Several DNA correction strategies developed and refined over the past few years hold great promise for the definitive cure of genetic diseases such as cystic fibrosis (CF).
CRISPR-Cas9 technology allows the correction of DNA mutations with high efficiency and precision.  While some mutations can be directly corrected with CRISPR-Cas9, there are mutations such as F508del that cannot be repaired with standard CRISPR-Cas9 strategies. However, that same technology can be exploited to introduce “neutralizing” mutations which can revert the effect of the F508del main mutation.
To correct the mutations the research team exploited CRISPR-base editors, advanced tools to precisely modify DNA. An alternative indirect strategy was used for F508del mutation, introducing “neutralizing” mutations which can revert the effect of F508del, while a direct correction was performed on 2789+5G>A mutation.
The previously identified neutralizing mutations have been inserted into F508del-CFTR cell lines, evaluating the editing efficiency and the rescue of CFTR channel function. Then the editing efficiency was evaluated also in primary human bronchial epithelial cells.
A correction strategy has been developed also for the 2789+5G>A mutation. After initial tests in cell lines, researchers verified the correction efficiency both in patient-derived intestinal organoids and in bronchial cells. The rescue of the CFTR function was also evaluated in these models.
It was observed that the introduction of neutralizing mutations in the CFTR gene carrying F508del mutation induces a functional rescue of CFTR protein in cellular models. Neutralizing mutations were also efficiently inserted into primary cells.
The use of base editors to correct the 2789+5G>A mutation restored the function of CFTR protein in both primary cells and organoids.
These results demonstrate that base editors are a promising strategy for future CF treatment.
This research has demonstrated that mutations, even the most difficult to correct, can be repaired with CRISPR-Cas9 technology.
The success of these strategies now depends on effective delivery to the target tissues, particularly the lungs of CF patients. to move forward with clinical testing.