MUSCLE TROPIC RAAV
Recombinant myotropic AAV rh.74 capsids are described herein. In addition, plasmids, viral vectors, dual vector systems, cells, and compositions comprising or encoding such recombinant capsids are further described. Such recombinant polynucleotides, plasmids, viral vectors, dual vector systems, cells, and compositions may be used to treat muscle diseases or disorders, including muscular dystrophies and cardiomyopathies.
1 . A modified AAV particle comprising (i) a capsid protein comprising an AAVrh74 capsid and a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2; and (ii) a transfer cassette comprising a 5′ ITR, a kazak sequence, a chimeric intron, an MHCK7 promoter, a microdystrophin gene, a polyA sequence, and a 3′ ITR,
wherein the microdystrophin gene comprises the nucleotide sequence as set forth in SEQ ID NO: 8.
2 . The modified AAV particle of claim 1 , wherein the peptide is located between amino acids corresponding to amino acids 587 and 591 of SEQ ID NO: 4 and amino acids corresponding to amino acids 588, 589, and 590 of SEQ ID NO: 4 are absent.
3 . The modified AAV particle of claim 1 or 2 , wherein the capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 1.
4 . The modified AAV particle of claim 3 , wherein the capsid protein is encoded by the nucleotide sequence as set forth in SEQ ID NO: 3.
5 . (canceled)
6 . The modified AAV particle of claim 1 , wherein the transfer cassette comprises the nucleotide sequence as set forth in SEQ ID NO: 17.
7 . A pharmaceutical composition comprising the modified AAV particle of claim 1 , and a pharmaceutically acceptable carrier.
8 .- 9 . (canceled)
10 . A method of treating a muscular disease comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 7 .
11 . The method of claim 10 , wherein the muscular disease is Duchenne muscular dystrophy, Becker's muscular dystrophy, Myotonic muscular dystrophy, Facioscapulohumeral muscular dystrophy, a limb-girdle muscle dystrophy, or a cardiomyopathy.
12 . (canceled)
13 . A pharmaceutical composition comprising (i) about 20 mM Tris, (ii) about 1 mM MgCl 2 , (iii) about 180-about 200 mM NaCl, (iv) about 0.01%-about 0.2% poloxamer, (v) an AAV particle, and (vi) optionally about 1%-about 3% sucrose.
14 . (canceled)
15 . The pharmaceutical composition of claim 13 , wherein (i) the NaCl is at a concentration of about 180 mM, about 190 mM, or about 200 mM, (ii) the poloxamer is at a concentration of about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.2%, and/or (iii) the sucrose is at a concentration of about 1%, about 1.5%, about 2%, about 2.5%, or about 3%.
16 .- 18 . (canceled)
19 . The pharmaceutical composition of claim 13 , comprising (i) about 20 mM Tris, (ii) about 1 mM MgCl 2 , (iii) about 180 NaCl, (iv) about 0.01% poloxamer, and (v) an AAV particle.
20 . The pharmaceutical composition of claim 13 , comprising (i) about 20 mM Tris, (ii) about 1 mM MgCl 2 , (iii) about 180 NaCl, (iv) about 0.01% poloxamer, (v) about 2% sucrose, and (vi) an AAV particle.
21 .- 26 . (canceled)
27 . The pharmaceutical composition of claim 13 , wherein the AAV particle comprises a capsid protein and a transfer cassette, wherein the capsid protein comprises a peptide targeting muscle comprising the amino acid sequence as set forth in SEQ ID NO: 2.
28 . The pharmaceutical composition of claim 27 , wherein the peptide is located in Hyper Variable Region VIII of a Rh74 capsid protein or Hyper Variable Region IV of a Rh74 capsid protein.
29 . The pharmaceutical composition of claim 27 , wherein the peptide is located (i) between amino acids corresponding to amino acids 587 and 591 of a wild type AAVRh74 capsid protein, and amino acids corresponding to amino acids 588, 589, and 590 of a wild type AAVRh74 are absent or (ii) between amino acid residues corresponding to amino acids 451 and 462 of a wild type AAVRh74 capsid protein, and wherein amino acid residues corresponding to amino acids 452-461 of a wild type AAVRh74 capsid protein are absent.
30 .- 32 . (canceled)
33 . A method of treating a disease or condition in a subject in need thereof comprising administering to the subject the pharmaceutical composition of claim 13 .
34 . The method of claim 33 , wherein the disease or condition comprises a muscular disease.
35 . The method of claim 34 , wherein the muscular disease is Duchenne muscular dystrophy, Becker's muscular dystrophy, Myotonic muscular dystrophy, Facioscapulohumeral muscular dystrophy, a limb-girdle muscle dystrophy, or a cardiomyopathy.
36 . The method of claim 33 , wherein the subject is a human.
37 . A modified AAV particle comprising (i) a capsid protein comprising the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 11 and (ii) a transfer cassette comprising the nucleotide sequence as set forth in SEQ ID NO: 17.
38 .- 40 . (canceled)