Tumor-Targeted Salmonella: Strain Development and Expression of the HSV-tK Effector Gene

Gene therapy approaches to cancer treatment have been limited by the ability of the delivery vectors to achieve specific high-level expression within tumor cells or the tumor environment following systemic administration. Numerous physical barriers exist in the delivery of therapeutic agents (including drugs, viruses, and liposomes) to solid tumors that can compromise the effectiveness (1 ), thus stimulating the search for alternative methods of delivery. Whereas it has been known for some time that spores of anaerobic Clostridium can germinate within the necrotic spaces of human tumors, they are limited to larger hypoxic tumors and are inaccessible to smaller metastases (2 ,3 ). The ability of motile, facultatively anaerobic Salmonella to target tumors following systemic administration, preferentially amplify within them, and express effector genes such as the herpes simplex virus thymidine kinase (HSV-TK ) makes them an attractive alternative to Clostridia , liposome and viral-based delivery vectors (4 ). These Salmonella were attenuated by poly-auxotrophic mutations, which limited their pathogenesis in normal tissues, but retained high-level replication within tumors, resulting in tumor suppression of both primary and metastatic tumors (4 ,5 ). The attenuating mutations were added stepwise following in vitro and in vivo selection and screening methods. Although live-attenuated vectors for use in humans requires defined genetic mutations, our experience has shown that combinations of point-mutations and frame-shift mutations allows for rapid isolation of strains with multiple mutations having desirable properties, which can later be defined and/ or stabilized. Bearing this in mind, we present the basic methodology for the development of tumor-targeting facultative anaerobes with effector gene delivery capabilities that we applied to Salmonella.