Using a newly enhanced gene-editing technology called prime editing, researchers in the U.S. have efficiently corrected the most common mutation that causes cystic fibrosis (CF) in human lung cells.
By correcting this mutation, known as F508del, in the CFTR gene, scientists at the Broad Institute of MIT and Harvard, and the University of Iowa say their findings may support the development of a one-time, permanent CF treatment.
“We are hopeful that the use of prime editing to correct the predominant cause of cystic fibrosis might lead to a one-time, permanent treatment for this serious disease,” David Liu, PhD, director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute and the study’s senior author, said in a university news story.
Liu, who is also a professor at Harvard University and a Howard Hughes Medical Institute investigator, and colleagues described the advance in the journal Nature Biomedical Engineering, in the study “Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells.”
In CF, mutations in the CFTR gene lead to a malfunctioning or completely absent CFTR protein. Because this protein functions as a channel to control the flow of salt and water in and out of cells, these mutations disrupt the balance of water on cell surfaces. As a result, thick mucus builds up in various organs, such as the lungs.
Prime editing developed by Liu’s lab in 2019
In 2019, Liu’s lab developed prime editing, a type of gene editing in which precise changes can be made in DNA. More recently, Liu showed the method could insert or substitute entire genes in human cells in their natural location within the genome, demonstrating the potential to treat several genetic diseases.
Now, the team has optimized prime editing to selectively correct F508del, the most common CF-causing mutation, in cells that line the airways.
“Developing a strategy to efficiently correct this challenging mutation also provided a blueprint for optimizing prime editing to precisely correct other mutations that cause devastating disorders,” Liu said.
Liu’s team applied six optimizations to enhance prime editing’s efficacy, including improving the RNA molecule that guides the DNA-editing enzyme to its proper location in the genome. In addition, the editing enzyme itself was modified, and the target site was made more accessible.
Before optimization, prime editing corrected the F508del mutation in less than 0.5% of lung cells, whereas after applying the six enhancements, 58% of cells were edited — a 140 times improvement.
In airway cells isolated from three CF patients with F508del, the mean F508del correction rate across all treated cells was 25% — a 59 times improvement with optimization.
To test their functional abilities, edited cells were grown in an air-liquid interface (ALI) that modeled human airways. The team confirmed that the proportion of corrected cells remained constant over the three-week ALI growth period, demonstrating “the corrective edit persists through cellular proliferation and differentiation,” the researchers wrote.
CFTR protein was then stimulated, and the flow of salt was measured. Compared to unedited CF cells, stimulation substantially increased the flow of salt in prime-edited CF cells to levels exceeding 50% of those found in healthy non-CF airway cells.
Flow of salt in prime-edited cells was comparable to treatment with Trikafta
Notably, the flow of salt in prime-edited CF cells was comparable to treatment with Trikafta, an approved CFTR modulator therapy designed to address the mutation-driving defects in the production and function of CFTR protein.
“These results demonstrate substantial rescue of CFTR channel activity following correction by [prime editing],” the researchers wrote.
Because prime editing creates breaks in both strands of DNA’s double helix, it carries so-called off-target risks, such as “indels,” where DNA fragments are unintentionally lost or inserted into the genome.
Experiments demonstrated the optimizations applied to the prime-editing system resulted in minimal off-target editing. Edit-to-indel ratios were 3.5 times better than those achieved by Cas9, the enzyme used to cut DNA in the CRISPR-Cas9 gene-editing system.
The researchers noted a “critical challenge to realizing the therapeutic potential of our [prime-editing] strategy for CFTR F508del correction will be the development of technologies to deliver our composition of matter to relevant airway tissues.”