A Versatile Reporter Platform for Evaluating HDR- and NHEJ-Based Genome Editing in Airway Epithelial Cell Cultures Using an rAAV Vector
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...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Sprog: | engelsk |
| Udgivet: |
MDPI AG
2025-06-01
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| Serier: | Viruses |
| Fag: | |
| Online adgang: | https://www.mdpi.com/1999-4915/17/6/821 |
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| Summary: | 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 ROSA<sup>mTmG</sup> 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. |
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| ISSN: | 1999-4915 |