Importantly, it was the unique combination of existing natural domains into a new combined DESynR human AP-1 TF, rather than overexpression of each domain individually or each contributing natural AP-1 TF individually, that programmed the full extent of improved functional behavior. (8/10)

Excitingly, to some extent top DESynR AP-1 TFs appeared to access these unique functional patterns by programming new transcriptional states not seen in base CAR T cells or with natural TF overexpression. (7/10)

Across a much wider array of functional assays in primary human T cells, DESynR AP-1 TFs showed unique and potentially improved patterns of functional behavior compared to natural AP-1 TFs or otherwise unmodified CAR T cells. (6/10)

For instance, a synthetic combination of BATF3’s N terminal effector domain, ATF3’s b-zip DNA binding domain, and BATF2’s C terminal effector domain, drove much greater proliferation in a long term repetitive stimulation assay than any natural AP-1 TF. (5/10)

Functional screening of these DESynR AP-1 TFs revealed numerous new human AP-1 TFs that outperformed overexpression of any natural AP-1 TF. (4/10)

We focused on the AP-1 family of basic lucine zipper TFs - they are both some of the most highly expressed in the genome (think Jun, Fos), as well as enriched in previous knockout and overexpression hits from prior natural gene screening in human T cell models. There are 24 natural human AP-1 TFs. We built over 13,000 new “DESynR” AP-1 TFs that the human genome could have constructed from existing AP1 TF domains. (3/10)

Eukaryotic genomes most commonly acquire new genes through duplication of existing genes followed by recombination. We set out to experimentally mimic and accelerate this process, building, integrating into a primary human T cell’s genome, and functionally testing over 10,000 new human transcription factors in a single experiment. (2/10)

Transcription Factors are central to cellular identity and function. The human genome currently contains ~1600 of them. But could there be productive human transcription factors that have not evolved? Out today in Cell, a tremendously talented graduate student Oliver Takacsi-Nagy @Oli_TN, together with the @Satpathology Lab, determined to find out. (1/10) authors.elsevier.com/sd/arti…

More broadly beyond ex vivo electroporation based T cell and iPSC editing, we characterized head-to-head toxicity, delivery, editing efficiency, and manufacturability properties for a comprehensive series of linear and circular, single stranded and double stranded DNA templates, hopefully of use as a reference across applications requiring intracellular DNA delivery. (9/11)

Ultra-large DNA integrations were compatible with clinical T cell manufacturing, and these cells were functional in vitro and in vivo, which will allow for clinical translation of more powerful genetically engineered cellular therapies. (8/11)