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Journal of Virology logoLink to Journal of Virology
. 1996 Dec;70(12):8944–8960. doi: 10.1128/jvi.70.12.8944-8960.1996

Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo.

A Lieber 1, C Y He 1, I Kirillova 1, M A Kay 1
PMCID: PMC190992  PMID: 8971024

Abstract

In vivo gene transfer of recombinant E1-deficient adenoviruses results in early and late viral gene expression that elicits a host immune response, limiting the duration of transgene expression and the use of adenoviruses for gene therapy. The prokaryotic Cre-lox P recombination system was adapted to generate recombinant adenoviruses with extended deletions in the viral genome (referred to here as deleted viruses) in order to minimize expression of immunogenic and/or cytotoxic viral proteins. As an example, an adenovirus with a 25-kb deletion that lacked E1, E2, E3, and late gene expression with viral titers similar to those achieved with first-generation vectors and less than 0.5% contamination with E1-deficient virus was produced. Gene transfer was similar in HeLa cells, mouse hepatoma cells, and primary mouse hepatocytes in vitro and in vivo as determined by measuring reporter gene expression and DNA transfer. However, transgene expression and deleted viral DNA concentrations were not stable and declined to undetectable levels much more rapidly than those found for first-generation vectors. Intravenous administration of deleted vectors in mice resulted in no hepatocellular injury relative to that seen with first-generation vectors. The mechanism for stability of first-generation adenovirus vectors (E1a deleted) appeared to be linked in part to their ability to replicate in transduced cells in vivo and in vitro. Furthermore, the deleted vectors were stabilized in the presence of undeleted first-generation adenovirus vectors. These results have important consequences for the development of these and other nonintegrating vectors for gene therapy.

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Selected References

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  1. Akira S., Isshiki H., Sugita T., Tanabe O., Kinoshita S., Nishio Y., Nakajima T., Hirano T., Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990 Jun;9(6):1897–1906. doi: 10.1002/j.1460-2075.1990.tb08316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armentano D., Sookdeo C. C., Hehir K. M., Gregory R. J., St George J. A., Prince G. A., Wadsworth S. C., Smith A. E. Characterization of an adenovirus gene transfer vector containing an E4 deletion. Hum Gene Ther. 1995 Oct;6(10):1343–1353. doi: 10.1089/hum.1995.6.10-1343. [DOI] [PubMed] [Google Scholar]
  3. Barr D., Tubb J., Ferguson D., Scaria A., Lieber A., Wilson C., Perkins J., Kay M. A. Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: comparisons between immunocompetent and immunodeficient inbred strains. Gene Ther. 1995 Mar;2(2):151–155. [PubMed] [Google Scholar]
  4. Berkner K. L. Development of adenovirus vectors for the expression of heterologous genes. Biotechniques. 1988 Jul-Aug;6(7):616–629. [PubMed] [Google Scholar]
  5. Bett A. J., Haddara W., Prevec L., Graham F. L. An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8802–8806. doi: 10.1073/pnas.91.19.8802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boucher R. C. Current status of CF gene therapy. Trends Genet. 1996 Mar;12(3):81–84. doi: 10.1016/0168-9525(96)81410-7. [DOI] [PubMed] [Google Scholar]
  7. Brody S. L., Metzger M., Danel C., Rosenfeld M. A., Crystal R. G. Acute responses of non-human primates to airway delivery of an adenovirus vector containing the human cystic fibrosis transmembrane conductance regulator cDNA. Hum Gene Ther. 1994 Jul;5(7):821–836. doi: 10.1089/hum.1994.5.7-821. [DOI] [PubMed] [Google Scholar]
  8. Crystal R. G., McElvaney N. G., Rosenfeld M. A., Chu C. S., Mastrangeli A., Hay J. G., Brody S. L., Jaffe H. A., Eissa N. T., Danel C. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat Genet. 1994 Sep;8(1):42–51. doi: 10.1038/ng0994-42. [DOI] [PubMed] [Google Scholar]
