Intravitreous Injection of VEGF-R2antisense Oligonucleotides Limits the Growth of Retinal New Vessels in the Mouse Rop Model

Abstract

Purpose: : To study the effects of intravitreous delivery of VEGF-R2 (KDR) antisense oligonucleotide in a cationic nanoemulsion on retinal neovascularization in the mouse ROP model.

Methods: : The effect of different sequences of partial phosphorothioate antisense oligonucleotides (ODN) directed at VEGFR-2 were evaluated on VEGF-induced HUVEC proliferation. The down-regulation of VEGFR-2 by specific anti VEGFR-2 antisense oligonucleotide was also evaluated by RT-PCR and western blot analysis. The tolerance of different formulations of cationic nanoemulsions was evaluated on rat retinal pigment epithelial and retinal glial Müller cells in vitro using the MTT test. For in vivo studies, one µl of cationic nanoemulsion containing anti VEGFR-2 ODN (25µM), sense and scrambled ODN sequences and vehicle emulsion were injected in the vitreous of ROP neonatal mice at PN13 and 15. At PN17, retinal vascular endothelial cell proliferation was quantified by direct cell count.

Results: : In vitro, a specific and dose-dependent (0.5 to 5µM) inhibition of VEGF-induced HUVEC proliferation was observed when antisense oligonucleotide directed at VEGFR-2 was added to the cultures. Both RPE and RMG cells tolerated best a 2.5%MCT, DOTAP oil cationic formulation without evident cell toxicity up to10µl/ml. In the ROP model, an inhibition of 23% retinal neovascularization was observed with the free ASODN This inhibition increased to 64% when the AS ODN was vehicled by the MCT/DOTAP cationic nanoemulsion. No inhibition was observed when either the scrambled ODN sequence or the emulsion were used. No histological damages were observed after the intravitreous injections of the cationic nanoemulsion.

Conclusions: : Antisense (ODN) directed at VEGFR-2 (KDR) can be used as specific inhibitor of endothelial cell proliferation. The formulation of specific ODNs in a cationic nanoemulsion may be a safe alternative for ocular gene therapeutic strategies.

Keywords: inhibitory receptors • gene transfer/gene therapy • neovascularization