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Cited by 468Immunotherapy and Immune ResponsesRNA Interference and Gene DeliveryMicrobial Inactivation Methods

Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells

Open access
Van Tendeloo, Viggo F. I., Ponsaerts, Peter, Lardon, Filip, Nijs, Griet, Lenjou, Marc, Van Broeckhoven, Christine, Van Bockstaele, Dirk R., Berneman, Zwi N.·2001·Blood
DOI 10.1182/blood.v98.1.49
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Abstract

Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP+ cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34+ progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A–specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.

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  • Antigen-encoding RNA constructs used currently for preclinical studies have poly(A) tails measuring 30 to 70 nucleotides, whereas those administered in clinical trials measure 64 nucleotides.

    “Antigen-encoding RNA constructs used currently for preclinical studies have poly(A) tails measuring 30 to 70 nucleotides in length, 4,5 whereas those administered in clinical trials measure 64 nucleotides in length.8,12”

    From Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells (2006) · cites this paper as [4]

    The ‘Production of in vitro-transcribed mRNA’ section describes the RNA templates and transcription method but gives no poly(A)-tail length. Searches for 64-nucleotide and poly(A) tails found no passage addressing the length of RNA constructs administered in clinical trials. (LLM, conf=0.76)

    Experimental — accuracy not yet measured

  • In vitro-transcribed RNA used in such studies typically consists of a 5' cap structure followed by the sequence encoding the antigen of interest and a poly(A) tail 30 to 70 nucleotides in length.

    “In vitro-transcribed RNA generated from plasmid templates and used in such studies typically consists of a 5Ј cap structure followed by the sequence encoding the antigen of interest and a poly(A) tail 30 to 70 nucleotides in length.4,5”

    From Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells (2006) · cites this paper as [4]

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Cited by

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Authors
Van Tendeloo, V. F. I., Ponsaerts, P., Lardon, F., Nijs, G., Lenjou, M., Van Broeckhoven, C., Van Bockstaele, D. R., & Berneman, Z. N.
Affiliations
  • From the Laboratory of Experimental Hematology, Antwerp University Hospital, University of Antwerp; the Laboratory of Molecular Genetics, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), University of Antwerp; and the Laboratory of Cancer Research and Clinical Oncology, University of Antwerp, Antwerp, Belgium.
  • University of Antwerp (BE)
ORCID
Venue
Blood
Publisher
American Society of Hematology
Published
2001-07-01
Volume
98
Issue
1
Pages
49-56
ISSN
  • 0006-4971 (ISSN-L)
  • 1528-0020
DOI
10.1182/blood.v98.1.49
OpenAlex
W2046718813
Cited by
468
References
0
Citations by year
2026: 10 · 2025: 7 · 2024: 8 · 2023: 14 · 2022: 15 · 2021: 18 · 2020: 18 · 2019: 10 · 2018: 13 · 2017: 13 · 2016: 15 · 2015: 16 · 2014: 24 · 2013: 21 · 2012: 26
FWCI
6.70
Type
journal article
Language
en
Open access
Locations
  • Blood (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Ludwig Institute for Cancer Research
  • Vlaamse regering
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Microbial Inactivation Methods
Keywords
mRNA electroporation, dendritic cells, tumor antigen loading, monocyte-derived dendritic cells, Langerhans cells, Melan-A, cytotoxic T lymphocytes, dendritic cell maturation, lipofection, K562 cells, transfection efficiency, non-viral gene delivery
MeSH terms
Antigens, Neoplasm, Dendritic Cells, Humans, Lymphocyte Culture Test, Mixed, Neoplasm Proteins, RNA, Messenger, T-Lymphocytes, Cytotoxic, Transfection, Immunophenotyping, Genes, Reporter, Antigen Presentation, Gene Transfer Techniques, DNA, Complementary, Electroporation, Cell Culture Techniques, K562 Cells, MART-1 Antigen

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