相关实验视频
Updated: Sep 9, 2025

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Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
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针对目标集成和多重基编辑的正交式CRISPR系统可实现异构CAR T细胞的非病毒工程
Nanna S Mikkelsen1, Sujan Ravendran1, Amalie D Broksø1
1Department of Biomedicine, Aarhus University, 8000 Aarhus C., Denmark.
概括
这项研究引入了一种更安全的,双链无断的CRISPR基编辑方法,用于工程化学抗原受体 (CAR) T细胞,增强它们的癌症治疗潜力.
科学领域:
- 基因编辑技术
- 细胞免疫疗法
- 基因组工程
背景情况:
- 开发强大的异构抗原受体 (CAR) T 细胞疗法需要复杂的基因组修改.
- 传统的CRISPR/Cas系统存在由于双链断裂 (DSB) 的基因组重组和基因毒性风险.
研究的目的:
- 通过使用无DSB基因编辑,开发一种更安全的多重基因编辑策略用于CAR T细胞治疗.
- 将基因淘汰的基因编辑与针对性的转基因整合相结合,以提高CAR T细胞的发育.
主要方法:
- 使用S. aureus Cas9 (SaCas9) mRNA基编辑器进行B2M和REGNASE-1的无DSB淘汰.
- 使用S. pyogenes Cas9 (SpCas9) 核酶在TRAC位点进行针对性的抗CD19 CAR转基因整合.
- 使用非病毒性ssDNA或病毒载体 (AAV6) 模板在原始人类T细胞中进行多重基因编辑.
主要成果:
- 实现了B2M (66%) 和REGNASE-1 (84%) 的高基数编辑频率.
- 在多达71%的细胞中成功集成抗CD19的CAR转基因.
- 与传统方法相比,证明平衡染色体转位减少了210倍,没有观察到对CAR T细胞功能的有害影响.
结论:
- 这种正交CRISPR/Cas方法为CAR T细胞的非病毒性多重基因工程提供了一种新且更安全的策略.
- 没有DSB的基因编辑显著降低了与CAR T细胞发育相关的基因毒性风险.
- 开发的方法增强了创造更安全,更有效的CAR T细胞疗法的潜力.
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