Identification of Stabilized Small Interfering RNAs (siRNAs) for the Potential Treatment of Age–related Macular Degeneration (AMD)

Abstract

Abstract: : Purpose: siRNAs are duplex RNA molecules that can be designed to interfere specifically with the expression of disease–related genes. Studies were carried out to identify stabilized siRNAs with improved ocular residence time that simultaneously inhibit multiple members of the vascular endothelial growth factor (VEGF) pathway. Methods: siRNAs were screened in cell culture using a luciferase–target gene fusion plasmid where siRNAs complementary to the target gene reduce luciferase expression. Potent siRNAs were identified from 64 stabilized siRNAs specific for each of the three target mRNAs (VEGF, VEGFR–1 and VEGFR–2). Lead siRNAs were then tested in combination to evaluate the efficacy of siRNA mixtures. In addition, bifunctional siRNAs that target two gene sequences were made by chemically linking two siRNAs or by creating siRNAs in which each strand of the duplex actively silences a different target. Chemically optimized bifunctional siRNAs were also tested in vitro on endogenous targets. In parallel, the effect of siRNA stabilizing modifications were compared in cell culture and in ocular pharmacokinetic (PK) studies following intravitreal injection in rabbit eyes. Results: VEGF, VEGFR–1 and VEGFR–2 targeted siRNAs induced >90% silencing in a cell culture reporter system. Ten stabilized siRNAs were identified for each target with IC₅₀s of 0.2–0.7 nM. Twenty–one pairs of these siRNAs, each targeting VEGF and one of its receptors, were tested to identify the combination having the greatest effect on both targets. For nine combinations, a >70% reduction of both targets was observed at low concentrations (1.2 nM each siRNA). Bifunctional siRNAs were designed based on the most potent in vitro combinations, and many demonstrated silencing activity equivalent to that observed as single siRNAs. Evaluation of the ocular PK of chemically stabilized siRNAs resulted in a >50–fold increase in ocular exposure compared to that of non–modified siRNAs. Conclusions: Stabilized siRNAs complementary to one or more members of the VEGF pathway specifically inhibit the expression of their respective gene targets in cell culture. In addition, the ocular residence time of stabilized siRNAs is significantly prolonged over that of non–modified siRNAs. These data support the continued development of stabilized, multi–targeted siRNAs as potential therapeutics to inhibit neovascularization (NV) in ocular diseases such as AMD and diabetic retinopathy. Inhibitory effects of bifunctional siRNAs on ocular NV in vivo are being assessed.

Keywords: age-related macular degeneration • retinal degenerations: cell biology • gene transfer/gene therapy