Compositions and methods 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.

What is claimed is: 
     
         1 . A method for eliminating or minimizing the formation of replication-competent virus during the production of a replication-deficient recombinant virus vector having a recombinant virus genome with one or more defective viral genes, said method comprising:
 infecting a host cell with a carrier virus having a carrier virus genome encoding one or more trans factors or variants thereof, wherein said carrier virus is a cytoplasmic virus that retains said carrier virus genome in the cytoplasm of said host cell, wherein said one or more trans factors compensate one or more functions of said one or more defective genes in said recombinant virus genome, and wherein said one or more trans factors comprise a structure protein of said replication-deficient recombinant virus;   incubating the infected host cell for a desired period of time; and   isolating said replication-deficient recombinant virus vector,   wherein said host cell contains said recombinant viral genome and retains said recombinant viral genome in a nucleus of said host cell.   
     
     
         2 . The method of  claim 1 , wherein said carrier virus carries a carrier virus genome encoding two or more structure proteins of said replication-deficient recombinant virus and expresses said two or more structure proteins at a ratio suitable for the production of said replication-deficient recombinant virus. 
     
     
         3 . The method of  claim 1 , wherein said cytoplasmic virus is a vaccinia virus or a vesicular stomatitis virus (VSV). 
     
     
         4 . The method of  claim 1 , wherein said replication-deficient recombinant virus vector is a virus vector in the Parvoviridae family. 
     
     
         5 . The method of  claim 1 , wherein said replication-deficient recombinant virus vector is a recombinant adenovirus vector. 
     
     
         6 . The method of  claim 1 , wherein said replication-deficient recombinant virus vector is a recombinant herpes virus vector. 
     
     
         7 . A method for producing a replication-deficient recombinant virus vector having a recombinant virus genome with one or more defective viral genes, said method comprising:
 infecting a host cell with a carrier virus having a carrier virus genome encoding one or more trans factors or variants thereof, wherein said carrier virus is a cytoplasmic virus that retains said carrier virus genome in the cytoplasm of said host cell and wherein said one or more trans factors compensate one or more functions of said one or more defective genes in said recombinant virus genome;   incubating the infected host cell for a desired period of time; and   isolating said replication-deficient recombinant virus vector,   wherein said host cell contains said recombinant viral genome and retains said recombinant viral genome in a nucleus of said host cell, and   wherein said replication-deficient recombinant virus vector is selected from the group consisting of recombinant virus vectors from the Adenoviridae family, the Herpesviridae family, the Hepadnaviridae family, and recombinant parvovirus vectors.   
     
     
         8 . The method of  claim 7 , wherein said replication-deficient recombinant virus vector is a recombinant adenovirus vector. 
     
     
         9 . The method of  claim 7 , wherein said replication-deficient recombinant virus vector is a recombinant herpes virus vector. 
     
     
         10 . The method of  claim 7 , wherein said replication-deficient recombinant virus vector is a recombinant parvovirus vector. 
     
     
         11 . The method of  claim 7 , wherein said carrier virus is a vaccinia virus or vesicular stomatitis virus (VSV). 
     
     
         12 . A method for producing a replication-deficient recombinant AAV vector having a recombinant virus genome that is defective in producing capsid proteins, said method comprising:
 infecting a host cell with one or more carrier viruses, wherein said carrier viruses are cytoplasmic viruses that encode 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 AAV vector;   incubating the infected host cell for a desired period of time; and   isolating said replication-deficient recombinant virus vector,   wherein said host cell contains said recombinant AAV genome and retains said recombinant AAV genome in a nucleus of said host cell.   
     
     
         13 . The method of  claim 12 , wherein said two or more AAV capsid proteins are expressed under the control of vaccinia virus p7.5 promoter. 
     
     
         14 . The method of  claim 12 , wherein said two or more AAV capsid proteins comprise VP2 and VP3 proteins expressed from a first vaccinia virus and VP1 expressed from a second vaccinia virus. 
     
     
         15 . The method of  claim 12 , wherein the infecting step comprises infecting said host cell with a single vaccinia virus that expresses AAV VP1, VP2 and VP3 proteins at a ratio suitable for the production of said replication-deficient AAV vector. 
     
     
         16 . The method of  claim 15 , wherein said single vaccinia virus contains a first expression cassette having AAV vp1 ggen under the control of a first vaccinia virus p7.5 promoter and a second expression cassette having AAV vp2 gene under the control of a second vaccinia virus p7.5 promoter. 
     
     
         17 . A carrier virus, comprising:
 a capsid;   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 capsid 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.