Adeno-associated virus serotype i nucleic acid sequences, vectors and host cells containing same

Abstract

The nucleic acid sequences of adeno-associated virus (AAV) serotype 1 are provided, as are vectors and host cells containing these sequences and functional fragments thereof. Also provided are methods of delivering genes via AAV-1 derived vectors.

1 . A hybrid virus particle comprising: an adeno-associated virus (AAV) serotype-1 capsid; and a nucleic acid comprising at least one AAV serotype 2 inverted terminal repeat packaged within said AAV serotype-1 capsid. 
     
     
         2 . The hybrid virus particle of  claim 1 , wherein said nucleic acid comprises at least one heterologous nucleotide sequence. 
     
     
         3 . The hybrid virus particle of  claim 2 , comprising two AAV inverted terminal repeats that flank said at least one heterologous nucleotide sequence. 
     
     
         4 . The hybrid virus particle of  claim 2 , wherein at least one heterologous nucleotide sequence encodes a protein or peptide. 
     
     
         5 . The hybrid virus particle of  claim 4 , wherein said protein or peptide is a therapeutic protein or peptide. 
     
     
         6 . The hybrid virus particle of  claim 4 , wherein said protein or peptide is an immunogenic protein or peptide. 
     
     
         7 . The hybrid virus particle of  claim 4 , wherein said at least one heterologous nucleotide sequence encodes erythropoietin, a cytokine, a hormone, or a growth factor. 
     
     
         8 . The hybrid virus particle of  claim 1 , wherein said nucleic acid does not comprise AAV cap genes or AAV rep genes. 
     
     
         9 . A pharmaceutical formulation comprising the hybrid virus particle of  claim 1  in a pharmaceutically-acceptable carrier. 
     
     
         10 . An isolated nucleic acid for producing the hybrid virus particle of  claim 1 , wherein said isolated nucleic acid comprises AAV serotype-1 cap genes and AAV rep genes, wherein the serotypes of said AAV cap genes and said AAV rep genes are different. 
     
     
         11 . The isolated nucleic acid of  claim 10 , wherein said AAV cap genes are operably associated with an authentic AAV promoter. 
     
     
         12 . The isolated nucleic acid of  claim 11 , wherein said authentic AAV promoter is an AAV p40 promoter. 
     
     
         13 . The isolated nucleic acid of  claim 10 , wherein said AAV rep genes are AAV serotype-2 rep genes. 
     
     
         14 . The isolated nucleic acid of  claim 10 , wherein said AAV rep genes are of a serotype selected from the group consisting of AAV serotypes 2, 3, 4, 5 and 6. 
     
     
         15 . A vector comprising the isolated nucleic acid of  claim 10 . 
     
     
         16 . The vector of  claim 15 , wherein said vector is selected from the group consisting of plasmids and viral vectors. 
     
     
         17 . The vector of  claim 16 , wherein said vector is a plasmid. 
     
     
         18 . A cell comprising the vector of  claim 15 . 
     
     
         19 . The cell of  claim 18 , wherein said cell is a mammalian cell. 
     
     
         20 . A cell comprising a vector comprising: adeno-associated virus (AAV) serotype-1 cap genes, AAV rep genes, and a nucleic acid comprising at least one AAV serotype 2 inverted terminal repeat. 
     
     
         21 . The cell of  claim 20 , wherein said cell is a mammalian cell. 
     
     
         22 . A cell comprising adeno-associated virus (AAV) serotype-1 cap genes and AAV rep genes stably integrated into the genome of the cell, wherein the serotypes of said AAV rep genes stably integrated into the genome of the cell, wherein the serotypes of said AAV cap genes and said AAV rep genes are different. 
     
     
         23 . The cell of  claim 22 , further comprising a nucleic acid comprising at least one AAV serotype 2 inverted terminal repeat. 
     
