Recombinant vectors
This disclosure provides modified cytosine deaminases (CDs). The disclosure further relates to cells and vector expressing or comprising such modified CDs and methods of using such modified CDs in the treatment of disease and disorders.
1. A recombinant replication competent retrovirus comprising:
a retroviral GAG protein;
a retroviral POL protein;
a retroviral envelope;
a retroviral RNA polynucleotide comprising 3′ untranslated region (U3) and repeat region (R) sequences at the 3′ end of the retroviral polynucleotide sequence, an R and 5′ untranslated region (U5) sequence at the 5′ end of the retroviral polynucleotide, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain located between the U5 and U3 regions;
a cassette comprising a pol III promoter upstream and operably linked to a heterologous polynucleotide, wherein the cassette is positioned 5′ to the U3 region and 3′ to the env nucleic acid domain; and
cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell;
wherein the retroviral RNA polynucleotide provides a proviral DNA sequence comprising a gag nucleic acid domain comprising a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19 or 22, the pol nucleic acid domain comprising the sequence from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO:19 or 22, the env domain comprising the sequence from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO:19 or 22; and the cassette, and
wherein the virus can infect a target cell multiple times resulting in an average number of copies/diploid genome of 5 or greater.
2. The retrovirus of claim 1 , wherein the retroviral polynucleotide sequence is recombinantly 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, Xenotropic murine leukemia virus-related virus (XMRV), avian reticuloendotheliosis virus (REV), or Gibbon ape leukemia virus (GALV).
3. The retrovirus of claim 1 , wherein the retroviral envelope is an amphotropic MLV envelope.
4. The retrovirus of claim 1 , wherein the retrovirus is a gammaretrovirus.
5. The retrovirus of claim 1 , wherein the target cell is a cell having a cell proliferative disorder.
6. The retrovirus of claim 1 , wherein the target cell is a neoplastic cell.
7. The retrovirus of claim 5 , 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, rheumatoid arthritis or other autoimmune disease.
8. The retrovirus of claim 1 , wherein the promoter sequence is associated with a growth regulatory gene.
9. The retrovirus of claim 1 , wherein the pol III promoter is an H1 or U6 promoter.
10. The retrovirus of claim 9 , wherein the pol III promoter is an H1 promoter.
11. The retrovirus of claim 1 , wherein the 3′ U3-R domain is derived from a gammaretrovirus.
12. The retrovirus according to claim 1 , wherein the heterologous nucleic acid sequence comprises an inhibitory polynucleotide.
13. The retrovirus according to claim 12 , wherein the inhibitory polynucleotide comprises an miRNA, RNAi or siRNA sequence.
14. A method of treating a cell proliferative disorder comprising administering a retrovirus 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.
15. The method of claim 14 , 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, P1-88, 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, or ZK-304709.
16. The method of claim 14 , wherein the retrovirus is administered from about 10 3 to 10 7 TU/g brain weight.
17. The method of claim 16 , wherein the retrovirus is administered from about 10 4 to 10 6 TU/g brain weight.
18. The method of claim 14 , wherein the retrovirus is administered from about 10 5 to 10 11 TU/g brain weight.
19. The retrovirus of claim 1 , further comprising an IRES cassette downstream of the env gene.
20. The retrovirus of claim 19 , wherein the IRES cassette comprises IRES operably linked to a heterologous gene.
21. A recombinant replication competent retrovirus (RCR) comprising:
a retroviral GAG protein;
a retroviral POL protein;
a retroviral envelope;
a retroviral RNA polynucleotide comprising a 3′ untranslated region (U3) and repeat region (R) sequences at the 3′ end of the retroviral polynucleotide sequence, an R and 5′ untranslated region (U5) sequence at the 5′ end of the retroviral polynucleotide, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain located between the U5 and U3 regions;
a cassette comprising an primary precursor miRNA (pri-miRNA) for an miRNA or siRNA sequence operably linked to a pol III promoter, wherein the cassette is positioned 5′ to the U3 region and 3′ to the env nucleic acid domain;
wherein the retroviral RNA polynucleotide provides a proviral DNA sequence consisting of a gag nucleic acid domain comprising a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19 or 22, a pol nucleic acid domain comprising the sequence from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO:19 or 22, an env domain comprising the sequence from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO:19 or 22, and the cassette; and
cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell.
22. The RCR of claim 21 , wherein the miRNA is selected from the group consisting of miR-142-3p, miR-181, miR-223, miR 128-1 and miR 128-2.
23. The RCR of claim 22 , wherein the retroviral polynucleotide further comprises an IRES cassette operably linked to a heterologous polynucleotide.
24. The RCR of claim 23 , wherein the IRES cassette is located immediately 3′ to the env gene.