Base editing and nanoparticle transfection of airway cell types essential for treatment of cystic fibrosis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE324177 | GEO | 2026/03/11
REPOSITORIES: GEO
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Project description:Purpose of experiment :To 1) evaluate for the presence of pulmonary ionocytes, 2) investigate the role of the CFTR in airway epithelium. Description of samples: 1564 (CF-repaired), 1565 (CF DF508homozygous) iPSC-airway epithelium through basal cell intermediate. iPSC-D15CD47/CD26sort- NGFR sort x 2
2023-03-02 | GSE203008 | GEO
Project description:Purpose: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single cell RNA-seq (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. Single cell-based RNA in situ hybridization (scRNA-ISH) and quantitative RT-PCR (scqRT-PCR) were performed for validation. In vitro culture systems correlated CFTR function to cell types. Lentiviruses were used for cell-type specific transduction of wild-type CFTR in CF cells. Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and particularly small airways, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, while expression in ciliated cells was infrequent and low. scRNA-ISH, and scqRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to normal controls. CFTR-mediated Cl- secretion tracked secretory cell, not ionocyte, densities. Further, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.
2020-12-16 | GSE160674 | GEO
Project description:Purpose: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single cell RNA-seq (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. Single cell-based RNA in situ hybridization (scRNA-ISH) and quantitative RT-PCR (scqRT-PCR) were performed for validation. In vitro culture systems correlated CFTR function to cell types. Lentiviruses were used for cell-type specific transduction of wild-type CFTR in CF cells. Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and particularly small airways, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, while expression in ciliated cells was infrequent and low. scRNA-ISH, and scqRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to normal controls. CFTR-mediated Cl- secretion tracked secretory cell, not ionocyte, densities. Further, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.
2020-12-16 | GSE160673 | GEO
Project description:Speciation leads to adaptive changes in organ cellular anatomy and physiology. These evolutionary changes create challenges for studying rare cell type functions that diverge between human and mice. Rare CFTR-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of novel conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO), and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models to cystic fibrosis (CF) ferrets, we demonstrate that ionocytes control airway surface liquid (ASL) absorption, secretion, pH, and mucus viscosity—leading to reduced ASL volume and impaired mucociliary clearance in CF, FOXI1-KO, and FOXI1-CreERT2::CFTRL/L ferrets. These processes were regulated by CFTR- dependent ionocyte transport of Cl– and HCO3–, as determined electrophysiologically and by single-cell imaging with a conditionally activated halide fluorescent sensor. Single-cell transcriptomics, using pulse-seq of lineage-traced ionocyte-enriched cultures, revealed three subtypes of pulmonary ionocytes with unique biologic functions and a common rare cell progenitor of ionocytes, tuft, and neuroendocrine cells. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of CF airway disease. These studies provide a road map for using conditional genetics in the first non- rodent mammal to address gene function, cell biology, and disease processes that have greater evolutionary conservation between humans and ferrets.
2023-08-01 | GSE233654 | GEO
Project description:Elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) is highly effective treatment for many cystic fibrosis patients, at least partly because it increases CFTR mediated Cl- and HCO3- secretion by airway epithelial cells leading to improved lung function and less frequent exacerbations and hospitalizations. However, little is known about how ETI affects airway epithelial cells in ways not related to CFTR mediated Cl- and HCO3- secretion, for example how ETI affects the expression of genes other than CFTR or how ETI might affect airway cells’ response to infection. It is established that CF airway cells bearing the delta F 508 mutation the CFTR gene respond characteristically differently from wild type CFTR cells, and we hypothesized that, as a highly effective CFTR modulator, ETI might make airway cells from CF donors respond to pathogen stimulation (Pseudomonas aeruginosa PA14 or outer membrane vesicles isolated from these bacteria) in more the same way that cells from wild type, healthy control cells do. We tested this hypothesis by measuring gene expression responses in polarized primary CF airway cells exposed to ETI alone or ETI in the presence of a pathogen challenge (PA14 or outer membrane vesicles). Responses of CFTR wild type primary CF airway cells to PA14 or outer membrane vesicles was also measured for comparison
2024-06-25 | GSE268718 | GEO
Project description:Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how loss of CFTR first disrupts airway host defense has remained uncertain. We asked what abnormality impairs elimination when a bacterium lands on the pristine surface of a newborn CF airway? To investigate this defect, we interrogated the viability of individual bacteria immobilized on solid grids and placed on the airway surface. As a model we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly killed bacteria in vivo, when removed from the lung, and in primary epithelial cultures. Lack of CFTR reduced bacterial killing. We found that ASL pH was more acidic in CF, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defense defect to loss of CFTR, an anion channel that facilitates HCO3- transport. Without CFTR, airway epithelial HCO3- secretion is defective, ASL pH falls and inhibits antimicrobial function, and thereby impairs killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF and that assaying ASL pH or bacterial killing could report on the benefit of therapeutic interventions. 11 samples of trachea primary airway epithelial cultures representing CFTR+/+ and CFTR-/- pigs. Pig samples representing 14 bronchus and 12 trachea tissue samples submitted in GSE21071.
