art. Sustained-release capsules, depending on their chemical nature, release the vector for several weeks to over 100 days after administration.

[0301] Pharmaceutical carriers, diluents, or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, and includes, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0302] Sterile injectable solution is prepared by incorporating the required amount of rAAV into a suitable solvent with various other ingredients as listed above, if necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which allows the powder of active ingredient plus any additional desired ingredients to be obtained from the solution that has been previously sterile-filtered.

[0303] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used, where these cells do not generate an inappropriate immune response in the subject.

[0304] Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro, for example, by mixing rAAV with muscle cells in an appropriate medium and screening for cells containing the DNA of interest using conventional techniques, for example, Southern blot and / or PCR, or using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, which can be introduced into the subject by various techniques, for example, intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or injection into smooth muscle and cardiac muscle, for example, using a catheter.

[0305] Transduction of cells with the rAAV of the present invention results in sustained co-expression of the one or more additional coding sequences and micro-dystrophin. The present invention therefore provides methods for administering / delivering rAAVs co-expressing the one or more additional coding sequences and micro-dystrophin to animals, preferably humans. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of the present invention. Transduction can be performed using gene cassettes containing tissue-specific regulatory elements. For example, one embodiment of the present invention provides a method for transducing muscle cells and muscle tissues directed by muscle-specific regulatory elements, including, but not limited to, those from the actin and myosin gene families, such as those from the myoD gene family (see Weintraub et al., Science 251:761-766, 1991), muscle cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11:4854-4862, 1991), the human skeletal actin gene (Muscat et al., Mol Cell Biol 7:4089-4099, 1987), the cardiac actin gene, and the muscle creatine kinase sequence element (Johnson et al., Mol Cell Biol 9:3393-3399, 1989), and regulatory elements derived from the mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene; hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA 88:5680-5684, 1991), steroid-inducible factor, and glucocorticoid response element (GRE)-containing promoters (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607, 1993), as well as other regulatory elements.

[0306] Muscle tissue is an attractive target for DNA delivery in vivo because it is a non-vital organ and is easily accessible. The present invention contemplates sustained co-expression of miRNA and micro-dystrophin from transduced muscle fibers.

[0307] As used herein, "muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, e.g., derived from the gastrointestinal tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, e.g., myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0308] The term "transduction" is used to refer to the administration / delivery of the one or more additional coding sequences and the micro-dystrophin coding region to a recipient cell, either in vivo or in vitro, via a replication-deficient rAAV of the present invention, resulting in co-expression of the one or more additional coding sequences and micro-dystrophin by the recipient cell.

[0309] Thus, the present invention provides methods of administering an effective dose (or multiple doses, administered essentially simultaneously or spaced apart) of an rAAV encoding the one or more additional coding sequences and micro-dystrophin to a patient in need thereof.

[0310] Generation of AAVs The genes encoding the necessary replication (rep) and structural (cap) proteins of AAV vectors have been deleted from the AAV vector, allowing the sequence to be delivered to be inserted between the remaining terminal repeat sequences. Thus, propagation of AAV vectors requires not only a helper virus but also the genes encoding the rep and cap proteins, which must be delivered to infected cells. Alternatively, the genes encoding the rep and cap proteins must be present in the cells used for production.

[0311] AAV vectors suitable for the methods of the present invention can be produced using any method recognized in the art.In a recent review, Penaud-Budloo et al. (Molecular Therapy: Methods & Clinical Development Vol.8, pages 166-180, 2018) provided a review of the most commonly used upstream methods for producing rAAV.The methods described in this review are incorporated herein by reference.

[0312] Transient transfection of packaging cell line (HEK293) In particular, in certain embodiments, the AAV vector is produced by using the transient transfection of packaging cell line, for example, HEK293 cell.This is the most established AAV production method, which involves the transfection of plasmid into human embryonic HEK293 cell.Usually, HEK293 cell is transfected simultaneously with vector plasmid (comprising the polynucleotide of the present subject that codes for both the gene of interest, for example, the dystrophin minigene and one or more additional coding sequences) and one or two helper plasmids, using calcium phosphate or cationic polymer polyethyleneimine (PEI).

[0313] The helper plasmid(s) allow expression of the four Rep proteins, the three AAV structural proteins VP1, VP2, and VP3, AAP, and the adenoviral accessory functions E2A, E4, and VA RNA. The additional adenoviral E1A / E1B cofactors required for rAAV replication are expressed in HEK293 producer cells. Rep-cap and adenoviral helper sequences are cloned into two separate plasmids or combined into a single plasmid, allowing for both three-plasmid and two-plasmid transfection systems. The three-plasmid protocol offers versatility, with the cap gene easily switchable from one serotype to another.

[0314] The plasmids are usually produced by conventional techniques in E. coli using antibiotic resistance genes of bacterial origin or by the minicircle technique. Transient transfection in adherent HEK293 cells has been used for large-scale production of rAAV vectors, and recently, HEK293 cells have also been adapted to suspension conditions, which makes them economically viable for long-term production.

[0315] HEK293 cells are typically grown in DMEM containing L-glutamine, 5%–10% fetal bovine serum (FBS), and 1% penicillin-streptomycin, except for suspension HEK293 cells, which are maintained in serum-free suspension F17, Expi293, or other manufacturer-specific media. For adherent cells, the percentage of FBS can be reduced during AAV production to limit contamination with animal-derived components.

[0316] Generally, rAAV vectors are recovered from the cell pellet and / or supernatant 48-72 hours after transfection of the plasmid, depending on the serotype. Infection of insect cells with recombinant baculovirus The baculovirus-Sf9 platform has been established as an alternative, GMP-compliant, scalable AAV production method in mammalian cells, with crude yields of up to 2 x 10 per cell. 5 A vector genome (vg) can be generated.

[0317] The current protocol involves infection of Sf9 insect cells with two recombinant baculoviruses: a baculovirus expression vector (BEV) that allows the synthesis of Rep78 / 52 and Cap, and a recombinant baculovirus carrying a gene of interest flanked by the ITRs of AAV. Several serum-free media are adapted for the growth of Sf9 cells in suspension.

[0318] The dual baculovirus-Sf9 production system has many advantages over other production platforms regarding these safety concerns: (1) the use of serum-free medium, (2) despite the discovery of foreign viral transcripts in Sf cell lines, most viruses that infect insects do not actively replicate in mammalian cells, and (3) other than baculovirus, no helper virus is required for rAAV production in insect cells.

[0319] In certain embodiments, a stable Sf9 insect cell line expressing Rep and Cap proteins is used, such that infection with only one recombinant baculovirus is required to produce high yields of infectious rAAV vector.

[0320] Infection of mammalian cells with rHSV vectors HSV is a helper virus for AAV replication in permissive cells, and therefore can serve both as a helper and as a shuttle to deliver essential AAV functions that support AAV genome replication and packaging into producer cells.

[0321] AAV production based on co-infection with rHSV allows efficient production of large amounts of rAAV with high total yields (up to 1.5 × 10 5 In addition to the increased viral titer (vg / cell), the method has the further advantage of producing rAAV stocks of demonstrably improved quality as measured by improved viral titer.

