Effect Of Cilary Muscle Plasmid Electrotransfert Of Tnf-alpha Soluble Receptor Variants In A Rat Model Of Choroidal Neovascularization

Abstract

Purpose: : Anti-VEGF proteins are the treatment of choice for choroidal neovascularization (CNV). However repeated injections are required to maintain their effect and tachyphylaxis begins to be described. There is a need for other anti angiogenic strategies and drug delivery solutions. We have evaluated the sustained release of soluble receptors for TNF-alpha using plasmid electrotransfer in the ciliary muscle of rats with CNV.

Methods: : CNV was induced in Brown-Norway rats using argon laser photocoagulation. 6 days before, plasmid electrotransfer was performed in the ciliary muscle by application of 8 square-wave electrical pulses (15V, 10ms, 5.3Hz) using two platinum / iridium electrodes, an internal wire and an external sheet, in order to transduce soluble receptors for TNF. Both pVAX2 hTNFR-Is and pVAX2 hTNFR-Is/mIgG1 were used. CNV were analyzed in vivo with confocal fluorescein angiography at 14 days and after sacrifice at day 16 on choroidal flatmount and histological section. Cytokine analysis was performed on retina/ choroid using qPCR. Control eyes were untreated or treated with empty plasmids using same electric conditions.

Results: : A significant reduction of CNV was observed in vivo and on choroidal flatmounts in the 2 groups of rats treated by both variants. No significant difference was observed between rats treated with pVAX2 hTNFR-Is or pVAX2 hTNFR-Is/mIgG1. The number of microglia/ macrophages (IBA-1 positive) was significantly reduced in the choroidal flatmounts of rats treated with TNF alpha soluble receptors. Moreover, a significant reduction of VEGF, IL6 and iNOs was noticed in anti TNF treated rats as compared to naked plasmids treated ones.

Conclusions: : Ciliary muscle electrotransfer of plasmids encoding for TNF alpha soluble receptors is efficient to reduce the leakage and the size of laser-induced CNV. The reduction of macrophage/ microglia cell infiltration in the choroid is accompanied by a lowering of VEGF and also IL6 and iNOS, suggesting that anti TNF could synergistically be associated to existing anti VEGF therapies. Sustained protein release in the ocular media using ciliary muscle electrotransfer avoids repeated intravitreous injections.

Keywords: age-related macular degeneration • gene transfer/gene therapy • ciliary muscle