DIRECTED GENE TRANSFER INTO RPE CELLS

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The retinal pigment epithelium (RPE) plays a critical role in maintaining the health and function of the neurosensory retina. Inherited and acquired RPE dysfunction is responsible for a number of important blinding diseases including certain forms of retinitis pigmentosa, retinal dystrophy and age-related macular degeneration. We believe that the greatest promise in the potential treatment of inherited and acquired retinal degeneration lies in the development and refinement of directed gene therapy to the host retinal pigment epithelium. Our working hypothesis is that directed liposome-mediated gene transfer can transfect RPE cells in vivo with high efficiency and can deliver functional genes which are capable of sustained expression in host RPE. The conceptual basis for this hypothesis is that the intrinsic phagocytic function of the RPE makes this cell an ideal host recipient for liposome-mediated gene transfer. Liposome-mediated gene transfer allows transfection of large molecular weight DNA s, potentially translatable genes capable of replacing the defective genes which result in retinal degeneration. Specific Aims and Methods 1: Develop and optimize an efficient liposome- mediated gene transfer protocol for mammalian retinal pigment epithelial cells in vitro: i) assess and optimize conditions of in vitro endocytosis of liposomes in human and rabbit RPE cells, ii) determine relative delivery efficiencies of different liposome preparations in humans and rabbit RPE cells, and iii) assess relative toxicity of different liposome preparations on cells in vitro. 2: Assess exogenous DNA uptake and expression frequencies in RPE cells by liposome-mediated gene transfer utilizing the reporter gene beta-galactosidase or the selective marker gene neomycin resistance: i) quantitate uptake of exogenous DNA sequences, ii) determine in vitro culture and transfection conditions which maximize DNA uptake in the human and rabbit RPE cell lines, iii) quantitate transcription and translation of exogenous DNA sequences. 3: Optimize this liposome-mediated gene transfer system for mammalian retinal pigment epithelial cells in vivo: I) assess toxicity of liposome/DNA complexes in the living retina, ii) assess transfection efficiencies in the retinal pigment epithelium, iii) optimize conditions for gene transfer to RPE cells in vivo and assess duration of expression of transfected genes in the living retina.