Lentiviral packaging cells and uses therefor
Novel packaging cell lines which produce recombinant retrovirus, free of detectable helper-virus are disclosed. Also disclosed are methods of making the cell lines and methods of producing recombinant retroviruses from the cell lines. Retroviruses produced by the cell lines include lentiviruses, such as HIV, capable of transfering heterologous DNA to a wide range of non-dividing cells. The packaging cells contain at least three vectors which collectively encode retroviral gag, pol, and env proteins, wherein the gag and pol genes are separated, in part, onto two or more different vectors. This is made possible by fusing Vpr or Vpx to pol proteins separated from gag so that the proteins are targeted to assembling virions. Among other advantages, the packaging cells provide the benefit of increased safety when used in human gene therapy by virtually eliminating the possibility of molecular recombination leading to production of replication competent helper virus.
1. A method of producing a packaging cell line capable of producing a recombinant, helper-free retrovirus, comprising transfecting a host cell with:
a first vector comprising a retroviral gag gene, wherein said gag gene encodes retroviral matrix, capsid and nucleocapsid proteins;
a second vector comprising a portion of a retroviral pol gene, fused to a Vpr or Vpx gene, wherein said portion of said pol gene encodes a retroviral protease;
a third vector comprising the remainder of the retroviral pol gene not contained in said second vector, fused to a Vpr or a Vpx gene, wherein said remainder of said pol gene encodes a retroviral reverse transcriptase and a retroviral integrase; and
a fourth vector comprising a viral env gene.
2. The method of claim 1 , wherein transcription of any one of said genes within said vectors is directed by an inducible promoter.
3. The method of claim 2 further comprising the step of contacting said host cell with an agent which induces said promoter.
4. The method of claim 1 wherein said retrovirus is a lentivirus.
5. The method of claim 4 , wherein said lentivirus is selected from the group consisting of HIV-1, HIV-2, SIV, FIV and EIV.
6. The method of claim 1 , further comprising transfecting the host cell with retroviral accessory genes.
7. The method of claim 1 , wherein said remainder of said retroviral pol gene further comprises a protease cleavage site upstream from said reverse transcriptase and said integrase.
8. The method of claim 1 , wherein said third vector further comprises a retroviral Vif gene.
9. The method of claim 1 , wherein said viral env gene within said third vector encodes an envelope protein selected from a virus selected from the group consisting of a Type C retrovirus, a lentivirus and Vesicular Stomatitis Virus.
10. The method of claim 1 , wherein any of the first, second, third or fourth vectors further comprise an RNA export element.
11. The method of claim 10 , wherein said RNA export element is the reverse response element (RRE) from human immunodeficiency virus (HIV).
12. The method of claim 1 , wherein any of the first, second, third or fourth vectors further comprise a marker gene.
13. The method of claim 4 , wherein said lentivirus is HIV.
14. The method of claim 1 , wherein transcription of any one of said genes within said vectors is induced by contact of Cre recombinase with one or more Lox sites contained in said vector.