美国加州大学旧金山分校Justin Eyquem等研究人员合作发现，SEED-Selection促进高效富集多个基因座编辑的原代T细胞。相关论文于2025年2月5日发表在《自然—生物技术》杂志上。

研究人员开发了一种一步法，通过使用一种名为合成外显子表达干扰物（SEED）的修复模板家族，富集含有多个靶点基因座敲入的未标记细胞。SEED将外源基因整合与配对的靶向内源性表面蛋白的破坏关联，同时保持未修改和部分编辑细胞的靶向表达，从而便于其去除（SEED-Selection）。

研究人员设计SEED以修改三个关键基因座，分别编码T细胞特异性、共受体表达和主要组织相容性复合体表达。

结果表明，在选择单一修改后纯度高达98%，对于六个同时编辑的细胞（包括三个敲入和三个敲除），纯度可达90%。该方法与现有的临床制造工作流兼容，并且可以轻松适应其他基因座，有助于复杂基因编辑细胞疗法的生产。

据悉，在多个基因座编辑T细胞的特异性和功能可以生成更有效的细胞疗法，但当前的制造方法会产生部分编辑细胞的异质混合物。

附：英文原文

Title: SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci

Author: Chang, Christopher R., Vykunta, Vivasvan S., Lee, Jae Hyun J., Li, Ke, Kochendoerfer, Clara, Muldoon, Joseph J., Wang, Charlotte H., Mazumder, Thomas, Sun, Yang, Goodman, Daniel B., Nyberg, William A., Liu, Chang, Allain, Vincent, Rothrock, Allison, Ye, Chun J., Marson, Alexander, Shy, Brian R., Eyquem, Justin

Issue&Volume: 2025-02-05

Abstract: Engineering T cell specificity and function at multiple loci can generate more effective cellular therapies, but current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here we develop a one-step process to enrich unlabeled cells containing knock-ins at multiple target loci using a family of repair templates named synthetic exon expression disruptors (SEEDs). SEEDs associate transgene integration with the disruption of a paired target endogenous surface protein while preserving target expression in nonmodified and partially edited cells to enable their removal (SEED-Selection). We design SEEDs to modify three critical loci encoding T cell specificity, coreceptor expression and major histocompatibility complex expression. The results demonstrate up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). This method is compatible with existing clinical manufacturing workflows and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

DOI: 10.1038/s41587-024-02531-6

Source: https://www.nature.com/articles/s41587-024-02531-6