Avgerinou, Afroditi;
(2020)
Investigating a combined cell and gene therapy for cystic fibrosis: Working towards a proof of principle.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
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.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Investigating a combined cell and gene therapy for cystic fibrosis: Working towards a proof of principle |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10101496 |
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