Expression of Therapeutic Proteins in the Whole Lens

描述（由申请人提供）：我们实验室的最新研究表明，大分子（如质粒DNA）可进入完整的透镜，并定位于透镜上皮细胞中。基于这一观察，该建议的总体假设是，在整个透镜中内化的质粒DNA可以表达治疗上有用量的蛋白质，例如酶超氧化物歧化酶（SOD）和透镜上皮衍生生长因子（LEDGF）。这两种成分都可以防止氧化应激引起的间隙连接活性变化和随后的白内障发生。具体的目的是1）确定是否可以在培养物中用LEDGF或SOD转染完整的透镜，使用具有各种启动子的质粒，所述启动子包括驱动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在整个透镜中的表达是否将防止暴露于LEDGF或SOD后的白内障形成，3）确定是否可以在培养物中用LEDGF或SOD转染完整的晶状体。 过氧化氢，氧化性白内障模型，或高葡萄糖，糖尿病模型，3）确定完整的透镜是否可以通过活体大鼠的玻璃体内注射用这些相同的载体转染。 总之，这些研究将首次测试使用质粒改变完整透镜的基本代谢的可行性，通过表达治疗上重要的大分子，其将抑制被认为导致透镜混浊的氧化过程。

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.