Engineering Aerotolerance and Quorum Sensing into Clostridium for Use as a Live Biotherapeutic Product against Solid Tumors

Abstract

Tumor targeting remains a significant challenge in cancer therapy, particularly for solid tumors, which constitute approximately 80% of all cancer types. Hypoxia, a hallmark of solid tumors, plays a crucial role in tumor progression and metastasis. However, hypoxia also serves as a distinguishing feature of tumor cells compared to healthy tissue, presenting an opportunity for targeted therapeutic strategies. One such approach involves the use of anaerobic bacteria, particularly clostridia, which have been known for over 60 years to selectively colonize the hypoxic and necrotic regions of tumors. Studies have demonstrated that infection with wild-type clostridia strains can induce tumor lysis and regression. However, despite their ability to degrade hypoxic tumor regions, tumor regrowth from the viable outer rim often leads to incomplete tumor elimination, limiting the efficacy of clostridia-based tumor therapy. This study aims to address the challenge of tumor regrowth by developing an aerotolerant strain of Clostridium sporogenes that cannot grow in healthy tissue but can proliferate in the viable outer rim of tumors. As a part of this effort, the water-forming NADH oxidase gene (noxA) from Clostridium aminovalericum was cloned downstream of the thl promoter from Clostridium acetobutylicum and introduced into C. sporogenes. Experimental analysis confirmed that the heterologous expression of noxA conferred aerotolerance, allowing C. sporogenes to grow in the presence of oxygen. To couple aerotolerance with tumor colonization, the expression of this gene should be regulated. We chose to employ a quorum sensing (QS) promoter. In parallel, a QS regulatory system was designed to enable density-dependent control of gene expression in C. sporogenes. The well-characterized agr operon from Staphylococcus aureus, a Gram-positive bacterium, was introduced into C. sporogenes. Growth and fluorescence assays were conducted to characterize the behavior of the QS system, demonstrating its potential to function as a switch-like regulatory mechanism in C. sporogenes. Additionally, a mathematical model based on kinetic principles was developed and calibrated using experimental data to describe the dynamic of this synthetic QS. While the final integration of noxA under QS control was not completed within the scope of this study, the successful development of both aerotolerance and a functional QS circuit establishes a strong foundation for future assembly and implementation of a regulated-aerotolerant strain of C. sporogenes.