Selection of human hematopoietic stem cells bearing the intended functional edit by transient AND-gate reporters

Targeted genomic integration of gene-sized cassettes into hematopoietic stem and progenitor cells (HSPCs) for genetic disease treatment is constrained by the low efficiency of homology-directed repair (HDR) and frequent unintended genetic changes at the editing site. Here, to overcome these challenges, we introduce selection by means of artificial transactivators (SMArT), which transiently implements AND reporter gates to achieve templated integration of a functional cassette at the target site. HDR-edited HSPCs were enriched to 80-100% purity through transient selector expression, whereas cells carrying undesired and potentially genotoxic on-target edits were preferentially depleted. Xenotransplantation of SMArT-enriched HSPCs in immunodeficient mice resulted in fully HDR-edited human grafts with the selector no longer detectable. SMArT strategies were implemented through clinically compliant manufacturing and selectors. They support both safe harbor integration and gene correction, can preserve physiological transcriptional regulation and are portable across loci also with polyfunctional editors. Overall, SMArT strategies may broaden the therapeutic applicability of gene-sized editing while reducing its genotoxic burden.