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K. Stieger, G. Le Meur, O. Mabon, M. Weber, D. Nivard, A. Mendes–Madeira, J.Y. Deschamps, P. Moullier, F. Rolling; Doxycycline–regulated Transgene Expression in the Retina of Nonhuman Primates Following Subretinal Injection of Recombinant AAV Vectors . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5205.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the ability of the tetracycline–regulatable (tetR) system to establish long–term transgene regulation in the retina of nonhuman primates. Recombinant rAAV–2/4 and rAAV–2/5 vectors harboring both the tetracycline–dependent transactivator (rtTA) and the macaque Epo cDNA were tested. Methods: Three rAAV vectors expressing the rtTA under the control of either the ubiquitous CAG promoter or the specific RPE65 promoter, AAV2/5.CAGrtTA–TetOn.Epo, AAV2/4.CAGrtTA–TetOn.Epo and AAV2/4.RPE65rtTA–TetOn.Epo, were generated and administered subretinally in 6 macaques (n=2 per construct). The doxycycline (dox) was given intravenously. The induction protocol started two months after subretinal injection and consisted of a 3–day induction pulse repeated once every 2 months. Anterior chamber fluid and vitreous were sampled and Epo was measured by ELISA. Results: All treated macaques showed successful initial inductions with a tight regulation and a rapid deinduction state upon dox withdrawal. An AAV2/5.CAGrtTA–TetOn.Epo pilote macaque, exhibited regulation of Epo secretion over a time period of 18 months. Following each induction, a peak of Epo (1200–1400mU/ml) was observed 48h after dox initiation, returning to baseline level (60–70mU/ml) within 10 days upon dox withdrawal. For the two AAV2/4.RPE65rtTA–TetOn.Epo injected macaques, regulation of Epo showed a similar profile of induction and deinduction with a peak at 14 mU/ml and a baseline level at 2 mU/ml. Conclusions: Repeated inductions of transgene expression in the nonhuman primate retina can be achieved using a tet–inducible system via rAAV vector administration over a long time period. Maximum Epo secretion in the anterior chamber depends upon the rAAV serotype and the nature of the promoter driving rtTA expression.
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