Study On A New Oncolytic Adenovirus For The Targeting Therapy To Malignancies

Background: Oncolytic adenovirus (also known as tumor specific replicating adenovirus or conditionally replicating adenovirus), based on the differences between tumor cells and normal tissue in cell structure and metabolic pathways, can target tumor cells and produce new infectious viral particles, and then eliminate the tumor cells in the end. At the same time,the expression of a therapeutic gene within a replication– competent adenovirus vector could carry between and express therapeutic genes in the tumor cells which will be treated by two efficient means. Adenovirus-infected cell types are mainly decided by the fibers on the viral capsid.Now human adenovirus type 5(Ad5) has been widely used as gene therapy vector. It has the merits of small size, great diffusion capacity and low pathogenicity. However, under the help of Coxsakie and Adenovirus Receptor (CAR) on the surface of the cells, Ad5 could interact with the cytomembrane and penetrates it, that's why Ad5 also has the demerit of low infection efficiency. But the viruses will be limited when they get in touch with the cells which have not much CAR receptor. Human adenovirus serotype 11(Ad11) can infect cells independent of CAR receptor, stem cells and primary tumor cells, so a constructed chimeric adenovirus Ad5/F11 can effectively transfer many important target cells with inadequate CAR expression, especially have a higher infection efficiency in tumor cells, hematopoietic stem cells, mesenchymal stem cells and so on.RNA interference (RNAi), also known as post-transcriptional gene silencing, can be triggered by small interfering RNAs (siRNAs) that inhibit the expression of the target gene in an effective and specific way to mediate sequence-specific mRNA degradation and induce cells to show the phenotype of the specific gene silencing. It is an important protective mechanism against external infection in vivo. Since RNAi was found, it has been widely used in the research of systematic functional genomics, virus inhibition and oncotherapy, which shows a bright application prospect.Bmi-1(B-cell-specific Moloney murine leukemia virus insertion site 1), a polycomb gene family member, was found in 1991. It is not only an oncogene that plays an important role in cell cycle regulation, cell immortalization and cell senescence, but also a necessary factor that involves in the regulation of self-renewal and differentiation of stem cells. Many studies have shown that aberrant Bmi-1 expression is associated with the initiation and progression of many cancers; therefore, it is expected to become a new tumor molecular marker.Aim: In order to realize success in the therapy of leukemia and study the function of Bmi-1 gene in the pathogenesis of leukemia, we use RNAi technology to construct the chimeric oncolytic adenovirus virus vector carrying shRNA, which can have an efficient and long expression of siRNAs, so as to down-regulate Bmi-1 expression. In addition, the chimeric oncolytic adenovirus has the advantage of gene-virotherapy, all of which will give support to the leukemia therapy.Methods: We designed a shRNA to interfere with the expression of Bmi-1 gene. First, we cloned the shRNA into a shuttle plasmid, and then recombined it with pCN103-11fiber, so we obtain the chimeric oncolytic adenovirus AdCN205-F11-Bmi-1shRNA, subsequently; we packaged it in HEK293 cells. After identification, purification and amplification of the virus, we did some experiments to assess whether adenovirus together with RNAi can inhibit the proliferation of the leukemia cells or the expression of Bmi-1 protein.Results: After the recombination of shuttle plasmid and pCN103-11fiber in E.coli BJ5183, and the transfection of it in HEK293 cells, we obtain the chimeric oncolytic adenovirus which can express shRNA and splice into effective siRNAs. The results showed that AdCN205-F11-Bmi-1-shRNA can express shRNA and produce siRNAs as well. Western Blot result revealed that the virus was able to inhibit the expression of the target gene Bmi-1 in leukemia cells. Besides,MTT results also showed that the proliferation of the leukemia cells was suppressed effectively by the virus. Conclusions: According to the results above, we concluded that the chimeric oncolytic adenovirus AdCN205-F11-Bmi-1shRNA we constructed can inhibit the expression of Bmi-1 as well as the proliferation of the leukemia cells, which will give hope to the therapy of leukemia.