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Cystic Fibrosis (CF) is an autosomal recessive genetic disorder, caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel protein that conducts chloride ions, bicarbonate, and regulates the function of other channels. This CFTR protein, when defective, results in excessive pulmonary and systemic inflammation, recurring bacterial infections and progressive lung disease. Autophagy is an essential cellular process by which damaged organelles, as well as misfolded and aggregated proteins, are cleared from the cell. This mechanism is postulated to be defective in CF, as a result of the mutated ΔF508 CFTR protein; however, the current model as to how autophagy is dysregulated remains a subject of debate. In this thesis I have investigated the state of the autophagy-lysosomal axis in CF, using human bronchial epithelial cell lines (HBECs), as well as monocytes and M1 macrophages from patients with homozygous ΔF508 CFTR mutations. I have also examined the effects of currently available CFTR modulators on modifying this axis. The state of mitochondria was also investigated to establish if dysregulated autophagy leads to an accumulation of damaged mitochondria, thereby exacerbating the NLRP3 inflammasome-driven inflammatory phenotype in CF. The impact of this dysregulation on metabolism was also investigated. This study found autophagy to be dysregulated in cells with CF mutations. In CF HBECs, there was an increased turnover of autophagy markers in HBECs homozygous for ΔF508 CFTR. CF monocytes and M1 macrophages, however, showed an increase in a ubiquitin binding protein, p62, with no clearance of the protein via autophagy, but normal clearance of autophagosomes. CFTR modulators, Symdeko and ivacaftor, failed to show an improvement in autophagic function in all cell types. CF monocytes were found to have damaged and damage-prone mitochondria, as well as having an increased metabolic profile. These studies suggest that autophagy is dysregulated in cells that are homozygous for ΔF508 CFTR, and that the mitochondria in these cells are more prone to damage, with an increased metabolic profile.

Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for the CFTR anion channel. ΔF508, the most common CF-associated mutation, causes both a gating and a trafficking defect in the CFTR protein. This thesis describes the optimisation and use of two fluorescence assays, capable of measuring the two defects caused by the ΔF508 mutation. Yellow fluorescent protein (YFP)-CFTR, in which halide sensitive YFP is tagged to the Nterminal of the CFTR coding sequence, is a functional assay, used to test for ΔF508-CFTR potentiator activity. CFTR-pHTomato, in which the pH sensor is tagged to the fourth extracellular loop of CFTR, is used to measure membrane density and internal CFTR expression. Human embryonic kidney cells (HEK293 cells), expressing YFP-ΔF508-CFTR were used to screen a pilot library of compounds with some structural similarity to known potentiator VX- 770. Ligand-based virtual screening was then used to construct two further libraries, based on VX-770 and the lead hit compound from the pilot screen. A number of novel ΔF508-CFTR potentiators were identified in each of the screens. Recently published studies suggest that chronic treatment with VX-770 decreases ΔF508- CFTR density at the plasma membrane, potentially limiting its clinical effectiveness. ΔF508- CFTR-pHTomato was used to show that a number of hit compounds from our screens did not decrease membrane density of ΔF508-CFTR. The YFP-CFTR assay was also used for studies investigating the mechanism of potentiation by VX-770. We provide evidence that WT-CFTR does not require phosphorylation for potentiation by VX-770, which has not been reported previously. Additionally, studies using gating mutations do not support the current hypothesis that VX-770 stabilises the posthydrolytic open state in the CFTR gating cycle.

