The Study On TRAIL/Mn-SOD Gene Armed Oncolytic Adenovirus Targeting Leukemia Stem Cells Inhibition Of Leukemia

Leukemia is a kind of common hematopoietic system malignant proliferation and clonal diseases, which serious harm to human health.Chemotherapy and bone marrow transplantation are the dominated therapeutic means of leukemia,60%~80%of patients by combined treatment can obtain complete remission,Unluckly,most of patients could only obtain short-term remission and eventually died of relapse.Numerous studies show that there is a group of cell with very low percentage in leukemia patients called leukemia stemcells(LSCs),which is similar with normal hematopoietic stem cells (HSCs),not only having unlimited proliferation and self-renewal capacity and playing an important role in the development and progression of leukemia.Currently, LSCs is considered as the major source of leukemia relapse and drug resistance.Therefore, targeting Clear LSCs can be expected to eradicate leukemia.Many studies showed that, CD123, IL-3receptor alpha chain that expression in acute myeloid leukemia stem cell surface, whereas in the hematopoietic stem cell surface almost not expressed, this subject based on the above characteristics.We designed and constructed the oncolytic adenovirus Ad.-sCAR-IL-3-TRAIL，Ad.-sCAR-IL-3-MnSOD in whichcontains three important regulatory elements: fused gene sCAR-IL-3, survivin promoter and TRAIL/MnSOD gene. The oncolytic adenovirus can easily eradicate LSCs when the three elements take functions together.This study is base on the strategy of Targeting Gene-Virotherapy of Cancer,still withoncolytic adenovirus as Gene carrier,by changing the adenovirus leukemia cell affinity toachieve the targeted therapy of leukemia stem cells. There are three new ideas of thissubject.(1) Fused gene sCAR-IL-3could provide a bridge for adenovirus knob and CD123on AML stem cells surface，which could mediate adenovirus into AML stem cellseffectively.(2) Utilization of survivin promoterto control the E1A gene (necessary for viral replication and proliferation) which can make adenovirus replicate in the survivin-positive leukemia stem cells and not in survivin-negative nomal cells,improved the oncolytic adenovirus targeting property and safety.(3) Tumor-suppressor genes TRAIL/MnSOD caninhibit a variety of tumor cell growth and induce its apoptosis, but has no effect on normal cells. Main methods:(1) Construction of the shuttle vector that contained two exogenous gene expression cassettes(hCMV-sCAR-IL-3and hCMV-TRAILor hCMV-MnSOD) for adenovirus package.(2) The oncolytic adenovirus were packaged in HEK293cells by using homologous recombinant method.(3) Identification,amplification and purification of the recombinant virus.(4) The tumor-killing effects and the safety evaluation were proved through the cell viability assay in vitro.(5) Analyzation of the apoptosis way of AML-LSCs through cell staining, flow cytometry for detection of apoptosis and protein leveldetection.The experimental results show that successfully constructed targeted AML stem cellsoluble tumor adenovirus Ad.-sCAR-IL-3-TRAIL, Ad.-sCAR-IL-3-MnSOD. In vitro cellexperiments show that Ad.-sCAR-IL-3-TRAIL, Ad.-sCAR-IL-3-MnSOD to specific targetAML stem cells, and can significantly inhibit the proliferation and induce AML stem cells apoptosis, and has higher security.This study used oncolytic adenovirus targeting AML stem cells has opened a new way for the future treatment of leukemia, and provided the experimental basis for Targeting Gene-Virotherapy of leukemia.