肿瘤细胞治疗研究
英文原题：Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.
Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.
未标注：经嵌合抗原受体 (CAR) 重编程的免疫细胞在肿瘤学、自身免疫性疾病、移植医学和感染领域具有重要的治疗潜力。
未标注：经嵌合抗原受体（CAR）重编程的免疫细胞在肿瘤、自身免疫病、移植医学和感染治疗中具有重要潜力。所有获批CAR-T疗法均依赖个体化制备，并采用非定向病毒基因转移；这会造成CAR信号的非生理性调控，并因物流困难、成本高和生物安全要求而限制治疗可及性。我们提出一种新方法，利用CRISPR-Cas基因编辑以CAR重新定向T细胞和自然杀伤（NK）细胞。将缺少主要激活结构域的较短截短CAR转基因导入人CD3（CD247）基因，可生成利用内源CD3基因作为CAR激活结构域的功能性CAR融合基因。重新利用这一T/NK细胞谱系基因，可实现CAR表达的生理性调控，并重编程多种免疫细胞类型，包括常规T细胞、TCRαβ阴性T细胞、调节性T细胞和NK细胞。在T细胞中，CD3同框融合可消除TCR表面表达，降低异基因现货型治疗中的移植物抗宿主病风险。CD3ζ-CD19-CAR-T细胞在体内控制白血病的效果与T细胞受体α恒定区（TRAC）替换及慢病毒转导的CAR-T细胞相当。调节CD3ζ-CAR表达水平可显著提高体内疗效。与TRAC编辑CAR-T细胞相比，将Her2-CAR整合入CD3可产生相似的体外肿瘤裂解能力，但对活化诱导细胞死亡和分化的易感性降低，推测与CAR表达水平较低有关。值得注意的是，CD3基因编辑可重编程NK细胞而不损害其典型功能。因此，CD3基因编辑是开发利用重新定向杀伤淋巴细胞的异基因现货型细胞疗法的有前景平台。 要点：将缺失ζ结构域的CAR整合进CD3基因，可生成功能性且TCR失活的CAR-T细胞，用于异基因现货型治疗；CD3ζ编辑平台可重编程NK细胞，且不影响其典型功能。
UNLABELLED: Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCR / T cells, regulatory T cells, and NK cells. In T cells, CD3 in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 -CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant ( TRAC )-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 -CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. KEY POINTS: Integration of -deficient CARs into CD3 gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 -editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.
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