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Lassa-VSV targets and kills glioma, and is not neurotoxic

Lassa-VSV 靶向并杀死神经胶质瘤，并且不具有神经毒性

基本信息

批准号：
8888841
负责人：
ANTHONY N VAN DEN POL
金额：
$ 35.32万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8888841
关键词：
Address Adoptive Transfer Adverse effects Animal Model Animals Antibodies Attenuated Binding Brain Brain Neoplasms CD8B1 gene Cell Transplants Cells Cessation of life Clinical Trials Code Complement Coupled Data Derivation procedure Diagnosis Ebola virus Electron Microscopy Frankfurt-Marburg Syndrome Virus G Protein Gene Genes Glioblastoma Glioma Glycoproteins Green Fluorescent Proteins Human Image Immune Immune Targeting Immune response Immune system Immunocompetent Immunodeficient Mouse Implant In Vitro Infection Injection of therapeutic agent Injury Intravenous Lassa virus Lead Life Lymphocytic Choriomeningitis Malignant neoplasm of brain Malignant neoplasm of lung Mediating Microscope Mus Neuroglia Neurologic Neurons Normal Cell Oncolytic Pathway interactions Patients Phase Play Primates Proteins Quality of life Quantitative Reverse Transcriptase PCR Rabies Rattus Recombinants Recruitment Activity Recurrence Reporter Reporter Genes Reverse Transcriptase Polymerase Chain Reaction Rodent Role SCID Mice Safety Slice Symptoms T-Lymphocyte Testing Time Toxic effect Transplantation Variant Vesicular stomatitis Indiana virus Virus Virus Diseases Virus Receptors Work cancer cell glioma cell line immunocytochemistry implantation in vivo killings melanoma neoplastic cell neurotoxic neurotoxicity novel outcome forecast prevent public health relevance receptor research study tumor vesicular stomatitis virus G protein virus development

项目摘要

DESCRIPTION (provided by applicant): Glioblastomas are aggressive and invasive brain tumors that generally lead to death within a year of diagnosis. No cure is available. Current treatments prolong life by only a few months, often at the expense of quality of life. Here we test the general hypothesis that a novel recombinant chimeric virus will target and kill gliomas with no detectable adverse effect to the brain. Of the large number of viruses we have tested, vesicular stomatitis virus (VSV) appears to be one of the most effective for targeting and destroying brain tumors. However, VSV has the potential unwanted side effect of infecting neurons, and half of our efforts in the last few years have focused on reducing or controlling potential neurotoxicity of VSV. To avoid complications and toxicity of the VSV G-protein, particularly its binding to neurons, we have compared a number of recombinant chimeric viruses in which the VSV G-protein gene was replaced by genes coding for binding glycoproteins of non-related viruses including rabies, lymphocytic choriomeningitis, Marburg, Ebola, and Lassa viruses. Of these chimeric viruses tested, one stood out as a clear superlative safe oncolytic candidate: a chimeric virus consisting of a gene coding for the Lassa glycoprotein together with genes coding for the VSV N,P,M, and L proteins, and a GFP reporter gene which further attenuates the virus. Lassa-VSV is safe, both in rodents and primates. Of considerable importance, our direct injection of Lassa-VSV into the brains of normal mice or rats, or even into the brains of immunodeficient mice has not resulted in any detectable adverse effects, whereas injections of native or other attenuated VSVs generated neurological complications sometimes resulting in death. Equally important, in our preliminary experiments in vitro and in vivo, Lassa-VSV targets and destroys gliomas completely without damage to the host brain, and extends survival of tumor-bearing mice indefinitely. In the first set of experiments, we test the hypothess that Lassa-VSV successfully targets and kills glioblastoma cells that are transplanted into the brain, both after an intratumoral virus injection, and after intravenous inoculation. We use both glioma cell lines and primary human gliomas transplanted into immunodeficient mice. We also test syngeneic mouse glioma implanted into immunocompetent mouse host brains. Tumors are detected by expression of a red fluorescent reporter and virally infected cells are detected by expression of a reporter gene coding for green fluorescent protein. In the second Aim, we address the hypothesis that the mechanism behind the safety and selectivity of Lassa-VSV in the brain is that the virus either does not bind to receptors on neurons or normal glia, or is not internalized, whereas binding and internalization in glioma is robust. The lack of virus infection of neurons is studied by blocking or enhancing various steps in the infectious pathway coupled with reverse transcriptase quantitative PCR, and corroborated with electron microscopy, and in additional species and in human brain slices. A key hypothesis we test in Aim 3 is that Lassa-VSV initiates an attack by the systemic immune system, particularly by CD8+ T cells, on the glioma that continues even after the virus is eliminated, thereby preventing the recurrence of tumor. This is tested by infection of glioma in the brain; after the virus is eliminated, we examin the potential of newly implanted glioma to grow in the presence of the enhanced immune response. Immune targeting is complemented with adoptive transfer, CD8-T cell elimination, and immunocytochemistry to detect immune cells recruited to the infected tumor. Lassa-VSV is remarkable in that it can completely kill glioma with no detectable adverse side effects in the brain or elsewhere. If our experiments are successful, we think this virus would be a top priority candidate for clinical trials.

