Mucociliary clearance (MCC) is an innate defense mechanism that normally keeps airways clean but is dysfunctional in cystic fibrosis (CF) and other muco-obstructive pulmonary diseases. Previously we discovered that activating adenyl cyclase in combination with a cholinergic agonist increased MCC velocity (MCCV) synergistically in ex vivo WT and CF ferret and WT piglets. For what we believe is the first time, we show in vivo synergistic MCC using FDA approved β-adrenergic and cholinergic drugs delivered to the apical surface of WT and CF rats and a CF sheep model. Also, a single dose of the combined drugs is tolerated by humans. As for mechanisms, via ex vivo experiments, we show the combined agonists increased net fluid secretion mainly by stimulating gland secretion and by inhibiting surface absorption, consequently increasing airway surface liquid depth. They also increased net base secretion and increased ciliary beat frequency. Additional ex vivo and in vitro experiments show that the combined agonists had additive effects when combined with highly effective CF transmembrane conductance regulator modulator therapy. The synergistic increase in MCCV induced by this combination of agonists offers therapeutic potential for treating muco-obstructive pulmonary diseases, including CF.

CRISPR-based gene editing holds promise for treating genetic diseases, yet its application to lung disorders has been hindered by the challenges of pulmonary delivery. Inspired by the modularity and biocompatibility of amino acid-derived chemistries, we report the combinatorial synthesis of 960 ionizable lipids incorporating chemically diverse backbones from both proteinogenic and non-proteinogenic α-amino acids. Through high-throughput screening and structure-function analysis, we identify CHCha-10, a cyclohexyl amino acid-derived lipid that forms biodegradable nanoparticles capable of efficiently delivering mRNA-based gene editors to lung epithelial cells. Following intratracheal administration, CHCha-10 nanoparticles exhibit enhanced mucus penetration and epithelial-specific transfection in both mice and ferrets. Here, as a functional application, we demonstrate in vivo base editing in the lung via inhalation. Delivery of adenine base editor mRNA and guide RNA targeting the CFTR G542X mutation restores CFTR expression and chloride channel function in G542X human airway epithelial cells, mouse-derived intestinal organoids and the lungs of cystic fibrosis mice. This work establishes a chemically modular design framework for ionizable lipids and a translatable platform for RNA-based pulmonary gene correction.

In 2024-2025, the Cystic Fibrosis Foundation (US) and Cystic Fibrosis Trust (UK) hosted an International CFRD Consortium round-table webinar series for basic science, translational, and clinical researchers with the goal of sharpening mechanistic understanding of CFRD pathogenesis and prioritizing therapeutic development. This review summarizes the research priorities identified in the International CFRD Consortium, including (i) further investigation into the role of pancreatic fibrosis, vascular abnormalities, and α-cell dysfunction in the development of CFRD; (ii) the creation and refinement of novel animal and human cell- and tissue-based models to understand the complex interplay of exocrine and endocrine cells in the CF pancreas; (iii) development and validation of circulating and imaging biomarkers, together with dynamic glucose testing to explore β-cell function and kinetics in people with CF across the dysglycemia spectrum; and (iv) prospective clinical studies to guide CFRD treatment options and investigate the changing landscape of aging, increasing prevalence of obesity and diabetes and their complications in the era of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. Collectively, these priorities aim to accelerate transition from mechanism to intervention and expand evidence-based care for people with CF at risk of, or living with, CFRD.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). While gene therapy holds promise as a cure, the cell-type-specific heterogeneity of CFTR expression in the lung presents significant challenges. Current CF ferret models closely replicate the human disease phenotype but have limitations in studying functional complementation through cell-type-specific CFTR restoration. To address this, we developed a new transgenic ferret line, CFTRint1-eGFP(lsl), in which a Cre-recombinase (Cre)-excisable enhanced fluorescent protein (eGFP) reporter cassette is knocked in (KI) to intron 1 of the CFTR locus. Breeding this reporter line with CFTRG551D CF ferret resulted in a novel CF model, CFTRint1-eGFP(lsl)/G551D, with disease onset manageable via the administration of CFTR modulator VX770. In this study, we confirmed two key properties of the CFTRint1-eGFP(lsl)/G551D CF ferrets: (1) cell-type-specific expression of the CFTR(N-24)-eGFP fusion protein, driven by the intrinsic CFTR promoter, in polarized epithelial cultures and selected tissues, and (2) functional reversion of the KI allele via Cre-mediated excision of the reporter cassette. This model provides a valuable tool for studying the effects of targeted CFTR reactivation in a cell-type-specific manner, which is crucial for enhancing our understanding of CFTR's roles in modulating airway clearance and innate immunity, and for identifying relevant cellular targets for CF gene therapy.

