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Title: Novel prodrug-converting enzymes and a directed DNA integration method for clostridia
Author: Heap, John Timothy
ISNI:       0000 0004 2748 2169
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2007
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Cancer is a major cause of mortality and morbidity that is likely to both increase in incidence and persist with humankind indefinitely. Conventional therapies are not effective against all types and stages of cancer, representing a huge present and future unmet medical need. Clostridial Directed Enzyme Prodrug Therapy (CDEPT) is a promising new experimental approach to the treatment of solid tumours, whose hypoxic (low-oxygen) regions make them resistant to conventional therapies. These same hypoxic regions can be exploited in therapies using clostridia; strictly-anaerobic bacteria that can grow in hypoxic tumours, but not elsewhere. In CDEPT, the clostridial cells are engineered to produce an enzyme that converts a subsequently-administered, non-toxic prodrug into a highly-toxic drug only at the tumour site, resulting in highly tumour- specific cytotoxicity; an important shortfall of existing therapies. The prodrug 5- (aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) has excellent properties, and is safe. However, CDEPT progress has been hindered by poor CB 1954-activating enzymes and the unavailability of techniques to chromosomally-localise the enzyme-encoding gene, a safety feature required for clinical use. Here, two novel CB 1954-activating enzymes have been isolated and characterised, one of which has kinetic properties with respect to CB 1954 substantially better than previously-reported enzymes, and may prove to be ideal for CDEPT and related therapies. Both separately, and in collaboration with structural biologists, progress has been made towards understanding, and therefore potentially improving, the interactions between CB 1954 and several enzymes. Finally, a method for the integration of DNA into clostridial chromosomes has been developed, which facilitates the construction of clinical CDEPT strains. This method has profound implications beyond CDEPT, particularly because it allows the construction of isogenic mutants, important research tools that were previously difficult or impossible to construct in major pathogens such as Clostridium difficile and Clostridium botulinum, and industrially-important strains such as Clostridium acetobutylicum.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available