Preparation of Helper-Dependent Adenoviral Vectors

  • Philip Ng
  • Robin J. Parks
  • Frank L. Graham
Part of the Methods in Molecular Medicine book series (MIMM, volume 69)


Adenoviruses (Ads) are excellent mammalian gene transfer vectors because of their ability to infect efficiently a wide variety of quiescent and proliferating cell types from various species to direct high-level gene expression. Consequently, Ad vectors are extensively used as potential recombinant viral vaccines, for high-level protein production in cultured cells and for gene therapy (1, 2, 3, 4). First-generation Ad vectors typically have foreign DNA inserted in place of early region 1 (E1). E1-deleted vectors are replication deficient and are propagated in E1-complementing cells such as 293 (5). Although these vectors remain very useful for many applications, it has become clear that transgene expression in vivo is only transient. Several factors contribute to this, including strong innate and inflammatory responses to the vector (6,7), acute and chronic toxicity caused by low-level viral gene expression from the vector backbone (8), and generation of anti-Ad cytotoxic T-lymphocytes caused by de novo viral gene expression (9, 10, 11, 12) or processing of virion proteins (13). Although high-level transient transgene expression afforded by first-generation Ad vectors may be adequate, or even desirable, for many gene transfer and gene therapy applications, the transient nature of expression kinetics renders these vectors unsuitable when prolonged, stable expression is required.


