Adeno-associated virus (aav) clades, sequences, vectors containing same, and uses therefor

Abstract

Sequences of novel adeno-associated virus capsids and vectors and host cells containing these sequences are provided. Also described are methods of using such host cells and vectors in production of rAAV particles. AAV-mediated delivery of therapeutic and immunogenic genes using the vectors of the invention is also provided.

1 . An AAV comprising an AAV9 capsid, wherein the AAV9 capsid comprises an amino acid sequence having at least 95% identity with amino acids 203 to 736 of SEQ ID NO: 123, and wherein said AAV further comprises a minigene having AAV inverted terminal repeats and a transgene comprising a heterologous gene operably linked to regulatory sequences which direct expression of the heterologous gene in an host cell. 
     
     
         2 . The AAV according to  claim 1 , wherein the AAV9 capsid comprises a sequence selected from the group consisting of an amino acid sequence having at least 95% identity with amino acids 138-736 of SEQ ID NO: 123 and an amino acid sequence having at least 95% identity with amino acids 1-736 of SEQ ID NO: 123. 
     
     
         3 . The AAV according to  claim 1 , wherein the AAV9 capsid comprises a sequence selected from the group consisting of an amino acid sequence having at least 99% identity with amino acids 203-736, an amino acid sequence having at least 99% identity with amino acids 138-736, and an amino acid sequence having at least 99% identity with amino acids 1-736. 
     
     
         4 . A composition comprising an AAV vector according to  claim 1  and a physiologically compatible carrier. 
     
     
         5 . An adeno-associated virus (AAV) comprising an AAV capsid selected from the group consisting of: serotype hu.35, amino acids 204 to 734 of SEQ ID NO: 186; serotype hu.37, amino acids 204 to 738 of SEQ ID NO: 88; serotype pi.2, amino acids 203 to 731 of SEQ ID NO: 95; serotype rh.64, SEQ ID NO: 99; and serotype rh.65R2, SEQ ID NO: 233;
 and wherein the AAV further comprises a minigene having AAV inverted terminal repeats and a heterologous gene operably linked to regulatory sequences which direct its expression in a host cell.   
     
     
         6 . An adeno-associated virus (AAV) clade F comprising AAV9, AAV hu.14/AAV9 (SEQ ID Nos: 3 and 123), hu.31 (SEQ ID NOs: 1 and 121) and hu.32 (SEQ ID Nos: 2 and 122), wherein each member of said AAV clade is phylogenetically related as determined using a Neighbor-Joining heuristic by a bootstrap value of at least 75% per 1000 isolates and a Poisson correction distance measurement of no more than 0.05. 
     
     
         7 . The AAV clade F according to  claim 6  wherein said clade comprises at least AAV hu.14/AAV9 (SEQ ID Nos: 3 and 123), hu.31 (SEQ ID NOs: 1 and 121) and hu.32 (SEQ ID Nos: 2 and 122). 
     
     
         8 . A composition comprising a pharmaceutical carrier and a protein comprising an AAV9/HU.14 capsid protein or a fragment thereof selected from the group consisting of:
 vp1 capsid protein, amino acids (aa) 1 to 736, SEQ ID NO:123;   an AAV capsid comprising a vp2 capsid protein, aa 138 to 736, SEQ ID NO: 123;   an AAV capsid comprising a vp3 capsid protein, aa 203 to 736, SEQ ID NO: 123;   an AAV capsid comprising a fragment encompassing at least one hypervariable region (HVR)1 through 12 selected the group consisting of: aa 146 to 152; aa 182 to 187; aa 262 to 264; aa 263 to 266; aa 263 to 266; aa 381 to 383; 383 to 385; aa 450 to 474; aa 451 to 475; aa 490 to 495; aa 491 to 496; aa500 to 504; aa 501 to 505; aa 514 to 522; aa 533 to 554; aa 534 to 555; aa 581 to 594; aa 583 to 596; aa 658 to 667; aa 660 to 669; and aa 705 to 719; aa 707 to 723;   aa 24 to 42, aa 25 to 28; aa 81 to 85; aa133 to 165; aa 134 to 165; aa 137 to 143; aa 154 to 156; aa 194 to 208; aa 261 to 274; aa 262 to 274; aa 171 to 173; aa 185 to 198; aa 413 to 417; aa 449 to 478; aa 494 to 525; aa 534 to 571; aa 581 to 601; aa 660 to 671; aa 709 to 723; and   aa 1 to 184, aa 199 to 259; aa 274 to 446; aa 603 to 659; aa 670 to 706; aa 724 to 736; aa 185 to 198; aa 260 to 273; aa447 to 477; aa495 to 602; aa603 to 659; aa 660 to 669; and aa707 to 723, wherein the amino acid numbers are those of the AAV2 capsid, SEQ ID NO:4 and corresponding regions in the capsid of AAV9/HU.14, SEQ ID NO:123.   
     
     
         9 . A vector containing a nucleic acid sequence encoding a protein according to  claim 8 . 
     
     
         10 . The vector according to  claim 9 , wherein said nucleic acid sequence is selected from the group consisting of: the nucleotides (nt) 1 to 2211 of SEQ ID NO: 3; the nt 411 to 2211 of SEQ ID NO: 3; and the nt 609 to 2211 of SEQ ID NO: 3. 
     
     
         11 . The vector according to  claim 10 , wherein said molecule comprises an AAV sequence encoding an AAV capsid protein and a functional AAV rep protein. 
     
     
         12 . The vector according to  claim 10 , wherein said molecule is a plasmid. 
     
     
         13 . A method of generating a recombinant adeno-associated virus (AAV) comprising an AAV capsid comprising the steps of culturing a host cell containing: (a) a molecule encoding an AAV capsid protein; (b) a functional rep gene; (c) a minigene comprising AAV inverted terminal repeats (ITRs) and a transgene; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein, wherein said host cell comprises a nucleic acid sequence according to  claim 10 . 
     
     
         14 . A host cell transfected with an adeno-associated virus according to  claim 1 . 
     
     
         15 . A host cell transfected with the vector according to  claim 9 .