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A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: S.M.R. provided consulting services and received grants from Novartis, TranslateBio, Galapagos/Abbvie, Synedgen/Synspira, Eloxx, Vertex Pharmaceuticals, Ionis, Astra Zenica, Renovion, Cystetic Medicines, and Arcturus. S.M.R. is the inventor or co-inventor of several patents and held stock or stock options of Synedgen/Synspira and Renovion. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cystic fibrosis (CF) is a genetic disease hallmarked by aberrant ion transport that results in delayed mucus clearance, chronic infection, and progressive lung function decline. Several animal models have been developed to study the airway anatomy and mucus physiology in CF, but they are costly and difficult to maintain, making them less accessible for many applications. A more available CFTR-/- rat model has been developed and characterized to develop CF airway abnormalities, but consistent dosing of pharmacologic agents and longitudinal evaluation remain a challenge. In this study, we report the development and characterization of a novel ex vivo trachea model that utilizes both wild type (WT) and CFTR-/- rat tracheae cultured on a porcine gelatin matrix. Here we show that the ex vivo tracheae remain viable for weeks, maintain a CF disease phenotype that can be readily quantified, and respond to stimulation of mucus and fluid secretion by cholinergic stimulation. Furthermore, we show that ex vivo tracheae may be used for well-controlled pharmacological treatments, which are difficult to perform on freshly excised trachea or in vivo models with this degree of scrutiny. With improved interrogation possible with a durable trachea, we also established firm evidence of a gland secretion defect in CFTR-/- rat tracheae compared to WT controls. Finally, we demonstrate that the ex vivo tracheae can be used to generate high mucus protein yields for subsequent studies, which are currently limited by in vivo mucus collection techniques. Overall, this study suggests that the ex vivo trachea model is an effective, easy to set up culture model to study airway and mucus physiology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Harris et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Pulmonary artery (PA) enlargement, defined as pulmonary artery to ascending aorta diameter ratio (PA:A)>1 on computed tomography (CT), is a marker of pulmonary vascular disease in chronic lung diseases. PA enlargement is prevalent in cystic fibrosis (CF), but its relationship to hemodynamics and prognostic utility in severe CF are unknown. We hypothesized that the PA:A would have utility in identifying pulmonary hypertension (PH) in severe CF and that PA enlargement would be associated with reduced transplant-free survival., Methods: We conducted a retrospective study of adults with CF undergoing lung transplant evaluation at a single center between 2000 and 2015. CT, right heart catheterization (RHC), and clinical data were collected. The PA:A was measured from a single CT slice. We measured associations between PA:A and invasive hemodynamic parameters including PH defined as a mPAP ≥25mmHg using adjusted linear and logistic regression models. Kaplan-Meier and adjusted Cox regression models were used to measure associations between PA:A>1, RHC-defined PH, and transplant-free survival in severe CF., Results: We analyzed 78 adults with CF that had CT scans available for review, including 44 that also had RHC. RHC-defined PH defined as a mPAP ≥25mmHg was present in 36% of patients with CF undergoing transplant evaluation. The PA:A correlated with mPAP (r = 0.73; 95% CI 3.87-7.80; p<0.001) and PVR (r = 0.42, p = 0.005) and the PA:A>1 was an independent predictor of PH (aOR 4.50; 95% CI 1.05-19.2; p = 0.042). PA:A>1 was independently associated with increased hazards for death or transplant (aHR 2.69; 95% CI 1.41-5.14; P = 0.003). The presence of mPAP ≥25mmHg was independently associated with decreased survival in this cohort., Conclusions: PA enlargement is associated with pulmonary hemodynamics and PH in severe CF. PA enlargement is an independent prognostic indicator of PH and decreased survival in this population., Competing Interests: JMW reports grants from the US National Institutes of Health (NIH) and the National Heart, Lung, and Blood Institute (NHLBI), and the Cystic Fibrosis Foundation during the conduct of the study; contracts to conduct clinical trials from AstraZeneca, GlaxoSmithKline, Bayer, and Mereo BioPharma; consulting fees from Boehringer Ingelheim, GlaxoSmithKline, Mereo BioPharma, Quintiles and Mylan outside the submitted work. SMR reports grants from the NIH and the Cystic Fibrosis Foundation during the conduct of the study; non-financial support from Vertex Pharmaceuticals, grants from Bayer, NIH, CFFT, Synedgen, Celtaxsys, and Translate Bio grants for projects outside of the submitted work; non-financial support from Synedgen and Celtaxsys; and personal fees from Celtaxsys for projects outside of the submitted work. These relationships do not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors declare no competing interests. The Cystic Fibrosis Foundation Therapeutics Inc. (CFFT), the Cystic Fibrosis Foundation's nonprofit drug development affiliate, has contractual agreements with Vertex and certain other pharmaceutical companies to receive royalties from the approval and/or the sales of any drugs (e.g., Kalydeco, etc.) that are developed because of CFFT funding. Any royalties CFFT receives are reinvested in support of the Cystic Fibrosis Foundation's mission. Relationships between the Cystic Fibrosis Foundation, the CFFT, and industry partners do not affect adherence to PLOS ONE policies on sharing data and materials.

