Abstract
Nonsense mutations arise from single nucleotide substitutions that result in premature termination codons (PTCs). PTCs result in little to no full-length protein production and decreased mRNA stability due to the nonsense-mediated mRNA decay (NMD) pathway. We provide evidence that anticodon-edited (ACE-) tRNAs efficiently suppress the most prevalent cystic fibrosis (CF)-causing PTCs, promoting significant rescue of endogenous cystic fibrosis transmembrane conductance regulator (CFTR) transcript abundance and channel function in different model systems. We show that our best-performing ACE-tRNA, which decodes all UGA PTCs to a leucine amino acid, markedly rescues CFTR function from the most prevalent CF-causing PTCs, all of which arose from nonleucine encoding codons. Using this single ACE-tRNA variant, we demonstrate significant rescue of CFTR function in an immortalized airway cell line and two different primary CF patient-derived intestinal cell models with CFTR nonsense mutations. Further, we demonstrate that leucine substitution CFTR variants are highly functional. Thus, ACE-tRNAs have promise as a platform therapeutic for CF and other nonsense-associated diseases.