Systematic deletion of symmetrical CFTR exons reveals new therapeutic targets for exon skipping antisense oligonucleotides

Abstract

There is a major need for therapeutics that treat diseases caused by pathogenic gene variants that disrupt protein open-reading frames. Splice-switching antisense oligonucleotides (ASOs) offer a potential solution by inducing the skipping of exons containing these variants, removing them from the mRNA and correcting the open-reading frame. Cystic fibrosis (CF), caused by disruption of the CF transmembrane regulator (CFTR) gene, is one such disease that has many chain-terminating variants, which are untreatable with standard protein-targeted modulator therapies. Using CFTR as a model, we demonstrate the utility of ASOs in engineering protein isoforms through exon skipping to rescue protein function disrupted by truncating variants. We functionally screened all CFTR isoforms generated by the deletion of symmetrical exons, which can be skipped without disrupting the open-reading frame. We identified exons that can be removed and produce CFTR isoforms that remain functionally responsive to modulators. We screened for ASOs that induce skipping of these exons and show that they recover CFTR function in airway cells derived from individuals with terminating CFTR variants. This study demonstrates that systematic functional analysis of in-frame exon-deleted protein isoforms can identify targets for ASO-based splice-switching therapies, a concept that can be broadly applied to any multi-exon protein-coding gene.