Suppression of disease causingnonsense mutations by targeted mRNA pseudouridylation

通过靶向 mRNA 假尿苷化抑制引起无义突变的疾病

• 批准号: 9805188
• 负责人: YI-TAO YU
• 金额: $ 16.75万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9805188
• 关键词: Aminoglycoside Antibiotics; Anticodon; Applications Grants; Attention; Breast Carcinoma; Cancer Etiology; Cancer cell line; Cell Line; Cells; Clinical; Code; Codon Nucleotides; Coupled; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Development; Disease; Elements; Engineering; Enzymes; Epithelial Cells; Genes; Genetic Diseases; Genetic Transcription; Goals; Human; Human Genetics; Inherited; Length; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Modification; Monitor; Mutation; Nonsense Codon; Nonsense Mutation; Nucleoplasm; Nucleotides; Primer Extension; Production; Protein p53; Proteins; Pseudouridine; RNA; Reporter Genes; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal RNA; Ribosomes; Signal Transduction; Site; Small Nuclear RNA; Specificity; Step Tests; Structure; System; TP53 gene; Terminator Codon; Testing; Transfection; Transfer RNA; Translations; Untranslated RNA; Uridine; Work; Yeasts; beta Globin; cancer type; chemical property; clinically relevant; cystic fibrosis patients; established cell line; experience; human disease; improved; mRNA Decay; novel strategies; novel therapeutics; premature; release factor

In this exploratory grant application, we propose to fully develop a novel approach, namely, targeted RNA pseudouridylation, to suppress nonsense mutations in genes that cause diseases (e.g., nonsense mutations in the CFTR gene that cause Cystic Fibrosis, and nonsense mutations in p53 gene that cause various types of cancer). This project, if successful, will constitute a giant step toward our ultimate goal of developing novel therapeutic treatment for a number of genetic disorders and certain types of cancer caused by nonsense mutations. We propose to carry out this project under two specific aims. Aim 1. To improve the efficiency of RNA-guided pseudouridylation in human cells. We will improve the efficiency of targeted mRNA pseudouridylation by identifying a better modifying enzyme (specifically, a better box H/ACA guide RNA). We will focus on the three elements within the guide RNA that have been identified (by our lab and other labs) to be important for directing pseudouridylation, and construct a perfect box H/ACA guide RNA. We will also engineer the guide RNA by adding a nucleoplasmic localization signal, thus targeting the guide RNA (and RNP) to the nucleoplasm where mRNA is synthesized and matures. Finally, we will increase box H/ACA RNA expression level, thus raising its concentration in the nucleoplasm of human cells. In doing so, we believe we will be able to create a super guide RNA that can efficiently direct site-specific mRNA pseudouridylation. Aim 2. To directly target the PTC within a disease gene in human disease cell lines. Using an designer box H/ACA RNA (especially when the improved super box H/ACA RNA identified in Aim 1 is available), we will directly target the premature termination codon (PTC, resulting from nonsense mutations) within the CFTR mRNA in a CF cell line and within the p53 mRNA in a human cancer cell line. The efficiency of guide RNA transfection and its expression level in transfected cells will be measured. Site-specific pseudouridylation at the PTC of mRNAs will also be quantified. Furthermore, nonsense suppression, including the suppression of NMD (nonsense-mediated mRNA decay) and PTC read-through, will be monitored. In short, this proposal presents a potential novel approach to treat many genetic diseases and certain types of cancer associated with nonsense mutations.

在这个探索性的资助申请中，我们建议充分开发一种新方法，即有针对性的 RNA 假尿苷化，抑制导致疾病的基因中的无义突变（例如， 导致囊性纤维化的 CFTR 基因中的无义突变和 p53 中的无义突变 导致各种癌症的基因）。该项目如果成功，将迈出一大步 实现我们的最终目标，即开发针对多种遗传性疾病的新型治疗方法 以及由无义突变引起的某些类型的癌症。我们建议开展这个项目 在两个具体目标下。 目的 1. 提高人类细胞中 RNA 引导的假尿苷化的效率。我们会改进 通过鉴定更好的修饰酶来提​​高靶向 mRNA 假尿苷化的效率 （具体来说，更好的盒 H/ACA 引导 RNA）。我们将重点关注指南中的三个要素 已（由我们的实验室和其他实验室）鉴定出对指导很重要的 RNA 假尿苷化，并构建完美的盒式 H/ACA 引导 RNA。我们还将设计指南 RNA 通过添加核质定位信号，从而将向导 RNA（和 RNP）靶向 核质是 mRNA 合成和成熟的地方。最后，我们将增加盒H/ACA RNA 表达水平，从而提高其在人体细胞核质中的浓度。这样做，我们 相信我们将能够创造出一种超级引导RNA，可以有效地指导位点特异性mRNA 假尿苷化。 目标 2. 直接靶向人类疾病细胞系中疾病基因内的 PTC。使用 设计盒 H/ACA RNA（特别是当目标 1 中鉴定出改进的超级盒 H/ACA RNA 时） 可用），我们将直接针对提前终止密码子（PTC，由废话产生） CF 细胞系中的 CFTR mRNA 内和人类癌细胞中的 p53 mRNA 内的突变） 线。指导RNA转染效率及其在转染细胞中的表达水平为 测量。 mRNA PTC 处的位点特异性假尿苷化也将被量化。此外， 无义抑制，包括抑制 NMD（无义介导的 mRNA 衰减）和 PTC 通读将受到监控。简而言之，该提案提出了一种潜在的新颖方法 治疗许多遗传疾病和与无义突变相关的某些类型的癌症。