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Nat Biotechnol. 2019 Sep 23. doi: 10.1038/s41587-019-0246-4. [Epub ahead of print]

In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma.

Ye L1,2,3, Park JJ1,2,3,4, Dong MB1,2,3,4,5,6, Yang Q1,2,3, Chow RD1,2,3,4, Peng L1,2,3, Du Y1,2,3, Guo J1,2,3, Dai X1,2,3, Wang G1,2,3, Errami Y1,2,3, Chen S7,8,9,10,11,12,13,14,15,16.

Author information

1
System Biology Institute, Integrated Science & Technology Center, West Haven, CT, USA.
2
Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
3
Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT, USA.
4
Yale M.D.-Ph.D. Program, Yale University School of Medicine, New Haven, CT, USA.
5
Immunobiology Program, Yale University School of Medicine, New Haven, CT, USA.
6
Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.
7
System Biology Institute, Integrated Science & Technology Center, West Haven, CT, USA. sidi.chen@yale.edu.
8
Department of Genetics, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
9
Center for Cancer Systems Biology, Integrated Science & Technology Center, West Haven, CT, USA. sidi.chen@yale.edu.
10
Yale M.D.-Ph.D. Program, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
11
Immunobiology Program, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
12
Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
13
Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
14
Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
15
Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.
16
Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA. sidi.chen@yale.edu.

Abstract

Targeting membrane proteins could improve the efficacy of T cell-based immunotherapies. To facilitate the identification of T cell targets, we developed a hybrid genetic screening system where the Sleeping Beauty (SB) transposon and single guide RNA cassette are nested in an adeno-associated virus (AAV). SB-mediated genomic integration of the single guide RNA cassette enables efficient gene editing in primary murine T cells as well as a screen readout. We performed in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8+ T cells in mouse models of glioblastoma (GBM). We validated screen hits by demonstrating that adoptive transfer of CD8+ T cells with Pdia3, Mgat5, Emp1 or Lag3 gene editing enhances the survival of GBM-bearing mice in both syngeneic and T-cell receptor transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.

PMID:
31548728
DOI:
10.1038/s41587-019-0246-4

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