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Frontiers of Medicine

, Volume 11, Issue 4, pp 554–562 | Cite as

CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells

  • Yongping Zhang
  • Xingying Zhang
  • Chen Cheng
  • Wei Mu
  • Xiaojuan Liu
  • Na Li
  • Xiaofei Wei
  • Xiang Liu
  • Changqing Xia
  • Haoyi Wang
Research Article

Abstract

T cells engineered with chimeric antigen receptor (CAR) have been successfully applied to treat advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of Tcell activity is lymphocyte activation gene-3 (LAG-3). We successfully generated LAG-3 knockout Tand CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

Keywords

CAR-T CRISPR-Cas9 LAG-3 

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Notes

Acknowledgements

We would like to thank Junning Wei and Yi Yang (Beijing Cord Blood Bank) for their help in preparing the cord blood samples. This work was supported by the National Natural Science Foundation of China (No. 31471215), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA01010409), and the National High Technology Research and Development Program of China (863 Program, No. 2015AA020307). Haoyi Wang is supported by the “Young Thousand Talent Project.”

Supplementary material

11684_2017_543_MOESM1_ESM.pdf (147 kb)
Supplementary material, approximately 146 KB.

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Copyright information

© Higher Education Press and Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Yongping Zhang
    • 1
  • Xingying Zhang
    • 2
    • 3
  • Chen Cheng
    • 2
    • 4
  • Wei Mu
    • 2
    • 3
  • Xiaojuan Liu
    • 2
  • Na Li
    • 2
  • Xiaofei Wei
    • 6
  • Xiang Liu
    • 2
  • Changqing Xia
    • 1
    • 5
  • Haoyi Wang
    • 2
    • 3
  1. 1.Department of Hematology, Xuanwu HospitalCapital Medical UniversityBeijingChina
  2. 2.State Key Laboratory of Stem Cell and Reproductive Biology, Institute of ZoologyChinese Academy of SciencesBeijingChina
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.Graduate SchoolUniversity of Science and Technology of ChinaHefeiChina
  5. 5.Department of Pathology, Immunology and Laboratory MedicineUniversity of FloridaFloridaUSA
  6. 6.Beijing Cord Blood BankBeijingChina

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