To systematically conduct a pooled knock-in screen for gene constructs capable of improving therapeutic T cell fitness, Roth et al. CRISPR-modified human T cells in the TRAC locus with a stably barcoded, NY-ESO-1-specific TCRɑ/β and 36 native or modified candidate genes. Single-cell RNA sequencing revealed the phenotypes of the engineered cells. A TGFβR2-41BB chimeric receptor increased T cell proliferation and cell killing in TGFβ-containing media, induced an effector-type phenotype enriched in cytokine (IFNγ, IL-2) expression, and improved tumor infiltration and growth control in a melanoma xenograft model.
Contributed by Alex Najibi
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 beta (TGF-beta) 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.
Author Info: (1) Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francis
Author Info: (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) 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) 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. (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. (6) 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. (7) 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. (8) 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. (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. (10) 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. (11) 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. (12) 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. (13) 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. (14) 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. (15) 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. (16) 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. (17) 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. (18) 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. (19) 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.
