Abstract
Purpose: :
Choroidal neovascularization is the leading cause of blindness in age related macular degeneration (AMD). Several lines of evidence implicate increased levels of Vascular Endothelial Growth Factor (VEGF) in retinal pigment epithelium (RPE) from AMD patients. Current approaches to attenuate VEGF or its receptors, including the use of small interfering RNA (siRNA) show promise but have limited efficacy and require repeat administrations using procedures associated with multiple complications including endophthalmitis, retinal detachment and cataract. Therapies currently under investigation also suffer from leakage of the anti–VEGF agent from the ocular environment into systemic circulation. The goal of this study was to develop an approach for long–term endogenous expression of short hairpin RNA (shRNA) that may significantly attenuate VEGF in specific ocular tissues such as the RPE and hence act as a potential therapy for AMD.
Methods: :
We developed several shRNAs expressed from recombinant adenovirus vectors. These shRNAs were expressed in human RPE cells in the presence of adenovirus vectors over–expressing VEGF and the amount of VEGF attenuation evaluated with each of the shRNAs. Having identified potent shRNA constructs, adenovirus vectors over expressing VEGF were injected into the subretinal space of mice to induce choroidal neovascularization which was characterized by several approaches including histology, lectin staining, fluorescein angiography and quantitative RT–PCR. Inhibition of choroidal neovascularization was measured after co administration of adenovirus vectors expressing VEGF and shRNA targeting VEGF.
Results: :
We identified potent shRNA sequences able to silence high levels of VEGF in human RPE cells. When expressed from adenovirus backbones, these shRNA constructs silenced VEGF by 93.55±0.07% at a 1:5, VEGF:shRNA molar ratio and 64.30±1.98% at 1:0.05 molar ratio. Adenovirus vectors expressing supraphysiological levels of VEGF could induce CNV in mice within 5 days. Co injection of VEGF–expressing viruses into mice with adenovirus vectors expressing shRNA targeting VEGF led to a reduction in size of choroidal neovascularization by 83.82±7.65%.
Conclusions: :
Our results are consistent with the hypothesis that shRNA targeting VEGF from adenovirus vectors allows potent attenuation of high levels of VEGF and prevents choroidal neovascularization. This novel approach shows promise as a therapy for AMD.
Keywords: choroid: neovascularization • adenovirus