     
         24 . A method of producing a hybrid virus particle, comprising: providing a cell with adeno-associated virus (AAV) rep genes, AAV serotype-1 cap genes, a nucleic acid comprising at least one AAV serotype 2 inverted terminal repeat, and helper functions for generating a productive AAV infection; and allowing assembly of the hybrid virus particles. 
     
     
         25 . The method of  claim 24 , further comprising collecting the hybrid virus particles. 
     
     
         26 . The method of  claim 24 , wherein the nucleic acid comprises at least one heterologous nucleotide sequence. 
     
     
         27 . The method of  claim 24 , wherein the AAV cap genes and AAV rep genes are provided by one or more transcomplementing packaging vectors. 
     
     
         28 . The method of  claim 24 , wherein the AAV cap genes and AAV rep genes are provided by a plasmid. 
     
     
         29 . The method of  claim 24 , wherein the AAV cap genes and AAV rep genes are provided by an adenovirus vector. 
     
     
         30 . The method of  claim 24 , wherein the AAV cap genes and AAV rep genes are stably integrated into the genome of the cell. 
     
     
         31 . A hybrid virus particle produced by the method of  claim 24 . 
     
     
         32 . An isolated AAV-1 nucleic acid molecule comprising a sequence selected from the group consisting of: (a) SEQ ID NO: 1; (b) a DNA sequence complementary to SEQ ID NO: 1; (c) cDNA complementary to (a) or (b); and (d) RNA complementary to any of (a) to (c). 
     
     
         33 . A nucleic acid molecule comprising an AAV-1 inverted terminal repeat (ITR) sequence selected from the group consisting of: (a) nt 1 to 143 of SEQ ID NO: 1; (b) nt 4576 to 4718 of SEQ ID NO: 1; (c) a nucleic acid sequence complementary to (a) or (b); and (d) a functional fragment of (a), (b), or (c). 
     
     
         34 . A recombinant vector comprising a 5′ AAV-1 inverted terminal repeat (ITR) and a selected transgene, wherein said ITR has the sequence selected from the group consisting of: (a) nt 1 to 143 of SEQ ID NO: 1; (b) a nucleic acid sequence complementary to (a); and (c) a functional fragment of (a) or (b). 
     
     
         35 . The recombinant vector according to  claim 34 , wherein said vector further comprises a 3′ AAV-1 ITR. 
     
     
         36 . A recombinant vector comprising a 3′ AAV-1 inverted terminal repeat (ITR) and a selected transgene, wherein said ITR has the sequence selected from the group consisting of: (a) nt 4576 to 4718 of SEQ ID NO: 1; (b) a nucleic acid sequence complementary to (a); and (c) a functional fragment of (a) or (b). 
     
     
         37 . The recombinant vector according to  claim 36 , wherein said vector further comprises a 5′ AAV-1 ITR. 
     
     
         38 . A pharmaceutical composition comprising a carrier and a virus comprising the vector according to  claim 36 . 
     
     
         39 . A method for producing a selected gene product comprising transfecting a mammalian cell with the molecule according to  claim 32  or a functional fragment thereof and culturing said cell under conditions suitable to express said gene product. 
     
     
         40 . The recombinant vector according to  claim 34 , wherein said vector further comprises AAV-1 capsid proteins having the sequence of SEQ ID NO: 13, 15 or 17 or functional fragments thereof. 
     
     
         41 . The recombinant vector according to  claim 34 , wherein said vector further comprises adenovirus sequences. 
     
     
         42 . The host cell transduced with a recombinant viral vector according to  claim 34 . 
     
     
         43 . The host cell transduced with a nucleic acid molecule according to  claim 32 . 
     
     
         44 . The host cell transduced with a nucleic acid molecule according to  claim 33 . 
     
     
         45 . The pharmaceutical composition comprising a carrier and a virus comprising the vector according to  claim 34 . 
     
     
         46 . The method for delivery of a transgene comprising delivering to a host cell a recombinant virus comprising a recombinant vector according to  claim 34 . 
     
