Mechanism of nonsense mutation suppression therapy

无义突变抑制疗法的机制

• 批准号: 6955226
• 负责人: Allan S Jacobson
• 金额: $ 36.42万
• 依托单位: PTC THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-23 至 2008-05-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6955226
• 关键词: combinatorial chemistry; drug design /synthesis /production; drug screening /evaluation; gene expression; gene mutation; genetic disorder; genetic translation; genetically modified animals; gentamicins; high throughput technology; immunofluorescence technique; laboratory mouse; microarray technology; pathologic process; posttranslational modifications; tissue /cell culture; transcription termination

DESCRIPTION (provided by applicant): Nonsense mutations inactivate gene function by promoting synthesis of truncated polypeptides and accelerating mRNA decay. These mutations are a significant cause of genetic disorders, giving rise to 15- 30% of all disease-causing alleles. Recent studies have shown that the antibiotic, gentamicin, can suppress translation termination at premature nonsense codons and produce limited but functionally significant quantities of essential proteins in mouse models of cystic fibrosis and muscular dystrophy. While this has led to initial clinical evaluations of gentamicin therapy in human inherited disorders, long-term application of this approach is compromised by the considerable adverse effects of this drug. PTC Therapeutics, Inc. (PTC) has pursued the concept of a pharmaceutical approach to treating diseases caused by nonsense mutations and has identified a novel, low molecular weight compound (PTC124) that is considerably more potent than gentamicin in both cell and animal models. PTC124 has the potential for near-term broad application in numerous genetic disorders, but such widespread utility requires mechanism of action analyses which demonstrate that it: i) promotes insertion of near-cognate tRNAs at premature termination codons; ii) principally targets premature translational termination, not normal translational termination; iii) has limited host-transcriptional effects on non-targeted mRNAs; iv) does not elicit toxic side effects in whole animals; tnd v) promotes nonsense suppression in additional animal models of disease states. This fast-track STTR proposal describes molecular biological approaches to all of these issues. In Phase I, we will use cultured =ells to address items i-iii and to establish the technological approaches required for addressing the remaining issues in animal models in Phase II. More specifically, we will treat HeLa cells with PTC124 and: I) define the events promoting nonsense suppression and II) determine whether PTC124-mediated nonsense suppression has global consequences for cellular mRNA decay and translation.

描述（由申请人提供）：无义突变通过促进截短多肽的合成和加速mRNA降解来抑制基因功能。这些突变是遗传疾病的重要原因，占所有致病等位基因的15- 30%。最近的研究表明，抗生素庆大霉素，可以抑制翻译终止在过早的无义密码子，并产生有限的，但功能显着数量的必需蛋白质在小鼠模型的囊性纤维化和肌营养不良症。虽然这导致了庆大霉素治疗人类遗传性疾病的初步临床评价，但这种方法的长期应用受到这种药物相当大的不良反应的影响。PTC Therapeutics，Inc. (PTC)研究了治疗无义突变引起的疾病的药物方法的概念，并鉴定了一种新的低分子量化合物（PTC 124），其在细胞和动物模型中比庆大霉素有效得多。PTC 124具有在许多遗传疾病中的近期广泛应用的潜力，但是这种广泛应用需要作用机制分析，其证明：i）促进近同源tRNA在提前终止密码子处的插入; ii）主要靶向提前翻译终止，而不是正常翻译终止; iii）对非靶向mRNA具有有限的宿主转录作用; iv）在整个动物中不引起毒性副作用; tnd v）在疾病状态的其它动物模型中促进无义抑制。这个快速通道STTR提案描述了所有这些问题的分子生物学方法。在第一阶段，我们将使用培养的=细胞来解决项目i-iii，并建立解决第二阶段动物模型中剩余问题所需的技术方法。更具体地，我们将用PTC 124处理HeLa细胞，并且：I）定义促进无义抑制的事件和II）确定PTC 124介导的无义抑制是否对细胞mRNA衰变和翻译具有全局后果。