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.

在这个探索性的赠款申请中，我们建议充分开发一种新的方法，即有针对性的 RNA假尿苷酸化，以抑制引起疾病的基因中的无义突变（例如， 导致囊性纤维化的CFTR基因无义突变，以及p53基因无义突变 导致各种癌症的基因）。这个项目，如果成功，将是一个巨大的进步， 我们的最终目标是为一些遗传性疾病开发新的治疗方法， 和某些由无义突变引起的癌症。我们建议实施这个项目 有两个具体目标。 目标1.提高人细胞中RNA引导的假尿苷酸化的效率。完善 通过鉴定更好的修饰酶来提高靶向mRNA假尿苷化的效率 （具体地，更好的盒H/ACA引导RNA）。我们将重点介绍指南中的三个要素 RNA已被确定（由我们的实验室和其他实验室）是重要的指导 假尿苷酸化，并构建完美盒H/ACA引导RNA。我们还将设计指南 通过添加核质定位信号，从而将引导RNA（和RNP）靶向至靶向RNA， 核质，mRNA在此合成并成熟。最后，我们将增加盒H/ACA RNA 表达水平，从而提高其在人细胞核质中的浓度。这样做 我相信我们将能够创造一种超级指导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 设计者盒H/ACA RNA（特别是当Aim 1中鉴定的改进的超级盒H/ACA RNA 可用），我们将直接靶向提前终止密码子（PTC，由无义产生 在CF细胞系中的CFTR mRNA内和在人癌细胞中的p53 mRNA内 线指导RNA转染的效率和其在转染细胞中的表达水平将被评估。 测定了还将定量mRNA PTC处的位点特异性假尿苷化。此外，委员会认为， 无义抑制，包括抑制NMD（无义介导的mRNA衰变）， 将监测PTC通读。简而言之，该提案提出了一种潜在的新方法， 治疗许多与无义突变相关的遗传疾病和某些类型的癌症。