title: High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation.
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.
authors.affiliation: Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Yangbing
authors.initials: Y
authors.lastname: Zhao
authors.forename: Zhili
authors.initials: Z
authors.lastname: Zheng
authors.forename: Cyrille J
authors.initials: CJ
authors.lastname: Cohen
authors.forename: Luca
authors.initials: L
authors.lastname: Gattinoni
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Palmer
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Restifo
authors.forename: Steven A
authors.initials: SA
authors.lastname: Rosenberg
authors.forename: Richard A
authors.initials: RA
authors.lastname: Morgan
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism; D017209:Apoptosis; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D018274:Electroporation; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D019041:L-Selectin / Q000235:genetics; D058965:MART-1 Antigen; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis / Q000235:genetics; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; C104948:CTAG1B protein, human; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008565:Membrane Proteins; C548668:Mlana protein, mouse; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D016159:Tumor Suppressor Protein p53; D019041:L-Selectin; D049452:Green Fluorescent Proteins