DIRECTED GENE TRANSFER INTO RPE CELLS

视网膜色素上皮(RPE)在 维持神经感觉性视网膜的健康和功能。 遗传性和获得性RPE功能障碍是许多 重要的致盲疾病，包括某些形式的视网膜炎 色素变性、视网膜营养不良和老年性黄斑变性。我们 相信在潜在的治疗上最大的希望是 遗传性和获得性视网膜变性的发生和发展 针对宿主视网膜色素的定向基因治疗的改进 上皮组织。我们的工作假设是直接脂质体介导的 基因转移可在体内高效、高效地转染RPE细胞 可以传递能够持续表达的功能基因 在主机RPE中。 这一假说的概念基础是内在的 RPE的吞噬功能使该细胞成为理想的宿主 用于脂质体介导的基因转移。脂质体介导的基因转移 允许大分子量脱氧核糖核酸的转染S，潜在地 能够取代缺陷基因的可翻译基因 视网膜退行性变。 具体目标和方法1：开发和优化高效脂质体-- 哺乳动物视网膜色素上皮介导的基因转移方案 体外培养细胞：I)评价和优化体外培养条件 脂质体在人和兔RPE细胞中的内吞作用，II)测定 不同脂质体制剂在体内的相对释药效率 人和兔RPE细胞，以及iii)评估 不同脂质体制剂对体外细胞的影响。2：评估外生性 脂质体介导的RPE细胞DNA摄取和表达频率 利用报告基因β-半乳糖苷酶或 选择标记基因新霉素抗性：I)定量摄取 外源DNA序列，II)体外培养和转染法 人和兔RPE细胞最大摄取DNA的条件 品系，III)外源DNA的定量转录和翻译 序列。3：优化脂质体介导的基因转移系统 活体哺乳动物视网膜色素上皮细胞：I)毒性评估 活体视网膜中脂质体/DNA复合体的研究，II)评价转染性 视网膜色素上皮的效率，III)优化条件 在体内将基因转移到RPE细胞并评估表达持续时间 活着的视网膜中的转基因基因。

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.