Recombinant vectors
This disclosure provides recombinant replication competent retroviral vectors having increased stability. The disclosure further relates compositions and uses of such vectors 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 polynucleotide sequence comprising a Long-Terminal Repeat (LTR) sequence at the 3′ end of the retroviral polynucleotide sequence, wherein the 3′ LTR comprises a sequence that is at least 98% identical to the sequence from about nucleotide 9405 to about 9998 of SEQ ID NO:19 or 22, a promoter sequence at the 5′ end of the retroviral polynucleotide sequence, 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, wherein the promoter, gag, pol and env domains comprise a sequence that is at least 98% identical to the sequence from about 1 to about 8323 of SEQ ID NO:19 or 22 and wherein the retroviral polynucleotide sequence lacks 70 base pairs of MLV sequence downstream from the 3′LTR compared to a pACE vector of SEQ ID NO:21;
a cassette comprising an internal ribosome entry site (IRES) comprising a sequence that is at least 98% identical to the sequence from about 8327 to 8876 of SEQ ID NO:19 or 22, operably linked to a heterologous polynucleotide, wherein the cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope and wherein SEQ ID NO: 19 and 22 lack small repeats on either side of the cassette compared to the pACE vector of SEQ ID NO:21; and
cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell,
wherein the RCR maintains higher replication competency after 6 passages compared to a pACE vector comprising SEQ ID NO:21.
2. The recombinant replication competent retrovirus of claim 1 , wherein the virus is capable of infecting a target cell multiple times resulting in an average number of copies/diploid genome of 5 or greater.
3. The retrovirus of claim 1 , wherein the retroviral polynucleotide sequence is derived 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).
4. The retrovirus of claim 1 , wherein the retroviral envelope is an amphotropic MLV envelope.
5. The retrovirus of claim 1 , wherein the retrovirus is a gammaretrovirus.
6. The retrovirus of claim 1 , wherein the promoter comprises a CMV promoter having a sequence as set forth in SEQ ID NO:19, 20 or 22 from nucleotide 1 to about nucleotide 582.
7. The retrovirus of claim 1 , wherein the promoter comprises a CMV-R-U5 domain polynucleotide.
8. The retrovirus of claim 7 , wherein the CMV-R-U5 domain comprises the immediately early promoter from human cytomegalovirus linked to an MLV R-U5 region.
9. The retrovirus of claim 8 , wherein the CMV-R-U5 domain polynucleotide comprises a sequence as set forth in SEQ ID NO:19, 20 or 22 from about nucleotide 1 to about nucleotide 1202 or sequences that are at least 99% identical to a sequence as set forth in SEQ ID NO:19, 20 or 22, wherein the polynucleotide promotes transcription of a nucleic acid molecule operably linked thereto.
10. The retrovirus of claim 1 , wherein the gag polynucleotide is derived from a gammaretrovirus.
11. The retrovirus of claim 10 , wherein the gag nucleic acid domain comprises a sequence from about nucleotide number 1203 to about nucleotide 2819 of SEQ ID NO: 19 or 22 or a sequence having at least 99% or 99.8% identity thereto.
12. The retrovirus of claim 1 , wherein the pol domain of the polynucleotide is derived from a gammaretrovirus.
13. The retrovirus of claim 12 , wherein the pol domain comprises a sequence from about nucleotide number 2820 to about nucleotide 6358 of SEQ ID NO:19 or 22 or a sequence having at least 99% or 99.9% identity thereto.
14. The retrovirus of claim 1 , wherein the env domain comprises a sequence from about nucleotide number 6359 to about nucleotide 8323 of SEQ ID NO:19 or 22 or a sequence having at least 99% or 99.8% identity thereto.
15. The retrovirus of claim 1 , wherein the IRES is derived from an encephalomyocarditis virus.
16. The retrovirus of claim 15 , wherein the IRES comprises a sequence from about nucleotide number 8327 to about nucleotide 8876 of SEQ ID NO:19 or 22 or a sequence having at least 99% identity thereto.
17. The retrovirus of claim 1 , wherein the heterologous polynucleotide comprises a polynucleotide having a sequence as set forth in SEQ ID NO:3, 5, 11, 13, 15 or 17.
18. The retrovirus of claim 1 , wherein the heterologous polynucleotide encodes a polypeptide comprising a sequence as set forth in SEQ ID NO:4.
19. The retrovirus of claim 1 , wherein the heterologous polynucleotide is human codon optimized and encodes a polypeptide as set forth in SEQ ID NO:4.
20. The retrovirus of claim 1 , wherein the heterologous polynucleotide comprises a sequence as set forth in SEQ ID NO: 19 or 22 from about nucleotide number 8877 to about 9353.
21. The retrovirus of claim 1 , wherein the 3′ LTR is derived from a gammaretrovirus.
22. The retrovirus of claim 1 , wherein the retroviral polynucleotide comprises a sequence as set forth in SEQ ID NO:19, 20 or 22.
23. The retrovirus of claim 22 , wherein the 3′ LTR comprises a U3-R-U5 domain.
24. The retrovirus of claim 23 , wherein the 3′ LTR comprises a sequence as set forth in SEQ ID NO:19 or 22 from about nucleotide 9405 to about 9998 or a sequence that is at least 99.5% identical thereto.
25. The retrovirus of claim 1 , wherein the heterologous polynucleotide encodes an immunopotentiating cytokine.
26. The retrovirus according to claim 25 , 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).
27. The retrovirus according to claim 1 , wherein the heterologous polynucleotide encodes a polypeptide that converts a nontoxic prodrug into a toxic drug.
28. The retrovirus according to claim 27 , wherein the polypeptide that converts a nontoxic prodrug into a toxic drug is thymidine kinase, purine nucleoside phosphorylase (PNP), or cytosine deaminase.
29. The retrovirus according to claim 1 , wherein the heterologous polynucleotide encodes a receptor domain, an antibody, or antibody fragment.
30. The retrovirus according to claim 1 , wherein the heterologous polynucleotide comprises an inhibitory polynucleotide.
31. The retrovirus according to claim 30 , wherein the inhibitory polynucleotide comprises an miRNA, RNAi or siRNA sequence.
32. The retrovirus of claim 1 , wherein the heterologous polynucleotide has been human codon optimized.
33. The retrovirus of claim 1 , wherein said retrovirus further comprises an miRNA, RNAi or siRNA sequence.