Lipid nanoparticle (LNP)-based delivery of CFTR mRNA holds promise for treating pulmonary manifestations of cystic fibrosis (CF). LUNAR-CFTR is a novel proprietary lipid nanoparticle (LNP) encapsulating codon-optimized human CFTR mRNA for CFTR replacement therapy. In this study, we examined the potential of LUNAR-CFTR for delivering mRNA and restoring CFTR function in primary human and ferret airway epithelia and mucus-laden CF ferret airways. LUNAR effectively delivered CFTR, Cre, and tdTomato mRNAs into multiple airway epithelial cell types of humans and ferrets in vitro and transgenic ferrets in vivo. The LUNAR-CFTR was able to restore ion transport in polarized CF human bronchial epithelia to levels comparable to those achieved with elexacaftor/tezacaftor/ivacaftor. Mucociliary clearance (MCC) assays in CF ferrets further showed that a single dose of LUNAR-mediated human CFTR mRNA delivery improved mean MCC values by 3-fold. The improved MCC was in line with an increase in CFTR-mediated transepithelial anion transport from the CF ferret trachea transfected with LUNAR-CFTR in vivo. These findings underscore the potential of LUNAR LNP as a robust vehicle for delivering CFTR-encoding therapeutic mRNA into airway epithelial cells and underscore the utility of CF ferrets as a reliable tool to evaluate the efficiency of gene therapy in mucus-laden airways.

Background Data from cystic fibrosis (CF) animal models and case studies suggests that in utero administration of CF transmembrane conductance regulator (CFTR) modulators (variant specific therapies, VST) can rescue CFTR-related pathophysiology in the fetus. Use of VST during pregnancy to prevent disease in infants has not been systematically studied. Through stakeholder engagement, we sought to determine if formal research evaluation is warranted. Methods We surveyed CF care center directors to assess their awareness of the potential off-label use of VST for in utero treatment of a fetus with CF. We then conducted a one-day, international multidisciplinary workshop to review available pre-clinical and clinical data, embryology principles and federal drug regulation considerations, identify knowledge gaps, and consider future clinical study designs. Results Sixty-two unique individuals responded to the survey; 92% were aware of use of VST to treat pregnant females who are CF carriers for the prevention of CF complications in the fetus. Expert workshop presentations suggested that use of VST in pregnant females carrying a fetus with CF to mitigate complications of CF is relatively safe and effective in animal models and human case series to date. Further research is needed to understand the optimal timing of VST initiation during pregnancy to improve clinical outcomes, to understand VST pharmacokinetics, and optimize dosing of VST during pregnancy and lactation, and to evaluate the long-term infant safety among those exposed to VST in utero. Conclusions Based on available data and knowledge gaps, stakeholders agreed that formal evaluation of in utero and early life VST therapy in a prospective trial is warranted.

