In vivo CRISPR screening in CD8 T cells with AAV–Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma
| Autor: | Quanjun Yang, Sidi Chen, Xiaoyun Dai, Lupeng Ye, Guangchuan Wang, Matthew B. Dong, Jianjian Guo, Ryan D. Chow, Lei Peng, Jonathan J. Park, Youssef Errami, Yaying Du |
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| Rok vydání: | 2019 |
| Předmět: |
Male
Adoptive cell transfer medicine.medical_treatment T cell Protein Disulfide-Isomerases Biomedical Engineering Transposases Receptors Cell Surface Bioengineering CD8-Positive T-Lymphocytes Biology N-Acetylglucosaminyltransferases Immunotherapy Adoptive Applied Microbiology and Biotechnology Article Mice 03 medical and health sciences 0302 clinical medicine Genome editing Antigen Antigens CD Cell Line Tumor medicine Animals Humans Cytotoxic T cell Guide RNA 030304 developmental biology Gene Editing 0303 health sciences Membrane Proteins Immunotherapy Dependovirus Xenograft Model Antitumor Assays Lymphocyte Activation Gene 3 Protein Neoplasm Proteins 3. Good health Cell biology medicine.anatomical_structure Molecular Medicine Female CRISPR-Cas Systems Glioblastoma 030217 neurology & neurosurgery CD8 RNA Guide Kinetoplastida Biotechnology |
| Zdroj: | Nature biotechnology |
| ISSN: | 1546-1696 1087-0156 |
| DOI: | 10.1038/s41587-019-0246-4 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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