Adeno-associated virus serotype I nucleic acid sequences, vectors and host cell 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 . 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).  
     
     
         2 . 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).  
     
     
         3 . 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).  
     
     
         4 . The recombinant vector according to  claim 3 , wherein said vector further comprises a 3′ AAV-1 ITR.  
     
     
         5 . 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).  
     
     
         6 . The recombinant vector according to  claim 5 , wherein said vector further comprises a 5′ AAV-1 ITR.  
     
     
         7 . A pharmaceutical composition comprising a carrier and a virus comprising the vector according to  claim 5 .  
     
     
         8 . A method for producing a selected gene product comprising the steps of transfecting a mammalian cell with the molecule according to  claim 1  or a functional fragment thereof and culturing said cell under conditions suitable to express said gene product.  
     
     
         9 . The recombinant vector according to  claim 3 , wherein said vector further comprises AAV-1 capsid proteins having the sequence of SEQ ID NO: 13, 15 or 17 or functional fragments thereof.  
     
     
         10 . The recombinant vector according to  claim 3 , wherein said vector further comprises adenovirus sequences.  
     
     
         11 . The host cell transduced with a recombinant viral vector according to  claim 3 .  
     
     
         12 . The host cell transduced with a nucleic acid molecule according to  claim 1 .  
     
     
         13 . The host cell transduced with a nucleic acid molecule according to  claim 2 .  
     
     
         14 . The pharmaceutical composition comprising a carrier and a virus comprising the vector according to  claim 3 .  
     
     
         15 . The method for delivery of a transgene comprising the step of delivering to a host cell a recombinant virus comprising a recombinant vector according to  claim 3 .  
     
     
         16 . A recombinant host cell transformed with a nucleic acid sequence expressing one or more AAV-1 rep proteins selected from among 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.  
     
     
         17 . A composition comprising a recombinant virus having an AAV-1 capsid comprising an AAV-1 protein selected from among 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.  
     
     
         18 . The composition of  claim 17  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.  
     
     
         19 . The composition of  claim 17  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.  
     
     
         20 . The composition of  claim 17  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 MacVector6.0.  
     
     
         21 . The composition of  claim 17  wherein the AAV 5′ ITR and 3′ ITR are of AAV serotype 2.  
     
     
         22 . The composition of  claim 21  wherein the recombinant virus further comprises a regulatable promoter which directs expression of the transgene.  
     
     
         23 . 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.  
     
     
         24 . 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.    
     
     
         25 . A host cell stably transduced with an AAV-1 P5 promoter having the sequence of nt 236 to 299 of SEQ ID NO: 1.  
     
     
         26 . A pharmaceutical composition comprising a carrier and a virus comprising the vector according to  claim 25 .  
     
     
         27 . A method for AAV-mediated delivery of a transgene comprising the step of 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.    
     
     
         28 . A method for AAV-mediated delivery of a transgene to a host comprising the steps of: 
 (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 step (a); and  
 (ii) a DNA molecule comprising a transgene under the control of regulatory sequences directing its expression.  
   
     
     
         29 . The method according to  claim 28 , comprising the additional step of repeating steps (a) and (b).  
     
     
         30 . The host cell transduced with a nucleic acid molecule according to  claim 24 .  
     
     
         31 . A method for transducing a muscle cell, said method comprising the step of infecting the cell with a recombinant AAV vector comprising an AAV1 capsid.  
     
     
         32 . A method for transducing a liver cell, said method comprising the step of infecting the cell with a recombinant AAV vector comprising an AAV1 capsid.  
     
     
         33 . 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.    
     
     
         34 . The method of  claim 33 , wherein said heterologous nucleic acid is a gene encoding a protein.  
     
     
         35 . The method of  claim 33 , wherein said heterologous nucleic acid is an antisense RNA.  
     
     
         36 . The method of  claim 33 , wherein said protein is a secreted protein.  
     
     
         37 . The method of  claim 35 , wherein said secreted protein is selected from the group consisting of cytokines, growth factors, and differentiation factors.  
     
