5' sequence of HMGB2 gene as a tumor specific sequence for transcriptional targeting of glioblastomas

Background: Glioblastoma multiforme (GBM) is a highly invasive brain tumor and one of the most lethal forms of human cancer (1). Present treatments for GBM, such as surgery, gamma-irradiation, and chemotherapy are ineffective in eradicating the cancer, evidenced by poor prognosis for glioma patients with a mean survival time of less than 1 year after diagnosis. Hence, there is an urgent need to improve the efficacy of therapies for this fatal disease. Method: Identification of the specific sequence by cDNA microarray followed by confirmation using real-time PCR. Cloning of sequence into a baculoviral vector to drive the expression of suicide gene thmidine kinase.Demonstration of in vitro efficacy using cell death assays in presence of ganciclovir and establishment of proof of concept using a mouse xenograft model for gliomas in vivo. Results: HMGB2 was identified as one of the genes having low expression level in normal human astrocytes, but was significantly up-regulated in U251 glioblastoma cells by cDNA microarray analysis. Real-time PCR quantification confirmed 11 to 79 fold increase in HMBG2 expression in glioblastoma tissues vs normal human brain tissue. We cloned a 2kb 5' upstream region of the HMGB2 gene. By progressive truncation of the 2kb fragment, we identified a 500bp fragment displaying very high transcriptional activity in glioblastoma cells, but a low activity in normal brain cells (primary neurons and normal human astrocytes). Baculoviral constructs with 500bp sequence driving the expression of the HSVtk gene were lethal to glioblastoma cells(cell survival618% after 48Hrs) in the presence of ganciclovir, whereas normal human astrocytes and neurons were not affected. We further confirmed the efficacy of the baculoviral vector in suppressing the growth of human Glioblastoma cells after intra-tumor injection in a mouse xenograft model. Conclusion: We have demonstrated the identification of a novel 5'-upstream sequence of the HMGB2 gene which has a potential to be used as an efficient, tumor-selective promoter in targeted glioblastoma gene therapy.