Construction of an adenoviral vector encoding Ad5GM-CSF-IL-2 in human bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2013.14.006

Abstract

Abstract:

BACKGROUND: A problem needed to be solved is how to deliver activating factors for dendritic cells and T cells into the tumors. Owing to the characteristics of tumor tropism and weak immunogenicity, human bone marrow mensenchymal stem cells are used as a vehicle for transferring the activating factor genes of the dendritic cells and T cells to the tumor, which may be a protocol to solve the problem.
OBJECTIVE: To construct a type 5 adenoviral (Ad) vector encoding granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-2 (IL-2) genes, and detect the expression levels and duration of GM-CSF and IL-2 after infection of human bone marrow mesenchymal stem cells, providing experimental evidence for in vivo activation of dendritic cells and T cells.
METHODS: GM-CSF and IL-2 cDNAs from the total RNA extracted by human peripheral blood mononuclear cells were cloned by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.1/Myc-His(-)B. GM-CSF and IL-2 cDNAs linked by the internal ribosomal entry sites (IRES) from encephalomyocarditis virus were subcloned into the shuttle vector pDC515 for the construction of pDC515 GM-CSF-IRES-IL-2. By using AdMax^TM adenovirus vector system, the shuttle vector pDC515 GM-CSF-IRES-IL-2 and the backbone vector pBGHfrt△E1,3 FLP were cotransfected into 293 cells, and Ad5 GM-CSF-IL-2 was obtained by FLP recombinase-mediated site-specific recombination. The amounts of GM-CSF and IL-2 in the culture supernatants at different time points were measured by enzyme-linked immunosorbent assay after human bone marrow mesenchymal stem cells were infected by Ad5 GM-CSF-IL-2 and irradiated with γ-ray to lose proliferative activity.
RESULTS AND CONCLUSION: The sequences of GM-CSF and IL-2 cDNAs were identical with those provided by GenBank NM_000758 (435 bp) and GenBank NM_000586 (462 bp) by sequencing, respectively. AdMax^TM was an efficient and quick packaging system of adenoviral vectors. The GM-CSF and IL-2 linked by IRES in the adenovirus can efficiently be expressed in the human bone marrow mesenchymal stem cells at a high level for 7 days, suggesting a potential application to the tumor immunotherapy taking human bone marrow mesenchymal stem cells as a carrier expressing the activating factors for dendritic cells and T cells.

Key words: stem cells, bone marrow-derived stem cells, dendritic cells, granulocyte-macrophage colony stimulating factor, interleukin-2, bone marrow mesenchymal stem cells, adenoviral vector, tumor, gene therapy, National Natural Science Foundation of China, stem cell photographs-containing paper

CLC Number:

R394.2

Zhang Yu-ping, Zhang Xuan, Guo Zhi, Tan Xiao-hua. Construction of an adenoviral vector encoding Ad5GM-CSF-IL-2 in human bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(14): 2509-2516.

Figures/Tables 3

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