ANAEROBIC-BACTERIA AS A DELIVERY SYSTEM FOR CANCER GENE-THERAPY - IN-VITRO ACTIVATION OF 5-FLUOROCYTOSINE BY GENETICALLY-ENGINEERED CLOSTRIDIA

Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therap y in which genetically engineered anaerobic bacteria of the genus Clos tridium are used to achieve tumour-specific gene delivery. Our strateg y involves enzyme/prodrug therapy, in which the Escherichia coli enzym e cytosine deaminase is used to convert the non-toxic prodrug 5-fluoro cytosine to the active chemotherapeutic agent 5-fluorouracil. The E. c oli gene encoding cytosine deaminase has been cloned into a clostridia l expression vector and transformed into Clostridium beijerinckii, res ulting in constitutive expression of cytosine deaminase and significan t levels of active enzyme in the bacterial medium when added to an in vitro clonogenic survival assay, supematant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hy poxic regions of tumours, indicates a potential use in cancer gene the rapy.