Treatment > Gene Therapy

Meeting Abstract

New ribozymes targeting vegf production to inhibit neoangiogenic potential of brain tumor cell lines

Ciafré SA, Wannenes F and Farace MG

Glioblastoma multiforme is one of the most highly vascularized solid neoplasms, with the amount of neovasculature closely correlated with the degree of malignancy. 
Among angiogenic factors, vascular endothelial growth factor (VEGF) plays an important role in the neovascula rization and growth of human cancers, and particularly in gliomas, where up-regulation of VEGF is a common event. 
Thus, strategies to downregulate VEGF could be applied to reduce the preformed tumor vasculature in established gliomas. 
We have designed hammerhead ribozymes against VEGF mRNA, with the aim of strongly down-regulating expression and secretion of VEGF by glioma cells and inhibiting their proangiogenic potential. 
Our experimental strategy can be summarized into the following steps: 
a) Design of two new ribozymes targeting human VEGF mRNA specifically cutting its sequence around 5' end, thus yielding short pieces of mRNA completely unavailable to the translational machinery. 
b) Cloning of the designed ribozymes into a short region of Adenovirus type 2 (Ad2), VAI, owning several features making it an optimal carrier for efficient and specific delivery of ribozymes into the cytoplasm: VAI is transcribed by the RNA Polymerase III, which drives high levels of expression, its secondary and tertiary structure confers high stability to the transcribed molecule, and VAI is specifically sorted into the cytoplasm, where the activity of the ribozyme is required. 
c) Stable transfection of a human glioma cell line, U87, with plasmids harboring the anti-VEGF ribozyme-VAI "hybrid" molecules, and selection of single cell clones. 
d) ELISA measurement of VEGF secretion from transfected glioma cells. 
With this assay we have demonstrated a significant reduction (about 50% of wild type concentration) of VEGF secretion in at least two transfected clones. 
We are now looking at VEGF mRNA expression in correlation with ribozyme transcription, and we will then proceed to functional tests to assess the relative efficiency of the supernatants of transfected cell lines in the induction of the proliferation and/or migration of endothelial cells. 
These in vitro data are preliminary to further in vivo experiments, where we will study growth and vascularization of subcutaneously injected tumors in SCID mice, and of experimentally induced intracranial gliomas in rats.