2012-03-29 | E-GEOD-36906 | biostudies-arrayexpress
Project description:Our laboratory has held a long interest in the glycosylation changes seen on the surface of airway epithelia of patients with the disease cystic fibrosis (CF). Experiments from our laboratory have detailed a CF glycosylation phenotype of increased Fuca1,3/4 and decreased Fuca1,2 and sialic acid on the surfaces of immortalized and primary CF cells compared to non-CF cells. Further, we have shown that gene transfer and subsequent expression of a wild type CF plasmid in CF airway cells results in correction or reversal of this glycosylation phenotype. We hypothesize that the changes in glycosylation seen in CF cells are key in the pathophysiology of the cystic fibrosis airway disease. For example, it has been shown that Pseudomonas aeruginosa, a bacterium that has a predilection for colonizing CF airways, adheres to asialylated glycolipids and glycoconjugates with terminal Fuca1,3/4. One focus of our laboratory is to elucidate the etiology of the glycosylation changes seen in CF cells and the mechanism by which these changes are reversed by wild type CFTR gene transfer. We propose to study the gene expression of immortalized and primary CF and non-CF airway epithelial cells: 1. CF/T43 vs. BEAS-2B cells. These are two widely used immortalized airway cell lines that we have used extensively in the past. 2. C38 cells; C38 cells are IB3 cells expressing wtCFTR. The experimental focus is to elucidate the etiology of the glycosylation changes seen in Cystic Fibrosis (CF) cells and the mechanism by which these changes are reversed by wild type CFTR gene transfer. To do so, the gene expression of immortalized and primary CF and non-CF airway epithelial cells were compared and studied. Cell lines used were CF/T43 and BEAS-2B, both widely used immortalized airway cell lines. Other cell lines studied included C38 cell lines (clonal derivatives of IB3 cells expressing wtCFTR).
2011-01-07 | E-GEOD-26482 | biostudies-arrayexpress
Project description:The human airway epithelium contains specialized rare cells, including ionocytes and tuft cells, that have been associated with respiratory disease. Ionocytes express the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) whereas chemosensory tuft cells express asthma-associated alarmins. Surprisingly, exceedingly few mature tuft cells have been identified in human lung cell atlases. To enable the identification of rare lung cells and define their lineage specification, we generated a deep lung cell atlas of 311,748 cell profiles with careful anatomical sampling of the large and small human airways from entire lungs declined for transplant. In contrast to prior reports, ionocytes were present in similar numbers in both the large and small airways, suggesting that they may contribute to both large and small airways pathologies in CF, a finding which has important implications for gene therapy. We also identify mature tuft cells in a patient who died from an asthma flare. Computational analysis identified rare, bipotent Tuft-Ionocyte Progenitor cells (TIP cells), which have a comparable/higher fraction of cycling cells in the large airways as basal cells (18% vs 11%) and are predicted to give rise to either tuft cells or ionocytes based on the airway signaling milieu. Using an optimized in vitro system we show that the default pathway of TIP cell differentiation produces ionocytes, but in the presence of Type 2 and Type 17 cytokines, TIP cells are diverted to differentiate into tuft cells. Using scRNAseq and scATACseq we further predicted transcription factors and intermediate cell states that mark these rare cell lineages. Overall, our findings suggest that the immune signaling pathways that characterize asthma and CF may skew the composition of disease relevant rare cells and show how deep atlases are required to identify disease relevant but scarce cells and link them to molecular mechanisms driving pathology.
