eRF3a Protein May Be Target in Treating CF Nonsense Mutations

Investigative therapy seen to raise CFTR function in cells with W1282X mutation

Marisa Wexler, MS avatar

by Marisa Wexler, MS |

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Targeting a protein called eRF3a may offer a way of treating cystic fibrosis (CF) caused by nonsense mutations, potentially opening a treatment avenue to people whose rare disease-causing mutations do not respond to current CFTR modulators, researchers reported.

Their study, “Small molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough,” was published in The Journal of Clinical Investigation.

CF is caused by mutations in the CFTR gene, which provides instructions for making a protein with the same name. CFTR modulators work improve the protein’s functionality for the vast majority of patients with amenable mutations.

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With nonsense mutations, a “stop” signal occurs too early in the code of a gene, sort of like a period placed in the middle of a sentence. When a gene with a nonsense mutation is “read” to make protein, only part of the protein will be made, and this unstable product is rapidly degraded within cells.

The functional consequence of nonsense mutations is little to no CFTR protein being produced, so CFTR modulators that work to improve the functionality of the existing protein are of limited or no benefit to people with these mutations.

Study in cells with W1282X nonsense mutation

Scientists at the University of North Carolina at Chapel Hill generated new bronchial and nasal cell lines harboring CF-causing mutations (the bronchi connect the lungs to the windpipe). Cell lines are cells that have been genetically engineered so they continually grow and divide in a laboratory culture; they are useful research tools since they allow scientists to conduct many experiments from the same cell.

“The extension of CFTR modulators to 90% of people with CF represents a tremendous victory. However, developing targeted therapies for the remaining 10% will be challenging. This challenge is heightened by the limited availability of rare-genotype primary cells,” the researchers wrote. “For this reason, the field has turned to cell lines for drug discovery efforts.”

Several of the cell lines harbored a nonsense mutation in the CFTR gene called W1282X.

The researchers conducted a number of tests to ensure that their cell lines recapitulated CFTR function as expected. For example, they showed that CFTR activity increased after modulator treatment in lines harboring the F508del mutation, the most common CF-causing mutation and known to respond to CFTR modulators.

A series of experiments in the W1282X cell lines followed. The scientists showed that treatment with an experimental compound called CC-90009 improved CFTR function to about 19.1% of what is typically seen in people without CF.

“We concluded that CC-90009 can function as a single agent to rescue W1282X-CFTR to [approximately] 20% of wildtype function,” the researchers wrote.

Of note, CC-90009 is currently being tested in a Phase 1 clinical trial (NCT02848001) in people with acute myeloid leukemia, a type of blood and bone marrow cancer. That trial is sponsored by Celgene, which was not involved in this CF study.

CC-90009 works by reducing the levels of eRF3a, a protein that is part of the molecular machinery responsible for recognizing “stop” signals when genes are “read.” Further experiments confirmed that, by targeting eRF3a, CC-90009 causes the machinery to “read through” the erroneous signal generated by the stop codon, ultimately boosting CFTR production.

Unexpectedly, the researchers also found that CC-90009 treatment decreased the activity of another protein called epithelial sodium channel (ENaC). Like CFTR, the ENaC protein is involved in regulating the flow of salts and water in and out of cells.

“Even though eRF3a is the only reported target of CC-90009, our data suggest that ENaC or an ENaC regulator may be a previously undescribed target,” the team wrote.

Another compound that degrades eRF3a, called SJ6986, also improved CFTR activity while reducing ENaC activity in W1282X cell lines, though the magnitude of the effect was generally smaller than that of CC-90009.

“We concluded that SJ6986 rescues W1282X-CFTR by the same mechanism as CC-90009,” the researchers wrote.

Tests in cell lines harboring another nonsense mutation, called G542X, showed little improvement in CFTR activity after treatment with CC-90009 or SJ6986. However, both compounds showed a strong effect when given in combination with a readthrough-promoting compound called an aminoglycoside.

“We found that treatment with the eRF3a degraders CC-90009 and SJ6986 significantly rescued CFTR function in patient-derived cell lines carrying the W1282X- and G542X-CFTR variants. By demonstrating consistent findings between two distinct eRF3a degraders, we propose that eRF3a degradation is a generalizable mechanism for CFTR [nonsense mutation] rescue,” the scientists wrote.

They also noted their patient-derived cell lines are “highly representative” and useful in modeling “rare CFTR variants.”