A Versatile Reporter Platform for Evaluating HDR- and NHEJ-Based Genome Editing in Airway Epithelial Cell Cultures Using an rAAV Vector

TL;DR

This paper introduces a new reporter system to study gene editing in airway cells, which could help improve gene therapy for cystic fibrosis.

Contribution

A versatile reporter platform for evaluating HDR and NHEJ-based genome editing in airway epithelial cells using an rAAV vector.

Findings

A reporter system was developed using an exon trap strategy in ferret-derived airway cells.

A human airway basal cell line was created for real-time visualization and quantitative assessment of gene editing.

The platform supports optimization of gene editing strategies for post-mitotic airway epithelium.

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.

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