High-Efficiency, Selection-free Gene Repair in Airway Stem Cells from Cystic Fibrosis Patients Rescues CFTR Function in Differentiated Epithelia

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using in vivo gene therapy, and low correction levels have hindered ex vivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the ΔF508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%–50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients and observed 20%–50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF. [Display omitted] •Cas9 RNP and AAV can be used to efficiently gene edit human airway basal stem cells•This method yields >30% allelic correction without selection markers or antibiotics•Correction of >30% ΔF508 alleles restores CFTR function to near non-CF levels•Corrected stem cells can differentiate after embedding in the scaffold for engraftment Vaidyanathan et al. use Cas9 and AAV to correct the CF causing ΔF508 mutation in >30% of alleles in airway basal stem cells from CF patients and embed corrected cells on a scaffold for engraftment. This method restores physiologic CFTR function and provides an ex vivo strategy to treat cystic fibrosis.