肿瘤细胞治疗研究
英文原题：Imaging CRISPR-edited CAR-T cell therapies with optical and positron emission tomography reporters.
结果：T 细胞受体（TCR）敲除效率超过 85%，CAR 表达达到 70-80%。
理论依据：嵌合抗原受体（CAR）T细胞疗法在治疗血液癌症方面取得显著成功，并日益显示其用于实体瘤的潜力。CRISPR基因组编辑有望提高CAR-T细胞效力和安全性，但肿瘤归巢效率低、对正常组织产生毒性等问题限制了其更广泛应用。生物发光成像（BLI）和正电子发射断层扫描（PET）等先进成像技术，可实时观察体内CAR-T细胞行为。本研究开发了可追踪报告基因适配型CRISPR编辑CAR（tRACE-CAR）T细胞，这是一种模块化系统，可在特定位点整合CAR和成像报告基因。 方法：将荧光素酶报告基因AkaLuciferase（AkaLuc）或人钠碘同向转运体（NIS）克隆至腺相关病毒（AAV）供体中的CAR下游，用于成像。通过CRISPR编辑和AAV转导，将CAR—报告基因盒插入原代人T细胞的T细胞受体恒定区位点。采用流式细胞术评估编辑效率，并在多种效靶比下检测体外细胞毒性。体内实验中，使用BLI和PET成像追踪荷瘤免疫缺陷小鼠的CAR-T细胞。 结果：T细胞受体敲除效率超过85%，CAR表达达到70%–80%。工程化表达报告基因的CAR-T细胞表现出显著细胞毒性，优于初始T细胞。体内实验中，AkaLuc BLI和¹⁸F-四氟硼酸盐PET可无创追踪活的CAR-T细胞。给药途径（静脉、瘤周或腹腔内）显著影响CAR-T细胞分布和治疗效果。 结论：tRACE-CAR可在白血病和卵巢癌模型中对CRISPR编辑CAR-T细胞进行精准光学和PET追踪，无创动态监测细胞在肿瘤及脱靶组织中的分布。该成像平台有望促进更加个体化、有效的CRISPR编辑CAR细胞疗法。
Rationale: Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance CAR-T cell potency and safety, yet challenges such as inefficient tumor homing and toxicities in normal tissues, limit broader adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time visualization of CAR-T cell behavior in vivo . Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. Methods: The luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) was cloned downstream of the CAR in adeno-associated virus (AAV) donors for imaging. CAR-reporter cassettes were inserted into the T-cell receptor constant locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was assessed by flow cytometry. In vitro cytotoxicity was evaluated across multiple effector-to-target ratios. In vivo , BLI and PET imaging were used for tracking CAR-T cells in tumor-bearing immunodeficient mice. Results: T cell receptor (TCR) knockout efficiency exceeded 85% and CAR expression reached 70-80%. Reporter-engineered CAR-T cells exhibited significant cytotoxicity and outperformed na ve T cells. In vivo , AkaLuc BLI and 18 F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Administration route (intravenous, peritumoral, or intraperitoneal) significantly influenced CAR-T cell distribution and therapeutic effectiveness. Conclusion: tRACE-CAR enables precise optical and PET tracking of CRISPR-edited CAR-T cells in models of leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging-enabled platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.
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