     
         38 . The method of  claim 34 , wherein said protein is α1-antitrypsin or erthryopoietin.  
     
     
         39 . The method of  claim 33 , wherein said administering of said rAAV virions is by way of direct injection to said muscle cell of said mammalian subject.  
     
     
         40 . The method of  claim 39 , wherein said muscle cell is a skeletal muscle cell.  
     
     
         41 . The method of  claim 33 , wherein said administering of said rAAV virions is by way of administration to a vascular conduit of said mammalian subject.  
     
     
         42 . The method of  claim 41 , wherein said vascular conduit is a vein.  
     
     
         43 . A chimeric virus particle comprising: 
 (a) a chimeric adeno-associated virus serotype-2 (AAV2) capsid comprising a chimeric capsid protein comprising at least one capsid region from AAV1; and    (b) a nucleic acid comprising 5′ and 3′ AAV inverted terminal repeats and at least one heterologous nucleotide sequence, wherein said nucleic acid is packaged within the chimeric parvovirus capsid.    
     
     
         44 . The chimeric virus particle of  claim 43 , wherein said at least one heterologous nucleotide sequence encodes a protein or peptide.  
     
     
         45 . The chimeric virus particle of  claim 44 , wherein said protein or peptide is a therapeutic protein or peptide.  
     
     
         46 . The chimeric virus particle of  claim 45 , wherein said protein or peptide is dystrophin or a mini-dystrophin.  
     
     
         47 . The chimeric virus particle of  claim 44 , wherein said protein or peptide is an immunogenic protein or peptide.  
     
     
         48 . The chimeric virus particle of  claim 43 , wherein said at least one heterologous nucleotide sequence encodes an untranslated RNA sequence.  
     
     
         49 . The chimeric virus particle of  claim 43 , wherein said AAV1 capsid region is inserted into the chimeric capsid protein but does not replace a region of said chimeric capsid protein.  
     
     
         50 . The chimeric virus particle of  claim 43 , wherein said at least one AAV1 capsid region replaces a region within said chimeric capsid region.  
     
     
         51 . The chimeric virus particle of  claim 43 , wherein said at least one AAV1 capsid region is a loop region of the major vp1 capsid subunit.  
     
     
         52 . The chimeric virus particle of  claim 51 , wherein said loop region replaces a loop region in the major Vp1 capsid subunit.  
     
     
         53 . The chimeric virus particle of  claim 43 , wherein said 5′ and 3′ AAV inverted terminal repeats are AAV2 inverted terminal repeats.  
     
     
         54 . The chimeric virus particle of  claim 43 , wherein said nucleic acid does not comprise the AAV cap genes or the AAV rep genes.  
     
     
         55 . The chimeric virus particle of  claim 43 , wherein an antigenic property of said chimeric AAV2 capsid is reduced as compared with the wild-type AAV2 capsid.  
     
     
         56 . A chimeric virus particle comprising: 
 (a) a chimeric adeno-associated virus serotype-2 (AAV2) capsid comprising at least one AAV1 capsid region; and    (b) a nucleic acid comprising 5′ and 3′ AAV inverted terminal repeats and at least one heterologous nucleotide sequence, wherein said nucleic acid is packaged within the chimeric AAV2 capsid.    
     
     
         57 . The chimeric virus particle of  claim 56 , wherein said at least one heterologous nucleotide sequence encodes a protein or peptide.  
     
     
         58 . The chimeric virus particle of  claim 57 , wherein said protein or peptide is a therapeutic protein or peptide.  
     
     
         59 . The chimeric virus particle of  claim 58 , wherein said protein or peptide is dystrophin or a mini-dystrophin.  
     
     
         60 . The chimeric virus particle of  claim 57 , wherein said protein or peptide is an immunogenic protein or peptide.  
     
     
         61 . The chimeric virus particle of  claim 57 , wherein said at least one heterologous nucleotide sequence encodes an untranslated RNA sequence.  
     
