TRIPLE ANTI ANGIOGENIC THERAPY FOR BRIAN TUMORS

超过3mm的实体瘤的生长迫切需要新生血管。肿瘤细胞通过产生血管生成因子在这一过程中发挥积极作用。在这些分子中，血管内皮生长因子（VEGF）在胶质瘤的生长中起着关键作用。因此，VEGF在这些肿瘤中的表达是高度上调的。此外，将VEGF转染到大鼠胶质瘤细胞后，会出现血管异常大的高血管化肿瘤，VEGF的突然退出会导致预先形成的肿瘤血管的消退。此外，将反义VEGF转移到胶质瘤细胞可抑制致瘤性。这些观察结果表明，靶向VEGF是开发胶质瘤新疗法的准确策略。在本提案中，我们的目标是结合三种不同的策略开发一种针对胶质瘤中VEGF的病毒系统。具体来说，在Aim 1中，我们提出构建三种腺病毒载体，它们能够表达VEGF基因反义序列、ANG2和MMAC1基因的不同组合。在第一步，我们将描述MMAC1对血管生成和VEGF信息表达的调节作用。第二步，我们将构建多基因腺病毒载体。寻找外源基因的肿瘤选择性表达，在这些构建中，反义VEGF cDNA将由VEGF启动子驱动，MMAC1和ANG2基因将由E2F-1启动子驱动。VEGF和E2F-1这两种启动子被认为对肿瘤细胞具有高度选择性。一系列的体外实验将测试这些结构正确表达异位信息的能力。在特异性目标2中，我们将通过使用裸鼠皮下和原位胶质瘤模型来确定这些构建物在体内的抗胶质瘤作用。我们提出的动物模型涉及到异种移植的使用。因此，我们将研究腺病毒构建体在胶质瘤中的作用，这些胶质瘤将直接从患者身上移植到小鼠身上。该系统可能比异种移植模型更可靠，因为运输的血管将来自人类，至少在第一段中是这样。总之，在本提案中，我们将评估反义VEGF、MMAC1和ANG2的抗血管生成潜力和可能的协同作用；这些分子的作用将通过使用两种肿瘤选择性启动子直接作用于肿瘤细胞，此外，我们将在异种移植模型中挑战该系统的抗血管生成效率，该模型可能比目前的异种移植模型更可靠。

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.