Researchers Explore Mutation-Cluster Targeting to Unlock Treatment for CF Variants

Researchers at Cincinnati Children’s are exploring two gene editing strategies for restoring function to small clusters of variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis (CF) can be caused by any one of more than 650 different variants in the CFTR gene, each one capable of disrupting the function of an ion channel protein necessary for proper lung function. Developing gene editing-based treatments for each individual variant in the gene is inefficient and would be cost prohibitive.

To tackle this problem, gene editing techniques are being developed in the Division of Pulmonary Medicine by Patrick Harrison, PhD, and his lab that have the potential to target multiple variants at the same time.

This basic research currently being evaluated in human cell lines and primary cells with some of these variants could lead to gene editing treatments that “restore production of a functional CFTR protein for many people with CF,” Harrison says.

Targeting Clusters of CF-causing Variants in the CFTR Gene

The majority of people with CF have a variant in the CFTR gene that makes an ion channel protein that responds to a small molecule therapy known as a modulator, which improves lung function and increases life expectancy. But this treatment is not a cure.

Moreover, for an estimated 20% of people worldwide who have CF caused by one of hundreds of other variants, these modulator treatments do not work.

Over the last decade, many labs have developed gene editing strategies to correct about a dozen individual CF-causing variants. But with over 650 different variants, how do you develop editing strategies that could be turned into viable therapies? One challenge is that these variants are spread across the 27 exons and 26 introns that make up the CFTR gene.

Harrison’s team partnered with researchers at Johns Hopkins to complete a statistical analysis of the CFTR gene to determine the best sections of the gene to target. Their efforts have focused on Exon 12, which includes just over 100 nucleotides and 18 known variants that cause CF.

Testing Two Complementary Gene Editing Techniques

Developing 18 different editing strategies, one for each variant, would be challenging to translate for therapeutic use. “We have recently developed a strategy to correct two variants with one editing strategy, but we only have an editing window of about 10-20 nucleotides at once,” Harrison says. “This strategy won’t work for the entire exon.”

The team is now working on two new modifications to their strategy. One is to test a method that begins at both ends of Exon 12 and meets in the middle. This could enable editing of any variant in the 100 base-pair exon. The second starts after the exon and uses a novel strategy to extend editing repair windows toward that 100 base-pair target.

The next step: testing in primary human cells with a range of different variants in Exon 12 and pre-clinical models. Such studies can tell us a lot about the challenges of delivery, and give insights into the percentage of lung cells affected and how long these changes last.

“We know the gene editing mechanism we use can have different efficiencies in different cell types,” Harrison says. “But we yet don’t know what proportion of each cell type that needs to have CFTR protein activity restored to achieve a physiological benefit.”

‘No One Optimal Solution’

Efficiency and accuracy of both techniques is improving all the time, Harrison says. But his team doesn’t know which technique will ultimately prevail. “We also know there is no one optimal solution,” Harrison says.

While a therapy ready for evaluation in humans is at least three to five years away, Harrison is focused on restoring a functional CFTR protein for as many variants as possible.

“We aim to make as few changes in the genome sequence as we can while at the same time restoring function,” he says. “If we can get this to work on Exon 12 in cells in the lab, we can then test in more advanced models, and then look at options for clinical application.”

Harrison’s lab was the first to correct a CF-causing variant with a gene editing tool known as zinc finger nucleases. His research is funded by the CF Foundation USA and the CF Trust UK. Since the discovery of CRISPR gene editing technology in 2013, Harrison’s lab has published more than 20 papers related to CF, gene editing and other diseases.

(Published December 2024)

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