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 . A method of generating a recombinant adeno-associated virus (AAV) comprising culturing a host cell containing: (a) a molecule encoding the AAV vp1 capsid protein having a sequence of amino acids 1 to 736 of SEQ ID NO: 123, or a sequence which is at least 95% identical to the full length of amino acids 1 to 736 of SEQ ID NO: 123; (b) a functional rep gene; (c) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a host cell; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein under conditions which permit packaging of the minigene into the AAV capsid. 
     
     
         2 . The method of  claim 1 , wherein the sequence of the vp1 protein is at least 97% identical to the full length of amino acids 1 to 736 of SEQ ID NO: 123. 
     
     
         3 . The method of  claim 1 , wherein the sequence of the vp1 protein is at least 99% identical to the full-length of amino acids 1 to 736 of SEQ ID NO: 123. 
     
     
         4 . The method of  claim 1 , wherein the sequence of the vp1 protein is the full-length of amino acids 1 to 736 of SEQ ID NO: 123. 
     
     
         5 . The method of  claim 1 , wherein the gene product is a low density lipoprotein (LDL) receptor, a high density lipoprotein (HDL) receptor, a very low density lipoprotein (VLDL) receptor, Factor VIII, Factor IX, erythropoietin, ornithine transcarbamylase (OTC), vascular endothelial growth factor (VEGF), glucose-6-phosphatase, alpha-1 antitrypsin, or an antibody. 
     
     
         6 . The method of  claim 1 , wherein the molecule is a nucleic acid molecule comprising nucleotides 1 to 2208 of SEQ ID NO: 3, or a nucleotide sequence at least 99% identical to nucleotides 1 to 2208 of SEQ ID NO: 3. 
     
     
         7 . The method of  claim 1 , wherein the rep gene is from AAV2. 
     
     
         8 . A method of generating a recombinant adeno-associated virus (AAV) comprising culturing a host cell containing: (a) a molecule encoding the AAV vp2 capsid protein having a sequence of amino acids 138 to 736 of SEQ ID NO: 123, or a sequence which is at least 95% identical to the full length of amino acids 138 to 736 of SEQ ID NO: 123; (b) a functional rep gene; (c) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a host cell; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein under conditions which permit packaging of the minigene into the AAV capsid. 
     
     
         9 . The method of  claim 8 , wherein the sequence of the vp2 protein is at least 97% identical to the full length of amino acids 138 to 736 of SEQ ID NO: 123. 
     
     
         10 . The method of  claim 8 , wherein the sequence of the vp2 protein is at least 99% identical to the full-length of amino acids 138 to 736 of SEQ ID NO: 123. 
     
     
         11 . The method of  claim 8 , wherein the sequence of the vp2 protein is the full-length of amino acids 138 to 736 of SEQ ID NO: 123. 
     
     
         12 . The method of  claim 8 , wherein the gene product is a low density lipoprotein (LDL) receptor, a high density lipoprotein (HDL) receptor, a very low density lipoprotein (VLDL) receptor, Factor VIII, Factor IX, erythropoietin, ornithine transcarbamylase (OTC), vascular endothelial growth factor (VEGF), glucose-6-phosphatase, alpha-1 antitrypsin, or an antibody. 
     
     
         13 . The method of  claim 8 , wherein the molecule is a nucleic acid molecule comprising nucleotides 412 to 2208 of SEQ ID NO: 3, or a nucleotide sequence at least 99% identical to nucleotides 412 to 2208 of SEQ ID NO: 3. 
     
     
         14 . The method of  claim 8 , wherein the rep gene is from AAV2. 
     
     
         15 . A method of generating a recombinant adeno-associated virus (AAV) comprising culturing a host cell containing: (a) a molecule encoding the AAV vp3 capsid protein having a sequence of amino acids 203 to 736 of SEQ ID NO: 123, or a sequence which is at least 95% identical to the full length of amino acids 203 to 736 of SEQ ID NO: 123; (b) a functional rep gene; (c) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences which direct expression of the gene product in a host cell; and (d) sufficient helper functions to permit packaging of the minigene into the AAV capsid protein under conditions which permit packaging of the minigene into the AAV capsid. 
     
     
         16 . The method of  claim 15 , wherein the sequence of the vp3 protein is at least 97% identical to the full length of amino acids 203 to 736 of SEQ ID NO: 123. 
     
     
         17 . The method of  claim 15 , wherein the sequence of the vp3 protein is at least 99% identical to the full-length of amino acids 203 to 736 of SEQ ID NO: 123. 
     
     
         18 . The method of  claim 15 , wherein the sequence of the vp3 protein is the full-length of amino acids 203 to 736 of SEQ ID NO: 123. 
     
     
         19 . The method of  claim 15 , wherein the gene product is a low density lipoprotein (LDL) receptor, a high density lipoprotein (HDL) receptor, a very low density lipoprotein (VLDL) receptor, Factor VIII, Factor IX, erythropoietin, ornithine transcarbamylase (OTC), vascular endothelial growth factor (VEGF), glucose-6-phosphatase, alpha-1 antitrypsin, or an antibody 
     
     
         20 . The method of  claim 15 , wherein the molecule is a nucleic acid molecule comprising nucleotides 607 to 2208 of SEQ ID NO: 3, or a nucleotide sequence at least 99% identical to nucleotides 607 to 2208 of SEQ ID NO: 3. 
     
     
         21 . The method of  claim 15 , wherein the rep gene is from AAV2.