Targeted replacement of full-length CFTR in human airway stem cells by CRISPR-Cas9 for pan-mutation correction in the endogenous locus.

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the CF transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR-Cas9 and two adeno-associated viruses (AAVs) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial epithelial cells (HBECs). The modified cells were enriched to obtain 60%-80% tCD19 ⁺ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.
Competing Interests: Declaration of interests M.H.P. has equity and serves on the Scientific Advisory Board of CRISPR Therapeutics and Graphite Bio. J.V.N. is a consultant with COOK Medical, which manufactures the pSIS graft. Neither company had any input on the design, execution, interpretation, or publication of the work in this manuscript.
(Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)