Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1997 Nov;71(11):8902–8907. doi: 10.1128/jvi.71.11.8902-8907.1997

Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication.

J E Nelson 1, M A Kay 1
PMCID: PMC192362  PMID: 9343256

Abstract

Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.

Full Text

The Full Text of this article is available as a PDF (304.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. 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]
  3. Brough D. E., Lizonova A., Hsu C., Kulesa V. A., Kovesdi I. A gene transfer vector-cell line system for complete functional complementation of adenovirus early regions E1 and E4. J Virol. 1996 Sep;70(9):6497–6501. doi: 10.1128/jvi.70.9.6497-6501.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dai Y., Schwarz E. M., Gu D., Zhang W. W., Sarvetnick N., Verma I. M. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1401–1405. doi: 10.1073/pnas.92.5.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Engelhardt J. F., Ye X., Doranz B., Wilson J. M. Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6196–6200. doi: 10.1073/pnas.91.13.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Feldman L. T., Imperiale M. J., Nevins J. R. Activation of early adenovirus transcription by the herpesvirus immediate early gene: evidence for a common cellular control factor. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4952–4956. doi: 10.1073/pnas.79.16.4952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Gao G. P., Yang Y., Wilson J. M. Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J Virol. 1996 Dec;70(12):8934–8943. doi: 10.1128/jvi.70.12.8934-8943.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Hardy S., Kitamura M., Harris-Stansil T., Dai Y., Phipps M. L. Construction of adenovirus vectors through Cre-lox recombination. J Virol. 1997 Mar;71(3):1842–1849. doi: 10.1128/jvi.71.3.1842-1849.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heffelfinger S. C., Hawkins H. H., Barrish J., Taylor L., Darlington G. J. SK HEP-1: a human cell line of endothelial origin. In Vitro Cell Dev Biol. 1992 Feb;28A(2):136–142. doi: 10.1007/BF02631017. [DOI] [PubMed] [Google Scholar]
  14. Imperiale M. J., Feldman L. T., Nevins J. R. Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element. Cell. 1983 Nov;35(1):127–136. doi: 10.1016/0092-8674(83)90215-5. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Isom I., Georgoff I., Salditt-Georgieff M., Darnell J. E., Jr Persistence of liver-specific messenger RNA in cultured hepatocytes: different regulatory events for different genes. J Cell Biol. 1987 Dec;105(6 Pt 2):2877–2885. doi: 10.1083/jcb.105.6.2877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Kochanek S., Clemens P. R., Mitani K., Chen H. H., Chan S., Caskey C. T. A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5731–5736. doi: 10.1073/pnas.93.12.5731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kozarsky K. F., Wilson J. M. Gene therapy: adenovirus vectors. Curr Opin Genet Dev. 1993 Jun;3(3):499–503. doi: 10.1016/0959-437x(93)90126-a. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kumar-Singh R., Chamberlain J. S. Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells. Hum Mol Genet. 1996 Jul;5(7):913–921. doi: 10.1093/hmg/5.7.913. [DOI] [PubMed] [Google Scholar]
  23. Kwoh T. J., Kwoh D. Y., McCue A. W., Davis G. R., Patrick D., Gingeras T. R. Introduction and expression of the bacterial PaeR7 methylase gene in mammalian cells. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7713–7717. doi: 10.1073/pnas.83.20.7713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lieber A., He C. Y., Kirillova I., Kay M. A. Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo. J Virol. 1996 Dec;70(12):8944–8960. doi: 10.1128/jvi.70.12.8944-8960.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Nakabayashi H., Taketa K., Miyano K., Yamane T., Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982 Sep;42(9):3858–3863. [PubMed] [Google Scholar]
  28. Parks R. J., Chen L., Anton M., Sankar U., Rudnicki M. A., Graham F. L. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13565–13570. doi: 10.1073/pnas.93.24.13565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Schaack J., Maguire H. F., Siddiqui A. Hepatitis B virus X protein partially substitutes for E1A transcriptional function during adenovirus infection. Virology. 1996 Feb 15;216(2):425–430. doi: 10.1006/viro.1996.0079. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Spessot R., Inchley K., Hupel T. M., Bacchetti S. Cloning of the herpes simplex virus ICP4 gene in an adenovirus vector: effects on adenovirus gene expression and replication. Virology. 1989 Feb;168(2):378–387. doi: 10.1016/0042-6822(89)90279-1. [DOI] [PubMed] [Google Scholar]
  34. Tremblay M. L., Yee S. P., Persson R. H., Bacchetti S., Smiley J. R., Branton P. E. Activation and inhibition of expression of the 72,000-Da early protein of adenovirus type 5 in mouse cells constitutively expressing an immediate early protein of herpes simplex virus type 1. Virology. 1985 Jul 15;144(1):35–45. doi: 10.1016/0042-6822(85)90302-2. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Wang Q., Finer M. H. Second-generation adenovirus vectors. Nat Med. 1996 Jun;2(6):714–716. doi: 10.1038/nm0696-714. [DOI] [PubMed] [Google Scholar]
  37. Weinberg D. H., Ketner G. A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5383–5386. doi: 10.1073/pnas.80.17.5383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Worgall S., Wolff G., Falck-Pedersen E., Crystal R. G. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum Gene Ther. 1997 Jan 1;8(1):37–44. doi: 10.1089/hum.1997.8.1-37. [DOI] [PubMed] [Google Scholar]
  39. Yang Y., Ertl H. C., Wilson J. M. MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity. 1994 Aug;1(5):433–442. doi: 10.1016/1074-7613(94)90074-4. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Yeh P., Dedieu J. F., Orsini C., Vigne E., Denefle P., Perricaudet M. Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit. J Virol. 1996 Jan;70(1):559–565. doi: 10.1128/jvi.70.1.559-565.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES