A Ribozyme Rescue Strategy for Autosomal Dominant RP

描述(由申请人提供)：人类视杆蛋白基因包含许多导致视网膜色素变性(RP)的突变。折叠视蛋白基因是核酶基因治疗常染色体显性遗传性视网膜病变的靶点。研究表明，具有数百个潜在切割位点的折叠mRNAs几乎没有允许强大的Rz催化的可访问区域。长期目标是建立一种快速生成活性Rz设计的方法，作为对任何特定突变的人类遗传性视网膜变性等位基因的候选基因治疗。其目的是构建活性RZS，以切割人视杆蛋白adrp mRNAs，为临床试验提供潜在的用途。中心假设是，opsin mRNA的折叠结构将保护大多数潜在的位置不被Rz切割。要找到最佳的切割位点，需要用多种方法来筛选许多潜在的HRZ设计，使之与人类mRNA折叠的精确模型相对照。预测mRNA结构可及性的折叠算法和针对裸露或蛋白质包被的折叠视蛋白mRNA的Rz文库的经验筛选的组合提供了优化的研究策略。这样确定的活性Rz基因可以克隆到表达载体中，以确定当在培养细胞中表达时，Rz是否能够识别和切割其目标基因，沿着正常的mRNA加工/运输流。通过这种分层测试的设计将在表达人类视蛋白等位基因的小鼠视网膜退化模型中进行抢救和毒性测试。其基本原理是，在体外和细胞培养系统中，将能够快速、高通量地鉴定有效的Rz设计，这些设计具有切割体内任意显性疾病等位基因转录的靶人类mRNAs的功能。为了验证中心假说和实现目标，将追求三个特定的目标：目的1.定位折叠的人视杆蛋白基因中最容易接近的区域。目的2.开发有效的RZS来切割视蛋白mRNA可及区域的NUH。目的3.通过在表达人视杆蛋白转基因的小鼠模型上测试视网膜变性的挽救和毒性，对击倒Rz策略进行临床前测试。这项拟议的工作具有创新性，因为它开发了一种方法，可以快速完成针对新的任意人类显性疾病等位基因的候选Rz基因治疗药物，并提供了已知的针对一个已知ADRP疾病等位基因的击倒Rz策略的临床前测试。预期的结果是，视杆蛋白mRNA的折叠结构将严重限制可访问的Rz裂解位点的数量，可以构建对视蛋白表达有显著影响的高活性RZS，并且敲除策略或相关的修饰将挽救动物模型中的视网膜退化。其意义在于，这项工作产生的有效的敲除RZS，证明了从退化中拯救和光感受器毒性的缺乏，然后可以转化为ADRP的人类临床试验。

DESCRIPTION (provided by applicant): The human rod opsin gene harbors many mutations that cause retinitis pigmentosa (RP). The folded opsin mRNA is a target for ribozyme (Rz) gene therapy for autosomal dominant (ad) RP. Studies have shown that folded mRNAs with hundreds of potential cleavage sites have few accessible regions that permit robust Rz catalysis. The long-range goal is to establish a rapid means of generating active Rz designs as candidate gene therapies for any ad mutant human hereditary retinal degeneration allele. The objective is to build active Rzs that cut human rod opsin adRP mRNAs for potential use in clinical trials. The central hypothesis is that the folded structure of opsin mRNA will shield most potential sites from Rz cleavage. Finding the best cleavage sites requires methods to screen many potential hRz designs against precise models of folded human mRNA. A combination of folding algorithms to predict mRNA structural accessibility, and empirical screens of Rz libraries against naked or protein-coated folded opsin mRNA provide for an optimized research strategy. Active Rz genes so identified can be cloned into expression constructs to determine if the Rz can identify and cleave its target along the normal mRNA processing/trafficking streams when expressed in cultured cells. Designs passing this hierarchial test will be tested for rescue and toxicity in murine retinal degeneration models that express human opsin alleles. The rationale is that in vitro and cell culture systems will permit rapid, high-throughput identification of efficacious Rz designs that function to cleave target human mRNAs transcribed from arbitrary dominant disease alleles in vivo. To test the central hypothesis and accomplish the objective three Specific Aims will be pursued: Aim 1. Locate the most accessible regions in folded human rod opsin mRNA. Aim 2. Develop efficacious Rzs to cleave NUH( sites in accessible regions of opsin mRNA. Aim 3. Conduct a preclinical test of the Knockdown Rz strategy by testing for rescue of retinal degeneration and toxicity in murine models that express human rod opsin transgenes. The proposed work is innovative in that it develops an approach to rapidly accomplish candidate Rz gene therapy agents for new arbitrary human dominant disease alleles, and it provides the first known preclinical test of the knockdown Rz strategy for one known adRP disease allele. The expected results are that the folded structure of rod opsin mRNA will severely constrain the number of accessible Rz cleavage sites, that highly active Rzs can be constructed that have marked impact on opsin expression, and that the knockdown strategy or related modifications will rescue retinal degeneration in an animal model. The significance is that efficacious knockdown Rzs generated by this work, which demonstrate both rescue from degeneration and lack of photoreceptor toxicity, could then be translated into human clinical trials for adRP.