Project Summary Neovascular age-related macular degeneration (nAMD) with choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries. All currently approved anti-angiogenic drugs for nAMD, such as ranibizumab and aflibercept, target vascular endothelial growth factor (VEGF) but have limited therapeutic efficacy. Patients with poor response to one anti-VEGF drug are often switched to another VEGF inhibitors due to the lack of approved drugs against other angiogenic pathways. Furthermore, repeated intravitreal injections in anti-VEGF-resistant patients may increase the risk of injection-related ocular complications. A new strategy is to improve the efficacy is to develop novel therapies against VEGF-independent angiogenic factors for alternative or combination therapy. Another strategy to circumvent repetitive intravitreal injections is to develop anti-angiogenesis gene therapy that requires only a single injection for long-lasting efficacy. However, current approach of anti-VEGF gene therapy yielded marginal treatment benefit to complement ranibizumab, possibly because of their similar mechanisms of action. To circumvent these problems, we recently discovered a novel angiogenic factor with high disease selectivity and VEGF-independent mechanisms. We developed a monoclonal antibody (mAb) against this target and demonstrated its high efficacy for anti-angiogenic therapy of CNV. This project is to develop anti-angiogenesis gene therapy against this novel target using an adeno-associated viral (AAV) vector. In Aim 1, we will generate AAV vectors to express an antibody Fab fragments against the novel target and VEGF under the direction of constitutive or hypoxia-inducible promoters and characterize expression, binding and neutralizing activity of Fab fragments in vitro. In Aim 2, we will determine the therapeutic efficacy and safety of these AAV vectors to alleviate CNV in animal models. Additionally, we will investigate combination gene therapy by simultaneously targeting both factors in the same mouse models to define possible synergy and efficacy improvement. Successful implementation of this project will lead to a novel anti-angiogenesis gene therapy that will improve treatment efficacy of CNV and reduce the requirement for monthly intravitreal injection of anti-VEGF protein drugs.