     
         62 . The chimeric virus particle of  claim 57 , wherein said AAV1 capsid region is inserted into the chimeric capsid protein but does not replace a region of said chimeric capsid protein.  
     
     
         63 . The chimeric virus particle of  claim 57 , wherein said at least one AAV1 capsid region replaces a region within said chimeric capsid region.  
     
     
         64 . The chimeric virus particle of  claim 57 , wherein said at least one AAV1 capsid region is a loop region of the major Vp1 capsid subunit.  
     
     
         65 . The chimeric virus particle of  claim 64 , wherein said loop region replaces a loop region in the major Vp1 capsid subunit.  
     
     
         66 . The chimeric virus particle of  claim 56 , wherein said 5′ and 3′ AAV inverted terminal repeats are AAV2 inverted terminal repeats.  
     
     
         67 . The chimeric virus particle of  claim 56 , wherein said nucleic acid does not comprise the AAV cap genes or the AAV rep genes.  
     
     
         68 . The chimeric virus particle of  claim 56 , wherein an antigenic property of said chimeric AAV2 capsid is reduced as compared with the wild-type AAV2 capsid.  
     
     
         69 . The chimeric virus particle of  claim 56 , wherein said at least one AAV1 capsid region replaces a capsid subunit in said AAV2 capsid.  
     
     
         70 . A composition comprising the chimeric virus particle of  claim 43 .  
     
     
         71 . A composition comprising the chimeric virus particle of  claim 57 .  
     
     
         72 . An isolated nucleic acid encoding the adeno-associated virus serotype-2 (AAV2) cap genes and AAV rep genes, wherein the AAV2 cap genes encode a chimeric AAV2 capsid comprising at least one AAV1 capsid region.  
     
     
         73 . A vector comprising the isolated nucleic acid of  claim 43 .  
     
     
         74 . A cell comprising the vector of  claim 57 .  
     
     
         75 . A cell comprising the isolated nucleic acid of  claim 74  stably integrated into the genome of the cell.  
     
     
         76 . A method of producing a chimeric virus particle, the method comprising: 
 providing a cell with chimeric adeno-associated virus serotype-2 (AAV2) cap genes, AAV rep genes, a nucleic acid comprising 5′ and 3′ AAV inverted terminal repeats and at least one heterologous nucleotide sequence, and helper functions for generating a productive AAV infection, wherein the chimeric AAV cap genes comprise at least one nucleic acid sequence from AAV1 cap genes such that the chimeric AAV2 cap genes encode a chimeric AAV2 capsid comprising at least one AAV1 capsid region; and    allowing assembly of the chimeric virus particles.    
     
     
         77 . The method of  claim 76 , further comprising collecting the chimeric virus particles.  
     
     
         78 . The method of  claim 76 , wherein the chimeric AAV2 cap genes and AAV rep genes are provided by one or more transcomplementing packaging vectors.  
     
     
         79 . The method of  claim 76 , wherein the chimeric AAV2 cap genes and AAV rep genes are provided by a plasmid.  
     
     
         80 . The method of  claim 76 , wherein the chimeric AAV2 cap genes and AAV rep genes are stably integrated into the genome of the cell.  
     
     
         81 . The method of  claim 76 , wherein the AAV rep genes are AAV2 rep genes.  
     
     
         82 . A chimeric virus particle produced by the method of  claim 78 .  
     
     
         83 . A method of delivering a nucleotide sequence to a cell, the method comprising introducing into a cell the chimeric virus particle of  claim 57 .  
     
     
         84 . A method of administering a nucleotide sequence to a subject, the method comprising administering to a subject the chimeric virus particle of  claim 57 .  
     
     
         85 . A pharmaceutical formulation comprising the chimeric virus particle of  claim 57  in a pharmaceutically-acceptable carrier.  
     
     
         86 . A hybrid virus particle comprising: 
 a parvovirus capsid; and    a nucleic acid comprising at least one adeno-associated virus (AAV) serotype 5 inverted terminal repeat packaged within said parvovirus capsid, subject to the proviso that said parvovirus capsid is not an AAV serotype 5 capsid.    
     
