In cystic fibrosis (CF), patients’ organs become overwhelmed by thick mucus, which affects breathing and can result in serious bacterial infections. The outlook for patients with CF has improved significantly since scientists identified the gene mutation responsible for the disease in 1989. A majority of cases are caused by a three-base-pair mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This mutation, called F508del, results in ion channel defects throughout the patients’ bodies.
Gene therapies have huge potential for diseases like CF because they provide long-lasting treatments. However, genetic editing techniques used to develop these promising treatments, including the Nobel Prize-winning CRISPR-Cas9, have proved inefficient at correcting F508del. Now, published in Nature Biomedical Engineering, researchers used an optimized version of the genome editing technology prime editing to alter this mutation in lung cells from patients with CF. The approach restored function to the same degree as a powerful CF drug combination therapy. The findings could lead to CF treatments that permanently correct the disease’s genetic origin.
David Liu, a geneticist at the Broad Institute of MIT and Harvard and coauthor on the study, recognized that complex genetic diseases require flexible and precise gene therapies. In 2019, he developed prime editing, a gene editing technique that takes a different approach from CRISPR. The latter technique cuts the genome like scissors, snipping through both strands of the DNA, which can lead to unintended, off-target edits. In contrast, prime editors, said Liu, are like “DNA word processors in that we develop them to do true search and replace editing.” He added that this allows the technology to make more precise and controllable changes, making it ideal for the repair job needed to correct the F508del mutation: the addition of only a few DNA letters at specific locations.
In their 2019 paper, Liu’s team used prime editing to alter the gene mutations causing sickle cell disease and Tay-Sachs disease. Liu explained that they attempted to edit mutated CFTR as well but with limited success. “That was a very difficult correction to make,” said Liu. The genetic machinery that guided the prime editor proved unstable and corrected less than one percent of mutations.
Researchers have made improvements to prime editing over the last five years. In the latest paper, Liu’s team took another crack at editing CFTR using what he calls a “kitchen sink approach” that utilized six major enhancements to prime editing. These changes improved the accuracy and stability of the molecular machinery.
Liu said much effort went into incorporating an enhancement that allowed the system to make edits to the DNA without attracting the attention of cells’ built-in repair pathways. This protein machinery—the DNA mismatch repair system—reversed the prime editor’s changes if it noticed the rewriting. By adding in silent mutations near the edits they made to F508del, Liu’s team found that the mismatch repair system was less likely to undo edits.
By RJ Mackenzie
Article can be accessed on: The Scientist