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Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo

Gene Therapy volume 10, pages 459–466 (2003)Cite this article

Abstract

Regulated expression of therapeutic genes is required for long-term gene therapy applications for many disorders. Here we describe a doxycycline (dox)-regulated lentiviral vector system consisting of two HIV-1-based self-inactivating viruses. One of the vectors is constitutively expressing a novel improved version of the tetracycline reverse transactivator rtTA2S-M2 and the other has a rtTA responsive promoter driving the expression of β-galactosidase gene (lacZ). The rtTA2S-M2 has highly improved properties with respect to specificity, stability and inducibility. Functionality of the system by dox was confirmed after in vitro cotransduction of Chinese hamster ovary and human endothelial hybridoma (EAhy926) cells. Regulation of the system showed tight control of the gene expression. Dose dependence for dox was seen with concentrations that can be obtained in vivo with doses normally used in clinical practice. LacZ expression could be switched on/off during long-term (3 months) culturing of cotransduced cells. The system was next tested in vivo after cotransduction into rat brain and studying expression of the lacZ gene in dox-treated and control rats. Nested RT-PCR confirmed that the tight control of the gene expression was achieved in vivo. Also, X-gal staining showed positive cells in the dox-treated rats, but not in the controls 10 days after cotransduction with 4 days preceding treatment with dox. It is concluded that our doxycycline-regulated vector system shows significant potential for long-term gene therapy treatments.

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References

  1. Ylä-Herttuala S, Martin JF . Cardiovascular gene therapy. Lancet 2000; 355: 213–222.

    Article  PubMed  Google Scholar 

  2. 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 

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

    Article  CAS  PubMed  Google Scholar 

  4. Orth P et al. Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nat Struct Biol 2000; 7: 215–219.

    Article  CAS  PubMed  Google Scholar 

  5. Baron U, Bujard H . Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol 2000; 327: 401–421.

    Article  CAS  PubMed  Google Scholar 

  6. Urlinger S et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000; 97: 7963–7968.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 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 

  8. Miyoshi H et al. Development of a self-inactivating lentivirus vector. J Virol 1998; 72: 8150–8157.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Dull T et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72: 8463–8471.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Blomer U et al. Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol 1997; 71: 6641–6649.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kordower JH et al. Lentiviral gene transfer to the nonhuman primate brain. Exp Neurol 1999; 160: 1–16.

    Article  CAS  PubMed  Google Scholar 

  12. Naldini L et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996; 93: 11382–11388.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zufferey R et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997; 15: 871–875.

    Article  CAS  PubMed  Google Scholar 

  14. Lai Z, Brady RO . Gene transfer into the central nervous system in vivo using a recombinanat lentivirus vector. J Neurosci Res 2002; 67: 363–371.

    Article  CAS  PubMed  Google Scholar 

  15. Miyoshi H et al. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 1999; 283: 682–686.

    Article  CAS  PubMed  Google Scholar 

  16. Demaison C et al. High-level transduction and gene expression in hematopoietic repopulating cells using a human imunodeficiency virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther 2002; 13: 803–813.

    Article  CAS  PubMed  Google Scholar 

  17. Pawliuk R et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001; 294: 2368–2371.

    Article  CAS  PubMed  Google Scholar 

  18. Park F et al. Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat Genet 2000; 24: 49–52.

    Article  CAS  PubMed  Google Scholar 

  19. Park F, Ohashi K, Kay MA . Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver. Blood 2000; 96: 1173–1176.

    CAS  PubMed  Google Scholar 

  20. Kafri T et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 1997; 17: 314–317.

    Article  CAS  PubMed  Google Scholar 

  21. Johnson LG et al. Pseudotyped human lentiviral vector-mediated gene transfer to airway epithelia in vivo. Gene Ther 2000; 7: 568–574.

    Article  CAS  PubMed  Google Scholar 

  22. Gallichan WS et al. Lentivirus-mediated transduction of islet grafts with interleukin 4 results in sustained gene expression and protection from insulitis. Hum Gene Ther 1998; 9: 2717–2726.

    Article  CAS  PubMed  Google Scholar 

  23. Gusella GL et al. Lentiviral gene transduction of kidney. Hum Gene Ther 2002; 13: 407–414.

    Article  CAS  PubMed  Google Scholar 

  24. Frimpong K, Spector SA . Cotransduction of nondividing cells using lentiviral vectors. Gene Ther 2000; 7: 1562–1569.

    Article  CAS  PubMed  Google Scholar 

  25. Wang Y et al. A regulatory system for use in gene transfer. Proc Natl Acad Sci USA 1994; 91: 8180–8184.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pollock R et al. Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector. Proc Natl Acad Sci USA 2000; 97: 13221–13226.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rivera VM et al. A humanized system for pharmacologic control of gene expression [see comments]. Nat Med 1996; 2: 1028–1032.

    Article  CAS  PubMed  Google Scholar 

  28. No D, Yao TP, Evans RM . Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 1996; 93: 3346–3351.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Deglon N et al. Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease. Hum Gene Ther 2000; 11: 179–190.

    Article  CAS  PubMed  Google Scholar 

  30. Vigna E et al. Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors. Mol Ther 2002; 5: 252–261.

    Article  CAS  PubMed  Google Scholar 

  31. Lamartina S et al. Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators. Hum Gene Ther 2002; 13: 199–210.

    Article  CAS  PubMed  Google Scholar 

  32. Ylä-Herttuala S et al. Transfer of 15-lipoxygenase gene into rabbit iliac arteries results in the appearance of oxidation-specific lipid-protein adducts characteristic of oxidized low density lipoprotein. J Clin Invest 1995; 95: 2692–2698.

    Article  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. A.I.Virtanen Institute and Department of Medicine, University of Kuopio, Kuopio, Finland

    J K Koponen, H Kankkonen, J Kannasto, T Wirth & S Ylä-Herttuala

  2. Institut für Mikrobiologie, Universität Erlangen, Germany

    W Hillen

  3. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Germany

    H Bujard

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  1. J K Koponen
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  7. S Ylä-Herttuala
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Koponen, J., Kankkonen, H., Kannasto, J. et al. Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo. Gene Ther 10, 459–466 (2003). https://doi.org/10.1038/sj.gt.3301889

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