ASO Cocktail May Help in Treating CF Due to W1282X Mutation
A potential gene-specific therapy for cystic fibrosis (CF) may lie in antisense oligonucleotides, or ASOs, that enhance the production and activity of the truncated CFTR protein caused by the W1282X mutation, a cell study suggested.
In combination with approved CFTR modulator therapies, candidate ASOs were seen to raise CFTR-W1282X activity into the therapeutic range, supporting further developmental work, its researchers said.
The study, “Gene-specific nonsense-mediated mRNA decay targeting for cystic fibrosis therapy,” was published in the journal Nature Communications.
In CF, mutations in the CFTR gene lead to a defective or deficient CFTR protein, needed to maintain the balance of salt and water on many cell surfaces. Without a properly working CFTR protein, abnormally thick mucus builds in various organs, including the lungs and digestive tract.
W1282X, fairly common nonsense mutation
A CFTR mutation called W1282X is the sixth most common CF-causing mutation, present in 2.2% of CF patients in the U.S. and up to 40% in Israel. This nonsense mutation leads to a shortened, or truncated, version of the CFTR protein. Despite this defect, the protein retains some of its activity.
However, the messenger RNA (mRNA) for W1282X — mRNA being molecule that carries the troubled genetic instructions to make the truncated protein — is degraded by nonsense-mediated mRNA decay (NMD). This decay process prevents the production of potentially damaging shortened proteins as a quality control measure. As such, people who carry W1282X have very little truncated CFTR protein, resulting in severe CF disease.
A proposed way of treatment is selectively preventing NMD to allow a truncated CFTR protein to be made, providing some CFTR activity. Combining such treatments with approved CFTR modulator therapies that correct CFTR protein defects also may strengthen the modulators’ effectiveness in these patients.
However, “a clinically viable NMD-suppression approach does not exist yet,” the study noted.
Researchers at the Cold Spring Harbor Laboratory in New York aimed to suppress W1282X-related NMD in cells using ASOs — lab-made molecules that selectively target and bind to mRNA and help modify protein production.
“If more of this type of drug, ASOs, are approved, I wouldn’t be surprised if in the not-so-distant future ASOs become a routine way to make personalized medicines,” Adrian Krainer, PhD, a biochemist and molecular geneticist at Cold Spring Harbor and the study’s senior author, said in a press release.
The researchers began by screening various ASOs to identify those that effectively prevent the nonsense-mediated decay of the W1282X mRNA, finding several ASOs that suppress NMD in a dose-dependent manner.
Using human airways and colon cancer cells carrying CFTR-W1282X, a cocktail of three ASO candidates, called LC15-1, was seen to significantly increase CFTR mRNA levels over an untreated control cell group. Additional experiments confirmed that these ASOs did not affect normal CFTR mRNA and those with different types of mutations.
In human airway cells, the increase in total CFTR mRNA by this lead ASO cocktail was accompanied by higher levels of CFTR-W1282X protein compared to control cells. Combining the ASOs with lumacaftor, a CFTR modulator therapy that improves the folding of the CFTR protein, further increased the total CFTR-W1282X protein.
Potential ASO cocktail identified
Additional screening experiments identified a separate ASO cocktail, called LC15-2, as more potent than the previous candidate. Although both ASO cocktails increased CFTR-W1282X mRNA similarly at the highest concentration, “at a lower concentration, the new lead cocktail had significantly higher potency,” the team noted.
To assess the impact on CFTR protein activity, airway cells carrying the W1282X mutation were treated with the lead ASO cocktail in combination with CFTR modulators. This included lumacaftor and ivacaftor, combined in the approved therapy Orkambi, and Trikafta, which contains three CFTR modulating medicines: elexacaftor, tezacaftor, and ivacaftor.
Mutant airways cells treated with the ASO cocktail plus lumacaftor/ivacaftor increased CFTR activity compared to cells treated with the modulators only. LC15-2 plus Trikafta further significantly increased CFTR activity, to about 18% to 30% relative to the protein’s normal activity.
“This result suggests that the combination treatment may enhance CFTR activity up to a therapeutic range,” the researchers wrote.
Final experiments confirmed that the increase in CFTR activity was due to NMDÂ suppression.
According to the researchers, the next step for an ASO cocktail as a potential treatment of CF caused by the W1282X mutation is optimizing its safe and effective delivery to affected tissues in animal models.
“These results set the stage for clinical development of an allele [gene]-specific therapy for CF caused by the W1282X mutation,” the scientists wrote.
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