Genoplasty in Ocular Gene Therapy

描述：(申请者摘要)没有争论，最重要的目标 基因治疗的核心是将体细胞内源基因修复为基因缺陷。这 Goal回避了与当前基因策略相关的大多数主要问题 病毒或前病毒、反义寡核苷酸或 核酶。我们的目的是修复一个基因，就像正常的细胞过程一样 核苷酸切除修复或错配修复，这发生在一个完美的 在人类细胞生理学中日常发生的正常现象。我们的目标是雇佣 眼睛的正常DNA修复酶并提供适当的修复 连接到细胞核的底物。在提供了这种衬底之后，我们将 依靠细胞的正常DNA修复酶来识别单个 底物和内源基因的核苷酸不匹配和替换 带有野生型核苷酸的缺陷碱基。这个过程现在被称为 基因成形术。 我们的主要模型系统将是常见的 人眼皮肤酪氨酸酶阴性白化病、小白鼠和新西兰 白兔。使用基因成形术，我们计划修复确切已知的病变 酪氨酸酶基因在RPE细胞中的体细胞表达。 因此，这项研究将对基因的未来发展方向产生重大影响。 眼睛里的治疗。这是新奇的，没有其他人在眼睛里这样做，而且它 是创新的，因为它使用了内源基因和正常的DNA修复酶 让细胞自我修复。 我们的指导性假设是，基因整形是通过大规模行动起作用的。越多 将底物放入细胞中，基因修复发生的可能性就越大。我们 进一步假设几乎每个细胞都有正确的DNA修复酶 和辅助蛋白。因此，要实现基因修复，我们所需要做的就是 适量的底物进入原子核，自然界就会吸收它的 当然了。我们的目标是：1)建立更好的检测基因修复的方法 酪氨酸酶基因在体细胞RPE细胞中的表达，2)获得高水平的 眼组织中的转基因和核定位；3)设计 更好的衬底。

DESCRIPTION: (Applicant's Abstract) Without argument, the most important goal of gene therapy is to fix the somatic endogenous gene in a gene defect. This goal circumvents most major problems associated with current strategies of gene therapy mediated by viruses or proviruses, antisense oligonucleotides, or ribozymes. Our intent is to repair a gene much like the normal cellular process of nucleotide excision repair or mismatch repair, which occurs as a perfectly normal everyday occurrence in human cellular physiology. Our aims are to employ the normal DNA repair enzymes of the eye and to supply a suitable repair substrate to the nucleus of the cell. Having provided this substrate, we will rely on the normal DNA repair enzymes of the cell to recognize a single nucleotide mismatch between the substrate and the endogenous gene and replace the defective base with the wildtype nucleotide. This process is now known as genoplasty. Our principal model systems will be the commonly available animal models of human oculocutaneous tyrosinase-negative albinism, white mice and New Zealand white rabbits. Using genoplasty, we plan to repair precisely known lesions in the tyrosinase gene somatically in RPE cells. Thus, this research will have high impact on the future directions of gene therapy in the eye. It is novel, no one else is doing this in the eye, and it is innovative because it uses the endogenous gene and normal DNA repair enzymes to allow the cell to fix itself. Our guiding hypothesis is that genoplasty works by mass action. The more substrate put into the cell, the more likely that gene repair will occur. We further hypothesize that virtually every cell has the right DNA repair enzymes and ancillary proteins. Thus, to achieve gene repair, all we need to do is get an adequate amount of substrate into the nucleus, and nature will take its course. Our aims are: 1) to develop better assays for detecting gene repair of the tyrosinase gene in somatic RPE cells, 2) to achieve high levels of transfection and nuclear localization in ocular tissues, and 3) to design better substrates.