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

项目概要/摘要 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，因为在编辑之后，它们可以扩展到足够的免疫疗法。 用于人类的数字。最近的研究表明，在T细胞受体上精确的CRISPR/Cas敲入 具有靶向B细胞抗原CD 19的嵌合抗原受体（CAR）的恒定（TRAC）基因座， 在人B细胞的小鼠模型中随机整合基于病毒的工程化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预后，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同等的反应，或者是否有些患者会倾向于关闭- 目标，危及生命的副作用所需要的是能够监测 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。本申请的长期目标是开发 分子成像报告基因技术用于非侵入性可视化TRAC靶向CAR-T细胞， 患者具体来说，我们建议开发新的诊断相关的磁共振成像（MRI） 和正电子发射断层扫描（PET）报告基因测定，以非侵入性地跟踪TRAC编辑的 CAR-T细胞随着时间的推移，并在临床前小鼠模型中验证这些技术。具体目标是 新的应用是：比较小环和非整合慢病毒CRISPR/Cas供体载体的 能够用CD 19靶向CAR在TRAC基因座处有效编辑T细胞（Aim 1.1）; 共编码我们的CAR，一种生物发光报告基因，以及人类MRI报告有机阴离子， 转运蛋白多肽1B 3（OATP 1B 3）或PET报告分子钠碘同向转运蛋白（NIS），并验证 体外TRAC编辑和功能性报告基因表达（目的1.2）;评价基于OATP 1B 3的 MRI和基于NIS的PET用于在体内可视化CAR-T细胞，以及进行纵向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，开发双报告基因 CRISPR/Cas系统，并评估非侵入性监测CAR-T细胞的能力。 小鼠进行PET和MRI（目标2.2）。这项工作的意义将是提供强大的细胞跟踪 技术，以允许CRISPR/Cas编辑的CAR-T细胞或其他CRISPR/Cas编辑的细胞的命运， 在临床前模型和患者中进行非侵入性监测。这些信息将使更多 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， 将细胞生物分布与患者结果联系起来。

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.