Vesicular stomatitis VSVrp30 selectively destroys human metastatic melanoma

描述（由申请人提供）：转移性黑色素瘤患者的生存预后在过去20年中没有改变，尽管肿瘤检测和黑色素瘤特异性全身治疗的发展取得了进展，但仍然令人沮丧。由于目前的化疗和免疫治疗的失败，以根除黑色素瘤，我们提出了一种新的方法。在这个提议中，我们测试了耶鲁大学实验室产生的一种具有复制能力的溶瘤病毒VSVrp 30。在初步测试中，VSVrp 30在黑色素瘤的潜在治疗中显示出相当大的前景。在体外和体内实验表明，该病毒选择性和快速感染和破坏人类转移性黑色素瘤，与正常的人类黑色素细胞相对较少或没有感染。我们寻求资金进行实验，以确定病毒是否可以在动物和体外模型的多种条件下靶向和破坏黑色素瘤细胞。我们将首先在耶鲁大学提供的大量人类黑色素瘤和正常黑色素细胞上进行一系列体外实验，以测试病毒的溶瘤作用。另一组实验将使用稳定转染有珊瑚报告基因的人类黑素瘤，该报告基因在癌细胞中产生红色荧光。这些人类细胞将作为实体瘤和分散的转移样癌细胞移植到SCID小鼠中。将通过瘤内和静脉内给予病毒，以检验病毒将靶向并杀死红肿瘤细胞，同时对正常细胞的感染最小的假设。感染的细胞可以通过整合到病毒基因组中的GFP报告基因的表达容易地检测到。第三组平行实验将在具有正常免疫系统的同基因C57 B16小鼠中使用小鼠黑素瘤细胞系B16进行，使我们能够检验病毒可以在正常免疫系统存在的情况下选择性地检测和破坏黑素瘤并延长黑素瘤小鼠存活的假设;如果病毒不能完全清除黑色素瘤细胞，我们将检验第二个假设，即用免疫抑制药物暂时抑制全身或先天免疫系统将增强溶瘤作用。病毒。第四组实验将研究黑色素瘤细胞病毒感染增加的遗传机制，使用一系列广泛的人类黑色素瘤，其中外显子组已被测序。这些实验将通过实验进行补充，以检验黑色素瘤常见的涉及BRAF、PTEN和CDKN 2A的特异性诱导基因突变直接增加病毒感染的假设。最后一系列的实验将测试这样的假设，即病毒可以穿过血脑屏障并选择性地破坏小鼠大脑中的黑色素瘤，并且预免疫，可能随后是短暂的免疫抑制，将增强溶瘤作用并为大脑提供另一层保护。如果我们检测到对正常大脑的附带损害，那么我们将测试一种新病毒，1，2-VSV，我们最近产生的，这是我们研究过的所有VSV中最弱的，但仍然针对黑色素瘤。其高度减毒的性质减少了与正常脑组织感染有关的担忧。如果这些实验成功，它们将成为人类转移性黑色素瘤临床试验的重大进展。 公共卫生相关性：已扩散到皮肤以外的黑色素瘤通常会导致患者在诊断后不到一年内死亡。目前还没有成功的治疗方法。在一系列拟议的实验中，我们测试了一种有前途的病毒VSVrp 30，以确定它是否可以成功地靶向和杀死人类黑色素瘤细胞，而不会导致正常细胞的感染，使用体外和体内实验。我们包括一些替代的实验范例，以确定如何提高这种病毒对黑色素瘤的溶瘤疗效。

DESCRIPTION (provided by applicant): The prognosis for survival in patients with metastatic melanoma has not changed in the last 20 years and remains dismal despite advances in tumor detection and the development of melanoma-specific systemic therapies. Because of the failure of current chemotherapeutic and immunologically- based treatments to eradicate melanoma, we propose a new approach. In this proposal, we test a replication-competent oncolytic virus generated in this lab at Yale, VSVrp30. In preliminary tests VSVrp30 shows considerable promise in the potential treatment of melanoma. In vitro and in vivo experiments show that the virus selectively and rapidly infects and destroys human metastatic melanoma, with relatively little or no infection of normal human melanocytes. We seek funding to pursue experiments to determine if the virus can target and destroy melanoma cells in multiple conditions in animal and in vitro models. We will first test the oncolytic actions of the virus with a series of in vitro experiments on a large number of human melanomas and normal melanocytes available at Yale. Another set of experiments will employ human melanoma that is stably transfected with a coral reporter gene that generates a red fluorescence in the cancer cells. These human cells will be transplanted into SCID mice, both as a solid tumor, and as dispersed metastatic-like cancer cells. Virus will be given intratumorally and intravenously to test the hypothesis that the virus wll target and kill the red tumor cells with minimal infection of normal cells. Infected cells can be readily detected by the expression of a GFP reporter incorporated into the viral genome. A third set of parallel experiments will be done using the mouse melanoma line B16 in syngeneic C57Bl/6 mice with a normal immune system, allowing us to test the hypothesis that the virus can selectively detect and destroy melanoma in the presence of a normal immune system, and prolong mouse survival from melanoma; if the virus does not completely eliminate the melanoma cells, we will test the secondary hypothesis that temporarily depressing the systemic or innate immune systems with immunosuppressant drugs will enhance the oncolytic actions of the virus. A fourth set of experiments will examine the genetic mechanisms underlying the increased viral infection of melanoma cells, using an extensive series of human melanomas in which the exomes have been sequenced. These experiments will be complemented by experiments to test the hypothesis that specific induced gene mutations involving BRAF, PTEN, and CDKN2A that are common to melanoma, directly increase virus infection. A final series of experiments will test the hypothesis that the virus can cross the blood brain barrier and selectively destroy melanoma in the mouse brain, and that pre-immunization, potentially followed by transient immunosuppression, will enhance oncolysis and provide another layer of protection to the brain. If we detect collateral damage to normal brain, then we will test a new virus, 1,2-VSV, that we recently generated which is the most attenuated of any VSV we have worked with, yet still targets melanoma. Its highly attenuated nature reduces concerns relating to infection of normal brain tissue. If these experiments are successful, they will form a major advance toward clinical trials for metastatic melanoma in humans. PUBLIC HEALTH RELEVANCE: Melanoma that has spread beyond the skin generally results in death of the patient in less than one year from diagnosis. There is currently no successful treatment. In the series of proposed experiments, we test a promising virus, VSVrp30, to determine if it can successfully target and kill human melanoma cells without causing problematic infections of normal cells, using in vitro and in vivo experiments. We include a number of alternate experimental paradigms to determine how to enhance the oncolytic efficacy of this virus against melanoma.