Sep 25, 2019

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

Nature Biotechnology
Lupeng YeSidi Chen

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.

  • References52
  • Citations2

References

  • References52
  • Citations2

Citations

Mentioned in this Paper

In Vivo
LAG3
T-Lymphocyte
Membrane
Mandibular Right Second Primary Molar
Genetic Screening (Procedure)
Nucleic Acid Sequencing
Mutant Chimeric Proteins
CD8-Positive T-Lymphocytes
Lymphocyte-activation gene 3 protein, human

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