  9. Daniell E., Groff D. E., Fedor M. J. Adenovirus chromatin structure at different stages of infection. Mol Cell Biol. 1981 Dec;1(12):1094–1105. doi: 10.1128/mcb.1.12.1094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dooley T. P., Miranda M., Jones N. C., DePamphilis M. L. Transactivation of the adenovirus EIIa promoter in the absence of adenovirus E1A protein is restricted to mouse oocytes and preimplantation embryos. Development. 1989 Dec;107(4):945–956. doi: 10.1242/dev.107.4.945. [DOI] [PubMed] [Google Scholar]
  11. Duncan S. J., Gordon F. C., Gregory D. W., McPhie J. L., Postlethwaite R., White R., Willcox H. N. Infection of mouse liver by human adenovirus type 5. J Gen Virol. 1978 Jul;40(1):45–61. doi: 10.1099/0022-1317-40-1-45. [DOI] [PubMed] [Google Scholar]
  12. Déry C. V., Toth M., Brown M., Horvath J., Allaire S., Weber J. M. The structure of adenovirus chromatin in infected cells. J Gen Virol. 1985 Dec;66(Pt 12):2671–2684. doi: 10.1099/0022-1317-66-12-2671. [DOI] [PubMed] [Google Scholar]
  13. Engelhardt J. F., Simon R. H., Yang Y., Zepeda M., Weber-Pendleton S., Doranz B., Grossman M., Wilson J. M. Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: biological efficacy study. Hum Gene Ther. 1993 Dec;4(6):759–769. doi: 10.1089/hum.1993.4.6-759. [DOI] [PubMed] [Google Scholar]
  14. Engelhardt J. F., Yang Y., Stratford-Perricaudet L. D., Allen E. D., Kozarsky K., Perricaudet M., Yankaskas J. R., Wilson J. M. Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses. Nat Genet. 1993 May;4(1):27–34. doi: 10.1038/ng0593-27. [DOI] [PubMed] [Google Scholar]
  15. Fang B., Wang H., Gordon G., Bellinger D. A., Read M. S., Brinkhous K. M., Woo S. L., Eisensmith R. C. Lack of persistence of E1- recombinant adenoviral vectors containing a temperature-sensitive E2A mutation in immunocompetent mice and hemophilia B dogs. Gene Ther. 1996 Mar;3(3):217–222. [PubMed] [Google Scholar]
  16. Fisher K. J., Choi H., Burda J., Chen S. J., Wilson J. M. Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology. 1996 Mar 1;217(1):11–22. doi: 10.1006/viro.1996.0088. [DOI] [PubMed] [Google Scholar]
  17. Fredman J. N., Engler J. A. Adenovirus precursor to terminal protein interacts with the nuclear matrix in vivo and in vitro. J Virol. 1993 Jun;67(6):3384–3395. doi: 10.1128/jvi.67.6.3384-3395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ginsberg H. S., Horswood R. L., Chanock R. M., Prince G. A. Role of early genes in pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6191–6195. doi: 10.1073/pnas.87.16.6191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ginsberg H. S., Moldawer L. L., Sehgal P. B., Redington M., Kilian P. L., Chanock R. M., Prince G. A. A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1651–1655. doi: 10.1073/pnas.88.5.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gorziglia M. I., Kadan M. J., Yei S., Lim J., Lee G. M., Luthra R., Trapnell B. C. Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy. J Virol. 1996 Jun;70(6):4173–4178. doi: 10.1128/jvi.70.6.4173-4178.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gu H., Zou Y. R., Rajewsky K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 1993 Jun 18;73(6):1155–1164. doi: 10.1016/0092-8674(93)90644-6. [DOI] [PubMed] [Google Scholar]
  22. Hitt M. M., Graham F. L. Adenovirus E1A under the control of heterologous promoters: wide variation in E1A expression levels has little effect on virus replication. Virology. 1990 Dec;179(2):667–678. doi: 10.1016/0042-6822(90)90134-d. [DOI] [PubMed] [Google Scholar]
  23. Hsu W., Chen-Kiang S. Convergent regulation of NF-IL6 and Oct-1 synthesis by interleukin-6 and retinoic acid signaling in embryonal carcinoma cells. Mol Cell Biol. 1993 Apr;13(4):2515–2523. doi: 10.1128/mcb.13.4.2515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Imler J. L., Chartier C., Dreyer D., Dieterle A., Sainte-Marie M., Faure T., Pavirani A., Mehtali M. Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors. Gene Ther. 1996 Jan;3(1):75–84. [PubMed] [Google Scholar]
