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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年2月5日

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

摘要

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.