mRNA-Based Car T Cell Engineering: Unmodified mRNA Enables High CAR Expression Without Innate Immune Activation in T Cells

Authors

• Nourhan Kahwaji
• Niklas Kotzian
• Jasmin Melissa Prinz
• Yaolin Pu
• Jonas Kath
• Samira Picht
• Anna Luisa Hiller
• Antonia Klaas
• Charlotte Maeve Dunne
• Michael Launspach
• Andriko Palmowski
• Arnd Kleyer
• David Nils Simon
• Dimitrios Laurin Wagner
• Chantal Pichon
• Gerhard Krönke
• Michael Schmueck-Henneresse
• Hans-Dieter Volk
• Manfred Gossen
• Norman Michael Drzeniek

Language

English

Publication Date

3-12-2026

Journal

Molecular Therapy Nucleic Acids

DOI

10.1016/j.omtn.2025.102805

PMID

41834823

PMCID

PMC12984635

PubMedCentral® Posted Date

1-5-2026

PubMedCentral® Full Text Version

Post-print

Abstract

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.

Keywords

MT, Oligonucleotides, Therapies and Applications, CAR t cells, messenger RNA, unmodified mRNA, innate immune activation, T cell engineering, nucleoside modifications, N1-methylpseudouridine, nonviral gene transfer, lipid nanoparticles, DNA-free CAR T manufacturing, chimeric antigen receptor, immunogenicity, uridine, toll-like receptor

Published Open-Access

yes

Recommended Citation

Kahwaji, Nourhan; Kotzian, Niklas; Prinz, Jasmin Melissa; et al., "mRNA-Based Car T Cell Engineering: Unmodified mRNA Enables High CAR Expression Without Innate Immune Activation in T Cells" (2026). Faculty, Staff and Students Publications. 8691.
https://digitalcommons.library.tmc.edu/baylor_docs/8691