Novel cell models to study Cystic Fibrosis : from disease mechanisms to new therapeutic approaches

Cystic fibrosis (CF) is the most common life-shortening autosomal genetic disorder in Caucasians, affecting 90,000 people worldwide. This disease is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an anion channel expressed on the apical membrane of epithelial cells. More than 2,100 variants have been identified in CFTR gene, 360 of which have been confirmed as disease-causing. Although four CFTR modulators have been approved for clinical use, they only benefit approximately 85% of individuals with CF. Hence, effective treatment is needed for the remaining patients. Many CFTR mutations are uncommon variants, with an outcome of the disease difficult to predict, since the precise nature of the functional defect they cause has not been defined. Elucidation of the molecular and cellular effects of different mutations on CFTR protein structure and function can be useful to predict disease severity and can provide the scientific basis to uncover new drug targets specific for each mutation. The use of cellular models is valuable in the clarification of these mechanisms, particularly when access to patients’ tissue is difficult. Here, we proposed: 1) to create isogenic cell lines, homozygous for rare CF-causing mutations; 2) to define disease signatures specific for different CF mutations; 3) to evaluate genome editing tools as a therapeutic option for CF. We adapted the CFF lab gene editing pipeline using CRISPR/Cas9-mediated homology-directed repair (HDR) to create cell lines homozygous for rare CFTR mutations – I507del and I1234V – and for most frequent CFTR mutation – F508del – on the epithelial cell line (16HBE14o-) derived from lung. These cells lines were then analysed for CFTR expression and function. Since these cell lines have the same genetic background, differing only by their CFTR mutation, cells carrying common and rare mutations were used in a large scale transcriptomic and proteomic study. In this study, we were able to identify a number of differentially expressed genes and proteins resulting from the different CFTR mutations. Integration of transcriptomic and proteomic data allowed us to identify molecular signatures specific for each of the different CFTR mutations, which may serve as potential therapeutic targets for CF treatment. CRISPR gene editing tools can be of great value to permanently correct these mutations. We compared the ability of HDR-mediated by Cas9 or Cas12a to correct W1282X-CFTR mutation. Our data demonstrate that Cas9 introduces higher levels of correction at this genomic site than Cas12a. This correction at the DNA level results in increased levels of CFTR mRNA and protein, and partially restoring CFTR anion channel function in the pool of edited cells. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT-CFTR, demonstrating the potential of gene editing as a therapeutic strategy for CF