Serial Passage Helper Virus Packaging Signal Southern Blot Hybridization Analysis Vector Titer 
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  1. 1.
    Berkner K. L. (1988) Development of adenovirus vectors for expression of heterologous genes. Biotechniques 6, 616–629.PubMedCrossRefGoogle Scholar
  2. 2.
    Graham F. L. and Prevec L. (1992) Adenovirus-based expression vectors and recombinant vaccines, in Vaccines: New Approaches to Immunological Problems (Ellis R. W., ed.), Butterworth-Heinemann, Boston, pp. 363–389.Google Scholar
  3. 3.
    Hitt M., Addison C. L., and Graham F. L. (1997) Human adenovirus vectors for gene transfer into mammalian cells. Adv. Pharmacol. 40, 137–206.PubMedCrossRefGoogle Scholar
  4. 4.
    Hitt M. M., Parks R. J., and Graham F. L. (1999) Structure and genetic organization of adenovirus vectors, in The Development of Human Gene Therapy (Friedman T., ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 61–86.Google Scholar
  5. 5.
    Graham F. L., Smiley J., Russell W. C., and Nairn R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus 5. J. Gen. Virol. 36, 59–72.PubMedCrossRefGoogle Scholar
  6. 6.
    Wolf G., Worgall S., Van R. N., Song W. R., Harvey B. G., and Crystal R. G. (1997) Enhancement of in vivo adeno virus-mediated gene transfer and expression by prior depletion of tissue macrophages in the target organ. J. Virol. 71, 624–629.Google Scholar
  7. 7.
    Worgall S., Wolff G., Falck-Pedersen E., and Crystal R. G. (1997) Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum. Gene Ther. 8, 37–44.PubMedCrossRefGoogle Scholar
  8. 8.
    Morral N., O’Neal W., Zhou H., Langston C., and Beaudet A. (1997) Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors: comparison of E2a wildtype and E2a deleted vectors. Hum. Gene Ther. 8, 1275–1286.PubMedCrossRefGoogle Scholar
  9. 9.
    Dai Y., Schwartz E. M., Gu D., et al. (1995) 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. USA 92, 1401–1405.PubMedCrossRefGoogle Scholar
  10. 10.
    Yang Y., Nunes F. A., Berencsi K., et al. (1994) Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. USA 91, 4407–4411.PubMedCrossRefGoogle Scholar
  11. 11.
    Yang Y., Li Q., Ertl H. C., and Wilson J. M. (1995) Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69, 2004–2015.PubMedGoogle Scholar
  12. 12.
    Yang Y., Xiang Z., Ertl H. C., and Wilson J. M. (1995) Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo. Proc. Natl. Acad. Sci. USA 92, 7257–7261.PubMedCrossRefGoogle Scholar
  13. 13.
    Kafri T., Morgan D., Krahl T., et al. (1998) Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy. Proc. Natl. Acad. Sci. USA 95, 11377–11382.PubMedCrossRefGoogle Scholar
  14. 14.
    Chen H-H., Mack L. M., Kelly R., et al. (1997) Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc. Natl. Acad. Sci. USA 94, 1645–1650.PubMedCrossRefGoogle Scholar
  15. 15.
    Morral. N., Parks R. J., Zhou H., et al. (1998) High doses of a helper-dependent adenoviral vector yield supraphysiological levels of α1-antitrypsin with negligible toxicity. Hum. Gene Ther. 9, 2709–2716.PubMedCrossRefGoogle Scholar
  16. 16.
    Morsy M. A., Gu M., Motzel S., et al. (1998) An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc. Natl. Acad. Sci. USA 95, 7866–7871.PubMedCrossRefGoogle Scholar
  17. 17.
    Schiedner G., Morral N., Parks R. J., et al. (1998) Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat. Genet. 18, 180–183.PubMedCrossRefGoogle Scholar
  18. 18.
    Morral N., O’Neal W., Rice K., et al. (1999) Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver directed gene transfer in baboons. Proc. Natl. Acad. Sci. USA 96, 12816–12821.PubMedCrossRefGoogle Scholar
  19. 19.
    Balague C., Zhou J., Dai Y. et al. (2000) Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 95, 820–828.PubMedGoogle Scholar
  20. 20.
    Cregan S. P., MacLaurin J., Gendron T. F., et al. (2000) Helper-dependent adenovirus vectors: their use as a gene delivery system to neurons. Gene Ther. 14, 1200–1209.CrossRefGoogle Scholar
  21. 21.
    Maione D., Wiznerowicz M., Delmastro P., et al. (2000) Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector. Hum. Gene Ther. 11, 859–868.PubMedCrossRefGoogle Scholar
  22. 22.
    Kochanek S. (1999) High-capacity adenoviral vectors for gene transfer and somatic gene therapy. Hum. Gene Ther. 10, 2451–2459.PubMedCrossRefGoogle Scholar
  23. 23.
    Parks R. J. (2000) Improvements in adenoviral vector technology: overcoming barriers for gene therapy. Clin. Genet. 58, 1–11.PubMedCrossRefGoogle Scholar
  24. 24.
    Parks R. J., Chen L., et al. (1996) A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. USA 93, 13565–13570.PubMedCrossRefGoogle Scholar
  25. 25.
    Chen L., Anton M., and Graham F. L. (1996) Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre. Somat. Cell Mol. Genet. 22, 477–488.PubMedCrossRefGoogle Scholar
  26. 26.
    Sandig V., Youil R., Bett A. J., et al. (1999) Optimization of the helper-dependent adenovirus system for production and potency in vivo. Proc. Natl. Acad. Sci. USA 97, 1002–1007.CrossRefGoogle Scholar
  27. 27.
    Parks R. J. and Graham F. L. (1997) A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging. J. Virol. 71,3293–3298.PubMedGoogle Scholar
  28. 28.
    Bett A. J., Prevec L., and Graham F. L. (1993) Packaging capacity and stability of human adenovirus type 5 vectors. J. Virol. 67, 5911–5921.PubMedGoogle Scholar
  29. 29.
    Parks R. J., Bramson J. L., Wan Y., Addison C. L., and Graham F. L. (1999) Effects of stuffer DNA on transgene expression from helper-dependent adenovirus vectors. J. Virol. 73, 8027–8034.PubMedGoogle Scholar
  30. 30.
    Parks R. J., Evelegh C. M., and Graham F. L. (1999) Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration. Gene Ther. 6, 1565–1573.PubMedCrossRefGoogle Scholar
  31. 31.
    Maizel J. V., White D., and Scharff. M. D. (1968) The polypeptides of adenovirus. I. Evidence of multiple protein components in the virion and a comparison of types 2, 7a, and 12. Virology 36, 115–125.PubMedCrossRefGoogle Scholar
  32. 32.
    Graham F. L. and van der Eb A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456–467.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Philip Ng
    • 1
  • Robin J. Parks
    • 2
  • Frank L. Graham
    • 1
  1. 1.Deparment of BiologyMcMaster UniversityOntarioCanada
  2. 2.Ottawa Health Research InstituteOntarioCanada

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