AAVS1 site-specific integration of the CAR gene into human primary T cells using a linear closed-ended AAV-based DNA vector
| Autor: | Jianxiang Wu, Taiming Li, Wei Chen, Chun Zhang, Qungang Zhou, Liang Tan, WenSheng Li |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Cytotoxicity Immunologic medicine.medical_treatment T cell T-Lymphocytes Virus Integration Antigens CD19 Genetic Vectors Gene Expression Biology Cell Line Immunophenotyping 03 medical and health sciences 0302 clinical medicine Plasmid Cancer immunotherapy Antigens Neoplasm Transduction Genetic Drug Discovery Gene Order Genetics medicine Humans Vector (molecular biology) Transgenes Molecular Biology Gene Genetics (clinical) Receptors Chimeric Antigen Transfection Dependovirus Chimeric antigen receptor Cell biology 030104 developmental biology medicine.anatomical_structure 030220 oncology & carcinogenesis Molecular Medicine Cytokine secretion Genetic Engineering |
| Zdroj: | The journal of gene medicineREFERENCES. 22(4) |
| ISSN: | 1521-2254 |
| Popis: | Background Use of chimeric antigen receptor (CAR) T cells has become a promising strategy in cancer immunotherapy. However, safety in clinical application is also one of the most controversial issues. Methods In the present study, we investigated the application of a non-viral site-directed vector (CELiD [closed-ended linear duplex DNA]) dependent on adeno-associated virus (AAV) genomes for the purpose of safe CAR-T engineering. We co-electroporated CD19-CAR encoding "CELiD" vectors with plasmid pCMV-Rep into human T cells and ensured stably transfected CAR-T cells by G418 selection. The efficiency of AAVS1 site-specific integration was analyzed by a real-time polymerase chain reaction. Results CAR-T cells engineered by CELiD vectors could be established within 20 days with up to 22.8% AAVS1 site-specific integration efficiency. CAR expression and cytokine secretion of CAR modified T cells were evaluated in vitro. Abundant effector cytokines were produced by the CAR-T cells engineered by CELiD vectors compared to control T cells and the killing efficiency of target cells was estimated to as high as 75% in vitro. Conclusions With the help of the AAV-derived CELiD vector, CAR genes were preferentially integrated into the AAVS1 site. This technology could be utilized in human T cell modification and remove the safety constraints of CAR-T therapy. |
| Databáze: | OpenAIRE |
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