[0322] In this method, cells, typically the hamster BHK21 cell line or HEK293 and derivatives, are infected with two rHSVs: one carrying the gene of interest surrounded by AAV ITRs (rHSV-AVV) and the second carrying the rep and cap ORFs of an AAV of the desired serotype (rHSVrepcap). After 2–3 days, the cells and / or medium are harvested, and the rAAV is purified through multiple purification steps to remove cellular impurities, HSV-derived contaminants, and unpackaged AAV DNA.

[0323] Thus, in some embodiments, HSV serves as a helper virus for AAV infection. In some embodiments, propagation of AAV is achieved using a non-replicating mutant of HSV that is deleted for ICP27.

[0324] Certain methods for producing recombinant AAV viral particles in mammalian cells are known in the art and have been improved over the past decade. For example, U.S. Patent Application Publication No. 20070202587 describes the production of recombinant AAV in mammalian cells based on coinfection of the cells with two or more replication-deficient recombinant HSV vectors. U.S. Patent Application Publication No. 20110229971 and Thomas et al. (Hum. Gene Ther. 20(8):861-870, 2009) describe a scalable method for producing recombinant AAV using coinfection of recombinant HSV type 1 in suspension-adapted mammalian cells. Adamson-Small et al. (Hum. Gene Ther. Methods 28(1):1-14, 2017) describe an improved method for producing AAV in a serum-free suspension manufacturing platform using the HSV system.

[0325] Mammalian stable cell lines rAAV vectors can also be efficiently and scalably produced using stable mammalian producer cells that stably express the rep and cap genes. Such cells can be infected with wild-type Ad5 helper virus, which is genetically stable and can be easily produced at high titers, to induce high levels of rep and cap expression. Infectious rAAV vectors can be produced by infecting these packaging cell lines with wild-type Ad5 and providing the rAAV genome by transfection of plasmids or after infection with recombinant Ad / AAV hybrid viruses.

[0326] Alternatively, Ad can be replaced with HSV-1 as the helper virus. Suitable stable mammalian producer cells may include HeLa-derived producer cell lines, A549 cells, or HEK293 cells. A preferred HeLa cell line is HeLaS3 cells, a HeLa subclone adapted to suspension culture.

[0327] The methods described herein can be used to produce the subject AAV vectors in animal component-free media, preferably at a 250-L scale, or a 2,000-L commercial scale. [Example]

[0328] Example 1: Expression verification of microD5 (SGT001) construct in C2C12 cells To confirm that the microD5 (SGT-001) micro-dystrophin transgene can be expressed in vitro with or without additional coding sequences for microRNA (e.g., miR-29c coding sequence) or shRNA (e.g., shRNA against SLN) inserted into the same AAV vector, C2C12 cells were infected in vitro with three AAV viral constructs: one encoding a wild-type microD5 (SGT-001) construct, one encoding a fusion construct of microD5 (SGT-001) and miR-29c, where the miR-29c coding sequence was inserted into the heterologous intron region 5′ of the microD5 (SGT-001) coding sequence, and one encoding a fusion construct of microD5 (SGT-001) and shRNA against SLN, where the shRNA coding sequence was inserted into the heterologous intron region 5′ of the SGT-001 coding sequence (see Figure 3).

[0329] The microD5 (SGT-001) / miR-29c fusion construct was predicted to generate an initial transcript encoding both the dystrophin minigene product and miR-29c RNA. The microD5 (SGT-001) / shRNA fusion construct for SLN was also predicted to generate an initial transcript encoding both the dystrophin minigene product and shRNA.

[0330] Without wishing to be bound by any particular theory, Applicants also believe that subsequent processing of the fusion transcript may result in premature cleavage of the fusion mRNA (such as causing loss of the polyA tail). This may have contributed to the reduced expression of microdystrophin in C2C12 cells infected with this fusion construct compared to those infected with a wild-type microdystrophin construct lacking this additional coding sequence. See Figure 3.

[0331] However, this experiment confirmed that the microD5 (SGT-001) construct successfully expressed the desired microdystrophin protein (as evidenced by immunofluorescence staining using an antibody specific for the microdystrophin gene product) and that this expression, although slightly reduced / inhibited / suppressed, persisted in C2C12 cells infected with miR-29c or shRNA fusion constructs.

[0332] Similar experiments were performed with other fusion constructs in which the miR-29c coding sequence was inserted at different positions in the heterologous intron between the promoter and the microD5 (SGT-001) coding sequence (see Figure 11 for the insertion positions of these various fusion constructs, Imir2, Imir3, Imir4, and Imir5).

[0333] After PCR amplification of intron sequences with or without the inserted coding sequence, the insertion was confirmed and the amplified products were analyzed by electrophoresis. For example, insertion of the 88-bp miR-29c coding sequence increased the size of the PCR amplified product by just under 100 bp.

[0334] This fusion construct was subsequently used in several experiments to determine whether expression of microD5 (SGT-001) in infected C2C12 cells was affected. In this particular experiment, it is clear that the presence of the shRNA coding sequence initially prevented microdystrophin expression in transfected C2C12 cells one day after transfection. However, microdystrophin expression quickly caught up, and by day 6 post-transfection, microdystrophin expression in transfected C2C12 cells was virtually identical with or without the additional coding sequence for the shRNA in the AAV vector. See Figures 8A-8C.

[0335] This data demonstrates that the subject AAV constructs can include additional coding sequences for shRNAs or microRNAs without significantly affecting the expression of the micro-dystrophin gene.

[0336] Example 2 Functional assay of shRNA against sarcolipin (SLN) This example demonstrates that the coding sequence for shRNA against SLN (shSLN) inserted into the subject AAV vector can generate a functional shRNA that reduces the expression of SLN.

[0337] Figure 4 is a schematic diagram showing a sarcolipin-luciferase fusion reporter encoded by an AAV vector. After co-transfection of C2C12 cells with AAV encoding the luciferase-bearing reporter and microD5 with or without shSLN, the ability of the encoded shSLN to reduce expression of the SLN-luciferase fusion can be assessed based on the luciferase-generated signal.

[0338] Figure 5 shows representative results of such cotransfection experiments. Specifically, in one experiment, cotransfection of the SLN-luciferase fusion reporter construct with an AAV vector encoding only microD5 ("SGT001") resulted in a strong luciferase signal. In another experiment, cotransfection of the SLN-luciferase fusion reporter construct with an AAV vector encoding microD5 and shSLN ("SGT001+SLN") resulted in an 86.7% reduction in the luciferase signal. This suggests that shSLN was expressed and was effective in reducing the expression of the target gene (microD5-luciferase fusion).

[0339] Figure 6A shows the results of a functional test of shRNA (SLN) based on direct immunostaining of sarcolipin. In C2C12 cells transfected with an AAV9 vector encoding the microD5 (SGT-001) dystrophin minigene and the coding sequence of shRNA (shSLN), endogenous SLN expression was reduced by 55% compared to C2C12 cells transfected with the same vector encoding only the microD5 (SGT-001) dystrophin minigene. See the immunofluorescence image in Figure 6B.

