肿瘤细胞治疗研究
英文原题：Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy.
嵌合抗原受体（CAR）T 细胞的制备通常需要使用病毒递送系统以实现高转基因表达。
嵌合抗原受体（CAR）T细胞的制备通常使用病毒递送系统，以实现较高转基因表达。然而，该方法成本较高，并可能导致CAR随机整合至基因组，从而产生多种缺点，包括转基因表达差异、功能基因沉默及潜在致癌转化。本研究优化了利用大供体DNA递送的非病毒CRISPR/Cas9基因组编辑方法，将抗肿瘤单链可变片段（scFv）敲入CD3的N端，高效制备受体融合蛋白（FP）T细胞。这些细胞的FP整合进入TCR/CD3复合物；与CAR-T相比，其基因表达差异更小，转染后细胞扩增良好。CD3 FP T细胞主要为CD8+效应记忆T细胞，并在体内外表现出抗肿瘤活性。通过将scFv整合至其他CD3亚基及CD28，研究还制备了双靶点FP T细胞。与病毒介导的方法相比，该技术为制备表达肿瘤靶向受体、用于癌症免疫治疗的T细胞提供了一种灵活的替代方案。
Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3 and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3 FP T cells were predominantly CD8 + effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.
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