Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Nonsense mutations and premature termination codons (PTCs) cause over 7,000 genetic diseases, including cystic fibrosis (CF).
• Current therapeutic strategies for nonsense-associated diseases are limited, highlighting the need for novel approaches.
• The potential of suppressor tRNAs to inhibit nonsense-mediated mRNA decay (NMD) and restore protein translation in mammalian cells remains largely undetermined.

Purpose of the Study:

• To investigate the efficacy of anticodon edited (ACE)-tRNAs in suppressing CF-causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene.
• To evaluate the ability of ACE-tRNAs to inhibit NMD and restore endogenous CFTR mRNA and protein expression in human bronchial epithelial cells.
• To assess the functional rescue of CFTR channel activity following ACE-tRNA treatment.

Main Methods:

• Utilized gene-edited immortalized human bronchial epithelial (16HBEge) cells harboring common CFTR PTC mutations (G542X, R1162X, W1282X).
• Delivered engineered ACE-tRNAs designed to recognize and suppress specific PTCs.
• Measured NMD inhibition, endogenous mRNA rescue, and CFTR channel function using functional assays.

Main Results:

• ACE-tRNA delivery significantly inhibited NMD in cells with CFTR PTCs.
• Endogenous CFTR mRNA expression was rescued by ACE-tRNA treatment.
• A leucine-encoding ACE-tRNA successfully rescued W1282X-CFTR channel function to therapeutically relevant levels.

Conclusions:

• The ACE-tRNA approach effectively suppresses nonsense mutations in CFTR, inhibiting NMD and restoring protein function.
• ACE-tRNAs demonstrate potential as a standalone therapeutic strategy for cystic fibrosis and other nonsense-associated genetic diseases.
• This study establishes ACE-tRNAs as a viable method for rescuing both mRNA and full-length protein expression from endogenous PTC-containing genes.