Expression of Therapeutic Proteins in the Whole Lens

描述(申请人提供)：我们实验室最近的工作表明，大分子如质粒DNA可以进入完整的晶状体，在那里它们定位于晶状体上皮细胞。基于这一观察，这一建议的总体假设是，整个晶状体内的质粒DNA可以表达大量治疗有用的蛋白质，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缝隙连接活性的变化和随后由氧化应激引起的白内障发生。具体的目的将是：1)确定完整的晶状体是否可以在含有LEDGF或SOD的培养中进行转基因，使用带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LEDGF或SOD在整个晶状体中的表达是否可以防止暴露于 过氧化氢，一种氧化性白内障模型，或高血糖，一种糖尿病模型，3)确定是否可以通过玻璃体内注射活体大鼠的方式将这些相同的载体导入完整的晶状体。总之，这些研究将首次测试，通过表达具有治疗意义的重要大分子来改变完整晶状体的基本新陈代谢的可行性，这些大分子将抑制被认为导致晶状体混浊的氧化过程。

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.