Targeted insertion of partial CFTR cDNA replicates native CFTR expression with restored channel function in CF airway epithelium

Our objective is to develop a universal gene editing therapeutic for individuals with cystic fibrosis (CF). Due to the large number of CFTR variants, we sought to develop a correction methodology utilizing targeted integration of CFTR _2-27 sequence into CFTR intron 1 (TI-1). Following electroporation of CF primary airway basal stem cells with intron 1-targeted Cas9/gRNA, we delivered CFTR _2-27 donor, flanked by a splice acceptor and polyadenylation signal, via an integrase defective lentiviral vector. Molecular characterization revealed highly efficient integration of the CFTR _2-27 sequence into the native CFTR intron 1 with most insertion events reflecting the desired homology-directed repair. Fully differentiated airway epithelium, derived from TI-1 treated basal cells without enrichment for corrected cells, exhibited functional restoration of CFTR channel activity with significant improvement of mucus hydration and transport. ATAC-seq revealed that the native CFTR chromatin architecture was unaffected by the CFTR _2-27 sequence insertion, and importantly, scRNA-seq demonstrated that the cell-type-specific expression of the introduced transgene replicated that of the endogenous CFTR. In this report, we demonstrate an almost completely mutation-agnostic therapeutic approach that is capable of functional CFTR restoration. These results may also be relevant to CFTR _2-27 integration via other editing and delivery methodologies.