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

Nat Biotechnol. 2019 Nov;37(11):1302-1313. doi: 10.1038/s41587-019-0246-4. Epub 2019 Sep 23.

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.

Publication types

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

MeSH terms

  • Animals
  • Antigens, CD / genetics
  • CD8-Positive T-Lymphocytes / metabolism
  • CD8-Positive T-Lymphocytes / transplantation*
  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • Dependovirus / genetics
  • Female
  • Gene Editing / methods*
  • Glioblastoma / genetics
  • Glioblastoma / immunology
  • Glioblastoma / therapy*
  • Humans
  • Immunotherapy, Adoptive
  • Male
  • Membrane Proteins / genetics*
  • Mice
  • N-Acetylglucosaminyltransferases / genetics
  • Neoplasm Proteins / genetics
  • Protein Disulfide-Isomerases / genetics
  • RNA, Guide / genetics
  • Receptors, Cell Surface / genetics
  • Transposases / genetics*
  • Transposases / metabolism
  • Xenograft Model Antitumor Assays

Substances

  • Antigens, CD
  • CD223 antigen
  • Membrane Proteins
  • Neoplasm Proteins
  • RNA, Guide
  • Receptors, Cell Surface
  • epithelial membrane protein-1
  • Mgat5 protein, human
  • N-Acetylglucosaminyltransferases
  • Transposases
  • sleeping beauty transposase, human
  • Protein Disulfide-Isomerases
  • PDIA3 protein, human