Construction Of IL-18 Expressing Cancer-Specific Replication-Competent Adenoviruses And In Vitro Cytotoxic Effect In Hepatocellular Carcinoma Cells

Replication-selective virotherapy holds great promise for the treatment of cancer. Appealing features include tumor-selective targeting, viral self-spreading in cancer cells, and no cross-resistance to current treatments. Several types of conditionally replicative viruses have already been tested in clinical trials, including conditionally replicative adenovirus (CRAD), herpes simplex virus, vaccinia virus, reovirus and Newcastle disease virus. Interleukin18 ( IL-18) is an important cytokine recently discovered. It has been found to play an important role in tumor immunology. But it is not reported that IL-18 treats liver cancer up to now.In order to cure liver cancer, we further combined gene therapy with viral therapy and constructed a brand-new gene-viral therapeutic system CNHK600-IL18 carrying human interferon-18 gene. The ability of CNHK600-IL18 selectively killing tumor cells will be assessed according to the in vitro experiments.1. Construction of replication-selective adenovirus pSG600-IL18. The vector T-IL18 was digested with the endonuclease SalⅠand BamHⅠgenerated the DNA fragment 623bp.Then ligating the 623bp with the pClone25 which was digested by BamHⅠand SalⅠ,and generated pClone25-IL18. The vector pClone25-IL18 was digested with the endonucleases AgeⅠ,NotⅠand EcoO109Ⅰand obtained fragment 2570bp. Then ligating the fragment 2570bp with pSG502-ΔCR2 digested by AgeⅠand NotⅠproduced pSG600-IL18. The plasmid pSG600-IL18 was co-transfected with ppE3 into 293 cells to produce the new recombinant adenovirus CNHK600-IL18 by homologous recombination. SG600-IL18 adenovirus was propagated in 293 cells and purified by cesium chloride gradient centrifugation. The viral titer was 6.9×109 pfu/ml determined by TCID50 method.2. Selective replication and oncolysis of CNHK600-IL18 in vitro. (1)ELISA: Bioactivity of IL-18 generated by CNHK600-IL-18 was assayed according to IFN-γsecretion by T cells followed incubation with CNHK600-IL-18 infected BJ culture supernatant. (2)Replication Assay: Different kinds of tumor cells were infected with CNHK600-IL18 to observe the ability of replication at 0h, 48h and 96h. The results manifested that CNHK600-IL18 could proliferate largely in most liver cancer cells, especially in BEL-7404 and SMMC-7721, the CNHK600-IL18 proliferated 31658 and 125396 folds, respectively ,at 96h. which were 400 and 1787 folds as much as in BJ cell line. But it proliferated few in normal cells, which demonstrated it was very safe. (3)MTT Assay: MTT assay was applied to evaluate the killing efficacy of pSG600 and CNHK600-IL-18 at different MOI.We successfully constructed an efficient replication-selective adenovirus CNHK600-IL18, which selectively replicated in and lysed tumor cells. Moreover, CNHK600-IL18 was superior to ONYX015 in the selective replication in tumor cells. Collectively, we provided a new strategy for liver cancer biotherapy through the application of CNHK600-IL18.