GENE THERAPY FOR AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA

在所有神经源性组织中，遗传疾病的分子基础是 最好的理解是视网膜。该项目旨在了解和 在分子水平治疗常染色体显性视网膜色素变性（ADRP） 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）靶向的方式， 选择性地抑制突变蛋白的产生，和（3）有效动物 ADRP模型，用于测试和优化（1）和（2）。 (1)使用重组腺相关病毒（rAAV），其中表达 是由视杆视蛋白启动子的一部分驱动的，我们已经实现了 主要（但不是绝对）光受体特异性表达 通过眼部注射在小鼠、兔和豚鼠中表达报告基因。我们 建议系统地研究rAAV构建体的能力， 视蛋白调节序列的片段以实现最大可控， 病毒包装的过客基因的细胞类型特异性表达。 (2)我们已经合成了几种核酶（RNA分子）的基因 能够破坏特定的靶RNA。 这些核酶识别 一种ADRP中引起P23 H突变的核苷酸变化， S334ter突变。我们建议插入这些核酶（和 其他可能改进的核酶）进入rAAV，并将它们递送至 携带这些突变形式的视杆视蛋白的转基因大鼠的视网膜， 表现出RP样症状 (3)转基因大鼠P23H或S334ter突变的研究 在视蛋白启动子控制下的视蛋白基因表现出视网膜变性过程， 与在携带相同病毒的人类中观察到的疾病非常相似的疾病 突变。 我们建议将rAAV-核酶注射到P23 H的玻璃体中， 和S334 ter转基因大鼠，以确定其病程是否 动物模型中的RP样疾病可以用最少的 致病副作用分析包括视网膜色素变性的形态学分析。 变性、定量mRNA研究和视网膜电图。作为证据 原则上，也将测试含核酶的转基因小鼠。

Of all neural-derived tissue, the molecular basis of genetic disease is best understood in the retina. 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 targeted way to selectively inhibit production of the mutant protein, and (3) valid animal models of ADRP in which to test and optimize (1) and(2). (1) 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, rabbit, and guinea pig by ocular injection. We propose to systematically study the ability of rAAV constructs containing segments of the opsin regulatory sequence to achieve maximum controllable, cell-type specific expression of the virally- packaged passenger gene. (2) We have made synthetic genes for the several ribozymes (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 propose to insert these ribozymes (and other, potentially improved ribozymes) into a rAAV and deliver them to the retina of transgenic rats bearing these mutant forms of rod opsin and exhibiting RP-like symptoms. (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 the same mutations. We propose to inject rAAV-ribozymes into the vitreous of P23H and S334ter transgenic rats in order to determine whether the course of the RP-like disease in animal models can be ameliorated with a minimum of pathogenic side effects. Assays include morphological analysis of retinal degeneration, quantitative mRNA studies, and electroretinography. As proof of principle, ribozyme -containing transgene mice will also be tested.