DNA-PKcs Inhibition Improves Sequential Gene Insertion of the Full-LengthCFTRcDNA in Airway Stem Cells

Abstract

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although many people with CF (pwCF) are treated using CFTR modulators, some are non-responsive due to their genotype or other uncharacterized reasons. Autologous airway stem cell therapies, in which theCFTRcDNA has been replaced, may enable a durable therapy for all pwCF. Previously, CRISPR-Cas9 with two AAVs was used to sequentially insert two halves of theCFTRcDNA and an enrichment cassette into theCFTRlocus. However, the editing efficiency was <10% and required enrichment to restore CFTR function. Further improvement in gene insertion may enhance cell therapy production. To improveCFTRcDNA insertion in human airway basal stem cells (ABCs), we evaluated the use of the small molecules AZD7648 and ART558 which inhibit non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ). Adding AZD7648 alone improved gene insertion by 2-3-fold. Adding both ART558 and AZD7648 improved gene insertion but induced toxicity. ABCs edited in the presence of AZD7648 produced differentiated airway epithelial sheets with restored CFTR function after enrichment. Adding AZD7648 did not increase off-target editing. Further studies are necessary to validate if AZD7648 treatment enriches cells with oncogenic mutations.