Our proof of concept showing successful shRNA knockdown of the target gene through the lipofectamine assay with HeLa cells shows that our shRNA knockdown design would work when implemented within cancer cells. The HeLa cells are an immortal cervical cell line, so this cell line represents tumor cells in patients. The success of the RNA interference mechanism as shown by our proof of concept results illustrate that this mechanism will also be effective once the shRNA is delivered into cancer cells of a patient. We are still working on the delivery mechanism for the shRNA. Our plan is to use an invasin-listeriolysin O circuit to have the bacteria invade cancer cells after the bacteria is orally administered and then naturally congregates in tumors due to the anaerobic and immunosuppressed environment of tumors. We will make invasion specific by putting this invasion circuit under the control of a quorum sensing promoter, since bacteria would only reach this quorum level in extremely anaerobic environments like tumor cells. Since the gut also has an extremely anaerobic environment in which quorum will be reached, the next step in our plan is to incorporate a nitric-oxide promoter in conjunction with the quorum sensing promoter to increase specificity of invasion and prevent invasion in non-cancer sites in the anaerobic gut. The nitric oxide promoter will increase specificity because high concentrations of nitric oxide is only present in areas of inflammation. With this dual promoter system, the invasion circuit will only be activated in inflamed areas that are also very anaerobic (only tumor cells, not the gut).
