TRIPLE ANTI ANGIOGENIC THERAPY FOR BRIAN TUMORS

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The growth of solid tumors beyond 3 mm critically requires neovascularization. Tumor cells play an active role in this process by producing angiogenic factors. Among these molecules the vascular endothelial growth factor (VEGF) plays a pivotal role in the growth of gliomas. Thus, expression of VEGF in these tumors is highly up- regulated. Moreover, transfection of VEGF to rat glioma cells is followed by hypervascularized tumors with abnormally large vessels, and abrupt withdrawal of VEGF resulted in regression of the preformed tumor vessels. In addition, transfer of antisense VEGF to glioma cells inhibits tumorigenicity. These observations indicate that targeting VEGF represents an accurate strategy for the development of new treatments for gliomas. In this proposal, we aim to develop a viral system to target VEGF in gliomas combining three different strategies. Specifically, in Aim 1 we propose to construct three adenoviral vectors able to express different combinations of an anti-sense sequence of the VEGF gene, the Angiopoietin2 (ANG2), and the MMAC1 genes. In a first step, we will characterize the effect of MMAC1 on the regulation of angiogenesis and the expression of the VEGF message. In a second step, we will construct the multiple-gene adenoviral vector. Looking for a tumor-selective expression of the exogenous genes, in these construct, the antisense- VEGF cDNA will be driven by the VEGF promoter, and the MMAC1 and ANG2 genes by the E2F-1 promoter. Both promoters, VEGF and E2F-1, are thought to be highly selective for tumor cells. A series of in vitro experiments will test the ability of these constructs to express properly the ectopic messages. In Specific Aim 2, we will determine the anti-glioma effect of these constructs in vivo by using subcutaneous and orthotopic models of human gliomas in nude mice. The animal model that we propose involve the use if heterotransplants. Thus, we will examine the effect of the adenoviral construct in gliomas that will be transplanted directly from the patient to the mice. This system may be more reliable than the xenograft model since the vessels of the transport will be of human origin, at least in the first passage. In summary, in this proposal, we will assess the anti-angiogenesis potential and possible synergism of the anti-sense VEGF, MMAC1 and ANG2; the effect of these molecules will be directed to tumor cells by using two tumor-selective promoters, furthermore we will challenge the anti-angiogenesis efficiency of this system in a heterotransplant model that may be more reliable than the current xenograft model.