Abstract
A recombinant adenovirus with deleted E1 and E3, and E4-inactivated by replacing the E4 promoter with a synthetic promoter composed of a minimal TATA box and five consensus yeast GAL4-binding site elements was developed and used to express the human tumor suppresser gene p53. The toxicity and immunogenicity of this vector and vector-mediated p53 gene expression in vivo were studied in immunocompetent C3H and C57BL/6 mice. Expression of the late viral gene product, hexon protein, was observed in C3H and C57BL/6 mice injected with E4 wild-type adenovirus constructs Adv-cmv-β-Gal (BG), Adv-cmv-hp53 (WT), and empty E1− vector Adv-E4 (EW) 3 to 28 days after injection, but was undetectable in mice treated with E4 modified empty E1− vector Adv-GAL4 (EG) or Adv-cmv-hp53-GAL4 (G4). Expression of the p53 gene was observed in both WT- and G4-injected C3H and C57BL/6 mouse livers from days 3 to 28. Ten weeks after injection, p53 gene expression was still detected in G4-treated C57BL/6 mice at similar levels, but was not detectable in WT-treated mice. Vector-induced liver toxicity was evaluated by analyzing serum transaminases (SGOT and SGPT) activities. In all cases, SGOT and SGPT activities were markedly decreased in EG-treated C3H and C57BL/6 mice compared with those in EW-treated mice on days 3, 7 and 14 after injection. In C57BL/6 mice, the total anti-adenoviral CTL activities were two- to three-fold higher in animals treated with EW vector than in those treated with EG vector. These results suggest that inactivation of the E4 promoter efficiently diminished the viral replication and the late viral gene expression, reduced host immune response and consequently reduced toxicity and prolonged the duration of transgene expression in vivo.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Chengalvala MV et al. Adenovirus vectors for gene expression Curr Opin Biotechnol 1991 2: 718–722
Crystal RG . Transfer of genes to humans: early lessons and obstacles to success Science 1995 270: 404–410
Gao GP, Yang Y and Wilson JM . Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy J Virol 1996 70: 8934–8943
Taneja SS, Pang S, Cohan P, Belldegrun A . Gene therapy: principles and potential Cancer Surv 1995 23: 247–266
Trapnell BC, Gorziglia M . Gene therapy using adenoviral vectors Curr Opin Biotechnol 1994 5: 617–625
Bridge E et al. Adenovirus early region 4 and viral DNA synthesis Virology 1993 193: 794–801
Fang B, Koch P, Roth JA . Diminishing adenovirus gene expression and viral replication by promoter replacement J Virol 1997 71: 4798–4803
Yang Y et al. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy Proc Natl Acad Sci USA 1994 91: 4407–4411
Engelhardt JF, Litzky L, Wilson JM . Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a Hum Gene Ther 1994 5: 1217–1229
Lieber A, He CY, Kirillova I, Kay MA . 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 70: 8944–8960
Song WR, Kong HL, Traktman P, Crystal RG . Tytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors Hum Gene Ther 1997 8: 1207–1217
Yang Y, Ertl HC, Wilson JM . MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses Immunity 1994 1: 433–442
Morral N et al. Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors – comparison of E2a wild-type and E2a-deleted vectors Hum Gene Ther 1997 8: 1275–1286
Tripathy SK, Black HB, Goldwasser E, Leiden JM . Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nature Med 1996 ; 2: 545–550
Yang Y et al. Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo Gene Therapy 1996 3: 137–144
Yang YP, Haecker SE, Su Q, Wilson JM . Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle Hum Mol Genet 1996 5: 1703–1712
Armentano D et al. Effect of the e4 region on the persistence of transgene expression from adenovirus vectors J Virol 1997 71: 2408–2416
Dedieu JF et al. Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses J Virol 1997 71: 4626–4637
Falgout B, Ketner G . Adenovirus early region 4 is required for efficient virus particle assembly J Virol 1987 61: 3759–3768
Halbert DN, Cutt JR, Shenk T . Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff J Virol 1985 56: 250–257
Hemstrom C, Nordqvist K, Pettersson U, Virtanen A . Gene product of region E4 of adenovirus type 5 modulates accumulation of certain viral polypeptides J Virol 1988 62: 3258–3264
Huang C, Deibel R . Nucleic acid hybridization for detection of cell culture-amplified adenovirus J Clin Microbiol 1988 26: 2652–2656
Pilder S, Moore M, Logan J, Shenk T . The adenovirus E1B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs Mol Cell Biol 1986 6: 470–476
Bondesson M et al. Adenovirus E4 open reading frame 4 protein autoregulates E4 transcription by inhibiting E1A transactivation of the E4 promoter J Virol 1996 70: 3844–3851
Kleinberger T, Shenk T . Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down-regulates E1A-enhanced junB transcription J Virol 1993 67: 7556–7560
Dobner T, Horikoshi N, Rubenwolf S, Shenk T . Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor Science 1996 272: 1470–1473
Armentano D et al. Characterization of an adenovirus gene transfer vector containing an E4 deletion Hum Gene Ther 1995 6: 1343–1353
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 6: 1575–1586
Weinberg A et al. Enzyme linked immunosorbant assay: determination of anti-adenovirus antibodies in an infant population Revista do Instituto de Medicina Tropical de Sao Paulo 1989 31: 336–340
Yeh P et al. Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit J Virol 1996 70: 559–565
Shenk T, Jones N, Colby W, Fowlkes D . Functional analysis of adenovirus-5 host-range deletion mutants defective for transformation of rat embryo cells Cold Spring Harbor Symp Quant Biol 1980 44: 367–375
Li H et al. Adenovirus-mediated wild-type p53 gene transfer and overexpression induces apoptosis of human glioma cells independent of endogenous p53 status J Neuropathol Exp Neurol 1997 56: 872–878
Liu TJ et al. Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck Cancer Res 1995 55: 3117–3122
Brough DE et al. Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo J Virol 1997 71: 9206–9213
Tyson CA, Story DL, Green CE . Traditionally used indicators in relation to pathologic lesions. In: Tyson CA, Sawhney DS (eds) . Organ Function Tests in Toxicity Evaluation Noyes Publications: Park Ridge 1985 23–88
Barr D et al. Strain-related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: comparisons between immunocompetent and immunodeficient inbred strains Gene Therapy 1995 2: 151–155
Hardy S et al. Construction of adenovirus vectors throughCre-lox recombination J Virol 1997 71: 1842–1849
Rittner K, Schultz H, Pavirani A, Mehtali M . Conditional repression of the E2 transcription unit in E1–E3-deleted adenovirus vectors is correlated with a strong reduction in viral DNA replication and late gene expression in vitro J Virol 1997 71: 3307–3311
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ji, L., Bouvet, M., Price, R. et al. Reduced toxicity, attenuated immunogenicity and efficient mediation of human p53 gene expression in vivo by an adenovirus vector with deleted E1-E3 and inactivated E4 by GAL4-TATA promoter replacement. Gene Ther 6, 393–402 (1999). https://doi.org/10.1038/sj.gt.3300825
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3300825