Optimization of Anticodon Edited Transfer RNAs (ACE-tRNAs) Function in Translation for Suppression of Nonsense Mutations

SUMMARY Nonsense suppressor tRNAs or AntiCodon-Edited tRNAs (ACE-tRNAs) have long been envisioned as a therapeutic approach to overcome genetic diseases resulting from the introduction of premature stop codons (PTCs). The ACE-tRNA approach for the rescue of PTCs has been hampered by ineffective delivery through available modalities for gene therapy. Here we have screened a series of ACE-tRNA expression cassette sequence libraries containing >1,800 members to optimize ACE-tRNA function and provide a roadmap for optimization in the future. By optimizing PTC suppression efficiency of ACE-tRNAs, we have decreased the amount of ACE-tRNA required by ∼16-fold for the most common cystic fibrosis-causing PTCs.