Abstract
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.