Correction of the ΔF508 mutation in the CFTR Gene by CRISPR/Cas9 system

Cystic Fibrosis (CF) is one of the most common autosomal recessive genetic diseases. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). CF causes chronic lung disease including thickened mucus, bacterial infection and inflammation, with a progressive loss of pulmonary function and, ultimately, death. Several clinical trials have been performed to date assessing the potential of gene therapy to limit the progression of CF lung disease. However, a clinically relevant treatment has yet to emerge. The major challenges in gene therapy for CF relate to the limited levels of gene transfer achieved in the lung airway epithelium, and the persistence of transgene expression. Here we are investigating the potential of genome editing to develop a genetic therapy for CF using the CRISPR/Cas system that allows for gene-specific, targeted correction of disease-related mutations to be introduced at the chromosomal level. In particular, we aim to investigate the therapeutic potential for ΔF508 mutation cystic fibrosis, which is the most common CF mutation and affects more than 70% of patients. Initially, multiple guide RNAs were screened for double strand break (DSB), targeting the CFTR gene close to the ΔF508 mutation on the CFBE41o- cell line. The efficient gRNAs were used for the mutation correcting through homology directed repair (HDR) after which the cells were cloned. The correction was confirmed at the molecular level, followed by restoring the electrophysiology function and the mRNA expression. This work was also extended to correct the mutation on primary CFBE cells where the editing was optimized with Cas9 mRNA/gRNA and ribonucleoprotein (RNP) was delivered by Receptor-Targeted Nanocomplex (RTN). To improve the editing efficiency, the homology-independent targeted integration (HITI) strategy as an alternative for HDR, was used to investigate the CFTR exon 10 in CFBE41o- being replaced with wildtype exon. The gene editing in vivo was explored successfully in a mice reporter model to restore tdTomato expression by paired gRNAs excision the stop cassette where targeting nanocomplex was used as a safe and non-immunogenic delivery method