mRNA-based CAR T cell engineering: Unmodified mRNA enables high CAR expression without innate immune activation in T cells
Unmodified, uridine-containing mRNA is known to trigger antiviral immune responses, inflammatory signaling, and apoptosis in transfected cells. To avoid this and enable high expression, modified nucleosides such as N1-methylpseudouridine have become the gold standard for mRNA applications including T cell engineering, albeit at increased cost. Here, immune responses toward mRNA were evaluated across five primary human cell types. Remarkably, T cells, unlike other immune and non-immune cell types tested, exhibited no immune activation by unmodified mRNA. T cell viability and cytokine secretion remained unaffected, regardless of mRNA delivery method via lipid nanoparticles or electroporation. The absence of nucleotide modifications improved expression of chimeric antigen receptor (CAR) in activated T cells and CAR-T cell cytotoxic potency. By eliminating the need for mRNA-nucleoside modification in CAR-T cell engineering, our findings challenge existing paradigms and position mRNA as a non-inflammatory, minimally invasive and highly efficient tool for T cell engineering, while simplifying and reducing manufacturing cost.
Authors
- Dimitrios L. Wagner (ORCID: https://orcid.org/0000-0002-2189-3579)
- Anna Hiller (ORCID: https://orcid.org/0000-0002-5252-0986)
- Chantal Pichon (ORCID: https://orcid.org/0000-0003-3161-3937)
- David Nils Simon (ORCID: https://orcid.org/0000-0002-4099-4738)
- Samira Picht (ORCID: https://orcid.org/0000-0002-7761-5711)
- Charlotte Maeve Dunne (ORCID: https://orcid.org/0000-0002-4430-5717)
- Nourhan Kahwaji (ORCID: https://orcid.org/0000-0002-9326-8529)
- Arnd Kleyer (ORCID: https://orcid.org/0000-0002-2026-7728)
- Jonas Kath (ORCID: https://orcid.org/0000-0001-7721-7978)
- Hans-Dieter Volk
- Andriko Palmowski (ORCID: https://orcid.org/0000-0002-3456-0597)
- Michael Launspach (ORCID: https://orcid.org/0000-0001-5337-5605)
- Norman Michael Drzeniek (ORCID: https://orcid.org/0000-0001-6562-2351)
- Niklas Kotzian
- Antonia M Klaas
- Yaolin Pu
- Jasmin Melissa Prinz
- Gerhard Krönke
- Manfred Gossen
- Michael Schmueck-Henneresse
Institutions
- Université d'Orléans (FR)
- Inserm (FR)
- German Cancer Research Center (DE)
- Baylor College of Medicine (US)
- Institut Universitaire de France (FR)
- Heidelberg University (DE)
- Robert Koch Institute (DE)
- Frederiksberg Hospital (DK)
- Humboldt-Universität zu Berlin (DE)
- Universitätsklinikum Erlangen (DE)
- Cleopa (Germany) (DE)
- German Rheumatism Research Centre (DE)
- German Cancer Society (DE)
- CRUK/MRC Oxford Institute for Radiation Oncology (GB)
- Fraunhofer Institute for Translational Medicine and Pharmacology (DE)
- Methodist Hospital (US)
- Berlin-Brandenburger Centrum für Regenerative Therapien (DE)
- Helmholtz-Zentrum Hereon (DE)
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin (DE)
- Texas Children's Hospital (US)
- Children's Cancer Center (US)
- Deutsches Konsortium für Translationale Krebsforschung (DE)
- Freie Universität Berlin (DE)
- Charité - Universitätsmedizin Berlin (DE)
Publication Details
- Journal
- Molecular Therapy — Nucleic Acids
- Published
- 2026-01-05
- DOI
- https://doi.org/10.1016/j.omtn.2025.102805
- Primary Topic
- CAR-T cell therapy research
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- University of Pennsylvania
- European Commission
- Bundesministerium für Bildung und Forschung
- Berlin Institute of Health
- HORIZON EUROPE Framework Programme
- Einstein Centrum für Regenerartive Therapien, Charité-Universitätsmedizin Berlin