A Ribozyme Rescue Strategy for Autosomal Dominant RP

常染色体显性 RP 的核酶救援策略

• 批准号: 6803082
• 负责人: JOHN M. SULLIVAN
• 金额: $ 27.95万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6803082
• 关键词: autosomal dominant trait; cytotoxicity; gene expression; gene therapy; genetically modified animals; laboratory mouse; messenger RNA; polymerase chain reaction; retina degeneration; retinitis pigmentosa; rhodopsin; ribozymes

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.

描述（由申请人提供）：人类视杆细胞视蛋白基因含有许多导致色素性视网膜炎（RP）的突变。折叠的视蛋白 mRNA 是常染色体显性 (ad) RP 核酶 (Rz) 基因治疗的靶标。研究表明，具有数百个潜在切割位点的折叠 mRNA 几乎没有可进行强 Rz 催化的可接近区域。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。目标是构建可切割人视杆细胞视蛋白 adRP mRNA 的活性 Rz，以用于临床试验。中心假设是视蛋白 mRNA 的折叠结构将保护大多数潜在位点免受 Rz 切割。寻找最佳切割位点需要根据折叠人类 mRNA 的精确模型筛选许​​多潜在的 hRz 设计。预测 mRNA 结构可及性的折叠算法与针对裸露或蛋白质包被的折叠视蛋白 mRNA 的 Rz 文库的经验筛选相结合，提供了优化的研究策略。如此鉴定的活性Rz基因可以克隆到表达构建体中，以确定当在培养细胞中表达时，Rz是否可以沿着正常的mRNA加工/运输流识别和切割其靶标。通过这一层次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、高通量地鉴定有效的 Rz 设计，这些设计的功能是在体内切割从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. 找到折叠的人杆视蛋白 mRNA 中最容易接近的区域。目标 2. 开发有效的 Rzs 来切割视蛋白 mRNA 可及区域中的 NUH( 位点。目标 3. 通过在表达人杆视蛋白转基因的小鼠模型中测试视网膜变性和毒性的挽救情况，对击倒 Rz 策略进行临床前测试。拟议的工作具有创新性，因为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，并且它提供了第一个已知的针对一种已知的 adRP 疾病等位基因的敲低 Rz 策略的临床前测试，预期结果是视杆视蛋白 mRNA 的折叠结构将严重限制可到达的 Rz 切割位点的数量，可以构建对视蛋白具有显着影响的高活性 Rz。表达，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，其意义在于本工作产生的有效敲低Rzs，这表明可以挽救视网膜变性。并且缺乏光感受器毒性，然后可以转化为 adRP 的人体临床试验。