High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation

Yangbing Zhao, Zhili Zheng, Cyrille J. Cohen, Luca Gattinoni, Douglas C. Palmer, Nicholas P. Restifo, Steven A. Rosenberg, Richard A. Morgan

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)151-159
Number of pages9
JournalMolecular Therapy
Volume13
Issue number1
DOIs
StatePublished - Jan 2006

Keywords

  • Electroporation
  • T lymphocytes
  • Transfection
  • mRNA

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Pharmacology
  • Drug Discovery

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