Adeno-Associated Virus Vector Delivery of Muscle Specific Micro-Dystrophin to Treat Muscular Dystrophy

Abstract

The invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, expressing a miniaturized human micro-dystrophin gene and method of using these vectors to express micro-dystrophin in skeletal muscle including diaphragm and cardiac muscle and to protect muscle fibers from injury, increase muscle strength and reduce and/or prevent fibrosis in subjects suffering from muscular dystrophy.

1 . A method of treating muscular dystrophy comprising administering i) a therapeutically effective amount of a recombinant AAV vector expressing micro-dystrophin and expression of micro-dystrophin is controlled by a muscle specific control element nucleotide sequence and ii) a therapeutically effective amount of a recombinant AAV vector expressing miR-29c and expression of miR-29c is controlled by a muscle-specific control element nucleotide sequence. 
     
     
         2 . A method of increasing muscular force or muscle mass in a subject suffering from muscular dystrophy comprising administering i) a therapeutically effective amount of a recombinant AAV vector expressing micro-dystrophin and expression of micro-dystrophin is controlled by a muscle specific control element nucleotide sequence and ii) a therapeutically effective amount of recombinant AAV vector expressing miR-29c and expression of miR-29c is controlled by a muscle-specific control element nucleotide sequence. 
     
     
         3 . A method of reducing or preventing fibrosis in a subject suffering from muscular dystrophy comprising administering i) a therapeutically effective amount of i) a recombinant AAV vector expressing micro-dystrophin and expression of micro-dystrophin is controlled by a muscle specific control element nucleotide sequence and ii) a therapeutically effective amount of recombinant AAV vector expressing miR-29c and expression of miR-29c is controlled by a muscle-specific control element nucleotide sequence. 
     
     
         4 . The method of  claim 1  wherein the muscular dystrophy is Duchenne muscular dystrophy. 
     
     
         5 . The method of  claim 1  wherein the nucleotide sequence encoding the micro-dystrophin protein comprises
 a) a nucleotide sequence that is at least 85% identical to the nucleotide sequence SEQ ID NO: 1 and encodes a functional micro-dystrophin protein, or 
 b) the nucleotide sequences of SEQ ID NO: 1. 
 
     
     
         6 . The method of  claim 1  wherein the recombinant AAV vector expressing miR-29c comprises:
 a) the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 9, 
 b) the nucleotide sequence of SEQ ID NO: 7, or 
 c) the nucleotide sequence of SEQ ID NO: 6. 
 
     
     
         7 . The method of  claim 1  wherein at least one of the muscle specific control 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), hybrid α-myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK7), C5-12, murine creatine kinase enhancer element, skeletal fast-twitch troponin c gene element, slow-twitch cardiac troponin c gene element, the slow-twitch troponin i gene element, hypoxia-inducible nuclear factors, steroid-inducible element or glucocorticoid response element (GRE). 
     
     
         8 . The method of  claim 1  wherein the muscle specific control element controlling expression of micro-dystrophin comprises SEQ ID NO: 2 (MHCK7). 
     
     
         9 . The method of  claim 1  wherein the muscle specific control element controlling expression of miR-29c comprises SEQ ID NO: 10 (CMV). 
     
     
         10 . The method of  claim 1  wherein the recombinant AAV vector expressing micro-dystrophin comprises i) the nucleotide sequences of SEQ ID NO: 1 (micro-dys) and ii) the nucleotide sequence of SEQ ID NO: 2 (MHCK7). 
     
     
         11 . The method of  claim 1  wherein the recombinant AAV vector expressing micro-dystrophin comprises the nucleotide sequence of SEQ ID NO: 3. 
     
     
         12 . The method of  claim 1  wherein the recombinant AAV vector expressing miR-29c comprises i) the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9 and ii) the nucleotide sequence of SEQ ID NO: 10 (CMV). 
     
     
         13 . The method of  claim 1  wherein the recombinant AAV vector expressing miR-29c comprises the nucleotide sequence of SEQ ID NO: 6. 
     
     
         14 . The method of  claim 1  wherein the recombinant AAV vector expressing micro-dystrophin comprises i) the nucleotide sequences of SEQ ID NO: 1 (micro-dys) and ii) the nucleotide sequence of SEQ 2 (MHCK7), and wherein the recombinant AAV vector expressing miR-29c comprises i) the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9 and ii) the nucleotide sequence of SEQ ID NO: 10 (CMV). 
     
     
         15 . The method of  claim 1  wherein the recombinant AAV vector expressing micro-dystrophin comprises the nucleotide sequence of SEQ ID NO: 3 and the recombinant AAV vector expressing miR-29c comprises the nucleotide sequence of SEQ ID NO: 6. 
     
     
         16 . The method of  claim 1  wherein at least one of the recombinant AAV vectors is the serotype AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13. 
     
     
         17 . The method of  claim 1  wherein at least one of the recombinant AAV vectors is administered by intramuscular injection or intravenous injection. 
     
     
         18 . The method of  claim 1  wherein at least one of the recombinant AAV vectors is administered systemically. 
     
     
         19 . The method of  claim 18 , wherein at least one of the recombinant AAV vectors is parenterally administered by injection, infusion or implantation. 
     
     
         20 - 57 . (canceled)