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Frontiers of Medicine

Front. Med.    2017, Vol. 11 Issue (4) : 554-562
CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells
Yongping Zhang1, Xingying Zhang2,3, Chen Cheng2,4, Wei Mu2,3, Xiaojuan Liu2, Na Li2, Xiaofei Wei6, Xiang Liu2, Changqing Xia1,5(), Haoyi Wang2,3()
1. Department of Hematology, Xuanwu Hospital, Capital Medical University, Beijing 100053, China
2. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing100190, China
3. University of Chinese Academy of Sciences, Beijing 100049, China
4. Graduate School, University of Science and Technology of China, Hefei 230026, China
5. Department of Pathology, Immunology and Laboratory Medicine, University of Florida, FL 32611, USA
6. Beijing Cord Blood Bank, Beijing100176, China
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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 T cell activity is lymphocyte activation gene-3 (LAG-3). We successfully generated LAG-3 knockout T and 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     
Corresponding Authors: Changqing Xia,Haoyi Wang   
Just Accepted Date: 10 May 2017   Online First Date: 19 June 2017    Issue Date: 04 December 2017
 Cite this article:   
Yongping Zhang,Xingying Zhang,Chen Cheng, et al. CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells[J]. Front. Med., 2017, 11(4): 554-562.
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Yongping Zhang
Xingying Zhang
Chen Cheng
Wei Mu
Xiaojuan Liu
Na Li
Xiaofei Wei
Xiang Liu
Changqing Xia
Haoyi Wang
Fig.1  Gene editing of LAG-3 in human primary T cells using CRISPR-Cas9. (A) Schematic of sgRNA5 targeting site at LAG-3 locus. The red color represents the sgRNA targeting sequence, and the green color represents the PAM sequence. (B) LAG-3 knockout efficiency using different sgRNAs. Column plot shows the indel frequency (mean±SEM, n = 2) of LAG-3 analyzed by TIDE analysis using either forward (F) or reverse (R) sequencing primer. Experiments are performed in two biological replicates. (C) Representative mutant alleles in RNP-transfected cells compared with wild-type sequence (WT). sgRNA targeting sites are colored in red, PAM sequence in green, and mutations in blue; PCR products from each sample are sub-cloned, and each cloned allele was sequenced. 30/49 indicates the number of clones containing mutant alleles out of total clones sequenced.
Fig.2  Analysis of proliferation and phenotype of LAG-3 knockout T cells. (A) Fold expansion of control and LAG-3 knockout T cells. Total cell numbers were counted on days 3 and 7. (B) Immunophenotype of the gene-edited T cell was assessed 10 days after electroporation by the expression of CD4 and CD8, as well as the characteristics of naïve (CD45RO/CD62L+, TN), central memory (CD45RO+/CD62L+, TCM), and effector memory (CD45RO+ /CD62L, TEM) T cell subsets. Data shown are mean±SEM of two independent experiments. T-CTRL-UE and T-CTRL-E indicate T cells with or without mock electroporation, and LAG-3-KO-T indicates RNP-treated T cells.
Fig.3  Generation of LAG-3 knockout CAR-T cells. (A) LAG-3 knockout efficiency in CAR-T cells from three donors. (B) Representative sequences of mutated alleles in RNP-transfected CAR-T cells compared with wild-type CAR-T sequence (WT). sgRNA targeting sites are colored in red, PAM sequence in green, and mutations in blue; 60/71 indicates the number of clones containing mutant alleles out of total clones sequenced. (C) Flow cytometric analysis (mean±SEM, n = 2) of LAG-3 surface expression of control and LAG-3-KO-CAR-T cells from two donors, at day 3 post-electroporation. (D) Fold expansion (mean±SEM, n = 2) of RNP-treated CAR-T cells and control CAR-T cells from three donors. Data shown are mean±SEM of two independent experiments. CAR-T-CTRL-UE and CAR-T-CTRL-E indicate T cells with or without mock electroporation, and LAG-3-KO-CAR-T indicates RNP-treated cells.
Fig.4  In vitro characterization of LAG-3 knockout CD19 CAR-T cells. (A) Immunophenotype of the gene-edited CAR-T cell was assessed 10 days after electroporation by the expression of CD4 and CD8 as well as the characteristics of naïve, central memory, and effector memory T cell subsets. Data shown are mean±SEM of three independent experiments. (B) IL-2 and IFN-g production (mean±SEM, n = 2). (C) Cytotoxicity assay evaluating the cell lytic activity of T, CAR-T, and LAG-3-KO-CAR-T cells against K19 cells.
Fig.5  Evaluation of the antitumor activity of LAG-3 knockout CAR-T cellsin vivo. On day 0, NPG mice were injected intraperitoneally with 2×105Raji-luciferase cells. On day 3, mice received 1×107 CAR-T cells, LAG-3-KO-CAR-T cells, T cells, or PBS intraperitoneally. (A) Bioluminescent imaging result of NPG mice treated with CAR-T and LAG-3-KO-CAR-T cells on days 3, 10, and 31 (n = 4). (B) Bioluminescent signal (mean±SEM, n = 4) of NPG mice treated with CAR-T cells, LAG-3-KO-CAR-T cells, and T cells. (C) Survival curve of 50-day post treatment.
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