(619) Antisense Oligonucleotide Splicing Modulation as a Novel Cystic Fibrosis Therapeutic Approach for the W1282X Nonsense Mutation

Background: An important goal of the CF research and care community is to provide CFTR-based therapies to every individual with CF. Despite enormous progress, CFTR modulators cannot assist all patients with CF. Therefore, further strategies of drug development are essential to address the unmet needs of patients with non-responsive mutations. These mutations include stop mutations as well as missense mutations leading to folding defects. SpliSense aims to address the unmet need of a specific sub-group of CF patients carrying the W1282X mutation, a stop mutation located at exon23 of the CFTR gene associated with a severe CF phenotype.

SpliSense develops SPL23-2 as an ASO treatment for Cystic Fibrosis patients carrying the W1282X CFTR mutation by inducing skipping over exon 23 of the CFTR transcript. Skipping over exon 23 eliminates the Premature Termination Codon (PTC) generated by the W1282X mutation and avoids RNA degradation induced by the Nonsense Mediated Decay (NMD) mechanism, allowing the production of active CFTR proteins lacking exon 23.

Methods: Screen for ASO effect on CFTR RNA splicing and protein was performed in 16HBEge W1282X cells. 24h following transfection with the ASOs, RNA was extracted and the levels of transcripts with Ex23 were measured by RT-PCR and RT-qPCR. CFTR protein levels and maturation were analyzed by Western Blot. Selected lead candidates were evaluated for their effect on CFTR function, first on the 16HBEge W1282X cell line followed by primary HNE and HBE cells from patients carrying the W1282X mutation. In these experiments the cells were treated with the lead ASOs by free uptake. The analysis of CFTR activity was performed with the chronic addition of VX-661, VX-445 for 48 hours and acute addition of VX-770, by the Ussing Chambers system.

Results: Cumulative results indicate that exon skipping generated by SPL23-2 leads to the production of mature CFTR proteins. These proteins, missing exon 23, are the molecular basis for the functional response measured by the Ussing Chamber experiments with the addition of the CFTR modulators. Further analysis in HNE and HBE cells from several W1282X patients on different genetic backgrounds (heterozygous and homozygous) revealed that treatment with SPL23-2 leads to significant levels of CFTR activation reaching ~40% of WT. This level of activity in primary respiratory cells predicts a clinical benefit to patients.

Conclusions: These results highlight the potential therapeutic and clinical benefit of ASO-based exon 23 skipping SPL23–2, supplemented by CFTR modulators, for CF patients carrying the W1282X nonsense mutation aiming to enable the initiation of clinical study by next year.

Acknowledgements:

References: