Expression of Therapeutic Proteins in the Whole Lens

描述（由申请人提供）：我们实验室最近的工作表明，质粒DNA等大分子可以进入完整的晶状体，并定位在晶状体上皮中。基于这一观察结果，本提议的总体假设是，整个晶状体内化的质粒DNA可以表达治疗上有用的蛋白质，如超氧化物歧化酶（SOD）和晶状体上皮衍生生长因子（LEDGF）。这两种成分都可以防止由氧化应激引起的间隙连接活性变化和随后的白内障发生。具体目的将是：1)确定完整的晶状体是否可以用LEDGF或SOD转染培养物，使用含有各种启动子的质粒，包括驱动LEDGF- gfp和SOD- gfp融合蛋白表达的晶状体特异性启动子；2)确定整个晶状体中LEDGF或SOD的表达是否会防止暴露于

DESCRIPTION (provided by applicant): Recent work from our laboratory has demonstrated that macromolecules such as plasmid DNA can pass into the intact lens, where they are localized in the lens epithelium. Based upon this observation, the overall hypothesis of this proposal is that plasmid DNA internalized in the whole lens can express therapeutically useful quantities of proteins such as the enzyme superoxide dismutase (SOD) and the lens epithelial derived growth factor (LEDGF). Both of these components protect against changes in gap junction activity and subsequent cataractogenesis that are caused by oxidative stress. The specific aims will be to 1) determine if the intact lens can be transfected in culture with LEDGF or SOD, using plasmids with various promoters including a lens-specific promoter that drives expression of LEDGF-GFP and SOD-GFP fusion proteins, 2) determine if expression of LEDGF or SOD in the whole lens will prevent cataract formation following exposure to hydrogen peroxide, an oxidative cataract model, or high glucose, a model for diabetes, 3) determine if the intact lens can be transfected with these same vectors by intravitreal injection of living rats. Together, these studies will test for the first time, the feasibility of using plasmids to alter the fundamental metabolism of the intact lens, through expression of therapeutically important macromolecules that will inhibit the oxidative processes thought to cause lens opacification.