无病毒 CRISPR 将嵌合抗原受体敲入 KLRC1 产生强效 GD2 特异性 NK 细胞

英文原题：Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.

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Virus-free CRISPR knockin of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.

PubMed 2025/01/14（内容时间） Mol Ther Q1 · IF 11.4（JCR 2025）

靶点：GD2 技术：CAR-NK 技术：NK 细胞技术：基因编辑黑色素瘤地区：美国

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研究概要

本研究证明，通过CRISPR-Cas9精确破坏NK细胞内的抑制性信号传导，同时表达CAR，从而生成针对HLA-E+实体瘤的强效同种异体细胞疗法，是可行的。

中文摘要

自然杀伤（NK）细胞因其细胞毒性特征，成为一种颇具吸引力的即用型异体细胞疗法。然而，其针对实体瘤的活性仍不理想，部分原因在于肿瘤微环境中NK抑制性配体（如HLA-E）的上调。在此，我们利用CRISPR-Cas9破坏KLRC1基因（编码与HLA-E结合的NKG2A受体），并在从人外周血分离的NK细胞中非病毒插入靶向GD2的嵌合抗原受体（CAR）。使用CRISPR-Cas9核糖核蛋白复合物进行基因组编辑，可实现KLRC1基因的高效基因组破坏，敲除效率达98%，GD2 CAR转基因的定点敲入率高达23%，且通过CHANGE-seq、in-out PCR、扩增子测序和长读长全基因组测序显示脱靶活性极低。KLRC1-GD2 CAR NK细胞表现出高活力和增殖能力，以及对GD2+人肿瘤细胞的精准细胞靶向和效力。值得注意的是，KLRC1-GD2 CAR NK细胞在体外可克服表达HLA-E的GD2+黑色素瘤细胞中基于HLA-E的抑制作用。本研究采用单步、无病毒基因组编辑工作流程，证明通过CRISPR-Cas9精确破坏NK细胞内的抑制性信号，同时表达CAR，以生成针对HLA-E+实体瘤的有效异体细胞疗法的可行性。

展开英文摘要原文

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR-Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knockin of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-seq, in-out PCR, amplicon sequencing, and long-read whole-genome sequencing. KLRC1-GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2 + human tumor cells. Notably, KLRC1-GD2 CAR NK cells overcome HLA-E-based inhibition in vitro against HLA-E-expressing, GD2 + melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR-Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E + solid tumors.

论文信息

作者: Shankar K、Zingler-Hoslet I、Tabima DM、Zima S、Shi L、Gimse K、Forsberg MH、Katta V
第一作者单位: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA.United States
通讯作者单位: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA; Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA; Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53715, USA. Electronic address: ksaha@wisc.edu.United States
期刊: Molecular therapy : the journal of the American Society of Gene Therapy2025 Mar 5

原文标识: PubMed 39815622　·　DOI 10.1016/j.ymthe.2025.01.024

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