Generation of replication competent viruses for therapeutic use
View Patent ↗The present invention relates to the generation of replication-competent viruses having therapeutic utility. The replication-competent viruses of the invention can express proteins useful in the treatment of disease.
1. A method of identifying functional insertion sites within the genome of a replication competent target virus, said method comprising the steps of:
(a) mixing the genomic DNA of said target virus with a donor DNA comprising a transposon under conditions that allow transposition, wherein said transposon comprises a eukaryotic splice acceptor sequence comprising SEQ ID NO:1, wherein the splice acceptor sequence is located upstream from and is operably linked to at least one genetic element of interest and wherein the transposon inserts into said viral genome in a non-biased manner; and
(b) isolating replication competent viruses from said step (a) which express said genetic element of interest.
2. The method of claim 1 , wherein said isolation step comprises assaying for expression of said genetic element of interest in animal cells transfected with said replication competent viruses.
3. The method of claim 1 , wherein said target virus is an animal virus.
4. The method of claim 3 , wherein said animal virus is oncolytic.
5. The method of claim 4 , wherein said animal virus is adenovirus, VSV, NDV, HSV, or vaccinia virus.
6. The method of claim 5 , wherein said adenovirus is Ad5.
7. The method of claim 1 , wherein said genetic element is a gene which encodes a reporter molecule, a therapeutic protein or an RNA molecule.
8. The method of claim 7 , wherein said reporter molecule is GFP, LacZ, renilla luciferase or firefly luciferase.
9. The method of claim 7 , wherein said therapeutic protein is an immunomodulatory protein, an antibody, a symporter or a pro-drug converting enzyme.
10. The method of claim 1 , wherein said transposon is a transposon which employs an ATP-utilizing regulatory proteins.
11. The method of claim 10 , wherein said transposon is a Tn-7 based transposon.
12. The method of claim 1 , wherein said transposon further comprises a selectable gene.
13. The method of claim 12 , wherein said selectable gene is an antibiotic resistance gene, a methotrexate resistance gene, or a G418 resistance gene.
14. The method of claim 1 , wherein said donor DNA is a plasmid.
15. The method of claim 14 , wherein said donor DNA further comprises an origin of replication.
16. The method of claim 1 , wherein said target virus genomic DNA is present in a plasmid.
17. The method of claim 16 , wherein said target virus genomic DNA is flanked by restriction enzyme sites.
18. The method of claim 17 , wherein said restriction enzyme sites are identical.
19. A method of identifying functional insertion sites within the genome of a replication competent target virus, said method comprising the steps of:
(a) mixing a plasmid comprising the genomic DNA of said target virus with a plasmid donor DNA comprising a transposon and an origin of replication under conditions that allow transposition, wherein said transposon comprises a selectable gene and at least one genetic element of interest operably linked to a eukaryotic splice acceptor sequence comprising SEQ ID NO:1, wherein the splice acceptor sequence is located upstream from the element, and wherein the transposon inserts into said target viral genome in a non-biased manner; and
(b) isolating replication competent viruses from said step (a) which express said genetic element of interest.
20. The method of claim 19 , wherein said isolation step (b) comprises assaying for expression of said genetic element of interest in animal cells transfected with said replication competent viruses.
21. The method of claim 19 , wherein said target virus genomic DNA is an animal virus.
22. The method of claim 21 , wherein said animal virus is oncolytic.
23. The method of claim 22 , wherein said animal virus is an adenovirus, VSV, NDV, HSV, or vaccinia virus.
24. The method of claim 23 , wherein said adenovirus is Ad5.
25. The method of claim 19 , wherein said genetic element is a gene which encodes a reporter molecule, a therapeutic protein or an RNA molecule.
26. The method of claim 25 , wherein said reporter gene encodes GFP, LacZ, renilla luciferase or firefly luciferase.
27. The method of claim 25 , wherein said therapeutic protein is an immunomodulatory protein, an antibody, a symporter or a pro-drug converting enzyme.
28. The method of claim 19 , wherein said transposon is transposon which employs an ATP-utilizing regulatory protein.
29. The method of claim 28 , wherein said transposon is transposon is a Tn-7-based transposon.
30. The method of claim 19 , wherein said selectable gene is an antibiotic resistance gene or a drug resistance gene.
31. The method of claim 19 , wherein said target virus genomic DNA is flanked by restriction enzyme sites.
32. The method of claim 31 , wherein said restriction enzyme sites are identical.
33. A method of identifying functional insertion sites within the genome of a replication competent Ad5 virus, said method comprising the steps of:
(a) mixing a plasmid comprising the genomic DNA of said Ad5 virus with a plasmid donor DNA comprising a Tn-7 based transposon and an R6K origin of replication under conditions that allow transposition, wherein said transposon comprises a chloramphenicol resistance gene and a gene encoding GFP operably linked to a eukaryotic splice acceptor sequence comprising SEQ ID NO:1, wherein the splice acceptor sequence is located upstream from the gene encoding GFP, and wherein said transposon inserts into said target Ad5 genome in a non-biased manner; and
(b) isolating replication competent Ad5 viruses which express GFP from said step (a).
