[Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase genes expressed in vascular endothelial cells].

BACKGROUND & OBJECTIVE: Blocking tumor angiogenesis is a promising tumoricidal approach. Herpes simplex virus-thymidine kinase (HSV-tk) suicide gene could effectively damage neoplastic vascular endothelial cells. However, the killing effect of currently used HSV-tk system is non-specific due to employing cytomegalovirus (CMV) as its promoter. Kinase domain insert containing receptor (KDR), a kind of vascular endothelial growth factor receptor, is highly expressed on tumor vascular endothelial cells, but lowly expressed on normal tissues. This study was to construct a recombinant adenovirus containing KDR promoter-mediated HSV-tk suicide gene, and analyze its specific expression on vascular endothelial cells.

METHODS: KDR-tk fragment was subcloned into corresponding restriction sites of vector with directional cloning method using pAdeasy system to construct recombinant adenoviral plasmid containing KDR promoter-controlled HSV-tk gene (AdKDR-tk). After packaged and amplified in 293 cells, the virus was used to infect KDR-expressed human umbilical venous endothelial cells (HUVECs) and KDR-unexpressed liver cancer cell line HepG2. After administration of ganciclovir (GCV), proliferation of gene-transfected HUVECs and HepG2 cells was evaluated by MTT assay.

RESULTS: According to the results of restriction digestion, reverse transcription-polymerase chain reaction (RT-PCR) and PCR, the length, position and orientation of inserted genes were correct. The titer of the recombinant adenovirus was 1 x 10 (10) pfu/ml. Under infection index of 100, with the concentration of GCV increased from 0 to 50 microg/ml, the survival rates of AdKDR-tk-transfected HUVECs and HepG2 cells decreased from 100% to (28.94+/-5.67)% and (75.45+/-2.91)%, respectively (P<0.01).

CONCLUSION: Adenovirus-mediated transfection of KDR promoter-HSV-tk gene could direct endothelial specific gene expression.