Construction Of An Adenoviral Vector Encoding Human GM-CSF And IL-2and Its Expression Of In Human Bone Marrow Mesenchymal Stem Cells

BACKGROUND:Dendritic cell （DC） vaccine as one of the currently most strategies for cancer immunotherapy is commonly used to induce tumor-specific T cell response in which in situ activation of DC and T cells in-the location of tumors is an important direction in the field. A problem needed to be solved is how to delivery of activating factors for DC and T cells into the tumors. The human bone marrow mesenchymal stem cells （hBMSCs） have characteristics of tumor tropism and weak immunogenicity. Using hBMSCs as a vehicle for the activating factor genes transfer to and the local expression in the tumor may be a protocol to solve the problem.OBJECTIVE:To construct a type5adenoviral （Ad） vector encoding GM-CSF and IL-2（IL-2） genes, and detect expression levels and duration of GM-CSF and IL-2after the infection of hBMSCs, providing experimental evidence for in vivo activation of DC and T cells.METHODS:The experiments were conducted at the Laboratory of Biotherapy Center of Beijing General Hospital of PLA from October2011to October2012.GM-CSF and IL-2cDNAs from the total RNA extracted by human peripheral blood mononuclear cell （PBMCs） were cloned by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.1/Myc-His（-）B. GM-CSF and IL-2cDNAs linked by the internal ribosomal entry sites （IRES） from encephalomyocarditis virus were subcloned into the shuttle vector pDC515for the construction of pDC515GM-CSF-IRES-IL-2. By using AdMaxTM adenovirus vector system, the shuttle vector pDC515GM-CSF-IRES-IL-2and the backbone vector pBGHfrtΔE1,3FLP were cotransfected into293cells, and Ad5GM-CSF-IL-2was obtained by FLP recombinase-mediated site-specific recombination. The amounts of GM-CSF and IL-2in the culture supernatants at different time points were measured by enzyme-linked immunosorbent （ELISA） assay after hBMSCs infected by Ad5GM-CSF-IL-2and irradiated with γ-ray to lose proliferative activity.RESULTS:The sequences of GM-CSF and IL-2cDNAs were identical with those provided by GenBank NM₀00758（435bp） and GenBank NM₀00586（462bp） by sequencing, respectively. The formation of plagues began10days after the cotransfection of pDC515GM-CSF-IL-2and PBGHfrtΔE1,3FLP into293cells. PCR verified there existed the GM-CSF and IL-2fragments in the lysates. After the infection of hBMSCs with Ad5GM-CSF-IL-2at the MOIs of100pfu per cell, the analysis of GM-CSF and IL-2by ELISA showed that the production of GM-CSF and IL-2began at12h, reached to the peak between48～96h, and kept at least for7days post-infection.CONCLUSION:AdMaxTM was an efficient and quick packaging system of adenoviral vectors. The GM-CSF and IL-2linked by IRES in the adenovirus can efficiently be expressed in the hBMSCs on a high level for7days, suggesting a potential application to the tumor immunotherapy by hBMSCs as a carrier expressing the activating factors for DC and T cells.