  25. Imperiale M. J., Kao H. T., Feldman L. T., Nevins J. R., Strickland S. Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity. Mol Cell Biol. 1984 May;4(5):867–874. doi: 10.1128/mcb.4.5.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jones N., Shenk T. An adenovirus type 5 early gene function regulates expression of other early viral genes. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3665–3669. doi: 10.1073/pnas.76.8.3665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kay M. A., Graham F., Leland F., Woo S. L. Therapeutic serum concentrations of human alpha-1-antitrypsin after adenoviral-mediated gene transfer into mouse hepatocytes. Hepatology. 1995 Mar;21(3):815–819. [PubMed] [Google Scholar]
  28. Kay M. A., Holterman A. X., Meuse L., Gown A., Ochs H. D., Linsley P. S., Wilson C. B. Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat Genet. 1995 Oct;11(2):191–197. doi: 10.1038/ng1095-191. [DOI] [PubMed] [Google Scholar]
  29. Kay M. A., Li Q., Liu T. J., Leland F., Toman C., Finegold M., Woo S. L. Hepatic gene therapy: persistent expression of human alpha 1-antitrypsin in mice after direct gene delivery in vivo. Hum Gene Ther. 1992 Dec;3(6):641–647. doi: 10.1089/hum.1992.3.6-641. [DOI] [PubMed] [Google Scholar]
  30. Krougliak V., Graham F. L. Development of cell lines capable of complementing E1, E4, and protein IX defective adenovirus type 5 mutants. Hum Gene Ther. 1995 Dec;6(12):1575–1586. doi: 10.1089/hum.1995.6.12-1575. [DOI] [PubMed] [Google Scholar]
  31. La Thangue N. B., Rigby P. W. An adenovirus E1A-like transcription factor is regulated during the differentiation of murine embryonal carcinoma stem cells. Cell. 1987 May 22;49(4):507–513. doi: 10.1016/0092-8674(87)90453-3. [DOI] [PubMed] [Google Scholar]
  32. Li Q., Kay M. A., Finegold M., Stratford-Perricaudet L. D., Woo S. L. Assessment of recombinant adenoviral vectors for hepatic gene therapy. Hum Gene Ther. 1993 Aug;4(4):403–409. doi: 10.1089/hum.1993.4.4-403. [DOI] [PubMed] [Google Scholar]
  33. Lieber A., Kay M. A. Adenovirus-mediated expression of ribozymes in mice. J Virol. 1996 May;70(5):3153–3158. doi: 10.1128/jvi.70.5.3153-3158.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lieber A., Vrancken Peeters M. J., Kay M. A. Adenovirus-mediated transfer of the amphotropic retrovirus receptor cDNA increases retroviral transduction in cultured cells. Hum Gene Ther. 1995 Jan;6(1):5–11. doi: 10.1089/hum.1995.6.1-5. [DOI] [PubMed] [Google Scholar]
  35. Matsumoto K., Okuwaki M., Kawase H., Handa H., Hanaoka F., Nagata K. Stimulation of DNA transcription by the replication factor from the adenovirus genome in a chromatin-like structure. J Biol Chem. 1995 Apr 21;270(16):9645–9650. doi: 10.1074/jbc.270.16.9645. [DOI] [PubMed] [Google Scholar]
  36. McCoy R. D., Davidson B. L., Roessler B. J., Huffnagle G. B., Janich S. L., Laing T. J., Simon R. H. Pulmonary inflammation induced by incomplete or inactivated adenoviral particles. Hum Gene Ther. 1995 Dec;6(12):1553–1560. doi: 10.1089/hum.1995.6.12-1553. [DOI] [PubMed] [Google Scholar]
  37. Mitani K., Graham F. L., Caskey C. T., Kochanek S. Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3854–3858. doi: 10.1073/pnas.92.9.3854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Murphy W. J., Kumar V., Bennett M. Immunobiology of bone marrow transplantation: studies using scid mice. Curr Top Microbiol Immunol. 1989;152:251–258. doi: 10.1007/978-3-642-74974-2_30. [DOI] [PubMed] [Google Scholar]
  39. Phelps W. C., Yee C. L., Münger K., Howley P. M. The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E1A. Cell. 1988 May 20;53(4):539–547. doi: 10.1016/0092-8674(88)90570-3. [DOI] [PubMed] [Google Scholar]
  40. Poli V., Mancini F. P., Cortese R. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell. 1990 Nov 2;63(3):643–653. doi: 10.1016/0092-8674(90)90459-r. [DOI] [PubMed] [Google Scholar]
  41. Rosenfeld M. A., Yoshimura K., Trapnell B. C., Yoneyama K., Rosenthal E. R., Dalemans W., Fukayama M., Bargon J., Stier L. E., Stratford-Perricaudet L. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 1992 Jan 10;68(1):143–155. doi: 10.1016/0092-8674(92)90213-v. [DOI] [PubMed] [Google Scholar]
  42. Sauer B., Henderson N. Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome. Nucleic Acids Res. 1989 Jan 11;17(1):147–161. doi: 10.1093/nar/17.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sauer B., Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5166–5170. doi: 10.1073/pnas.85.14.5166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sauer B. Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol. 1993;225:890–900. doi: 10.1016/0076-6879(93)25056-8. [DOI] [PubMed] [Google Scholar]
