High-Efficiency Transfection of Primary Human and Mouse T Lymphocytes Using RNA Electroporation

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• 作者：Yangbing Zhao ; Zhili Zheng ; Cyrille J. Cohen ; Luca Gattinoni ; Douglas C. Palmer ; Nicholas P. Restifo ; Steven A. Rosenberg and Richard A. Morgan
• 关键词：mRNA ; electroporation ; transfection ; T lymphocytes
• 刊名：Molecular Therapy
• 出版年：2006
• 出版时间：January 2006
• 年：2006
• 卷：13
• 期：1
• 页码：151-159
• 全文大小：521 K

文摘

The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90 % transgene expression with >80 % viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56 % GFP⁺ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes.