title: Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia.
abstract: 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 therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant 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 hypoxic regions of tumours, indicates a potential use in cancer gene therapy.
authors.affiliation: Department of Radiation Oncology, Stanford University School of Medicine, CA 94305, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Fox
authors.forename: M J
authors.initials: MJ
authors.lastname: Lemmon
authors.forename: M L
authors.initials: ML
authors.lastname: Mauchline
authors.forename: T O
authors.initials: TO
authors.lastname: Davis
authors.forename: A J
authors.initials: AJ
authors.lastname: Giaccia
authors.forename: N P
authors.initials: NP
authors.lastname: Minton
authors.forename: J M
authors.initials: JM
authors.lastname: Brown
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic; D001483:Base Sequence; D002277:Carcinoma / Q000628:therapy*; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics*; D043525:Cytosine Deaminase; D004926:Escherichia coli / Q000201:enzymology; D005437:Flucytosine / Q000633:toxicity*; D005472:Fluorouracil; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D008325:Mammary Neoplasms, Experimental / Q000628:therapy*; D051379:Mice; D008969:Molecular Sequence Data; D009700:Nucleoside Deaminases / Q000235:genetics / Q000378:metabolism; D011355:Prodrugs / Q000633:toxicity*; D014169:Transformation, Bacterial; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D011355:Prodrugs; D005437:Flucytosine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005472:Fluorouracil