Exon Skipping May Open Nonsense CF Mutations to Current Therapies

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by Patricia Inácio, PhD |

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Exon skipping may be a promising approach for cystic fibrosis (CF) patients carrying nonsense mutations in the CFTR gene, which render current CF therapies less effective, a study reported.

Researchers were able to raise CFTR protein function and protein response to approved CF therapies in cells isolated from a patient with the common nonsense mutation CFTR–W1282X.

The study, “Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis,” was published in the journal PNAS.

CF is caused by mutations in the CFTR gene that affects the levels and workings of the CFTR protein, a cell membrane protein that allows the passage of chloride ions in and out of cells. Currently, over 2,000 CFTR mutations have been described, with different effects on the resulting protein.

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While approved CF modulators treat many of these people, those with nonsense or frameshift mutations are less likely to benefit.

Nonsense mutations lead to a premature stop in protein production and a shorter and defective protein, and frameshift mutations change the way the gene sequence is read. While less common CF-causing mutations, together they “account for [about] 20% of disease-associated mutations in CF,” the researchers wrote.

“There is a critical need for therapies to treat patients with rare CFTR mutations, in particular, nonsense variants that create premature termination codons resulting in low CFTR expression [activity],” they added.

Genes are organized as stretches of exons and introns, in which exons are pieces of DNA carrying instructions to make a protein, and introns are regions that do not code for protein.

This genetic information is transcribed into a messenger RNA (mRNA), an intermediate molecule used to guide protein production. Much like pieces of a puzzle, production depends on exons being “stitched” together while introns are removed in a process known as mRNA splicing.

The loss of one or more exons, however, often prevents the remaining exons from linking together properly, impairing the production of a working protein.

Researchers at the Rosalind Franklin University of Science and Medicine in Illinois wondered if exon skipping using antisense oligonucleotides might help patients with these rarer CF mutations.

Exon skipping therapy, developed to treat Duchenne muscular dystrophy, works to overcome this broken link by forcing the cell’s machinery to skip one or more exons near the missing piece in a gene’s mRNA, so the remaining exons fit together — binding to produce a smaller, but functional, protein.

This is achieved with antisense oligonucleotides or ASOs, lab-made molecules designed to be complementary to a specific region in the premature mRNA, altering the mature mRNA and the resulting protein.

Researchers tested this approach in correcting the CFTR–W1282X nonsense mutation the CFTR gene, the fifth most common CF-causing mutation worldwide. As a result of the premature stop sign in exon 23, its mRNA is degraded and little CFTR protein produced.

Experiments in a thyroid cell line from rats showed that CFTR protein produced from an mRNA lacking exon 23 was responsive to therapies.

Specifically, they used a compound that mimics the action of lumacaftor (VX-809), the combination of elexacaftor (VX-445) plus tezacaftor (VX-661), or of ivacaftor (VX-770). Ivacaftor is marketed as Kalydeco as a single therapy, in combination with lumacaftor as Orkambi, and in combo with elexacaftor and tezacaftor as Trikafta; all are marketed by Vertex Pharmaceuticals.

Ivacaftor is a CFTR potentiator, meaning it enhances protein activity by keeping the CFTR gate open for longer at the cell surface. The others are CFTR correctors, as they work by binding to the faulty CFTR protein and helping it to achieve a proper 3D structure.

The therapeutic potential of ASO-induced exon 23 skipping to rescue CFTR was then tested in airway cells isolated from a patient with the CFTR-W1282X mutation in both CFTR gene copies. This approach is “the gold standard for preclinical testing of CF therapeutics, as the functional response to drugs in this assay has been shown to accurately predict efficacy in the clinic,” the scientists noted.

ASO-mediated exon skipping increased the levels of CFTR mRNA. The protein produced from this mRNA also showed greater chloride secretion and led to a dose-dependent increase in conductance the flow of electric current when in combination with CFTR modulators.

“We demonstrate that elimination of the relatively common nonsense mutation, CFTR-W1282X, by ASO-induced skipping of CFTR exon 23, which encodes the mutation, recovers CFTR expression,” the researchers wrote. With modulators, it does so “in a manner expected to be therapeutic in CF patients who don’t currently have effective treatment options.”

Overall, “this ASO approach in combination with current CF drugs offers a potential therapeutic for individuals with the CFTR-W1282X mutation and opens the door for similar strategies to treat other terminating mutations, both in CF and other diseases,” the researchers concluded.