A Versatile Reporter Platform for Evaluating HDR- and NHEJ-Based Genome Editing in Airway Epithelial Cell Cultures Using an rAAV Vector
Year: 2025
Type: article
Abstract: Therapeutic gene editing strategies utilize endogenous DNA repair pathways—nonhomologous end joining (NHEJ) or homology-directed repair (HDR)—to introduce targeted genomic modifications. Because HDR is restricted to dividing cells, whereas NHEJ functions in both dividing and non-dividing cells, NHEJ-based approaches are better suited for in vivo gene editing in the largely post-mitotic airway epithelium. Homology-independent targeted insertion (HITI), an NHEJ-based method, offers a promising strategy for cystic fibrosis (CF) gene therapy. Here, we applied HITI to drive the expression of a promoterless reporter through an exon trap strategy in both proliferating airway basal cells and well-differentiated primary airway epithelial cultures derived from transgenic ROSAmTmG ferrets. We also established a versatile human gene editing reporter (GER) airway basal cell line capable of multipotent differentiation, enabling real-time visualization of editing outcomes and the quantitative assessment of HDR- and NHEJ-based editing efficiencies. Together, these platforms provide easily accessible tools for optimizing genome editing strategies in the respiratory epithelium and advancing clinically relevant delivery strategies for CF gene therapy.
Source: Viruses
DOI: 10.3390/v17060821
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,137
Funders: Cystic Fibrosis Foundation, National Institutes of Health, Holden Comprehensive Cancer Center, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Environmental Health Sciences
Awards: G1147578840, G1504126945, G2505900161, G2666572476, G336615015, G4118404268, G414168849, Development of myotropic human bocavirus vectors for gene therapy, G5651769939, G5703857491, G5879425825, G6637812794, G6727933168, G7544451984, Viral and Host Determinants of Parvovirus Replication, Environmental Health Sciences Research Center, Development of novel approaches for gene editing therapies of cystic fibrosis
OpenAlex ID: W4411101377