A NOVEL-APPROACH TO GLIOMA GENE-THERAPY - DOWN-REGULATION OF THE VASCULAR ENDOTHELIAL GROWTH-FACTOR IN GLIOMA-CELLS USING RIBOZYMES

Glioblastoma multiforme is one of the most highly vascularized solid n eoplasms, therefore treatments that target neovascularization process would be of great clinical importance. Studies of glioblastoma angioge nesis have revealed that expression of the vascular endothelial growth factor (VEGF) is up-regulated in these tumors. Previous reports have shown that down-regulation of VEGF correlates with modification in the glioma growth. To examine this phenomenon further, in this study we c onstructed two hammerhead ribozymes (RZI and RZII) to target the 5' co mmon region of VEGF mRNA. Both ribozymes exhibited site-specific cleav age to a 318-nucleotide VEGF transcript and showed a high digestion ef ficiency in vitro (65-95%). After the transfection of glioma cells wit h two expression vectors carrying the ribozyme sequence, Northern blot analyses detected high levels of ribozyme expression. Treatment of th e glioma cells with the ribozymes resulted in a reduction in VEGF mRNA in six of eight clones. Furthermore, the anti-VEGF effect was confirm ed at protein level. Thus, enzyme-linked immunoabsorbent analyses (ELI SA) showed a >70% reduction in the VEGF(165) expression level. These r esults indicate that hammerhead ribozymes may be useful in downregulat ing VEGF expression and suggest that anti-VEGF strategies may be used to potentiate other gene therapies targeting tumor suppressor genes.