Cystic fibrosis (CF) is one of the most common autosomal recessive disorders affecting Caucasian populations. Seventy percent of CF alleles produce the protein ΔF508 CFTR, in which a single phenylalanine residue is missing from position 508 of the polypeptide chain. This is a processing mutant: ΔF508 CFTR is functional, but retained in the endoplasmic reticulum (ER) and degraded by the ubiquitin-proteasome pathway. The release of wild-type CFTR from the ER through the secretory pathway requires the formation of a stable form of the protein that is no longer a substrate for ubiquitination ("stable B"), ΔF508 CFTR is unable to reach this stable form and this is the basis for its premature degradation in the ER. The rabbit reticulocyte lysate (RRL) system has been used to measure the ubiquitination of the wild-type and mutant proteins. Following translation at 24^oC, when the incubation temperature is increased to 30^oC, approximately 20% of translated wild-type CFTR reaches a form indistinguishable from stable B (a similar proportion to that observed in the cell), whereas all ΔF508 is ubiquitinated. Interactions with the molecular chaperones hsp90 and hsc70 cease when stable B is reached, indicating they are involved both in the folding, and possibly ubiquitination, of the wild-type protein. ΔF508 CFTR remains associated with hsc70 and hsp90 throughout its lifetime in the RRL. Addition of the hsp90 binding agent geldanamycin to ΔF508 CFTR-expressing RRL post-translationally (but not co-translationally) stabilises approximately 25% of the mutant protein with respect to ubiquitination. Again, the form of the protein produced is indistinguishable from stable B, and stabilisation coincides with its release from the molecular chaperones hsc70 and hsp90. The timing of release from hsc70 indicates a role for this chaperone in particular in the ubiquitination of the protein.

Cystic fibrosis (CF) is the most common heritable disease among people with Northern European ancestry. CF is a life-limiting condition affecting multiple systems, however its morbidity and mortality are associated with progressive lung disease followed by respiratory failure. Currently, there is no available cure for CF, as most drugs work only for patients with specific mutations and show limited efficacy. We believe that a combined cell and gene therapy approach (ex vivo gene therapy) where the patient’s own airway cells are corrected by CFTR gene editing in vitro and subsequently transplanted to the lungs, could provide a cure for all patients with CF. Here, we show that we can successfully expand and differentiate primary human airway epithelial cells while maintaining physiological marker expression, electrical responses and CFTR expression. We optimised nucleofections and CRISPR/Cas9 RNP mediated double strand breaks in order to correct the ΔF508 CFTR mutation in patient cells. Correction was achieved through the homology directed repair (HDR) pathway with the help of a donor repair plasmid with a puromycin selection cassette. A second correction approach which included scarless removal of the selection cassette was not successful. We also created an isogenic CFTR knockout in Normal Human Bronchial Epithelial (NHBE) cells, which, after single cell cloning, maintains the ability to form a monolayer and differentiate towards different cell types such as ciliated and mucus cells. Furthermore, we have optimised a protocol for full decellularisation of rat tracheas via a system, which includes perfusion of solutions through the trachea lumen with a pump. CF primary airway epithelial cells engraft in these acellular scaffolds and form a cell layer positive for airway epithelial cell markers. Finally, we have successfully created injury in mouse lungs by intratracheal instillation of polidocanol, which will promote engraftment of delivered airway epithelial cells among the native epithelium.