[0340] The function of the shRNA(SLN) coding sequence in this AAV vector was further investigated based on measurements of calcium reuptake into the sarcoplasmic reticulum. Sarcoplasmic reticulum (endoplasmic reticulum) Ca 2+ ERCA transports Ca from the cytosol into the lumen of the sarcoplasmic reticulum in muscle cells. 2+ Sarcolipin, encoded by the SLN gene, is a transmembrane protein that catalyzes the ATP-dependent transport of Ca in the sarcoplasmic reticulum without affecting the rate of ATP hydrolysis. 2+ Sarcolipin is a small transmembrane proteolipid that regulates some SERCAs by reducing the accumulation of Ca. Sarcolipin depletion reduces atrial Ca 2+The transient amplitude and force of atrial contraction are increased. Furthermore, atria from sarcolipin null mice exhibit a blunted response to isoproterenol stimulation, indicating that sarcolipin is a mediator of beta-adrenergic responses in the atria.

[0341] Therefore, functional shRNA(SLN) is expected to reduce the expression level of endogenous SLN, reduce SLN binding to SERCA, and ultimately reduce the impairment of calcium reuptake into the sarcoplasmic reticulum. Phenotypically, the effect of expression of functional shRNA(SLN) is similar to that of overexpression of SERCA, such as SERCA2a, and overexpression in rats has been shown to shorten the relaxation time of right ventricular papillary muscle, suggesting faster calcium reuptake into the sarcoplasmic reticulum.

[0342] Indeed, the faster calcium reuptake into the sarcoplasmic reticulum is demonstrated in Figure 7, which shows the normalized fluorescence signal emitted by the calcium-binding dye Fluo-8.

[0343] Fluo-8 (Abcam) is a cell-permeable, medium-affinity, green fluorescent calcium-binding dye. It has a K of approximately 390 nM. d Binds to intracellular calcium at Ca 2+ The binding of the β-glucan increases its fluorescence intensity.

[0344] The faster decrease in Fluo-8 signal represents a faster decrease in cytoplasmic calcium in C2C12 cells infected with the AAV vector encoding microD5(SGT-001)-shSLN compared with untransfected C2C12 cells and C2C12 cells infected with the AAV vector encoding only the microD5(SGT-001) dystrophin minigene.

[0345] On the other hand, expression of the microD5 (SGT-001) dystrophin minigene was not significantly affected by the presence of the shSLN coding sequence or shSLN expression, even several days (e.g., 6 days) after infecting cells with the AAV construct, whereas expression of the microD5 (SGT-001) dystrophin minigene was initially decreased (i.e., 1 day after infection) (see Figures 8A-8C).

[0346] In comparison, 6 days after infection, endogenous SLN expression is reduced by 55% (see Figure 6A), consistent with the 86% reduction seen in the SLN-luciferase reporter assay in Figure 5.

[0347] These data support the view that the subject AAV vectors co-expressing both microdystrophin and shSLN can efficiently express both transgenes in the same AAV vector, while expression of shSLN does not adversely affect microdystrophin minigene expression over the long term.

[0348] Furthermore, the expressed shSLN appears to be functional based on both luciferase reporter assays and direct measurements of endogenous SLN expression. Example 3 In vitro expression of coding sequences from fusion constructs The fusion viral vector of the present invention can express not only a functional gene or protein of interest (GOI), but also one or more coding sequences of a specific RNAi, antisense, sgRNA, miRNA, or their inhibitors. A representative, non-limiting configuration of the recombinant viral vector of the present invention is shown in Figure 12. For example, the recombinant viral vector of the present invention can be a fusion AAV vector, such as an AAV9 vector designed to express a type of functional dystrophin gene, such as any one of the aforementioned μDys genes. The same fusion vector can also express one or more additional coding sequence(s) within the intron of the initial transcript, within the 3'-UTR, or after the polyA signal, for example, after the polyA signal of the recombinant AAV9 vector but before the transcription termination sequence, or after the transcription termination sequence.

[0349] When the additional coding sequence encodes an miRNA, such as miR-29c, the backbone sequence of the miR-29c coding sequence can be modified so that the sequences surrounding the mature miR-29c sequence are obtained from other miRNAs, e.g., miR-30, -101, -155, or -451 (see above). It has been found that replacing the surrounding sequences of native miR-29c with those from miR-30, -101, -155, or -451 can increase the production of one strand of miR-29c (i.e., the guide strand) that is designed to target the miR-29c target sequence (i.e., decrease the production of its complementary passenger strand, which is not useful for targeting the miR-29c target sequence).

[0350] As a control, several so-called solo expression constructs were generated in the same vector background, which do not express the μDys gene but instead may express a reporter gene such as EGFP or GFP.

[0351] For example, one such solo vector might express a miR-29c coding sequence inserted into an intron sequence upstream of an EGFP coding sequence, all from the EF1A promoter. The backbone sequence of the miR-29c coding sequence may also be modified by that of miR-30, -101, -155, or -451.

[0352] Another such solo vector may express an shRNA, for example, shSLN, which targets / downregulates the expression of SLN. The expression of this shRNA may be driven by a U6 promoter, which can be used by RNA Pol III to generate strong transcription of short RNA transcripts. The shRNA coding sequence may be inserted into the intron of the U6 transcription cassette before the coding sequence of GFP.

[0353] Several such representative fusion or solo vectors were used to transfect human iPS-derived cardiomyocytes in vitro, and the expression of miR-29c in the infected cardiomyocytes was measured, the results of which are shown in FIG.

[0354] Specifically, five solo constructs, five fusion constructs, and a control μDys expression construct were transfected into human iPS-derived cardiomyocytes according to standard procedures. Mature miR-29c levels were measured by Taqman stem-loop QPCR. The five solo constructs examined included those with U6- or EF1A-driven miR-29c expression cassettes designed in the miR-30 backbone (EF1A-29c-M30E and U6-29c-M30E) and those designed in the miR-155 backbone (EF1A-29c-19nt and EF1A-29c-155). The five fusion constructs examined included miR-29c expression cassettes designed in the miR-101 backbone (μDys-29c-101-i2 and μDys-29c-3UTR-101), miR-30 backbone (μDys-29c-M30E-i2), and miR-155 backbone (29c-19nt-μDys-3UTR and 29c-19nt-μDys-pa), inserted at the intron (i2), 3'UTR (3UTR), and post-pA (pa) sites relative to the μDys expression cassette.

[0355] It is clear that the fusion constructs of the present invention generally overexpressed miR-29c by 2- to 11-fold in infected human iPS-derived cardiomyocytes compared to controls using similar constructs expressing only μDys (and thus presenting only background levels of endogenous miR-29c expression).

[0356] The specific fusion constructs used to generate the data in Figure 13 include: 29c-19nt-μDys-3UTR: modified miR29c contained in the miR-155 backbone and inserted into the 3′-UTR region (before the polyA adenylation signal sequence) of the μDys expression cassette.

