Optimized mini-dystrophin genes and expression cassettes and their use

Abstract

This invention relates to polynucleotides encoding mini-dystrophin proteins, viral vectors comprising the same, and methods of using the same for delivery of mini-dystrophin to a cell or a subject.

1 - 93 . (canceled) 
     
     
         94 . A recombinant AAV particle, comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein. 
     
     
         95 . The recombinant AAV particle of  claim 94 , wherein the human mini-dystrophin protein comprises the following subdomains from full-length human dystrophin protein in order from amino-terminus to carboxy-terminus: N-terminal Actin-Binding Domain, H1, R1, R2, H3, R22, R23, R24, H4, Cysteine Rich Domain, and a portion of the Carboxy-Terminal Domain not including the last 3 amino acids in wildtype human muscle dystrophin. 
     
     
         96 . The recombinant AAV particle of  claim 95 , wherein the human mini-dystrophin protein comprises the amino acid sequence of SEQ ID NO:7. 
     
     
         97 . The recombinant AAV particle of  claim 95 , wherein said human codon-optimized nucleic acid encoding the human mini-dystrophin protein comprises the nucleic acid sequence of SEQ ID NO:1, or a nucleic acid sequence at least 95% identical thereto. 
     
     
         98 . The recombinant AAV particle of  claim 95 , wherein said vector genome comprises, in 5′ to 3′ order: a first AAV2 ITR, a muscle-specific transcriptional regulatory element operably linked to said human codon-optimized nucleic acid encoding the human mini-dystrophin protein, a transcription termination sequence, and a second AAV2 ITR. 
     
     
         99 . The recombinant AAV particle of  claim 98 , wherein said muscle-specific transcriptional regulatory element is derived from the human or mouse creatine kinase gene. 
     
     
         100 . The recombinant AAV particle of  claim 99 , wherein said muscle-specific transcriptional regulatory element comprises the nucleotide sequence of SEQ ID NO:16. 
     
     
         101 . The recombinant AAV particle of  claim 100 , wherein said transcription termination sequence comprises a polyadenylation signal sequence. 
     
     
         102 . The recombinant AAV particle of  claim 101 , wherein said transcription termination sequence comprises the nucleotide sequence of SEQ ID NO:17. 
     
     
         103 . The recombinant AAV particle of  claim 102 , wherein said vector genome comprises the nucleotide sequence of SEQ ID NO:18 or the reverse-complement thereof. 
     
     
         104 . A recombinant AAV particle, comprising an AAV9 capsid and a vector genome consisting of the nucleotide sequence of SEQ ID NO:18 or the reverse complement thereof. 
     
     
         105 . A pharmaceutical composition comprising a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein and a pharmaceutically acceptable carrier. 
     
     
         106 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein. 
     
     
         107 . The method of  claim 106 , wherein the composition is administered with a least a second agent effective for treating DMD. 
     
     
         108 . The method of  claim 107 , wherein said second agent is selected from the group consisting of: an antisense oligonucleotide that causes exon skipping of the DMD gene, an anti-myostatin antibody, an agent that promotes ribosomal read-through of nonsense mutations, an agent that suppresses premature stop codons, an anabolic steroid and a corticosteroid. 
     
     
         109 . The method of  claim 106 , wherein the recombinant AAV particles are administered in a dose selected from the group consisting of: 1×10 12  vg/kg, 2×10 12  vg/kg, 3×10 12  vg/kg, 4×10 12  vg/kg, 5×10 12  vg/kg, 6×10 12  vg/kg, 7×10 12  vg/kg, 8×10 12  vg/kg, 9×10 12  vg/kg, 1×10 13  vg/kg, 2×10 13  vg/kg, 3×10 13  vg/kg,  4 × 10   13  vg/kg, 5×10 13  vg/kg, 6×10 13  vg/kg, 7×10 13  vg/kg, 8×10 13  vg/kg, 9×10 13  vg/kg, 1×10 14  vg/kg, 1.5×10 14  vg/kg, 2×10 14  vg/kg, 2.5×10 14  vg/kg, 3×10 14  vg/kg, 3.5×10 14  vg/kg, 4×10 14  vg/kg, 4.5×10 14  vg/kg, 5×10 14  vg/kg, 5.5×10 14  vg/kg, 6×10 14  vg/kg,  6 . 5 × 10   14  vg/kg,  7 × 10   14  vg/kg,  7 . 5 × 10   14  vg/kg, 8×10 14  vg/kg, 8.5×10 14  vg/kg, and 9×10 14  vg/kg. 
     
     
         110 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method is effective to reduce the average of at least one of ALT, AST, or LDH levels in the blood of the subject to within about 7-, 6-, 5-, 4-, 3-, or 2-fold greater than the average level of ALT, AST, or LDH in the blood of healthy controls. 
     
     
         111 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method is effective to reduce the total CK level, in the blood of the subject to within about 50-, 48-, 46-, 44-, 42-, 40-, 38-, 36-, 34-, 32-, 30-, 28-, 26, 24-, 22-, 20-, 18-, 16-, 14-, 12-, 10-, 9-, 8-, 7-, 6-, 5-, 4-, 3-, or 2-fold greater than the average total CK level in the blood of healthy controls. 
     
     
         112 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method is effective to increase the average 6 minute walk distance (6MWD) of the subject by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 meters compared to the average 6MWD of untreated DMD controls, or of the subject before treatment. 
     
     
         113 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method is effective to reduce the average time required for the subject to perform the 4 stair climb test by at least 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0 seconds compared to the average time of untreated DMD controls, or the subject before treatment. 
     
     
         114 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method is effective to reduce the average proportion of treated subjects that have lost ambulation by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% compared to the average proportion of untreated DMD controls that have lost ambulation. 
     
     
         115 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method is effective to reduce the fat fraction in the lower extremities of the subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% compared to the average fat fraction in the lower extremities of untreated DMD controls, or the subject before treatment. 
     
     
         116 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method is effective to cause at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the muscle fibers in a skeletal muscle biopsy from said subject to produce detectable levels of said mini-dystrophin protein. 
     
     
         117 . A method for treating Duchenne muscular dystrophy (DMD) comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, wherein said method causes a cellular immune response against, said mini-dystrophin protein, or muscle inflammation, in less than or equal to about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of treated subjects. 
     
     
         118 . A method of increasing muscle mass or strength comprising administering to a subject in need thereof a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein in an amount sufficient to increase muscle mass or strength in the subject. 
     
     
         119 . A method of preventing loss of muscle mass or strength comprising administering to a subject in need thereof a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein in an amount sufficient to prevent loss of muscle mass or strength in the subject. 
     
     
         120 . A method of making a recombinant AAV particle comprising an AAV9 capsid and a vector genome comprising a human codon-optimized nucleic acid sequence encoding a human mini-dystrophin protein, comprising:
 introducing into a cell a polynucleotide comprising the AAV vector genome, a polynucleotide comprising an AAV rep gene, a polynucleotide comprising an AAV9 cap gene and, optionally, a polynucleotide encoding one or more viral helper genes,   incubating said cell; and   purifying the recombinant AAV particles produced thereby.   
     
     
         121 . A recombinant AAV particle produced by the method of  claim 120 . 
     
     
         122 . A recombinant AAV particle, comprising an AAV9 capsid and a vector genome comprising the nucleotide sequence of SEQ ID NO:18 or the reverse complement of.