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Updated: Mar 16, 2026
Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study
Published on: April 11, 2018
Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell
Christien Bednarski1,2, Katja Tomczak3, Beate Vom Hövel1,2
1Institute for Cell and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.
Abstract:
In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5' end of exon 11.
Insights
This study developed a simple method to correct cystic fibrosis (CF) by integrating a large super-exon into the CFTR gene in bronchial cells, restoring normal ion transport.
Area of Science:
- Biotechnology
- Genetic Engineering
- Cell Biology
Background:
- In vitro models are crucial for understanding human diseases and testing therapies like genome engineering.
- Existing cystic fibrosis (CF) models, such as iPSC-derived organoids, often involve complex and costly culturing, limiting high-throughput screening.
- There is a need for simpler, more accessible in vitro models for CF research and therapeutic validation.
Purpose of the Study:
- To establish a straightforward in vitro model for validating CFTR functional correction.
- To demonstrate the efficacy of a novel genome engineering strategy for correcting CFTR in a CF cell line.
- To assess the restoration of CFTR channel function after gene editing.
Main Methods:
- Utilized the CFBE41o- bronchial epithelial cell line, a model for CF.
- Employed an engineered nuclease for targeted integration of a CFTR super-exon (exons 11-27) into exon 11.
- Activated endogenous CFTR expression using a demethylating agent.
- Validated gene correction through mRNA expression analysis and short-circuit current measurements in Ussing chambers.
Main Results:
- Successfully integrated a CFTR super-exon into the target locus in CFBE41o- cells.
- Demonstrated expression of corrected CFTR mRNA transcribed from the endogenous promoter.
- Confirmed restoration of ion transport function in the edited CFTR channels via Ussing chamber assays.
Conclusions:
- Targeted integration of a large super-exon into CFTR exon 11 effectively corrects CFTR function.
- This genome engineering strategy offers a viable approach for functionally correcting CFTR mutations downstream of the 5' end of exon 11.
- The developed simple cellular model is suitable for validating CFTR correction strategies.
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