PEDF and Photoreceptor Protection: A Naked DNA Approach

EXCEED提供的空间。PEDF与光感受器保护：裸露DNA方法。感光细胞死亡在许多致盲疾病中是不可逆转的事件，包括视网膜黑色素性视网膜炎、老年性黄斑变性和视网膜脱离。色素上皮衍生因子(PEDF)是一种分泌型蛋白质，具有神经营养和抗血管生成的特性，在胚胎和成年动物体内由多种细胞合成。在视网膜内，它由视网膜色素上皮(RPE)细胞分泌到光感受器间基质中，将其置于最佳物理位置，以影响潜在的光感光细胞。我们最近报道了玻璃体内注射PEDF对光感受器细胞的持续光损伤的保护作用。另一种将蛋白质因子输送到细胞和组织的方法是只使用编码蛋白质的DNA。我们的初步数据表明，在光感受器细胞特异性启动子鼠视黄醇结合蛋白的控制下，可以将编码增强型绿色荧光蛋白的裸露DNA导入原代培养的大鼠视网膜神经细胞。我们已经进行了类似的实验，在其他感光器细胞特异性启动子的控制下表达其他报告基因。这些实验表明，我们能够将PEDF Edna导入视网膜神经元，然后视网膜神经元将表达PEDF蛋白。在这项提案中，我正在寻求三年的资金，以开发一种使用表达PEDF的质粒DNA盒的新方法，并将其应用于视网膜，通过延缓或防止视网膜退化来延长有用的视力。这一新方法实际上被用于I期临床试验，将编码血管生成因子的裸露DNA注射到心脏病患者的心脏中。在几个II期试验中，直接注射DNA对癌症患者也显示出显著的治疗效果。该建议的具体目的是：1)证实编码PEDF的裸质粒DNA在培养的啮齿动物视网膜细胞中的表达和保护效果。2)通过优化给药途径，如局部给药、前房注射、玻璃体腔内注射等，使裸质粒PEDF在眼内的表达最大化。3)评价该方案对防止由光或遗传引起的视网膜变性的保护效果。建议的研究是创新的，作为一种延缓或预防视网膜变性和延长有用视力的新的治疗方法，可能会产生重大的临床影响。俄克拉何马大学眼科中心主任A·麦基眼科研究所608斯坦顿·L·杨大道。俄克拉荷马城，俄克拉荷马州73104 KEYPERSONELS========================================Section End===========================================

EXCEEDTHE SPACE PROVIDED. PEDF and photoreceptor protection: a naked DNA approach. Photoreceptor cell death is an irreversible event in many blinding diseases including retinitis pigrnentosa, age-related macular degeneration and retinal detachment. Pigment epithelium derived factor (PEDF), a secreted protein with both neurotrophic and anti-angiogenic properties, is made in rive by a variety of cells in both fetal and adult animals. Within the retina, it is secreted by retinal pigment epithelial (RPE) cells into the interphotoreceptor matrix, placing it in a prime physical location to affect the underlying photorecoptor cells. We recently reported that the intravitreal injection of PEDF protected photoroceptor cells against constant light induced damage in a rat modcl. An alternative mot_hodfor delivering a protein factor to cells and tissues is to use only the DNA which codes for the protein. Our preliminary data show that primary cell cultures of rat retinal neurons can be transfected with a naked DNA coding for enhanced green fluorescent protein under the control of the photoreceptor cell-specific promoter, mouse interphotoreceptor retinol binding protein, We have performed similar experiments expressing other reporter genes under control of other photoreceptor cell specific promoters. These experiments suggest that we are able to tran_sfeot PEDF eDNA into retinal neurons which will then express the PEDF protein. I am seeking three years of funding in this proposal to develop a novel approach using plasmid DNA cassettes expressing PEDF and apply it to the retina to prolong useful vision by delaying or preventing retinal degeneration. This novel method is actually being employed in phase I clinical trials with the injection of a naked DNA encoding the angiogenesis factor, VEGF, into the hearts of cardiac patients. In several phase II trials, direct injection of DNA has also demonstrated significant therapeutic effects in cancer patients. The specific aims of this proposal are: 1) To demonstrate the expression and protection efficiency of transfected naked plasmid DNA, encoding PEDF, in cultured rodent retinal cells. 2) To maximize the m rive expression of naked plasmid PEDF in the eye, through the optimization of the route of administration, such as topical application, anterior chamber/intravitreal]subretinal injection. 3) To evaluate the protective efficacy of this protocol to prevent the retinal degeneration induced by light or by heredity. The proposed studies are innovative and could have a significant clinical impact as a novel therapeutic approach to delay or prevent retinal degeneration and to prolong useful vision. PERFORMANCSEITE(S)(organizatiocni,ty,state) Departmentof Ophthalmology The Universityof Oklahoma Health sciencesCenter Dean A. McGee Eye Institute 608 Stanton L. Young Blvd. Oklahoma City, Oklahoma 73104 KEYPERSONNELS ========================================Section End===========================================