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，每年超过100万例。在美国，其发病率从每10万人1.4例（1976- 1980年）增加到每10万人2.4例（1991- 1995年），这可能与慢性丙型肝炎感染的增加有关，2002年估计有16 600个新病例和14 100人死亡。未经治疗的HCC患者中位生存期仅为7.8个月，3年生存率为10%。目前HCC的治疗方式为肝切除、瘤内消融和原位肝移植，但效果不佳，患者预后较差。靶向肿瘤的有条件复制病毒是一类新型的溶瘤药物。水疱性口炎病毒（VSV）是一种负链RNA病毒，由于其抗病毒反应减弱，具有在肿瘤细胞中复制的固有特异性。VSV作为一种溶瘤病毒尤其具有吸引力，因为它在肿瘤细胞内的复制速度极快，1-2小时，从而在宿主体内潜在中和的抗病毒免疫反应发生之前，溶瘤作用就可以最大限度地发挥出来。我们成功地挽救了一个表达绿色荧光蛋白（rVSV-GFP）的重组VSV载体，并在培养的Morris大鼠肝癌细胞和在同基因Buffalo大鼠肝脏中建立的肝癌孤立结节中证明了其细胞病变作用和复制。我们拟探讨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在免疫能力强的宿主中治疗多灶性HCC的杀瘤潜力，我们通过肠系膜静脉输注Morris大鼠肝癌细胞，在同基因水牛大鼠肝脏中建立了这种模型。肝动脉输注rVSV-GFP 3天后，多个肝病变明显出现广泛坏死，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，周围的正常肝组织没有VSV染色和任何病理迹象。利用这种具有免疫能力的肝脏多灶性HCC同质动物模型，我们提出验证以下假设：rVSV通过肝动脉输注可作为一种有效的溶瘤剂，以最小的毒性延长荷瘤动物的生存期；2. 将HSV-VP22/TK融合基因植入载体并及时给予更昔洛韦，可增强其溶瘤潜能；3. 其有效性可通过与异源病毒融合的融合膜糖蛋白基因进一步增强，该基因可诱导与邻近细胞形成合胞体。通过将溶瘤病毒的G基因替换为不同血清型vsv的G基因，可以在免疫能力强的动物中有效地重复给药。这些临床前研究的成功开展将为未来开发重组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.