Targeted Insertion of Corrective CFTR Transgene Replicates Native Expression Pattern with Functional Restoration in Cystic Fibrosis Airway Epithelium

Our objective is to develop a universal gene editing therapeutic for individuals with Cystic Fibrosis (CF). Because of the large number of CFTR variants, we sought to develop a mutation-agnostic methodology capable of efficient CFTR2-27 integration into CFTR intron 1. Following electroporation of CF primary airway epithelial cells with intron 1-targeted Cas9/gRNA, we delivered CFTR2-27 donor, flanked by a splice acceptor and polyadenylation signal, via integrase defective lentivirus vector. Molecular characterization revealed highly efficient integration of CFTR2-27 into the native CFTR intron 1 with the majority of insertion events reflecting the desired homology directed repair. Integration resulted in functional restoration of CFTR channel activity in fully differentiated airway epithelium with significant improvement of mucus hydration and transport. ATAC-seq revealed that the native CFTR chromatin architecture was unaffected by the CFTR2-27 insertion and importantly, scRNA-seq demonstrated that the cell type-specific expression of the introduced transgene replicated that of the endogenous CFTR.