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Title: An investigation into the impact of early development and manufacturing processes on cell product quality
Authors: Kay, Charlotte
Advisors: Themis, M
Roberts, T
Keywords: chimeric antigen receptor;cell and gene therapy;Retrogenix;T cells;transduction enhancers
Issue Date: 2023
Publisher: Brunel University London
Abstract: Cell and Gene Therapy (CGT) now offers curative treatment for several genetic disorders and haematological malignancies; however, development and manufacturing still hamper broader clinical application. This thesis investigates early development and manufacturing processes to reduce cost, whilst keeping product safety, quality and efficacy in mind. In Chapter 1, Retrogenix™ was assessed for its suitability as an early development screening platform to reduce the number of candidate constructs progressed by screening for off-target binding, which was observed with CAR-X binding to the Netrin1 receptor. However, a missed off-target binding hit with CAR-A, the unsuitability for engineered TCR-T cells and high costs, all limited the suitability of Retrogenix™ for screening large numbers of candidates. In Chapters 2 and 3, methods to reduce “vein-to-vein” time for apheresis, treatment, and re-infusion into the patient, were explored. With limitations on cell expansion rate focussed upon within chapter 2, and T cell transduction efficiency enhancement investigated within chapter 3. As monocyte contamination of peripheral blood mononuclear cell (PBMC) populations is believed to reduce cell product expansion, these were removed, however, this did not significantly impact T cell expansion and bulk PBMCs were transduced by lentiviral vectors (LVV) at higher levels than isolated T cells. To investigate culture vessel impact on T cell expansion, G-REX® vessels, that provide increased oxygen perfusion and nutrient availability to cells, were compared to standard flat-bottom culture plates and found to significantly improve T cell expansion. Additionally, cytokines were investigated on cell expansion using IL-2 alone or a combination of IL-7/IL-15. This resulted in neither being able to increase T cell expansion or transduction efficiency, however, IL-7/IL-15 did impact the CD4:CD8 T cell ratio. Finally, using small molecule compounds, T cell transduction was investigated with two candidate compounds identified that improved LV transduction efficiency. To conclude, this work identified and enabled implementation of several process improvements that have benefitted GSKs CGT development process. These findings will be discussed with consideration of ways to reduce the cost of gene therapy products thereby making them more attractive to the marketplace.
Description: This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London
Appears in Collections:Biological Sciences
Dept of Life Sciences Theses

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