Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.780487
Title: Telomerase-targeting radiopharmaceuticals for the selective treatment of cancer
Author: Bavelaar, Bas
ISNI:       0000 0004 7966 129X
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Abstract:
Background Telomerase is a ribonucleoprotein that is expressed in the majority of malignancies, whilst being largely absent in differentiated tissue. Previous research has shown that short-term inhibition of telomerase can lead to radiosensitisation of cancer cells. It has been shown previously that enhanced retention and nuclear localisation of transfected Indium-111(111In)-labelled oligonucleotides, targeted to the RNA template of telomerase (hTR), resulted in a telomerase-dependent cytotoxic induction of DNA double-strand breaks. Aim The aim of this DPhil project was to develop an in vitro and in vivo delivery system for 111In-labelled anti-hTR oligonucleotides. To this end, we set out to synthesise and characterise 111In-labelled anti-telomerase gold nanoparticle (AuNP) constructs that can be targeted to the tumour site with ultrasound-activatable cavitation nuclei. Results hTR complementary (Match) or non-complementary (Scramble) oligonucleotides were conjugated to DTPA for labelling with the Auger electron-emitter 111In. AuNP, 15.5 nm in diameter, were synthesised by citrate reduction and decorated with a monofunctional layer of oligonucleotides (ON-AuNP) or a multifunctional layer of oligonucleotides, PEG800-SH and the cell-penetrating peptide Tat (ON-AuNP-Tat). The synthesised AuNP constructs were found to greatly enhance the cellular uptake of radiolabelled oligonucleotides, whilst retaining the ability to inhibit telomerase activity. The addition of Tat on the AuNP surface resulted in increased nuclear localisation, as shown in internalisation and fractionation experiments, as well as confocal and transmission electron microscopy. This uptake resulted in a significant dose-dependent impact of 111In-Match-AuNP-Tat on the clonogenic survival of a telomerase-positive cell line, but not telomerase-negative cells, and corresponded with an increase in DNA double-strand breaks. 111In-Match-AuNP did not reduce clonogenic survival, which is partly attributed to the lower nuclear uptake of the construct. For this reason, only 111In-Match-AuNP-Tat was taken forward into the animal studies. It was found in a biodistribution study of 111In-Match-AuNP-Tat and 111In-Scramble-AuNP-Tat that the AuNP constructs accumulated in the liver with low tumour uptake. To mitigate this effect, nanocups, ultrasound-activatable cavitation agents, were used to direct the AuNP constructs to the tumour. Nanocups significantly increased extravasation of Cy5-Match-AuNP-Tat in an in vitro model, and was therefore trialled in an in vivo setting. A pilot study demonstrated promising tumour uptake, prompting ongoing research of this delivery strategy. Conclusions This DPhil study has delivered a solution for the subcellular targeting of radiolabelled anti-hTR oligonucleotides by using an AuNP-based system. This novel approach of combined modality telomerase-targeting may be a start for future in vivo research with ultrasound-mediated delivery. The techniques described in this thesis may provide a building block for nucleic acid-based radiopharmaceutical research.
Supervisor: Vallis, Katherine Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.780487  DOI: Not available
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