Non-Invasive Monitoring of CRISPR/Cas-Edited Chimeric Antigen Receptor T (CAR-T) Cells with Reporter Gene-Based Magnetic Resonance Imaging and Positron Emission Tomography

使用基于报告基因的磁共振成像和正电子发射断层扫描对 CRISPR/Cas 编辑的嵌合抗原受体 T (CAR-T) 细胞进行无创监测

• 批准号: 10447329
• 负责人: John Andrew Ronald
• 金额: $ 32.4万
• 依托单位: UNIVERSITY OF WESTERN ONTARIO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447329
• 关键词: Non; Invasive; Monitoring; CRISPR; Cas

PROJECT SUMMARY/ABSTRACT Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease (CRISPR/Cas) genome editing has enabled scientists to design and build next-generation cell-based therapies. T cell-based immunotherapies are particularly suitable CRISPR/Cas tools as after editing they can be expanded to sufficient numbers for use in humans. Recent efforts have shown that precise CRISPR/Cas knock-in at the T cell receptor a constant (TRAC) locus with a chimeric antigen receptor (CAR) targeted towards the B cell antigen CD19 is more effective over randomly integrating viral-based engineered CAR-T cells in mouse models of human B cell leukemia. Therefore, while CRISPR/Cas cellular therapies may improve patient outcomes, like traditional CAR- T cell therapy, it is not known if every patient will respond equivalently or if some patients will be prone to off- target, life-threatening side effects. What is needed are translationally-relevant technologies that can monitor the fate of CRISPR/Cas-edited CAR-T cells in vivo to provide the information needed to understand patient treatment response/non-response, relapse and/or toxicity. The long-term objective of this application is to develop molecular imaging reporter gene technologies for non-invasive visualization of TRAC-targeted CAR-T cells in patients. Specifically, we propose to develop novel translationally-relevant magnetic resonance imaging (MRI) and positron emission tomography (PET) reporter gene assays to noninvasively track the fate of TRAC-edited CAR-T cells over time and to validate these technologies in preclinical mouse models. The specific aims of this new application are: to compare minicircle and non-integrating lentiviral CRISPR/Cas donor vectors for their ability to efficiently edit T cells at the TRAC loci with a CD19-targeted CAR (Aim 1.1); to develop donor vectors co-encoding our CAR, a bioluminescence reporter gene, and either the human MRI reporter organic anion transporter polypeptide 1B3 (OATP1B3) or the PET reporter sodium iodide symporter (NIS), and to validate TRAC-editing and functional reporter expression in vitro (Aim 1.2); to evaluate the sensitivity of OATP1B3-based MRI and NIS-based PET for visualizing CAR-T cells in vivo, as well as perform longitudinal PET and MRI of CAR-T cells in mouse models of B cell malignancies (Aim 2.1); and finally, to develop a dual-reporter translationally-relevant CRISPR/Cas system and evaluate the ability to noninvasively monitor CAR-T cells in mice with both PET and MRI (Aim 2.2). The significance of this work will be to provide powerful cell tracking technologies to allow the fate of CRISPR/Cas-edited CAR-T cells, or other CRISPR/Cas-edited cellular therapies, to be monitored noninvasively in preclinical models and patients. This information will allow more precise monitoring of these transformative therapies in individual patients to better assess safety as well as to relate cell biodistribution to patient outcomes.

项目概要/摘要 成簇规则间隔短回文重复序列/CRISPR相关核酸酶 (CRISPR/Cas) 基因组 编辑使科学家能够设计和构建下一代基于细胞的疗法。基于T细胞的 免疫疗法是特别合适的 CRISPR/Cas 工具，因为编辑后它们可以扩展到足够的范围 用于人类的数字。最近的研究表明，T 细胞受体上的精确 CRISPR/Cas 敲入 具有针对 B 细胞抗原 CD19 的嵌合抗原受体 (CAR) 的恒定 (TRAC) 基因座是 比在人类 B 细胞小鼠模型中随机整合基于病毒的工程 CAR-T 细胞更有效 白血病。因此，虽然 CRISPR/Cas 细胞疗法可以改善患者的治疗效果，就像传统的 CAR- T 细胞疗法，目前尚不清楚是否每个患者都会有相同的反应，或者某些患者是否容易出现副作用 目标，危及生命的副作用。我们需要的是能够监控转化的相关技术 CRISPR/Cas 编辑的 CAR-T 细胞在体内的命运，以提供了解患者治疗所需的信息 反应/无反应、复发和/或毒性。该应用程序的长期目标是开发 分子成像报告基因技术，用于 TRAC 靶向 CAR-T 细胞的非侵入性可视化 患者。具体来说，我们建议开发新型平移相关磁共振成像（MRI） 和正电子发射断层扫描 (PET) 报告基因检测，以无创追踪 TRAC 编辑的命运 随着时间的推移，CAR-T 细胞并在临床前小鼠模型中验证这些技术。本次活动的具体目标 新应用是：比较小环和非整合慢病毒 CRISPR/Cas 供体载体的性能 使用 CD19 靶向 CAR 有效编辑 TRAC 位点的 T 细胞的能力（目标 1.1）；开发供体载体 共同编码我们的 CAR、生物发光报告基因和人类 MRI 报告有机阴离子 转运蛋白多肽 1B3 (OATP1B3) 或 PET 报告碘化钠同向转运蛋白 (NIS)，并验证 TRAC 编辑和功能性报告基因体外表达（目标 1.2）；评估基于 OATP1B3 的敏感性 MRI 和基于 NIS 的 PET，用于体内可视化 CAR-T 细胞，并对细胞进行纵向 PET 和 MRI B 细胞恶性肿瘤小鼠模型中的 CAR-T 细胞（目标 2.1）；最后，开发一个双报告器 翻译相关的 CRISPR/Cas 系统并评估非侵入性监测 CAR-T 细胞的能力 对小鼠进行 PET 和 MRI 检查（目标 2.2）。这项工作的意义将是提供强大的细胞追踪 允许 CRISPR/Cas 编辑的 CAR-T 细胞或其他 CRISPR/Cas 编辑的细胞命运的技术 治疗，在临床前模型和患者中进行无创监测。这些信息将允许更多 对个体患者的这些变革性疗法进行精确监测，以更好地评估安全性并 将细胞生物分布与患者结果联系起来。