将CRISPR/Cas9介导的TRAC敲除与基于mRNA的CAR表达相结合，可实现异体CAR T细胞的灵活制备
Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells.
摘要(中文译文)
自体嵌合抗原受体（CAR）T细胞疗法在血液系统恶性肿瘤中已展现出显著疗效，但其应用仍受限于复杂的生产流程。来源于健康供者的异体即用型CAR T细胞是一种有前景的替代方案；然而，其安全实施需要消除内源性T细胞受体（TCR）表达并采用灵活的CAR表达策略。本研究旨在通过将CRISPR/Cas9介导的TCR敲除与基于mRNA的CAR表达相结合，开发一种优化的异体CAR T细胞生产流程，并评估能够实现按需生成CAR T细胞的冷冻保存策略。使用CRISPR/Cas9对健康供者T细胞的TRAC基因座进行编辑，以生成TCR缺陷型T细胞。将这些细胞冷冻保存，随后转染编码CD117、BCMA或CD19 CAR的mRNA。在不同冷冻保存-转染条件下评估CAR表达、细胞活力、免疫表型、细胞因子分泌及抗原特异性细胞毒性。TCR敲除T细胞表现出高效的TCR破坏，且同种异体反应性增殖降低。来源于TCR缺陷型T细胞的CD117 mRNA CAR T细胞展现出与野生型CAR T细胞相当的CAR表达动力学、免疫表型特征及抗原特异性细胞毒性。对两种冷冻保存策略的评估显示，在mRNA电穿孔前进行冷冻保存可保持细胞活力、表型及细胞毒功能，而在mRNA转染后进行冷冻保存则与功能活性降低相关。该优化方案成功扩展至靶向CD19和BCMA的CAR mRNA。总体而言，这些发现建立了一个利用mRNA技术生产异体CAR T细胞的模块化平台，为快速、按需的CAR T细胞治疗提供了一种实用方法。
Abstract
Autologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable efficacy in hematologic malignancies but remain limited by complex manufacturing processes. Allogeneic, off-the-shelf CAR T cells derived from healthy donors represent a promising alternative; however, safe implementation requires elimination of endogenous T-cell receptor (TCR) expression and flexible CAR expression strategies. This study aimed to develop an optimized manufacturing workflow for allogeneic CAR T cells by combining CRISPR/Cas9-mediated TCR knockout with mRNA-based CAR expression, and to evaluate cryopreservation strategies enabling on-demand CAR T-cell generation. Healthy donor T cells were edited at the TRAC locus using CRISPR/Cas9 to generate TCR-deficient T cells. These cells were cryopreserved and subsequently transfected with mRNA encoding CD117, BCMA, or CD19 CARs. CAR expression, cell viability, immunophenotype, cytokine secretion, and antigen-specific cytotoxicity were assessed under different cryopreservation-transfection conditions. TCR knockout T cells exhibited efficient TCR disruption with reduced alloreactive proliferation. CD117 mRNA CAR T cells derived from TCR-deficient T cells demonstrated CAR expression kinetics, immunophenotypic profiles, and antigen-specific cytotoxicity comparable to wild-type CAR T cells. Evaluation of two cryopreservation strategies revealed that cryopreservation prior to mRNA electroporation preserved cell viability, phenotype, and cytotoxic function, whereas cryopreservation after mRNA transfection was associated with reduced functional activity. The optimized protocol was successfully extended to CD19- and BCMA-targeting CAR mRNAs. Collectively, these findings establish a modular platform for producing allogeneic CAR T cells using mRNA technology, offering a practical approach for rapid, on-demand CAR T-cell therapy.
如何引用
AMA
Thanyaporn Buakaew, Rattapoom Thaiwong, Thananya Inthanachai, Tanapat Palaga, Drew Weissman, Koramit Suppipat, et al. Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells.. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2026; doi:10.1016/j.biopha.2026.119300.
APA
Thanyaporn Buakaew, Rattapoom Thaiwong, Thananya Inthanachai, Tanapat Palaga, Drew Weissman, Koramit Suppipat, et al (2026). Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells.. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. https://doi.org/10.1016/j.biopha.2026.119300
GB/T 7714
Thanyaporn Buakaew, Rattapoom Thaiwong, Thananya Inthanachai, Tanapat Palaga, Drew Weissman, Koramit Suppipat, et al. Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells.[J]. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 2026 doi:10.1016/j.biopha.2026.119300.
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