Retroviral vector with mini-promoter cassette
This disclosure provides a retroviral replicating vector for gene delivery comprising a therapeutic cassette containing at least one mini-promoter linked to a gene to be expressed.
1. A recombinant replication competent gammaretrovirus comprising:
a retroviral GAG protein;
a retroviral POL protein;
a retroviral envelope;
a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain;
a therapeutic cassette comprising at least one mini-promoter cassette having a mini-promoter that is regulated by an RNA polymerase II, wherein the mini-promoter is about 70-500 bp in length and operably linked to a heterologous polynucleotide, wherein the therapeutic cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope, and wherein when only one mini-promoter cassette is present the heterologous polynucleotide is 1.2kb to 2.0 kb in length; and
cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell, and wherein the mini-promoter comprises an RSV promoter.
2. The recombinant replication competent gammaretrovirus of claim 1 , wherein the retroviral polynucleotide sequence is engineered from a virus selected from the group consisting of murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus (FeLV), Baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), the cat derived retrovirus RD114, squirrel monkey retrovirus, avian reticuloendotheliosis virus(REV), or Gibbon ape leukemia virus (GALV).
3. The recombinant replication competent gammaretrovirus of claim 1 , wherein the retroviral envelope is an amphotropic MLV envelope.
4. The recombinant replication competent gammaretrovirus of claim 1 , wherein the target cell is a cancer cell.
5. The recombinant replication competent gammaretrovirus of claim 1 , wherein the target cell is a neoplastic cell.
6. The recombinant replication competent gammaretrovirus of claim 4 , wherein the cancer cell is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer, lymphoma, and leukemia.
7. The recombinant replication competent gammaretrovirus of claim 1 , wherein the promoter sequence is a growth regulatory gene promoter.
8. The recombinant replication competent gammaretrovirus of claim 1 , wherein the promoter sequence comprises a tissue-specific promoter sequence.
9. The recombinant replication competent gammaretrovirus of claim 1 , wherein the promoter comprises a CMV-R-U5 domain polynucleotide.
10. The recombinant replication competent gammaretrovirus of claim 1 , wherein the mini-promoter is a core promoter.
11. The recombinant replication competent gammaretrovirus of claim 1 , wherein the mini-promoter is an optimized core promoter.
12. The recombinant replication competent gammaretrovirus of claim 1 , wherein the therapeutic cassette comprises (a) at least two mini-promoter cassettes, (b) at least one minipromoter cassette and a polIII promoter cassette or (c) at least one mini-promoter cassette and an IRES cassette.
13. The recombinant replication competent gammaretrovirus of claim 1 , wherein the mini promoter comprises a core promoter and further comprises an enhancer element.
14. The recombinant replication competent gammaretrovirus of claim 1 , wherein the mini-promoter comprises a TATA box, and initiator site, a Motif Ten Element (MTE), a Downstream promoter element (DPE) and at least one additional element selected from the group consisting of: (a) TFIIB recognition element, upstream (BREu); (b) TFIIB recognition element downstream (BREd); (c) HBV X core promoter element 1 (XCPE1); (d) HBV X core promoter element 2 (XCPE2); (d) downstream core element site I (CDE SI); (e) downstream core element site II (CDE SII); and (f) downstream core element site III (CDE SIII).
15. The recombinant replication competent gammaretrovirus of claim 14 , wherein the mini-promoter further comprises an enhancer element.
16. The recombinant replication competent gammaretrovirus of claim 1 , wherein the heterologous nucleic acid comprises a polynucleotide having a sequence as set forth in SEQ ID NO:3, 5, 11, 13, 15 or 17.
17. The recombinant replication competent gammaretrovirus of claim 1 , wherein the heterologous nucleic acid is human codon optimized and encodes a polypeptide as set forth in SEQ ID NO:4.
18. The recombinant replication competent gammaretrovirus of claim 1 , wherein the 3′ LTR is engineered from a gammaretrovirus.
19. The recombinant replication competent gammaretrovirus of claim 18 , wherein the 3′ LTR comprises a U3-R-U5 domain.
20. The recombinant replication competent gammaretrovirus of claim 1 , wherein the heterologous nucleic acid sequence encodes a biological response modifier or an immunopotentiating cytokine.
