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的多肽的合成并加速mRNA的衰变来失活基因功能。这些突变是遗传性疾病的重要原因，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过早无义密码子的翻译终止，并在囊性纤维化和肌营养不良的小鼠模型中产生数量有限但功能显着的必需蛋白质。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估，但这种方法的长期应用因该药物的相当大的副作用而受到损害。 PTC Therapeutics, Inc. (PTC) 一直致力于通过药物方法来治疗无义突变引起的疾病，并发现了一种新型低分子量化合物 (PTC124)，该化合物在细胞和动物模型中比庆大霉素更有效。 PTC124 具有在许多遗传性疾病中短期广泛应用的潜力，但如此广泛的用途需要作用机制分析来证明它： i) 促进在过早终止密码子处插入近同源 tRNA； ii) 主要针对过早翻译终止，而不是正常翻译终止； iii) 对非靶向 mRNA 的宿主转录作用有限； iv) 不会对整个动物产生毒副作用； tnd v) 在其他疾病状态动物模型中促进无意义抑制。这个快速通道 STTR 提案描述了解决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第 i-iii 项，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。更具体地说，我们将用 PTC124 处理 HeLa 细胞，并且：I) 定义促进无义抑制的事件，II) 确定 PTC124 介导的无义抑制是否对细胞 mRNA 衰减和翻译具有全局影响。