Inhibition of VEGF‐Induced Angiogenesis and Vascular Leakage by Bicistronic Co‐Expression of Aflibercept and COMP‐Ang1

ABSTRACT

Current anti‐VEGF monotherapies for neovascular age‐related macular degeneration (AMD) and diabetic retinopathy (DR) are limited by incomplete therapeutic responses, drug resistance, and the high burden of frequent intravitreal injections (IVTs). Although extensive research is underway to improve therapeutic outcomes, there remains a critical need for more effective strategies. To address this limitation, a bicistronic rAAV8 vector, Afb/cAng1, was developed to co‐express aflibercept (Afb) and COMP‐Ang1 (cAng1), simultaneously inhibiting VEGF signaling and activating the Tie2 pathway. In vitro characterization using conditioned medium containing secreted Afb/cAng1 showed that the treatment effectively suppresses VEGF‐induced VEGFR2 phosphorylation to below basal levels while robustly activating Tie2. Consequently, the conditioned medium significantly inhibited endothelial cell migration and tube formation, and restored VE‐cadherin expression compromised by VEGF stimulation or hyperglycemic conditions. Afb/cAng1 in a 3D angiogenesis‐on‐a‐chip model reduces neovascular area and vascular permeability, showing superior efficacy compared to Afb (Eylea). In vivo choroidal neovascularization (CNV) analysis confirmed dose‐dependent anti‐angiogenic efficacy of rAAV8‐Afb/cAng1, comparable to that of Eylea. These findings highlight Afb/cAng1 as a dual‐targeting gene therapy strategy that concurrently suppresses pathological angiogenesis and promotes vascular stabilization through coordinated VEGF inhibition and Tie2 activation in AMD and DR.