PURPOSE: Choroidal neovascularization (CNV) 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 patients with AMD. Current approaches to attenuate VEGF or its receptors, including the use of small interfering (si)RNA, show significant promise, but still have limited efficacy and require repeat administrations, using procedures associated with multiple complications. The goal of this study was to develop an approach for long-term endogenous expression of short hairpin (sh)RNA that would significantly attenuate VEGF and hence act as a potential therapy for AMD. METHODS: Several shRNAs expressed from recombinant adenovirus were developed. These shRNAs were expressed in human RPE cells in the presence of adenovirus vectors overexpressing VEGF, and the amount of VEGF attenuation was evaluated. Adenovirus vectors expressing VEGF were subsequently injected into the subretinal space of mice, and induction of CNV was measured in the presence of adenovirus vectors expressing shRNA targeting VEGF. RESULTS: Potent shRNA sequences were identified that were able to silence VEGF in human RPE cells. When expressed from adenovirus backbones, these shRNA constructs silenced VEGF by 94% at a 1:5 molar ratio (VEGF to shRNA) and 64% at a 1:0.05 molar ratio. Adenovirus vectors expressing high levels of VEGF could induce CNV in mice within 5 days. Co-injection of VEGF-expressing viruses into mice with shRNA targeting VEGF led to a substantial (84%) reduction in CNV. CONCLUSIONS: shRNA targeting VEGF from adenovirus vectors allows potent attenuation of VEGF and prevents CNV. This approach shows promise as a therapy for AMD.
PURPOSE: Choroidal neovascularization (CNV) 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 patients with AMD. Current approaches to attenuate VEGF or its receptors, including the use of small interfering (si)RNA, show significant promise, but still have limited efficacy and require repeat administrations, using procedures associated with multiple complications. The goal of this study was to develop an approach for long-term endogenous expression of short hairpin (sh)RNA that would significantly attenuate VEGF and hence act as a potential therapy for AMD. METHODS: Several shRNAs expressed from recombinant adenovirus were developed. These shRNAs were expressed in human RPE cells in the presence of adenovirus vectors overexpressing VEGF, and the amount of VEGF attenuation was evaluated. Adenovirus vectors expressing VEGF were subsequently injected into the subretinal space of mice, and induction of CNV was measured in the presence of adenovirus vectors expressing shRNA targeting VEGF. RESULTS: Potent shRNA sequences were identified that were able to silence VEGF in human RPE cells. When expressed from adenovirus backbones, these shRNA constructs silenced VEGF by 94% at a 1:5 molar ratio (VEGF to shRNA) and 64% at a 1:0.05 molar ratio. Adenovirus vectors expressing high levels of VEGF could induce CNV in mice within 5 days. Co-injection of VEGF-expressing viruses into mice with shRNA targeting VEGF led to a substantial (84%) reduction in CNV. CONCLUSIONS: shRNA targeting VEGF from adenovirus vectors allows potent attenuation of VEGF and prevents CNV. This approach shows promise as a therapy for AMD.
Authors: Karen L Posey; Peiman Liu; Huiqiu R Wang; Alka C Veerisetty; Joseph L Alcorn; Jacqueline T Hecht Journal: PLoS One Date: 2010-04-22 Impact factor: 3.240