使用 CRISPR 和 AAV 优化和验证 CAR 转导到人类原代 NK 细胞。
Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.
机构信息
Center for Childhood Cancer and Blood Diseases, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.
Department of Pediatrics, The Ohio State University, Columbus, OH, USA.
出版信息
Cell Rep Methods. 2022 Jun 13;2(6):100236. doi: 10.1016/j.crmeth.2022.100236. eCollection 2022 Jun 20.
Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.
人类原代自然杀伤 (NK) 细胞正被广泛应用于癌症免疫疗法。然而，这些细胞的基因编辑方法存在转导效率低、细胞死亡率高以及在扩增后转基因表达丢失等问题。在这里，我们开发了一种使用 CRISPR（Cas9/RNP）和 AAV 对 NK 细胞进行定点基因插入的高效方法。我们比较了设计用于通过不同 DNA 修复机制、同源臂长度和病毒浓度介导基因插入的 AAV 载体。然后，我们验证了该方法用于将 CD33 特异性 CAR 定点插入原代人 NK 细胞。CAR 的转导效率高，其表达在扩增后保持稳定，并提高了对 AML 靶标的疗效。
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