Study On Targeting Oncolytic Adenovirus With E1b Gene Deletion And Mda-7/il-24 For Hepatocellular Carcinoma Therapy

Cancer is one of the diseases that seriously threat human's life and health. Liver cancer is one of the most commonly occurred malignant tumors. There are many hepatitis b patients in China .That induced the highest incidence of carcinoma happened in our country. Liver cancer (hepatocellular carcinoma, HCC), known as "The king of cancer", because of its high incidence, high mortality ,difficultly diagnosis and easily recurrence. Nearly 55% of global HCC patients and 45% of total death number happened in China. Thus the research of tumor therapy is very necessary and pressing.Tumor including HCC are induced by gene disorder ,so Gene Therapy be general considered as the hope of tumor therapy. The research of Targeting Gene Therapy is especially significative. The choice of vector is most important in Gene Therapy. At present, virus vector is the first choice in tumor Gene Therapy. virus vector has many merits such as high transfection, gene prolong expression and so on.Modified oncolytic adenoviruses are one of the most promising vectors for Gene Therapy. Since the E1A is the key protein of adenoviruses, we used alpha-fetoprotein promoter(AFP) which high-specific expression in HCC cells to instead of E1A promoter.Ad-enAFP- E1A was constructed . E1B is compsosed with 55kDa and 19kDa proteins. 55kDa deletion could target tumor cells which has P53 pathway mutation , and selectively inhibit nuclear export. The adenoviral protein E1B-19 kDa is a Bcl-2 homologue. Current reports showed that 19kDa deletion could active many signal pathways to promote celluar apoptosis and enhance viral spread in tumor tissues. Double-deletion oncolytic adenovirus E1B 19 kDa and 55 kDa , which could not only enhance virus targeting, but also improve the activity of apoptosis. This constructed method is a new way to modify oncolytic adenovirus. Ad.enAFP-E1A- _△E1B was successfully constructed. This HCC-specific oncolytic adenovirus has tri-targeting , which make adenovirus replicate in the HCC cells and not in nomal cells.Targeting Gene-Virotherapy was applicated by Prof. Liu initially. Than is Dual Targeting Gene-Virotherapy . In these therapies, a good adenovirus vector should take along with a therapeutic gene. Combined virus selective replication in tumor cells with anti-tumor activity of gene, we can achieve the aim of virus tumor-specific proliferation and therapeutic gene antitumor effect .Mda-7/IL-24 (melanoma differentiation associated gene-7/interleukin-24 )be chosen as therapeutic gene for the new constructed adenovirus vector in this study . IL-24 is a novel multi-functional cytokine, which induces apoptosis in tumor cells .Since IL-24 has strong antitumor activity and high selectivity in normal cells, it is considered as the"star"of tumor therapeutic gene and broadly applied in Gene Therapy of cancer.In this research, IL-24 was inserted in the new constructed adenovirus vector Ad.enAFP-E1A-_△E1B, which named Ad.enAFP-E1A-_△E1B-IL-24. Vitro experiment showed that Ad.enAFP-E1A-_△E1B-IL-24 could induce apoptosis in HCC cells but not in normal cells. The safety and antitumor effectivity of objective adenovirus is excellent . Vivo experiment in an animal model of hepatoma carcinoma Huh-7 xenograft proved that Ad.enAFP-E1A-_△E1B-IL-24 can near complete inhibit (although not elimination) the Huh-7 liver carcinoma growth and the virus has potential spread activity.Both can drama- tically improved the survival rate of animals. In exploring the mechanism of Ad.enAFP- E1A-_△E1B-IL-24 in tumor cells, it was found that Ad.enAFP-E1A-_△E1B-IL-24 induced apoptosis by both death receptor pathway and mitochondrion / cytochrome C pathway.The study preliminarily practised multi-Targeting Gene-Virotherapy strategy, successfully constructed the tumor-specific oncolytic adenovirus vector Ad.enAFP-E1A-_△E1B-IL-24, and set up a solid foundation for hepatoma- specific tumor-targeted clinical therapy of HCC. Both the apoptosis of tumor cells and virus spread activity were actived by E1B 19kDa. All the fingings provided new thoughts for viral modification and clinical application with Targeting Gene-Virotherapy.