  45. Schneider-Gädicke A., Schwarz E. Different human cervical carcinoma cell lines show similar transcription patterns of human papillomavirus type 18 early genes. EMBO J. 1986 Sep;5(9):2285–2292. doi: 10.1002/j.1460-2075.1986.tb04496.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  47. Spergel J. M., Chen-Kiang S. Interleukin 6 enhances a cellular activity that functionally substitutes for E1A protein in transactivation. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6472–6476. doi: 10.1073/pnas.88.15.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Spergel J. M., Hsu W., Akira S., Thimmappaya B., Kishimoto T., Chen-Kiang S. NF-IL6, a member of the C/EBP family, regulates E1A-responsive promoters in the absence of E1A. J Virol. 1992 Feb;66(2):1021–1030. doi: 10.1128/jvi.66.2.1021-1030.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sternberg N., Hamilton D. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol. 1981 Aug 25;150(4):467–486. doi: 10.1016/0022-2836(81)90375-2. [DOI] [PubMed] [Google Scholar]
  50. Sussenbach J. S., van der Vliet P. C. Studies on the mechanism of replication of adenovirus DNA. I. The effect of hydroxyurea. Virology. 1973 Jul;54(1):299–303. doi: 10.1016/0042-6822(73)90142-6. [DOI] [PubMed] [Google Scholar]
  51. Vrancken Peeters M. J., Perkins A. L., Kay M. A. Method for multiple portal vein infusions in mice: quantitation of adenovirus-mediated hepatic gene transfer. Biotechniques. 1996 Feb;20(2):278–285. doi: 10.2144/96202rr05. [DOI] [PubMed] [Google Scholar]
  52. Wang Q., Jia X. C., Finer M. H. A packaging cell line for propagation of recombinant adenovirus vectors containing two lethal gene-region deletions. Gene Ther. 1995 Dec;2(10):775–783. [PubMed] [Google Scholar]
  53. Wegner M., Cao Z., Rosenfeld M. G. Calcium-regulated phosphorylation within the leucine zipper of C/EBP beta. Science. 1992 Apr 17;256(5055):370–373. doi: 10.1126/science.256.5055.370. [DOI] [PubMed] [Google Scholar]
  54. Wong M. L., Hsu M. T. Linear adenovirus DNA is organized into supercoiled domains in virus particles. Nucleic Acids Res. 1989 May 11;17(9):3535–3550. doi: 10.1093/nar/17.9.3535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wong M. L., Hsu M. T. Psoralen-cross-linking study of the organization of intracellular adenovirus nucleoprotein complexes. J Virol. 1988 Apr;62(4):1227–1234. doi: 10.1128/jvi.62.4.1227-1234.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wrighton C. J., Hofer-Warbinek R., Moll T., Eytner R., Bach F. H., de Martin R. Inhibition of endothelial cell activation by adenovirus-mediated expression of I kappa B alpha, an inhibitor of the transcription factor NF-kappa B. J Exp Med. 1996 Mar 1;183(3):1013–1022. doi: 10.1084/jem.183.3.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yang Y., Li Q., Ertl H. C., Wilson J. M. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol. 1995 Apr;69(4):2004–2015. doi: 10.1128/jvi.69.4.2004-2015.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yang Y., Nunes F. A., Berencsi K., Furth E. E., Gönczöl E., Wilson J. M. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4407–4411. doi: 10.1073/pnas.91.10.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yang Y., Nunes F. A., Berencsi K., Gönczöl E., Engelhardt J. F., Wilson J. M. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet. 1994 Jul;7(3):362–369. doi: 10.1038/ng0794-362. [DOI] [PubMed] [Google Scholar]
  60. Yang Y., Trinchieri G., Wilson J. M. Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nat Med. 1995 Sep;1(9):890–893. doi: 10.1038/nm0995-890. [DOI] [PubMed] [Google Scholar]
  61. Zabner J., Couture L. A., Gregory R. J., Graham S. M., Smith A. E., Welsh M. J. Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic fibrosis. Cell. 1993 Oct 22;75(2):207–216. doi: 10.1016/0092-8674(93)80063-k. [DOI] [PubMed] [Google Scholar]
  62. Zhou H., O'Neal W., Morral N., Beaudet A. L. Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted. J Virol. 1996 Oct;70(10):7030–7038. doi: 10.1128/jvi.70.10.7030-7038.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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