[0357] 29c-19nt-μDys-pA: The same modified miR29c coding sequence contained in the miR-155 backbone and inserted after the polyA adenylation signal sequence of the μDys expression cassette.

[0358] μDys-29c-M30E-i2: A modified miR29c coding sequence contained in a miR-30E backbone and inserted into the intron region of the μDys expression cassette. μDys-29c-101-i2: A modified miR29c coding sequence contained in a miR-101 backbone and inserted into the intron region of the μDys expression cassette.

[0359] μDys-29c-3UTR-101: A modified miR29c coding sequence contained in a miR-101 backbone and inserted into the 3′-UTR region of the μDys expression cassette. In contrast, solo constructs expressing miR-29c generally overexpressed miR-29c by 6- to 73-fold in infected human iPS-derived cardiomyocytes compared to the same control vector expressing μDys alone.

[0360] The specific solo constructs used to generate the data in Figure 13 include: EF1A-29c-M30E: A modified miR29c coding sequence in a miR-30E backbone and driven by the EF1A promoter.

[0361] U6-29c-M30E: A modified miR29c coding sequence contained in a miR-30E backbone and driven by the Pol III U6 promoter. U6-29c-v1: miR29c coding sequence driven by Pol III U6 promoter.

[0362] EF1A-29c-19nt: A modified miR29c coding sequence in a miR-155 backbone and driven by the EF1A promoter. EF1A-29c-155: another modified miR29c coding sequence contained in a miR-155 backbone and driven by the EF1A promoter.

[0363] Similar trends indicating (preferential) production of miR-29c from these constructs were obtained when these constructs were evaluated in other in vitro cell lines, including Mouly human healthy primary myoblasts and the mouse C2C12 immortalized myoblast cell line (data not shown). Insertion of the miR-29c element into the μDys expression cassette does not result in a significant decrease in μDys mRNA production.

[0364] AAV9 viral particles containing several selected fusion recombinant viral vectors were similarly used to infect differentiated C2C12 myotubes and primary mouse cardiomyocytes, and miR-29c expression was also confirmed in these cells (see Figure 14, which includes results expressed as relative miR-29c expression after normalization to a control expressing μDys alone).

[0365] In this experiment, μDys production did not appear to be significantly affected compared to the control group, and the levels of the passenger strand of miR-29c did not show any increase. Meanwhile, the expression of shSLN from the subject fusion construct and the results of approximately 50% downregulation of SLN in mouse cells infected with such fusion construct are shown in Figures 16 and 15, respectively.

[0366] Specifically, three solo constructs expressing μDys alone and two fusion constructs expressing μDys and shSLN were transfected into mouse C2C12 cells stably overexpressing SLN-Myc / DDK (Myc-DDK-tagged SLN; DDK is the same as the proprietary Sigma-Aldrich FLAG® tag, and the Myc-DDK tag can be detected using anti-Myc or anti-DDK antibodies). This mouse C2C12 stable cell line was generated by lentiviral transduction of a vector encoding SLN-Myc / DDK followed by selection of stable cell lines. One of the fusion constructs, "fusion-v2" or μDys-shmSLN-v2, showed approximately 50% knockdown of SLN protein expression levels as detected using a Myc tag-specific antibody. See Figure 15.

[0367] The various constructs used in Figure 15 are shown below. μDys: control AAV9 vector encoding only the μDys GOI. EF1A-mSLN: A solo construct expressing only an shRNA targeting mouse SLN. Transcription of this shRNA coding sequence is driven by the EF1A promoter.

[0368] EF1A-mSLN(V2): Another solo construct expressing only shRNA targeting mouse SLN. Transcription of this shRNA coding sequence is driven by the EF1A promoter.

[0369] EF1A-mSLN(V4): Yet another solo construct expressing only an shRNA targeting mouse SLN. Transcription of this shRNA coding sequence is driven by the EF1A promoter.

[0370] Fusion-v1: A fusion construct of the invention expressing both the GOI of μDys and the coding sequence of an shRNA targeting mouse SLN. Fusion-v2: Another fusion construct of the invention expressing both the GOI of μDys and the coding sequence of an shRNA targeting mouse SLN.

[0371] It is evident that expression of mSLN was reduced by approximately 50% upon infection of mouse cells with the subject fusion construct encoding a form of mSLN shRNA. Figure 16 shows the relative expression levels of siSLN (processed siRNA product derived from transcribed shSLN) in differentiated C2C12 myotubes or primary mouse cardiomyocytes for various recombinant AAV9 vectors encoding shSLN, either as the sole coding sequence of this viral vector ("solo") or as part of the fusion constructs of the present disclosure ("fusion"). siRNA production was quantified using a conventional Taqman stem-loop QPCR system. Relative siSLN expression levels of solo and fusion constructs were normalized to the μDys control level; however, the apparent high expression ratio may not be informative, as the control produced little or no siSLN-like RNA. Nevertheless, it is clear that in both cell types examined, the solo constructs expressed approximately 1000-fold higher levels of siSLN from the strong U6 Pol III promoter compared to the control. Meanwhile, the fusion constructs examined expressed 1-2 orders of magnitude higher levels of siSLN compared to the control.

[0372] Numerous additional solo and fusion constructs expressing shRNAs targeting human SLN were also tested in human iPS-derived cardiomyocytes. These included six solo and 12 fusion constructs targeting human SLN. These fusion constructs contained shSLN sequences in miR-29 and miR-155 backbones and were inserted into introns, 3'-UTRs, or post-pA sites relative to the μDys expression cassette. The results of these experiments are summarized in Figure 17.

[0373] Specifically, several negative controls (e.g., multiple μDys and GFP plasmids) and positive controls were used in the experiment in Figure 17. The negative controls included two constructs expressing μDys alone (μDys1 and μDys2), which did not affect SLN mRNA expression levels; a construct expressing GFP under the muscle-specific promoter CK8 (CK8-GFP), which also did not affect SLN mRNA expression; and a construct expressing "Sigma Scramble," a scrambled version of hSLN-targeted shSLN (which was not expected to affect SLN mRNA expression). The positive control was "Sigma shRNA," a commercially available shRNA plasmid from Sigma that encodes an hSLN-targeted shSLN that downregulates hSLN mRNA by approximately 80%.

[0374] Six solo constructs, each expressing a type of shRNA targeting hSLN and each under the transcriptional control of the strong Pol III U6 promoter, were examined and found to generally downregulate approximately 80-90% of hSLN mRNA expression.

[0375] Across six solo constructs and four fusion constructs of the present invention, we also observed up to 90% down-down of hSLN mRNA expression. For example, the c2-m30e-i2 construct is a fusion construct co-expressing μDys and an shRNA targeting hSLN. This shRNA is embedded in the M30E backbone sequence (see above) and inserted into an intron of the μDys expression cassette. Transfection of human iPS-derived cardiomyocytes with this construct resulted in up to 90% knockdown of hSLN mRNA.

