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Tetracycline-inducible transgene expression mediated by a single AAV vector

Gene Therapy volume 10, pages 84–94 (2003)Cite this article

Abstract

Regulated gene delivery systems are usually made of two elements: an inducible promoter and a transactivator. In order to optimize gene delivery and regulation, a single viral vector ensuring adequate stoichiometry of the two elements is required. However, efficient regulation is hampered by interferences between the inducible promoter and (i) the promoter used to express the transactivator and/or (ii) promoter/enhancer elements present in the viral vector backbone. We describe a single AAV vector in which transcription of both the reverse tetracycline transactivator (rtTA) and the transgene is initiated from a bidirectional tetracycline-responsive promoter and terminated at bidirectional SV40 polyadenylation sites flanking both ITRs. Up to 50-fold induction of gene expression in human tumor cell lines and 100-fold in primary cultures of rat Schwann cells was demonstrated. In addition an 80-fold induction in vivo in the rat brain has been obtained. In vitro, the autoregulatory vector exhibits an induced expression level superior to that obtained using the constitutive CMV promoter. Although extinction of the transgene after removal of tetracycline was rapid (less than 3 days), inducibility after addition of tetracycline was slow (about 14 days). This kinetics is suitable for therapeutic gene expression in slowly progressive diseases while allowing rapid switch-off in case of undesirable effects. As compared to previously described autoregulatory tet-repressible (tetOFF) AAV vectors, the tet-inducible (tetON) vector prevents chronic antibiotic administration in the uninduced state.

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References

  1. Mayo KE, Palmiter RD . Glucocorticoid regulation of the mouse metallothionein I gene is selectively lost following amplification of the gene. J Biol Chem 1982; 257: 3061–3067.

    CAS  PubMed  Google Scholar 

  2. Palmiter RD . Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc Natl Acad Sci USA 1994; 91: 1219–1223.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Baim SB, Labow MA, Levine AJ, Shenk T . A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside. Proc Natl Acad Sci USA 1991; 88: 5072–5076.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lee F et al. Functional analysis of the steroid hormone control region of mouse mammary tumor virus. Nucleic Acids Res 1984; 12: 4191–4206.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wang Y, O'Malley Jr BW, Tsai SY, O'Malley BW . A regulatory system for use in gene transfer. Proc Natl Acad Sci USA 1994; 91: 8180–8184.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gossen M, Bujard H . Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992; 89: 5547–5551.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Solera JA . Treatment of human brucellosis with netilmicin and doxycycline. Clin Infect Dis 1996; 22: 441–445.

    Article  CAS  PubMed  Google Scholar 

  8. Kringstein AM, Rossi FM, Hofmann A, Blau HM . Graded transcriptional response to different concentrations of a single transactivator. Proc Natl Acad Sci USA 1998; 95: 13 670–13 675.

    Article  Google Scholar 

  9. Gossen M et al. Transcriptional activation by tetracyclines in mammalian cells. Science 1995; 268: 1766–1769.

    Article  CAS  PubMed  Google Scholar 

  10. Smith-Arica JR, et al. Cell-type-specific and regulatable transgenesis in the adult brain: adenovirus-encoded combined transcriptional targeting and inducible transgene expression. Mol Ther 2000; 2: 579–587.

    Article  CAS  PubMed  Google Scholar 

  11. Mohammadi S, Hawkins RE . Efficient transgene regulation from a single tetracycline-controlled positive feedback regulatory system. Gene Ther 1998; 5: 76–84.

    Article  Google Scholar 

  12. Strathdee CA, McLeod MR, Hall JR . Efficient control of tetracycline-responsive gene expression from an autoregulated bi-directional expression vector. Gene 1999; 229: 21–29.

    Article  CAS  PubMed  Google Scholar 

  13. Gallia GL, Khalili K . Evaluation of an autoregulatory tetracycline regulated system. Oncogene 1998; 16: 1879–1884.

    Article  CAS  PubMed  Google Scholar 

  14. Corti O et al. A single adenovirus vector mediates doxycycline-controlled expression of tyrosine hydroxylase in brain grafts of human neural progenitors. Nat Biotechnol 1999; 17: 349–354.

    Article  CAS  PubMed  Google Scholar 

  15. Haberman RP, McCown TJ, Samulski RJ . Inducible long-term gene expression in brain with adeno-associated virus gene transfer. Gene Ther 1998; 5: 1604–1611.

    Article  CAS  PubMed  Google Scholar 

  16. Hofmann A, Nolan GP, Blau HM . Rapid retroviral delivery of tetracycline-inducible genes in a single autoregulatory cassette. Proc Natl Acad Sci USA 1996; 93: 5185–5190.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ho DY, McLaughlin JR, Sapolsky RM . Inducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system. Brain Res Mol Brain Res 1996; 41: 200–209.

    Article  CAS  PubMed  Google Scholar 

  18. Unsinger J, Kroger A, Hauser H, Wirth D . Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes. Mol Ther 2001; 4: 484–489.

    Article  CAS  PubMed  Google Scholar 

  19. Haberman RP, McCown TJ, Samulski RJ . Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element. J Virol 2000; 74: 8732–8739.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Witzgall R et al. The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc Natl Acad Sci USA 1994; 91: 4514–4518.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. McGee Sanftner LH et al. Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina. Mol Ther 2001; 3: 688–696.

    Article  CAS  PubMed  Google Scholar 

  22. Bell AC, West AG, Felsenfeld G . The protein CTCF is required for the enhancer blocking activity of vertebrate insulators. Cell 1999; 98: 387–396.

