Biology subjects

Bear, C.

Publications and source records attributed to Bear, C..

9 recordsLinked to original sources

bioRxiv2026

A Dual-Locus-Targeting Strategy to Enhance CRISPR/Cas9-mediated CFTR Replacement via Helper-Dependent Adenoviral vector in porcine genome

Gene therapy has been the subject of extensive research following the advent of gene-editing technologies. Genetic disorders with difficult-to-target tissues, such as cystic fibrosis (CF), still face many challenges in developing efficacious gene therapy. The potential universal approach of gene replacement involves inserting a functional CFTR gene after generating DNA double strand breaks using gene editors such as CRISPR/Cas9. However, this strategy has not achieved clinical significance, as CRISPR/Cas9-mediated integration of CFTR is limited primarily by the infrequent activity of the homology-directed repair (HDR) pathway. To circumvent this limitation and improve CFTR transgene integration and expression, we explored a method of adding a second integration site, which we termed the dual-locus-targeting method. Using a helper-dependent adenoviral vector (HDAd)-delivered CRISPR/Cas9 system in porcine epithelial cells, we found that sequential delivery of two vectors, one targeting the CFTR locus and the other the genomic safe harbour site GGTA1, enhanced the integration efficiency of lacZ and CFTR donor genes to 16.5% and 3.4%, respectively. These results demonstrated a potential strategy to improve the efficacy of CFTR replacement for the development of a universal and permanent gene therapy treatment for CF lung disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/731381v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@14c8583org.highwire.dtl.DTLVardef@d1862eorg.highwire.dtl.DTLVardef@1c0594eorg.highwire.dtl.DTLVardef@1271c10_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Bioengineered 3D hPSC-cholangiocyte ducts with physiological signals for biliary disease modelling

The progression of intrahepatic biliary diseases remains poorly understood, underscoring the urgent need to develop physiologically relevant human intrahepatic cholangiocyte disease models. Current approaches lack the complexity and throughput to capture how diverse biliary microenvironmental signals shape cholangiocyte behavior. To address this gap, we created a fully epithelialized and perfusible 3D bile duct from human pluripotent stem cell-derived cholangiocytes characterized by robust primary ciliation, apical-basal polarity and CFTR-mediated chloride conductance. Our results revealed physiologically relevant fluid flow and biliary stroma cells as essential components for sustaining cholangiocyte epithelial barrier integrity and ciliation. From here, we interrogated a broad spectrum of biliary signals to study bile acid toxicity and cytokine-driven injury, offering an unprecedented view into intrahepatic cholangiocyte stress responses and potential pathological mechanisms. By integrating physiologically relevant signals, fidelity and functional resolution, this platform provides the foundation needed to precisely decode pathogenic drivers and accelerate therapeutic development for devastating cholangiopathies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/686855v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11c1255org.highwire.dtl.DTLVardef@1f78d11org.highwire.dtl.DTLVardef@14e9398org.highwire.dtl.DTLVardef@1bc08d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

A technique for evaluating calcium flux in primary airway tissue at single cell resolution

Calcium signaling is critical in a multitude of biological processes, reinforcing the need to develop methods to study calcium flux in primary tissue. The study of live calcium mobilization into the cytosol is commonly studied by intracellular calcium dyes. However, the study of calcium mobilization at a single-cell resolution in differentiated, primary human tissue remains challenging. Here we present methodology to study calcium flux at single cell resolution in situ on primary tissue seeded on trans wells. As well, we developed a machine learning software that simplifies and streamlines analysis. SUMMARYLive intracellular calcium dyes enable our group to study cell type specific responses as fluorescent readouts in real time. Utilizing microscopic techniques, we devised methods to study calcium flux in individual cells on complex, differentiated primary tissues seeded on trans well inserts.

cell biology↗

bioRxiv2024

Benchmarking organ-specific responses to therapies in tissues differentiated from Cystic Fibrosis patient derived iPSCs

Cystic Fibrosis (CF) is a life-shortening disease that is caused by mutations in the CFTR gene, a gene that is expressed in multiple organs. There are several primary tissue models of CF disease, including nasal epithelial cultures and rectal organoids, that are effective in reporting the potential efficacy of mutation-targeted therapies called CFTR modulators. However, there is the well-documented variation in tissue dependent, therapeutic response amongst CF patients, even those with the same CF-causing mutation. Hence, there is an interest in developing strategies for benchmarking therapeutic efficacy in different organs relative to isogenic controls. In this study, we evaluated the CFTR chloride channel response to the highly effective CFTR modulator: Trikafta, in CF patient specific, iPSC-derived colonic and airway cultures relative to mutation-corrected (non-CF) tissues from that same individual. We measured pharmacological rescue in both tissues, but interestingly, Trikafta treatment resulted in different levels of functional rescue in the two tissues relative to the relevant isogenic control. This proof-of-concept study lays the groundwork for future comparisons of patient-specific CF therapeutic responses in both pulmonary and extra-pulmonary systems.

pharmacology and toxicology↗

bioRxiv2024

Comparison of a novel potentiator of CFTR channel activity to ivacaftor in ameliorating mucostasis caused by cigarette smoke in primary human bronchial airway epithelial cells

BackgroundCystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis. MethodsA novel small molecule potentiator (SK-POT1), previously identified in CFTR binding studies, was tested for its activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement. ResultsWe showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures. ConclusionTogether, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD.

physiology↗

bioRxiv2023

Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity.

