AAV-mediated multiple gene therapy combining VEGFA-targeting miR-agshRNAs and PEDF for the suppression of choroidal...

Common ophthalmic diseases, including age-related macular degeneration (AMD), generally have a complex pathogenesis involving multiple pathways and varying involvement of specific cell types. This provides a strong rationale for developing novel gene therapy platforms that allow cell-specific up- and down-regulation of multiple targets while contained within standard adeno-associated viral vectors (AAVs). Hence, we engineered a tunable expression cassette with two pri-miR-embedded, Ago2-dependent shRNAs (miR-agshRNAs) units enabling dual target silencing, and intron embedment allowing downstream protein expression. With this platform, we demonstrated additive Vegfa knockdown, concurrent silencing of Vegfa and mTOR, and simultaneous expression of pigment epithelium-derived factor (PEDF) from a single promoter. Following the subretinal injection of AAV5 vectors encoding Vegfa-targeting miR-agshRNAs and PEDF into the murine retina, profound Vegfa suppression and strong PEDF expression were observed. Notably, laser-induced choroidal neovascularization (CNV) was significantly reduced in the therapeutic groups, with the multi-targeting vector achieving the highest level of CNV suppression. Collectively, our data demonstrated robust anti-angiogenic effects of multiple gene therapies, suggesting a "one-and-done" AAV-based delivery of cross-species anti-VEGFA RNAi therapeutics together with PEDF as a valuable tool for the management of neovascular AMD (nAMD) and other complex neovascular ocular diseases.