     
         87 . The hybrid virus particle of  claim 86 , wherein said nucleic acid comprises at least one heterologous nucleotide sequence.  
     
     
         88 . The hybrid virus particle of  claim 86 , wherein said parvovirus capsid is an autonomous parvovirus capsid.  
     
     
         89 . The hybrid virus particle of  claim 86 , wherein said parvovirus capsid is a B 19 capsid.  
     
     
         90 . The hybrid virus particle of  claim 86 , wherein said parvovirus capsid is an AAV capsid.  
     
     
         91 . The hybrid virus particle of  claim 90 , wherein said AAV capsid is of a serotype selected from the group consisting of AAV serotypes 1, 2, 3, 4 and 6.  
     
     
         92 . The hybrid virus particle of  claim 91  selected from the group consisting of: 
 (a) a hybrid virus particle comprising an AAV serotype-1 capsid and at least one AAV serotype-5 inverted terminal repeat,    (b) a hybrid virus particle comprising an AAV serotype-2 capsid and at least one AAV serotype-5 inverted terminal repeat, and    (c) a hybrid virus particle comprising an AAV serotype-6 capsid and at least one AAV serotype-5 inverted terminal repeat.    
     
     
         93 . The hybrid virus particle of  claim 86 , wherein said nucleic acid does not comprise the AAV cap genes or the AAV rep genes.  
     
     
         94 . The hybrid virus particle of  claim 86  comprising two AAV inverted terminal repeats that flank said at least one heterologous nucleotide sequence.  
     
     
         95 . The hybrid virus particle of  claim 87 , wherein said at least one heterologous nucleotide sequence encodes a protein or peptide.  
     
     
         96 . The hybrid virus-particle of  claim 95 , wherein said protein or peptide is a therapeutic protein or peptide.  
     
     
         97 . The hybrid virus particle of  claim 95 , wherein said protein or peptide is an immunogenic protein or peptide.  
     
     
         98 . The hybrid virus particle of  claim 95 , wherein said at least one heterologous nucleotide sequence encodes dystrophin, a mini-dystrophin, a clotting factor, β-glucocerebrosidase, erythropoietin, cystic fibrosis transmembrane regulator protein, a cytokine, β-globin, a hormone, α-globin or a growth factor.  
     
     
         99 . The hybrid virus particle of  claim 87 , wherein said at least one heterologous nucleotide sequence encodes an untranslated RNA.  
     
     
         100 . A pharmaceutical formulation comprising the hybrid virus particle of  claim 86  in a pharmaceutically-acceptable carrier.  
     
     
         101 . An isolated nucleic acid for producing the hybrid virus particle of  claim 86 , wherein said isolated nucleic acid comprises parvovirus cap genes and adeno-associated virus (AAV) rep genes, subject to the proviso that said parvovirus cap genes are not AAV serotype 5 cap genes.  
     
     
         102 . The isolated nucleic acid of  claim 101 , wherein said parvovirus cap genes are operably associated with an authentic parvovirus promoter.  
     
     
         103 . The isolated nucleic acid of  claim 101 , wherein said parvovirus cap genes are B19 cap genes.  
     
     
         104 . The isolated nucleic acid of  claim 103 , wherein said AAV rep genes are AAV serotype-5 rep genes.  
     
     
         105 . The isolated nucleic acid of  claim 103 , wherein said AAV cap genes are of a serotype selected from the group consisting of AAV serotypes 1, 2, 3, 4 and 6.  
     
     
         106 . The isolated nucleic acid of  claim 105  selected from the group consisting of: 
 (a) an isolated nucleic acid comprising AAV serotype-1 cap genes and AAV serotype-5 rep genes,    (b) an isolated nucleic acid comprising AAV serotype-2 cap genes and AAV serotype-5 rep genes,    (c) an isolated nucleic acid comprising AAV serotype-3 cap genes and AAV serotype-5 rep genes,    (d) an isolated nucleic acid comprising AAV serotype-4 cap genes and AAV serotype-5 rep genes, and    (e) an isolated nucleic acid comprising AAV serotype-6 cap genes and AAV serotype-5 rep genes.    
     
