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Developing generic and modular approaches to targeted cancer cell therapeutics

Mills, Emily 2021. Developing generic and modular approaches to targeted cancer cell therapeutics. PhD Thesis, Cardiff University.
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Abstract

The intricacies and complexities of cancer render it a difficult disease to treat, and existing treatments are frequently non-selective. This project investigated two selective cancer therapeutic approaches: organelle-targeted drug delivery, and genetic re-wiring to achieve a switchable CAR-T model. Cancer mitochondria are different to healthy cells, including a higher mitochondrial membrane potential (MMP) which can be exploited for selective delivery of a therapeutic. Cyanine dyes Cy3 and Cy5, along with a dimer Cy3- Cy5 and a CPP conjugate Cy3-Cy5-R8 were characterised, and all constructs stained the mitochondria of HeLa in an MMP-dependent manner. Staining capacities of Cy3, Cy5 and Cy3-Cy5 were not hindered by serum proteins or endocytosis inhibition. Conversely, serum proteins reduced Cy3-Cy5-R8 staining capacities, and its uptake was endocytosis-dependent. Cy3 was subsequently tested as a mitochondrial-drug delivery vehicle, and Cy3 conjugation to mitochondrial toxins improved EC50 values by up to 1000-fold. The Cy3-drug conjugates were more toxic to cancerous (HeLa) vs noncancerous cells (HEK293), but toxicity was still present in HEK293. Further studies are therefore needed to enhance Cy3-drug selectivity to cancer cells. Cancers can alternatively be targeted via CAR-T cell immunotherapy; however, CAR-T frequently over-activate and bring unwanted toxicity to the patient. Through genetic code expansion, a new logic gates approach was developed. 11 quadruplet-decoding pyrrolysyl tRNA variants that incorporate BocK were analysed. Unlike their literature representation in E. coli, only five variants were functional in HEK293. Increasing tRNA copy number from 1 to 4 improved BocK-incorporation, and PylRS/tRNA was found to function orthogonally alongside a mutant TyrRS/tRNA pair. A split GFP reporter system was subsequently developed, where AND and OR logic operations were successfully generated whereby GFP output can be controlled via the unnatural amino acid makeup of the cellular media. The cell circuits developed here provide a new approach to mammalian cell logic operations, and can potentially be translated into a switchable ON/OFF CAR-T model.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Funders: KESS2, Tenovus Cancer Care, Cardiff University
Date of First Compliant Deposit: 21 October 2021
Last Modified: 21 Oct 2021 14:49
URI: https://orca.cardiff.ac.uk/id/eprint/144993

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