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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 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.