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. A lentiviral packaging construct for use in generating lentiviral vectors comprising a sequence encoding a lentiviral protease gene and a deletion in a lentiviral packaging signal sequence, wherein said sequence encoding said protease includes a substitution wherein the T in the DTGAD (residues 25–29 of SEQ ID NO:1) motif is replaced with an S.
2. The construct of claim 1 , wherein said sequence encoding said protease comprises a lentiviral pol gene.
3. The packaging construct of claim 1 wherein said lentiviral protease is an HIV protease.
4. The packaging construct of claim 1 wherein said lentiviral protease is a BIV protease.
5. The packaging construct of claim 1 wherein said lentiviral protease is an EIAV protease.
6. The packaging construct of claim 1 wherein said lentiviral protease is an SIV protease.
7. The packaging construct of claim 1 wherein said lentiviral protease is an FIV protease.
8. The construct of claim 2 , further comprising a lentiviral gag gene.
9. The packaging construct of claim 1 , 2 or 8 which is obtained from an HIV genome.
10. The packaging construct of claim 1 , 2 or 8 which is obtained from a BIV genome.
11. A pre-packaging cell line comprising said packaging construct of claim 1 , 2 or 8 .
12. The packaging construct of claim 9 further comprising a deletion of, or mutation in, a vif gene, a vpr gene or both.
13. The packaging construct of claim 10 further comprising a deletion of, or mutation in, one or more of a vif gene, a W gene, a Y gene or a Tat gene.
14. The pre-packaging cell line of claim 2 or 8 wherein expression of the lentiviral pol gene is constitutive.
15. The pre-packaging cell line of claim 8 wherein expression of the lentiviral gag gene is constitutive.
16. The pre-packaging cell line of claim 11 wherein expression of the lentiviral protease gene is constitutive.
17. A packaging cell line comprising the pre-packaging cell line of claim 11 and further comprising an env gene.
18. A packaging cell line comprising the prepackaging cell line of claim 16 and further comprising an env gene.
19. A packaging cell line comprising the prepackaging cell line of claim 14 and further comprising an env gene.
20. A packaging cell line comprising the prepackaging cell line of claim 15 and further comprising an env gene.
21. A producer cell line comprising the packaging cell line of claim 17 and further comprising a lentiviral vector.
22. A producer cell line comprising the packaging cell line of claim 18 and further comprising a lentiviral vector.
23. A producer cell line comprising the packaging cell line of claim 19 and further comprising a lentiviral vector.
24. A producer cell line comprising the packaging cell line of claim 20 and further comprising a lentiviral vector.