Reverting the F508del-CFTR defect in Cystic Fibrosis with CRISPR-Cas technology

Cystic Fibrosis (CF) is a common life-shortening autosomal recessive disease that affects over 100.000 people worldwide people worldwide. It is caused by mutations in the CF trans-membrane conductance regulator (CFTR) gene, that encodes for a membrane channel localized at the apical surface of epithelial cells where it has a crucial role in the secretion of chloride and bicarbonate. Over 2100 different CFTR mutations have been reported and among the pathogenic once the most common is F508del, located in the nucleotide-binding domain 1 (NBD1). F508del is a three-nucleotide deletion that results in the loss of a phenylalanine at position 508 in the protein and in the consequent CFTR degradation by the ubiquitin-proteasome system. Different attempts to correct F508del-CFTR gene were made using genome editing approaches, however deletions like F508del remain difficult to be repaired. Several studies reported that additional mutations (revertant mutations) in the F508del-CFTR gene can rescue both CFTR folding and activity, suggesting a potential novel strategy to correct F508del. For this reason, the first aim of this work was the identification of novel F508del-CFTR revertants that can rescue CFTR localization and function. We generated a library of mutants introducing random substitutions into the F508del-CFTR gene. Revertant mutations were isolated based on their ability to rescue the presence of CFTR at the plasma membrane (PM) in HEK293T cells and identified by Sanger sequencing. Restoration of CFTR maturation, localization, and function of the identified revertants was evaluated by western blot, flow cytometry analysis and YFP assay, reaching levels similar to the wild type CFTR. Then we used CRISPR-Cas technology to introduce selected revertant mutations, such as I539T, R553Q, G550E, R555K and R1070W, in the endogenous F508del-CFTR gene. Adenine and cytosine base editors (ABE and CBE) allow the insertion of the desired base conversion without the formation of double strand breaks. Efficient editing was evaluated through Sanger sequencing, reaching up to 60% of base conversion. CFTR rescue at the PM in edited cells was analyzed by flow cytometry showing different degrees of recovery compared to the wild type CFTR. In this work, we confirmed that revertant mutations can rescue F508del CFTR localization and function. In addition, we demonstrated that CRISPR-base editors are valid tools to introduce these mutations in the endogenous F508del-CFTR gene, leading to a permanent correction. The proposed strategy could overcome the limits that genome editing strategies faced till now in the correction of F508del, providing a new potential therapeutic approach to treat CF.