Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection.
Ultra-high efficiency T cell reprogramming at multiple loci with SEED-Selection.
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通过 SEED-Selection 在多个位点进行超高效 T 细胞重编程。
DOI：
10.1101/2024.02.06.576175
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Eyquem,Justin
中科院分区：
文献类型：
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作者：
Chang,ChristopherR;Vykunta,VivasvanS;Goodman,DanielB;Muldoon,JosephJ;Nyberg,WilliamA;Liu,Chang;Allain,Vincent;Rothrock,Allison;Wang,CharlotteH;Marson,Alexander;Shy,BrianR;Eyquem,Justin
T细胞特异性和功能的多重重编程可以产生强大的下一代细胞疗法。然而，目前的制造方法产生部分工程细胞的异质混合物。在这里，我们开发了一个一步的过程，利用一个名为合成外显子/表达干扰物（SEEDs）的修复模板家族，在多个目标位点富集具有敲入蛋白的未标记细胞。SEED工程将转基因整合与一对内源性表面蛋白的破坏联系起来，允许未修饰和部分编辑的细胞被免疫磁耗尽（SEED- selection）。我们设计的SEEDs可以完全重编程编码T细胞特异性、共受体表达和MHC表达的三个关键位点，在选择单个修饰后纯度高达98%，在6次同时编辑（3次敲入和3次敲除）时纯度高达90%。这些方法简单，与现有的临床生产工作流程兼容，并且可以很容易地适应其他基因座，以促进复杂基因编辑细胞疗法的生产。
Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.