Sustained, Non-viral Ocular Therapy Using Nanoparticles

描述（由申请人提供）：我们的研究目标是使用DNA纳米技术和非辅助分子依赖性睡美人转座子-转座酶基序来开发一种新颖有效的眼科疾病治疗策略;特别是与视网膜和视网膜色素上皮（RPE）中的功能丧失突变相关的眼科疾病。眼部治疗的成功应用取决于治疗剂以细胞特异性方式的有效递送和靶向。我们建议测试以下假设：压实的DNA纳米颗粒是一种有效、高效和耐受性良好的方法，用于治疗性基因递送和靶向视网膜和RPE细胞，以分别对抗视网膜和RPE疾病。由于纳米颗粒很小且浓缩，它们可以安全地穿过细胞膜和核膜并进入细胞核。我们的实验计划需要三个组成部分：工程纳米颗粒，基因工程治疗和眼部疾病的动物模型，所有这些都是目前在我们的实验室。将使用视网膜电图（ERG）和多焦ERG在功能上评估视网膜疾病的治疗性挽救，将使用免疫荧光、光学和电子显微镜评估结构分析，将使用真实的时间RT-PCR、RNA酶保护、蛋白质印迹和ELISA分析进行生化分析。这些研究将为非病毒基因治疗作为治疗遗传性致盲性疾病的前瞻性方法提供有价值的见解。

DESCRIPTION (provided by applicant): Our research objective is to use DNA nanotechnology and helper-independent Sleeping Beauty Trasposon-Transposase motif to develop a novel and effective therapeutic strategy for eye diseases; particularly those associated with loss-of-function mutations in the retina and retinal pigment epithelium (RPE). Successful application of ocular therapy is contingent upon the efficient delivery and targeting of therapeutic agents in a cell-specific manner. We propose to test the hypothesis that compacted DNA nanoparticles is an effective, efficient, and well-tolerated method for therapeutic gene delivery and targeting to the retina and RPE cells to respectively battle diseases of the retina and RPE. Since nanoparticles are small and condensed, they can pass safely through the cell membrane as well as the nuclear membrane and into the nucleus. Our experimental plan requires three components: engineered nanparticles, genetically engineered therapy, and animal models of ocular diseases, all of which are currently available in our laboratory. Therapeutic rescue of retinal disease will be assessed functionally using electroretinography (ERG) and multifocal ERG, structural analysis will be assessed using immunofluorescence, light and electron microscopy, and biochemical analysis will use real time RT-PCR, RNase protection, Western blot, and ELISA analysis. When taken together, these studies will provide valuable insight into non-viral gene therapy as a prospective method for the treatment of inherited blinding diseases.