Harnessing engineered T regulatory cells to promote beta cell health in T1D

总结 1型糖尿病（T1 D）的特征是β细胞的破坏，通过自身免疫攻击驱动， 在胰岛上。与β细胞破坏相一致，β细胞应激和损伤通过启动细胞凋亡而导致疾病。 - 损害β细胞功能并产生新抗原的酶活性。这个项目的研究人员 将整合临床T1 D，β细胞应激，胰岛反应性T细胞，调节性T细胞（TCRs）和基因的知识 工程制造出能回到胰岛并抑制自身免疫和炎症的Tendon， 这将创造一个环境，使β细胞恢复和促进β细胞健康。这些经过改造的THEORY （EngTdR）将利用基于同源性定向修复（HDR）的基因编辑来产生，以介导组成型 FOXP 3的表达与特异性T细胞受体（TCR）序列的慢病毒转导组合， 在β细胞应激期间胰岛中呈递的抗原。此外，我们建议开发EngTUNK与 共同投放有效载荷的能力（有或没有TCR参与），将起到抑制正在进行的 炎症和/或促进β细胞存活和生长。目的1将产生应激胰岛特异性CD 4+， CD 8 + EngT细胞通过基因编辑，同时通过TCR的表达赋予胰岛特异性 其识别瓜氨酸化或脱酰胺化的β细胞衍生的新表位。目标1的核心假设是， 靶向在β细胞应激期间产生的新抗原将增强EngT 3的靶向递送 组织损伤部位新表位发现研究将鉴定限制于新β细胞的TCR序列 强调新表位。功能性研究将评估在存在以下物质的情况下应激胰岛特异性EngTdR活化： 细胞应激以及在CD 4+和CD 8+效应T细胞的存在下。目的2将产生胰岛特异性CD 4 + 介导靶向免疫抑制并共同递送胰岛保护性治疗剂的CD 8 + EngT细胞 货物.同时，我们将开发在TCR识别抗原后释放其货物的EngTcells。的 提出的研究将原代人细胞研究与相关的T1 D小鼠模型相结合， 鉴定有希望的候选工程化T细胞产物。拟议的研究是直接回应 RFA-DK-21-005，要求1）工程化抗原特异性Treg细胞， 胰岛或胰腺引流淋巴结并抑制这些区室中效应T细胞; 2）工程化胰岛- 归巢合成抑制细胞，如CD 4 + T细胞，经工程改造，局部产生抑制因子， 炎症，刺激效应T细胞，或促进胰岛组织修复;和3）工程化T细胞， 对β细胞具有营养作用以促进功能、免疫保护和/或复制的因子。

Summary Type 1 diabetes (T1D) is characterized by the destruction of β-cells, driven through autoimmune attack directed at the pancreatic islet. In concert with β-cell destruction, β-cell stress and injury contribute to disease by initiating enzymatic activities that compromise β-cell function and generate neoantigens. The investigators of this project will integrate knowledge of clinical T1D, β-cell stress, islet reactive T cells, regulatory T cells (Tregs) and gene engineering to create Tregs that will home to the islet and suppress autoimmunity and inflammation in a manner that will create an environment that will allow β-cell recovery and promote β-cell health. These engineered Tregs (EngTregs) will be generated utilizing homology-directed repair (HDR)-based gene editing to mediate constitutive expression of FOXP3 combined with lentiviral transduction of T cell receptor (TCR) sequences specific to antigens presented in the islet during periods of β-cell stress. Further, we propose develop EngTregs with the ability to co-deliver a payload (with or without TCR engagement) that will function to suppress ongoing inflammation and/or promote β-cell survival and growth. Aim 1 will generate stressed-islet-specific CD4+ and CD8+ EngTregs via gene editing and, in parallel, confer specificity to the pancreatic islet via expression of TCRs that recognize citrullinated or deamidated β-cell-derived neoepitopes. The central hypothesis for Aim 1 is that targeting neoantigens generated during periods of β-cell stress will enhance the targeted delivery of EngTregs to the site of tissue injury. Neoepitope discovery studies will identify TCR sequences restricted to novel β-cell stress neoepitopes. Functional studies will assess stressed-islet-specific EngTregs activation in the presence of β-cell stress as well as in the presence of CD4+ and CD8+ effector T cells. Aim 2 will generate islet-specific CD4+ and CD8+ EngTregs that mediate targeted immune suppression and co-deliver an islet-protective therapeutic cargo. In parallel, we will develop EngTregs that release their cargo upon TCR recognition of antigen. The proposed studies integrate primary human cell studies with relevant T1D murine models, facilitating more rapid identification of promising candidate engineered T cell products. The proposed studies are directly responsive to RFA-DK-21-005 with its request to 1) engineer antigen-specific Treg cells that can home to the pancreatic islet or pancreatic draining lymph nodes and inhibit effector T cells in these compartments; 2) engineer islet- homing synthetic suppressor cells, such as CD4+ T cells engineered to locally produce factors that dampen inflammation, inactivate effector T cells, or promote islet tissue repair; and 3) engineer T cells that produce factors with trophic effects on β-cells to promote function, immunoprotection and/or replication.