Sustained, Non-viral Ocular Therapy Using Nanoparticles

描述(申请人提供)：我们的研究目标是使用DNA纳米技术和非依赖助手的睡美人转座酶基序来开发一种新颖而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)功能丧失突变相关的疾病。眼科治疗的成功应用取决于以细胞特异性的方式有效地输送和靶向治疗剂。我们建议检验这一假设，即致密的DNA纳米颗粒是一种有效、高效和耐受性良好的治疗性基因输送方法，并靶向视网膜和RPE细胞，分别对抗视网膜和RPE疾病。由于纳米粒子很小且浓缩，它们可以安全地穿过细胞膜和核膜，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、基因工程疗法和眼部疾病的动物模型，所有这些目前都在我们的实验室提供。视网膜疾病的治疗抢救将使用视网膜电图(ERG)和多焦点ERG从功能上进行评估，结构分析将使用免疫荧光、光学和电子显微镜进行评估，生化分析将使用实时RT-PCR、核糖核酸酶保护、Western印迹和ELISA分析。综上所述，这些研究将为非病毒基因疗法作为治疗遗传性致盲疾病的一种有前景的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.