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Published February 21, 2020 | Submitted + Supplemental Material
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Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Abstract

Non-invasive epigenome editing is a promising strategy for engineering gene expression programs, yet potency, specificity, and persistence remain challenging. Here we show that effective epigenome editing is gated at single-base precision via 'keyhole' sites in endogenous regulatory DNA. Synthetic repressors targeting promoter keyholes can ablate gene expression in up to 99% of primary cells with single-gene specificity and can seamlessly repress multiple genes in combination. Transient exposure of primary T cells to keyhole repressors confers mitotically heritable silencing that persists to the limit of primary cultures in vitro and for at least 4 weeks in vivo, enabling manufacturing of cell products with enhanced therapeutic efficacy. DNA recognition and effector domains can be encoded as separate proteins that reassemble at keyhole sites and function with the same efficiency as single chain effectors, enabling gated control and rapid screening for novel functional domains that modulate endogenous gene expression patterns. Our results provide a powerful and exponentially flexible system for programming gene expression and therapeutic cell products.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. Posted February 20, 2020. We thank J. Halow and K. Lee for assistance with cell culture; M. Diegel and F. Neri for assistance with sequencing; J. Lazar for input on statistical analysis, Stanley Riddell (Fred Hutchinson Cancer Research Center) for generously providing the NALM-6 tumor cell line, and Teri Blevins (Fred Hutch Comparative Medicine) for all animal handling. This study was funded in part by NIH grants R33HL120752 and UM1HG009444 to J.A.S. and by a charitable contribution to the Altius Institute from GlaxoSmithKline PLC (M.S.W., C.C, J.P., E.S., J.B., H.L., B.V.B., R.A., S.V., E.O., D.D., H.W., P.Z, V.N., D.B., R.S., A.F, F.D.U., S.G, J.A.S.). D.B., Z.C., and S.E.B. were supported by the Howard Hughes Medical Institute (D.B.); the Schmidt Futures program (D.B. and Z.C.); IPD-WA State funding Y5 / 07-5568 (D.B.); NIH BTRR Yeast Resource Grant Y8-12 / 61-3650 (Z.C.); Bruce and Jeannie Nordstrom / Patty and Jimmy Barrier Gift for the Institute for Protein Design (Z.C.); Spark ABCA4 / 63-3819 (Z.C.); Open Philanthropy (D.B.); and a Burroughs Wellcome Fund Career Award at the Scientific Interface (S.E.B.). Author contributions: M.S.W., C.C., J.P., A.F., F.D.U., S.G. and J.A.S. designed the research. M.S.W., C.C., J.P., E.S., J.B., H.L., B.V.B., K.Q, G.H, A.F., R.A., S.V., E.O., and A.F. performed cell engineering experiments. Z.C., S.B., and D.B. designed obligate orthogonal heterodimer pairs. D.D., H.W., and D.B. performed RNA-seq and CUT&RUN experiments. P.Z. and V.N. performed imaging experiments. M.S.W., C.C., J.P., J.B., R.S., P.V., and V.N. analyzed data. M.S.W., C.C., S.G., and J.A.S. wrote the manuscript with input from other co-authors. Competing interests: M.S.W., C.C., S.G, A.F., F.D.U., and J.AS. are listed as inventors on patent applications related to the subject matter of the paper; D.B., Z.C., and S.E.B. are listed as inventors on patent applications related to obligate heterodimers; J.P. and S.E.B. are employees of Lyell Immunopharma, a for-profit biotechnology company. D.B. is a scientific advisor to Lyell Immunopharma. Data and materials availability: All RNA-seq and imaging data, software code used for analysis, protein sequences, protocols, and materials used in the experiments and data analysis will be made freely available.

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Submitted - 2020.02.19.956730v1.full.pdf

Supplemental Material - media-1.pdf

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Additional details

Created:
August 19, 2023
Modified:
October 19, 2023