Minimal Effective Dosing of a Novel Recombinant Form of VEGF Soluble Receptor 1 (sFlt-1) Delivered via AAV Vector in Inhibiting Choroidal Neovascularization in a Murine Model

Abstract

Purpose: : Choroidal neovascularization (CNV) occurs in variety of chorioretinal diseases, including age-related macular degeneration (AMD) and is the major cause of severe visual loss in patients with AMD. VEGF is known to be a critical stimulus for CNV. In a previous study, we showed that AAV-mediated delivery of a novel genetically engineered soluble form of VEGF receptor 1 (sFLT01) effectively inhibited CNV in an experimental animal model. In this study, we further investigated the minimal effective dose of the vector as a function of either intravitreal or subretinal routes of vector delivery in the mouse laser induced CNV model.

Methods: : The cDNA for sFLT01 was packaged into an AAV serotype-2 vector and the vector serially diluted in saline ranging from full dose (~5X10⁸ vg/eye) to 1000 fold dilution (~5X10⁵ vg/eye). Each dilution was injected either intravitreously or subretinally into the right eyes of 10-15 adult C57BL/6 mice. 532nm laser photocoagulation was performed 4-6 weeks after injection to focally rupture Bruch’s membrane in both eyes (4-5 sites each eye). The development of CNV at each laser burn was evaluated 14 days later by fluorescein angiographic choroidal flat-mount image analysis, as well as by histology of transverse retinal sections. The expression of the sFlt01 in the retina was confirmed by immunocytochemistry.

Results: : sFLT01was primarily expressed in the retinal ganglion cells when the AAV2-CBA-sFLT01 vector was injected intravitreously, and in photoreceptors and RPE cells when injected subretinally. The minimum effective dose was approximately 4X10⁷ vg/eye when the vector was injected intravitreously, and resulted in about 40% reduction of the CNV area per laser burn. In contrast, minimum effective dose for subretinal vector was more than ten fold lower (< 4X10⁶ vg/eye) than for intravitreal vector.

Keywords: choroid: neovascularization • gene transfer/gene therapy • age-related macular degeneration