Bovine immunodeficiency virus (BIV) based vectors
This invention pertains to BIV constructs encompassing BIV combination vectors, BIV vectors and BIV packaging vectors and particularly the invention pertains to a three vector system comprising: a) a BIV vector construct including a DNA segment from a BIV genome, a packaging sequence to package RNA into virions; a promoter operably linked to the DNA segment; and a transgene operably linked to a second promoter; b) a BIV packaging vector construct comprising a BIV DNA sequence fragment comprising at least a gag gene or pol gene of BIV; a promoter operably linked to the BIV DNA fragment; and a polyadenylation sequence located downstream of the BIV DNA fragment; and c) an expression vector construct comprising a gene encoding a viral surface protein. Also provided is a method for transferring a gene of interest into a mammalian cell.
1. A method of transferring a transgene to a mammalian cell comprising:
transfecting a eukaryotic host cell with a vector system comprising:
(a) a BIV vector construct comprising:
a DNA segment from a BIV genome,
a packaging sequence to package RNA into vinons;
a promoter operably linked to said segment; and
a transgene operably linked to a second promoter, wherein at least one of vpw, vpy and tat is deleted from said BIV construct;
(b) a BIV packaging construct comprising:
a BIV sequence comprising a gag gene, a pol gene or both of BIV operably linked to a promoter, and
a downstream polyadenylation sequence; and
(c) an expression construct comprising a gene encoding a viral surface protein;
culturing the transfected host cell and collecting the virions produced; and
administering the collected virions to a mammalian cell to allow infection of the mammalian cell and thereby transferring the transgene.
2. The method of claim 1 , wherein the mammalian cell is located in vitro.
3. The method of claim 1 , wherein the mammalian cell is located in vivo.
4. A method of transferring a transgene to a mammalian cell comprising:
transfecting a eukaryotic host cell with a vector system comprising:
a) a BIV vector construct comprising:
a promoter linked to a first BIV R region;
a BIV U 5 element linked to the first BIV R region;
a packaging sequence;
a transgene; and
a BIV U 3 element linked to a second BIV R region, wherein the promoter initiates RNA transcription of the vector construct, and at least one of vpw, vpy and tat is deleted from said construct;
b) a BIV packaging construct comprising:
a BIV sequence comprising a gag gene, a pol gene or both of BIV operably linked to a promoter, and
a downstream polyadenylation sequence; and
c) an expression construct comprising a gene encoding a viral surface protein;
culturing the transfected host cell and collecting the virions produced; and
administering the collected virions to a mammalian cell to allow infection of the mammalian cell and thereby transferring the transgene.
5. The method of claim 1 , wherein said viral surface protein is a VSV-G protein.
6. The method of claim 1 , wherein said packaging sequence is a BIV packaging sequence.
7. The method of claim 1 , wherein said transgene is operably linked to a mammalian promoter.
8. The method of claim 1 , wherein said BIV vector construct further comprises a rev-response element (RRE).
9. The method of claim 1 , wherein said BIV vector construct further comprises a central polypurine tract (cPPT).
10. The method of claim 4 , wherein said transgene is operably linked to an internal promoter.
11. The method of claim 4 , wherein one or more nucleotides in said U 3 element are mutated or deleted to diminish or eliminate U 3 -mediated transcription.
12. The method of claim 4 , wherein said packaging sequence is a BIV packaging sequence.
13. The method of claim 4 , wherein any start codons in said packaging sequence are eliminated by deletion or mutation.
14. The method of claim 4 , wherein said U 3 element further comprises a sequence that enhances polyadenylation.
15. The method of claim 4 , wherein said vector construct further comprises a cPPT.
16. The method of claim 15 , wherein said cPPT is a BIV cPPT.
17. The method of claim 4 , wherein said vector construct further comprises a 3′ polypurine tract.
18. The method of claim 4 , wherein said vector construct further comprises an RNA transport element.
19. The method of claim 18 , wherein said RNA transport element is a lentiviral rev response element (RRE).
20. The method of claim 19 , wherein said lentiviral RRE is a BIV RRE.
21. The method of claim 18 , wherein said RNA transport element is a constitutive transport element (CTE).