肿瘤细胞治疗研究
英文原题：Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression.
本研究利用无病毒基因组编辑技术生成具有TRAC靶向CAR的CAR T细胞，这可为制造用于治疗癌症（包括实体瘤）的CAR T细胞提供参考。
嵌合抗原受体（CAR）T细胞已证明对血液系统恶性肿瘤（如CD19+癌症）具有高临床缓解率，但在实体瘤患者中活性有限。近期研究表明，在CD19 CAR的背景下，将CAR精确插入特定位点可改善治疗结局；然而，尚不清楚该策略是否也能影响实体瘤的结局。此外，CAR制备通常依赖病毒载体进行基因递送，而这是制备供应链中复杂且资源密集的环节。
抗GD2 CAR T细胞在9天内使用重组Cas9蛋白和核酸通过CRISPR/Cas9生成，未使用任何病毒载体。CAR被特异性靶向至T细胞受体α恒定基因（TRAC）。分别通过CHANGE-seq、靶向下一代测序、scRNA-seq、光谱流式细胞术和ELISA测定，在基因组、转录组、蛋白质组和分泌组水平对T细胞产物进行了表征。在NSG异种移植神经母细胞瘤模型中评估了体内功能性。
与逆转录病毒 CAR T 细胞相比，无病毒 CRISPR CAR（VFC-CAR）T 细胞表现出 CAR 转基因在 TRAC 靶向的基因组整合、与记忆样表型相关的转录和蛋白特征的提升，以及输注前较低的 tonic signaling，这部分源于 T 细胞受体的敲除。在暴露于 GD2 靶抗原后，抗 GD2 VFC-CAR T 细胞在体外对 GD2+ 细胞表现出特异性细胞毒性，并在体内诱导实体瘤消退。在针对人神经母细胞瘤异种移植模型时，VFC-CAR T 细胞相对于逆转录病毒 CAR T 细胞表现出强劲的归巢和持久性以及降低的耗竭。
BACKGROUND: Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain. METHODS: Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene ( TRAC ). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model. RESULTS: In comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC -targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo . VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model. CONCLUSIONS: This study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC -targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.
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