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（由适用提供）：胶质母细胞瘤是侵略性和侵入性脑肿瘤，通常在诊断后一年内导致死亡。无法治愈。当前的治疗方法仅延长了几个月的时间，通常以牺牲生活质量为代价。在这里，我们检验了一个普遍的假设，即新型的重组嵌合病毒将靶向并杀死神经胶质瘤，而对大脑没有可检测的不良影响。在我们已经测试的大量病毒中，囊泡气孔病毒（VSV）似乎是靶向和破坏脑肿瘤的最有效的病毒。但是，VSV具有感染神经元的潜在不良副作用，在过去几年中，我们的一半努力集中在降低或控制VSV的潜在神经毒性。为了避免VSV G蛋白的并发症和毒性，尤其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蛋白基因被编码编码的基因代替，编码用于结合无关病毒的糖蛋白蛋白，包括狂犬病，淋巴细胞，淋巴细胞，ebolg，marburg，ebola，lasbur，lasbura，lasbura，lass，lass，ebola，ebola，laka and lasha，la abura，laaa，laa。在经过测试的这些嵌合病毒中，一种是一种清晰的安全性溶瘤候选者：一种由编码LASSA糖蛋白编码的基因组成的嵌合病毒以及编码VSV N，P，M和L蛋白的基因，以及GFP报告基因，以进一步吸收病毒素。 Lassa-VSV在啮齿动物和私人方面都是安全的。值得注意的是，我们直接注射LASSA-VSV对正常小鼠或大鼠的大脑，甚至是免疫缺陷小鼠的大脑，并未导致任何可检测的不良反应，而注射天然或其他减弱的VSVS产生的神经学上的并发症有时会导致死亡。同样重要的是，在我们在体外和体内的初步实验中，LASSA-VSV靶标并完全破坏神经胶质瘤，而不会损害宿主大脑，并无限期地扩展了含有肿瘤小鼠的存活。在第一组实验中，我们测试了LASSA-VSV成功靶向并杀死肿瘤内病毒后和静脉内接种后将其杀死被移植到大脑中的胶质母细胞瘤细胞的假设。我们还测试了植入免疫功能的小鼠宿主大脑中的合成小鼠胶质瘤。通过表达红色荧光报告基因的表达检测到扭结者，并通过表达编码绿色荧光蛋白的报告基因来检测几乎感染的细胞。在第二个目的中，我们解决了以下假设：大脑中LASSA-VSV的安全性和选择性背后的机制是该病毒要么与神经元或正常神经胶质的受体结合，要么不与受体结合，或者未内在化，而胶质瘤中的结合和内在化是可靠的。通过阻止或增强感染性途径的各种步骤以及逆转录酶定量PCR，并与电子显微镜以及其他物种和人类脑切片中的逆转录酶进行核对来研究神经元的病毒感染。我们在AIM 3中检验的一个关键假设是，LASSA-VSV启动了系统性免疫系统的攻击，尤其是CD8+ T细胞对神经胶质瘤的攻击，即使在消除病毒后仍会继续，从而防止了肿瘤的复发。这是通过大脑中神经胶质瘤感染来检验的。消除病毒后，我们检查了新植入的神经胶质瘤在增强的免疫响应存在下生长的潜力。免疫靶向通过适应性转移，CD8-T细胞的演化和免疫细胞化学来完成，以检测募集到感染肿瘤的免疫细胞。 LASSA-VSV非常出色，因为它可以完全杀死神经胶质瘤，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，我们认为该病毒将是临床试验的首要候选者。