Functional rescue of F508del-CFTR through revertant mutations introduced by CRISPR base editing

Abstract

SummaryCystic Fibrosis (CF) is a life-shortening autosomal recessive disease caused by mutations in theCFTRgene, resulting in functional impairment of the encoded ion channel. F508del mutation, a trinucleotide deletion, is the most frequent cause of CF affecting approximately 80% of patients. Even though current pharmacological treatments alleviate the F508del-CF disease symptoms there is no definitive cure. Here we leveraged revertant mutations (RMs) inciswith F508del to rescue CFTR protein folding and restore its function. We developed CRISPR base editing strategies to efficiently and precisely introduce the desired mutations in the F508del locus. Both editing and CFTR function recovery were verified in CF cellular models including primary epithelial cells derived from CF patients. The efficacy of the CFTR recovery strategy was validated in cultures of pseudostratified epithelia from patients’ cells showing full recovery of ion transport. Additionally, we observed an additive effect by combining our strategy with small molecules that enhance F508del activity, thus paving the way to combinatorial therapies.