Pooled Knock-In Targeting for Genome Engineering of Cellular Immunotherapies

Adoptive transfer of genetically-modified immune cells holds great promise for cancer immunotherapy. CRISPR knock-in targeting can improve cell therapies, but more high-throughput methods are needed to test which knock-in gene constructs most potently enhance primary cell functions in vivo . We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates, and applied it to perform pooled knock-in screens in primary human T cells. Pooled knock-in of dozens of unique barcoded templates into the TCR-locus revealed gene constructs that enhanced fitness in vitro and in vivo . We further developed Pooled Knock-In Sequencing (PoKI-Seq), combining single-cell transcriptome analysis and pooled knock-in screening to measure cell abundance and cell state ex vivo and in vivo . This platform nominated a novel TGFβR2–41BB chimeric receptor that improved solid tumor clearance. Pooled knock-in screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knock-in programs for cell therapies. Development of a platform to assess the functional effects of pools of knock-in constructs targeting one locus allows discovery of constructs promoting anti-tumor activity in T cells.