Recombinant Adeno-Associated Virus Vector Mediated Gene Editing in Proliferating and Polarized Cultures of Human Airway Epithelial Cells.

Authors

Soo Yeun Park, Zehua Feng, Soon H Choi, Xiujuan Zhang, Yinghua Tang, Grace N Gasser, Donovan Richart, Feng Yuan, Jianming Qiu, John F Engelhardt, Ziying Yan

Published in

Human gene therapy. Volume 36. Issue 15-16. Pages 1067-1082. Epub May 13, 2025.

Abstract

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. While CRISPR-based CFTR editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and organoid cultures derived from patients with CF, their efficacy remains suboptimal. Here, we developed the CuFi^{Cas9(Y66S)eGFP} reporter system by integrating spCas9 and a non-fluorescent Y66S eGFP mutant into CuFi-8 cells, an immortalized human airway epithelial cell line derived from a patient with CF with homozygous F508del mutations. These cells retain the basal cell phenotype in proliferating cultures and can differentiate into polarized airway epithelium at an air-liquid interface (ALI), enabling both visualized detection of gene editing and electrophysiological assessment of CFTR functional restoration. Using this system, recombinant adeno-associated virus (rAAV)-mediated homology-directed repair (HDR) was evaluated in proliferating cultures. A correction rate of 13.5 ± 0.8% was achieved in a population where 82.3 ± 5.6% of cells were productively transduced by AAV.eGFP630g2-CMVmCh, an rAAV editing vector with an mCherry reporter. Dual-editing of F508del CFTR and Y66S eGFP was explored using AAV.HR-eGFP630-F508(g03) to deliver two templates and single guide RNAs. eGFP⁺ (Y66S-corrected) cells and eGFP^- (non-corrected) cells were sorted via fluorescence-activated cell sorting and differentiated at an ALI to assess the recovery of CFTR function. Despite a low F508 correction rate of 2.8%, ALI cultures derived from the eGFP^- population exhibited 25.2% of the CFTR-specific transepithelial Cl^- transport observed in CuFi-ALI cultures treated with CFTR modulators. Next-generation sequencing revealed frequent co-editing at both genomic loci, with sixfold higher F508 correction rate in the eGFP⁺ cells than eGFP^- cells. In both populations, non-homology end joining predominated over HDR. This reporter system provides a valuable platform for optimizing editing efficiencies in proliferating airway basal cells, particularly for development of strategies to enhance HDR through modulation of DNA repair pathways.

PMID:
40359132
Bibliographic data and abstract were imported from PubMed on 12 Sep 2026.

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