Study On Targeting Oncolytic Poxvirus With IL-24and Smac Dual-gene For Cancer Therapy

Targeting Gene-Viro Therapy is a new strategy for cancer treatment. The oncolytic virus can be specifically targeted tumor cells and their proliferation has highly increased in a hundred times. Meanwhile,the cancer genes inserted in oncolytic virus is correspondingly increased in hundred times,so the tumor cell killing effect is improved.Oncolynic poxvirus can be used as an ideal vector in therapeutic gene therapy because of its following characteristics, such as high safety, large genome capacity, rapid replicate ability and injected intravenously and so on.IL-24is a novel cytokine,which can induces caspase activation or apoptosis of cancer cells. It became a star in the field of gene therapy and the ING241(Ad-IL-24) is currently in clinical2. Smac gene is an apoptosis-inducing factor, which can be released from mitochondria with cytochrome C,when cells are stimulated. It can be combined to IAP family,and then induced cancer cell apoptosis and inhibitd cell growth.Besides, IL-24and Smac are the important candidate genes for the targeting multi-genes therapy of cancer. In the construction of IL-24and Smac dual gene expression vector, a stable and efficient linker is critical. We use three different linkers such as IETD, EEED and F2A to obtain three eukaryotic expression plasmids which are pcDNA3.1(+)-IL-24-IETD-Smac, pcDNA3.](+)-IL-24-EEED-Smac,pcDNA3.1(+)-IL-24-F2A-Smac, and then combined with5-fluorouracil (5-FU) treatment to study the cleavage efficiency of the three linker peptides. The results showed that among three IL-24-linker-Smac dual gene expression vectors, the F2A linker is superior to IETD and EEED linkers. Based on the above results, in this paper, IL-24and Smac gene mediated by F2A linker peptide were inserted into the vaccinia virus vector to construct dual-gene oncolytic vaccinia virus,such as Oncopox-IL-24-F2A-SMAC and Oncopox-SMAC-F2A-IL-24.The expression of target protein is normal by Western Blot detection. The preliminary test results show that dual-gene oncolytic vaccinia virus which are Oncopox IL-24-F2A-SMAC and Oncopox-SMAC-F2A-IL-24have better tumor killing effect than the single-gene oncolytic vaccinia virus by MTT assay in the cellular level. This also provide a reference for application of dual-gene vaccinia virus vector in cancer treatment therapy.