Abstract
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5′ splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.
Highlights
Cystic fibrosis (CF) is a fatal, autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]
A previous study reported on antisense oligonucleotides (ASOs)-mediated inhibition of CFTR c.3718-2477T aberrant splicing in a CFTR expressing mini-gene system with the mutation, though no further work on this mutation has been done to show that ASOs can be used to recover CFTR activity in epithelial cells [25]
We demonstrate that ASO, an ASO designed to block splicing to the aberrant -Ex splice site created by the CFTR c.3718-2477C>T mutation, redirects splicing to the correct, wild-type splice site, increases forskolin-stimulated apical membrane conductance and restores chloride secretion in CF patient-derived bronchial epithelial cells
Summary
Cystic fibrosis (CF) is a fatal, autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]. Pharmacotherapy for CF has focused on the development of two main categories of drugs: correctors that improve the expression of CFTR, and potentiators that stimulate channel function [4, 5]. These therapeutics have proven effective in treating a large proportion of CF patients with mutations that disrupt protein expression and channel function, such as the common F508del-CFTR ( Δ F508) and G551D mutations [6,7,8,9,10]. These types of therapeutics are less effective in treating patients with mutations that affect pre-mRNA splicing, as these types of mutations often result in the disruption of the CFTR reading frame
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