Inhibition of Retinal Neovascularization by Intravitreous Delivery of HIF1a.siRNA in a Mice Model of Oxygen Induced Retinopathy(OIR)

Abstract

Abstract: : Purpose: The purpose of this study is to test the capacity of an intravitreally administered siRNA directed against hypoxia inducible factor 1– alpha ( HIF1–a) to inhibit experimental retinal neovascularization in a mouse. Methods: We induced retinal neovascularization in Balb/c mice by exposing p7 mice to 75% O2 for 5 days followed by removal into room air for 6 days. We performed intravitreal injections of HIF1a.siRNA, BSS or GFP.siRNA at p12 upon removal of the mice to room air. Vascular endothelial nuclei counts and measurement of clock hours of pre retinal neovascularization in ADPase stained retinal flat mounts was performed to quantify retinal neovascularization. Immunhistochemical staining and real–time RT–PCR for HIF1a and VEGF at post injection 1, 3, 6 days was also performed. Results: When compared to BSS injected eyes, HIF1a siRNA injected eyes had 58% reduced neovascular nuclei count and 58% reduction in clock hours of neovascularization. Immunohistochemistry shows HIF1–a protein is decreased in HIF–1alpha siRNA injected eyes as compared with BSS injected eyes. Real–time PCR show that HIF1–alpha and VEGF mRNA are reduced in eyes injected with HIF1–a siRNA when compared to BSS injected eyes.When HIF1–a siRNA injected eyes are compared with GFP siRNA injected eyes, there was no significant difference in retinal neovascularization or HIF–1 alpha and VEGF mRNA levels. Conclusions: This data show that siRNA targeting HIF1a or GFP are both capable of inhibiting retinal NV, HIF1a and VEGF levels. Inhibition of retinal neovascularization and protein _expression may be mediated by targeted reduction of HIF–1 alpha but may also be mediated by non specific means.

Keywords: gene transfer/gene therapy