Sequence-Specific Gene Correction of Cystic Fibrosis Airway Basal Cells

Cystic fibrosis (CF) is a lethal monogenic disease resulting from mutations in the CFTR gene which encodes a protein involved in regulating anion trans-epithelial transport. A three-base deletion in CFTR (termed as ΔF508 mutation), wherein CFTR protein is misfolded leading to its pre-mature degradation in the endoplasmic reticulum (ER), is the most common cause of this debilitating disease. Since CFTR is expressed in multiple body systems, CF affects different organs, but lung pathology is the greatest cause of death in affected patients. We achieved site-specific gene correction with an efficiency of ~10 % in CF airway basal cells homozygous for the ΔF508 mutation. Basal cells are a multipotent stem cell population of the respiratory epithelium and therefore, their gene correction could provide a long-term, permanent remedy for CF. Delivery of engineered sequence-specific zinc finger nucleases (ZFNs) and single-stranded oligo DNA (ssODN) carrying the correcting sequence via electroporation facilitated the correction. The gene-corrected cells upon in vitro differentiation using air-liquid interface showed presence of fully-glycosylated mature CFTR protein as opposed to differentiated mutant cells which synthesized only the core-glycosylated immature form. Most importantly, we demonstrated CFTR ion channel activity in the gene-corrected cells by Ussing chamber electrophysiology