Lentiviral packaging constructs
The present invention provides novel lentiviral packaging constructs that are useful for the establishment of stable packaging cell lines and producer cell lines. In particular, the present invention provides novel packaging cell lines that are capable of constitutively expressing high levels of lentiviral proteins.
1. An isolated nucleic acid comprising a coding region for a lentiviral protease, wherein said protease comprises a Thr to Ser substitution of the Thr residue in the Asp-Thr-Gly motif of said protease corresponding to amino acid number 26 of SEQ ID NO: 1, and wherein said nucleic acid does not comprise a lentiviral packaging signal.
2. The nucleic acid of claim 1 , comprising SEQ ID NO:17.
3. The nucleic acid of claim 1 , wherein said nucleic acid does not comprise a coding region for a BIV vif, W, Y or Tat gene.
4. The nucleic acid of claim 1 , further comprising a scaffold attachment region (SAR).
5. The nucleic acid of claim 4 , wherein said SAR comprises an IFN SAR or a β-IFN SAR.
6. The nucleic acid of claim 1 , wherein said coding region comprises one or more recoded codons that are preferred codons of an organism.
7. The nucleic acid of claim 6 , wherein said organism is human.
8. The nucleic acid of claim 1 , wherein said nucleic acid does not comprise a BIV RRE.
9. An isolated cell line comprising the nucleic acid of claim 1 .
10. The cell line of claim 9 , wherein said cell line expresses at least 5 ng/ml of RT (reverse transcriptase).
11. The cell line of claim 9 , wherein said cell line expresses an envelope (env) protein.
12. The cell line of claim 11 , wherein said env protein is selected from the group consisting of a VSV-G env, an LCMV env and a mutant LCMV env.
13. The cell line of claim 9 , further comprising a lentiviral vector.
14. The cell line of claim 13 , wherein said line produces at least 10 5 cfu/ml of virus particles.
15. The cell line of claim 9 , wherein said cell line produces a lentiviral vector virus particle comprising a non-lentiviral gene.
16. A method of producing a lentivirus particle comprising the steps of:
(a) transfecting the cell line of claim 9 with a lentiviral vector;
(b) propagating said transfected cell line of step (a) in a suitable culture medium; and
(c) obtaining said lentiviral particle from said culture medium.
17. The nucleic acid of claim 1 , wherein the lentiviral protease is a BIV protease.
18. The cell line of claim 13 , wherein the lentiviral vector is a BIV vector.
19. The nucleic acid of claim 1 , wherein said lentiviral protease is an HIV protease.
20. The cell line of claim 13 , wherein said lentiviral vector is an HIV vector.
21. The nucleic acid of claim 1 , wherein said lentiviral protease is an SIV protease.
22. The cell line of claim 13 , wherein said lentiviral vector is an SIV vector.
23. The nucleic acid of claim 1 , wherein said lentiviral protease is an EIAV protease.
24. The cell line of claim 13 , wherein said lentiviral vector is an EIAV vector.