Complementary adenoviral systems and cell lines

• US 5,851,806 A
• Filed: 12/14/1995
• Issued: 12/22/1998
• Est. Priority Date: 06/10/1994
• Status: Expired due to Term

• Current Assignee: GenVec, Inc. (Precigen, Inc.)
• Original Assignee: GenVec, Inc. (Precigen, Inc.)
• Inventors: Bruder, Joseph T., Brough, Douglas E., Kovesdi, Imre, McVey, Duncan L., Lizonova, Alena
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/572,126
• Time in Patent Office: 1,104 Days
• Field of Search: 435/69.1, 435/91.1, 435/235.1, 435/246.1, 435/320.1, 435/91.41, 435/172.3, 435/325, 435/366, 536/23.1, 536/24.1, 536/24.2
• US Class Current: 435/91.41
• CPC Class Codes: A61K 2039/5256 expressing foreign proteins
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/4712 Cystic fibrosis
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/10352 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2810/60 from viruses
• CPC Class Codes: C12N 2810/6081 rhabdoviridae, e.g. VSV
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 2840/44 being a specific part of the splice mechanism, e.g. donor, acceptor
• 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

Abstract

The present invention provides multiply replication deficient adenoviral vectors having a spacer in at least one replication deficient adenoviral region, as well as complementing cell lines therefor. Also provided are means of constructing the multiply replication deficient adenoviral vectors and methods of use thereof, e.g., in gene therapy.

Claims

1. An adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome,, (b) a spacer of at least 15 base pairs in the E4 region of the adenoviral genome, and (c) a passenger gene, wherein said spacer comprises a sequence that is non-native to the E4 region of the adenoviral genome, and wherein the viral production level of said adenoviral vector is greater than the viral production level of said adenoviral vector without said spacer.

2. The adenoviral vector of claim 1, wherein the viral production level of said adenoviral vector without said spacer and without said passenger gene is lower than the viral production level of said adenoviral vector without said spacer, said passenger gene, and said deficiency in the E4 region of the adenoviral genome.

3. A host cell comprising the adenoviral vector of claim 1.

4. A composition comprising the adenoviral vector of claim 1, and a carrier therefor.

5. A replication competent adenovirus-free stock of the adenoviral vector of claim 1, wherein said spacer comprises said passenger gene, wherein said adenoviral vector is prepared in a cell line which will support the growth of said adenoviral vector, and wherein the genome of said cell line is free of overlapping sequences with said adenoviral vector such that said cell genome will not mediate a recombination event which would produce a replication competent adenoviral vector.

6. A method of genetically modifying a cell in vitro, which comprises contacting said cell with the adenoviral vector of claim 1.

7. The adenoviral vector of claim 1, wherein said adenoviral genome is deficient in one or more essential gene functions of the E4 region of the adenoviral genome.

8. The adenoviral vector of claim 7, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

9. The adenoviral vector of claim 8, wherein said another region is the E1 region of the adenoviral genome.

10. The adenoviral vector of claim 1, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome.

11. The adenoviral vector of claim 10, wherein said adenoviral genome is deficient in one or more other essential gene functions of another region of the adenoviral genome.

12. The adenoviral vector of claim 1, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome except the E4 promoter and the E4 polyadenylation sequence.

13. The adenoviral vector of claim 12, wherein said adenoviral genome is deficient in one or more other essential gene functions of another region of the adenoviral genome.

14. The adenoviral vector of claim 1, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome except the E4 polyadenylation sequence.

15. The adenoviral vector of claim 14, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

16. The adenoviral vector of claim 1, wherein said adenoviral genome is further deficient in the E2A region of the adenoviral genome.

17. The adenoviral vector of claim 16, wherein said E2A region comprises a portion of the adenoviral genome which encodes amino acids of the Nt domain of the DBP sufficient to allow for viral production, and wherein said E2A region is deficient in the portion of the adenoviral genome which encodes amino acid sequences of the Ct domain that are necessary for DNA binding.

18. The adenoviral vector of claim 1, wherein said spacer is about 100 to about 12,000 base pairs.

19. A method of genetically modifying a cell in vitro, which comprises contacting said cell with the adenoviral vector of claim 18.

20. The adenoviral vector of claim 18, wherein said spacer is about 500 to about 8,000 base pairs.

21. The adenoviral vector of claim 20, wherein said spacer is about 1,500 to about 6,000 base pairs.

22. The adenoviral vector of claim 1, wherein said spacer comprises a polyadenylation sequence other than an E4 adenoviral polyadenylation sequence.

23. The adenoviral vector of claim 22, wherein said spacer comprises an SV40 polyadenylation sequence.

24. A method of genetically modifying a cell in vitro, which comprises-contacting said cell with the adenoviral vector of claim 22.

25. A replication competent adenovirus-free stock of the adenoviral vector of any of claims 1, 2 and 7-17, wherein said spacer comprises a polyadenylation sequence other than an E4 adenoviral polyadenylation sequence.

26. The replication competent adenovirus-free stock of claim 25, wherein said spacer comprises an SV40 polyadenylation sequence.

27. A replication competent adenovirus-free stock of the adenoviral vector of claim 1, wherein said spacer comprises a polyadenylation sequence other than an E4 adenoviral polyadenylation sequence, wherein said adenoviral vector is prepared in a cell line which will support the growth of said adenoviral vector, and wherein the genome of said cell line is free of overlapping sequences with said adenoviral vector such that said cell genome will not mediate a recombination event which would produce a replication competent adenoviral vector.

