TX OF AUTOSOMAL DOMINANT EYE DISEASE USING CATALYTIC RNA

该项目旨在了解和治疗常染色体 显性视网膜色素变性（ADRP）在分子水平。 成功的ADRP基因治疗需要：（1）有效的， 细胞类型特异性基因递送/表达系统，（2）选择性 抑制突变蛋白质产生的手段，以及（3）有效 ADRP的动物模型，其中测试和优化（1）和（2）。 (1)我们已经合成了几种核酶的基因 （催化RNA分子能够破坏特定的目标， RNA）。 这些核酶识别核苷酸的变化， 一种ADRP中的P23 H突变和S334 ter 另一种变异。 我们将在体外和培养的 细胞，并计划提供最活跃的版本， 核酶的视网膜转基因率携带这些突变 视杆视蛋白的形式并表现出RP样症状。 (2)使用 重组腺相关病毒（rAAV），其中表达 是由视杆细胞视蛋白启动子的一部分驱动的， 实现了主要的（但不是绝对的）光受体特异性 报告基因在小鼠中的表达和通过眼部注射的速率。 我们提出了一个系统的研究rAAV结构， 视蛋白调节序列的片段， 病毒包装乘客的细胞类型特异性表达 基因 (3)携带P23 H或S334 ter的转基因大鼠系 视蛋白控制下的视杆细胞视蛋白基因突变 启动子显示视网膜疾病过程与 在携带这种突变的人类身上观察到的。 我们建议 在转基因大鼠中测试P23 H和S334 ter中的rAAV-核酶， 确定RP样疾病的病程是否可以 以最小的致病副作用改善。 作为 在这些模型中，我们对核酶的功效进行了独立测试， 还将制备表达P23 H核酶的转基因小鼠 并在与P23 H转基因小鼠交配后测定其活性 小鼠 活性的测定包括以下的形态学分析： 视网膜变性，定量mRNA研究， 视网膜电图 我们正在验证病毒介导视网膜病变的特定假设， 核糖酶的传递以减少显性阴性的表达 基因将是ADRP的治疗剂。 从更广泛的角度来看， 系统的方法来优化调控表达， 分化的组织应该在多个层面上 人类基因疗法

This project is aimed at understanding and treating autosomal dominant retinitis pigmentosa (ADRP) at a molecular level. Successful gene therapy for ADRP requires: (1) an efficient and cell-type specific gene delivery/expression system, (2) a selective means of inhibiting production of the mutant protein, and (3) valid animal models of ADRP in which to test and optimize (1) and (2). (1) We have made synthetic genes for the several ribozymes (catalytic RNA molecules capable of destroying specific target RNAs). These ribozymes recognize the nucleotide change causing the P23H mutation in one form of ADRP and the S334ter mutation in another. We will test these in vitro and in cultured cells and plan to deliver the most active versions of these ribozymes to the retinas of transgenic rates bearing these mutant forms of rod opsin and exhibiting RP-like symptoms. (2) Using a recombinant Adeno-associated virus (rAAV) in which expression is driven by a portion of the rod opsin promoter, we have achieved predominant (but not absolute) photoreceptor-specific expression of reporter genes in mouse and rate by ocular injection. We propose a systematic study of rAAV constructs containing segments of the opsin regulatory sequence to achieve controllable,, cell-type specific expression of the virally-packaged passenger gene. (3) Transgenic rat lines carrying the P23H or S334ter mutation in the rod opsin gene under control of the opsin promoter exhibit a course of retinal disease remarkably similar to that observed in humans bearing such mutations. We propose to test rAAV-ribozymes in P23H and S334ter in transgenic rats to determine whether the course of the RP-like disease can be ameliorated with a minimum of pathogenic side effects. As an independent test of the efficacy of ribozymes in these models, we will also prepare transgenic mice expressing the P23H ribozymes and measure its activity following matings with P23H transgenic mice. Assays for activity include morphological analysis of retinal degeneration, quantitative mRNA studies, and electroretinography. We are testing the specific hypothesis that viral mediated retinal delivery of ribozymes to reduce expression of a dominant negative gene will be therapeutic for ADRP. In a broader view, this systematic approach to optimizing regulated expression in a differentiated tissue should contribute at many levels toward human gene therapy.