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New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo

Gene Therapy volume 12, pages 504–511 (2005)Cite this article

Abstract

Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand (FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available.

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Acknowledgements

We thank Dr James S Norris, Dr H Bujard and Dr Maria Trojanowska for their support. This research was supported by grants from the National Institutes of Health, R01 DK57997, the National Cancer Institute, 1R24 CA82933, and a start up fund provided to Jian-yun (John) Dong by the Medical University of South Carolina.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA

    S Rubinchik, J Woraratanadharm, H Yu & J-y Dong

  2. GenPhar Inc., Mount Pleasant, SC, USA

    J-y Dong

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  1. S Rubinchik
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Rubinchik, S., Woraratanadharm, J., Yu, H. et al. New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo. Gene Ther 12, 504–511 (2005). https://doi.org/10.1038/sj.gt.3302437

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