Human T cell engineering via serial delivery of mRNA encapsulated within lipid nanoparticles
Year: 2026
Type: article
Abstract: Human T cells modified with nucleic acids constitute a powerful and emerging therapeutic modality for cancer, autoimmune disorders, and aging-related diseases. However, delivering nucleic acids, such as mRNA encoding synthetic receptors, transcription factors, cytokines, or genome editors to T cells can be challenging, as nucleic acids can unintentionally reduce viable T cell yield and function, particularly when delivered sequentially ex vivo . To address this challenge, we evaluated the efficiency of serial non-viral delivery of synthetic mRNA encapsulated within lipid nanoparticles (LNPs) incorporating cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and fusogenic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). DOTAP/DOPE-containing LNPs delivered mRNA more efficiently than a clinically benchmarked 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-containing LNP formulation, resulting in significantly higher protein expression in primary human T cells. Furthermore, DOTAP/DOPE-LNP delivery resulted in 100% higher yield of live cells compared to electroporation, an advantage that compounded over serial rounds, with LNP-treated cultures maintaining substantially more viable cells through all four transfections. Additionally, serial transfection of mRNAs encoding a therapeutically relevant chimeric antigen receptor (CAR) payload produced functional CAR T cells. These results demonstrate that LNPs can be a viable platform for serial and iterative T cell engineering using multiple mRNA payloads, providing an alternative to electroporation for the engineering of therapeutic T cells.
Institution: University of Wisconsin–Madison
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,900
Funders: National Science Foundation, Wisconsin Alumni Research Foundation, University of Wisconsin-Madison, University of Wisconsin Carbone Cancer Center, National Institutes of Health, Office of the Vice Chancellor for Research and Graduate Education, University of Wisconsin-Madison, National Cancer Institute, National Institute of General Medical Sciences, Division of Materials Research, Division of Graduate Education
Awards: G1712026730, G187035202, Wisconsin MRSEC, G4004214011, G4305155978, NSF Engineering Research Center for Cell Manufacturing Technologies (CMaT), G5813506420, G6420496198, G6832210154, G9000352744, Label-free imaging of CAR T cell metabolism, UW Comprehensive Cancer Center Support, Assembly of Novel Gene Editing Particles to Understand Genome Surgery in Patient-Derived Cells
OpenAlex ID: W7206213539