Rescue of CFTR function impaired by mutations in exon 15

Abstract

Introduction:2000 different mutations have been reported in patients with Cystic Fibrosis (CF) and occur in all CFTR exons and introns. Some mutations are not amenable to current therapies, and new drugs must be developed. Antisense oligonucleotides (AO) are synthetic nucleic acid analogues whose binding to pre-mRNA modulates exon selection. We hypothesise that skipping in frame CFTR exon 15 in patients with intra-exonic mutations such as p.Phe861Leufsx3 studied here, the induced isoform may retain residual function.

Methods: AO sequences were initially optimised using 2’-O-Methyl modified bases on a phosphorothioate backbone (2OMe) and transfected into monolayer primary non-CF and p.Phe861Leufsx3/p.Phe508del CF airway epithelial cells. The most effective 2OMe AO sequence was re-synthesised as the clinically validated phosphorodiamidate morpholino (PMO) chemistry. Monolayer transfections were repeated and modification of protein was detected by western blot analysis. Residual CFTR function was measured using Ussing Chamber.

Results: The 2OMe sequence produced 50% skipping in p.Phe861Leufsx3/p.Phe508del CF airway epithelial cells The PMO induced skipping of exon 15 from non-CF (49%) and p.Phe861Leufsx3/p.Phe508del CF (88%) cells after 7 days in culture. Western blot was then used to determine the effect on the induced CFTR protein. In addition, airway epithelial cells from children with CF prior to and following AO treatment were mucocillary differentiated at the air-liquid interface for 28 days and CFTR function assessed using Ussing chamber.

Conclusion: Exon 15 can be skipped from CFTR in airway epithelial cells. We propose that exon skipping to remove disease-causing mutations in selected in-frame exons can improve CFTR function.