Abstract
Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2s-M2) alone or in combination with Tet-repressor (tTS-Kid) were explored in the context of helper-dependent adenovirus vector. Various genetic elements were assembled in a series of vectors and the ability to control secreted alkaline phosphatase expression evaluated in vitro in HeLa cells and in vivo by intramuscular injection in both C57/B6 and Balb/C nude mice. The results allow us to draw some general conclusions about the combination of transcription regulators and their relative orientation to the transgene to achieve maximal induction, while minimizing leakiness of expression.
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References
Agha-Mohammadi S et al. Regulatable systems: applications in gene therapy and replicating viruses J Clin Invest 2000 105: 1177–1183
Baron U, Bujard H . Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances Meth Enzymol 2000 327: 401–421
Gossen M et al. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters Proc Natl Acad Sci USA 1992 89: 5547–5551
Gossen M et al. Transcriptional activation by tetracyclines in mammalian cells Science 1995 268: 1766–1769
Deuschle U, Meyer WK, Thiesen HJ . Tetracycline-reversible silencing of eukaryotic promoters Mol Cell Biol 1995 15: 1907–1914
Freundlieb S, Schirra-Muller C, Bujard H . A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells J Gene Med 1999 1: 4–12
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
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
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
Kochanek S et al. High-capacity adenoviral vectors for gene transfer and somatic gene therapy Hum Gene Ther 1999 10: 2451–2459
Burcin MM et al. Adenovirus-mediated regulable target gene expression in vivo Proc Natl Acad Sci USA 1999 96: 355–360
Aurisicchio L et al. Regulated and prolonged expression of mIFN(alpha) in immunocompetent mice mediated by a helper-dependent adenovirus vector Gene Therapy 2001 8: 1817–1825
Schiedner G et al. Variables affecting in vivo performance of high-capacity adenovirus vectors J Virol 2002 76: 1600–1609
Klein NC, Cunha BA . Tetracyclines Med Clin North Am 1995 79: 789–801
Steinwaerder DS, Lieber A . Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo Gene Therapy 2000 7: 556–567
Guo ZS et al. Evaluation of promoter strength for hepatic gene expression in vivo following adenovirus-mediated gene transfer Gene Therapy 1996 3: 802–810
Abruzzese RV et al. Ligand-dependent regulation of plasmid-based transgene expression in vivo Hum Gene Ther 1999 10: 1499–1507
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
Rivera VM et al. Long-term regulated expression of growth hormone in mice after intramuscular gene transfer Giampaoli S, Nicolaus G, Delmastro P, Cortese R. Adeno-cosmid cloning vectors for regulated gene expression. J Gene Med (in press).
Sandig V et al. Optimization of the helper-dependent adenovirus system for production and potency in vivo Proc Natl Acad Sci USA 2000 97: 1002–1007
Rizzuto G et al. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation Proc Natl Acad Sci USA 1999 96: 6417–6422
Acknowledgements
We thank Fabrizio Fiore for animal care, Nicola La Monica and Riccardo Cortese for critical reading of the manuscript. We also thank J Clench for editorial assistance and Manuela Emili for graphics.
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Salucci, V., Scarito, A., Aurisicchio, L. et al. Tight control of gene expression by a helper-dependent adenovirus vector carrying the rtTA2s-M2 tetracycline transactivator and repressor system. Gene Ther 9, 1415–1421 (2002). https://doi.org/10.1038/sj.gt.3301813
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DOI: https://doi.org/10.1038/sj.gt.3301813
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