Mechanistic Basis Underlying Protein Repair of CFTR Nonsense Mutations

描述（由申请人提供）： 用氨基糖苷类和其他药物抑制过早终止密码子（PTC）具有治疗囊性纤维化（CF）和其他遗传疾病（包括杜氏肌营养不良症、赫尔勒综合征和脊髓性肌萎缩症）的潜在原因的潜力。虽然对庆大霉素、阿米卡星和新型小分子PTC 124的初步研究表明该方法具有重大前景，但在CF和其他疾病中成功采用该策略将需要克服我们实验室确定的重要挑战，包括需要更好地了解基本作用机制，确定对治疗最敏感的患者人群（特别是，W1282 X囊性纤维化跨膜传导调节因子（CFTR）突变观察到的应答增加的分子基础），并鉴定增强CFTR无义密码子拯救的途径。在多中心临床试验中，修复CFTR突变（与PTC引起的功能障碍相反）的方法也受到越来越多的关注。VX-770是一种新型的小分子和CFTR调节剂，可增强定位于细胞表面的突变CFTR的门控，测试的中期结果表明，携带G551 D CFTR的CF患者的CFTR活性和肺功能得到改善（G551 D CFTR是一种位于细胞表面但由于通道门控缺陷而失活的突变）。这些结果表明，通过蛋白质修复疗法纠正基本CF缺陷是可行的，并且即使在短时间（2周）内也可以产生临床有益效果。我们实验室最近的证据表明，CFTR增效剂（如VX-770）也能有效激活W1282 X CFTR，这是一种相对常见的过早终止突变。此外，当CFTR增效剂与氨基糖苷类组合使用以诱导CFTR提前终止密码子的翻译通读时，我们观察到显著增强的Cl-转运。这些结果为治疗CFTR中携带提前终止密码子的CF患者的创新治疗方法提供了初步数据。该提案将测试某些CFTR无义突变体（如W1282 X）即使在截短状态下也表现出残留活性的假设，并可能对增强通道门控和恢复CFTR活性的小分子增效剂产生反应。拟议的研究将利用在CF患者中测试终止密码子抑制剂PTC 124的国际临床试验（由Rowe博士领导）中出现的数据，并将在实验模型中检查终止密码子抑制和CFTR通读产物增强的组合，包括原代人支气管上皮细胞和G542 X CFTR人转基因小鼠。结果将提供有关提前终止密码子抑制的机制基础的见解，这是一种适用于各种遗传性疾病患者的新兴治疗策略，并提供必要的初步数据，以证明恢复突变CFTR功能和改善疾病基本缺陷的新方法的临床试验的合理性。囊性纤维化跨膜传导调节因子（CFTR）中无义突变的纠正代表了一种新的治疗策略，以纠正囊性纤维化（CF）的根本缺陷。然而，需要更好地理解该方法的机制基础，以克服人类临床试验中发现的重要挑战。这些实验将定义对终止密码子抑制的应答的潜在机制，并检查CFTR截短突变体（和这些突变抑制后的通读产物）的增强是否代表恢复CFTR功能并因此改善疾病的可行方法。

DESCRIPTION (provided by applicant): Suppression of premature termination codons (PTCs) with aminoglycosides and other agents has the potential to treat an underlying cause of cystic fibrosis (CF) and other genetic diseases including Duchenne's muscular dystrophy, Hurler's syndrome, and spinal muscular atrophy. While initial studies with gentamicin, amikacin, and the novel small molecule PTC124 indicate significant promise to the approach, successfully employing the strategy in CF and other diseases will require overcoming important challenges identified by our laboratory, including the need for a better understanding of basic mechanisms of action, defining patient populations most responsive to treatment (in particular, the molecular basis underlying increased response observed for the W1282X cystic fibrosis transmembrane conductance regulator (CFTR) mutation), and identifying pathways to augment rescue of CFTR nonsense codons. Approaches to repair CFTR mutations that confer dysfuction at the plasma membrane (as opposed to those caused by PTCs) have also received increasing attention in multicenter clinical trials. Interim results testing VX-770, a novel small molecule and CFTR modulator that potentiates gating of mutant CFTR localized to the cell surface, indicated improved CFTR activity and lung function in CF patients harboring G551D CFTR (a mutation resident at the cell surface but inactive due to a channel gating defect). These results indicate that correction of basic CF defects by protein repair therapy is feasible, and can result in clinically beneficial effects, even in a short (2 week) time frame. Recent evidence from our laboratory establishes that CFTR potentiators (such as VX-770) also potently activate W1282X CFTR, a relatively common premature stop mutation. Moreover, we observed significantly enhanced Cl- transport when a CFTR potentiator was used in combination with an aminoglycoside to induce translational readthrough of CFTR premature termination codons. These results provide preliminary data for an innovative therapeutic approach to treat CF patients harboring premature termination codons in CFTR. This proposal will test the hypothesis that certain CFTR nonsense mutants such as W1282X exhibit residual activity, even in the truncated state, and may respond to small molecule potentiators that enhance channel gating and restore CFTR activity. The proposed studies will capitalize on data emerging from an international clinical trial testing the stop codon suppressor PTC124 in CF patients (led by Dr. Rowe), and will also examine the combination of stop codon suppression and potentiation of CFTR readthrough product in experimental models, including primary human bronchial epithelial cells and G542X CFTR human transgenic mice. Results will lend insight regarding the mechanistic underpinnings of premature stop codon suppression, an emerging treatment strategy applicable to patients with a variety of genetic diseases, and provide preliminary data necessary to justify clinical testing of a novel approach to restore mutant CFTR function and ameliorate the basic defect in the disease. Correction of nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) represents a novel treatment strategy to correct a fundamental defect underlying cystic fibrosis (CF). However, a better understanding of the mechanistic basis of the approach is needed to overcome important challenges identified in human clinical trials. These experiments will define mechanisms underlying response to stop codon suppression, and examine whether potentiation of CFTR truncation mutants (and readthrough product following suppression of these mutations) represents a viable approach to restore CFTR function and thus ameliorate disease.