(557b) Engineering Salmonella Typhimurium for Controlled Therapeutic Delivery in Solid Tumors

To effectively treat cancer, therapeutics must specifically target tumor tissue and impart minimal toxicity to healthy tissues. Genetically engineered bacteria, such as Salmonella typhimurium, can accomplish this targeting specificity and thus be exploited as drug delivery vectors. By using cellular engineering techniques, the innate genetic machinery of bacteria can be used to produce toxic peptides in situ. In addition to spatial control, temporal control can be achieved by using an externally inducible promoter. A plasmid construct that expresses TNF-related apoptosis-inducing ligand (TRAIL) under a radiation inducible promoter, P_RecA, has been tested in vivo. In a syngeneic murine tumor model, induction of P_RecA controlled TRAIL expression by 2Gy γ-irradiation caused a significant delay in tumor growth and reduced risk of death by 76%. A repeated dosing and irradiation scheme improved thirty-day survival from 0% to 100%. These results demonstrate that radiation induced temporal control of gene expression, combined with the targeting capabilities of bacteria, can effectively enhance cancer survival.

However, the use of P_RecA for tightly controlled release of a cytotoxic peptide is not ideal. As RecA is a self-regulated component of the SOS radiation response pathway, it has a high level of basal expression. An ideal therapeutic would have no constitutive expression and high levels of induced expression. Our lab has engineered non-pathogenic Salmonella to secrete TRAIL under the control of a radiation inducible promoter (P_RI). The promoter is tightly repressed under normal conditions and is highly activated after exposure to cellular radiation damage. By using a Green Fluorescent Protein (GFP) construct with P_RI control, it was shown that this inducible gene system can be used to tightly control expression in response to radiation. An ELISA assay was used to confirm secretion of TRAIL from bacteria into culture supernatant following induction. Additionally, the ability of TRAIL secreted from this bacterial system to induce apoptosis in mammalian carcinoma cells was quantified by using monolayer experiments and an in vitro 3-D tumor model. These data indicate that a S. typhimurium system utilizing this construct has the potential to augment the tumor targeting properties of bacteria with high-level temporal control, increasing specificity and efficacy in cancer treatment.