Rationale: Asthma is a chronic inflammatory lung disease caused by inflammation of airway after exposure to an allergen, which is characterized by variable and recurring symptoms including intermittent airflow obstruction, coughing, bronchial hyperresponsiveness wheezing and chest tightness. The ovalbumin (OVA)-induced allergic airway inflammation of rodents are the most characterized asthma models. However, rodents failed to recapitulate clinical symptoms of coughing, airway hypersecretion, and glandular remodeling observed in humans, in part due to the differences in lung cell biology, distribution of airway submucosal glands (SMGs), and cellular structure of the respiratory zone (lack of respiratory bronchioles in rodents). Ferrets are excellent models of many pulmonary diseases, including influenza infection, cystic fibrosis, and COPD. We thus hypothesized that an OVA-induced allergic airway inflammation ferret model could recapitulate key clinical and pathohistological features of asthma seen in human but lacking in OVA rodents. Methods: Ferrets were sensitized by intraperitoneally injecting 3 doses of OVA at 1-week intervals before they were boosted by weekly dosing OVA at 5 mg/mL/kG via nasal instillation for 9 weeks. The asthmatic lung phenotype of OVA-challenged ferret lungs was assessed by histopathology, expression of Th2 cytokines (IL-4, IL-5 and IL13), cytology of BALF, OVA-specific IgG, IgA, IgM and IgE, and pulmonary function tests (PFTs), as compared to that of saline controls. Results: Ferrets were hyperresponsive with wheezing and chest tightness in response to OVA exposure. Pathohistological analysis found a predominant infiltration of inflammatory cells in the lung of ferrets that received OVA with a score of 3.3±0.5, compared to the controls 0.6±0.3. Cytological analysis showed a significant increase of eosinophils in BALF of OVA ferrets (27.8±10.6%), compared to the control ferrets (1.42±1.64%). Serological evaluation demonstrated an antigen-specific immune response, with increased levels of OVA-specific IgE, IgA and IgG in plasma of OVA ferrets by 6.1 ± 1.0, 5.0 ± 0.6 and 65.4 ± 7.6 folds, compared to the controls, respectively. PFT further revealed a significant OVA-induced allergic reduction of inspiratory capacity, FEV0.4, and FVC, but increased airway resistance (Rrs) and elastance of the respiratory system (Ers). Conclusion: We characterized an OVA-induced allergic airway inflammation model of ferret, which recapitulated many features of asthma in humans, including eosinophil infiltration, elevated IgE, and increased airway obstruction. The OVA ferret model thus may offer a tool for delineating the pathogenesis of allergic airway inflammation and airway remodeling with human-relevant features.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited treatment options, partly due to a lack of effective disease models. This study presents a ferret model of pulmonary fibrosis (PF) induced by bleomycin, which replicates key characteristics of human IPF. The ferret model demonstrates an irreversible loss of pulmonary compliance, increased opacification, and structures resembling honeycomb cysts. Using single-nucleus RNA sequencing, we observed a significant shift in the distal lung epithelium toward a proximal phenotype. Cell trajectory analysis showed that AT2 cells transition into KRT8high/KRT7low/SOX4+ cells, and eventually into KRT8high/KRT7high/SFN+/TP63+/KRT5low “basaloid-like” cells. These cells, along with KRT7 and KRT8 populations, are located over myofibroblasts in fibrotic areas, suggesting a role in fibrosis progression similar to that in human IPF. This model accurately reproduces the pathophysiological and molecular features of human IPF, making it a valuable tool for future research and therapeutic development.

Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air-liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets (Mustela putorius furo) have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis. Key features • A protocol for isolating ferret airway basal cells and generating air-liquid interface (ALI) cultures for electrophysiologic research. • Detailed procedures for propagating ferret airway basal cells and culturing in vitro well-differentiated airway epithelium. • A protocol for measuring ion transport, conductance, and immunofluorescence to identify airway cell types.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by irreversible scarring of the distal lung, leading to respiratory failure and death. To date, only Pirfenidone and Nintedanib have been approved by the FDA for the treatment for IPF patients. However, neither drug has shown a significant improvement in patient survival. Therefore, it is crucial to develop novel therapeutic agents and strategies for mitigating or even curing the disease. Ivacaftor (VX770) is a potentiator of Cystic Fibrosis Transmembrane Regulator (CFTR) and has been for treatment of cystic fibrosis (CF) patients. In addition to its potency of CFTR function, VX770 also show an activity of anti-inflammation and effect of mitigation of smoking-induced bronchitis/bronchiolitis in experimental animals. Here we hypothesize that VX770 may have an alternative function in the pathogenesis of pulmonary fibrosis (PF), as IPF shares some characteristics of CF lung diseases including aberrant epithelial cell function and tissue scarring. Methods: To investigate the effect and underlying mechanism of VX770 in PF pathogenesis, changes of histopathology, expression of fibrotic biomarkers, epithelial cell type composition of distal lung and senescence of alveolar type 2 (AT2) cells were evaluated by histochemical and immunostaining assays, qRT-PCR and organoid cultures. Results: Our results demonstrated that VX770 was able to reduce the fibrotic burden in lung tissues of BLM-injured mice, as evidenced by the reduction of the collagen and fibroblast markers, including Sma, Col1a1, Ng2 and Vimentin, Runx1 and Cthrc1. In addition, VX770 also inhibited the aberrant change of epithelial cells populations, including Ager-, Sftpc-, Krt7- and Krt8-positive cells in BLM mouse lung, qRT-PCR further revealed a dramatical reduced expression of genes related to cell senescence, p16 and p21, and genes involved in inflammatory response, Col1a1, TGF-β, IL-6 and Ccl2 in BLM mice treated with VX770. Importantly, the impact of VX770 on epithelial cell senescence and aberrant epithelial cell differentiation under a fibrotic microenvironment was corroborated using AT2 organoid cultures. This result inplies the restoration of epithelial cell homeostasis. Conclusion: Our results demonstrate that VX770 mitigates the BLM-induced pulmonary fibrosis in mice in part by inhibiting inflammation and AT2 cell senescence. There are controversies about whether VX770 potentiates murine CFTR, thus the mechanism of action in these studies remains to be determined. This study thus provides evidence of the anti-fibrotic role of VX770 and warrants for further investigations of its therapeutic potential in treatment of IPF.

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.

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 CuFiCas9(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.

Rationale: Cystic fibrosis (CF) is a genetic disorder caused by mutations in the CFTR channel. While the involvement of CFTR in regulating salt and fluid movement by conducting airway epithelia has been well-studied, its role in alveolar epithelial type II (AT2) cells remains poorly understood. Given that ferrets are excellent models of CF disease, we investigated CFTR-mediated transepithelial ion and fluid transport of ferret primary AT2 epithelia in air-liquid interface (ALI) and organoid cultures. Methods: Primary AT2 cells were isolated from wild-type (WT) and CFTR-G551D/G551D ferret lungs by FACS and propagated in organoid cultures. CFTR-mediated transepithelial chloride transport was assessed in ALI cultures by measuring short-circuit current (Isc), while fluid transport was evaluated using organoid swelling assays. Results: Primary ferret AT2 cells retained their phenotype in proliferative culture conditions as assessed by immunostaining and qRT-PCR for AT2 and AT1 markers (SFTPC, AGER, HOXP1). Following forskolin induction of cAMP to stimulate CFTR, WT AT2 organoid swelled by 18.8 ± 3.8% of their starting volume, while G551D AT2 organoids shrunk in volume by 20.8 ± 5.9% of their initial volumes. These findings suggest that under cAMP stimulatory conditions, CFTR facilitates fluid secretion by AT2 cells and in its absence fluid absorption dominates. In the presence of amiloride and DIDS to inhibit ENaC and non-CFTR anion channels, respectively, IBMX/forskolin induced chloride currents by WT AT2 ALI cultures (ΔIsc 33.57 ± 9.65 µA/cm2), but not G551D cultures (ΔIsc 0.13 ± 0.69 µA/cm2). Conclusion: We demonstrate the ability to propagate ferret AT2 cells and maintain them as undifferentiated epithelia in ALI and organoid cultures. chloride movement by AT2 cells facilitates salt-mediated fluid movement, which counterbalances CFTR-independent fluid absorption. The mechanisms by which these processes interact in native mixed epithelial populations containing both AT2 and AT1 cells remain to be determined. These findings highlight the crucial role of CFTR in sustaining fluid homeostasis in the alveoli.