     
         47 . A recombinant host cell transformed with a nucleic acid sequence expressing one or more AAV-1 rep proteins selected from the group consisting of rep78 having the amino acid sequence of SEQ ID NO: 7, rep 68 having the amino acid sequence of SEQ ID NO: 7, rep 52 having the amino acid sequence of SEQ ID NO: 9, and rep 40 having the amino acid sequence of SEQ ID NO: 11. 
     
     
         48 . A composition comprising a recombinant virus having an AAV-1 capsid comprising an AAV-1 protein selected from the group consisting of AAV-1 vp1 having the amino acid sequence of SEQ ID No: 13; AAV-1 vp2 having the amino acid sequence of SEQ ID NO: 15 and AAV-1 vp3 having the amino acid sequence of SEQ ID NO: 17 and a heterologous molecule which comprises an AAV 5′ inverted terminal repeat sequence (ITR), a transgene, and an AAV 3′ ITR. 
     
     
         49 . The composition of  claim 48  wherein the AAV-1 protein vp1 is encoded by a nucleic acid having at least about 98% identity to nucleotides 2223-4431 of SEQ ID NO: 1, as measured by MacVector 6.0. 
     
     
         50 . The composition of  claim 48  wherein the AAV-1 protein vp2 is encoded by a nucleic acid having at least about 98% identity to nucleotides 2634-4432 of SEQ ID NO: 1, as measured by MacVector 6.0. 
     
     
         51 . The composition of  claim 48  wherein the AAV-1 protein vp3 is encoded by a nucleic acid having at least about 98% identity to nucleotides 2829-4432 of SEQ ID NO: 1, as measured by MacVector 6.0. 
     
     
         52 . The composition of  claim 48  wherein the AAV 5′ ITR and 3′ ITR are of AAV serotype 2. 
     
     
         53 . The composition of  claim 52  wherein the recombinant virus further comprises a regulatable promoter which directs expression of the transgene. 
     
     
         54 . A recombinant vector comprising an AAV-1 P5 promoter having the sequence of nt 236 to 299 of SEQ ID NO: 1 or a functional fragment thereof. 
     
     
         55 . A nucleic acid molecule encoding AAV-1 helper functions, said molecule comprising an AAV rep coding region and an AAV cap coding region, wherein said cap coding region comprises at least one member is selected from the group consisting of:
 (a) vp1, nt 2223 to 4431 of SEQ ID NO: 1;   (b) vp2, nt 2634 to 4432 of SEQ ID NO: 1; and   (c) vp3, nt 2829 to 4432 of SEQ ID NO: 1.   
     
     
         56 . A host cell stably transduced with an AAV-1 P5 promoter having the sequence of nt 236 to 299 of SEQ ID NO: 1. 
     
     
         57 . A pharmaceutical composition comprising a carrier and a virus comprising the vector according to  claim 56 . 
     
     
         58 . A method for AAV-mediated delivery of a transgene comprising delivering to a host cell an AAV virion which comprises:
 (a) a capsid comprising at least one capsid protein encoded by an AAV-1 cap gene; and   (b) a DNA molecule comprising a transgene under the control of regulatory sequences directing its expression.   
     
     
         59 . A method for AAV-mediated delivery of a transgene to a host comprising:
 (a) assaying a sample from the host to determine the presence of neutralizing antibodies specific against any serotype of AAV; and   (b) delivering to the host an AAV virion which comprises:
 (i) a capsid comprising at least one capsid protein encoded by a cap gene of an AAV serotype against which the host has no antibodies as determined in (a); and 
 (ii) a DNA molecule comprising a transgene under the control of regulatory sequences directing its expression. 
   
     
     
         60 . The method according to  claim 59 , comprising repeating (a) and (b). 
     
     
         61 . The host cell transduced with a nucleic acid molecule according to  claim 55 . 
     
     
         62 . A method for transducing a muscle cell, said method comprising infecting the cell with a recombinant AAV vector comprising an AAV1 capsid. 
     
