Oncolytic VSV for Hepatocellular Carcinoma

描述(申请人提供)：肝细胞癌是世界上第三大癌症死亡原因，每年超过100万例。在美国，其发病率从每10万人中1.4例(1976-1980年)增加到2.4例(1991年-1995年)，这可能与慢性丙型肝炎感染增加有关，2002年估计有16,600例新病例和14,100例死亡。未经治疗的肝癌患者中位生存期仅为7.8个月，3年生存率为10%。目前肝细胞癌的治疗方法有肝切除、瘤内消融、原位肝移植等，但疗效不佳，患者预后较差。以肿瘤为靶点的有条件复制病毒正被开发为一类新型的溶瘤药物。水泡性口炎病毒(VSV)是一种负链RNA病毒，由于其抗病毒反应减弱，在肿瘤细胞中具有固有的复制特异性。VSV作为一种溶瘤病毒，由于其在肿瘤细胞中1-2小时的异常快速复制而特别吸引人，因此在宿主潜在的中和抗病毒免疫反应开始之前，溶瘤效应可以最大限度地表现出来。我们成功地挽救了表达绿色荧光蛋白的重组VSV载体(rVSV-GFP)，并在培养的Morris大鼠肝癌细胞和同基因水牛大鼠肝脏建立的孤立肝癌结节中证明了其细胞病变和复制作用。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(S)。为了评估VSV在免疫活性宿主中治疗多灶性肝癌的杀瘤潜力，通过肠系膜静脉注射Morris大鼠肝癌细胞，在同基因水牛大鼠的肝脏中建立了这样的模型。肝动脉注射rVSV-GFP后3d，荧光分析显示GFP阳性，免疫组织化学显示VSV阳性的多发性肝病变可见广泛坏死。重要的是，周围的正常肝组织VSV染色和任何病理迹象均为阴性。利用这种免疫活性的同种异体肝细胞癌动物模型，我们提出了以下假设：1.经肝动脉灌注的rVSV可以作为一种有效的溶瘤剂，以最小的毒性延长荷瘤动物的存活：2.将HSV-VP22/TK融合基因整合到载体中，并适时给予更昔洛韦，可以增强其溶瘤能力；3.加入异源病毒融合膜糖蛋白基因，可诱导与邻近细胞形成合胞体；4.用溶瘤病毒的G基因替代不同血清型VSV的G基因，可有效地应用于具有免疫功能的动物体内。这些临床前研究的成功将为重组VSV作为一种新的治疗多灶性肝癌的药物的未来发展提供科学基础。

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.