Zygouropoulou, Maria
(2019)
Advancing the clinical implementation of Clostridial cancer therapies.
PhD thesis, University of Nottingham.
Abstract
Although tumour hypoxia and necrosis are a nemesis to the efficacy of conventional cancer therapies, they can be exploited by anaerobic bacteria for the selective targeting of solid tumours. Clostridium sporogenes lends itself well to this purpose, being a non-pathogenic, spore-forming and proteolytic obligate anaerobe. Intravenously injected spores of this organism profusely infiltrate, selectively germinate and naturally thrive in the hypoxic and necrotic regions of tumours, whilst non-germinated spores are eliminated. Capitalising on its inherent tumor-targeting capabilities, C. sporogenes can be genetically engineered to enhance its oncolytic properties as well as to incorporate safety features, cooperatively accelerating its translation towards clinical applications.
In the present thesis, C. sporogenes NCIMB 10696 was engineered to express nitroreductases which can co-metabolise a clinical-stage, mustard-based prodrug and 2-nitroimidazole PET probe analogues, thus enabling cancer cell killing as well as non-invasive imaging of the clostridial vector. Importantly, a highly expressed nitroreductase candidate was identified and its crystal structure was determined, facilitating the design of optimised prodrug derivatives. The strain was additionally engineered to secrete biologically-active Granulocyte Macrophage-Colony Stimulating Factor in high titres, expanding hitherto explored functionalities and providing proof-of-concept for clostridial-mediated cancer immunotherapy. Furthermore, empowered by the CRISPR/Cas9 technology, a multicopy genome integration strategy was implemented, generating significant improvements in protein yield, beyond those afforded by transcriptional and translational fine-tuning. Novel genomic loci for insertions of therapeutic cargos were characterised, revealing another layer of gene expression control that may be advantageously incorporated in the design of clostridial vectors.
Safety considerations permeated all genetic manipulations undertaken, culminating in the elimination of the biosynthetic gene cluster of the haemolytic clostridiolysin toxin, to render the strain safer and regulatorily compliant. Highlighting the versatility of the vector, intelligibly, safety and efficacy deliverables were closely interlinked (for instance, imaging via the therapeutic functionality, cargo insertion with concomitant aberration of key genes). Overall, the advancements generated here constitute significant progress in the development of a clinically relevant end-product.
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