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

项目摘要/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建造下一代基于细胞的疗法。T细胞为基础 免疫疗法特别适合CRISPR/CAS工具，因为经过编辑后，它们可以扩展到足够的 用于人类的数字。最近的研究表明，精确的CRISPR/Cas敲入T细胞受体 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 在人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细胞疗法可能会改善患者的预后，但与传统的治疗方法一样-- T细胞治疗，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- 靶向，危及生命的副作用。需要的是与翻译相关的技术，可以监控 CRISPR/Cas编辑的CAR-T细胞在体内的去向提供了解患者治疗所需的信息 反应/无反应、复发和/或毒性。这个应用程序的长期目标是开发 应用分子成像报告基因技术无创显示TRAC靶向的CAR-T细胞 病人。具体地说，我们建议开发新的翻译相关磁共振成像(Mri)。 以及正电子发射断层扫描(PET)报告基因分析，以非侵入性地跟踪TRAC编辑的命运 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。这样做的具体目的是 新的应用是：比较微环和非整合慢病毒CRISPR/Cas供体载体对其 能够使用CD19靶向CAR有效编辑TRAC基因上的T细胞(目标1.1)；开发供体载体 共同编码我们的汽车，一个生物发光报告基因，以及人类核磁共振报告有机阴离子 转运蛋白多肽1B3(OATP1B3)或PET报告钠碘转运蛋白(NIS)，并验证 体外Trac编辑和功能报告表达(Aim 1.2)；评价OATP1B3为基础的敏感性 MRI和基于NIS的PET用于活体显示CAR-T细胞，以及执行纵向PET和MRI CAR-T细胞在B细胞恶性肿瘤小鼠模型中的作用(目标2.1)；最后，开发一种双报告基因 翻译相关CRISPR/Cas系统的建立及对CAR-T细胞非侵入性监测能力的评价 同时接受正电子发射计算机断层扫描和核磁共振成像的小鼠(目标2.2)。这项工作的意义将是提供强大的细胞跟踪 允许CRISPR/CAS编辑的CAR-T细胞或其他CRISPR/CAS编辑的细胞的命运的技术 治疗，在临床前模型和患者中进行非侵入性监测。这一信息将允许更多 对个别患者的这些变革性治疗进行精确监测，以更好地评估安全性以及 将细胞生物分布与患者预后联系起来。

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.