     
         63 . A method for transducing a liver cell, said method comprising infecting the cell with a recombinant AAV vector comprising an AAV1 capsid. 
     
     
         64 . A method of delivering a heterologous nucleic acid to at least one muscle cell in a mammalian subject, comprising:
 (a) providing at least one recombinant adeno-associated virus (rAAV) virion, said rAAV virion comprising an AAV-1 capsid and a heterologous nucleic acid operably linked to expression control elements; and   (b) administering said rAAV virions to said muscle cell, whereby expression of said heterologous nucleic acid provides for a therapeutic effect.   
     
     
         65 . The method of  claim 64 , wherein said heterologous nucleic acid is a gene encoding a protein. 
     
     
         66 . The method of  claim 64 , wherein said heterologous nucleic acid is an antisense RNA. 
     
     
         67 . The method of  claim 64 , wherein said protein is a secreted protein. 
     
     
         68 . The method of  claim 67 , wherein said secreted protein is selected from the group consisting of cytokines, growth factors, and differentiation factors. 
     
     
         69 . The method of  claim 65 , wherein said protein is α1-antitrypsin or erythropoietin. 
     
     
         70 . The method of  claim 64 , wherein said administering of said rAAV virions is by way of direct injection to said muscle cell of said mammalian subject. 
     
     
         71 . The method of  claim 70 , wherein said muscle cell is a skeletal muscle cell. 
     
     
         72 . The method of  claim 64 , wherein said administering of said rAAV virions is by way of administration to a vascular conduit of said mammalian subject. 
     
     
         73 . The method of  claim 72 , wherein said vascular conduit is a vein. 
     
     
         74 . A method of delivering a nucleotide sequence to a cell, in vitro comprising introducing into a cell the hybrid virus particle according to  claim 2 . 
     
     
         75 . The method of  claim 74 , wherein the heterologous nucleotide sequence is expressed in the cell. 
     
     
         76 . The method of  claim 75 , wherein the protein or peptide is an immunogenic protein or peptide. 
     
     
         77 . The method of  claim 74 , wherein the parvovirus capsid is a B19 capsid. 
     
     
         78 . The method of  claim 74 , wherein the at least one heterologous nucleotide sequence encodes a protein or peptide. 
     
     
         79 . The method of  claim 78 , wherein the protein or peptide is a therapeutic protein or peptide. 
     
     
         80 . The method of  claim 78 , wherein the at least one heterologous nucleotide sequence encodes dystrophin, a mini-dystrophin, a clotting factor, β-glucocerebrosidase, or a growth factor. 
     
     
         81 . The method of  claim 74 , wherein the heterologous nucleotide sequence encodes an untranslated RNA. 
     
     
         82 . The method of  claim 74 , wherein the cell is selected from the group consisting of a neural cell, lung cell, retinal cell, epithelial cell, muscle cell, pancreatic cell, hepatic cell, myocardial cell, bone cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, germ cell, progenitor cell, and a stem cell. 
     
     
         83 . The method of  claim 74 , wherein the parvovirus capsid is an AAV capsid. 
     
     
         84 . The method of  claim 83 , wherein: the AAV capsid is of a serotype selected from the group consisting of AAV serotypes 1, 3, 4, 5 and 6. 
     
     
         85 . The method of  claim 84 , wherein the hybrid virus particle is selected from the group consisting of: (a) a hybrid virus particle comprising an AAV serotype-1 capsid and at least one AAV serotype-2 inverted terminal repeat, (b) a hybrid virus particle comprising an AAV serotype-3 capsid and at least one AAV serotype-2 inverted terminal repeat, (c) a hybrid virus particle comprising an AAV serotype-4 capsid and at least one AAV serotype-2 inverted terminal repeat, (d) a hybrid virus particle comprising an AAV serotype-5 capsid and at least one AAV serotype-2 inverted terminal repeat, (e) a hybrid virus particle comprising an AAV serotype-6 capsid and at least one AAV serotype-2 inverted terminal repeat.