Engineered translation release factor 1 suppresses disease-causing nonsense mutations and enables multi-site nonstandard amino acids incorporation

Abstract

Nonsense mutations convert sense codons into premature termination codons (PTC), resulting in early termination of translation of mRNAs, underlie [~]11% of human genetic diseases. Restoring translation of genes carrying nonsense mutations remains a major therapeutic challenge. Here we present OPENER (Overexpression of Protein-engineered eRF1 for Nonsense Elision and Readthrough), a strategy to efficiently suppress nonsense mutations based on engineered variants of the eukaryotic translation termination factor eRF1. Through saturation mutagenesis of the human eRF1 N-domain, we identified variants, notably eRF1S77E, that promote efficient readthrough of all three PTCs (UAG, UGA, UAA) and rescue 12 pathogenic PTCs reporters in multiple disease contexts. Systemic delivery of OPENER-S77E via AAV8 in a Duchenne muscular dystrophy mouse model (DmdQ995X) restored dystrophin expression, improved muscle function, and ameliorated pathology without detected toxicity. Furthermore, when combined with orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs, OPENER-S77E enabled efficient, multi-site incorporation of nonstandard amino acids (BocK and AzF) into a single protein in mammalian cells, achieving efficiency of 27.77% and 19.83%, respectively. The incorporation efficiency of BocK was further enhanced to 32.80% in conditional eRF1-knockout cells. OPENER thus provides a platform for treating nonsense-mediated diseases and expands the synthetic biology toolkit for precise protein engineering.