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Researchers corrected mutations that cause cystic fibrosis in cultured human stem cells. In collaboration with the UMC Utrecht and Oncode Institute, they used a technique called prime editing to replace the 'wrong' piece of DNA with a healthy sequence. The study, published August 9 in Life Science Alliance, found that prime editing is safer than conventional CRISPR/Cas9 technology. "For the first time, we have shown that this technique really works and can be safely applied in human stem cells to correct cystic fibrosis."

Cystic fibrosis (CF) is one of the most common genetic diseases worldwide and has major consequences for patients. Although treatments are available to reduce the mucus and prevent inflammation typical of the disease, CF is not yet curable. CF is not yet curable. However, a new study by the Hans Clevers group (Hubrecht Institute) in collaboration with the Jeffrey Beekman group (UMC Utrech)  and Oncode Institute offers new hope.

Correction of the CF mutations 

The researchers succeeded in correcting the mutations that cause CF in human gut organoids. These organoids, also called mini-organs, are small 3D structures that mimic the intestinal function of CF patients. They were previously developed by the same research group starting from stem cells of patients with CF and stored in a biobank in Utrecht. For the study, published in Life Science Alliance, a technique called prime editing was used to replace the pathogenic mutation with a healthy DNA sequence.

Safer than CRISPR/Cas9
Prime editing is a new version of the better known CRISPR/Cas9 gene editing technique. The CRISPR/(Cas9 system acts as molecular scissors, cutting the DNA before correcting it. Although it can correct mutations of the DNA, it also causes damage in other places in the genome. "In our study, prime editing is safer than the conventional CRISPR/Cas9. It can correct DNA without causing damage elsewhere in the genome. This makes the technique promising for applications in patients," said Maarten Geurts, first author on the publication.

Swell up 

The mutations that cause CF are localized in the CFTR channel, which is present in the cells of various organs, including the lungs. Due to the mutations, the canal does not function properly, so the mucus layer that covers the cells gets too little water: the mucus becomes tough. In organoids without mutations, the addition of a substance called forskolin leads to the swelling of the mini-organ, but this response does not occur in organoids with mutations in the CFTR channel. "We applied prime editing to the mutations and saw that the treated organoids then gave the same response as the healthy organoids: they swelled up. That was proof that our technique had worked and replaced the mutated DNA," Geurts said.

Curing genetic disorders 

Now that the researchers have shown that the mutations that cause CF can be corrected in a safe way, applications in the clinic are one step closer. "Newer variants of CRISPR/Cas9, such as prime editing, can correct mutations without causing damage at any other place in the DNA. This will hopefully allow us to cure or even prevent genetic disorders in the future." Before that happens, there are also some challenges ahead for the researchers. For example, the technique still needs to be adjusted before it can also be used safely in humans. "But this is a nice step towards the successful application of prime editing in the clinic" concludes Geurts.

Another example

Last year, the group of Sabine Fuchs (UMCU) has already proved prime editing to be a successful strategy to repair genes in liver organoids.

Liver organoids were grown from Wilson's disease patients. These patients cannot process copper properly, copper accumulates in the liver and becomes toxic. The same happens to mini-livers derived from such patients, as they die when exposed to copper. After genetic repair with prime editing, patients' organoids survive and grow, even in the presence of copper.

The same approach was used with intestinal organoids from patients with DGAT1, a disease in which fats are not properly processed and result toxic. Thanks to genetic correction, intestinal organoids derived from these patients were able to survive after exposure to fat.