Rationale: The CFTR (cystic fibrosis transmembrane conductance regulator) channel contributes to the precise control of airway surface liquid volume by regulating fluid absorption and secretion, both of which are disrupted in cystic fibrosis (CF). CFTR is highly expressed in pulmonary ionocytes, yet conflicting evidence has emerged regarding how pulmonary ionocytes regulate ion and fluid transport. Different groups have shown that pulmonary ionocytes mediate chloride and fluid absorption, but whether ionocytes and/or secretory cells mediate chloride secretion remains unclear. This question has important therapeutic implications. Thus, there is a need to improve our understanding of cell type-specific CFTR functions on which to rationally design therapeutic approaches for CF. Objectives: To elucidate how CFTR in ionocytes mediates chloride absorption and secretion using conditional ionocytespecific CFTR deletion or reactivation. Methods: A novel transgenic ferret model (ROSA-TG::FOXI1-CreERT2::CFTRL/L) was developed to specifically inactivate CFTR in ionocytes while simultaneously enabling fate mapping of ionocyte lineages. This model was used to investigate the mechanisms of ion and fluid transport by pulmonary ionocytes and their regeneration. A second transgenic ferret model (FOXI1-CreERT2::CFTR-cKI), which reactivates CFTR in CF ionocytes, was used to further study therapeutically relevant cellular mechanisms of CFTR gene correction. Measurements and Main Results: Ionocyte-specific CFTR facilitates both chloride absorption and secretion, supporting the dual role of ionocytes in airway fluid homeostasis. Deletion of CFTR within ionocytes led to rapid renewal of CFTR-competent ionocytes from lineage-negative progenitors. Conclusions: Ionocyte-expressed CFTR is required for both chloride absorption and secretion, enabling salt and fluid transport in the large airways.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by irreversible scarring of the distal lung, leading to respiratory failure and death. The lack of clinically relevant and highly predictive models has greatly impeded our understanding of IPF pathogenesis, and consequently, the development of new treatments for IPF. Domestic ferrets (Mustela putorius furo) have been recognized as excellent models for medical research on a variety of respiratory diseases owing to similarities of airway anatomy, lung cell biology, and physiology to humans. We thus hypothesized that bleomycin-injured ferrets might be a good model of human IPF. Methods: Ferrets were injured with three doses of bleomycin (BLM) at 1-week intervals via the laryngotracheal route. The histopathology, pulmonary function, radiographic phenotypes of pulmonary fibrosis were comprehensively evaluated. The cellular and molecular features of the distal lungs of BLM-injured ferret were also analyzed by a single-nucleus RNA sequencing (snRNA-Seq) method using the 10X platform. Results: Histopathological analysis showed pulmonary fibrosis with honeycomb cyst structure and the predominant deposition of collagen and extracellular matrix (ECM) in the lungs of BLM-injured ferrets, compared to control ferrets. Pulmonary function tests and flash CT scanning further demonstrated a significantly decreased pulmonary compliance and increased opacification in BLM ferret lungs, typical phenotypes of human IPF. snRNA-seq analysis revealed a significant shift in distal lung epithelium towards a proximal epithelial phenotype, and identified a previously undescribed subpopulation of epithelial cells that highly upregulated KRT7 in BLM ferret lungs. Furthermore, immunostaining revealed the presence of atypical epithelial cells, and the persistence of KRT7+ cells co-expressing markers of other proximal airway epithelial cell types, including KRT5, acetylated α-tubulin, SCGB1A1, SCGB3A2 and MUC5B. Importantly, a histopathological pattern of bronchiolization encompassing divergent atypical epithelial cells, and KRT17+/TP63+/KRT5low"basaloid-like” cells, were present in the distal fibrotic lung lesions. Trajectory analysis revealed AT2 cells transition through multiple cell-states in bleomycin ferret lungs, particularly AT2 to KRT8high/KRT7low/SOX4+ to eventual KRT8high/KRT7high/SFN+/TP63+/KRT5low"basaloid-like” cells. Further, immunofluorescence and in silico cell-cell communication analyses demonstrated KRT7 and KRT8 populations reside in close proximity to the ACTA2 positive myofibroblasts in fibrotic foci. Conclusion: Collectively, our results provide evidence that BLM-induced lung injury in ferrets can reproduce pathophysiological, cellular, and molecular features of human IPF, suggesting that they may be a reliable model for understanding mechanisms of IPF pathogenesis and for testing therapeutic strategies for treatment of IPF.