Lentiviral triplex DNA, and vectors and recombinant cells containing lentiviral triplex DNA
The present invention provides nucleic acid, vectors, viruses, and recombinant cells comprising triple-stranded structures, such as those resulting from central initiation and termination of HIV-1 reverse transcription at the center of HIV-1 linear DNA genomes. These triplex structures can act as a cis-determinant of HIV-1 DNA nuclear import, allowing infection of non-dividing target cells. In one aspect, the presence of the DNA triplex sequence in an HIV vector strongly stimulates gene transfer in hematopoietic stem cells. The invention also provides methods of using these triplex structures for making recombinant cells, as well as methods of using the recombinant cells to express proteins of interest both in vitro and in vivo.
1. A method for producing a lentiviral gene transfer vector that promotes nuclear import of a gene of interest comprising generating a gene transfer vector comprising:
(a) at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure,
(b) a gene of interest,
(c) an HIV-1 LTR that is deleted for the promoter and the enhancer of U3, and
(d) a cis-acting lentiviral encapsidation sequence.
2. The method of claim 1 , wherein the cPPT and CTS regions are lentiviral cPPT and CTS regions.
3. The method of claim 2 , wherein the cPPT and CTS regions are human immunodeficiency virus (HIV) cPPT and CTS regions.
4. The method of claim 2 , wherein the cPPT and CTS regions are HIV-1 cPPT and CTS regions.
5. The method of claim 2 , wherein the cPPT and CTS regions are HIV-2 cPPT and CTS regions.
6. The method of claim 2 , wherein the cPPT and CTS regions are VISNA, EIAV, FIV, or CAEV cPPT and CTS regions.
7. A method for producing a lentiviral gene transfer vector that promotes nuclear import of a gene of interest comprising:
(a) inserting into the gene transfer vector at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure,
(b) inserting into the gene transfer vector a gene of interest,
(c) inserting into the gene transfer vector an HIV-1 LTR that is deleted for the promoter and the enhancer of U3,
wherein the gene transfer vector comprises a cis-acting lentiviral encapsidation sequence.
8. The method of claim 7 , wherein the cPPT and CTS regions are lentiviral cPPT and CTS regions.
9. The method of claim 8 , wherein the cPPT and CTS regions are human immunodeficiency virus (HIV) cPPT and CTS regions.
10. The method of claim 8 , wherein the cPPT and CTS regions are HIV-1 cPPT and CTS regions.
11. The method of claim 8 , wherein the cPPT and CTS regions are HIV-2 cPPT and CTS regions.
12. The method of claim 8 , wherein the cPPT and CTS regions are VISNA, EIAV, FIV, or CAEV cPPT and CTS regions.
13. A method for producing a lentiviral gene transfer vector that promotes nuclear import of a gene of interest comprising inserting into a gene transfer vector comprising a cis-acting lentiviral encapsidation sequence and an HIV-1 LTR that is deleted for the promoter and the enhancer of U3:
(a) at least one copy of the cPPT and CTS cis-acting regions of a retrovirus, wherein said cPPT and CTS regions induce a three-stranded DNA structure; and
(b) a gene of interest.
14. The method of claim 13 , wherein the cPPT and CTS regions are lentiviral cPPT and CTS regions.
15. The method of claim 14 , wherein the cPPT and CTS regions are human immunodeficiency virus (HIV) cPPT and CTS regions.
16. The method of claim 14 , wherein the cPPT and CTS regions are HIV-1 cPPT and CTS regions.
17. The method of claim 14 , wherein the cPPT and CTS regions are HIV-2 cPPT and CTS regions.
18. The method of claim 14 , wherein the cPPT and CTS regions are VISNA, EIAV, FIV, or CAEV cPPT and CTS regions.