Development of an inter-kingdom delivery and expression system for therapeutic molecules against cancer

Abstract

Background: Several bacterial species such as Clostridium, E.coli and Salmonella can colonize within solid tumors and potentially be used as anticancer agents. The naturally occurring antitumor activity of Salmonella Typhimurium SL7207 strain can be further enhanced by genetic manipulations, making it a promising system for targeted delivery and expression of therapeutic molecules in cancer therapy.

Methodology/Principal Findings: The engineered strain, termed ST1, was generated in the SL7207 background using the λ Red-mediated ‘long homology arm’ recombineering technology. Firstly, T7 RNA polymerase (T7 RNAP) gene was integrated into the bacterial genome to enable the transcription of small RNA or exogenous gene. Secondly, an essential gene asd was replaced by a pore-forming listeriolysin O gene hlyA, which encodes a bacterial factor required for a maximal transfer of therapeutic factors in vivo. Subsequently, the asd gene with anaerobic control was cloned back at the htrA gene locus. The double mutation of asd and htrA made bacteria to grow only under anaerobic conditions while being disabled for growth in normal tissues. The final step was to relocate a small essential gene infA (coding for translation initiation factor 1) from chromosome to plasmid for stable plasmid maintenance. After genetic manipulations, a series of functional assays have been performed to test the safety and efficiency. In tumor-bearing nude mice, ST1 could target the hypoxic/ necrotic regions of inside the solid tumors without detectable cytotoxicity. To facilitate gene expression, an inter-kingdom dual expression (IKDE) system, containing T7 RNAP-based cytoplasmic expression system and a cytomegalovirus (CMV)-based nuclear system, has been developed. Compared to the standard nuclear expression system, this system produced more than 50–fold higher gene expression in MDA-MB-231 cells following in vitro infection.

The anticancer effects of ST1 can be exerted in different approaches: (a) Bacterial protein delivery: as a protein delivery vehicle, ST1 expressed the extracellular domain of TNF-related apoptosis-inducing ligand (TRAIL) directly in extracellular environment or in tumor cells to induce apoptosis. In the MDA-MB-231 xenograft tumor model, the delivery of biologically active proteins by ST1 significantly inhibited tumor growth. (b) Bactofection: ST1 has been used as a vector to transfer DNA encoding therapeutic gene in tumors through an IKDE system. After intravenous injection of ST1 carrying a gene encoding diphtheria toxin A chain, high levels of plasmid-derived mRNA and protein were detected inside the tumors. Significant tumor shrinkage was observed with no gross sign of toxicity. (c) Inter-kingdom RNA interference (ikRNAi): ST1-mediated ikRNAi system can transfer RNAi effectors between bacteria and mammals. Systematic administration of ST1 harboring RNAi system against tumor-related genes induced a specific and efficient silencing inside the tumor and retarded its development without apparent side effects.

Significance: We believe that our present study is the first to provide a proof of concept for the engineered tumor-targeting Salmonella ST1 as a tumor-targeted delivery vehicle of antitumor molecules and as a therapeutic ‘factory’ for use in an innovative cancer therapy, and anticipate that it will serve as a platform to bring the technology closer from bench to bedside.