1. Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies
- Author
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Roth, Theodore L, Li, P Jonathan, Blaeschke, Franziska, Nies, Jasper F, Apathy, Ryan, Mowery, Cody, Yu, Ruby, Nguyen, Michelle LT, Lee, Youjin, Truong, Anna, Hiatt, Joseph, Wu, David, Nguyen, David N, Goodman, Daniel, Bluestone, Jeffrey A, Ye, Chun Jimmie, Roybal, Kole, Shifrut, Eric, and Marson, Alexander
- Subjects
Biomedical and Clinical Sciences ,Oncology and Carcinogenesis ,Immunology ,Biotechnology ,Human Genome ,Immunization ,Genetics ,Gene Therapy ,Vaccine Related ,Cancer ,5.2 Cellular and gene therapies ,Underpinning research ,Development of treatments and therapeutic interventions ,1.1 Normal biological development and functioning ,Animals ,Blood Cells ,CRISPR-Cas Systems ,Clustered Regularly Interspaced Short Palindromic Repeats ,Gene Knock-In Techniques ,Genetic Engineering ,Humans ,Immunotherapy ,Mice ,Mice ,Inbred NOD ,Mice ,SCID ,RNA ,Guide ,Kinetoplastida ,Single-Cell Analysis ,T-Lymphocytes ,Transcriptome ,CRISPR ,cell therapy ,human T cell ,knockins ,pooled screen ,single-cell RNA-seq ,Biological Sciences ,Medical and Health Sciences ,Developmental Biology ,Biological sciences ,Biomedical and clinical sciences - 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.
- Published
- 2020