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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：
9043833
负责人：
ANTHONY N VAN DEN POL
金额：
$ 38.26万
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/9043833
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 Health 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 Thinking 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 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）：胶质母细胞瘤是一种侵袭性和侵袭性脑肿瘤，通常会在诊断后一年内导致死亡。没有治愈方法。目前的治疗只能延长生命几个月，而且往往以牺牲生活质量为代价。在这里，我们测试了一般假设，即一种新型重组嵌合病毒将靶向并杀死神经胶质瘤，并且对大脑没有可检测到的不利影响。在我们测试的大量病毒中，水泡性口炎病毒（VSV）似乎是针对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，过去几年我们一半的努力都集中在减少或控制 VSV 的潜在神经毒性。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蛋白基因被编码与非相关病毒（包括狂犬病、淋巴细胞脉络膜脑膜炎、马尔堡病毒、埃博拉病毒和拉沙病毒）的糖蛋白结合的基因取代。在这些测试的嵌合病毒中，有一种明显是最安全的溶瘤候选病毒：一种嵌合病毒，由编码拉沙糖蛋白的基因、编码VSV N、P、M和L蛋白的基因以及进一步减毒病毒的GFP报告基因组成。拉沙病毒 VSV 对啮齿动物和灵长类动物都是安全的。相当重要的是，我们将拉沙-VSV直接注射到正常小鼠或大鼠的大脑中，甚至注射到免疫缺陷小鼠的大脑中，并没有导致任何可检测到的副作用，而注射天然或其他减毒的VSV会产生神经系统并发症，有时会导致死亡。同样重要的是，在我们的体外和体内初步实验中，Lassa-VSV 完全靶向并破坏神经胶质瘤，而不损害宿主大脑，并无限期延长荷瘤小鼠的生存期。在第一组实验中，我们测试了拉沙-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代人类神经胶质瘤移植到免疫缺陷小鼠体内。我们还测试了植入免疫活性小鼠宿主大脑中的同基因小鼠神经胶质瘤。通过红色荧光报告基因的表达来检测肿瘤，通过编码绿色荧光蛋白的报告基因的表达来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：Lassa-VSV 在大脑中的安全性和选择性背后的机制是该病毒不与神经元或正常神经胶质细胞上的受体结合，或者不被内化，而神经胶质瘤中的结合和内化是强大的。通过阻断或增强感染途径中的各个步骤并结合逆转录酶定量 PCR 来研究神经元不存在病毒感染，并通过电子显微镜、其他物种和人脑切片进行证实。我们在目标 3 中测试的一个关键假设是，Lassa-VSV 会启动系统免疫系统（特别是 CD8+ T 细胞）对神经胶质瘤的攻击，即使在病毒被消除后这种攻击仍会持续，从而防止肿瘤复发。这是通过大脑中神经胶质瘤的感染来测试的；病毒被消除后，我们检查了新植入的神经胶质瘤在免疫反应增强的情况下生长的潜力。免疫靶向辅以过继转移、CD8-T 细胞消除和免疫细胞化学来检测招募到受感染肿瘤的免疫细胞。 Lassa-VSV 的非凡之处在于它可以完全杀死神经胶质瘤，并且在大脑或其他部位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成为临床试验的首要候选者。