(620) Antisense Oligonucleotide Splicing Modulation as a Novel Cystic Fibrosis Therapeutic Approach for the N1303K Nonsense Mutation

Background: SpliSense aims to address the unmet need of CF patients carrying the N1303K mutation for which CFTR modulators are not approved. The N1303K is a missense class II mutation in exon 24 which causes a folding defect, resulting in a reduced number of N1303K CFTR channels in the apical membrane, which also have channel gating defects. SpliSense proposes a novel ASO-based therapy for CF patients carrying this mutation by skipping over exon 24. Based on previous published work (1,2), the proteins translated from this variant are expected to mature and form partially active channels on the cell membrane. Addition of modulators is likely to enhance the maturation and improve the function of these CFTR channels. This approach is expected to provide treatment for about 2500 patients carrying the N1303K mutation.

Methods: Lead ASO selection was performed by transfecting ASOs designed to skip over exon 24 into 16HBEge N1303K cells. Following transfection, RNA was extracted and analyzed for exon 24 skipping by RT-qPCR and RT-PCR. The selected lead ASO was assessed by the Ussing Chamber system in 16HBEge N1303K and in Human Nasal Epithelial (HNE) cells derived from patients carrying the N1303K mutation, in comparison to TRIKAFTA®.

Results: Proof of concept studies showed that proteins lacking exon 24 are mature and functional. Several lead ASO candidates which efficiently promoted exon 24 skipping were identified. The effect of TRIKAFTA® on 16HBE and HNE cells containing the N1303K mutation was assessed alone and in combo with SpliSense ASOs in the Ussing chamber system.

Conclusions: Lead candidate ASOs promoting skipping over exon 24 have promising potential for production of functional CFTR channels that can be further enhanced by the addition of correctors. This strategy has potential to have a clinical benefit for CF patient carrying the N1303K mutation that currently have no available therapy.

Acknowledgements:

References: 1. Cui L, Aleksandrov L, Chang X-B, Hou Y-X, He L, Hegedus T, et al. Domain interdependence in the biosynthetic assembly of CFTR. J Mol Biol 365, 981–94 (2007).
2. Molinski S V., Ahmadi S, Hung M, Bear CE. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening. J Biomol Screen 20, 1204–17 (2015).