Identifying the underlying cause of limitations in Salmonella mediated gene transfer for the development of a successful cancer gene therapy

Many novel treatment options have been invented to improve on serious side effects experienced by cancer patients commonly administered with chemotherapy drugs. In the recent decade, salmonella-mediated cancer therapy has gained prominence in its ability to efficiently target and penetrate tumor tissue. Many therapeutics agents including proteins and chemotherapy drugs have been successfully delivered to the tumor microenvironment, however, relatively limited success has been observed in salmonella-mediated gene transfer therapy. Despite a successful bacterial invasion into the mammalian cancer cell, poor efficiency in regards to the number of cells expressing the plasmid is observed. The purpose of this project is to examine how a nuclear localization signal on a plasmid impacts DNA transport from salmonella to the nucleus. Attenuated salmonella strain VNP20009 with overexpressing flhDC increases invasion efficiency. In order to ensure the intracellular occupancy of the vector, optimizing the invasion of salmonella carrying the plasmid in the tumor cell is crucial. A plasmid containing lysis cassette and EGFP gene driven by the CMV promoter with absence or presence of SV40 origin is bactofected into mammalian cancer cells to observe the effect of SV40 origin on nuclear localization of the plasmid. However, no EGFP expression was observed in mammalian cells, which led to the hypothesis that the entrapment of plasmid in salmonella containing vacuoles (SCV) inhibits the plasmid expression in mammalian cells. Understanding the mobility of the delivered plasmid in the cancer cell through in situ hybridization could provide insights critical for the successful development of salmonella-mediated gene transfer therapy for treating cancer.