The Development of Proliferating Endothelial Cell–Specific Promoters for the Expression of Ribozymes

Abstract

Abstract: : Purpose:Angiogenesis requires the action of growth factors binding to specific receptors on endothelial cells. We examined the pathways of angiogenesis with hammerhead ribozymes that reduce the expression of specific receptors. In an attempt to only influence areas of neovascularization while not altering the resident endothelial cells we designed enhancer/promoter elements that limit the expression of these ribozymes to proliferating endothelial cells. Methods: Two proliferating endothelial cell–specific enhancer/promoters were produced. These enhancer/promoters both contain the cell division cycle 6 (cdc6) promoter preceded by either four or seven endothelial cell–specific enhancer elements (ET/cdc6). These enhancer/promoters drive the expression of a luciferase reporter gene. Luciferase activity was used to confirm cell specificity in human retinal endothelial cells (HRECs) compared to human fibroblasts. Luciferase immunolocalization was used to determine cell specificity in vivo. The insulin–like growth factor–1 receptor (IGF–1R) ribozyme was cloned downstream of the luciferase gene, and transcription produces a single contiguous transcript containing both luciferase and the ribozyme. The ability of the ribozyme to inhibit neovascularization while not injuring the established or developing vasculature was examined using two models: the oxygen–induced retinopathy (OIR) mouse model and an adult mouse model of laser–induced neovascularization. Results: The ET/cdc6 promoter drove the expression of luciferase in HRECs but not in human fibroblasts. The expression of luciferase in vivo was limited to the endothelial cells in areas of neovascularization in both the OIR and the adult mouse models. The expression of the IGF–1R ribozyme by the ET/cdc6 promoter resulted in reduced neovascularization in the oxygen–induced retinopathy (OIR) mouse model (51 ± 15%). The adult mouse model of laser–induced neovascularization also showed comparable reduction of neovascularization. Conclusions: Proliferating endothelial cell–specific promoters allow targeting of the ribozyme expression to the rapidly proliferating chorioretinal endothelial cells. This should allow a safer approach for gene delivery to the ocular vasculature.

Keywords: retinal culture • retinal neovascularization • receptors: pharmacology/physiology