Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10kb C-to-T splicing mutation

Abstract

Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently been approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10kb C-to-T splicing mutation in the CFTR gene. We have screened, in FRT cells expressing this mutation, ~30 ASOs chemically modified with 2'-O-Methyl on a phosphrothioate backbone, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The screening identified five ASO candidates able to promote CFTR correct splicing and rescue channel activity. Further analyses in well differentiated primary human nasal and bronchial epithelial cells (HNEs, HBEs), derived from patients carrying at least one 3849+10kb C-to-T allele, led to the identification of a highly potent lead ASO. The ASO was efficiently delivered by free uptake into patients HNEs and HBEs and completely restored CFTR function to wild type levels in cells from a homozygous patient and led to 43{+/-}8% of wild type levels in cells from various heterozygous patients. Optimized efficiency was further obtained with 2-Methoxy Ethyl chemical modification. The results demonstrate the therapeutic potential and clinical benefit of ASO-based splicing modulation for genetic diseases caused by splicing mutations.