CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro
Abstract CAR T cell therapy has been successfully used in the treatment of hematological malignancies, and the strategy that deletion of inhibitory receptor on the CAR T cell surface, such as PD-1, greatly enhance the antitumor effects. Here, we describe a one-step electroporation for the co-transfe...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-10-01
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| Series: | Experimental Hematology & Oncology |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s40164-023-00450-x |
| _version_ | 1797577565867606016 |
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| author | Ming Liu Linlin Zhang Mingtian Zhong Yihao Long Wenhui Yang Ting Liu Xingxu Huang Xiaodong Ma |
| author_facet | Ming Liu Linlin Zhang Mingtian Zhong Yihao Long Wenhui Yang Ting Liu Xingxu Huang Xiaodong Ma |
| author_sort | Ming Liu |
| collection | DOAJ |
| description | Abstract CAR T cell therapy has been successfully used in the treatment of hematological malignancies, and the strategy that deletion of inhibitory receptor on the CAR T cell surface, such as PD-1, greatly enhance the antitumor effects. Here, we describe a one-step electroporation for the co-transfection of Cas9:sgRNA and CAR plasmids on primary T cells to demonstrate the effect of SHP-1 deletion in CAR T cells. By using PiggyBac Transposase system, we can achieve more than 90% of T cells express CAR gene and nearly 60% SHP-1 knockout efficiency in T cells. We show that knockout of SHP-1 in CD133 CAR T cells resulted in significantly improve the cytolysis effect on CD133 positive glioma cell lines. We further demonstrate that the enhanced antitumor efficacy of SHP-1 deletion is due to the increased release of TNF-α, IL-2 and IFN-γ in vitro. Finally, we evaluated the biosafety of Cas9 genome editing and did not find any insertions of Cas9 and obvious editing in off-target sites in CAR T cells. These data provide an approach for achieving both intracellular inhibitory molecule, SHP-1 deletion and CD133 CAR gene over-expression in human T cells. And SHP-1 could be a new potential target for adoptive CAR T cells immunotherapy. |
| first_indexed | 2024-03-10T22:11:11Z |
| format | Article |
| id | doaj.art-37f639e56a5e468a964cca835dbf34f1 |
| institution | Directory Open Access Journal |
| issn | 2162-3619 |
| language | English |
| last_indexed | 2024-03-10T22:11:11Z |
| publishDate | 2023-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Experimental Hematology & Oncology |
| spelling | doaj.art-37f639e56a5e468a964cca835dbf34f12023-11-19T12:36:10ZengBMCExperimental Hematology & Oncology2162-36192023-10-011211510.1186/s40164-023-00450-xCRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitroMing Liu0Linlin Zhang1Mingtian Zhong2Yihao Long3Wenhui Yang4Ting Liu5Xingxu Huang6Xiaodong Ma7National Center for Respiratory Medicine, National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical UniversityModel Animal Research Center, MOE Key Laboratory of Model Animal for Disease Study, Nanjing UniversityKey Laboratory of Brain, Cognition and Education Sciences, Institute for Brain Research and Rehabilitation, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, South China Normal UniversityKey Laboratory of Brain, Cognition and Education Sciences, Institute for Brain Research and Rehabilitation, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, South China Normal UniversityKey Laboratory of Brain, Cognition and Education Sciences, Institute for Brain Research and Rehabilitation, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, South China Normal UniversityNational Center for Respiratory Medicine, National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical UniversityZhejiang LabKey Laboratory of Brain, Cognition and Education Sciences, Institute for Brain Research and Rehabilitation, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, South China Normal UniversityAbstract CAR T cell therapy has been successfully used in the treatment of hematological malignancies, and the strategy that deletion of inhibitory receptor on the CAR T cell surface, such as PD-1, greatly enhance the antitumor effects. Here, we describe a one-step electroporation for the co-transfection of Cas9:sgRNA and CAR plasmids on primary T cells to demonstrate the effect of SHP-1 deletion in CAR T cells. By using PiggyBac Transposase system, we can achieve more than 90% of T cells express CAR gene and nearly 60% SHP-1 knockout efficiency in T cells. We show that knockout of SHP-1 in CD133 CAR T cells resulted in significantly improve the cytolysis effect on CD133 positive glioma cell lines. We further demonstrate that the enhanced antitumor efficacy of SHP-1 deletion is due to the increased release of TNF-α, IL-2 and IFN-γ in vitro. Finally, we evaluated the biosafety of Cas9 genome editing and did not find any insertions of Cas9 and obvious editing in off-target sites in CAR T cells. These data provide an approach for achieving both intracellular inhibitory molecule, SHP-1 deletion and CD133 CAR gene over-expression in human T cells. And SHP-1 could be a new potential target for adoptive CAR T cells immunotherapy.https://doi.org/10.1186/s40164-023-00450-xSHP-1CD133CARCRISPR/Cas9PiggyBac Transposase |
| spellingShingle | Ming Liu Linlin Zhang Mingtian Zhong Yihao Long Wenhui Yang Ting Liu Xingxu Huang Xiaodong Ma CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro Experimental Hematology & Oncology SHP-1 CD133 CAR CRISPR/Cas9 PiggyBac Transposase |
| title | CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro |
| title_full | CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro |
| title_fullStr | CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro |
| title_full_unstemmed | CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro |
| title_short | CRISPR/Cas9-mediated knockout of intracellular molecule SHP-1 enhances tumor-killing ability of CD133-targeted CAR T cells in vitro |
| title_sort | crispr cas9 mediated knockout of intracellular molecule shp 1 enhances tumor killing ability of cd133 targeted car t cells in vitro |
| topic | SHP-1 CD133 CAR CRISPR/Cas9 PiggyBac Transposase |
| url | https://doi.org/10.1186/s40164-023-00450-x |
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