Sterner Rosalie M, Cox Michelle J, Sakemura Reona, Kenderian Saad S
Mayo Clinic Medical Scientist Training Program, Mayo Clinic College of Medicine and Science; Department of Immunology, Mayo Clinic.
Division of Hematology, Mayo Clinic.
J Vis Exp. 2019 Jul 22(149). doi: 10.3791/59629.
Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSF CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.
嵌合抗原受体T（CAR-T）细胞疗法是一种前沿且可能具有革命性的癌症新治疗选择。然而，其在癌症治疗中的广泛应用存在重大局限性。这些局限性包括出现独特的毒性，如细胞因子释放综合征（CRS）和神经毒性（NT），以及实体瘤中CAR-T细胞的扩增、效应功能和抗肿瘤活性有限。增强CAR-T疗效和/或控制CAR-T细胞毒性的一种策略是在CAR-T细胞制造过程中编辑CAR-T细胞本身的基因组。在此，我们描述了通过转导含有针对粒细胞巨噬细胞集落刺激因子（GM-CSF）的引导RNA和Cas9的慢病毒构建体，在CAR-T细胞中使用CRISPR/Cas9基因编辑。作为一个例子，我们描述了CRISPR/Cas9介导的GM-CSF基因敲除。我们已经表明，与野生型CAR-T细胞相比，这些GM-CSF CAR-T细胞在维持关键T细胞功能的同时，有效减少了GM-CSF的产生，并在体内增强了抗肿瘤活性。
