Oral Administration of Doxycycline Allows Tight Control of Transgene Expression: A Key Step Towards Gene Therapy of Retinal Diseases

Abstract

Purpose: : Expression of neurotrophic or antiangiogenic factors after gene transfer requires the use of a regulatory system to control transgene expression in order to avoid unwanted side effects in cases of overexpression. In a previous study we demonstrated that rAAV mediated gene transfer of the tetracycline-regulatable (tetR) system allows transgene regulation in the retina of nonhuman primates after intravenous administration of doxycycline (Dox). The purpose of this study was to evaluate oral administration of Dox to control transgene expression in the retina, since the pharmacokinetics after oral administration of the inducer drug represent a key factor when considering advancing to clinical trials.

Methods: : Two rAAV vectors expressing the rtTA under the control of the ubiquitous CAG promoter, AAV2/5.CAGrtTA-TetOn.Epo and AAV2/4.CAGrtTA-TetOn.Epo, were generated and administered subretinally in 4 macaques (n=2 per construct). Intravenous administration was performed by a 3-day Doxycycline pulse and oral administration consisted of the daily uptake of Dox hidden in dry fruits. Anterior chamber fluid and vitreous were sampled and Epo was measured by ELISA.

Results: : We report on the outcome of this evaluation and demonstrate that oral administration of Dox at a dose that is clinically used in humans (5mg/kg/day) is capable to continuously induce transgene expression in all macaques tested for 6 months. Moreover, control of transgene expression persists up to 4 years post-subretinal injection, with maximal induced levels of transgene product remaining stable over time.

Conclusions: : We demonstrated, here, for the first time that oral administration of Doxycycline: (i) is about as efficient as the intravenous delivery of Dox; (ii) can induce stable EPO expression in the retina over a 6 month period; and (iii) is not associated with detectable side effects due to either the long-term Dox administration or the elevated local EPO concentrations.

Keywords: gene transfer/gene therapy • retina • gene/expression