A Synthetic Biology Approach to Bacteria Mediated Tumor Targeting

Abstract

Development of a drug delivery agent that selectively targets and destroys tumor cells with minimal toxicity to normal tissues is a major challenge in cancer therapy. It has been known for more than 60 years that anaerobic bacteria such as Clostridium can selectively colonize inside the necrotic core of solid tumors. Inoculation of a tumor by wild type Clostridium results in colonization of the necrotic core and consequently significant tumor destruction. This treatment strategy is hampered by the fact that the outer rim of the tumor is typically viable, and so does not present an anaerobic environment. As a result, colonization by Clostridium is unlikely to lead to complete tumor regression, since tumor regrowth occurs from the remaining outer viable rim, as evidenced by clinical trials. This project aims to address the problem of regrowth by developing a novel selectively aerotolerant strain of Clostridium that cannot colonize inside healthy tissue, but that could grow in the viable rim of an infected tumor. We have engineered a gene coding for an aerotolerance enzyme into Clostridium sporogenes. To couple the selective expression of this gene to tumor colonization, it can be placed under the control of a promoter activated by a synthetic quorum sensing circuit. This document describes the foundational work that will allow this system to be implemented. A suitable strain of C. sporogenes was selected, and a cloning technique (via conjugation with E. coli) was implemented. Expression of the aerotolerance enzyme and a synthetic quorum sensing circuit were verified in engineered colonies, and appropriate function was confirmed in both cases. Additionally, a model-based design exercise was carried out in order to better understand the system behavior and to identify key parameters for controlling the bacterial population. This analysis was based on mathematical models of the quorum-sensing circuit and of bacterial growth in the tumor environment. Sensitivity analysis reveals the design parameters that have the most significant impact on the extent and specificity of colonization of the viable rim, and thus provides insights into efficient design of the synthetic mechanism.