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INVESTING THE ROLES OF E26 TRANSFORMATION-SPECIFIC HOMOLOGOUS FACTOR IN CYSTIC FIBROSIS LUNG DISEASE https://doi.org/10.17863/CAM.99854 Pinte, Laetitia Vallier, Ludovic Cicuta, Pietro CF (Cystic Fibrosis) is the most prevalent genetic disease among Caucasians. Here, a sodiumbicarbonate channel, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), is impaired, dehydrating all epithelia. The principal cause of morbimortality of CF is CF lung disease (CFLD) where CF airways have impaired mucociliary clearance and undergo constant remodelling and chronic inflammation, ultimately leading to irreversible loss of lung function. Interestingly, within CF patients carrying the same CFTR mutation resides a high variability in terms of CFLD severity and response to treatments. These variabilities are thought to be due to differentially expressed non-CFTR genes. To locate these genes, Genome-Wide Association Studies (GWAS) collected and analysed data from large patient cohorts, identifying four loci (5p15, 3q29, 11p12-13, Xq22-23) associated with more severe clinical manifestations of CFLD. These loci could sit close by genes potentially impacting CFLD penetrance, and patient responsiveness to therapeutics. We aim to understand how genes outlined by GWAS influence CFLD. To this end, we first optimized a model system which relies on human induced pluripotent stem cells (hiPSC)-derived Airways Epithelial Cells (AECs). We first explored two differentiation protocols that generate lung AECs as organoids and Air-Liquid-Interface (ALI) cultures and outlined their respective advantages and limitations. Then, using the most robust differentiation protocol, we explored the expression of GWAS-outlined genes and observed that E26 Transformation-Specific Homologous Factor (EHF) was expressed at comparable levels to primary Human Bronchial Epithelial Cells. Thus, we decided to study EHF in our AECs in-vitro model. First, we knocked out EHF using CRISPR-Cas9 from two non-CF hiPSC lines and selected, within the pool of edited cells, homozygous and heterozygous knock-out (KO) clones alongside wild-type clones, then used as internal controls. The KO was confirmed by finding decreased or absent levels of EHF mRNA and protein in all clones. Second, the resulting hiPSCs lined were differentiated into AECs differentiation. The absence of EHF led to a decreased expression of MUC5B in organoids and alterations in the transcriptomic signature of Basal Cell (BCs) in ALI cultures. Finally, we assessed physiological functions (trans-epithelial electrical resistance (TEER), cilia motility, and cell migration) and functions known to be faulty in CFLD (CFTR function, survival to infection, and response to hypoxia). EHF-/- clones had significantly increased TEER values and forskolin-induced swelling while reducing the HIF1a-induced response to hypoxia, thereby modifying the pH of EHF-/- epithelia, compared to EHF+/+ clones. Taken together, our data outlined several mechanisms by which EHF could be linked to CFLD. In conclusion, we hypothesise that any polymorphisms increasing the expression of EHF could lead to more severe CFLD. Our study sheds light on how a single gene can impact the severity of disease through multiple mechanisms and draws perspectives onto broader targets for personalized therapies against CF. Cambridge university 2023-04-01 Thesis Doctoral Doctor of Philosophy (PhD) eng Cystic Fibrosis Trust grant SRC-016 https://www.repository.cam.ac.uk/handle/1810/353821 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/549cd67b-9947-4144-9b93-7f7691362c29/download 19c7852cc1de3ac25385556cf3793834 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f8b84368-46fd-41cc-90a9-396e0feb4e3a/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ Biology Cystic Fibrosis Genetic modifiers Induced pluripotent stem cells Lung disease Stem Cells