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Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs
Wooree Ko1, Joseph J Porter1, Matthew T Sipple1
1Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Abstract:
Nonsense mutations or premature termination codons (PTCs) comprise ∼11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases; however, their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here, we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542XUGA-, R1162XUGA-, and W1282XUGA-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine-encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential standalone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC-containing endogenous genes.
Insights
Anticodon edited (ACE)-tRNAs effectively suppress nonsense mutations causing cystic fibrosis (CF) by inhibiting mRNA decay and restoring CFTR protein function. This approach shows promise as a standalone therapy for genetic diseases linked to premature termination codons.
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.
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