Ocular Sublocalization and Pharmacokinetics of Expression of Pigment Epithelium-Derived Factor in Murine Eyes Following Adenovirus-Based Intravitreous Gene Delivery

Abstract

Abstract: : Purpose: Intravitreous delivery of AdPEDF.11D has been shown to block and cause regression of neovascular lesions in multiple animal models. In order to determine the levels of pigment-epithelium derived factor (PEDF) associated with this activity, we set out to assess the pharmacokinetics of expression and ocular sub-localization of this anti-angiogenic protein. Methods: Two ul of a recombinant adenovector, Ad_GVPEDF.11D (E1-, partial E3-, E4-), with the human cytomegalovirus (CMV) promoter driving the expression of human PEDF was injected into adult female C57BL/6 mice intravitreously, at doses of 1e7, 1e8, and 1e9 particles (pu). Control animals were given buffer vehicle or used as naïves. Eyes were harvested at various intervals and sub-fractionated (for 1e9 pu dose only) or processed as whole eyes. Quantification was achieved with a GenVec generated ELISA specific for human PEDF. Results: Expression of PEDF following intravitreous administration of AdPEDF.11D was evaluated at 1, 7, 14, and 28 days in murine whole eyes. The mean peak ocular concentration for all doses occurs at day one. When compared to vehicle-injected animals, a dose of 1e7 pu, results in a 3 fold increase (p= 0.01) in PEDF levels and is only detectable at day 1. Also at day 1, a dose of 1e8 pu shows a 17- fold increase (p< 0.001) in PEDF, and 1e9 pu gives a 19- fold increase in PEDF levels (p< 0.001). At day 7, doses of 1e8 and 1e9 pu give levels of PEDF 2-3 fold above vehicle (p= 0.001, for both). At day 14, only animals given 1e9 pu have measurable levels of PEDF (p= 0.01) above vehicle. No detectable levels of PEDF were observed at day 28 above vehicle for any of the vector doses used. Sub fractionated eyes (cornea, aqueous humor, iris/ciliary body, lens, retina, RPE/choroid, sclera, and conjunctiva) were evaluated at days 1, 4, 7, and 14. In this study, measurable levels of hPEDF were detected at day 1 in every fraction except the lens. Conclusions: Intravitreous delivery of 1e9 pu of AdPEDF.11D results in measurable levels of protein for 14 days in whole eyes, with measurable levels of hPEDF in the retina and RPE/choroid. Our data suggest that expression of only a small amount of PEDF at the right location may be sufficient to achieve a relevant therapeutic effect.

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