Modular pooled discovery of synthetic knockin sequences to program durable cell therapies

Franziska Blaeschke; Yan Yi Chen; Ryan Apathy; Bence Daniel; Andy Y Chen; Peixin Amy Chen; Katalin Sandor; Wenxi Zhang; Zhongmei Li; Cody T Mowery; Tori N Yamamoto; William A Nyberg; Angela To; Ruby Yu; Raymund Bueno; Min Cheol Kim; Ralf Schmidt; Daniel B Goodman; Tobias Feuchtinger; Justin Eyquem; Chun Jimmie Ye; Julia Carnevale; Ansuman T Satpathy; Eric Shifrut; Theodore L Roth; Alexander Marson

doi:10.1016/j.cell.2023.08.013

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies

Cell. 2023 Sep 14;186(19):4216-4234.e33. doi: 10.1016/j.cell.2023.08.013.

Authors

Franziska Blaeschke¹, Yan Yi Chen¹, Ryan Apathy¹, Bence Daniel², Andy Y Chen³, Peixin Amy Chen¹, Katalin Sandor⁴, Wenxi Zhang⁴, Zhongmei Li¹, Cody T Mowery¹, Tori N Yamamoto¹, William A Nyberg¹, Angela To¹, Ruby Yu¹, Raymund Bueno⁵, Min Cheol Kim⁵, Ralf Schmidt¹, Daniel B Goodman⁶, Tobias Feuchtinger⁷, Justin Eyquem⁸, Chun Jimmie Ye⁹, Julia Carnevale¹⁰, Ansuman T Satpathy¹¹, Eric Shifrut¹, Theodore L Roth¹², Alexander Marson¹³

Affiliations

¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
² Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Pathology, Stanford University, Stanford, CA 94305, USA; Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA 94305, USA.
³ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Pathology, Stanford University, Stanford, CA 94305, USA; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁴ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Pathology, Stanford University, Stanford, CA 94305, USA.
⁵ Institute for Human Genetics (IHG), University of California, San Francisco, San Francisco, CA 94143, USA.
⁶ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94129, USA.
⁷ Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, Dr. von Hauner Children's Hospital, University Hospital, LMU Munich, Munich 80337, Germany; German Cancer Consortium (DKTK), Partner Site Munich, Munich 80336, Germany; National Center for Infection Research (DZIF), Munich 81377, Germany.
⁸ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Institute for Human Genetics (IHG), University of California, San Francisco, San Francisco, CA 94143, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94129, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA.
⁹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Institute for Human Genetics (IHG), University of California, San Francisco, San Francisco, CA 94143, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94129, USA; Institute for Computational Health Sciences, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁰ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94129, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA.
¹¹ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Pathology, Stanford University, Stanford, CA 94305, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94129, USA; Program in Immunology, Stanford University, Stanford, CA 94305, USA.
¹² Department of Pathology, Stanford University, Stanford, CA 94305, USA. Electronic address: troth@stanford.edu.
¹³ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Institute for Human Genetics (IHG), University of California, San Francisco, San Francisco, CA 94143, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94129, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94720, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: alexander.marson@ucsf.edu.

Abstract

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Keywords: CRISPR; chimeric antigen receptor; chronic stimulation; human T cells; immunotherapy; knockins; pooled screens; synthetic surface receptor; transcription factor.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

Cell- and Tissue-Based Therapy*
Exercise*
Gene Library
Humans
Immunotherapy
Research

Abstract

Publication types

MeSH terms

Grants and funding