Brezovjakova, Helena;
(2023)
Universal genome-edited skin grafts.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Failure to repair wounds and the resultant chronically non-healing ulcers present a clinically severe consequence of several conditions, including diabetes, vascular disorders, severe burn injuries, and genetic diseases like epidermolysis bullosa (EB). In the UK, managing chronic wounds costs the NHS over £2 billion annually with bulk of the budget spent on district nurse visits followed by costs of dressings and compression bandages [1]. While skin grafts can be used to treat chronic wounds, patients often have inadequate quantities of healthy skin usable as autografts and allogeneic skin grafting is hindered by human leukocyte antigen (HLA) barriers as donor skin is acutely rejected [2]. Generating universal epidermal sheets from healthy donor keratinocytes may provide a possible strategy to bypass these limitations. The aim of this project was to evaluate the possibility of generating universal skin grafts from healthy donor keratinocytes genome-engineered to reduce immunogenicity. Initial investigation of HLA class I (HLA-I) and class II (HLA-II) expression disruption utilised third-generation cytosine base editor (CBE3)-mediated knockout of B2M and RFXANK. Significantly reduced HLA-I and HLA-II expression in primary human keratinocytes was confirmed by flow cytometry and Sanger sequencing of the edited loci. Purification of this population by magnetic bead separation yielded primary keratinocytes devoid of surface HLAs (HLA-dKO). Study of naïve T cell responses to HLA-dKO keratinocytes showed reduced activation and proliferation. Moreover, primed CD8+ T cells mounted strong cytotoxicity against unedited but not HLA-dKO keratinocytes. Importantly, HLA-I removal in primary keratinocytes did not trigger NK cell-mediated lysis. Functional assessment of cell migration, proliferation, and differentiation demonstrated equal migratory ability of unedited and HLA-dKO keratinocytes in vitro and multiple epidermal layer formation with the corresponding expressions of related protein markers in 3D skin equivalent cultures derived from HLA-dKO keratinocytes. Downstream potential of HLA-dKO keratinocytes was exemplified by generating HLA-dKO epidermal sheets.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Universal genome-edited skin grafts |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/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 > School of Life and Medical 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-pp.ucl.ac.uk/id/eprint/10176888 |
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