Methods and compositions for the production of recombinant virus vectors

Abstract

A method for the production of a replication-deficient recombinant virus vector is disclosed. The replication-deficient recombinant virus vector has a recombinant virus genome with one or more defective viral genes. The method comprises infecting a host cell with a carrier virus having a carrier virus genome encoding one or more trans factors or variants thereof, incubating the infected host cell for a desired period of time, and isolating the replication-deficient recombinant virus vector. The carrier virus is a cytoplasmic virus that retains the carrier virus genome in the cytoplasm of the host cell. The host cell contains the recombinant viral genome and retains the recombinant viral genome in a nucleus of the host cell. Also disclosed is a carrier virus for the production of a replication-deficient recombinant virus vector.

1 - 16 . (canceled) 
     
     
         17 . A carrier virus, comprising:
 an envelope;   a carrier virus genome comprising:
 a nucleic acid sequence from a cytoplasmic virus; and 
 a nucleotide sequence that encodes two or more AAV structure proteins or variants thereof; 
   wherein said envelope and said nucleic acid sequence from a cytoplasmic virus allow said carrier virus genome to retain in the cytoplasm after entering a host cell, and wherein said carrier virus genome expresses said two or more AAV structure proteins or variants thereof in the cytoplasm at a ratio suitable for the production of a recombinant AAV vector that is defective in said two or more AAV structure proteins.   
     
     
         18 . The carrier virus of  claim 17 , wherein said carrier virus is a vaccinia carrier virus or vesicular stomatitis virus (VSV), and wherein said virus genome comprises an expression cassette having an AAV vp1 gene under the control of a vaccinia virus p7.5 promoter when the carrier virus is a vaccinia virus. 
     
     
         19 . The carrier virus of  claim 17 , wherein said carrier virus genome further comprises:
 a nucleotide sequence that encodes one or more AAV non-structure proteins or variants thereof.   
     
     
         20 . The carrier virus of  claim 18 , comprising a first expression cassette having an AAV vp1 gene under the control of a first vaccinia virus p7.5 promoter and a second expression cassette having an AAV vp2 gene under the control of a second vaccinia virus p7.5 promoter. 
     
     
         21 . An isolated cytoplasmic carrier virus, comprising:
 a virus genome comprising a nucleotide sequence encoding one or more structural proteins of an adeno-associated virus (AAV), wherein said nucleotide sequence is constructed to express said structural proteins in the cytoplasmic compartment upon a host cell is infected with said carrier cytoplasmic virus.   
     
     
         22 . The carrier virus of  claim 21 , wherein said carrier virus is a vaccinia carrier virus and wherein said virus genome comprises an expression cassette having an AAV vp1 gene under the control of a vaccinia virus promoter when the carrier virus is a vaccinia virus. 
     
     
         23 . The carrier virus of  claim 21 , wherein said carrier virus is a vaccinia carrier virus and wherein said virus genome comprises a first expression cassette having an AAV vp1 gene under the control of a first vaccinia virus promoter and a second expression cassette having an AAV vp2 gene under the control of a second vaccinia virus promoter. 
     
     
         24 . The carrier virus of  claim 21 , wherein said carrier virus genome further comprises:
 a nucleotide sequence that encodes one or more AAV non-structure proteins or variants thereof.   
     
     
         25 . The carrier virus of  claim 21 , wherein said carrier virus is a member of the poxviridae family. 
     
     
         26 . The carrier virus of  claim 21 , wherein said carrier virus is a vesicular stomatitis virus (VSV). 
     
     
         27 . A method for producing a replication-deficient recombinant AAV vector, said method comprising:
 infecting a host cell with the cytoplasmic carrier viruse of  claim 21 , wherein said cytoplasmic carrier virus encodes two or more AAV capsid proteins and express said two or more capsid proteins in the cytoplasm of said host cell in a ratio suitable for the production of said replication-deficient recombinant AAV vector;   incubating the infected host cell for a desired period of time; and   isolating said replication-deficient recombinant AAV vector.   
     
     
         28 . The method of  claim 27 , wherein said host cell comprises AAV vector sequences integrated into a chromosome of said host cell. 
     
     
         29 . The method of  claim 27 , further comprising:
 infecting said host cell with a nuclear carrier virus comprising AAV vector sequences.   
     
     
         30 . The method of  claim 27 , wherein said two or more AAV capsid proteins are expressed under the control of a vaccinia virus promoter. 
     
     
         31 . The method of  claim 27 , wherein said two or more AAV capsid proteins comprise VP2 and VP3 proteins expressed from a first cytoplasmic vaccinia carrier virus and VP1 expressed from a second cytoplasmic vaccinia carrier virus. 
     
     
         32 . The method of  claim 27 , wherein the infecting step comprises infecting said host cell with a single cytoplasmic vaccinia carrier virus that expresses AAV VP1, VP2 and VP3 proteins at a ratio suitable for the production of said replication-deficient AAV vector. 
     
     
         33 . The method of  claim 32 , wherein said single cytoplasmic vaccinia carrier virus contains a first expression cassette having AAV vp1 gene under the control of a first vaccinia virus promoter and a second expression cassette having AAV vp2 gene under the control of a second vaccinia virus promoter. 
     
     
         34 . The method of  claim 26 , wherein said cytoplasmic carrier virus is a vaccinia virus or vesicular stomatitis virus (VSV). 
     
     
         35 . The method of  claim 26 , wherein said cytoplasmic carrier virus is a member of the poxviridae family.