Background: Reduced growth and osteopenia are common in individuals with cystic fibrosis (CF). Additionally, improved weight and height are associated with better lung function and overall health in the disease. Mechanisms for this reduction in growth are not understood. We utilized a new CFTR knockout rat to evaluate growth in young CF animals, via femur length, microarchitecture of bone and growth plate, as well as serum IGF-I concentrations., Methods: Femur length was measured in wild-type (WT) and SD-CFTRtm1sage (Cftr-/-) rats, as a surrogate marker for growth. Quantitative bone parameters in Cftr-/- and WT rats were measured by micro computed tomography (micro-CT). Bone histomorphometry and cartilaginous growth plates were analyzed. Serum IGF-I concentrations were also compared., Results: Femur length was reduced in both Cftr-/- male and female rats compared to WT. Multiple parameters of bone microarchitecture (of both trabecular and cortical bone) were adversely affected in Cftr-/- rats. There was a reduction in overall growth plate thichkness in both male and female Cftr-/- rats, as well as hypertrophic zone thickness and mean hypertrophic cell volume in male rats, indicating abnormal growth characteristics at the plate. Serum IGF-I concentrations were severely reduced in Cftr-/- rats compared to WT littermates., Conclusions: Despite absence of overt lung or pancreatic disease, reduced growth and bone content were readily detected in young Cftr-/- rats. Reduced size of the growth plate and decreased IGF-I concentrations suggest the mechanistic basis for this phenotype. These findings appear to be intrinsic to the CFTR deficient state and independent of significant clinical confounders, providing substantive evidence for the importance of CFTR on maintinaing normal bone growth.

Limited success achieved in translating basic science discoveries into clinical applications for chronic airway diseases is attributed to differences in respiratory anatomy and physiology, poor approximation of pathologic processes, and lack of correlative clinical endpoints between humans and laboratory animal models. Here, we discuss advantages of using ferrets (Mustela putorus furo) as a model for improved understanding of human airway physiology and demonstrate assays for quantifying airway epithelial ion transport in vivo and ex vivo, and establish air-liquid interface cultures of ferret airway epithelial cells as a complementary in vitro model for mechanistic studies. We present data here that establishes the feasibility of measuring these human disease endpoints in ferrets. Briefly, potential difference across the nasal and the lower airway epithelium in ferrets could be consistently assessed, were highly reproducible, and responsive to experimental interventions. Additionally, ferret airway epithelial cells were amenable to primary cell culture methods for in vitro experiments as was the use of ferret tracheal explants as an ex vivo system for assessing ion transport. The feasibility of conducting multiple assessments of disease outcomes supports the adoption of ferrets as a highly relevant model for research in obstructive airway diseases.

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41-a compound that inhibits the E1 ubiquitin activating enzyme-was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR., Competing Interests: Dr. Sorscher serves as an advisor to Proteostasis Therapeutics, Inc. This not not alter our adherence to PLOS ONE policies on sharing data and materials.

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.

Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/-) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR-/-) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR-/- rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR-/- males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR-/- animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities.

Intestinal current measurements (ICM) from rectal biopsies are a sensitive means to detect cystic fibrosis transmembrane conductance regulator (CFTR) function, but have not been optimized for multicenter use. We piloted multicenter standard operating procedures (SOPs) to detect CFTR activity by ICM and examined key questions for use in clinical trials. SOPs for ICM using human rectal biopsies were developed across three centers and used to characterize ion transport from non-CF and CF subjects (two severe CFTR mutations). All data were centrally evaluated by a blinded interpreter. SOPs were then used across four centers to examine the effect of cold storage on CFTR currents and compare CFTR currents in biopsies from one subject studied simultaneously either at two sites (24 hours post-biopsy) or when biopsies were obtained by either forceps or suction. Rectal biopsies from 44 non-CF and 17 CF subjects were analyzed. Mean differences (µA/cm(2); 95% confidence intervals) between CF and non-CF were forskolin/IBMX=102.6(128.0 to 81.1), carbachol=96.3(118.7 to 73.9), forskolin/IBMX+carbachol=200.9(243.1 to 158.6), and bumetanide=-44.6 (-33.7 to -55.6) (P<0.005, CF vs non-CF for all parameters). Receiver Operating Characteristic curves indicated that each parameter discriminated CF from non-CF subjects (area under the curve of 0.94-0.98). CFTR dependent currents following 18-24 hours of cold storage for forskolin/IBMX, carbachol, and forskolin/IBMX+carbachol stimulation (n=17 non-CF subjects) were 44%, 47.5%, and 47.3%, respectively of those in fresh biopsies. CFTR-dependent currents from biopsies studied after cold storage at two sites simultaneously demonstrated moderate correlation (n=14 non-CF subjects, Pearson correlation coefficients 0.389, 0.484, and 0.533). Similar CFTR dependent currents were detected from fresh biopsies obtained by either forceps or suction (within-subject comparisons, n=22 biopsies from three non-CF subjects). Multicenter ICM is a feasible CFTR outcome measure that discriminates CF from non-CF ion transport, offers unique advantages over other CFTR bioassays, and warrants further development as a potential CFTR biomarker.

