Antisense-oligonucleotide-directed inhibition of nonsense-mediated mRNA decay

反义寡核苷酸定向抑制无义介导的 mRNA 衰减

• 批准号: 8835766
• 负责人: Tomoki T Nomakuchi
• 金额: $ 2.71万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-15 至 2016-02-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835766
• 关键词: Accounting; Aminoglycosides; Antisense Oligonucleotides; Antisense Technology; Attenuated; Binding; Biological Assay; Cell Line; Cells; Clinical; Code; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Deposition; Development; Disease; Dominant-Negative Mutation; Exons; Fibroblasts; Gene Expression Profile; Gene Targeting; Genes; Genetic; Globin; Half-Life; Hereditary Disease; Immunoprecipitation; Individual; Knowledge; Lead; Length; Lesion; Mediating; Messenger RNA; Metabolism; Modeling; Monitor; Mucopolysaccharidoses; Mucopolysaccharidosis I; Mus; Mutation; Nonsense Codon; Nonsense Mutation; Outcome; Pathology; Patients; Pharmaceutical Preparations; Phenotype; Play; Positioning Attribute; Process; Proteins; Quality Control; RNA; RNA Splicing; Reading; Research Project Grants; Ribosomes; Role; Site; Techniques; Terminator Codon; Testing; Therapeutic; Transcript; Translations; base; beta Globin; clinically relevant; crosslink; design; functional restoration; gene function; improved; insight; interest; mRNA Decay; mRNA Precursor; mRNA Transcript Degradation; mouse model; mutant; prevent; public health relevance; research study; technology/technique; therapy development; tool

 DESCRIPTION (provided by applicant): The objective of this two-year dissertation research project is to inhibit the degradation of mRNA triggered by nonsense mutations so as to restore gene function and ameliorate the pathology caused by the mutations. RNA-based therapeutic approaches have become increasingly promising, as demonstrated by recent remarkable progress in the development of antisense oligonucleotides (ASOs) and stop-codon read-through drugs as therapeutics for various genetic diseases. Nonsense mutations, which account for a large proportion of pathogenic genetic lesions, can be partially overcome by suppressing premature stop codons (PTCs) using read-through drugs, such as ataluren or aminoglycosides. Nonsense-mediated mRNA decay (NMD) is a cellular quality-control mechanism that degrades mRNAs with PTCs. In the context of genetic diseases caused by PTCs, NMD can worsen the clinical outcome by degrading transcripts that code for truncated but semi- functional proteins, or by reducing the efficacy of read-through therapy. NMD is highly dependent on the exon- junction complex (EJC), which assembles upstream of exon-exon junctions at the completion of pre-mRNA splicing. I have designed ASOs that target presumptive EJC sites and suppress NMD of β-globin and MECP2 transcripts with PTCs. To develop and apply this technique more broadly, and to pursue therapeutic applications of NMD-inhibiting ASOs, I will systematically test EJC-targeting ASOs on transcripts with clinically- relevant PTCs, including nonsense mutations in IDUA and CFTR genes that cause type I mucopolysaccharidosis and cystic fibrosis, respectively. I will design and test ASOs that target the EJCs of these transcripts, and test the lead ASOs in patient fibroblasts and murine models for their therapeutic potential. My ASO designs will be guided by the predicted EJC positions, as well as by using public transcriptome-wide data on EJC crosslinking or binding to RNA. To validate the mechanism of action, I will monitor the effects of individual ASOs on EJC assembly using an RNA-immunoprecipitation-based assay. This project seeks to develop therapies potentially applicable to numerous genetic diseases, while also providing fundamental mechanistic insights into the precise roles and functions of EJCs in NMD.

 描述（由申请人提供）：这个为期两年的论文研究项目的目标是抑制无义突变引发的mRNA降解，以恢复基因功能并改善突变引起的病理。基于RNA的治疗方法已经变得越来越有前途，如最近在开发反义寡核苷酸（ASO）和终止密码子通读药物作为各种遗传疾病的治疗剂方面的显著进展所证明的。无义突变占致病性遗传病变的很大比例，可以通过使用通读药物（如阿他卢仑或氨基糖苷类）抑制过早终止密码子（PTC）来部分克服。无义介导的mRNA衰变（NMD）是一种细胞质量控制机制，它通过PTC降解mRNA。在由PTC引起的遗传疾病的背景下，NMD可通过降解编码截短但半功能性蛋白质的转录物或通过降低通读疗法的功效而使临床结果恶化。NMD高度依赖于外显子-连接复合物（EJC），其在前体mRNA剪接完成时在外显子-外显子连接的上游组装。我设计了靶向假定EJC位点并抑制β-珠蛋白和MECP 2转录物与PTC的NMD的ASO。为了更广泛地开发和应用该技术，并追求NMD抑制性ASO的治疗应用，我将在具有临床相关PTC的转录物上系统地测试靶向EJC的ASO，包括分别引起I型粘多糖样变性和囊性纤维化的IDUA和CFTR基因中的无义突变。我将设计和测试靶向这些转录本的EJC的ASO，并在患者成纤维细胞和小鼠模型中测试其治疗潜力。我的阿索设计将由预测的EJC位置指导，以及通过使用关于EJC交联或结合RNA的公共转录组范围的数据。为了验证作用机制，我将使用基于RNA免疫沉淀的测定来监测单个ASO对EJC组装的影响。该项目旨在开发可能适用于许多遗传疾病的疗法，同时也为EJC在NMD中的确切作用和功能提供基本的机制见解。