A Ribozyme Rescue Strategy for Autosomal Dominant RP

描述（由申请人提供）：人类视蛋白杆状蛋白基因包含许多导致视网膜色素变性（RP）的突变。折叠的视蛋白mRNA是核酶（Rz）基因治疗常染色体显性（ad） RP的靶标。研究表明，具有数百个潜在切割位点的折叠mrna几乎没有允许强大Rz催化的可访问区域。长期目标是建立一种快速产生活性Rz设计的方法，作为任何突变的人类遗传性视网膜变性等位基因的候选基因疗法。目的是构建能够切割人视杆蛋白adRP mrna的活性Rzs，用于临床试验。核心假设是，视蛋白mRNA的折叠结构将保护大多数潜在位点免受Rz切割。为了找到最佳的切割位点，需要针对折叠人类mRNA的精确模型筛选许多潜在的hRz设计。结合折叠算法预测mRNA的结构可及性，以及Rz文库针对裸或蛋白包被折叠视蛋白mRNA的经验筛选，提供了优化的研究策略。这样鉴定的活性Rz基因可以克隆到表达构建体中，以确定Rz在培养细胞中表达时是否能够沿正常mRNA加工/运输流识别和切割其靶标。通过这一等级测试的设计将在表达人类视蛋白等位基因的小鼠视网膜变性模型中进行拯救和毒性测试。其基本原理是，体外和细胞培养系统将允许快速，高通量鉴定有效的Rz设计，其功能是切割从体内任意显性疾病等位基因转录的目标人类mrna。为了检验中心假设并实现目标，将追求三个具体目标：目标1。定位折叠的人视蛋白棒状mRNA中最易接近的区域。目标2。开发有效的Rzs来切割视蛋白mRNA可达区域的NUH位点。目标3。通过在表达人视杆蛋白转基因的小鼠模型中测试对视网膜变性的拯救和毒性，对Knockdown Rz策略进行临床前测试。这项工作的创新之处在于，它开发了一种快速完成针对新的任意人类显性疾病等位基因的候选Rz基因治疗药物的方法，并且它提供了已知的第一个针对已知adRP疾病等位基因的敲除Rz策略的临床前测试。预期的结果是杆视蛋白mRNA的折叠结构将严重限制可访问的Rz切割位点的数量，可以构建对视蛋白表达有显著影响的高活性Rz，以及敲低策略或相关修饰将挽救动物模型中的视网膜变性。重要的是，这项工作产生的有效敲除Rzs，证明了从变性中拯救和缺乏光感受器毒性，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.