[0376] These fusion constructs significantly affected hSLN mRNA expression but did not appear to adversely affect μDys expression in the same vector. As shown in Figure 18, six solo and 12 fusion constructs targeting human SLN were transfected into human iPS-derived cardiomyocytes. Most fusion constructs showed μDys mRNA expression similar (>50%) to that of the control μDys-only construct.

[0377] Denaturing agarose gel analysis of selected solo and two fusion constructs also confirmed that the AAV9 genome in these miR-29c constructs was largely intact. See Figure 19.

[0378] Example 4 In vivo expression of coding sequences from fusion constructs This experiment demonstrates that the subject fusion constructs can be used to simultaneously express μDys and one or more additional coding sequence(s) that affect another pathway (e.g., downregulation of SLN and / or upregulation of miR-29c), achieving more than solo, if not synergistic, therapeutic effects.

[0379] In this series of experiments, AAV9 encoding the μDys gene and a second coding sequence, i.e., miR-29c or shSLN constructs targeting mouse SLN, were fused together. The various fusion constructs were injected into 6-week-old male mdx mice via the tail vein at a dose of approximately 5E13vg / kg (except for one group, i.e., U6-29c-v1, at 1E14vg / kg). The expression of μDys, miR-29c, and SLN mRNA was then monitored for 28 days after injection. The detailed experimental setup is summarized below:

[0380] [Table 1]

[0381] In the miR-29c experimental group, the two fusion constructs tested—one in the M30E backbone and inserted into the intron of the μDys expression cassette, and one in the miR-101 backbone and inserted into the 3'-UTR region of the μDys expression cassette—were found to result in 1.4- to 2.8-fold upregulation of miR-29c in the left gastrocnemius muscle (Figure 20A), diaphragm (Figure 20B), and left ventricle (Figure 20C). The miR-29c-μDys fusion AAV9 construct was administered at a dose of 5E13 vg / kg. The solo U6 promoter-driven miR-29c construct in AAV9 resulted in 2- to 11-fold upregulation at a dose of 5E13 vg / kg, and 6- to 16-fold at a dose of 1E14 vg / kg.

[0382] In contrast, upregulation of miR-29c by the fusion AAV9 construct did not result in a decrease in μDys production in the gastrocnemius muscle (Figure 21), diaphragm (data not shown), or left ventricle (data not shown). The fusion AAV9 construct showed μDys expression at both the RNA and protein levels similar to that of the control μDys-only AAV9 construct. The solo construct expressing only miR-29c showed a lack of μDys levels, as it does not produce μDys.

[0383] In this shSLN experimental group, the shSLN-fusion AAV9 constructs tested were found to result in up to 50% downregulation of mSLN mRNA in the diaphragm, left gastrocnemius muscle, and atrium (Figure 22), as well as the tongue (data not shown). Similarly, downregulation of mSLN mRNA by the fusion AAV9 constructs did not reduce μDys production at both the RNA and protein levels in the gastrocnemius muscle (Figure 23), diaphragm (data not shown), and left ventricle (data not shown) compared to that of the control AAV9 expressing μDys alone. The solo construct expressing shmSLN alone did not produce μDys, thus demonstrating a lack of μDys levels. Results are shown for the diaphragm. Similar results were observed in the tongue and atrium.

[0384] These data demonstrate that the subject fusion constructs can simultaneously express both the μDys gene and at least one additional coding sequence, e.g., miR-29c or shRNA against SLN, and thus achieve better therapeutic results compared to viral vectors that express only one coding sequence, e.g., μDys.

[0385] Example 5 Coding sequences expressed in vivo from fusion constructs are biologically active This experiment demonstrates that the coding sequences expressed from the fusion constructs of the present invention are biologically active.

[0386] Dystrophin provides structural stability to the sarcolemma, and increased permeability of the sarcolemma sheath leads to the release of creatine kinase (CK) from muscle fibers. Therefore, elevated CK levels are a hallmark of muscle damage. In DMD patients, CK levels are significantly increased above the normal range (e.g., 10-100 times normal levels since birth). Similarly, serum CK levels are considered a general measure of muscle health in the mdx mouse model.

[0387] The data from this experiment showed that both AAV9 miR-29c solo (administered at a high dose of 1E14 vg / kg) and miR-29c-μDys fusion (administered at a dose of 5E13 vg / kg) constructs reduced serum CK levels to a similar extent in the mdx mouse model compared to μDys controls, suggesting the therapeutic utility of expressing miR-29c in DMD patients.

[0388] Specifically, serum CK levels were also measured in various mouse groups in the in vivo experiments of Example 4. Figure 24 shows that expression of μDys alone resulted in a significant reduction in serum CK levels. Coexpression of μDys and miR-29c in both fusion constructs tested also significantly reduced serum CK levels. Interestingly, expression of miR-29c alone significantly reduced serum CK levels, especially when higher viral doses (miR-29c-expressing solo constructs) were used.

[0389] Meanwhile, tissue inhibitor of metalloproteinase 1 (TIMP-1) has been proposed as a serum biomarker for monitoring disease progression and / or treatment efficacy in patients with Duchenne muscular dystrophy (DMD). This is because serum levels of TIMP-1 are significantly higher in DMD patients compared with healthy controls. Similarly, TIMP-1 is also a serum marker of muscle health in the mdx mouse model.

[0390] Therefore, serum TIMP1 levels were also measured in various mdx mouse groups in the in vivo experiments of Example 4. The left panel of Figure 25 shows that expression of μDys alone resulted in a significant reduction in serum TIMP1 levels. Coexpression of μDys and miR-29c in both fusion constructs tested similarly significantly reduced serum TIMP1 levels. In contrast, expression of miR-29c alone did not reduce serum TIMP1 levels, even when higher viral doses (solo constructs expressing miR-29c) were used.

[0391] Similarly, the right panel of Figure 25 shows that expression of μDys alone significantly reduced serum TIMP1 levels. Coexpression of μDys and shRNA directed against mSLN in the fusion constructs examined also significantly reduced serum TIMP1 levels. In contrast, expression of shRNA directed against mSLN alone did not reduce serum TIMP1 levels.

[0392] These data suggest that the coding sequences expressed in vivo from the fusion constructs of the present invention are biologically active. Example 6 Fusion constructs do not alter the biodistribution of viral vectors In the in vivo experiments in Example 4, the biodistribution of the fusion viral vectors was compared with that of a solo viral vector expressing only μDys. The biodistribution of all viral vectors used was found to be similar in the gastrocnemius muscle, regardless of whether the fusion construct expressed miR-29c or shSLN. See Figure 26.

[0393] Quantification of viral titers in gastrocnemius muscle also showed similar viral titers for fusion and control AAV9. Example 7 Fusion constructs do not alter the hepatic biodistribution of viral vectors In the in vivo experiments in Example 4, the liver levels of fusion viral vectors were compared with those of a solo viral vector expressing only μDys. The viral titers of all viral vectors used were found to be similar in the liver, regardless of whether the fusion construct expressed miR-29c or shSLN. See Figure 27.

