Lentiviral vectors featuring liver specific transcriptional enhancer and methods of using same
View Patent ↗Recombinant lentiviruses and transfer vectors for transgene delivery as well as methods for gene therapy using such vectors are disclosed. The invention provides a third generation lentiviral packaging system and a set of vectors for producing recombinant lentiviruses, as well as novel tissue specific enhancer and promoter elements useful for optimizing liver specific transgene delivery. The transgene is preferably a blood clotting factor such as human factor IX (hFIX) or human factor VIII (hFVIII) and can be used for treatment of hemophilia.
1. A lentiviral producer cell comprising:
(a) a gag gene, or a pol gene, or gag and pol genes;
(b) an env gene; and
(c) a lentiviral transfer vector that comprises a transgene operably linked to an expression control sequence, wherein the expression control sequence comprises the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
2. The producer cell of claim 1 , wherein the expression control sequence comprises the nucleotide sequence of SEQ ID NO: 10.
3. The producer cell of claim 1 , wherein the expression control sequence consists of the nucleotide sequence of SEQ ID NO: 10.
4. The producer cell of claim 1 , wherein the expression control sequence further comprises a mouse transthyretin (mTTR) promoter.
5. The producer cell of claim 1 , wherein the expression control sequence further comprises an α-1-microglobulin/bikunin enhancer and a human factor VIII endogenous promoter (L-F8).
6. The lentiviral producer cell of claim 1 , wherein the elements of (a), (b) and (c) are stably integrated into the genome of the lentiviral producer cell.
7. The lentiviral producer cell of claim 1 , wherein the lentivirus is a human immunodeficiency virus (HIV).
8. The lentiviral producer cell of claim 7 , wherein the HIV is HIV-1.
9. The lentiviral producer cell of claim 1 , further comprising a rev gene.
10. The lentiviral producer cell of claim 1 , which lacks functional genes selected from tat, vif, vpr, vpu, vpx and nef, or a combination thereof.
11. The lentiviral producer cell of claim 1 , wherein the expression control sequence further comprises a liver specific promoter.
12. The lentiviral producer cell of claim 11 , wherein the liver specific promoter comprises a mouse transthyretin (mTTR) promoter.
13. The lentiviral producer cell of claim 1 , wherein the expression control sequence further comprises an α-1-microglobulin/bikunin enhancer and a human factor VIII endogenous promoter (L-F8).
14. A set of lentiviral vectors comprising:
(a) a first packaging vector comprising a gag gene, or a pol gene, or gag and pol genes operably linked to an expression control sequence;
(b) a second packaging vector comprising a heterologous env gene operably linked to an expression control sequence; and
(c) a lentiviral transfer vector that comprises a transgene operably linked to an expression control sequence;
wherein the expression control sequence comprises the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
15. The set of claim 14 , wherein the expression control sequence comprises the nucleotide sequence of SEQ ID NO: 10.
16. The set of claim 14 , wherein the expression control sequence consists of the nucleotide sequence of SEQ ID NO: 10.
17. The set of claim 14 , wherein the expression control sequence further comprises a mouse transthyretin (mTTR) promoter.
18. The set of claim 14 , wherein the expression control sequence further comprises an α-1-microglobulin/bikunin enhancer and a human factor VIII endogenous promoter (L-F8).
19. The set of claim 14 , wherein the transgene encodes a blood clotting factor.
20. The set of claim 14 , wherein the transgene encodes human factor IX (hFIX).
21. The set of claim 14 , wherein the transgene encodes human factor VIII (hFVIII).
22. The set of claim 14 , wherein the lentivirus is a human immunodeficiency virus (HIV).
23. The set of claim 22 , wherein the HIV is HIV-1.
24. The set of claim 14 , further comprising a fourth vector that comprises a rev gene.
25. The set of claim 14 , which lacks functional genes selected from tat, vif, vpr, vpu, vpx and net or a combination thereof.
26. A method of producing a lentiviral producer cell comprising transforming a cell with the set of vectors of claim 14 .
27. A method of producing a recombinant lentivirus comprising culturing a producer cell of claim 1 in a medium and recovering recombinant lentivirus from the medium.
28. A method of delivering a transgene to a cell comprising contacting the cell with a recombinant lentivirus produced by the lentiviral producer cell of claim 1 under conditions permitting transformation of the cell.