Abstract:
Current systems for delivering therapeutic genes to tumours suffer from a number of serious deficiences, most notably a lack of specificity for cancer cells. Here we propose an innovative solution to the problem based on the use of the spores of a harmless, nonpathogenic Clostridium species. Intravenously injected clostridial spores localise to, and exclusively germinate in, the hypoxic regions of solid tumours.

The spores are incapable of germinating in healthy tissue. We will engineer this harmless organism such that it produces a variety of prodrug converting enzymes and assess their anti-tumour effects in a rat tumour model. New, more efficient enzymes and prodrugs will be developed. The clostridial host will be further disabled through defined proprietary to improve its biosafety.

Objectives:
It is the objective of this project to develop a new and safe therapy for treating solid tumours based on a tumour-specific clostridial delivery vehicle. The organism to be used will be Clostridium spp., which is entirely non-pathogenic to man. Using rhabdomyosarcoma-bearing rats as the in vivo model, this organism will be used to deliver the two classes of prodrug converting enzymes, and their effects on tumour growth assessed following prodrug administration. To increase the system's effectiveness, new enzymes and prodrugs with improved characteristics will be developed. A non-invasive procedure will be developed to monitor tumour colonisation. To enhance the safety of the system, further defined alteration will be made to disable the host system employed.

Description of the Work:
The organism to be employed is a harmless, industrial Clostridium spp.

During reproduction it colonises tumours, where it achieves high cell numbers, and is readily amenable to genetic modification. Two types of therapeutic enzymes will be delivered which are able to turnover innocuous circulatory prodrugs into highly toxic species. To maximise their affects, new more potent prodrugs will be synthesised.

The expression and secretion of enzymes will be optimised, through the analysis and eventually deployment of different promoters, signal peptides and vectors.

The therapeutic effect of these derivatised strains on tumours will be evaluated in vivo using an appropriate animal model.

For proof of principal studies, initial strains will carry the genes on the systems currently available. In the final strains, new strategies will be employed to maximise segregational stability, and defined alterations made to the host genotype to disable the strain and enhance biosafety.

Further improvements on specificity will be made by using additional therapeutic agents to increase the degree of colonisation. Non-invasive, procedures will also be devised to estimate the extent of tumour colonisation, a prerequisite to prodrug administration. Finally, the system will be tested with respect to side effects and efficacy. As part of this process, an assessment of biosafety of the system will be undertaken. This will allow an assessment of the potential risks to human health and the environment and provide a framework for the derivation of the clinical regimes to be imposed when the system moves into the developmental phase.

Deliverables:
We will have established the feasibility of using clostridia as an anti-cancer drug delivery system. It will circumvent current problems associated with existing gene delivery systems, and will be a highly selective and safe tumour targeting therapy.

Major milestones:

• Discovery of more effective prodrug converting enzymes/prodrugs.
• Generation of disabled strains producing therapeutic agents.
• Assessment of their effectiveness and safety in an in vivo tumour model