Generation of Replication Competent Viruses for Therapeutic Use
The present invention relates to the generation or 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 at least one genetic element of interest and 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 8 , 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 further comprises an expression element, wherein said element is located upstream from, and is operably linked to, said genetic element of interest.
11 . The method of claim 10 , wherein said expression element is a eukaryotic promoter.
12 . The method of claim 11 , wherein said eukaryotic promoter is a CMV, SV40, thymidine kinase, ubiquitin or actin promoter.
13 . The method of claim 12 , wherein said expression element is a eukaryotic splice acceptor sequence.
14 . The method of claim 13 , wherein said splice acceptor sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
15 . The method of claim 1 , wherein said transposon is a transposon which employs an ATP-utilizing regulatory proteins.
16 . The method of claim 15 , wherein said transposon is a Tn-7 based transposon.
17 . The method of claim 1 , wherein said transposon further comprises a selectable gene.
18 . The method of claim 17 , wherein said selectable gene is an antibiotic resistance gene, a methotrexate resistance gene, or a G418 resistance gene.
19 . The method of claim 1 , wherein said donor DNA is a plasmid.
20 . The method of claim 19 , wherein said donor DNA further comprises an origin of replication.
21 . The method of claim 1 , wherein said target virus genomic DNA is present in a plasmid.
22 . The method of claim 21 , wherein said target virus genomic DNA is flanked by restriction enzyme sites.
23 . The method of claim 22 , wherein said restriction enzyme sites are identical.
24 . 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 at least one genetic element of interest operably linked to an expression element and a selectable gene and 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.
25 . The method of claim 24 , wherein said isolation step (b) comprises assaying for expression of said genetic element of interest in animal cells transfected with said replication competent viruses
26 . The method of claim 24 , wherein said target virus genomic DNA is an animal virus.
27 . The method of claim 26 , wherein said animal virus is oncolytic.
28 . The method of claim 27 , wherein said animal virus is an adenovirus, VSV, NDV, HSV, or vaccinia virus.
29 . The method of claim 28 , wherein said adenovirus is Ad5.
30 . The method of claim 24 , wherein said genetic element is a gene which encodes a reporter molecule, a therapeutic protein or an RNA molecule.
31 . The method of claim 30 , wherein said reporter gene encodes GFP, LacZ, renilla luciferase or firefly luciferase.
32 . The method of claim 31 , wherein said therapeutic protein is an immunomodulatory protein, an antibody, a symporter or a pro-drug converting enzyme.
33 . The method of claim 24 , wherein said expression element is a eukaryotic promoter or a eukaryotic splice acceptor sequence.
34 . The method of claim 33 , wherein said eukaryotic promoter is a CMV, SV40, thymidine kinase, ubiquitin or actin promoter.
35 . The method of claim 34 , wherein said eukaryotic promoter is the early SV40 promoter.
36 . The method of claim 33 , wherein said splice acceptor sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
37 . The method of claim 24 , wherein said transposon is transposon which employs an ATP-utilizing regulatory protein.
38 . The method of claim 37 , wherein said transposon is a Tn-7-based transposon.
39 . The method of claim 24 , wherein said selectable gene is an antibiotic resistance gene or a drug resistance gene
40 . The method of claim 24 , wherein said target virus genomic DNA is flanked by restriction enzyme sites.
41 . The method of claim 40 , wherein said restriction enzyme sites are identical.
42 . 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 gene encoding GFP operably linked to an expression element and a chloramphenicol resistance gene 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).
43 . The method of claim 42 , wherein said isolation step (b) comprises the steps of:
(a′) transforming bacterial cells with the mixture of said step (a);
(b′) isolating replication competent Ad5 viruses from said transformed cells;
(c′) transfecting eukaryotic cells with said Ad5 viruses; and
(d′) identifying plaques derived from plating of said cells from said step (c′).
44 . The method of claim 42 , wherein said expression element is a eukaryotic promoter or a eukaryotic splice acceptor sequence.
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 45 , wherein said eukaryotic promoter is an early SV40 promoter.
47 . The method of claim 44 , wherein said splice acceptor sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
48 . A replication competent virus containing a functional insertion site identified by the method of claim 1 , claim 24 , or claim 42 .
49 . 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 genetic element of interest and 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.
50 . The method of claim 49 , wherein said target virus is an animal virus.
51 . The method of claim 50 , wherein said animal virus is oncolytic.
52 . The method of claim 51 , wherein said animal virus is adenovirus, VSV, NDV, HSV, or vaccinia virus.
53 . The method of claim 52 , wherein said adenovirus is Ad5.
54 . The method of claim 49 , wherein said genetic element is a gene which encodes a reporter molecule, a therapeutic protein or an RNA molecule.
55 . The method of claim 54 , wherein said reporter molecule is GFP, LacZ, renilla luciferase or firefly luciferase.
56 . The method of claim 54 , wherein said therapeutic protein is an immunomodulatory protein, an antibody, a symporter or a pro-drug converting enzyme.
57 . The method of claim 49 , 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.
58 . The method of claim 57 , wherein said expression element is a eukaryotic promoter.
59 . The method of claim 58 , wherein said eukaryotic promoter is a CMV, SV40, thymidine kinase, ubiquitin or actin promoter.
60 . The method of claim 57 , wherein said expression element is a eukaryotic splice acceptor sequence.
61 . The method of claim 60 , wherein said splice acceptor sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
62 . The method of claim 49 , wherein said transposon is a transposon which employs an ATP-utilizing regulatory proteins.
63 . The method of claim 62 , wherein said transposon is a Tn-7 based transposon.
64 . The method of claim 49 , wherein said transposon further comprises a selectable gene.
65 . The method of claim 64 , wherein said selectable gene is an antibiotic resistance gene, a methotrexate resistance gene, or a G418 resistance gene.
66 . The method of claim 49 , wherein said donor DNA is a plasmid.
67 . The method of claim 66 , wherein said donor DNA further comprises an origin of replication.
68 . The method of claim 49 , wherein said target virus genomic DNA is present in a plasmid.
69 . The method of claim 68 , wherein said target virus genomic DNA is flanked by restriction enzyme sites.
70 . The method of claim 69 , wherein said restriction enzyme sites are identical.