Search tips
Search criteria 


Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. 1996 October; 70(10): 7030–7038.
PMCID: PMC190753

Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted.


Although adenovirus vectors offer many advantages, it would be desirable to develop vectors with improved expression and decreased toxicity. Toward this objective, an adenovirus vector system with deletion of both the El and E2a regions was developed. A 5.9-kb fragment of the adenovirus type 5 (Ad5) genome containing the E2a gene and its early and late promoters was transfected into 293 cells. A complementing cell line, designated 293-C2, expressed the E2a mRNA and protein and was found to complement the defect in Ad5 viruses with temperature-sensitive or deletion mutations in E2a. A deletion of 1.3 kb removing codons 40 to 471 of the 529 amino acids of E2a was introduced into plasmids for preparation of viruses and vectors. An Ad5 virus with disruption of the El gene and deletion of E2a grew on 293-C2 cells but not on 293 cells. Vectors with E1 and E2a deleted expressing Escherichia coli beta-galactosidase or human alpha1-antitrypsin were prepared and expressed the reporter genes after intravenous injection into mice. This vector system retains sequences in common between the complementing cell line and the vectors, including 3.4 kb upstream and 1.1 kb downstream of the deletion. These vectors have potential advantages of increased capacity for insertion of transgene sequences, elimination of expression of E2a, and possibly reduction in expression of other viral proteins. Although the titers of the vectors with deleted are about 10- to 30-fold below those of vectors with E2a wild-type regions, the former vectors are suitable for detailed studies with animals to evaluate the effects on host immune responses, on duration of expression, and on safety.