[0394] Example 8. Improved therapeutic efficacy using fusion constructs compared to μDys monotherapy To determine whether co-expression of μDys and miR-29c leads to better therapeutic efficacy and / or reduced complications such as fibrosis, the expression levels of two fibrotic marker genes, Col3a1 and Fn1, were examined in mice treated with the various fusion, solo, or control constructs described in Example 4. Col3A1 expression and FN1 expression are used as markers of fibrotic activity.

[0395] Solo AAV9 vectors expressing only miR-29c resulted in decreased expression of Col3A1 and FN1 at a low dose of 5E13vg / kg and a high dose of 1E14vg / kg. Fusion AAV9 vectors also resulted in decreased marker gene expression in the diaphragm. See Figure 28.

[0396] These results demonstrate the additive effect of the fusion constructs of the present invention over the μDys construct alone in the diaphragm based on their effect on these two fibrotic marker genes.

[0397] Example 9 Delivery of enzyme-based gene editing: CRISPR / Cas and sgRNA / crRNA The subject viral vectors, e.g., rAAV viral vectors, can be used to deliver CRISPR / Cas9 or CRISPR / Cas12a (or other engineered or modified Cas enzymes or homologs thereof) to target cells along with one or more sgRNAs (for Cas9) or one or more crRNAs (for Cas12a) for simultaneous knockdown of target genes in the target cells. The tropism of the target cells can be controlled in part by the tropism of the viral particles in which the sequences encoding the CRISPR / Cas and sgRNA / crRNA are present.

[0398] For example, in the case of AAV-mediated delivery, the GOI in the subject viral vector can be the coding sequence of CRISPR / Cas9 or CRISPR / Cas12a. One or more sgRNAs or crRNAs that can be loaded onto Cas9 or Cas12a, respectively, can be expressed from an intron, 3'-UTR, or other location in the Cas9 / Cas12a expression cassette.

[0399] Upon infection of target cells with the subject viral vectors, e.g., AAV vectors, the Cas proteins and sgRNA / crRNA are co-expressed in the target cells and mediate gene editing. The present invention includes, but is not limited to, the following aspects. [Aspect 1] Recombinant viral vectors, including: a) a polynucleotide encoding a functional gene or protein of interest (GOI), e.g., one effective in treating muscular dystrophy, wherein said polynucleotide comprises a 3'-UTR coding region and is immediately 3' to a heterologous intron sequence that enhances expression of said functional protein encoded by said polynucleotide; b) a regulatory element (e.g., a muscle-specific regulatory element) operably linked to the polynucleotide and driving its expression; and c) one or more coding sequences inserted into the intron sequence or the 3'-UTR coding region; Here, the one or more coding sequences independently encode an RNAi sequence (siRNA, shRNA, miRNA), an antisense sequence, a guide sequence for a gene editing enzyme, a microRNA (miRNA) and / or a miRNA inhibitor. [Aspect 2] The recombinant viral vector according to aspect 1, wherein the recombinant viral vector is a recombinant AAV (adeno-associated virus) vector. [Aspect 3] The recombinant viral vector of embodiment 1 or 2, wherein the one or more coding sequences are inserted into the 3'-UTR coding region or after a polyadenylation (polyA) signal sequence (e.g., AATAAA). [Aspect 4] 4. The recombinant viral vector of any one of aspects 1 to 3, wherein expression of said functional GOI is substantially unaffected in the presence of said one or more coding sequences (e.g., compared to an otherwise identical control construct in which said one or more coding sequences have not been inserted). [Aspect 5] 5. The recombinant viral vector according to any one of embodiments 1 to 4, comprising: a) the polynucleotide is a dystrophin microgene or minigene encoding a functional dystrophin protein; and / or b) The recombinant viral vector, wherein the regulatory element is a muscle-specific promoter operably linked to and driving expression of the dystrophin minigene. [Aspect 6] 6. The recombinant viral vector of embodiment 5, wherein the functional dystrophin protein is microD5 and / or the muscle-specific promoter is a CK promoter. [Aspect 7] 7. The recombinant viral vector according to any one of aspects 1 to 6, wherein the one or more coding sequences comprise an exon-skipping antisense sequence that induces skipping of a defective dystrophin exon, for example, skipping of any one of dystrophin exons 45 to 55, or skipping of dystrophin exons 44, 45, 51, and / or 53. [Aspect 8] Aspect 8. The recombinant viral vector according to any one of aspects 1 to 7, wherein the microRNA is miR-1, miR-133a, miR-29c, miR-30c, and / or miR-206. [Aspect 9] 9. The recombinant viral vector of embodiment 8, wherein the microRNA is miR-29c and optionally has modified flanking backbone sequences that facilitate processing of the guide strand of miR-29c designed for the target sequence. [Aspect 10] 10. The recombinant viral vector of embodiment 9, wherein the modified flanking backbone sequences are derived from or based on miR-30, -101, -155, or -451. [Aspect 11] 11. The recombinant viral vector of any one of aspects 8 to 10, wherein expression of the microRNA in a host cell is upregulated by at least about 1.5 to 15 fold (e.g., about 2 to 10 fold, about 1.4 to 2.8 fold, about 2 to 5 fold, about 5 to 10 fold, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 fold) compared to endogenous expression of the microRNA in the host cell. [Aspect 12] 12. The recombinant viral vector according to any one of aspects 1 to 11, wherein the RNAi sequence is an shRNA (shSLN) against sarcolipin. [Aspect 13] 13. The recombinant viral vector according to any one of aspects 1 to 12, wherein the one or more coding sequences encode one or more identical or different shRNAs (shSLNs) against sarcolipins. [Aspect 14] 14. The recombinant viral vector of embodiment 12 or 13, wherein the shRNA reduces expression of sarcolipin mRNA and / or sarcolipin protein by at least about 50%. [Aspect 15] 15. The recombinant viral vector according to any one of aspects 1 to 14, wherein the GOI is CRISPR / Cas9 and the guide sequence is sgRNA (single-stranded guide RNA), or the GOI is CRISPR / Cas12a and the guide sequence is crRNA. [Aspect 16] The RNAi sequence (siRNA, shRNA, miRNA), the antisense sequence, the CRISPR / Cas9 sgRNA, the CRISPR / Cas12a crRNA, and / or the microRNA target one or more target genes, such as inflammatory genes, activators of the NF-κB signaling pathway (e.g., TNF-α, IL-1, IL-1β, IL-6, receptor activator of NF-κB (RANK), and Toll-like receptors (TLRs)), NF-κB, and downstream inflammatory sites induced by NF-κB. 16. The recombinant viral vector of any one of aspects 1 to 15, which antagonizes the function of kine, histone deacetylase (e.g., HDAC2), TGF-β, connective tissue growth factor (CTGF), collagen, elastin, a component of the extracellular matrix, glucose-6-phosphate dehydrogenase (G6PD), myostatin, phosphodiesterase-5 (PED-5) or ACE, VEGF decoy receptor type 1 (VEGFR-1 or Flt-1), and hematopoietic prostaglandin D synthase (HPGDS). [Aspect 17] 2. The recombinant viral vector of embodiment 1, comprising: In patients with Fukuyama congenital muscular dystrophy (FCMD), a) the polynucleotide encodes a functional Fukutin (FKTN) protein, and / or b) The recombinant viral vector, wherein the one or more coding sequences encode an exon-skipping antisense sequence that restores correct splicing of exon 10 of a defective FKTN gene. [Aspect 18] 2. The recombinant viral vector of embodiment 1, comprising: In patients with merosin-deficient congenital muscular dystrophy type 1A (MDC1A), a) the polynucleotide encodes a functional LAMA2 protein, and / or b) The recombinant viral vector, wherein the one or more coding sequences encode an exon-skipping antisense sequence that restores expression of the C-terminal G domain (exons 45 to 64) of the defective LAMA2 gene, particularly G4 and G5. [Aspect 19] 2. The recombinant viral vector of embodiment 1, comprising: In DM1 patients, a) the polynucleotide encodes a functional DMPK protein or a CLCN1 gene; and / or b) The recombinant viral vector, wherein the RNAi sequence (siRNA, shRNA, miRNA), the antisense sequence, or the microRNA (miRNA) targets an expanded repeat of a mutant transcript of a defective DMPK gene or encodes an exon skipping antisense sequence that leads to skipping of exon 7A of the CLCN1 gene. [Aspect 20] 2. The recombinant viral vector of embodiment 1, comprising: In patients with dysferlinopathy (LGMD2B or MM), a) the polynucleotide encodes a functional DYSF protein, and / or b) The recombinant viral vector, wherein the one or more coding sequences encode an exon skipping antisense sequence that leads to skipping of exon 32 of the defective DYSF gene. [Aspect 21] 2. The recombinant viral vector of embodiment 1, comprising: In patients with LGMD2C, a) the polynucleotide encodes a functional SGCG protein, and / or b) The recombinant viral vector, wherein one or more coding sequences encode an exon skipping antisense sequence that leads to skipping of exons 4 to 7 of a defective LGMD2C gene (e.g., one having a Δ-521T SGCG mutation). [Aspect 22] 22. The recombinant viral vector according to any one of aspects 1 to 21, wherein the heterologous intron sequence is SEQ ID NO:1. [Aspect 23] 23. The recombinant viral vector according to any one of aspects 1 to 22, wherein the one or more coding sequences are inserted into the intron sequence. [Aspect 24] 24. The recombinant viral vector of any one of aspects 1 to 23, wherein expression of the functional protein is not adversely affected by the insertion of the one or more coding sequences. [Aspect 25] 25. The recombinant viral vector according to any one of aspects 1 to 24, wherein the vector is a recombinant AAV vector of the AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 serotype. [Aspect 26] 26. The recombinant viral vector of any one of aspects 1 to 25, wherein the regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer-binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), C5-12, a mouse creatine kinase enhancer element, a fast skeletal troponin c gene element, a slow cardiac troponin c gene element, a slow troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible factor, or a glucocorticoid response element (gre). [Aspect 27] 27. The recombinant viral vector of any one of aspects 1 to 26, wherein the regulatory element comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11 of WO2017 / 181015. [Aspect 28] A composition comprising the recombinant viral vector according to any one of embodiments 1 to 27. [Aspect 29] 29. The composition of embodiment 28, which is a pharmaceutical composition, further comprising a therapeutically compatible carrier, diluent, or excipient. [Aspect 30] 30. The composition of embodiment 29, wherein the therapeutically acceptable carrier, diluent, or excipient is a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride. [Aspect 31] At least 1.6 x 10 13 31. The composition of embodiment 29 or 30, wherein the composition is in the form of about 10 mL of an aqueous solution having the vector genome of [Aspect 32] At least 2 x 10 per milliliter 12 32. The composition according to any one of aspects 29 to 31, wherein the composition has the efficacy of a vector genome of [Aspect 33] A method of producing the composition of any one of aspects 28 to 32, comprising producing the recombinant viral vector (e.g., the recombinant AAV vector) in a cell and lysing the cell to obtain the vector. [Aspect 34] 34. The method of embodiment 33, wherein the vector is a recombinant AAV vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. [Aspect 35] 1. A method of treating muscular dystrophy or dystrophinopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the recombinant viral vector of any one of aspects 1 to 27 (e.g., the recombinant AAV vector), or the composition of any one of aspects 28 to 32. [Aspect 36] 36. The method of embodiment 35, wherein the recombinant AAV vector or the composition is administered by intramuscular injection, intravenous injection, parenteral administration, or systemic administration. [Aspect 37] 37. The method of embodiment 35 or 36, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy, myotonic dystrophy, and merosin-deficient congenital muscular dystrophy type 1A, facioscapulohumeral muscular dystrophy (FSHD), congenital muscular dystrophy (CMD), or limb-girdle muscular dystrophy (LGMDR5 or LGMD2C).