Background: Cystic fibrosis (CF) is characterized by acute pulmonary exacerbations (APE). The CF nasal airway exhibits a similar ion transport defect as the lung, and colonization, infection, and inflammation within the nasal passages are common among CF patients. Nasal lavage fluid (NLF) is a minimally invasive means to collect upper airway samples., Methods: We collected NLF at the onset and resolution of CF APE and compared a 27-plex cytokine profile to stable CF outpatients and normal controls. We also tested IP-10 levels in the bronchoalveolar lavage fluid (BALF) of CF patients. Well-differentiated murine sinonasal monolayers were exposed to bacterial stimulus, and IP-10 levels were measured to test epithelial secretion., Results: Subjects hospitalized for APE had elevated IP-10 (2582 pg/mL [95% CL of mean: 818,8165], N=13) which significantly decreased (647 pg/mL [357,1174], P<0.05, N =13) following antimicrobial therapy. Stable CF outpatients exhibited intermediately elevated levels (680 pg/mL [281,1644], N=13) that were less than CF inpatients upon admission (P=0.056) but not significantly different than normal controls (342 pg/mL [110,1061]; P=0.3, N=10). IP-10 was significantly increased in CF BALF (2673 pg/mL [1306,5458], N=10) compared to healthy post-lung transplant patients (8.4 pg/mL [0.03,2172], N=5, P<0.001). IP-10 levels from well-differentiated CF murine nasal epithelial monolayers exposed to Pseudomonas PAO-1 bacteria-free prep or LPS (100 nM) apically for 24 hours were significantly elevated (1159 ± 147, P<0.001 for PAO-1; 1373 ± 191, P<0.001 for LPS vs. 305 ± 68 for vehicle controls). Human sino-nasal epithelial cells derived from CF patients had a similar response to LPS (34% increase, P<0.05, N=6)., Conclusions: IP-10 is elevated in the nasal lavage of CF patients with APE and responds to antimicrobial therapy. IP-10 is induced by airway epithelia following stimulation with bacterial pathogens in a murine model. Additional research regarding IP-10 as a potential biomarker is warranted.

Nasal potential difference (NPD) is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco™; also known as VX-770) in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1) the average of both nostrils; (2) the most-polarized nostril at each visit; and (3) the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity), the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity), and the delta NPD (measuring CFTR and ENaC activity). The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (± 2.8 mV). Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified.

Background: Mucus stasis in chronic obstructive pulmonary disease (COPD) is a significant contributor to morbidity and mortality. Potentiators of cystic fibrosis transmembrane conductance regulator (CFTR) activity pharmacologically enhance CFTR function; ivacaftor is one such agent approved to treat CF patients with the G551D-CFTR gating mutation. CFTR potentiators may also be useful for other diseases of mucus stasis, including COPD., Methods and Findings: In primary human bronchial epithelial cells, exposure to cigarette smoke extract diminished CFTR-mediated anion transport (65.8±0.2% of control, P<0.005) and mucociliary transport (0.17±0.05 µm/sec vs. 2.4±0.47 µm/sec control, P<0.05) by reducing airway surface liquid depth (7.3±0.6 µm vs. 13.0±0.6 µm control, P<0.005) and augmenting mucus expression (by 64%, P<0.05) without altering transepithelial resistance. Smokers with or without COPD had reduced CFTR activity measured by nasal potential difference compared to age-matched non-smokers (-6.3±1.4 and -8.0±2.0 mV, respectively vs. -15.2±2.7 mV control, each P<0.005, n = 12-14/group); this CFTR decrement was associated with symptoms of chronic bronchitis as measured by the Breathlessness Cough and Sputum Score (r = 0.30, P<0.05) despite controlling for smoking (r = 0.31, P<0.05). Ivacaftor activated CFTR-dependent chloride transport in non-CF epithelia and ameliorated the functional CFTR defect induced by smoke to 185±36% of non-CF control (P<0.05), thereby increasing airway surface liquid (from 7.3±0.6 µm to 10.1±0.4 µm, P<0.005) and mucociliary transport (from 0.27±0.11 µm/s to 2.7±0.28 µm/s, P<0.005)., Conclusions: Cigarette smoking reduces CFTR activity and is causally related to reduced mucus transport in smokers due to inhibition of CFTR dependent fluid transport. These effects are reversible by the CFTR potentiator ivacaftor, representing a potential therapeutic strategy to augment mucociliary clearance in patients with smoking related lung disease.