Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies

Theodore L Roth; P Jonathan Li; Franziska Blaeschke; Jasper F Nies; Ryan Apathy; Cody Mowery; Ruby Yu; Michelle L T Nguyen; Youjin Lee; Anna Truong; Joseph Hiatt; David Wu; David N Nguyen; Daniel Goodman; Jeffrey A Bluestone; Chun Jimmie Ye; Kole Roybal; Eric Shifrut; Alexander Marson

doi:10.1016/j.cell.2020.03.039

Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies

Cell. 2020 Apr 30;181(3):728-744.e21. doi: 10.1016/j.cell.2020.03.039. Epub 2020 Apr 16.

Authors

Theodore L Roth¹, P Jonathan Li², Franziska Blaeschke², Jasper F Nies², Ryan Apathy², Cody Mowery³, Ruby Yu², Michelle L T Nguyen², Youjin Lee², Anna Truong², Joseph Hiatt³, David Wu⁴, David N Nguyen⁵, Daniel Goodman², Jeffrey A Bluestone⁶, Chun Jimmie Ye⁷, Kole Roybal⁸, Eric Shifrut², Alexander Marson⁹

Affiliations

¹ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA. Electronic address: theodore.roth@ucsf.edu.
² Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
³ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
⁴ Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
⁵ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
⁶ Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA; Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, USA.
⁷ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA; Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA; Institute of Computational Health Sciences, University of California, San Francisco, San Francisco, CA, USA; Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, CA, USA.
⁸ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA; Sean N. Parker Autoimmune Research Laboratory, University of California, San Francisco, San Francisco, CA, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
⁹ Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. Electronic address: alexander.marson@ucsf.edu.

Abstract

Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin 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 knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor β (TGF-β) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.

Keywords: CRISPR; cell therapy; human T cell; knockins; pooled screen; single-cell RNA-seq.

Publication types

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

MeSH terms

Animals
Blood Cells
CRISPR-Cas Systems / genetics
Clustered Regularly Interspaced Short Palindromic Repeats / genetics
Gene Knock-In Techniques / methods*
Genetic Engineering / methods*
Humans
Immunotherapy / methods*
Mice
Mice, Inbred NOD
Mice, SCID
RNA, Guide, CRISPR-Cas Systems / genetics
Single-Cell Analysis / methods
T-Lymphocytes
Transcriptome / genetics

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

Publication types

MeSH terms

Substances

Grants and funding