Study On Introduction Of Oncolytic Adenovirus With IL-24 Gene Into Acute Myeloid Leukeima Stem Cell Through CD123

Leukemia is one of common malignant diseases of the hematopoietic system that is severe harm to human health. Chemotherapy and bone marrow transplantation are the primary therapeutic means of leukemia, but most of patients could only obtain short-term remission and eventually died of relapse or drug resistance. The lastest research have shown that, there is a group of cell with very low percentage in leukemia patients called leukemia stem cells(LSCs),which is similar with normal hematopoietic stem cells (HSCs) 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, only eradicating LSCs is possible to completely cure leukemia.CD123, which is widely reported to be overexpressed on AML blasts, CD34+ leukemic progenitors, and AML-LSCs in comparison with normal HSCs, seems to be an excellent target for the therapy of leukemia.In this study, the oncolytic adenovirus Ad.hCMV-sCAR-IL-3-sp-E1A-△E 1B-hCMV-IL-24 was constructed,in which contains three important regulatory elements: bifunction-fused gene sCAR-IL-3, survivin promoter and IL-24 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, utilization of bifuction-fused protein sCAR-IL-3 as a bridge to mediate adenovirus into leukemia stem cells effectively.There are three new ideas of this study:(1) Transformate the target of the traditional adenovirus type 5: The expression of bifuction-fused gene sCAR-IL-3 could provide a bridge for adenovirus knob and CD123 on AML stem cells surface, which could mediate adenovirus into AML stem cells effectively. (2) With the characteristics of high-specific expression in leukemia stem cells of survivin, utilization of survivin promoter to control the E1A gene (necessary for viral replication and proliferation) can make adenovirus replicate in the survivin-positive leukemia stem cells and not in survivin-negative nomal cells. (3) IL-24 is an antitumor gene, which has a potential "bystander effect". It can inhibit the proliferation of tumor cells and induce apoptosis efficiently, but had no effect on normal cells.Main methods: (1) Construction of the shuttle vector that contained two exogenous gene expression cassettes(hCMV-sCAR-IL-3 and hCMV-IL-24) for adenovirus package (2) The oncolytic adenovirus were packaged in HEK293 cells by using homologous recombinant method (3) Identification 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 apoptosis staining assay and western blot.The results showed that the targeting AML-LSC oncolytic adenovirus Ad.hCMV-sCAR-IL-3-sp-E1A-△E 1B-hCMV-IL-24 was successfully constructed and it could target AML-LSCs specifically, and induce AML-LSCs apoptosis effectively in vitro assay, but had no effect on HSCs. This study is a new idea to cure AML patients through targeting CD123. Our results suggest that a targeted oncolytic adenovirus might be a valuable agent for AML patients.