Retrogenic humanized mice for the study of T1D

描述（由申请人提供）：1型糖尿病（T1 D）直接在人体中进行研究特别具有挑战性。当患者被诊断患有T1 D时，免疫系统对β细胞的破坏几乎完成，因此难以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的人源化小鼠模型的T1 D，允许在体内的人类胰岛自身反应性T细胞的生成和功能的研究。这种新模式是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，使人造血干细胞（HSC）的有效转导成为可能。3)新型NOD-scid IL 2 rgnull（NSG）小鼠的可用性，其支持在用人HSC移植后产生功能性人免疫系统。4)人胎肝/胸腺/胰腺模型的开发，其中在存在自体功能性人胰腺的情况下产生人免疫系统。结合这些新的尖端技术，我们将产生移植有富含人TCR转基因（Tg）自身反应性T细胞的功能性人免疫系统的人TCR转基因（“逆转录”）小鼠。为了实现这一目标，我们开发了“下一代”遗传修饰的NSG小鼠，包括HLA Tg NSG小鼠。我们已经在我们的实验室中建立了人HSC的慢病毒转导，并且已经将（a）来自对人IGRP 265 -273特异性的HLA-A2限制性CD 8 T细胞克隆的TCR;（B）来自对人GAD 555 -567特异性的HLA-DR 4限制性CD 4 T细胞克隆的TCR和（c）阳性对照HIV特异性TCR克隆到病毒中。我们还开发了一种自发性糖尿病NRG-秋田小鼠品系，其可以与人胎肝、胸腺和胰腺移植，以产生在自体人胎胰腺移植物存在下发育的人免疫系统，所述自体人胎胰腺移植物成熟并调节葡萄糖稳态。我们将使用这些新技术来产生移植有表达针对IGRP 265 -273或GAD 555 -567的Tg TCR的人免疫系统的“逆转录”NSG-HLA Tg小鼠。我们将检验人TCR Tg自身反应性CD 4和CD 8 T细胞单独或一起将在人源化小鼠中诱导糖尿病的总体假设。Specific Aim 1将组装和验证创建具有人类T1 D反应性免疫系统的转基因小鼠所需的组件。具体目标2将确定人类TCR退变模型是否发展为胰岛炎或T1 D，并将分析环境扰动调节该过程的能力。这些研究将导致开发T1 D的人源化小鼠模型，其中所有人免疫组分都存在以参与疾病的起始和进展。本研究的结果将为T1 D发展的研究提供重要的临床前模型系统，而不会使患者处于风险之中。这些新动物模型的产生、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，并提供对治疗剂如何调节这一过程的见解。

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.