21. The recombinant replication competent gammaretrovirus of claim 20 , wherein the immunopotentiating cytokine is selected from the group consisting of interleukins 1 through 15, interferon, tumor necrosis factor (TNF), and granulocyte-macrophage-colony stimulating factor (GM-CSF).
22. The recombinant replication competent gammaretrovirus of claim 20 , wherein the immunopotentiating cytokine is interferon gamma.
23. The recombinant replication competent gammaretrovirus of claim 1 , wherein the heterologous nucleic acid encodes a polypeptide that converts a nontoxic prodrug in to a toxic drug.
24. The recombinant replication competent gammaretrovirus of claim 23 , wherein the polypeptide that converts a nontoxic prodrug in to a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase.
25. The recombinant replication competent gammaretrovirus of claim 1 , wherein the heterologous nucleic acid sequence comprises an inhibitory polynucleotide.
26. The recombinant replication competent gammaretrovirus of claim 25 , wherein the inhibitory polynucleotide comprises an miRNA, siRNA sequence or other RNAi construct.
27. The recombinant replication competent gammaretrovirus of claim 26 , wherein the therapeutic cassette comprises a mini-promoter operably linked to heterologous nucleic acid and a polIII promoter operably linked to the miRNA, siRNA sequence or other RNAi coding domain.
28. A method of delivering a therapeutic molecule to a subject comprising contacting the subject with a recombinant replication competent gammaretrovirus of claim 1 .
29. A method of treating a cell proliferative disorder comprising contacting the subject with the recombinant replication competent gammaretrovirus of claim 16 under conditions such that the polynucleotide is expressed and contacting the subject with 5-fluorocytosine.
30. The method of claim 29 , wherein the cell proliferative disorder is glioblastoma multiforme.
31. The method of claim 29 , wherein the cell proliferative disorder is selected from the group consisting of lung cancer, colon-rectum cancer, breast cancer, prostate cancer, urinary tract cancer, uterine cancer, brain cancer, head and neck cancer, pancreatic cancer, melanoma, stomach cancer and ovarian cancer.
32. A method of treating a cell proliferative disorder in a subject comprising contacting the subject with the recombinant replication competent gammaretrovirus of claim 1 , wherein the heterologous nucleic acid sequence encodes a therapeutic protein that inhibits proliferation of a neoplastic cell.
33. The method of claim 32 , wherein the therapeutic protein comprises a polypeptide that converts a non-cytotoxic drug to a cytotoxic drug.
34. The method of claim 33 , wherein the polypeptide has cytosine deaminase activity.
35. The method of claim 34 , wherein the polypeptide comprises a sequence as set forth in SEQ ID NO:4, 12, 14, 16, or 18.
36. The method of claim 33 , wherein the non-cytotoxic drug is 5-fluorocytosine.
37. A method of treating a cell proliferative disorder comprising administering the recombinant replication competent gammaretrovirus of claim 1 to a subject having a cell proliferative disorder under conditions such that the retrovirus infects cells with the disorder and contacting the subject with an anti-cancer agent or chemotherapeutic agent.
38. The method of claim 37 , wherein the anti-cancer agent is selected from the group consisting of bevacizumab, pegaptanib, ranibizumab, sorafenib, sunitinib, AE-941, VEGF Trap, pazopanib, vandetanib, vatalanib, cediranib, fenretinide, squalamine, INGN-241, oral tetrathiomolybdate, tetrathiomolybdate, Panzem NCD, 2-methoxyestradiol, AEE-788, AG-013958, bevasiranib sodium, AMG-706, axitinib, BIBF-1120, CDP-791, CP-547632, PI-88, SU-14813, SU-6668, XL-647, XL-999, IMC-1121B, ABT-869, BAY-57-9352, BAY-73-4506, BMS-582664, CEP-7055, CHIR-265, CT-322, CX-3542, E-7080, ENMD-1198, OSI-930, PTC-299, Sirna-027, TKI-258, Veglin, XL-184, and ZK-304709.
39. The method of claim 37 , wherein the gammaretrovirus is administered from about 10 3 to 10 7 TU/g brain weight.
40. The method of claim 39 , wherein the gammaretrovirus is administered from about 10 4 to 10 6 TU/g brain weight.