(638) Splice-switching Antisense Oligonucleotides for the Treatment of Class I CFTR Mutations

Background: Cystic Fibrosis (CF) is an autosomal recessive disease affecting more than 70,000 people worldwide. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when mutated, results in the buildup of mucus in tissues such as the lung and pancreas disrupting proper organ function. The majority of CF therapies in the clinic or in development target only the most abundant CFTR mutations, leaving patients with rare mutations in need of more personalized therapies. CFTR mutations that introduce premature termination codons (PTC) account for ~10% of CF cases. These mutations are associated with a severe form of the disease resulting from low CFTR mRNA and protein expression due to nonsense mediated mRNA decay (NMD). Because of the low CFTR expression, current therapeutics for CF, which work to increase protein function, are not effective in patients with these types of mutations. One approach that holds promise as a therapeutic strategy involves antisense technology. We tested splice-switching antisense oligonucleotides (ASOs) that modify gene expression by directly modulating pre-mRNA splicing. We examined the effects of ASOs designed to induce skipping of symmetrical exons, which can be deleted from the mRNA without disrupting the open reading frame, that contain PTCs introduced by nonsense mutations, thereby stabilizing CFTR mRNA and allowing for translation to the natural C-terminus. This approach holds promise for the W1282X-CFTR mutation, present in 2.4% of CF patients, which resides in exon 23 of CFTR mRNA and triggers NMD of the CFTR transcript. Because complete removal of exon 23 does not disrupt the open-reading frame, we hypothesized that ASO-mediated skipping of CFTR exon 23 will recover CFTR mRNA levels and channel activity.

Methods: To analyze the activity of CFTR isoforms lacking symmetrical exons, including exon 23, plasmids designed to express CFTR cDNA lacking symmetrical exons were created and transfected into Fischer Rat Thyroid (FRT) cells. Cells were treated with CFTR modulators C18, VX-661+VX-445, and VX-770 and cAMP-activated conductance was analyzed utilizing a transepithelial current clamp (TECC-24) system. Additionally, ASOs were designed to induce exon skipping and analyzed in T84 cells, expression WT-CFTR. To test the effect of ASO-induced skipping of CFTR exons containing PTC mutations, ASOs were transfected into primary patient bronchial epithelial cells (hBE) and CFTR function was analyzed as cAMP-dependent equivalent currents in the same TECC-24 system.

Results: We found that CFTR lacking the amino acids encoding exon 23 retains conductance activity in cells. The functionality of this CFTR isoform is enhanced by corrector and potentiator drugs currently in use clinically to treat certain CFTR mutations, as well as with co-potentiator therapies in development. Further, ASO treatment in primary CF-patient derived hBE cells successfully induces exon 23 skipping and recovers CFTR function disrupted by the common W1282X-CFTR mutation that resides in exon 23.

Conclusions: These results support the use of ASOs in treating CF patients with CFTR class I mutations that result in truncations of the CFTR protein, who currently do not have available treatment options. On a broader scale, our results further validate ASO-mediated exon skipping strategies for open reading frame correction disrupted by mutations in other genetic diseases.

Acknowledgements: This work was supported by the CFF.

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