Onoclytic VSV for Hepatocellular Carcinoma

描述（由申请人提供）：肝细胞癌（HCC）是世界上第三大癌症死亡原因，每年有超过100万例病例。在美国，其发病率从1.4（1976-80）增加到2.4（1991-95）每100，000人，这可能与慢性丙型肝炎感染的增加有关，2002年估计有16，600例新病例和14，100例死亡。未经治疗的HCC患者的中位生存期仅为7.8个月，3年生存率为10%。目前，肝癌的治疗方法主要有肝切除、肿瘤内消融和原位肝移植，这些方法疗效不佳，患者预后差。靶向肿瘤的连续复制病毒正在被开发为一类新的溶瘤剂。水泡性口炎病毒（VSV）是一种负链RNA病毒，由于其减弱的抗病毒反应，对肿瘤细胞中的复制具有固有的特异性。VSV作为溶瘤病毒特别吸引人，因为其在肿瘤细胞中1-2小时的异常快速的复制速率，使得溶瘤作用可以在宿主中潜在中和抗病毒免疫应答开始之前最大限度地显现。我们已经成功地挽救了表达绿色荧光蛋白的重组VSV载体（rVSV-GFP），并证明了其在培养的Morris大鼠肝癌细胞和在同基因布法罗大鼠肝脏中建立的肝癌孤立结节中的细胞病变效应和复制。我们建议研究VSV固有的肿瘤特异性在大鼠肝癌细胞中表现的分子机制。为了评估VSV治疗免疫活性宿主中多灶性HCC的杀肿瘤潜力，已经通过Morris大鼠肝癌细胞的肠系膜静脉输注在同系布法罗大鼠的肝脏中开发了这样的模型。肝动脉灌注rVSV-GFP后3天，在多个肝脏病变中出现明显的广泛坏死，荧光分析显示GFP阳性，免疫组织化学染色显示VSV阳性。重要的是，周围的正常肝组织对VSV染色和任何病理学体征均呈阴性。利用这种免疫功能正常的同基因动物模型，我们提出了以下假设：1。结论1. rVSV经肝动脉输注给药，能有效地溶瘤，延长荷瘤动物的生存期，且毒副作用小;将HSV-VP 22/TK融合基因整合到载体中并适时给予更昔洛韦可增强其溶瘤能力; 3.它的有效性可以通过掺入异源病毒融合膜糖蛋白基因来进一步放大，该基因将诱导与邻近细胞的合胞体形成;通过将其G基因替换为来自不同血清型的VSV的G基因，可以将溶瘤病毒的重复施用有效地应用于免疫活性动物。这些临床前研究的成功进行将为将来开发重组VSV作为多灶性HCC患者的新型治疗剂提供科学基础。

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the world, accounting for over 1 million cases annually. In the United States its incidence has increased from 1.4 (1976-80) to 2.4 (1991-95) per 100,000, which may be related to an increase in chronic hepatitis C infection, with an estimated 16,600 new cases and 14,100 deaths in 2002. Median survival of untreated HCC patients is only 7.8 months, with a 3-year survival rate of 10%. The current treatment modalities for HCC are hepatic resection, intra-tumoral ablation and orthotopic liver transplantation, which are not very effective and patient's prognosis remains poor. Conditionally replicating viruses targeted to tumors are being developed as a novel class of oncolytic agents. Vesicular Stomatitis Virus (VSV) is a negative-strand RNA virus with inherent specificity for replication in tumor cells due to their attenuated anti-viral responses. VSV as an oncolytic virus is particularly appealing for its exceptionally rapid replication rate of 1-2 hours in tumor cells, such that the oncolytic effects could be maximally manifested before the onset of potentially neutralizing anti-viral immune responses in the host. We have successfully rescued a recombinant VSV vector expressing the green fluorescent protein (rVSV-GFP) and demonstrated its cytopathic effects and replication in cultured Morris rat hepatoma cells and in solitary nodules of HCC established in the livers of syngeneic Buffalo rats. We propose to investigate the molecular mechanism(s) by which VSV's inherent tumor specificity is manifested in the rat hepatoma cells. In order to assess the tumoricidal potential of VSV to treat multi-focal HCC in an immune-competent host, such a model has been developed in the livers of syngeneic Buffalo rats through mesenteric vein infusion of the Morris rat hepatoma cells. Three days after hepatic artery infusion of rVSV-GFP, extensive necrosis was apparent in multiple hepatic lesions that were also positive for GFP by flouresence analyses and VSV by immuno-histochemical staining. Importantly, the surrounding normal liver tissues were negative for VSV staining and any signs of pathology. Using this immune-competent syngeneic animal model with multi-focal HCC in the liver, we propose to test the following hypotheses: 1. rVSV, delivered through hepatic artery infusion, can be an effective oncolytic agent and prolong survival of the tumor-bearing animals with minimal toxicities; 2. Its oncolytic potential can be enhanced by incorporation of the HSV-VP22/TK fusion gene into the vector with timely administration of ganciclovir; 3. Its effectiveness can be further amplified by incorporation of a heterologous viral fusogenic membrane glycoprotein gene that will induce syncytia formation with neighboring cells and 4. Repeated administration of the oncolytic virus can be effectively applied in immune-competent animals by substituting its G gene for those from VSVs of different serotypes. Successful conduct of these pre-clinical studies will provide the scientific foundation for future development of recombinant VSV as a novel therapeutic agent for multi-focal HCC in patients.