SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci
Christopher Chang, Vivasvan S. Vykunta, Jae Hyun J. Lee, Lian 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 Ye, Alexander Marson, Brian R. Shy, Justin Eyquem
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.