Department of Physiology and Pharmacology, University of Rochester School of Medicine and Dentistry, Rochester, NY, 14642, USA.
CFFT Lab, Cystic Fibrosis Foundation Therapeutics, Lexington, 02421, MA, USA.
Nat Commun. 2019 Feb 18;10(1):822. doi: 10.1038/s41467-019-08329-4.
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. Here 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 identify 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（PTCs）负责所有遗传疾病的 10-15%。在翻译过程中抑制 PTC 是治疗各种遗传疾病的一种很有前途的方法，但在临床试验中，促进 PTC 通读的小分子的表现参差不齐。在这里，我们提出了一种高通量、基于细胞的测定方法来鉴定反密码子工程转移 RNA（ACE-tRNA），它可以有效地抑制框架内 PTC 并忠实编码其同源氨基酸。总共，我们鉴定了 ACE-tRNA，它们针对最常见的人类致病无义密码子具有高度抑制活性。表达 ACE-tRNA 的细胞的全基因组转录组核糖体谱分析表明，与翻译终止密码子的相互作用有限。这些 ACE-tRNA 在哺乳动物细胞、非洲爪蟾卵母细胞和体内小鼠中表现出高抑制效力，可修复多个基因中的 PTC，包括囊性纤维化跨膜电导调节剂（CFTR）中的致病突变。
