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Richards, Danielle (2025) Engineering of Clostridium novyi to enhance efficacy in cancer therapy. PhD thesis, University of Nottingham.

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Abstract

Clostridium novyi-NT (non-toxic) is an attenuated clone derived from C. novyi, rendered non-pathogenic through the removal of a resident phage that carries the gene for the lethal alpha toxin. It is a Gram-positive spore-forming bacterium, which has gained significant attention for its potential use in cancer therapy. C. novyi-NT is a strictly anaerobic bacterium, enabling it to thrive in the hypoxic regions of tumours, a characteristic hallmark of solid malignancies. For therapy, C. novyi-NT can be delivered as dormant spores that germinate in hypoxic, necrotic tumours, inducing oncolysis through enzymatic activity and immune activation, while complementing treatments that target oxygen-rich tumour areas.

This study focused on genetically modifying C. novyi-NT to improve its safety profile for use in cancer therapeutics, and to improve its efficacy. Prior to modifying the organism for cancer therapeutics, gene transfer into C. novyi-NT and its molecular characteristics were assessed. Various strategies, including gene knockouts and altering donor methylation patterns, were used to bypass restriction-modification barriers, but showed no effect.

To enhance the safety profile of C. novyi-NT conditionally sporulating strains were created using CRISPR-Cas technology, whereby the strains were only able to sporulate when in the presence of specific ligands. This approach provided a solution for preventing the spread of spores beyond the targeted tumour region, thereby reducing the risk of toxicity and the dissemination of recombinant spores.

The promoters of C. novyi-NT were investigated, and reporter assays identified a suitable candidate for cytokine expression. C. novyi-NT was subsequently engineered to express the cytokines GM-CSF and IL-4, with the investigation of signal peptides resulting in the efficient secretion of GM-CSF.

Furthermore, a novel inducible xylose promoter was engineered for use in both E. coli and C. novyi-NT to control toxicity, which could facilitate the advancement of cytokine expression and cloning in future studies.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Minton, Nigel
Keywords:	Clostridium novyi-NT, cancer therapeutics, genetic engineering
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QT Physiology R Medicine > RM Therapeutics. Pharmacology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	82885
Depositing User:	Richards, Danielle
Date Deposited:	10 Dec 2025 04:40
Last Modified:	10 Dec 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/82885

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