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.

在这个探索性的拨款申请中，我们建议充分开发一种新颖的方法，即目标