Exon-Skipping Antisense Oligonucleotides for Cystic Fibrosis Therapy

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity, but is expressed at a very low level due to nonsense-mediated mRNA decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable; and read-through drugs are inefficient, partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression of CFTR mRNA without the PTC-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

Antisense Oligonucleotide Therapeutics for Cystic Fibrosis: Recent Developments and Perspectives

“…Alternatively, exon-skipping ASOs can restore CFTR function in HBE cells harboring the W1282X mutation by inducing the skipping of the exon containing the PTC. The resulting CFTR-Δ23 mRNA isoform, which lacks the in-frame exon 23 with the W1282X mutation, is not degraded by NMD (Kim et al, 2022b;Michaels et al, 2022;Oren et al, 2022). CFTR-Δ 23 protein lack residues 1240 through 1291, but retains func- tional domains important for CFTR function that are missing in CFTR-W1282X protein (Kim et al, 2022b).…”

“…The resulting CFTR-Δ23 mRNA isoform, which lacks the in-frame exon 23 with the W1282X mutation, is not degraded by NMD (Kim et al, 2022b;Michaels et al, 2022;Oren et al, 2022). CFTR-Δ 23 protein lack residues 1240 through 1291, but retains func- tional domains important for CFTR function that are missing in CFTR-W1282X protein (Kim et al, 2022b). Exon-skipping ASOs with uniform 2'-MOE/PS (Kim et al, 2022b;Oren et al, 2022) or PMO (Michaels et al, 2022) modification targeting the splice sites, exonic splicing enhancer (ESE) elements, or both, induced efficient exon 23 skipping in W1282X mutant CFF16HBEge cells (Fig.…”

“…3, Table 2). Exon 23 skipping ASOs combined with VX-770/809 or VX-770/445/661 led to higher CFTR protein levels and CFTR function in the W1282X mutant CFF16HBEge-cells, patient-derived HBE cells (heterozygous W1282X/F508del), and patient-derived human nasal epithelial (HNE) cells (homozygous W1282X/W1282X) (Kim et al, 2022b;Michaels et al, 2022;Oren et al, 2022). Oren et al (2022) estimated that one of their lead exon-skipping ASOs combined with VX-770/445/661 increased CFTR function to up to 20% of wild-type CFTR.…”

Splicing mutations in the CFTR gene as therapeutic targets

“…The transfection of these molecules in bronchial epithelial cells of patients with CF leads to an increase of CFTR expression and activity [79]. AON strategy was recently applied to promote exon skipping of exon 23 to eliminate the W1282X nonsense mutation and to avoid RNA degradation induced by the NMD mechanism [81][82][83].…”