Bahal, Sameer;
(2025)
Gene editing in haematopoietic stem cells for the treatment of
X-Linked Agammaglobulinaemia.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
This project was a pre-clinical feasibility study to design a gene editing strategy for X-Linked Agammaglobulinaemia (XLA). This affects roughly 1 in 250,000 births and is caused by a mutation in the BTK gene. This causes an absence of B Cells and an absolute Antibody Deficiency. As a result, patients have severe and frequent infections. Standard management is with Immunoglobulin replacement therapy; however, studies show 93% of patients still develop Bronchiectasis and have often have breakthrough infections. For patients with primary immunodeficiencies, gene editing has the potential to rescue genetic defects via the correction and transplantation of autologous haematopoietic stem cell (HSCs). Our universal strategy involves inserting a codon optimised BTK minigene via Homology Directed Repair using an Adeno Associated Virus-based donor vector in a Cas9 mediated break at the endogenous gene location. This would correct almost all XLA causing gene mutations with the transgene remaining under the control of local regulatory factors. Initially we trialled our optimised gene editing technique in a BTK knockout version of a B cell line called DG75 and showed successful integration and restored BTK expression to nearly wild type levels, while no expression was found in the Jurkat T cell line. This suggests the rescue of a physiological pattern of expression with the therapeutic transgene. We next recruited 3 XLA patients to undergo peripheral blood stem cell donation. We subsequently edited patient HSCs and using a specialist culture technique we demonstrated resorted ability of these cells to differentiate into B cells in vitro and in vivo using the NSG mouse model, as well as restoration of Immunoglobulin production. This work provides proof-of-concept of this strategy as a potential novel curative therapy for XLA.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Gene editing in haematopoietic stem cells for the treatment of X-Linked Agammaglobulinaemia |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2025. 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 > 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 UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10205852 |
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