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. 1996 Dec;70(12):8459–8467. doi: 10.1128/jvi.70.12.8459-8467.1996

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

K M Hehir 1, D Armentano 1, L M Cardoza 1, T L Choquette 1, P B Berthelette 1, G A White 1, L A Couture 1, M B Everton 1, J Keegan 1, J M Martin 1, D A Pratt 1, M P Smith 1, A E Smith 1, S C Wadsworth 1
PMCID: PMC190936  PMID: 8970968

Abstract

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

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Selected References

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  1. Aiello L., Guilfoyle R., Huebner K., Weinmann R. Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology. 1979 Apr 30;94(2):460–469. doi: 10.1016/0042-6822(79)90476-8. [DOI] [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. Caravokyri C., Leppard K. N. Constitutive episomal expression of polypeptide IX (pIX) in a 293-based cell line complements the deficiency of pIX mutant adenovirus type 5. J Virol. 1995 Nov;69(11):6627–6633. doi: 10.1128/jvi.69.11.6627-6633.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colby W. W., Shenk T. Adenovirus type 5 virions can be assembled in vivo in the absence of detectable polypeptide IX. J Virol. 1981 Sep;39(3):977–980. doi: 10.1128/jvi.39.3.977-980.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donahue R. E., Kessler S. W., Bodine D., McDonagh K., Dunbar C., Goodman S., Agricola B., Byrne E., Raffeld M., Moen R. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med. 1992 Oct 1;176(4):1125–1135. doi: 10.1084/jem.176.4.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ghosh-Choudhury G., Haj-Ahmad Y., Graham F. L. Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes. EMBO J. 1987 Jun;6(6):1733–1739. doi: 10.1002/j.1460-2075.1987.tb02425.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ginsberg H. S., Lundholm-Beauchamp U., Horswood R. L., Pernis B., Wold W. S., Chanock R. M., Prince G. A. Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc Natl Acad Sci U S A. 1989 May;86(10):3823–3827. doi: 10.1073/pnas.86.10.3823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  9. Hearing P., Samulski R. J., Wishart W. L., Shenk T. Identification of a repeated sequence element required for efficient encapsidation of the adenovirus type 5 chromosome. J Virol. 1987 Aug;61(8):2555–2558. doi: 10.1128/jvi.61.8.2555-2558.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Lochmüller H., Jani A., Huard J., Prescott S., Simoneau M., Massie B., Karpati G., Acsadi G. Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 + delta E3) during multiple passages in 293 cells. Hum Gene Ther. 1994 Dec;5(12):1485–1491. doi: 10.1089/hum.1994.5.12-1485. [DOI] [PubMed] [Google Scholar]
  13. Rich D. P., Couture L. A., Cardoza L. M., Guiggio V. M., Armentano D., Espino P. C., Hehir K., Welsh M. J., Smith A. E., Gregory R. J. Development and analysis of recombinant adenoviruses for gene therapy of cystic fibrosis. Hum Gene Ther. 1993 Aug;4(4):461–476. doi: 10.1089/hum.1993.4.4-461. [DOI] [PubMed] [Google Scholar]
  14. Spector D. J. The pattern of integration of viral DNA sequences in the adenovirus 5-transformed human cell line 293. Virology. 1983 Oct 30;130(2):533–538. doi: 10.1016/0042-6822(83)90107-1. [DOI] [PubMed] [Google Scholar]
  15. Vanin E. F., Kaloss M., Broscius C., Nienhuis A. W. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol. 1994 Jul;68(7):4241–4250. doi: 10.1128/jvi.68.7.4241-4250.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Zabner J., Petersen D. M., Puga A. P., Graham S. M., Couture L. A., Keyes L. D., Lukason M. J., St George J. A., Gregory R. J., Smith A. E. Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats. Nat Genet. 1994 Jan;6(1):75–83. doi: 10.1038/ng0194-75. [DOI] [PubMed] [Google Scholar]

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