Full Text

The Full Text of this article is available as a PDF (622K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Amalfitano A, Begy CR, Chamberlain JS. Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3352–3356. [PubMed]
  • Armentano D, Sookdeo CC, Hehir KM, Gregory RJ, St George JA, Prince GA, Wadsworth SC, Smith AE. Characterization of an adenovirus gene transfer vector containing an E4 deletion. Hum Gene Ther. 1995 Oct;6(10):1343–1353. [PubMed]
  • Barr D, Tubb J, Ferguson D, Scaria A, Lieber A, Wilson C, Perkins J, Kay MA. 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]
  • Berkner KL. Development of adenovirus vectors for the expression of heterologous genes. Biotechniques. 1988 Jul-Aug;6(7):616–629. [PubMed]
  • Bett AJ, Haddara W, Prevec L, Graham FL. 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. [PubMed]
  • Boucher RC. Current status of CF gene therapy. Trends Genet. 1996 Mar;12(3):81–84. [PubMed]
  • Bramson JL, Graham FL, Gauldie J. The use of adenoviral vectors for gene therapy and gene transfer in vivo. Curr Opin Biotechnol. 1995 Oct;6(5):590–595. [PubMed]
  • Brody SL, Metzger M, Danel C, Rosenfeld MA, Crystal RG. 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. [PubMed]
  • Brough DE, Cleghon V, Klessig DF. Construction, characterization, and utilization of cell lines which inducibly express the adenovirus DNA-binding protein. Virology. 1992 Oct;190(2):624–634. [PubMed]
  • Chang LS, Shenk T. The adenovirus DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters. J Virol. 1990 May;64(5):2103–2109. [PMC free article] [PubMed]
  • Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science. 1995 Oct 20;270(5235):404–410. [PubMed]
  • Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, Brody SL, Jaffe HA, Eissa NT, 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. [PubMed]
  • Engelhardt JF, Ye X, Doranz B, Wilson JM. 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. [PubMed]
  • Ensinger MJ, Ginsberg HS. Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J Virol. 1972 Sep;10(3):328–339. [PMC free article] [PubMed]
  • Fang B, Wang H, Gordon G, Bellinger DA, Read MS, Brinkhous KM, Woo SL, Eisensmith RC. 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]
  • Fisher KJ, Choi H, Burda J, Chen SJ, Wilson JM. Recombinant adenovirus deleted of all viral genes for gene therapy of cystic fibrosis. Virology. 1996 Mar 1;217(1):11–22. [PubMed]
  • French BA, Mazur W, Ali NM, Geske RS, Finnigan JP, Rodgers GP, Roberts R, Raizner AE. Percutaneous transluminal in vivo gene transfer by recombinant adenovirus in normal porcine coronary arteries, atherosclerotic arteries, and two models of coronary restenosis. Circulation. 1994 Nov;90(5):2402–2413. [PubMed]
  • Ginsberg HS, Williams JF, Doerfler WH, Shimojo H. Proposed nomenclature for mutants of adenoviruses. J Virol. 1973 Sep;12(3):663–664. [PMC free article] [PubMed]
  • Goldman MJ, Litzky LA, Engelhardt JF, Wilson JM. Transfer of the CFTR gene to the lung of nonhuman primates with E1-deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study. Hum Gene Ther. 1995 Jul;6(7):839–851. [PubMed]
  • Gorziglia MI, Kadan MJ, Yei S, Lim J, Lee GM, Luthra R, Trapnell BC. 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. [PMC free article] [PubMed]
  • Graham FL. Covalently closed circles of human adenovirus DNA are infectious. EMBO J. 1984 Dec 1;3(12):2917–2922. [PubMed]
  • Graham FL, Rudy J, Brinkley P. Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences. EMBO J. 1989 Jul;8(7):2077–2085. [PubMed]
  • Graham FL, Smiley J, Russell WC, 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. [PubMed]
  • Herrmann F. Cancer gene therapy: principles, problems, and perspectives. J Mol Med (Berl) 1995 Apr;73(4):157–163. [PubMed]
  • Kay MA, Graham F, Leland F, Woo SL. Therapeutic serum concentrations of human alpha-1-antitrypsin after adenoviral-mediated gene transfer into mouse hepatocytes. Hepatology. 1995 Mar;21(3):815–819. [PubMed]
  • Klessig DF, Brough DE, Cleghon V. Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell. Mol Cell Biol. 1984 Jul;4(7):1354–1362. [PMC free article] [PubMed]
  • Kochanek S, Clemens PR, Mitani K, Chen HH, Chan S, Caskey CT. 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. [PubMed]
  • Krougliak V, Graham FL. 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. [PubMed]
  • McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology. 1988 Apr;163(2):614–617. [PubMed]
  • Mittereder N, Yei S, Bachurski C, Cuppoletti J, Whitsett JA, Tolstoshev P, Trapnell BC. Evaluation of the efficacy and safety of in vitro, adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA. Hum Gene Ther. 1994 Jun;5(6):717–729. [PubMed]
  • Mizushima S, Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. [PMC free article] [PubMed]
  • Morgan JE. Cell and gene therapy in Duchenne muscular dystrophy. Hum Gene Ther. 1994 Feb;5(2):165–173. [PubMed]
  • Morsy MA, Alford EL, Bett A, Graham FL, Caskey CT. Efficient adenoviral-mediated ornithine transcarbamylase expression in deficient mouse and human hepatocytes. J Clin Invest. 1993 Sep;92(3):1580–1586. [PMC free article] [PubMed]
  • Neve RL. Adenovirus vectors enter the brain. Trends Neurosci. 1993 Jul;16(7):251–253. [PubMed]
  • Nicolas JC, Sarnow P, Girard M, Levine AJ. Host range temperature-conditional mutants in the adenovirus DNA binding protein are defective in the assembly of infectious virus. Virology. 1983 Apr 15;126(1):228–239. [PubMed]
  • O'Connor RJ, Hearing P. Mutually exclusive interaction of the adenovirus E4-6/7 protein and the retinoblastoma gene product with internal domains of E2F-1 and DP-1. J Virol. 1994 Nov;68(11):6848–6862. [PMC free article] [PubMed]
  • O'Neal WK, Beaudet AL. Somatic gene therapy for cystic fibrosis. Hum Mol Genet. 1994;3(Spec No):1497–1502. [PubMed]
  • Rice SA, Klessig DF. Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein. J Virol. 1985 Dec;56(3):767–778. [PMC free article] [PubMed]
  • Soriano P, Montgomery C, Geske R, Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. [PubMed]
  • Stratford-Perricaudet LD, Makeh I, Perricaudet M, Briand P. Widespread long-term gene transfer to mouse skeletal muscles and heart. J Clin Invest. 1992 Aug;90(2):626–630. [PMC free article] [PubMed]
  • Top FH, Jr, Buescher EL, Bancroft WH, Russell PK. Immunization with live types 7 and 4 adenovirus vaccines. II. Antibody response and protective effect against acute respiratory disease due to adenovirus type 7. J Infect Dis. 1971 Aug;124(2):155–160. [PubMed]
  • Tsuji M, van der Vliet PC, Kitchingman GR. Temperature-sensitive mutants of adenovirus single-stranded DNA-binding protein. Inability to support DNA replication is associated with an altered DNA-binding activity of the protein. J Biol Chem. 1991 Aug 25;266(24):16178–16187. [PubMed]
  • Van Der Vliet PC, Levine AJ, Ensinger MJ, Ginsberg HS. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis. J Virol. 1975 Feb;15(2):348–354. [PMC free article] [PubMed]
  • van Ormondt H, Galibert F. Nucleotide sequences of adenovirus DNAs. Curr Top Microbiol Immunol. 1984;110:73–142. [PubMed]
  • Voelkerding K, Klessig DF. Identification of two nuclear subclasses of the adenovirus type 5-encoded DNA-binding protein. J Virol. 1986 Nov;60(2):353–362. [PMC free article] [PubMed]
  • Wang Q, Jia XC, Finer MH. 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]
  • Yang Y, Nunes FA, Berencsi K, Furth EE, Gönczöl E, Wilson JM. 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. [PubMed]
  • Yang Y, Nunes FA, Berencsi K, Gönczöl E, Engelhardt JF, Wilson JM. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet. 1994 Jul;7(3):362–369. [PubMed]
  • Yei S, Mittereder N, Wert S, Whitsett JA, Wilmott RW, Trapnell BC. In vivo evaluation of the safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lung. Hum Gene Ther. 1994 Jun;5(6):731–744. [PubMed]
  • Zsengeller ZK, Wert SE, Hull WM, Hu X, Yei S, Trapnell BC, Whitsett JA. Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice. Hum Gene Ther. 1995 Apr;6(4):457–467. [PubMed]

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