34. The method of claim 33 , wherein said isolation step (b) comprises the steps of:
(a′) transforming bacterial cells with the mixture of said step (a) of claim 33 ;
(b′) isolating plasmid DNA from said transformed cells;
(c′) excising replication competent Ad5 viruses from said isolated plasmid DNA;
(d′) transfecting eukaryotic cells with said replication competent Ad5 viruses; and
(e′) identifying plaques derived from plating of said transfected cells from said step (d′).
35. A method of generating a replication competent virus comprising at least one genetic element of interest inserted into a functional insertion site within the genome of said virus, said method comprising the steps of:
(a) mixing the genomic DNA of a target replication competent virus with a donor DNA comprising a transposon, under conditions that allow transposition, wherein said transposon comprises a eukaryotic splice acceptor sequence comprising SEQ ID NO:1, wherein the splice acceptor sequence is located upstream from and is operably linked to at least one genetic element of interest and wherein the transposon inserts into said viral genome in a non-biased manner; and
(b) identifying product replication competent viruses formed in said step (a) which express said genetic element.
36. The method of claim 35 , wherein said target virus is an animal virus.
37. The method of claim 36 , wherein said animal virus is oncolytic.
38. The method of claim 37 , wherein said animal virus is adenovirus, VSV, NDV, HSV, or vaccinia virus.
39. The method of claim 38 , wherein said adenovirus is Ad5.
40. The method of claim 35 , wherein said genetic element is a gene which encodes a reporter molecule, a therapeutic protein or an RNA molecule.
41. The method of claim 40 , wherein said reporter molecule is GFP, LacZ, renilla luciferase or firefly luciferase.
42. The method of claim 40 , wherein said therapeutic protein is an immunomodulatory protein, an antibody, a symporter or a pro-drug converting enzyme.
43. The method of claim 35 , wherein said transposon further comprises an expression element, wherein said element is located upstream from, and is operably linked to, said genetic element of interest.
44. The method of claim 43 , wherein said expression element is a eukaryotic promoter.
45. The method of claim 44 , wherein said eukaryotic promoter is a CMV, SV40, thymidine kinase, ubiquitin or actin promoter.
46. The method of claim 35 , wherein said transposon is a transposon which employs an ATP-utilizing regulatory proteins.
47. The method of claim 46 , wherein said transposon is a Tn-7 based transposon.
48. The method of claim 35 , wherein said transposon further comprises a selectable gene.
49. The method of claim 48 , wherein said selectable gene is an antibiotic resistance gene, a methotrexate resistance gene, or a G418 resistance gene.
50. The method of claim 35 , wherein said donor DNA is a plasmid.
51. The method of claim 50 , wherein said donor DNA further comprises an origin of replication.
52. The method of claim 35 , wherein said target virus genomic DNA is present in a plasmid.
53. The method of claim 52 , wherein said target virus genomic DNA is flanked by restriction enzyme sites.
54. The method of claim 53 , wherein said restriction enzyme sites are identical.
55. A method of identifying functional insertion sites within the genome of a replication competent target virus, said method comprising the steps of:
(a) mixing the genomic DNA of said target virus with a donor DNA comprising a transposon under conditions that allow transposition, wherein said transposon comprises a eukaryotic splice acceptor sequence comprising SEQ ID NO:2, wherein the splice acceptor sequence is located upstream from and is operably linked to at least one genetic element of interest and wherein the transposon inserts into said viral genome in a non-biased manner; and
(b) isolating replication competent viruses from said step (a) which express said genetic element of interest.
56. A method of identifying functional insertion sites within the genome of a replication competent target virus, said method comprising the steps of:
(a) mixing a plasmid comprising the genomic DNA of said target virus with a plasmid donor DNA comprising a transposon and an origin of replication under conditions that allow transposition, wherein said transposon comprises a selectable gene and at least one genetic element of interest operably linked to a eukaryotic splice acceptor sequence comprising SEQ ID NO:2, wherein the splice acceptor sequence is located upstream from the element, and wherein the transposon inserts into said target viral genome in a non-biased manner; and
(b) isolating replication competent viruses from said step (a) which express said genetic element of interest.
57. A method of identifying functional insertion sites within the genome of a replication competent Ad5 virus, said method comprising the steps of:
(a) mixing a plasmid comprising the genomic DNA of said Ad5 virus with a plasmid donor DNA comprising a Tn-7 based transposon and an R6K origin of replication under conditions that allow transposition, wherein said transposon comprises a chloramphenicol resistance gene and a gene encoding GFP operably linked to a eukaryotic splice acceptor sequence comprising SEQ ID NO:2, wherein the splice acceptor sequence is located upstream from the gene encoding GFP, and wherein said transposon inserts into said target Ad5 genome in a non-biased manner; and
(b) isolating replication competent Ad5 viruses which express GFP from said step (a).
58. A method of generating a replication competent virus comprising at least one genetic element of interest inserted into a functional insertion site within the genome of said virus, said method comprising the steps of:
(a) mixing the genomic DNA of a target replication competent virus with a donor DNA comprising a transposon, under conditions that allow transposition, wherein said transposon comprises a eukaryotic splice acceptor sequence comprising SEQ ID NO:2, wherein the splice acceptor sequence is located upstream from and is operably linked to at least one genetic element of interest and wherein the transposon inserts into said viral genome in a non-biased manner; and
(b) identifying product replication competent viruses formed in said step (a) which express said genetic element.