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Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification

Wilken, Matthew S. and Ciarlo, Christie and Pearl, Jocelynn and Bloom, Jordan and Schanzer, Elaine and Liao, Hanna and Boyken, Scott E. and Van Biber, Benjamin and Queitsch, Konstantin and Heberlein, Gregory and Federation, Alexander and Acosta, Reyes and Vong, Shinny and Otterman, Ericka and Dunn, Douglass and Wang, Hao and Zrazhevskey, Pavel and Nandakumar, Vivek and Bates, Daniel and Sandstrom, Richard and Chen, Zibo and Urnov, Fyodor D. and Baker, David and Funnell, Alister and Green, Shon and Stamatoyannopoulos, John A. (2020) Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20200220-160637361

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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.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2020.02.19.956730DOIDiscussion Paper
ORCID:
AuthorORCID
Chen, Zibo0000-0003-2990-2895
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.
Funders:
Funding AgencyGrant Number
NIHR33HL120752
NIHM1HG009444
GlaxoSmithKline PLCUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Schmidt Futures ProgramUNSPECIFIED
IPD-WA State FundingY5/07-5568
NIHY8-12/61-3650
Institute for Protein DesignUNSPECIFIED
SparkABCA4/63-3819
Open PhilanthropyUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Record Number:CaltechAUTHORS:20200220-160637361
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200220-160637361
Official Citation:Promoter keyholes enable specific and persistent multi-gene expression programs in primary T cells without genome modification. Matthew S. Wilken, Christie Ciarlo, Jocelynn Pearl, Jordan Bloom, Elaine Schanzer, Hanna Liao, Scott E. Boyken, Benjamin Van Biber, Konstantin Queitsch, Gregory Heberlein, Alexander Federation, Reyes Acosta, Shinny Vong, Ericka Otterman, Douglass Dunn, Hao Wang, Pavel Zrazhevskey, Vivek Nandakumar, Daniel Bates, Richard Sandstrom, Zibo Chen, Fyodor D. Urnov, David Baker, Alister Funnell, Shon Green, John A. Stamatoyannopoulos. bioRxiv 2020.02.19.956730; doi: https://doi.org/10.1101/2020.02.19.956730
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101440
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Feb 2020 00:31
Last Modified:21 Feb 2020 00:31

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