肿瘤细胞治疗研究
英文原题：Integration of ζ-deficient CARs into the CD3ζ gene conveys potent cytotoxicity in T and NK cells.
嵌合抗原受体（CAR）重定向免疫细胞在肿瘤学、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。
嵌合抗原受体（CAR）重定向免疫细胞在肿瘤学、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。所有已获批的 CAR-T 疗法均依赖个性化生产，使用非定向病毒基因转移，这导致 CAR 信号传导的非生理性调控，并因物流挑战、高成本和生物安全要求而限制了其可及性。随机基因转移方式存在插入突变导致恶性转化的风险。在此，我们提出一种新方法，利用 CRISPR-Cas 基因编辑技术以 CAR 重定向 T 细胞和自然杀伤（NK）细胞。通过将缺乏主要激活结构域的较短截短 CAR 转基因导入人 CD3（CD247）基因，生成功能性 CAR 融合基因，利用内源性 CD3 基因作为 CAR 的激活结构域。重新利用这一 T/NK 细胞谱系基因，促进了 CAR 表达的生理性调控以及多种免疫细胞类型的重定向，包括常规 T 细胞、TCR / T 细胞、调节性 T 细胞和 NK 细胞。在 T 细胞中，CD3 框内融合消除了 TCR 表面表达，降低了异体通用型应用中移植物抗宿主病的风险。CD3 -CD19-CAR-T 细胞在体内表现出与 TCR 链恒定区（TRAC）替换和慢病毒转导的 CAR-T 细胞相当的白血病控制效果。调节 CD3 -CAR 表达水平显著改善了体内疗效。值得注意的是，CD3 基因编辑使 NK 细胞重定向成为可能，且不损害其经典功能。因此，CD3 基因编辑是利用重定向杀伤性淋巴细胞开发异体通用型细胞疗法的一个有前景的平台。
Chimeric antigen receptor (CAR)-redirected 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 nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. 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 redirection 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 TCR chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3 -CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3 gene editing enabled redirection 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.
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