Abstract
Introduction/Aim. Over 2000 different mutations have been reported in patients with Cystic Fibrosis (CF) and found to occur in all CFTR exons and introns. Many mutations are not amenable to current therapies, and therefore new drugs must be developed. Antisense oligonucleotides (AOs) are synthetic nucleic acid analogues designed to anneal to selected splice motifs within pre-mRNAs. AO binding alters the recognition of the splice site by the spliceosome and therefore modulates exon selection. c.2989-1G > A causes skipping of CFTR exon 19, which disrupts the reading frame, and abolishes CFTR protein production. We hypothesize by also skipping exon 18 in patients with c.2989-1G > A the reading frame will be restored, and the induced protein isoform may regain function or become amenable to CFTR modifying drugs.
Methods. Three AO sequences were initially optimised using 2’-OMethyl modified bases on a phosphorothioate backbone (2OMe) and transfected using lipofectamine into primary airway epithelial cells (AECs) from non-CF and child with CF causing c.2989-1G > A/ c.2989-1G > A. The transfection is left for 48 hours after which the cells are collected, and RNA extracted. PCR primers that amplify CFTR exons 17 to 21 are used to determine the efficacy of exon skipping. PCR bands are measured using densitometry and the density of the full-length band to the exon skipped band is compared.
Results. The 2OMe sequence produced 64% exon 18 skipping in c.2989-1G > A/ c.2989-1G > A CF airway epithelial cells. In non-CF AECs the efficiency was greatly reduced to only 11% exon 18 skipping.
Conclusion. Exon 18 can be efficiently skipped from the CFTR transcript in c.2989-1G > A/ c.2989-1G > A CF-derived airway epithelial cells. We propose that exon skipping to restore the reading frame of the CFTR gene, resulting in improved CFTR protein production. This new internally truncated CFTR protein could have improve function and/or become amenable to CFTR modifying drugs.