Unraveling Adverse Effects of Checkpoint Inhibitors Using iPSC-derived Cardiac Organoids

项目摘要 与小分子癌症治疗相关的心脏毒性是有据可查的。然而， 基于单抗的免疫检查点引起心脏不良反应的新报告 抑制剂(ICIS)是一个日益令人担忧的问题。这些ICI作用于程序性细胞死亡蛋白1(PD-1)受体或 它的配体编程细胞死亡配体1(PD-L1)增强抗肿瘤反应，这往往导致 这种与免疫相关的不良事件(IRAE)。一些研究指出了肿瘤之间的同源性 抗原和心脏蛋白是导致心肌炎、心脏病变等结局的原因之一 节律和脉管炎。 由于罕见的早期检测iRAE和患者样本的稀缺。脑缺血再灌注损伤的发生机制 心脏病仍然难以捉摸。因此，利用独特的多学科方法和专业知识 诱导多能干细胞(IPSC)技术、免疫学和生物工程策略I假设 因此，IPSC衍生的带有免疫细胞的3-D心脏器官(CoS)可以用来模拟ICI诱导的心脏 受伤。用简化论方法揭示ICI治疗心脏损伤的发病机制 可能导致发现新的靶点，为免疫疗法提供心脏保护。为了 为了实现这个目标，我定义了三个具体目标：(1)我将通过自组装生成CoS IPSC-心肌细胞、IPSC-内皮细胞和IPSC-心脏成纤维细胞，并测试两种常用的ICIS Pembrolizumab(PD-1抑制剂)和Atezolizumab(PD-L1抑制剂)。我将评估CO的变化 有无用于IPSCs的外周血单核细胞(PBMC)的功能 派生(2)我将在单个细胞水平上对CoS进行深入的分子分析，并确定潜在的 负责高免疫反应的靶点可能会导致心脏功能障碍(3)最后，我将使用尖端技术 基因组编辑技术敲除有助于挽救ICI后CO功能的基因靶点 治疗。在AIM 2中确定的靶点将通过向小鼠体内注射ICIS在小鼠模型中再次得到验证 有不同的免疫背景，随后评估心脏功能和分子变化在 组织。 在这个多学科项目中，在我尊敬的导师、顾问和 合作者们，我相信我将接受卓越的训练，以完成科学和职业生涯。 发展目标。鉴于我到目前为止的科学记录，以及通过这一点寻求的补充培训 K99/R00奖，共同帮助我开发和领导几个研究领域，朝着成功的方向发展 独立的事业。

Project Summary Cardiotoxicities associated with small molecule cancer therapeutics are well documented. However, emerging reports of adverse cardiac reactions due to monoclonal antibody-based immune checkpoint inhibitors (ICIs) is a growing concern. These ICIs act on programmed cell death protein 1 (PD-1) receptor or its ligand programmed cell death-ligand 1 (PD-L1) enhancing antitumor response, and these often lead to such immune-related adverse events (iRAEs). Several studies point toward the homology between tumor antigens and cardiac proteins as one of the reasons for outcomes such as myocarditis, changes in cardiac rhythm, and vasculitis. Due to the rare, early detection iRAEs and scarcity of patient samples. The mechanism of ICI-induced cardiac disease remains elusive. Hence, utilizing a unique multi-disciplinary approach and expertise in induced pluripotent stem cell (iPSC) technology, immunology and bioengineering strategies, I hypothesize that, iPSC-derived 3-D cardiac organoids (COs) with immune cells can be used to model ICI-induced cardiac injury. Unraveling the disease mechanism of cardiac injury due to ICI treatment using a reductionist approach may lead to discovery of new targets that can confer cardioprotection for immunotherapies. In order to accomplish this goal, I have defined three specific aims: (1) I will generate COs through self-assembly of iPSC-cardiomyocytes, iPSC-endothelial cells and iPSC-cardiac fibroblasts and test two commonly used ICIs Pembrolizumab (PD-1 inhibitor) and Atezolizumab (PD-L1 inhibitor). I will for assess for changes in CO function in the presence and absence of peripheral blood mononuclear cells (PBMCs) used for iPSCs derivation (2) I will conduct in-depth molecular analyses of the COs at a single cell level and identify potential targets responsible to hyper immune response can cardiac dysfunction (3) Finally, I will use cutting-edge genome editing techniques to knockdown the gene targets that help rescue the CO function after ICI treatment. The targets identified in Aim 2 will be validated again in mouse models by injecting ICIs into mice with diverse immune backgrounds followed by evaluation of heart function and molecular changes within the tissue. In this multidisciplinary project, with the support from my esteemed panel of mentors, advisors and collaborators, I am confident that I will receive par excellence training to accomplish both scientific and career development goals. Given my scientific track record to date, and complementary training sought through this K99/R00 award, together will help me develop and lead several research areas building toward a successful independent career.