Claims

1. A recombinant AAV (adeno-associated virus) viral vector comprising: a) a polynucleotide encoding a functional gene or protein of interest (GOI), wherein said polynucleotide comprises a 3'-UTR coding region and is 3' to a heterologous intron sequence that enhances expression of said functional protein encoded by said polynucleotide, wherein said GOI is directly linked to the 3' side of said heterologous intron sequence; b) a regulatory element operably linked to the polynucleotide that drives its expression; and c) one or more coding sequences inserted into the intron sequence, the 3'-UTR coding region, and / or after a polyadenylation (polyA) signal sequence; wherein the one or more coding sequences independently encode an RNAi sequence, an antisense sequence, a guide sequence for a gene editing enzyme, a microRNA (miRNA) and / or a miRNA inhibitor.

2. The recombinant AAV virus vector of claim 1, wherein the functional gene or protein of interest is effective in treating muscular dystrophy.

3. The heterologous intron sequence comprises the first intron of natural rGH, intron A of the rat insulin II (rIns-II) gene, intron B of the hβG gene, or the small t intron of SV40. The recombinant AAV virus vector of claim 1.

4. The recombinant AAV virus vector of any one of claims 1 to 3, wherein the one or more coding sequences are inserted into the 3'-UTR coding region or after a polyadenylation (polyA) signal sequence.

5. 5. The recombinant AAV viral vector of any one of claims 1 to 4, wherein expression of the functional GOI is substantially unaffected in the presence of the one or more coding sequences compared to an otherwise identical control construct in which the one or more coding sequences are not inserted.

6. The recombinant AAV viral vector according to any one of claims 1 to 5, comprising: a) the polynucleotide encodes a functional dystrophin protein; and / or b) the control element is a muscle-specific promoter operably linked to and driving expression of the polynucleotide; The recombinant AAV virus vector.

7. The recombinant AAV virus vector of claim 6, wherein the functional dystrophin protein is microD5 and / or the muscle-specific promoter is a CK promoter.

8. The recombinant AAV virus vector of any one of claims 1 to 7, wherein the one or more coding sequences comprise an exon skipping antisense sequence that induces skipping of an exon of defective dystrophin.

9. The recombinant AAV virus vector of claim 8, wherein the skipping is skipping of any one of dystrophin exons 45 to 55, or dystrophin exons 44, 45, 51, and / or 53.

