Targeted Inhibition of NMD to Enhance the Efficacy of Readthrough Drugs

描述(由申请人提供)：人类基因组计划的成功完成和许多疾病基因的绘制，加上对基因表达途径的分子理解的进展，为设计针对各种遗传病的基于机制的新疗法提供了前所未有的机会。在几乎所有遗传病的因果突变中，无义突变占很大一部分。根据定义，无义突变会引入提前终止密码子(PTCs)，导致蛋白质被截断，通常会出现严重的疾病症状。翻译通读药物，如阿托洛林，允许从无义突变的有缺陷的基因合成一些全长的功能蛋白。然而，无稽之谈介导的信使核糖核酸衰变(NMD)是一种普遍存在的信使核糖核酸质量控制途径，降低了通读药物的有效性。我们将有选择地取消含有PTC的mRNAs的NMD，以增加其用于通读药物的可用性。为了探索我们方法的可行性，我们首先将重点放在几个无意义的CFTR、MECP2、DMD和HBB基因的细胞培养实验上，这些基因分别导致囊性纤维化、Rett综合征、Duchenne肌营养不良和β-地中海贫血。我们将系统地检测抑制NMD对mRNA积累的影响。使用报告细胞系，然后我们将结合转录特异性NMD抑制和阿托洛林治疗，并确定与单独使用阿托洛林治疗相比，是否有增加全长蛋白质的合成。这项探索性研究的结果有望为靶向抑制NMD以提高通读药物疗效的有效性提供原则证据。拟议的实验可能导致一种广泛适用的治疗方法，这种方法将与直读药物结合使用，以治疗大量严重的遗传疾病。 与公共卫生相关：无义突变会导致蛋白质生产被截断，并导致许多严重的遗传病。像阿塔鲁伦这样的药物可以促进无义突变基因合成完整的蛋白质；然而，细胞有一个内在的机制，可以减少无义突变基因的输出。我们将针对单个突变基因开发一种绕过这一机制的方法，以增强阿托鲁仑和类似药物的有效性，从而为各种遗传性疾病提供更有效的治疗方法。

DESCRIPTION (provided by applicant): The successful completion of the human genome project and mapping of many disease genes, combined with advances in the molecular understanding of gene-expression pathways, provides unprecedented opportunities to design novel mechanism-based therapies for various genetic diseases. Nonsense mutations account for a large fraction of the causal mutations in nearly all genetic diseases. By definition, nonsense mutations introduce premature termination codons (PTCs), resulting in truncated proteins, and usually severe disease presentations. Translational readthrough drugs, such as ataluren, allow the synthesis of some full-length, functional protein from defective genes with nonsense mutations. However, nonsense-mediated mRNA decay (NMD)¿a ubiquitous mRNA quality-control pathway¿diminishes the effectiveness of readthrough drugs. We will selectively abrogate NMD of mRNAs harboring PTCs, so as to increase their availability for readthrough drugs. To explore the feasibility of our approach, we will initially focus on cell-culture experiments with several nonsense alleles of CFTR, MECP2, DMD, and HBB genes, which cause cystic fibrosis, Rett syndrome, Duchenne muscular dystrophy, and beta-thalassemia, respectively. We will systematically test the effect of inhibiting NMD on mRNA accumulation. Using reporter cell lines, we will then combine transcript-specific NMD inhibition with ataluren treatment, and determine whether there is increased synthesis of full-length protein, compared to ataluren treatment alone. The results of this exploratory study are expected to provide proof of principle for the effectiveness of targeted inhibition of NMD to enhance the efficacy of readthrough drugs. The proposed experiments could lead to a broadly applicable therapeutic approach that would be used in combination with readthrough drugs to treat a large number of severe genetic diseases. PUBLIC HEALTH RELEVANCE: Nonsense mutations result in truncated protein production and cause many severe genetic diseases. Drugs like ataluren can promote the synthesis of intact proteins from genes with nonsense mutations; however, cells have an intrinsic mechanism that diminishes the output of genes with nonsense mutations. We will develop a method to bypass this mechanism for individual mutant genes, so as to enhance the effectiveness of ataluren and similar drugs, and thereby provide more effective therapies for a variety of genetic diseases.