The emerging role of the multifunctional enzyme, Transglutaminase 2 (TG2) in Cystic Fibrosis (CF) has been linked to its increased expression and intracellular transamidating activity. However, a full understanding of the molecular mechanisms involved still remains unclear despite numerous studies that have attempted to delineate this process. These mechanisms include the NFκB and TGFβ1 pathway amongst others. This study reveals for the first time that the development of fibrosis in CF is due to a TG2-driven epithelial to mesenchymal transition (EMT) via a mechanism involving the activation of the pro-fibrotic cytokine TGFβ1. Using a human ΔF508/W1282X CFTR CF mutant bronchial cell (IB3-1), its CFTR corrected “add-back” cell (C38) as well as a primary human bronchial epithelial cell (HBEC), elevated TG2 levels in the CFTR mutant IB3 cell were shown to activate latent TGFβ1 leading to increased levels found in the culture medium. This activation process was blocked by the presence of cell-permeable and impermeable TG2 inhibitors while inhibition of TGFβ1 receptors blocked TG2 expression. This demonstrates the direct link between TG2 and TGFβ1 in CF. The presence of active cell surface TG2 correlated with an increase in the expression of EMT markers, associated with the CF mutant cells, which could be blocked by the presence of TG2 inhibitors. This was mimicked using the “addback” C38 cell and the primary human bronchial epithelial cell, HBEC, where an increase in TG2 expression and activity in the presence of TGFβ1 concurred with a change in cell morphology and an elevation in EMT marker expression. Conversely, a knockdown of TG2 in the CF mutant IB3 cells illustrated that an inhibition of TG2 blocks the increase in EMT marker expression as well as causing an increase in TEER measurement. This together with an increase in the migration profile of the CF mutant IB3 cell against the “add-back” C38 cell suggests that TG2 drives a mesenchymal phenotype in CF. The involvement of TG2 activated TGFβ1 in CF was further demonstrated with an elevation/inhibition of p- SMAD 2 and 3 activation in the presence of TGFβ1/TG2 cell-permeable/impermeable inhibitors respectively. The use of a comparative airway cell model where bronchial epithelial cells were cultured at the air liquid interface (ALI) confirmed the observations in submerged culture depicting the robustness of the model and reiterated the importance of TG2 in CF. Using a CFTR corrector combined with TG2 inhibitors, this study showed that the correction and stabilisation of the ΔF508 CFTR mutation in the mutant cell forged an increase in matured CFTR copies trafficking to the apical surface by circumventing proteosomal degradation. Thus the results presented here suggests that TG2 expression is elevated in the CFTR mutant bronchial cell via a TGFβ1 driven positive feedback cycle whereby activation of latent TGFβ1 by TG2 leads in turn to an elevation in its own expression by TGFβ1. This vicious cycle then drives EMT in CF ultimately leading to lung remodelling and fibrosis. Importantly, TG2 inhibition blocks TGFβ1 activation leading to an inhibition of EMT and further blocks the emerging fibrosis, thus stabilizing and supporting the maturation, trafficking and conductance of CFTR channels at the apical surface.

Cystic fibrosis (CF) is a life-threatening genetic disease arising from mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). The mutant ΔF508 CFTR accounts for more than 70% of the CF causing alleles and exhibits temperature sensitive export defect from the endoplasmic reticulum (ER). Hsp105/110 facilitates the nucleotide exchange of Hsp70 and possesses independent chaperone activity (holdase activity) in vitro. Human Hsp105α, the constitutively expressed isoform, has been shown to associate with CFTR in the ER but its role in CFTR biogenesis is as yet unclear. We show that Hsp105 plays versatile roles in CFTR biogenesis in the ER affecting synthesis, maturation and degradation. The nucleotide exchange activity of Hsp105 is necessary for the degradation of CFTR in the ER. Its holdase activity reduces synthesis and stabilizes CFTR in the ER. Hsp105 stabilizes CFTR in the ER and proportionally promotes its processing. Hsp105 preferentially associates with ΔF508 CFTR and promotes its stabilization and rescue at both reduced and physiological temperatures. In addition, Hsp105 escorts the mutant CFTR in post-ER compartments and prolongs its half-life. Hsp105’s versatile functions and its high affinity for misfolded ΔF508 CFTR make it a useful molecular target for the rescue of protein misfolding in CF.Hsp70 and Hsp90 regulate CFTR biogenesis but their functional relationship remains unknown. Using the temperature-dependent rescue of ΔF508 CFTR as a model system we provide evidence for functional cooperation between Hsp70 and Hsp90 mediated by cochaperones Hop and Hsp105 in conformational maturation in the ER. Hsp70 associates with ΔF508 CFTR earlier than Hsp90. Hsp90 directly promotes its maturation and the integrity of Hsp70-Hsp90 chaperone network is essential for maturation of ΔF508 CFTR at reduced temperature. Our data support Hsp105 as an integral player in the Hsp70-Hsp90 chaperone network mediated CFTR conformational maturation in the ER.