Advancing HLA-"Humanized" Models for T1D Therapy Development

项目摘要/摘要 1型糖尿病(T1D)是一种自身免疫性疾病，致病T细胞破坏产生胰岛素的β- 胰腺细胞。尽管NOD小鼠对我们理解T1D的发病机制做出了贡献，但它 事实证明，这并不是测试潜在临床适用的疾病疗法的理想选择。缺乏临床翻译 主张需要新的小鼠模型，为治疗开发提供更好的管道。自.以来 主要组织相容性复合体(小鼠的MHC，人类的HLA)基因是最有效的贡献者 T1D易感性，正在进行的人类白细胞抗原“人源化”NOD小鼠的创造代表了模型的下一个水平 制定可能的疾病干预措施。Serreze实验室已经开发出几种这样的小鼠，携带不同的 人类与糖尿病易感性相关的人类HLAI类基因：nod.β2M-/-.HHD(HLAA2.1)，nod.β2M-/-.B39 和Nod.β2M-/-.A24(HLAA24)。为了防止小鼠MHC I分子的表达，这些 小鼠携带β2M-/-突变。然而，由于β2M是FcRN复合体和Ig G的关键成分 在挽救途径上，这些小鼠在测试基于抗体的疗法方面受到限制。因此，我们使用了 CRISPR/Cas9技术直接消除小鼠MHC-I类分子在NOD小鼠体内的表达 (指定为NOD.MHCI-/-)。上述编码人类糖尿病I类转基因的转基因 已经进入了NOD.MHCI-/-STOCK。这项提议的总体假设是，在很大程度上 已有的人类白细胞抗原人源化NOD小鼠可以用来开发潜在的临床可翻译手段 减弱相当大比例的T1D具有致病意义的自体反应性CD8+T细胞群 病人。利用我在Aim 1的杰克逊实验室接受的小鼠遗传学培训 我建议利用CRISPR/Cas9介导的方法也敲除唯一的nod MHC II(H2-Ag7)变体 在最近开发的NOD.MHCI-/-小鼠中。人类白细胞抗原-A2.1、B39、A24 I类或DQ8或DR4的任意组合 已经在NOD背景上可用的II类转基因随后可以被杂交到NOD。MHC-/-STOCK 为人类白细胞抗原特异性治疗开发产生更多的新模型。此外，我建议测试两个 新开发的NOD.MHCI-/-.A2.1小鼠的潜在治疗方法。目标2将检验产生耐受性的假设 通过PLGA-载体运送人类白细胞抗原-A2.1限制性多肽(INS1B：5-14、INS1/2A：2-10、IGRP228-236和IGRP265-273) 微球可以减弱针对这些表位的非生理性CD8+T细胞反应。在AIM 3I中 将检验通过混合嵌合体的无辐射预适应方案进行诱导的假设 在MHC不匹配的情况下，供体细胞可以消除或灭活具有交叉反应的促糖尿病CD8+T细胞 捐赠者-MHC。综上所述，这项建议旨在改进现有的节点鼠标模型，以开发和验证 T1D的治疗方法，同时为我提供必要的小鼠遗传和基因组方面的培训 编辑技术最终为其他自身免疫性疾病创建人性化的小鼠模型。

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is an autoimmune disorder in which pathogenic T-cells destroy the insulin producing β- cells of the pancreas. Despite the NOD mouse contributing to our understanding of T1D pathogenesis, it has not proved ideal for testing potentially clinically applicable disease therapies. This lack of clinical translation argues for the need for new mouse models that will provide better pipelines for therapy development. Since the major histocompatibility complex (MHC in mice, HLA in humans) genes are the most potent contributors to T1D susceptibility, the on going creation of HLA-“humanized” NOD mice represents the next level of models to develop possible disease interventions. The Serreze lab has developed several such mice carrying different human HLA class I genes linked to diabetes susceptibility: NOD.β2m-/-.HHD (HLA-A2.1), NOD.β2m-/-.B39 (HLA-B39) and NOD.β2m-/-.A24 (HLA-A24). In order to prevent expression of murine MHC I molecules, these mice carry the β2m-/- mutation. However, since β2m is a critical component of the FcRn complex and IgG salvage pathways, these mice are limited for testing antibody-based therapies. Therefore, we have used CRISPR/Cas9 technology to directly eliminate expression of murine MHC class I molecules in NOD mice (designated NOD.MHCI-/-). The transgenes described above encoding human diabetogenic class I transgenes have been crossed into the NOD.MHCI-/- stock. The overall hypothesis of this proposal is that such largely already available HLA-humanized NOD mice can be utilized to develop potentially clinically translatable means to attenuate autoreactive CD8+ T-cell populations of pathogenic significance to a sizeable proportion of T1D patients. Taking advantage of the mouse genetics training I am receiving at The Jackson Laboratory, in Aim 1 I propose utilizing a CRISPR/Cas9-mediated approach to also knock out the sole NOD MHC II (H2-Ag7) variant in the recently developed NOD.MHCI-/- mice. Any combination of HLA-A2.1, B39, A24 class I or DQ8 or DR4 class II transgenes already available on the NOD background can then be crossed into the NOD.MHC-/- stock to generate additional new models for HLA-specific therapy development. Additionally, I propose to test two potential therapies in newly developed NOD.MHCI-/-.A2.1 mice. Aim 2 will test the hypothesis that tolerogenic delivery of HLA-A2.1 restricted peptides (INS1B:5-14, INS1/2A:2-10, IGRP228-236, and IGRP265-273) via PLGA- microspheres can attenuate diabetogenic CD8+ T cell responses directed against these epitopes. In Aim 3 I will test the hypothesis that induction through an irradiation free pre-conditioning regimen of mixed chimerism with MHC-mismatched donor cells can eliminate or inactivate diabetogenic CD8+ T-cells with cross-reactivity to the donor-MHC. Together, this proposal aims to improve current NOD mouse models to develop and validate curative therapies for T1D, while providing me with the necessary training in mouse genetic and genomic editing techniques to eventual create humanized mouse models for other autoimmune disorders.