PEDF and Photoreceptor Protection: A Naked DNA Approach

描述（由申请人提供）：PEDF和光受体保护：裸DNA方法。视网膜色素变性、年龄相关性黄斑变性和视网膜脱离等致盲性疾病的感光细胞死亡是不可逆的。 色素上皮衍生因子（PEDF）是一种具有神经营养和抗血管生成特性的分泌蛋白，由胎儿和成年动物的多种细胞在体内产生。在视网膜内，它由视网膜色素上皮（RPE）细胞分泌到感光细胞间基质中，将其置于影响底层感光细胞的主要物理位置。我们最近报道了玻璃体内注射PEDF保护大鼠模型中的感光细胞免受恒定光诱导的损伤。另一种将蛋白质因子递送至细胞和组织的方法是仅使用编码蛋白质的DNA。我们的初步数据表明，大鼠视网膜神经元的原代细胞培养物可以转染与裸DNA编码增强绿色荧光蛋白的控制下的感光细胞特异性启动子，小鼠interphotoreceptor视黄醇结合蛋白。我们已经进行了类似的实验，在其他感光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 cDNA转染到视网膜神经元中，然后表达PEDF蛋白。我正在寻求三年的资金在这个建议，以开发一种新的方法，使用质粒DNA盒表达PEDF，并将其应用于视网膜，以延长有用的视力，通过延迟或预防视网膜变性。这种新的方法实际上正在I期临床试验中使用，将编码血管生成因子VEGF的裸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在癌症患者中也显示出显著的治疗效果。本发明的具体目的是：I）证明转染的编码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率。2)为了最大化裸质粒PEDF在眼内的体内表达，通过优化给药途径，如局部应用、前房/玻璃体内/视网膜下注射。3)评价该方案对光诱导或遗传性视网膜变性的保护作用。拟议的研究是创新的，作为一种新的治疗方法，可能会产生重大的临床影响，以延迟或预防视网膜变性，并延长有用的视力。

DESCRIPTION (provided by applicant): PEDF and photoreceotor protection: a naked DNA approach. Photoreceptor cell death is an irreversible event in many blinding diseases including retinitis pigmentosa, 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 vivo 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 photoreceptor cells. We recently reported that the intravitreal injection of PEDF protected photoreceptor cells against constant light induced damage in a rat model. An alternative method for 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 transfect PEDF cDNA 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: I) To demonstrate the expression and protection efficiency of transfected naked plasmid DNA, encoding PEDF, in cultured rodent retinal cells. 2) To maximize the in vivo 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.