Small interfering RNA (siRNA) targeting VEGF effectivelyinhibits ocular neovascularization in a mouse model

[摘要] Purpose: RNA interference mediated by small interfering RNAs(siRNAs) is a powerful technology allowing the silencing of mamaliangenes with great specificity and potency. The purpose of this study wasto demonstrate the feasibility of RNA interference mediated by siRNA inretinal cells in vitro and in the murine retina in vivo.Methods: siRNAs specific for enhanced green fluorescent protein(EGFP) and murine and human vascular endothelial growth factor(VEGF) were designed. In vitro studies in human cell lines entailedmodulation of endogenous VEGF levels through chemically induced hypoxia.Effects of siRNA treatment on these levels were measured by ELISA. Invivo studies evaluating effects of siRNA on levels of EGFP and VEGF wereperformed by co-injecting recombinant viruses carrying EGFP orhVEGF cDNAs along with the appropriate siRNAs subretinally in mice.Additional studies aimed at blocking production of endogenous mVEGF wereperformed using laser-induced choroidal neovascularization (CNV) inmice. Effects of in vivo treatments were evaluated ophthalmoscopically.Retinal/choroidal flat mounts were evaluated after perfusion withdextran-fluorescein. Alternatively, retinas were evaluated inhistological sections or VEGF levels were measured in intact eyes usingELISA.Results: Successful delivery of siRNA to the subretinal space wasconfirmed by observing significantly reduced levels of EGFP in eyestreated with Ad.CMV.EGFP plus EGFP-directed siRNA. siRNAs directedagainst hVEGF effectively and specifically inhibit hypoxia-inducedVEGF levels in human cell lines and after adenoviral induced hVEGFtransgene expression in vivo. In addition, subretinal delivery of siRNAdirected against murine Vegf significantly inhibited CNV after laserphotocoagulation.Conclusions: Delivery of siRNA can be used in vitro and in vivo totarget specific RNAs and to reduce the levels of the specific proteinproduct in the targeted cells. This work suggests that RNA interferencehas potential for application to studies of retinal biology and for thetreatment of a variety of retinal diseases, including those involvingabnormal blood vessel growth.

[学科分类] 生物化学/生物物理