Generation of Replication Competent Viruses for Therapeutic Use

• US 20090208924A1
• Filed: 11/28/2005
• Published: 08/20/2009
• Est. Priority Date: 12/01/2004
• Status: Abandoned Application

• Current Assignee: PsiOxus Therapeutics Limited
• Original Assignee: Bayer Schering Pharma AG (Bayer AG)
• Inventors: Kretschmer, Peter, Hermiston, Terry, Jin, Fang
• Application Number:
  US11/791,856
• Publication Number:
  US 20090208924A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: A61K 35/13 Tumour cells, irrespective of tissue of origin tumour vaccines A61K39/00
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 48/0091 Purification or manufacturing processes for gene therapy compositions
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10321 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/10361 Methods of inactivation or attenuation
• CPC Class Codes: C12N 2770/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2800/90 Vectors containing a transposable element
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 1/701 Specific hybridization probes

Abstract

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.

Claims

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.

48. A replication competent virus containing a functional insertion site identified by the method of claim 1, claim 24, or claim 42.

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.

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.