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

摘要 1型糖尿病(T1D)的特征是通过自身免疫攻击导致β细胞的破坏 在胰岛。在β细胞破坏的同时，β细胞应激和损伤通过启动 破坏β细胞功能并产生新抗原的酶活性。这个项目的调查人员 将整合临床T1D、β细胞应激、胰岛反应性T细胞、调节性T细胞(Treg)和基因的知识 基因工程创造的树突状细胞将成为胰岛的家园，并以一种方式抑制自身免疫和炎症 这将创造一个允许β细胞恢复和促进β细胞健康的环境。这些经过改造的树 (EngTregs)将利用基于同源定向修复(HDR)的基因编辑来中介构成 FOXP3与慢病毒转导特异性T细胞受体(TCR)序列的联合表达 在β细胞应激期间，胰岛内出现抗原。此外，我们建议使用 能够联合交付有效负载(有或没有TCR参与)，以抑制持续进行 炎症和/或促进β细胞的生存和生长。AIM 1将产生应激性胰岛特异的CD4+和 CD8+EngTregs通过基因编辑，同时通过TCRs的表达赋予胰岛特异性 识别瓜氨酸化或去胺化的β细胞衍生的新表位。目标1的中心假设是 靶向β细胞应激期产生的新抗原将增强EngTregs的靶向递送 去组织损伤的地方。新表位发现研究将识别限制在新的β细胞的TCRs序列 强调新的表位。功能研究将评估在存在应激性胰岛特异性EngTregs的情况下 β-细胞应激以及存在CD_4~+和CD_8~+效应性T细胞。AIM 2将产生胰岛特异的CD4+ 和CD8+EngTregs介导靶向免疫抑制并共同提供胰岛保护性治疗 货物。同时，我们将开发EngTregs，在TCR识别抗原后释放它们的货物。这个 拟议的研究将原代人类细胞研究与相关的T1D小鼠模型相结合，有助于更快 有希望的候选工程T细胞产品的鉴定。建议的研究是直接回应的。 RFA-DK-21-005，其要求1)设计可定位于胰腺的抗原特异性Treg细胞 胰岛或胰腺引流淋巴结并抑制这些间隔内的效应T细胞；2)工程胰岛- 归巢合成抑制细胞，如经改造可在局部产生抑制因子的CD4+T细胞 炎症，灭活效应T细胞，或促进胰岛组织修复；以及3)设计T细胞，使其产生 具有营养作用的因子对β-细胞促进功能、免疫保护和/或复制。

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.