Mechanism of nonsense mutation suppression therapy

描述（由申请人提供）：无义突变通过促进截断多肽的合成和加速mRNA衰变来灭活基因功能。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过早无义密码子的翻译终止，并在囊性纤维化和肌肉萎缩症小鼠模型中产生有限但功能显著的必需蛋白。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受到该药物相当大的不良反应的影响。PTC Therapeutics, Inc. （PTC）一直在追求一种药物方法的概念，以治疗无义突变引起的疾病，并已确定了一种新的低分子量化合物（PTC124），在细胞和动物模型中都比庆大霉素更有效。PTC124具有短期内广泛应用于多种遗传疾病的潜力，但这种广泛的应用需要对其作用机制进行分析，以证明它：i)促进近同源trna在过早终止密码子上的插入；Ii)主要针对过早的翻译终止，而不是正常的翻译终止；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)在疾病状态的其他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治疗HeLa细胞，并：1)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衰变和翻译具有全局影响。

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.