ASO Cocktail Corrects Severe Cystic Fibrosis Mutation

Little girl receives breathing treatment
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In a pair of related PNAS articles, researchers have demonstrated the potential of splice-switching antisense oligonucleotides (ASOs) to eliminate a relatively common CFTR mutation that causes a severe form of the disease. This pair, or cocktail, of ASOs helps the genetic machinery skip over the region encoding the CFTR mutation during messenger RNA production. 

The researchers are from the Rosalind Franklin University of Science and Medicine, Cold Spring Harbor, and Stonybrook University.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel. More than 2000 of these mutations have been identified. Some of them lead to a truncated version of the CFTR protein, disrupting its function in multiple organs, including the lungs, gastrointestinal tract, and reproductive organs. These mutations can also cause low CFTR mRNA expression as a result of nonsense-mediated mRNA decay.

There are several CFTR-related treatments already available, but they do not address all the mutations. Still, the current worldwide CF market is estimated at about $5 billion and is expected to double within the next few years.

In particular, there is no treatment for CF caused by the CFTR-W1282X mutation, which is located on CFTR exon 23 and is estimated to be the fifth most common cause of cystic fibrosis. Nonsense-mediated messenger RNA (mRNA) decay (NMD) degrades the CFTR-W1282X mRNA, leading to low levels of functional CFTR protein. 

These teams determined that exon-skipping would be a viable approach and then developed a cocktail of two ASOs that promotes the skipping of exon 23 of the CFTR-W1282X mRNA. The resulting mRNA is NMD resistant and preserves the reading frame. Its translation produces CFTR-Δex23 protein that improves CFTR activity in human bronchial epithelial cells. 

The first team determined whether correction of the CFTR-W1282X open reading frame by removing exon 23 might be therapeutic (Michaels et al. PNAS, 2022). They tested whether an expressed CFTR isoform lacking the amino acids encoded by exon 23 had channel activity in Fischer rat thyroid (FRT) cells, which lack endogenous expression of cAMP-regulated, apical chloride channels.The cells were transfected with plasmids expressing CFTR without exon 23 (CFTR-Δ23) or with the W1282X mutation (CFTR-W1282X).  They next treated these cell lines with known modulators (including VRT-534) and found that CFTR-Δ23 has functional activity in the presence of CFTR modulator drugs.

This establishes that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use.

The authors of the second study (Kim et al, PNAS, 2022) introduced specifically designed ASOs into isolated human bronchial epithelial cells in the laboratory, and found that they induced the cells to produce partially functional CFTR proteins. Their findings raise the possibility that ASO-based treatments might be therapeutic for a subset of patients who currently lack effective alternatives.

Frameshift and nonsense mutations pose a major problem for disease therapeutic development. Eliminating these mutations from the messenger RNA by inducing exon skipping is a relatively unexplored treatment approach, though it has shown promise for some diseases. These teams have shown that eliminating a common stop mutation associated with cystic fibrosis (CF), by inducing the skipping of the exon it is located in, results in a restoration of the open reading frame and recovers CFTR protein function in a manner that may be therapeutic in a set of CF patients who don’t currently have effective treatment options.

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