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

Identification of T cell targets to improve immunotherapies is of prime interest. To facilitate large-scale CRISPR screens directly in T cells in vivo, here, we developed a hybrid genetic screening system with adeno associated virus (AAV) and the Sleeping Beauty (SB) transposon, where the transposon is nested in the viral vector. The approach enables efficient gene editing in primary murine T cells and genomic integration of the sgRNA cassette for screen readout. We performed focused in vivo AAV-SB-CRISPR screens in CD8(+) T cells in mouse models of glioblastoma (GBM) and identified membrane protein targets. Adoptive transfer of CD8(+) T cells with Pdia3, Mgat5, Emp1, or Lag3 gene editing enhance the survival of GBM-bearing mice in both syngeneic and TCR-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.