Westhaus, Adrian;
(2022)
New avenues of directing evolution in viral vector capsid bioengineering.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Gene therapy offers the treatment of diseases caused by mutations in the patient’s genome. The delivery of a healthy copy of the mutated gene to affected cells is central to this intervention. Recombinant adeno-associated viral vectors (rAAV) are rapidly becoming the vector of choice for gene delivery applications. One of the many distinguishing features of rAAV vectors is the large diversity of capsid variants enabling a variety of cells and tissues to be targeted for gene delivery. While gene replacement and addition approaches remain the most common strategies utilising rAAVs, refinements in the use of targeted nucleases have made rAAVs a promising gene editing tool. Regardless of the intended application, the capsid variant utilised is a key determinant of clinical efficacy. This thesis presents three endeavours undertaken investigating capsid development and characterisation. The first is an investigation of high-throughput tools for identifying high-performing capsid variants and comparing non-human pre-clinical models. The second is a directed evolutionbased selection platform enabling the reliable generation of novel capsid variants that enable high levels of transgene expression. This technology was validated in two unrelated targets – the human liver and the human retina – demonstrating its broad applicability. Finally, the concept of directed evolution was applied to create a platform for generating novel capsids with increased homology-directed repair outcomes in primary T-lymphocytes and haematopoietic stem cells. This was the primary aim of the thesis and it was achieved by incorporating the lessons learned from all previous studies. In summary, over the course of this project, three key technologies were developed and validated. All these technologies are highly impactful as they enable the choice of the optimal capsids and model system as well as the most functional generation of novel capsids. Moreover, novel capsids were generated in human hepatocytes, human retina, and cells of the immune system.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | New avenues of directing evolution in viral vector capsid bioengineering |
| 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 > Faculty of Population Health Sciences > UCL GOS Institute of Child Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10157789 |
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