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）：直接在人体中研究 1 型糖尿病 (T1D) 特别具有挑战性。当患者被诊断出患有 T1D 时，免疫系统对 β 细胞的破坏已接近完成，因此很难研究该疾病发生的机制。为了解决这个问题，我们建议开发一种新型 T1D 人源化小鼠模型，允许研究人胰岛自身反应性 T 细胞的体内生成和功能。这种新模型基于最近的技术突破。 1) 人胰岛自身反应性 T 细胞克隆的产生。 2）开发慢病毒技术，实现人类造血干细胞（HSC）的高效转导。 3) 新型 NOD-scid IL2rgnull (NSG) 小鼠的可用性，该小鼠支持在植入人类 HSC 后生成功能性人类免疫系统。 4) 开发人类胎儿肝脏/胸腺/胰腺模型，其中在存在自体、功能正常的人类胰腺的情况下产生人类免疫系统。结合这些新的尖端技术，我们将产生人类 TCR 转基因（“逆转录”）小鼠，该小鼠植入了富含人类 TCR 转基因（Tg）自身反应性 T 细胞的功能性人类免疫系统。为了实现这一目标，我们开发了“下一代”转基因 NSG 小鼠，包括 HLA Tg NSG 小鼠。我们在实验室建立了人 HSC 的慢病毒转导，并克隆到病毒中： (a) 来自 HLA-A2 限制性 CD8 T 细胞克隆的 TCR，对人 IGRP265-273 具有特异性； (b) 来自人类 GAD555-567 特异性 HLA-DR4 限制性 CD4 T 细胞克隆的 TCR 和 (c) 阳性对照 HIV 特异性 TCR。我们还开发了一种自发性糖尿病 NRG-Akita 小鼠品系，可以将其移植到人胎儿肝脏、胸腺和胰腺中，以产生人类免疫系统，该系统在自体人胎儿胰腺移植物存在的情况下发育，成熟并调节葡萄糖稳态。我们将利用这些新技术来生成“逆转录”NSG-HLA Tg 小鼠，这些小鼠植入了表达针对 IGRP265-273 或 GAD555-567 的 Tg TCR 的人类免疫系统。我们将测试以下总体假设：人类 TCR Tg 自体反应性 CD4 和 CD8 T 细胞单独或一起会在人源化小鼠中诱发糖尿病。具体目标 1 将组装并验证创建具有人类 T1D 反应性免疫系统的逆转录小鼠所需的组件。具体目标 2 将确定人类 TCR 逆转录模型是否会发展为胰岛炎或 T1D，并将分析环境扰动调节这一过程的能力。这些研究将导致 T1D 人源化小鼠模型的开发，其中所有的人类免疫成分都参与疾病的发生和进展。这项研究的结果将为研究 T1D 的发展提供一个重要的临床前模型系统，而不会将患者置于危险之中。这些新动物模型的生成、验证和使用将允许研究调节人类自身反应性 T 细胞发育和功能的机制，并为治疗如何调节这一过程提供见解。