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CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer.

ABSTRACT: The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy toward EBVaGC. These studies reveal possibility of generating PD-1-disrupted CTL by CRISPR-Cas9 system and demonstrate enhanced immune response of these PD-1-disrupted CTLs to the EBV-LMP2A antigen and superior cytotoxicity to the EBV-positive gastric cancer cell. In addition, when combined with low-dose radiotherapy, these PD-1-disrupted CTLs mediated an impressive antitumor effect in a xenograft mouse model of EBVaGC. Taken together, these studies illustrate PD-1/PD-L1-mediated immune tolerance of EBVaGC and provide a new strategy for targeting immune checkpoints to break the tolerance for the T cell-based adoptive therapy.

SUBMITTER: Su S

PROVIDER: S-EPMC5283615 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer.

Su Shu S Zou Zhengyun Z Chen Fangjun F Ding Naiqing N Du Juan J Shao Jie J Li Lin L Fu Yao Y Hu Bian B Yang Yang Y Sha Huizi H Meng Fanyan F Wei Jia J Huang Xingxu X Liu Baorui B

Oncoimmunology 20161122 1

The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy t ...[more]

PMID: 28197365

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Project description:BackgroundA barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery.MethodsA library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels.FindingsVirus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing.InterpretationThese results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing.FundingThe work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.

Dataset Information

CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer.

Publications

CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer.

Similar Datasets

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