Virus vectors and methods of making and administering the same

Abstract

The present invention provides genetically-engineered parvovirus capsids and viruses designed to introduce a heterologous gene into a target cell. The parvoviruses of the invention provide a repertoire of vectors with altered antigenic properties, packaging capabilities, and/or cellular tropisms as compared with current AAV vectors.

1 . 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.    
     
     
         2 . The chimeric virus particle of  claim 1 , wherein said at least one heterologous nucleotide sequence encodes a protein or peptide.  
     
     
         3 . The chimeric virus particle of  claim 2 , wherein said protein or peptide is a therapeutic protein or peptide.  
     
     
         4 . The chimeric virus particle of  claim 3 , wherein said protein or peptide is dystrophin or a mini-dystrophin.  
     
     
         5 . The chimeric virus particle of  claim 2 , wherein said protein or peptide is an immunogenic protein or peptide.  
     
     
         6 . The chimeric virus particle of  claim 1 , wherein said at least one heterologous nucleotide sequence encodes an untranslated RNA sequence.  
     
     
         7 . The chimeric virus particle of  claim 1 , wherein said AAV1 capsid region is inserted into the chimeric capsid protein but does not replace a region of said chimeric capsid protein.  
     
     
         8 . The chimeric virus particle of  claim 1 , wherein said at least one AAV1 capsid region replaces a region within said chimeric capsid region.  
     
     
         9 . The chimeric virus particle of  claim 1 , wherein said at least one AAV1 capsid region is a loop region of the major Vp1 capsid subunit.  
     
     
         10 . The chimeric virus particle of  claim 9 , wherein said loop region replaces a loop region in the major Vp1 capsid subunit.  
     
     
         11 . The chimeric virus particle of  claim 1 , wherein said 5′ and 3′ AAV inverted terminal repeats are AAV2 inverted terminal repeats.  
     
     
         12 . The chimeric virus particle of  claim 1 , wherein said nucleic acid does not comprise the AAV cap genes or the AAV rep genes.  
     
     
         13 . The chimeric virus particle of  claim 1 , wherein an antigenic property of said chimeric AAV2 capsid is reduced as compared with the wild-type AAV2 capsid.  
     
     
         14 . 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.    
     
     
         15 . The chimeric virus particle of  claim 14 , wherein said at least one heterologous nucleotide sequence encodes a protein or peptide.  
     
     
         16 . The chimeric virus particle of  claim 15 , wherein said protein or peptide is a therapeutic protein or peptide.  
     
     
         17 . The chimeric virus particle of  claim 16 , wherein said protein or peptide is dystrophin or a mini-dystrophin.  
     
     
         18 . The chimeric virus particle of  claim 15 , wherein said protein or peptide is an immunogenic protein or peptide.  
     
     
         19 . The chimeric virus particle of  claim 14 , wherein said at least one heterologous nucleotide sequence encodes an untranslated RNA sequence.  
     
     
         20 . The chimeric virus particle of  claim 14 , wherein said AAV1 capsid region is inserted into the chimeric capsid protein but does not replace a region of said chimeric capsid protein.  
     
     
         21 . The chimeric virus particle of  claim 14 , wherein said at least one AAV1 capsid region replaces a region within said chimeric capsid region.  
     
     
         22 . The chimeric virus particle of  claim 14 , wherein said at least one AAV1 capsid region is a loop region of the major Vp1 capsid subunit.  
     
     
         23 . The chimeric virus particle of  claim 22 , wherein said loop region replaces a loop region in the major Vp1 capsid subunit.  
     
     
         24 . The chimeric virus particle of  claim 14 , wherein said 5′ and 3′ AAV inverted terminal repeats are AAV2 inverted terminal repeats.  
     
     
         25 . The chimeric virus particle of  claim 14 , wherein said nucleic acid does not comprise the AAV cap genes or the AAV rep genes.  
     
     
         26 . The chimeric virus particle of  claim 14 , wherein an antigenic property of said chimeric AAV2 capsid is reduced as compared with the wild-type AAV2 capsid.  
     
     
         27 . The chimeric virus particle of  claim 14 , wherein said at least one AAV1 capsid region replaces a capsid subunit in said AAV2 capsid.  
     
     
         28 . A composition comprising the chimeric virus particle of  claim 1 .  
     
     
         29 . A composition comprising the chimeric virus particle of  claim 14 .  
     
     
         30 . 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.  
     
     
         31 . A vector comprising the isolated nucleic acid of  claim 30 .  
     
     
         32 . A cell comprising the vector of  claim 31 .  
     
     
         33 . A cell comprising the isolated nucleic acid of  claim 30  stably integrated into the genome of the cell.  
     
     
         34 . 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.    
     
     
         35 . The method of  claim 34 , further comprising collecting the chimeric virus particles.  
     
     
         36 . The method of  claim 34 , wherein the chimeric AAV2 cap genes and AAV rep genes are provided by one or more transcomplementing packaging vectors.  
     
     
         37 . The method of  claim 34 , wherein the chimeric AAV2 cap genes and AAV rep genes are provided by a plasmid.  
     
     
         38 . The method of  claim 34 , wherein the chimeric AAV2 cap genes and AAV rep genes are stably integrated into the genome of the cell.  
     
     
         39 . The method of  claim 34 , wherein the AAV rep genes are AAV2 rep genes.  
     
     
         40 . A chimeric virus particle produced by the method of  claim 34 .  
     
     
         41 . A method of delivering a nucleotide sequence to a cell, the method comprising introducing into a cell the chimeric virus particle of  claim 14 .  
     
     
         42 - 49 . (canceled)  
     
     
         50 . A method of administering a nucleotide sequence to a subject, the method comprising administering to a subject the chimeric virus particle of  claim 14 .  
     
     
         51 - 58 . (canceled)  
     
     
         59 . A pharmaceutical formulation comprising the chimeric virus particle of  claim 14  in a pharmaceutically-acceptable carrier.