描述（由申请人提供）：胶质母细胞瘤是侵袭性和侵袭性脑肿瘤，通常在诊断后一年内导致死亡。没有治愈方法。目前的治疗方法只能延长几个月的生命，通常是以牺牲生活质量为代价的。在这里，我们测试的一般假设，一种新的重组嵌合病毒将靶向和杀死神经胶质瘤，没有检测到的不利影响的大脑。在我们测试的大量病毒中，水泡性口炎病毒（VSV）似乎是靶向和破坏脑肿瘤最有效的病毒之一。然而，VSV具有感染神经元的潜在不良副作用，并且在过去几年中，我们的一半努力集中在减少或控制VSV的潜在神经毒性上。为了避免VSV G蛋白的并发症和毒性，特别是其与神经元的结合，我们比较了许多重组嵌合病毒，其中VSV G蛋白基因被编码非相关病毒（包括狂犬病、淋巴细胞性脉络丛脑膜炎、马尔堡、埃博拉和拉沙病毒）的结合糖蛋白的基因所取代。在这些测试的嵌合病毒中，一种作为明确的最高级安全溶瘤候选物而突出：由编码Lassa糖蛋白的基因连同编码VSV N、P、M和L蛋白的基因以及进一步减弱病毒的GFP报告基因组成的嵌合病毒。Lassa-VSV在啮齿动物和灵长类动物中均是安全的。相当重要的是，我们将Lassa-VSV直接注射到正常小鼠或大鼠的脑中，甚至注射到免疫缺陷小鼠的脑中，没有导致任何可检测到的不良反应，而注射天然或其他减毒的VSV产生神经系统并发症，有时导致死亡。同样重要的是，在我们的体外和体内初步实验中，Lassa-VSV完全靶向和破坏胶质瘤，而不损伤宿主大脑，并无限期延长荷瘤小鼠的生存期。在第一组实验中，我们测试了Lassa-VSV成功靶向并杀死移植到大脑中的胶质母细胞瘤细胞的假设，无论是在瘤内病毒注射后，还是在静脉注射后。我们使用神经胶质瘤细胞系和原发性人类神经胶质瘤移植到免疫缺陷小鼠。我们还测试了植入免疫活性小鼠宿主脑中的同基因小鼠神经胶质瘤。通过表达红色荧光报告基因检测肿瘤，通过表达编码绿色荧光蛋白的报告基因检测病毒感染的细胞。在第二个目标中，我们提出了这样的假设，即Lassa-VSV在脑中的安全性和选择性背后的机制是病毒不与神经元或正常神经胶质细胞上的受体结合，或者不内化，而在胶质瘤中的结合和内化是稳健的。通过阻断或增强感染途径中的各个步骤，结合逆转录酶定量PCR，并通过电子显微镜、其他物种和人脑切片证实，研究了神经元缺乏病毒感染。我们在目标3中测试的一个关键假设是，Lassa-VSV启动全身免疫系统，特别是CD 8 + T细胞对胶质瘤的攻击，即使在病毒被消除后仍继续，从而防止肿瘤复发。这是通过感染脑胶质瘤来测试的;在病毒被清除后，我们检查了新植入的胶质瘤在增强的免疫反应存在下生长的潜力。免疫靶向与过继转移、CD 8-T细胞消除和免疫细胞化学互补，以检测募集到感染肿瘤的免疫细胞。Lassa-VSV的显著之处在于它可以完全杀死胶质瘤，而在大脑或其他地方没有可检测到的不良副作用。如果我们的实验成功，我们认为这种病毒将是临床试验的首要候选者。