Retrogenic humanized mice for the study of T1D

用于 T1D 研究的逆基因人源化小鼠

• 批准号: 8728475
• 负责人: Michael Allen Brehm
• 金额: $ 27.14万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8728475
• 关键词: Address; Animal Model; Autologous; Beta Cell; Biological Models; Biomedical Research; CD34 gene; CD4 Positive T Lymphocytes; CD8B1 gene; Clinical Trials; Collaborations; Controlled Environment; Development; Diabetes Mellitus; Diagnosis; Disease; Disease Progression; Due Process; Engraftment; Environment; Fetal Liver; General Population; Generations; HIV; HLA-A2 Antigen; HLA-DR4 Antigen; Hematopoietic stem cells; Home environment; Human; Immune; Immune system; Insulin-Dependent Diabetes Mellitus; Intervention; Investigation; Laboratories; Lead; Modeling; Mouse Strains; Mus; Natural History; Organ; Pancreas; Pancreas Transplantation; Patients; Pre-Clinical Model; Principal Investigator; Process; Reagent; Recording of previous events; Research Design; Risk; T-Cell Development; T-Lymphocyte; TNFRSF10A gene; Technology; Testing; Therapeutic; Thymus Gland; Time; Transgenic Mice; Transgenic Organisms; Validation; Virus; Work; autoreactive T cell; base; blood glucose regulation; diabetic; diabetic patient; fetal; in vivo; insight; islet; mouse model; new technology; next generation; novel; public health relevance; research study; tool

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is particularly challenging to study directly in humans. By the time a patient is diagnosed with T1D, the destruction of beta cells by the immune system is nearly complete, rendering it difficult to investigate the mechanisms by which the disease develops. To address this, we propose to develop a novel humanized mouse model of T1D that permits study of the in vivo generation and function of human islet autoreactive T cells. This new model is based on recent technological breakthroughs. 1) Generation of human islet autoreactive T-cell clones. 2) Development of lentiviral technology enabling efficient transduction of human hematopoietic stem cells (HSC). 3) Availability of novel NOD-scid IL2rgnull (NSG) mice that support the generation of a functional human immune system following engraftment with human HSC. 4) Development of a human fetal liver/thymus/pancreas model in which a human immune system is generated in the presence of an autologous, functioning human pancreas. Combining these new cutting edge technologies, we will generate human TCR-transgenic ("retrogenic") mice engrafted with a functional human immune system enriched in human TCR transgenic (Tg) autoreactive T cells. To accomplish this, we have developed "next generation" genetically modified NSG mice, including HLA Tg NSG mice. We have established lentiviral transduction of human HSC in our laboratory, and have cloned into the virus (a) TCRs from HLA-A2-restricted CD8 T cell clones specific for human IGRP265-273; (b) TCRs from HLA-DR4-restricted CD4 T cell clones specific for human GAD555-567 and (c) a positive control HIV-specific TCR. We have also developed a spontaneously diabetic NRG-Akita mouse strain that can be engrafted with human fetal liver, thymus, and pancreas to generate a human immune system that develops in the presence of the autologous human fetal pancreas graft that matures and regulates glucose homeostasis. We will use these new technologies to generate "retrogenic" NSG-HLA Tg mice engrafted with a human immune system expressing Tg TCRs against IGRP265-273 or GAD555-567. We will test the overall hypothesis that human TCR Tg autoreactive CD4 and CD8 T cells either alone or together will induce diabetes in humanized mice. Specific Aim 1 will assemble and validate the components needed to create retrogenic mice with human T1D reactive immune systems. Specific Aim 2 will determine whether the human TCR retrogenic models develop insulitis or T1D and will analyze the ability of environmental perturbations to modulate this process. These studies will lead to the development of a humanized mouse model of T1D in which all of the human immune components are present to participate in disease initiation and progression. The results of this study will provide an important pre-clinical model system for investigation of T1D development without putting patients at risk. The generation, validation, and use of these new animal models will permit investigation of mechanisms that regulate human autoreactive T cell development and function and provide insights into how therapeutics may modulate this process.

描述（由申请人提供）：1型糖尿病（T1D）在人类中直接研究特别具有挑战性。到患者被诊断出患有T1D时，免疫系统对β细胞的破坏几乎完整，这使得很难研究疾病发展的机制。为了解决这个问题，我们建议开发一种新型的T1D人源化小鼠模型，该模型允许研究人类胰岛自动反应性T细胞的体内产生和功能。这个新模型基于最近的技术突破。 1）人类胰岛自动反应性T细胞克隆的产生。 2）开发慢病毒技术，有效地转导了人类造血干细胞（HSC）。 3）在与人类HSC植入后，可支持支持人类免疫系统的新型NOD-SCID IL2RGNULL（NSG）小鼠的可用性。 4）开发人胎儿肝脏/胸腺/胰腺模型，其中人类免疫系统是在自体，功能正常的人胰腺存在下产生的。结合了这些新的尖端技术，我们将产生人类TCR-转基因（“肾后源性”）小鼠，该小鼠与富含人TCR转基因（TG）自动反应性T细胞的功能性人免疫系统。为此，我们开发了包括HLA TG NSG小鼠在内的“下一代”转基因NSG小鼠。我们已经在实验室中建立了人类HSC的慢病毒转导，并从HLA-A2限制的CD8 T细胞克隆中克隆到病毒（a）TCR中，该克隆针对人IGRP265-273； （b）针对人GAD555-567和（c）阳性对照HIV特异性TCR的HLA-DR4限制CD4 T细胞克隆的TCR。我们还开发了一种自发的糖尿病NRG-AKITA小鼠菌株，可以与人类胎儿肝脏，胸腺和胰腺植入，以产生一种人类免疫系统，该系统在存在自体胎儿胰腺移植物的存在下发展并调节葡萄糖稳态。我们将使用这些新技术来生成“后源性” NSG-HLA TG小鼠，该NSG-HLA TG小鼠植入了针对IGRP265-273或GAD555-567的人类免疫系统。我们将测试人类TCR TG自动反应性CD4和CD8 T细胞的总体假设，或者可以一起诱导人源性小鼠的糖尿病。特定的目标1将组装和验证使用人T1D反应性免疫系统创建后源性小鼠所需的成分。具体目标2将确定人类TCR后源性模型是否患有胰岛炎或T1D，并将分析环境扰动调节此过程的能力。这些研究将导致T1D的人源化小鼠模型的发展，其中所有人类免疫成分都存在于疾病的启动和进展中。这项研究的结果将提供一个重要的临床前模型系统，用于研究T1D发育，而不会使患者处于危险之中。这些新动物模型的产生，验证和使用将允许调查调节人类自动反应性T细胞开发和功能的机制，并提供有关治疗方法如何调节此过程的见解。