10. The recombinant AAV virus vector according to any one of claims 1 to 9, wherein the miRNA is miR-1, miR-133a, miR-29c, miR-30c, and / or miR-206.

11. The recombinant AAV virus vector of claim 10, wherein the miRNA is miR-29c and optionally has a modified flanking backbone sequence that facilitates processing of the guide strand of miR-29c designed for the target sequence.

12. The recombinant AAV viral vector of claim 11, wherein the modified flanking backbone sequence is derived from or based on miR-30, -101, -155, or -451.

13. The recombinant AAV virus vector of any one of claims 10 to 12, wherein expression of the microRNA in a host cell is upregulated by 1.5 to 15 times compared to endogenous expression of the miRNA in the host cell.

14. The recombinant AAV virus vector according to any one of claims 1 to 13, wherein the RNAi sequence is an shRNA (shSLN) against sarcolipin.

15. The recombinant AAV virus vector of any one of claims 1 to 14, wherein the one or more coding sequences encode one or more identical or different shRNAs against sarcolipin (shSLN).

16. The recombinant AAV virus vector of claim 14 or 15, wherein the shRNA reduces the expression of sarcolipin mRNA and / or sarcolipin protein by at least 50%.

17. The recombinant AAV virus vector of any one of claims 1 to 16, wherein the GOI is CRISPR / Cas9 and the guide sequence is sgRNA (single-stranded guide RNA), or the GOI is CRISPR / Cas12a and the guide sequence is crRNA.

18. The recombinant AAV virus vector of any one of claims 1 to 17, wherein the RNAi sequence, the antisense sequence, the CRISPR / Cas9 sgRNA, the CRISPR / Cas12a crRNA and / or the miRNA antagonize the function of one or more target genes.

19. The recombinant AAV virus vector of claim 18, wherein the target is selected from the group consisting of an inflammatory gene, an activator of the NF-κB signaling pathway, NF-κB, a downstream inflammatory cytokine induced by NF-κB, histone deacetylase, TGF-β, connective tissue growth factor (CTGF), collagen, elastin, a component of the extracellular matrix, glucose-6-phosphate dehydrogenase (G6PD), myostatin, phosphodiesterase-5 (PED-5) or ACE, VEGF decoy receptor type 1 (VEGFR-1 or Flt-1), and hematopoietic prostaglandin D synthase (HPGDS).

20. 2. The recombinant AAV viral vector of claim 1, comprising: a) the polynucleotide encodes a functional fukutin (FKTN) protein, and / or b) the one or more coding sequences encode an exon-skipping antisense sequence that restores correct splicing of exon 10 of the defective FKTN gene in Fukuyama congenital muscular dystrophy (FCMD) patients; The recombinant AAV virus vector.

21. 2. The recombinant AAV viral vector of claim 1, comprising: a) the polynucleotide encodes a functional LAMA2 protein, and / or b) the one or more coding sequences encode exon-skipping antisense sequences that restore expression of the C-terminal G domain (exons 45-64) of the defective LAMA2 gene, particularly G4 and G5, in patients with merosin-deficient congenital muscular dystrophy type 1A (MDC1A); The recombinant AAV virus vector.

22. 2. The recombinant AAV viral vector of claim 1, comprising: a) the polynucleotide encodes a functional DMPK protein or a CLCN1 gene, and / or b) in DM1 patients, the RNAi sequence, the antisense sequence, or the miRNA targets an expanded repeat of a mutant transcript of a defective DMPK gene or encodes an exon skipping antisense sequence that leads to the skipping of exon 7A of the CLCN1 gene; The recombinant AAV virus vector.

23. 2. The recombinant AAV viral vector of claim 1, comprising: a) the polynucleotide encodes a functional DYSF protein, and / or b) one or more coding sequences encode an exon skipping antisense sequence that leads to skipping of exon 32 of the defective DYSF gene in dysferlinopathy patients; The recombinant AAV virus vector.

24. 2. The recombinant AAV viral vector of claim 1, comprising: a) the polynucleotide encodes a functional SGCG protein, and / or b) one or more coding sequences encode an exon skipping antisense sequence that leads to the skipping of exons 4 to 7 of the defective LGMD2C gene in LGMD2C patients; The recombinant AAV virus vector.

25. The recombinant AAV virus vector of any one of claims 1 to 24, wherein the heterologous intron sequence is SEQ ID NO:

1.

26. The recombinant AAV virus vector of any one of claims 1 to 25, wherein the one or more coding sequences are inserted into the intron sequence.

27. 27. The recombinant AAV viral vector of any one of claims 1 to 26, wherein expression of the functional protein is not adversely affected by the insertion of the one or more coding sequences.

28. The recombinant AAV virus vector of any one of claims 1 to 27, wherein the vector is a recombinant AAV vector of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

29. 29. The recombinant AAV virus vector of any one of claims 1 to 28, wherein the regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer-binding factor mef, a muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), C5-12, a mouse creatine kinase enhancer element, a fast skeletal troponin c gene element, a slow cardiac troponin c gene element, a slow troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible factor, or a glucocorticoid response element (gre).

30. The regulatory element has the following nucleotide sequence: SEQ ID NO: 10 of WO 2017 / 181015 【Chemistry 1】 or SEQ ID NO: 11 of WO 2017 / 181015 【Chemistry 2】 The recombinant AAV virus vector according to any one of claims 1 to 29, comprising any one of:

31. A composition comprising the recombinant AAV viral vector of any one of claims 1 to 30.

32. 31. The composition of claim 30, which is a pharmaceutical composition, further comprising a therapeutically compatible carrier, diluent, or excipient.

33. 33. The composition of claim 32, wherein the therapeutically acceptable carrier, diluent, or excipient is a sterile aqueous solution comprising 10 mM L-histidine at pH 6.0, 150 mM sodium chloride, and 1 mM magnesium chloride.

34. At least 1.6 x 10 １３ 34. The composition of claim 32 or 33, in the form of a 10 mL aqueous solution having a vector genome of

35. At least 2 x 10 per milliliter １２ The composition according to any one of claims 32 to 34, having a vector genome potency of

36. 36. A method of producing the composition of any one of claims 31 to 35, comprising producing the recombinant AAV viral vector in a cell and lysing the cell to obtain the vector.

37. 37. The method of claim 36, wherein the recombinant AAV viral vector has a serotype of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

38. A pharmaceutical composition for treating muscular dystrophy or dystrophinopathy in a subject in need thereof, the pharmaceutical composition comprising a recombinant AAV viral vector according to any one of claims 1 to 30, or a composition according to any one of claims 31 to 35.

39. 39. The pharmaceutical composition of claim 38, wherein the recombinant AAV viral vector or the composition is administered by intramuscular injection, intravenous injection, parenteral administration or systemic administration.

40. 40. The pharmaceutical composition of claim 38 or 39, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy, myotonic dystrophy, merosin-deficient congenital muscular dystrophy type 1A, facioscapulohumeral muscular dystrophy (FSHD), congenital muscular dystrophy (CMD), or limb-girdle muscular dystrophy.

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