Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.