GENE THERAPY FOR AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA

在所有神经源性组织中，遗传病的分子基础是 最容易理解的是视网膜。该项目的目的是了解和 常染色体显性遗传性视网膜色素变性的分子治疗 水平。成功的adrp基因治疗需要：(1)有效和 细胞类型特异性基因传递/表达系统，(2)靶向途径 选择性地抑制突变蛋白的产生，以及(3)有效动物 要测试和优化的adrp模型(1)和(2)。 (1)使用重组腺相关病毒(RAAV)，其中表达 是由一部分视蛋白驱动的杆状启动子，我们已经实现了 主要(但不是绝对)光感受器特异性表达 小鼠、兔和豚鼠眼内注射的报告基因。我们 建议系统地研究rAAV构建物的能力，包括 视蛋白调节序列的片段以实现最大的可控性， 病毒包装的乘客基因的细胞类型特异性表达。 (2)我们合成了几种核酶(RNA分子)的基因 能够摧毁特定的靶标RNA。这些核酶识别 导致一种形式的ADRP的P23H突变的核苷酸变化和 另一例S334ter突变。我们建议插入这些核酶(和 其他可能改进的核酶)转化为rAAV，并将它们传递给 携带这些突变型视蛋白和视蛋白的转基因大鼠的视网膜 表现出类似RP的症状。 (3)杆状突变型P23H或S334ter转基因大鼠品系 视蛋白启动子控制下的视蛋白基因呈现视网膜病程 这种疾病与在携带同样疾病的人身上观察到的非常相似 突变。我们建议将rAAV-ribozyme注射到P23H的玻璃体中 和S334ter转基因大鼠，以确定其病程是否 动物模型中的RP样病可以得到改善，只要使用最少的 致病的副作用。分析包括视网膜的形态分析。 变性、定量信使核糖核酸研究和视网膜电描记术。作为证据 原则上，含有核酶的转基因小鼠也将接受测试。

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.