Claims
- 1. A retroviral packaging cell line comprising:
a first vector encoding a retroviral gag polyprotein and a portion of a retroviral pol polyprotein; a second vector encoding the remainder of the retroviral pol polyprotein not encoded by said first vector, fused to a Vpr or a Vpx protein or peptide; and a third vector encoding a viral env protein.
- 2. The packaging cell line of claim 1 wherein said retroviral polyproteins are lentiviral polyproteins.
- 3. The packaging cell line of claim 2 wherein the lentivirus is selected from the group consisting of HIV-1, HIV-2, SIV, FIV and EIV.
- 4. The packaging cell line of claim 1 wherein said retroviral gag polyprotein encoded by said first vector comprises retroviral matrix, capsid and nucleocapsid proteins.
- 5. The packaging cell line of claim 1 wherein said portion of said pol polyprotein encoded by said first vector comprises a retroviral protease.
- 6. The packaging cell line of claim 1 wherein said remainder of said retroviral pol polyprotein encoded by said second vector comprises a retroviral reverse transcriptase and a retroviral integrase.
- 7. The packaging cell line of claim 6 wherein said remainder of said retroviral pol polyprotein further comprises a protease cleavage site upstream from said reverse transcriptase and said integrase.
- 8. The packaging cell line of claim 1 wherein said viral env protein encoded by said third vector comprises an envelope protein selected from a virus selected from the group consisting of a Type C retrovirus, a lentivirus and Vesicular Stomatitis Virus.
- 9. The packaging cell line of claim 1 wherein transcription of any one of said proteins encoded by said vectors is driven by an inducible promoter.
- 10. The packaging cell line of claim 1 wherein transcription of any one of said proteins encoded by said vectors is induced by contact of Cre recombinase with one or more Lox sites contained in said vector.
- 11. The packaging cell line of claim 1 further comprising a fourth vector containing a packaging signal, a viral long terminal repeat (LTR), and a transgene, wherein co-expression of said fourth vector with said first, second, and third vectors, in said packaging cell line, results in the production of a recombinant, helper-free retrovirus containing said transgene.
- 12. 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 encoding a retroviral gag polyprotein and a portion of a retroviral pol polyprotein; a second vector encoding the remainder of the retroviral pol polyprotein not encoded by said first vector, fused to a Vpr or a Vpx protein or peptide; and a third vector encoding a viral env protein.
- 13. The method of claim 12 wherein any one of said first, second or third vectors comprises an inducible promoter operably linked to a gene encoding said gag polyprotein, said pol polyprotein, said Vpr protein or peptide, said Vpx protein or peptide, or said env protein.
- 14. The method of claim 13 further comprising the step of contacting said host cell with an agent which induces said promoter.
- 15. The method of claim 12 wherein said retrovirus is a lentivirus.
- 16. The method of claim 15 wherein said lentivirus is selected from the group consisting of HIV-1, HIV-2, SIV, FIV and EIV.
- 17. The method of claim 12 wherein said retroviral gag polyprotein encoded by said first vector comprises lentiviral matrix, capsid and nucleocapsid proteins.
- 18. The method of claim 12 wherein said wherein said portion of said pol polyprotein encoded by said first vector comprises a retroviral protease.
- 19. The method of claim 12 wherein said remainder of said retroviral pol polyprotein encoded by said second vector comprises a retroviral reverse transcriptase and a retroviral integrase.
- 20. The method of claim 13 wherein said viral env protein encoded by said third vector comprises an envelope protein selected from a virus selected from the group consisting of a Type C retrovirus, a lentivirus and Vesicular Stomatitis Virus.
- 21. A method of producing a recombinant retrovirus comprising transfecting a host cell with:
a first vector comprising a retroviral gag gene and a portion of a retroviral pol gene operably linked to a promoter; a second vector comprising the portion of the retroviral pol gene not encoded by the first vector and a gene encoding all or a portion of a Vpr or a Vpx protein, wherein both of said genes are expressed as a single fusion protein and are operably linked to a promoter; a third vector comprising a viral env gene; and a fourth vector comprising a viral packaging signal, a viral long terminal repeat (LTR), and a transgene operably linked to a promoter; and recovering the recombinant virus.
- 22. The method of claim 21 wherein any one of said promoters contained within said first, second, third or fourth vectors is inducible.
- 23. The method of claim 22 further comprising the step of contacting said host cell with an agent which induces said promoter.
- 24. The method of claim 21 wherein said retrovirus is a lentivirus.
- 25. The method of claim 24 wherein said lentivirus is HIV.
- 26. The method of claim 21 wherein said portion of said pol gene contained in said first vector encodes a retroviral protease.
- 27. The method of claim 21 wherein said remainder of said pol gene contained in said second vector encodes a retroviral reverse transcriptase and a retroviral integrase.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 60/085,283, filed May 13, 1998, the entire contents of which are hereby incorporated by reference herein.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60085283 |
May 1998 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09311684 |
May 1999 |
US |
Child |
10112894 |
Apr 2002 |
US |