28. The replication competent adenovirus-free stock of claim 27, wherein said spacer comprises an SV40 polyadenylation sequence.

29. A replication competent adenovirus-free stock of the adenoviral vector of any of claims 1, 2 and 7-17, wherein said spacer comprises a polyadenylation sequence other than an E4 adenoviral polyadenylation sequence, wherein said adenoviral vector is prepared in a cell line which will support the growth of said adenoviral vector.

30. The replication competent adenovirus-free stock of claim 29, wherein said spacer comprises an SV40 polyadenylation sequence.

31. A replication competent adenovirus-free stock of the adenoviral vector of any of claims 1, 2 and 7-17, wherein said spacer comprises said passenger gene, wherein said adenoviral vector is prepared in a cell line which will support the growth of said adenoviral vector.

32. A method of increasing the propagation in a complementing cell line of an adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region and (b) a passenger gene, which method comprises incorporating into the E4 region of the adenoviral genome of said adenoviral vector a spacer comprising at least about 15 base pairs, wherein said spacer comprises a sequence that is non-native to the E4 region of the adenoviral genome, thereby resulting in an increase in the viral production level of said adenoviral vector.

33. The method of claim 32, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

34. The method of claim 32, wherein said adenoviral genome is deficient in one or more essential gene functions of the E4 region of the adenoviral genome.

35. The method of claim 34, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

36. The method of claim 34, wherein said adenoviral genome is further deficient in at least one or more essential gene functions of the E1 region of the adenoviral genome.

37. The method of claim 32, wherein said adenoviral genome is deleted of all the open reading frames of the E4 region of the adenoviral genome.

38. The method of claim 37, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

39. The method of claim 32, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome except the E4 promoter and the E4 polyadenylation sequence.

40. The method of claim 39, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

41. The method of claim 39, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome except the E4 polyadenylation sequence.

42. The method of claim 32, wherein said spacer is about 100 to about 12,000 base pairs.

43. The method of claim 42, wherein said spacer is about 500 to about 8,000 base pairs.

44. The method of claim 42, wherein said spacer is about 1,500 to about 6,000 base pairs.

45. The method of claim 32, wherein said spacer comprises a polyadenylation sequence other than an E4 adenoviral polyadenylation sequence.

46. The method of claim 45, wherein said spacer comprises an SV40 polyadenylation sequence.

47. An adenoviral vector comprising a portion of the E2A region of the adenoviral genome which encodes amino acids of the Nt domain of the DBP sufficient to allow for viral production, and wherein said E2A region is deficient in the portion of the adenoviral genome which encodes amino acid sequences of the Ct domain that are necessary for DNA bindings and further wherein the adenoviral vector comprises a passenger gene.

48. The adenoviral vector of claim 47, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

49. An adenoviral vector comprising (a) an adenoviral genome deficient in the E4 region of the adenoviral genome, and (b) a spacer of at least 15 base pairs in the E4 region of the adenoviral genome, wherein said spacer comprises a passenger gene that is non-native to the E4 region of the adenoviral genome, and wherein the viral production level of said adenoviral vector is greater than the viral production level of said adenoviral vector without said spacer.

50. The adenoviral vector of claim 49, wherein said adenoviral genome is deficient in one or more essential gene functions of the E4 region of the adenoviral genome.

51. The adenoviral vector of claim 49, wherein the viral production level of said adenoviral vector without said spacer and without said passenger gene is lower than the viral production level of said adenoviral vector without said spacer, said passenger gene, and said deficiency in the E4 region of the adenoviral genome.

52. The adenoviral vector of claim 50, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

53. The adenoviral vector of claim 52, wherein said another region is the E1 region of the adenoviral genome.

54. The adenoviral vector of claim 52, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome.

55. The adenoviral vector of claim 54, wherein said adenoviral genome is deficient in one or more other essential gene functions of another region of the adenoviral genome.

56. The adenoviral vector of claim 52, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome except the E4 promoter and the E4 polyadenylation sequence.

57. The adenoviral vector of claim 56, wherein said adenoviral genome is deficient in one or more other essential gene functions of another region of the adenoviral genome.

58. The adenoviral vector of claim 49, wherein said adenoviral genome is deleted of the E4 region of the adenoviral genome except the E4 polyadenylation sequence.

59. The adenoviral vector of claim 50, wherein said adenoviral genome is deficient in one or more essential gene functions of another region of the adenoviral genome.

60. The adenoviral vector of claim 49, wherein said adenoviral genome is further deficient in the E2A region of the adenoviral genome.

61. The adenoviral vector of claim 60, wherein said E2A region comprises a portion of the adenoviral genome which encodes amino acids of the Nt domain of the DBP sufficient to allow for viral production, and wherein said E2A region is deficient in the portion of the adenoviral genome which encodes amino acid sequences of the Ct domain that are necessary for DNA binding.

62. A replication competent adenovirus-free stock of the adenoviral vector of any of claims 49-61.

63. A method of increasing the propagation in a complementing cell line of an adenoviral vector comprising an adenoviral genome deficient in the E4 region, which method comprises incorporating into the E4 region of the adenoviral genome of said adenoviral vector a spacer comprising at least about 15 base pairs, wherein said spacer comprises a passenger gene that is non-native to the E4 region of the adenoviral genome, thereby resulting in an increase in the viral production level of said adenoviral vector.