Abstract

10−15% of mutations in the CFTR gene affect its correct splicing. The disease severity in patients carrying these mutations is highly variable, correlated with the level of aberrantly spliced transcripts. We aim to develop a splicing mutationspecific therapy using antisense oligonucleotides (AOs), to modulate the splicing pattern in patients carrying common splicing mutations. We focused on a common splicing mutation, the 3849+10kb C-to-T, which leads to inclusion of an 84 bp cryptic exon between exons 22−23 in the mature mRNA. This cryptic exon contains an in-frame stop codon that leads to degradation of a significant fraction of the mRNA by the NMD pathway as well as to the production of prematurely truncated nonfunctional proteins. We designed 4 2′-O-methyl phosphorothioatemodified AOs, targeted to prevent the recognition of enhancer splice motifs in the cryptic exon or to mask the junctions between this exon and its flanking sequences. Epithelial cell line established from a nasal polyp of a CF patient carrying the 3849+10kb C-to-T mutation was transfected with 2 of the designed AOs. AOstreated cells showed a highly significant decrease in the level of aberrantly spliced CFTR mRNA, along with a significant increase in normal spliced CFTR mRNA levels, reflecting transcripts that under normal conditions are degraded by the NMD pathway. These results indicate that AOs targeted to mask splicing motifs around the cryptic exon generated due to the 3849+10kb C-to-T splicing mutation, can modulate increase the correct splicing. Further studies are required to investigate whether these AOS can restore the CFTR function and improve patients’ clinical state. WS1.6 OligoG normalizes the CF mucus phenotype

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