Therapeutic Antitumor Effects Of Semliki Forest Virus-based DNA Vector Carrying Suicide Genes Via Salmonella

Suicide gene therapies is hot issue for killing tumor cells. The Escherichia coli purine nucleoside phosphorylase/2-fluoro-2-deoxyadenosine (ePNP/F-dAdo) suicide system has demonstrated a powerful killing on tumor cells. However, several drawbacks to this approache remain to be resolved, such as:(1) How to improve the efficiency of suicide gene,(2) how to stimulate the immune system to produce obvious antitumor effect in vivo, and (3) how to improve the specific effects for tumor therapy. Studies have shown that protein expression based on the alphavirus replicase complex is several times greater than the protein expression driven by the conventional CMV promoter. Because attenuated Salmonella typhimurium SL7207exhibits strong invasiveness and can target a variety of solid tumors remotely, it could be used as a carrier to introduce Eukaryotic expression plasmids into the tumor cells. The heat shock protein65of Mycobacterium tuberculosis (mHsp65) could be used due to its strong ability to act as a molecular chaperone which can carry mixtures or fusion peptides into the APCs. Therefore, tumor-specific CTL responses could be activated by MHC-I classes, as strong immune responses against tumors were apparent for a long time in vivo.In the part1, a Semliki Forest virus-based DNA vector PSC-ePNP-mHsp65with a high level expression of suicide/ePNP and immunomodulatory heat shock protein65of Mycobacterium tuberculosis (mHsp65) genes was constructed. Live attenuated Salmonella typhimurium 7207(SL7207) was used initially as a vehicle to targetly translocate the large alpha-virus vector into tumor cells for cancer treament. The in vitro expression efficiency was measured by GFP quantification, and the enzymatic conversion of F-Ado into F-Ade was tested by HPLC. The killing effect induced by SL/PSC-ePNP-mHsp65infection were detected by cytotoxicity assay, in combination with the ePNP precursor drug F-Ado administration. Following oral administration, recombinant bacteria allocated within the solid tumor. The live recombinant bacteria vaccine plus F-Ado were found to significantly induce obvious immunity, produce therapeutic effects against tumor growth, and prolong the survival time of tumor-bearing mice.In order to improve the specific effects of our recombinant bacteria vaccine, in the part2, a human telomerase reverse transcriptase promoter-driven Semliki Forest virus-based DNA vector (pShT-ePNP) with high expression of the ePNP gene was constructed. Live attenuated Salmonella typhimurium7207(SL7207) was used as a vehicle to targetly transfer the large alpha-virus vector into tumor cells. The targeted expressions of ePNP gene in tumor cells were also detected by RT-PCR. After F-dAdo addition, the enzymatic conversion of F-Ado into2-fluoroadmine (F-Ade) was tested by HPLC. Cell cytotoxicity assays showed that the obvious inhibitory effect of SL/pShT-ePNP system on tumor cells was dose and time-dependent. Following oral administration, recombinant bacteria SL/pShT-ePNP and F-dAdo were also found to exert powerful therapeutic effects in combination against tumor growth and for prolonging the life-span of tumor-bearing mice.