Adeno-associated virus type 8 vector?mediated expression of siRNA targeting vascular endothelial growth factor efficiently inhibits neovascularization in a murine choroidal neovascularization model

Choroidal neovascularization (CNV) is a major compli-cation that threatens the vision of patients with various retinal degenerative and inflammatory diseases, including patho-logic myopia, ocular histoplasmosis [1,2], and, especially, age-related macular degeneration (AMD) [3,4], which is the most frequent cause of visual impairment in individuals over the age of 40 years in developed countries [5,6]. Vascular endothelial growth factor (VEGF) is a 45-kDa homodi-meric glycoprotein that increases vascular permeability [7], stimulates angiogenesis [8], and is a specific endothelial cell mitogen [9]. VEGF also functions as a vasodilator and an anti-apoptotic, endothelial cell survival factor [ 10], expression of which is upregulated by hypoxia [11] and inflammatory mediators [ 12]. In addition, VEGF has pathogenic importance in cases of AMD, since VEGF contributes to the development of CNV in humans [13,14] and experimental CNV models [15]. The angiogenic actions of VEGF are mediated through its binding to two endothelium-specific receptor tyrosine kinases: Flt-1 (fms-like tyrosine kinase or VEGFR1) and Flk-1/KDR (fetal liver kinase or VEGFR2) [16,17], with the VEGF ligand showing at least 10-fold greater affinity for Flt-1 than Flk-1/KDR. We previously showed that neovas-cularization in a mouse CNV model is efficiently inhibited by type 8 adeno-associated viral (AAV) vectors mediating expression of flt-1 [18]. Clinically, moreover, inhibiting the VEGF pathway using a VEGF aptamer or an anti-VEGF antibody effectively suppresses the pathway’s CNV-related activity [19-21].The ability of siRNA to specially and potently down-regulate the expression of a target gene post-transcriptionally is based on the sequence-specific degradation of homologous target mRNA [22]. Intravitreous injections of siRNA have been shown to inhibit expression of selected genes, and specific siRNA targeting VEGF has been shown to prevent retinal or choroidal neovascularization in mice [23,24]. However, because of the short half-lives of these molecules in vivo, repeated intraocular injections of siRNA are frequently required for therapeutic benefit, which confers a high cumu -lative potential for local ocular complications. Gene transfer