P2.02Using modified Clostridium sporogenes as a delivery vehicle for anti-cancer therapeutics

ABSTRACT Background: Clostridium sporogenes is part of a highly diverse group of Gram positive, spore forming, anaerobic bacteria. C. sporogenes can be used as a delivery vehicle for chemotherapeutics in cancer treatment due to the inactive spore form of C. sporogenes only germinating in the microenvironment of the hypoxic tumour. The directed enzyme pro-drug therapy (DEPT) strategy has been tested with several different delivery vectors, and Clostridial DEPT (CDEPT) has previously been developed with one pro-drug converting enzyme (nitroreductase). In this treatment the non-toxic pro-drugs are administered to the patient, then a pro-drug converting enzyme is delivered through genetically altered C. sporogenes. This enzyme breaks the pro-drug down into a toxic component. The aim of this project is to develop the system further with an alternative pro-drug converting enzyme which digests its potential substrate into a 100 fold more toxic subcomponent. Materials and Methods: To efficiently implement the use of this enzyme in the CDEPT strategy, the delivery system needed to be optimised to allow for its effective export and binding to the cell wall of the bacteria to maintain site specificity. To do this, the use of a panel of different signal peptides was investigated. Concurrently to that different sortase signal motifs were identified in Clostridium species through psiBLAST, and their use to anchor the protein to the cell wall was investigated. Successful attachment and protein export was judged by assaying the presence of enzyme activity in the different fractions of bacterial cultures. Conclusion: Results showed an enhanced export of the enzyme into the culture supernatant, and significant cell wall anchoring. This data gives a good basis from which development of an optimized CDEPT system should be achievable, which would result in a highly site specific, hypoxic tumour therapy system.