WS17.2 Identification of CF mutations in deep intronic regions: Design of antisense oligonucleotides for a targeted therapeutic approach

Considering that the extensive study of the CFTR gene classically performed in molecular diagnosis does not detect all disease-causing mutations, we previously developed an approach for a complete resequencing of the CFTR locus to search for mutations deeply located in introns (Bonini et al. Genet Med, 2015). After identifying candidate intronic mutations, we now aim to restore full-length CFTR transcripts by using antisense oligonucleotides also named Target Site Blockers (TSB). DNA from 15 patients with only one CF mutation were collected through a national collaborative study. Enrichments of the CFTR locus (250 kb) by long-range PCR or targeted capture were sequenced using 454 GS Junior and MiSeq Illumina platforms. TSB (Exiqon) were specifically designed for the deleterious variants. Out of 200 variations detected, our in-house pipeline pointed out 10 intronic variations, per DNA in average. In silico analysis tools predicted that 13 variants (for the 15 patients) had a deleterious effect on CFTR splicing by promoting inclusion of pseudoexons. TSB were tested whether the splicing defect was confirmed. For instance, TSB used on bronchial and primary nasal cells transfected with minigene constructs, significantly restored the full-length CFTR transcript for the well-known mutation c.1680–886A>G and a new identified c.1680–883A>G. Two new mutations in introns 18 and 23 are being tested. Finally, our massively parallel sequencing strategy lead to the identification of new CF-causing mutations in introns. Our findings also demonstrate the efficiency of antisense oligonucleotides for a targeted therapeutic for cystic fibrosis. Work supported by Vaincre La Mucoviscidose.