     
         107 . The isolated nucleic acid of  claim 106 , wherein said cap genes are AAV cap genes.  
     
     
         108 . The isolated nucleic acid of  claim 107 , wherein said AAV cap genes are operably associated with an authentic AAV promoter.  
     
     
         109 . The isolated nucleic acid of  claim 108 , wherein said authentic AAV promoter is an AAV p40 promoter.  
     
     
         110 . A vector comprising the isolated nucleic acid of  claim 101 .  
     
     
         111 . The vector of  claim 110 , wherein said vector is selected from the group consisting of plasmids, naked DNA vectors, bacterial artificial chromosomes, yeast artificial chromosomes, and viral vectors.  
     
     
         112 . The vector of  claim 110 , wherein said vector is a plasmid.  
     
     
         113 . A cell comprising the vector of  claim 110 .  
     
     
         114 . The cell of  claim 113 , wherein said cell is selected from the group consisting of bacterial, protozoan, yeast, fungus, plant, and animal cells.  
     
     
         115 . A cell comprising a vector, the vector comprising: 
 parvovirus cap genes,    adeno-associated virus (AAV) rep genes, and    a nucleic acid comprising at least one AAV inverted serotype 5 terminal repeat, subject to the proviso that said parvovirus cap genes are not AAV serotype 5 cap genes.    
     
     
         116 . The cell of  claim 115 , wherein said cell is a mammalian cell.  
     
     
         117 . A cell comprising parvovirus cap genes and adeno-associated virus (AAV) rep genes stably integrated into the genome of the cell, subject to the proviso that if said parvovirus cap genes are not AAV serotype 2 cap genes, the serotypes of said AAV cap genes and said AAV rep genes are different.  
     
     
         118 . The cell of  claim 117  further comprising a nucleic acid comprising at least one AAV serotype 5 inverted terminal repeat, subject to the proviso that said parvovirus cap genes are not AAV serotype 5 cap genes.  
     
     
         119 . A method of producing a hybrid virus particle, the method comprising: providing a cell with adeno-associated virus (AAV) rep genes, parvovirus cap genes, a nucleic acid comprising at least one AAV serotype 5 inverted terminal repeat, and helper functions for generating a productive AAV infection; subject to the proviso that the parvovirus cap genes are not AAV serotype 5 cap genes, and allowing assembly of the hybrid virus particles.  
     
     
         120 . The method of  claim 119 , further comprising collecting the hybrid virus particles.  
     
     
         121 . The method of  claim 119 , wherein the nucleic acid comprises at least one heterologous nucleotide sequence.  
     
     
         122 . The method of  claim 119 , wherein the parvovirus cap genes and AAV rep genes are provided by one or more transcomplementing packaging vectors.  
     
     
         123 . The method of  claim 119 , wherein the parvovirus cap genes and AAV rep genes are provided by a plasmid.  
     
     
         124 . The method of  claim 119 , wherein the parvovirus cap genes and AAV rep genes are provided by an adenovirus vector.  
     
     
         125 . The method of  claim 119 , wherein the parvovirus cap genes and AAV rep genes are stably integrated into the genome of the cell.  
     
     
         126 . The method of  claim 119 , wherein the parvovirus cap genes are AAV cap genes.  
     
     
         127 . A hybrid virus particle produced by the method of  claim 119 .  
     
     
         128 . A method of delivering a nucleotide sequence to a cell, comprising introducing into a cell the hybrid virus particle according to  claim 87 .  
     
     
         129 . A method of administering a nucleotide sequence to a subject, comprising administering the cell of  claim 113  to a subject.  
     
     
         130 . A method of administering a nucleotide sequence to a subject, comprising administering to a subject the hybrid virus particle according to  claim 127.