    Article  CAS  PubMed  Google Scholar 

  23. Fitzsimons HL, McKenzie JM, During MJ . Insulators coupled to a minimal bidirectional tet cassette for tight regulation of rAAV-mediated gene transfer in the mammalian brain. Gene Ther 2001; 8: 1675–1681.

    Article  CAS  PubMed  Google Scholar 

  24. Rivera VM et al. Long-term regulated expression of growth hormone in mice after intramuscular gene transfer. Proc Natl Acad Sci USA 1999; 96: 8657–8662.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ye X et al. Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer. Science 1999; 283: 88–91.

    Article  CAS  PubMed  Google Scholar 

  26. Zolotukhin S et al. A ‘humanized’ green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J Virol 1996; 70: 4646–4654.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Lemke G, Lamar E, Patterson J . Isolation and analysis of the gene encoding peripheral myelin protein zero. Neuron 1988; 1: 73–83.

    Article  CAS  PubMed  Google Scholar 

  28. Messing A et al. P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice. Neuron 1992; 8: 507–520.

    Article  CAS  PubMed  Google Scholar 

  29. Tenenbaum L et al. Cellular contaminants of adeno-associated virus vector stocks can enhance transduction. Gene Ther 1999; 6: 1045–1053.

    Article  CAS  PubMed  Google Scholar 

  30. Brockes JP, Raff MC . Studies on cultured rat Schwann cells. II. Comparison with a rat Schwann cell line. In Vitro 1979; 15: 772–778.

    Article  CAS  PubMed  Google Scholar 

  31. Brockes JP, Fields KL, Raff MC . Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve. Brain Res 1979; 165: 105–118.

    Article  CAS  PubMed  Google Scholar 

  32. Grimm D, Kern A, Rittner K, Kleinschmidt JA . Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 1998; 9: 2745–2760.

    Article  CAS  PubMed  Google Scholar 

  33. Zolotukhin S et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 1999; 6: 973–985.

    Article  CAS  PubMed  Google Scholar 

  34. Yakobson B, Hrynko TA, Peak MJ, Winocour E . Replication of adeno-associated virus in cells irradiated with UV light at 254 nm. J Virol 1989; 63: 1023–1030.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Flotte TR et al. Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J Biol Chem 1993; 268: 3781–3790.

    CAS  PubMed  Google Scholar 

  36. Van Heuverswyn H, d Van V, Fiers W . Complete nucleotide sequence of the simian-virus 40 Hind-G fragment and localisation of the carboxyl terminus of the VP1 protein. Eur J Biochem 1978; 86: 325–334.

    Article  CAS  PubMed  Google Scholar 

  37. Van Heuverswyn H, d Van V, Fiers W . Nucleotide sequence of the simian-virus-40 DNA region coding for the carboxyl-terminal part of the T antigen. Eur J Biochem 1978; 86: 335–344.

    Article  CAS  PubMed  Google Scholar 

  38. Brown AM, Lemke G . Multiple regulatory elements control transcription of the peripheral myelin protein zero gene. J Biol Chem 1997; 272: 28 939–28 947.

    Article  Google Scholar 

  39. Baron U, Freundlieb S, Gossen M, Bujard H . Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res 1995; 23: 3605–3606.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mandel RJ, Spratt SK, Snyder RO, Leff SE . Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats. Proc Natl Acad Sci USA 1997; 94: 14 083–14 088.

    Article  Google Scholar 

  41. Smith-Arica JR et al. Cell-type-specific and regulatable transgenesis in the adult brain: adenovirus-encoded combined transcriptional targeting and inducible transgene expression. Mol Ther 2000; 2: 579–587.

    Article  CAS  PubMed  Google Scholar 

  42. Baron U, Gossen M, Bujard H . Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res 1997; 25: 2723–2729.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Szoka Jr FC, Papahadjopoulos D . Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA 1978; 75: 4194–4198.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Vassaux G, Hurst HC, Lemoine NR . Insulation of a conditionally expressed transgene in an adenoviral vector. Gene Ther 1999; 6: 1192–1197.

    Article  CAS  PubMed  Google Scholar 

  45. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates 3rd compact edition. Academic Press: Orlando, FL, 1997.

    Google Scholar 

Download references

Acknowledgements

We thank Drs Marc Peschanski, Jude Samulski and Thomas McCown for helpful suggestions, Dr M Azarkan for his help in fluorimetric analysis, Dr David Blum for his help in image analysis and Olivier Bockstael and Catherine Melas for technical help. AC and EL are recipients of predoctoral fellowships from the Belgian National Research Foundation (FNRS-Télévie). Plasmid pAC1: patent pending. This research was supported by a Biotech European Community grant no. BIO-CT97-2207, by grants of the Belgian National Research Foundation (FNRS-FRSM no. 3.4540.96 and no. 3.4565.98) and by grants from the Société Générale de Belgique, from ‘Bruxelles-Capitale’ and from the Belgian Loterie Nationale.

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Authors and Affiliations

  1. Interdisciplinary Research Institute (IRIBHM) ULB,

    A Chtarto, T Kemp, E Lehtonen, T Velu & L Tenenbaum

  2. Laboratory of Experimental Neurosurgery, ULB -Hôpital Erasme, Brussels, Belgium

    A Chtarto, T Kemp, E Lehtonen, M Levivier, J Brotchi & L Tenenbaum

  3. Department of Neurology Heinrich Heine University, Düsseldorf, Germany

    H U Bender

  4. Department of Neurology, Zentrum für klinische Forschung, University of Ulm, Germany

    C O Hanemann

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  1. A Chtarto
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Chtarto, A., Bender, H., Hanemann, C. et al. Tetracycline-inducible transgene expression mediated by a single AAV vector. Gene Ther 10, 84–94 (2003). https://doi.org/10.1038/sj.gt.3301838

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