Site-specific Human CFTR Gene Integration through CRISPR/Cas9-mediated Targeting

Abstract

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). It leads to multiple organ dysfunctions, where lung disease is the major cause of morbidity and mortality. Gene therapy for CF lung disease is still challenged with achieving sustained and sufficient therapeutic transgene expression. The advancement of site-specific endonuclease, CRISPR/Cas9, sheds light on permanent CFTR gene correction. We proposed a novel strategy that can precisely integrate therapeutic human CFTR (hCFTR) gene into genomic safe harbor through Cas9-induced homology directed repair (HDR), via an all-in-one helper-dependent adenoviral (HD-Ad) vector delivery. This strategy can target CFTR mutations regardless of mutation types. We have demonstrated a 10% integration efficiency in porcine CFTR-/- cells. The integrated hCFTR gene is sustainably and functionally expressed in cells. By co-delivering another HD-Ad vector to express HDR factor, Exo1 or CtIP, transgene integration efficiency is further enhanced by an average of 2-fold. We have also proven that the airway basal stem cells can be targeted by our hCFTR gene editing system in vitro. Moreover, functional hCFTR is expressed in primary CFTR-/- porcine airway epithelial cells. Overall, this study demonstrates that our HD-Ad vector-based hCFTR gene targeting system has the potential to be adapted for treating CF lung disease.