Cystic fibrosis Cell Models for Research

Cystic fibrosis (CF) is a life-limiting autosomal recessive disorder caused by mutations in the CFTR gene. According to the World Health Organization (WHO), the global incidence is approximately 1 in 2,500 to 1 in 6,000 live births, with higher prevalence in populations of European descent. The median predicted survival age has improved to over 50 years in some registries, but significant morbidity remains. The disease affects multiple organs, primarily the lungs, leading to chronic infections and progressive respiratory failure. The National Cancer Institute (NCI) does not track CF as a cancer, but the disease burden is significant, with a high economic cost and reduced quality of life. The CF Foundation Patient Registry reports over 30,000 individuals in the US alone. Research models are critical to understand disease mechanisms and develop therapies.

Cystic fibrosis is an ideal model for studying monogenic disorders, protein misfolding, and ion channel biology. The availability of well-characterized patient-derived cell lines and organoids, combined with CRISPR gene editing, allows for precise modeling of specific CFTR mutations. Public datasets such as the CFTR2 database and the Cystic Fibrosis Foundation's Therapeutics Development Network provide extensive clinical and genetic data. Open questions include the role of modifier genes, the impact of CFTR dysfunction on immune responses, and the development of resistance to current therapies. Gene-edited cell models enable functional studies of CFTR variants and the evaluation of novel therapeutic strategies.

While CF is not a cancer, the molecular pathways involved in CFTR dysfunction share similarities with oncogenic signaling. The primary defect is in the CFTR chloride channel, leading to dysregulated ion transport and subsequent multi-organ pathology. The key pathways include:

• • Defective CFTR processing and trafficking: The most common mutation, F508del, causes misfolding and degradation of the protein, leading to loss of function.
• • Chronic inflammation and infection: CFTR dysfunction impairs mucociliary clearance, leading to chronic bacterial colonization and neutrophil-dominated inflammation.
• • Activation of NF-κB and MAPK pathways: Chronic inflammation activates these pathways, contributing to tissue damage and remodeling.
• • Epithelial-mesenchymal transition (EMT): Chronic injury may promote EMT, leading to fibrosis and potentially increasing cancer risk in CF patients, though this is not well established.

The most common genetic alterations in CF are mutations in the CFTR gene. Data from the CFTR2 database and ClinVar indicate over 2,000 variants, with frequencies varying by population. The table below lists the most frequent mutations:

These mutations are catalogued in ClinVar and the CFTR2 database.

CFTR dysfunction leads to deregulation of several signaling networks:

• • NF-κB pathway: Chronic inflammation activates NF-κB, leading to increased expression of pro-inflammatory cytokines (IL-6, IL-8).
• • MAPK/ERK pathway: Inflammatory stimuli activate MAPK, contributing to cell proliferation and fibrosis.
• • PI3K/AKT pathway: CFTR may interact with PI3K, affecting cell survival and apoptosis.
• • Wnt/β-catenin pathway: Altered CFTR function may influence Wnt signaling, affecting epithelial repair and regeneration.

These networks are potential therapeutic targets and can be studied using gene-edited cell models.

Several cell lines are commonly used in CF research. The table below lists key cell lines and their characteristics:

Organoids derived from patient intestinal or lung tissue are increasingly used to model CFTR function and test therapeutics. They recapitulate disease phenotypes and can be gene-edited to create isogenic controls.

Animal models for CF include:

• • CFTR knockout mice: Developed by gene targeting, these mice exhibit severe intestinal obstruction but mild lung disease, limiting their utility for lung studies.
• • CFTR F508del mice: These mice carry the most common mutation and show residual function, better mimicking human disease.
• • CFTR knockout rats: Rats have more human-like lung physiology and are used for lung disease modeling.
• • Pig and ferret models: These large animals develop spontaneous lung disease and are valuable for studying pathogenesis and testing therapies.
• • Patient-derived xenografts (PDX): Not commonly used for CF, but organoids can be transplanted into mice to study CFTR function.

CRISPR-Cas9 gene editing has revolutionized CF research by enabling the creation of isogenic cell lines with specific CFTR mutations. These models are essential for studying mutation-specific effects and testing therapies. Examples include:

• • CFTR knockout cell lines: Generated by introducing frameshift mutations in CFTR, these lines are useful for studying the loss-of-function phenotype.
• • F508del knock-in cell lines: Created by introducing the F508del mutation into wild-type cells, these lines mimic the most common patient mutation.
• • G551D knock-in cell lines: These lines carry the gating mutation and are used to test potentiators.

These gene-edited models are commercially available and sequence-verified, ensuring reproducibility. They are used in drug screening, functional genomics, and biomarker discovery.

Gene-edited cell lines are used to validate the function of CFTR variants and identify modifier genes. For example, CRISPR knockout of CFTR in bronchial epithelial cells allows researchers to study the impact on ion transport and inflammation. Knock-in of specific mutations enables the study of mutation-specific responses to drugs. These models are also used in genome-wide CRISPR screens to identify genes that modulate CFTR function or rescue mutant CFTR.

Isogenic cell line pairs (wild-type vs. mutant) are used in high-throughput screening to identify compounds that correct CFTR folding or potentiate channel activity. For example, F508del knock-in cells are used to test correctors like lumacaftor and tezacaftor. Resistance to CFTR modulators can be studied by exposing cells to increasing drug concentrations and selecting for resistant clones, which can then be analyzed for secondary mutations.

CRISPR-based synthetic lethality screens in CFTR-deficient cells can identify genes that are essential for cell survival in the absence of CFTR. These genes may serve as therapeutic targets or biomarkers. Additionally, gene-edited cells can be used to identify biomarkers of CFTR function, such as changes in protein expression or post-translational modifications.