2025-04-28 | GSE240168 | GEO
Project description:Background: CFTR modulators will decrease some etiologies of inflammation in the CF airway; however, current data indicate that non-resolving airway infection and inflammation will persist in individuals with CF and chronic bacterial infections. Thus, identification of therapies that diminish CF airway inflammation without allowing unrestrained bacterial growth remains a critical research goal. Novel strategies for combatting deleterious airway inflammation require a better understanding of the cellular contributions to chronic CF airway disease, and how cells change after initiation of CFTR modulator therapy. Peripheral blood monocytes, which traffic to the CF airway, can develop both pro-inflammatory and inflammation-resolving phenotypes, and represent intriguing targets for focused therapies to dampen CF airway inflammation. Material and Methods: In order to characterize the inflammatory phenotype of CF blood monocytes, and how these cells change after initiation of CFTR modulator therapy, we studied adults (n=10) with CF, chronic airway infections, and the CFTR-R117H mutations before and 7 days after initiation of ivacaftor. Transcriptomes of freshly isolated blood monocytes were interrogated by RNA-sequencing (RNA-seq). Plasma concentrations of cytokines and chemokines were evaluated by multiplex ELISA. Results: RNAseq identified approximately 50 monocyte genes for which basal expression was significantly changed in all 10 subjects after 7 days of ivacaftor. Of these, the majority were increased in expression post ivacaftor, including many genes traditionally associated with enhanced inflammation and immune responses. Pathway analyses confirmed that transcriptional pathways were overwhelmingly up-regulated in monocytes after 7 days of ivacaftor, including transcriptional modules associated with immunity, cell cycle, oxidative phosphorylation, and the unfolded protein response. Ivacaftor increased plasma concentrations of CXCL2, a neutrophil chemokine secreted by monocytes and macrophages, and CCL2, a monocyte chemokine. These results demonstrate that ivacaftor causes acute changes in blood monocytes and plasma chemokines, and suggest that increased monocyte inflammatory signals and potentially improved trafficking to the lung contribute to changes in airway inflammation in people taking CFTR modulators. To our knowledge, this is the first report investigating the transcriptomic response of isolated blood monocytes in CF subjects.
2020-07-31 | GSE148076 | GEO
Project description:Cystic Fibrosis (CF) is a genetic disorder CF is caused by mutations of the gene encoding for the cystic fibrosis transmembrane conductance regulator protein (CFTR), a transmembrane anion channel expressed at the apical membrane of several organs, including the epithelial cells of the airway. CFTR mutations result in dysfunctional ion transport across the apical membrane at the surface of the epithelia, generating thickened and dehydrated secretions. In the lung, this leads to a decrease in the mucociliary clearance, favoring bacterial colonization and progressive obstruction of the duct. Although over 2000 CFTR variants have been identified so far, the most common mutation is a deletion of the phenylalanine in position 508 (F508del), which shows an allelic frequency of around 90% among CF patients. F508del-CFTR is incorrectly folded, causing its retention at the endoplasmic reticulum (ER) and subsequent proteasomal degradation. Among the several drugs available in CF pharmacological treatment, VX-809 (commercial name Lumacaftor) is the most used drug for patients carrying F508del-CFTR mutation. This drug corrects the aberrant folding of F508del-CFTR by favoring the correct intramolecular interactions, thus enabling a higher number of copies of the defective protein to reach the plasma membrane. We applied SWATH-based proteomics to understand if a pharmacological rescue of F508del-CFTR is associated with changes in global protein expression of the human bronchial epithelium by using the CFBE41o- cell model, a line that stably expresses F508del-CFTR.
2022-08-11 | PXD028355 | Pride