Efforts to develop treatments for diseases caused by nonsense mutations have focused on identification of small molecules that promote translational read-through of messenger RNAs (mRNAs) harboring nonsense stop codons to produce full-length proteins. However, to date, no small molecule read-through drug has received FDA approval, probably because of a lack of balance between efficacy and safety. Depletion of translation termination factors eukaryotic release factor (eRF) 1 and eRF3a in cells was shown to promote translational read-through of a luciferase reporter gene harboring a nonsense mutation. In this study, we identified antisense oligonucleotides (ASOs) targeting translation termination factors and determined if ASO-mediated depletion of these factors could be a potentially effective and safe therapeutic approach for diseases caused by nonsense mutations. We found that ASO-mediated reduction of either eRF1 or eRF3a to 30%-40% of normal levels in the mouse liver is well tolerated. Hemophilia mice that express a mutant allele of human coagulation factor IX (FIX) containing nonsense mutation R338X were treated with eRF1- or eRF3a-ASO. We found that although eRF1- or eRF3a-ASO alone only elicited a moderate read-through effect on hFIX-R338X mRNA, both worked in synergy with geneticin, a small molecule read-through drug, demonstrating significantly increased production of functional full-length hFIX protein to levels that would rescue disease phenotypes in these mice. Overall our results indicate that modulating the translation termination pathway in the liver by ASOs may provide a novel approach to improving the efficacy of small molecule read-through drugs to treat human genetic diseases caused by nonsense mutations.

中文翻译：

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针对由反义寡核苷酸引起的疾病的反义寡核苷酸靶向翻译终止机制。

开发针对无义突变引起的疾病的治疗方法的努力集中在鉴定促进携带无义终止密码子的信使RNA（mRNA）的翻译通读以产生全长蛋白质的小分子。但是，到目前为止，可能没有在小分子通读药物中获得FDA的批准，这可能是因为疗效和安全性之间缺乏平衡。显示细胞中翻译终止因子真核释放因子（eRF）1和eRF3a的耗竭可促进具有无义突变的荧光素酶报道基因的翻译通读。在这个研究中，我们确定了针对翻译终止因子的反义寡核苷酸（ASO），并确定了ASO介导的这些因子的消耗是否可能是针对无义突变所致疾病的潜在有效且安全的治疗方法。我们发现在小鼠肝脏中，ASO介导的eRF1或eRF3a降低至正常水平的30％-40％的耐受性良好。用eRF1或eRF3a-ASO处理表达含有无意义突变R338X的人凝血因子IX（FIX）突变等位基因的血友病小鼠。我们发现，虽然单独使用eRF1-或eRF3a-ASO只会对hFIX-R338X mRNA产生中等的通读效果，但两者均与小分子通读药物遗传霉素协同作用，证实功能性全长hFIX蛋白的产生显着增加至可以挽救这些小鼠疾病表型的水平。总的来说，我们的结果表明，通过ASO调节肝脏中的翻译终止途径可能为提高小分子通读药物治疗由无意义突变引起的人类遗传疾病的功效提供一种新颖的方法。