Genoplasty in Ocular Gene Therapy

描述：（申请人摘要）没有争论，最重要的目标 基因治疗的主要目的是将体细胞内源性基因修复在基因缺陷中。这 目标避免了与当前基因策略相关的大多数主要问题， 由病毒或前病毒、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 核苷酸切除修复或错配修复，这是一个完美的 在人体细胞生理学中的正常日常发生。我们的目标是雇用 眼睛的正常DNA修复酶，并提供合适的修复 细胞核的基质。在提供了这个基底之后，我们将 依靠细胞的正常DNA修复酶来识别单个 底物和内源基因之间的核苷酸错配， 有缺陷的碱基与野生型核苷酸。这个过程现在被称为 基因整形术 我们的主要模型系统将是常见的动物模型， 人眼皮肤酪氨酸酶阴性白化病、白色小鼠和新西兰 白色兔子。使用基因成形术，我们计划修复精确已知的病变， 酪氨酸酶基因在RPE细胞中的体细胞学作用。 因此，本研究将对基因工程的未来发展方向产生重大影响。 治疗眼睛。它是新颖的，没有其他人在做这件事，而且它 是创新的，因为它使用了内源性基因和正常的DNA修复酶， 让细胞自我修复 我们的指导假设是基因整形术是通过群体作用起作用的。越 底物进入细胞，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， 和辅助蛋白质。因此，要实现基因修复，我们所需要做的就是 足够数量的底物进入细胞核，自然界将采取其 当然了我们的目标是：1）开发更好的检测基因修复的方法， 体细胞RPE细胞中的酪氨酸酶基因，2）实现高水平的 眼组织中的转染和核定位，以及3）设计 更好的基板。

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.