While animal models have provided key insights into conserved mechanisms of how the lung forms during development, human-specific developmental mechanisms are not always captured. To fully appreciate how developmental defects and disease states alter the function of the lungs, studies in human lung models are important. Here, we sequenced >150,000 single single-cells from 19 healthy human fetal lung tissues from gestational weeks 10-19 and identified at least 58 unique cell types/states contributing to the developing lung. We captured novel dynamic developmental trajectories from various progenitor cells that give rise to club, ciliated, and pulmonary neuroendocrine cells. We also identified four CFTR-expressing progenitor cell types and pinpointed the temporal emergence of these cell types. These developmental dynamics reveal broader epithelial cell plasticity and novel lineage hierarchies that were not previously reported. Combined with spatial transcriptomics, we identified both cell autonomous and non-cell autonomous signalling pathways that may dictate the temporal and spatial emergence of cell lineages. Finally, we showed that human pluripotent stem cell-derived fetal lung models capture cell lineage trajectories specifically through CFTR-expressing progenitor cells, that were also observed in the native fetal tissue. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development. HighlightsO_LISingle-cell transcriptomics atlas from 19 human fetal lungs reveals cellular heterogeneity and previously unappreciated cellular plasticity in the epithelial compartment. C_LIO_LIIdentification of novel CFTR-expressing progenitor cells that gives rise to club, ciliated and PNEC. C_LIO_LINovel RNA velocity facilitated the identification of dynamic lineage trajectories in the epithelial compartment. C_LIO_LITemporally regulated cell signaling through promiscuous interactions between sender and receiving cells may dictate cell lineage fates. C_LIO_LIIntegration of human pluripotent stem cell (hPSC)-derived fetal lung cells and organoids with primary lung dataset show hPSC-differentiations captures key developmental trajectories of fetal epithelial cell states. C_LI

developmental biology↗

bioRxiv2021

A new platform for high-throughput therapy testing on iPSC-derived, immature airway from Cystic Fibrosis Patients

Induced pluripotent, stem cell (iPSC)-derived models of airway tissue have successfully modeled the primary defect in regulated chloride conductance caused by the major Cystic Fibrosis causing mutation, F508del. However, it remains unclear if iPSC-derived airway cultures can be used in high-throughput therapy development for F508del and rarer mutations. There is an urgent need for airway tissue models that reflect the variability of patient-specific responses and are scalable for therapy development. In the current work, we describe a robust, high-throughput fluorescence assay of mutant CFTR function in iPSCs differentiated to immature airway epithelium. This assay measures reproducible functional responses to modulators targeting either the major CF mutant F508del or the nonsense mutant: W1282X-CFTR. We show that the ranking of patient-specific responses to interventions in this stem-cell based model recapitulates the ranking observed in primary nasal epithelial cultures obtained from the same individuals. In summary, these proof-of-concept studies show that this novel platform has the potential to support therapy development and precision medicine for Cystic Fibrosis patients. One Sentence SummaryWe describe a fluorescence-based platform that enables high-throughput Cystic Fibrosis therapy testing using iPSCs differentiated to immature lung.

genomics↗

bioRxiv2021

Generation of functional ciliates cholangiocytes from human pluripotent stem cells

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) would provide a model for studying the pathogenesis of cholangiopathies and for developing novel therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we describe a new strategy to generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of CFTR and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of new cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies showed that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice providing the first indication that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

cell biology↗

bioRxiv2018

Lipid interactions enhance activation and potentiation of cystic fibrosis transmembrane conductance regulator (CFTR)

The recent cryo-electron microscopy structures of phosphorylated, ATP-bound CFTR in detergent micelles failed to reveal an open anion conduction pathway as expected on the basis of previous functional studies in biological membranes. We tested the hypothesis that interaction of CFTR with lipids is important for opening of its channel. Interestingly, molecular dynamics studies revealed that phospholipids associate with regions of CFTR proposed to contribute to its channel activity. More directly, we found that CFTR purified together with associated lipids using the amphipol: A8-35, exhibited higher rates of catalytic activity, channel activation and potentiation using ivacaftor, than did CFTR purified in detergent. Catalytic activity in CFTR detergent micelles was partially rescued by addition of phospholipids plus cholesterol, arguing that these lipids contribute directly to its modulation. In summary, these studies highlight the importance of lipids in regulated CFTR channel activation and potentiation.

biophysics↗