Humes Daryl, Rainwater Stephanie, Overbaugh Julie
Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Mol Ther Methods Clin Dev. 2020 Oct 27;20:30-38. doi: 10.1016/j.omtm.2020.10.020. eCollection 2021 Mar 12.
Efficient delivery of nucleic acids for the engineering of primary T cells is central to the study of the basic biology of these key immune effector cells and has clinical implications. To date, lentiviral vectors delivering guide RNAs for CRISPR-Cas9 editing are not optimal for use in primary cells. Herein, we describe the T cell optimized for packaging (TOP) vector for delivering guide RNAs and transgenes into primary T cells. The TOP vector produces high-titer virus compared to a routinely used guide RNA vector, resulting in a ~10-fold increase in transduction in T cells. Moreover, a TOP vector expressing a chimeric antigen receptor and a guide RNA targeting the T cell receptor showed an ~5- to 9-fold increased transduction efficiency with ~2- to 3-fold higher expression compared to the commonly used epHIV7 vector and was simultaneously able to mediate efficient knockout of the endogenous T cell receptor in >71% of transduced cells upon Cas9 electroporation. The increased packaging of the TOP vector genome into viral particles appears to contribute to its higher transduction efficiency. The TOP vector represents an optimal tool for tandem delivery of transgenes and guide RNAs to primary T cells for use in functional screens and immunotherapy applications.
将核酸高效递送至原代T细胞用于这些关键免疫效应细胞的基础生物学研究至关重要，且具有临床意义。迄今为止，用于CRISPR-Cas9编辑的递送导向RNA的慢病毒载体并不适合用于原代细胞。在此，我们描述了一种用于包装的T细胞优化（TOP）载体，用于将导向RNA和转基因递送至原代T细胞。与常规使用的导向RNA载体相比，TOP载体产生高滴度病毒，导致T细胞转导增加约10倍。此外，与常用的epHIV7载体相比，表达嵌合抗原受体和靶向T细胞受体的导向RNA的TOP载体转导效率提高约5至9倍，表达水平提高约2至3倍，并且在Cas9电穿孔后，能够在>71%的转导细胞中介导内源性T细胞受体的有效敲除。TOP载体基因组更多地包装到病毒颗粒中似乎有助于其更高的转导效率。TOP载体是用于将转基因和导向RNA串联递送至原代T细胞以用于功能筛选和免疫治疗应用的最佳工具。
