A Ribozyme Rescue Strategy for Autosomal Dominant RP

描述（由申请人提供）：人视杆细胞视蛋白基因含有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遗传（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Rz催化的可接近区域。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ad突变型人类遗传性视网膜变性等位基因的候选基因疗法。目的是构建切割人视杆视蛋白adRP mRNA的活性Rzs，以用于临床试验。核心假设是视蛋白mRNA的折叠结构将屏蔽大多数潜在位点不受Rz切割。找到最佳切割位点需要针对折叠的人mRNA的精确模型筛选许多潜在的hRz设计的方法。预测mRNA结构可及性的折叠算法的组合，以及针对裸的或蛋白质包被的折叠视蛋白mRNA的Rz文库的经验筛选提供了优化的研究策略。可以将如此鉴定的活性Rz基因克隆到表达构建体中，以确定当在培养的细胞中表达时，Rz是否可以沿着正常的mRNA加工/运输流鉴定和切割其靶标。将在表达人视蛋白等位基因的鼠视网膜变性模型中测试通过该分级测试的设计的拯救和毒性。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，其功能是在体内切割从任意显性疾病等位基因转录的靶人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。在折叠的人视杆视蛋白mRNA中定位最易接近的区域。目标二。开发有效的Rz以切割NUH（视蛋白mRNA的可接近区域中的位点）。目标3：通过在表达人视杆视蛋白转基因的鼠模型中测试视网膜变性和毒性的拯救，进行Knockdown Rz策略的临床前测试。所提出的工作是创新的，因为它开发了一种方法来快速实现新的任意人类显性疾病等位基因的候选Rz基因治疗剂，并且它提供了一种已知的adRP疾病等位基因的敲低Rz策略的第一个已知的临床前测试。预期的结果是视杆视蛋白mRNA的折叠结构将严重限制可接近的Rz切割位点的数量，可以构建对视蛋白表达具有显著影响的高活性Rz，并且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，通过这项工作产生的有效敲低Rzs，其证明了从变性中的拯救和缺乏感光细胞毒性，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.