Advancing HLA-"Humanized" Models for T1D Therapy Development

推进 T1D 治疗开发的 HLA“人性化”模型

• 批准号: 9256822
• 负责人: Jeremy J Racine
• 金额: $ 5.8万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2017-09-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9256822
• 关键词: Allogenic; Antibodies; Antigens; Attenuated; Autoimmune Diseases; Bone Marrow; CD3 Antigens; CD4 Positive T Lymphocytes; CD8B1 gene; CRISPR/Cas technology; Cells; Chimerism; Clinical; Complex; Development; Diabetes Mellitus; Disease; Engraftment; Epitopes; FDA approved; Future; Generations; Genes; Genetic; Genomics; Goals; Growth; HLA Class I Genes; HLA-A2.1; Health; Hematopoietic; Hematopoietic Stem Cell Transplantation; Histocompatibility Antigens Class I; Human; Human Genetics; Immunoglobulin G; Immunology; Inbred NOD Mice; Individual; Infusion procedures; Insulin; Insulin-Dependent Diabetes Mellitus; Intervention; Knock-out; Link; MHC Class II Genes; Major Histocompatibility Complex; Mediating; Microspheres; Modeling; Mus; Mutation; Pathogenesis; Pathway interactions; Patients; Peptides; Population; Predisposition; Radiation; Regimen; Source; Splenocyte; Staging; Structure of beta Cell of islet; System; T cell response; T-Lymphocyte; TNFRSF10A gene; Techniques; Technology; Testing; The Jackson Laboratory; Training; Transgenes; Translations; Vaccines; Variant; base; clinical efficacy; clinically relevant; conditioning; cross reactivity; curative treatments; design; diabetes mellitus therapy; diabetic patient; diabetogenic; genetic profiling; genome editing; graft vs host disease; humanized mouse; improved; irradiation; mouse model; neonatal Fc receptor; next generation; novel; prevent; response; therapy design; therapy development

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.

项目总结/摘要 1型糖尿病（T1 D）是一种自身免疫性疾病，其中致病性T细胞破坏胰岛素产生β- 胰腺细胞。尽管NOD小鼠有助于我们理解T1 D发病机制，但它 对于测试潜在的临床上可应用的疾病疗法来说，并不理想。缺乏临床翻译 认为需要新的小鼠模型，这将为治疗开发提供更好的管道。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， T1 D易感性，HLA-“人源化”NOD小鼠的持续创造代表了下一个水平的模型， 制定可能的疾病干预措施。Serreze实验室已经培养了几只这样的小鼠， 与糖尿病易感性相关的人类HLA-I类基因：NOD.β 2 m-/-.HHD（HLA-A2.1），NOD.β 2 m-/-. B39 A24（HLA-A24）。为了防止鼠MHC I分子的表达，这些 小鼠携带β 2 m-/-突变。然而，由于β 2 m是FcRn复合物和IgG的关键组分， 由于这些小鼠缺乏补救途径，因此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 CRISPR/Cas9技术直接消除NOD小鼠中鼠MHC I类分子的表达 （指定为NOD.MHCI-/-）。上述编码人致糖尿病I类转基因的转基因 已经进入了NOD的MHCI-/-库存这一建议的总体假设是， 已经可用的HLA-人源化NOD小鼠可用于开发潜在的临床上可转化的手段 将具有致病意义的自身反应性CD 8 + T细胞群减弱为相当大比例的T1 D 患者利用我在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-Ag 7）变体 在最近开发的NOD.MHCI-/-小鼠中。HLA-A2.1、B39、A24 I类或DQ 8或DR 4的任何组合 然后可以将NOD背景中已经存在的II类转基因杂交到NOD.MHC-/-原种中 以产生用于HLA特异性疗法开发的额外新模型。另外，我建议测试两个 在新开发的NOD.MHCI-/-.A2.1小鼠中的潜在疗法。目的2将检验耐受原性 HLA-A2.1限制性肽（INS 1B：5-14，INS 1/2A：2-10，IGRP 228 -236，和IGRP 265 -273）通过PLGA- 微球可以减弱针对这些表位的致糖尿病CD 8 + T细胞应答。目标3 I 将检验通过混合嵌合体的无辐射预处理方案诱导的假设 与MHC不匹配的供体细胞的免疫反应可以消除或抑制具有交叉反应性的致糖尿病CD 8 + T细胞， 供体MHC总之，该提案旨在改进当前的NOD小鼠模型，以开发和验证 T1 D的治愈性疗法，同时为我提供必要的小鼠遗传和基因组学培训， 编辑技术，以最终创建其他自身免疫性疾病的人源化小鼠模型。