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

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.