Antisense oligonucleotides to improve CFTR function for people with the INTRON 9 5T polymorphism

Introduction: Over 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes cystic fibrosis (CF) with variable clinical phenotypes. The length of the poly T tract in intron 9 influences exon 10 selection and can manifest as mild or severe disease depending on other CFTR mutations. Manipulation of CFTR pre-RNA splicing using antisense oligonucleotides (AOs) is a potential therapy for selected CF-causing mutations. We aim to develop splice modulating AOs 2019 Cystic Fibrosis Conference 218 to rescue CFTR function in CF patients that carry the shorter 5T polymorphism in intron 9. AOs could strengthen exon 10 selection or weaken the selection of flanking exons. As seen with specific cases of Duchenne muscular dystrophy, removing a block of exons can restore more functional dystrophin protein over the removal of a single exon. Methods: Multiple AOs targeting CFTR intron 9 and the flanking exons; 9 and 11 were designed and initially optimised using 2’-O-methyl modified bases on a phosphorothioate backbone (2OMe) and transfected into primary airway epithelial cells from a child with p.508del/ Arg117His;5T CF. After 48 hours RNA was collected, and PCR was used to determine the ratio of altered transcript compared to full-length product. CFTR protein size was determined by Western blot analysis. CFTR functional outcomes were measured using Ussing chamber studies utilising air-liquid interface primary airway cell cultures. Results: Of the 32 2OMe AOs tested for exon 10 inclusion, none reduced the intron 9 5T induced exon 10 skipping. Of the 8 AOs designed to skip exon 9, the highest efficiency was 24% from both the p.Phe508del allele and intron 9 5T allele. Of the 6 AOs designed to skip Exon 11, the highest efficiency was 22% from the intron 9 5T allele. CFTR protein size was determined on Western blot and CFTR function was determined by response to forskolin (change in Isc). Conclusion: We propose that skipping the exons flanking exon 10 (9 and/or 11) on the CFTR 5T allele could improve CFTR function in CF patients carrying selected mutations, either alone or in combination with current therapeutics. Acknowledgments: Supported by USCF; NHMRC; CFWA.