Engineered transfer RNAs for suppression of premature termination codons

Premature termination codons (PTCs) are responsible for 10-15% of all inherited disease. PTC suppression during translation offers a promising approach to treat a variety of genetic disorders, yet small molecules that promote PTC read-through have yielded mixed performance in clinical trials. We present a high-throughput, cell-based assay to identify anticodon engineered transfer RNAs (ACE-tRNA) which can effectively suppress in-frame PTCs and faithfully encode their cognate amino acid. In total, we identified ACE-tRNA with a high degree of suppression activity targeting the most common human disease-causing nonsense codons. Genome-wide transcriptome ribosome profiling of cells expressing ACE-tRNA at levels which repair PTC indicate that there are limited interactions with translation termination codons. These ACE-tRNAs display high suppression potency in mammalian cells, Xenopus oocytes and mice in vivo, producing PTC repair in multiple genes, including disease causing mutations within cystic fibrosis transmembrane conductance regulator (CFTR).

提前终止密码子（Premature Termination Codons, PTCs）约占所有遗传性疾病致病因素的10%-15%。翻译过程中的PTC抑制策略为多种遗传性疾病的治疗提供了极具前景的方案，但可促进PTC通读的小分子化合物在临床试验中表现参差不齐。本研究报道一种高通量细胞水平检测方法，用于筛选可有效抑制框内PTC并精准编码其同源氨基酸的反密码子工程化转运RNA（anticodon engineered transfer RNAs, ACE-tRNA）。最终，我们针对人类最常见的致病性无义密码子，筛选得到具有高抑制活性的ACE-tRNA。对表达可修复PTC水平的ACE-tRNA的细胞进行全转录组核糖体谱分析后发现，其与翻译终止密码子的互作极为有限。这些ACE-tRNA在哺乳动物细胞、非洲爪蟾卵母细胞以及活体小鼠中均展现出优异的抑制效能，可在包括囊性纤维化跨膜传导调节因子（cystic fibrosis transmembrane conductance regulator, CFTR）致病突变在内的多个基因中实现PTC修复。