The standard manufacturing workflow for approved CAR T cell therapies involves isolating a patient’s own T cells, introducing a CAR-encoding gene, expanding the cells, and reinfusing them. Viral vectors achieve stable CAR expression but carry well-known liabilities: the risk of insertional mutagenesis, potential immunogenicity, limited cargo capacity, labor-intensive production, and costs that restrict broader clinical access. Electroporation, the clinical standard for mRNA transfection of T cells, requires specialized equipment and compromises membrane integrity, reducing viability and altering cellular physiology in ways that demand additional safety evaluation.

Messenger RNA offers an attractive middle path. Because it does not integrate into the genome and drives only transient CAR expression, it confers a self-limiting safety profile, confining receptor expression to a defined window of days. mRNA-based CAR T cells have already shown efficacy against acute lymphoblastic leukemia, melanoma, and Hodgkin lymphoma comparable to virally transduced cells. The challenge has always been delivery: naked mRNA is inherently unstable and cannot cross the cell membrane on its own, so a carrier is essential.

The research team, led by investigators at Shanghai University and the Shanghai Cell Therapy Group, synthesized a library of eight novel ionizable lipids as structural analogs of previously reported candidates effective in immune cell transfection. Each lipid was formulated into a nanoparticle by microfluidic mixing with a fixed excipient composition: cholesterol for membrane integrity and stability, the phospholipid DOPE to facilitate endosomal escape, and the PEG-lipid DMG-PEG2000 to confer colloidal stability and reduce nonspecific uptake. Holding the excipient ratios constant allowed the researchers to isolate the effect of the ionizable lipid head group during screening.

Dynamic light scattering and RiboGreen assays confirmed that all eight formulations were monodisperse, with Z-average diameters between 79.6 and 97.2 nanometers, polydispersity indices between 0.107 and 0.180, and mRNA encapsulation efficiencies above 95 percent. When screened in the murine dendritic cell line DC2.4 and in activated human peripheral blood mononuclear cells using eGFP mRNA, four of the eight candidates transfected more than 90 percent of DC2.4 cells, and lipid 6 emerged as the clear leader, delivering the highest eGFP positivity rates and mean fluorescence intensity and surpassing the clinically validated benchmark lipid ALC-0315.

In vivo validation using firefly luciferase mRNA in mice showed that lipid 6 elicited significantly higher luciferase expression than the benchmark lipids SM-102 and ALC-0315 at the six-hour time point for both intravenous and intramuscular routes. Intravenous injection produced the strongest systemic signal, whereas intramuscular administration yielded a more prolonged, localized signal that remained detectable at the injection site 72 hours after dosing. The researchers then systematically optimized the formulation using a design-of-experiments approach, screening 26 formulations across two rounds by varying the stoichiometry of the four LNP components. Both the ionizable lipid and DMG-PEG2000 concentrations exerted the strongest influence on transfection outcomes, and the team selected a formulation with reduced PEG-lipid content to minimize potential immunogenicity.

Titrating the nitrogen-to-phosphate ratio revealed that eGFP positivity increased with the ratio and plateaued at a value of 5, while mean fluorescence intensity peaked at a ratio of 10 before declining. Supplementing the transfection with recombinant APOE4 boosted efficiency, which plateaued at 1 microgram per milliliter, and an mRNA dose of 150 nanograms per 500,000 cells proved saturating. Under the finalized protocol, more than 90 percent of primary human T cells, natural killer cells, and dendritic cells expressed eGFP, and tumor-infiltrating lymphocytes isolated from hepatocellular carcinoma tissues exceeded 97 percent positivity. Both activated and unactivated T cells were transfected efficiently, at greater than 95 percent and greater than 80 percent positivity respectively. Storage testing showed the nanoparticles remained remarkably stable at 4 degrees Celsius, maintaining high cell viability and roughly 100 percent eGFP expression for up to 110 days, but performance collapsed within two weeks at 25 or 37 degrees Celsius.

The pivotal demonstration came when the team encapsulated mRNA encoding a MUC1-targeting CAR in lipid 6. Roughly 90 percent of transfected T cells expressed the CAR protein 24 hours after transfection, with CD4-positive and CD8-positive subset ratios unchanged and no significant upregulation of the exhaustion markers PD-1, LAG-3, or TIM-3. In real-time cytotoxicity assays, the engineered cells showed significantly enhanced killing of MUC1-expressing MDA-MB-468 breast cancer cells and SKOV3 ovarian cancer cells at every effector-to-target ratio tested, accompanied by markedly increased secretion of the Th1-type cytokines IL-2, TNF-alpha, and IFN-gamma. Longitudinal tracking showed CAR expression peaked at 24 hours and declined to below 5 percent by day 4, yet the cells retained significantly higher tumor-killing capacity than controls at each of the first three days after transfection, all while maintaining normal proliferation and viability.

Transcriptomic sequencing of lipid 6-transfected T cells reinforced the mechanistic picture. Gene Ontology enrichment pointed to upregulation of gene sets involved in transmembrane transport, membrane dynamics, cell junction organization, and intracellular trafficking, consistent with enhanced endosomal escape and cytosolic mRNA release. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed robust activation of cytokine-cytokine receptor interactions, T cell receptor signaling, chemokine signaling, and Th1/Th2 differentiation pathways, indicating that the delivery process triggered physiological T cell activation without exhaustion or aberrant inflammatory signaling. Notably, unlike liver-tropic lipid nanoparticles that depend on APOE-mediated uptake, lipid 6 appears to operate in immune cells through alternative endocytic pathways, though the precise intracellular trafficking and endosomal escape mechanisms remain to be fully elucidated.

The authors are candid about the study’s limitations. All cellular assessments were confined to in vitro experiments, so long-term antitumor efficacy, biodistribution, and biosafety in tumor-bearing animal models remain unverified. Only a MUC1-targeted CAR was examined, leaving the platform’s generality across other tumor antigens untested, and scalable GMP-level production processes have not yet been established. The transient nature of mRNA expression, while a safety advantage, may also limit therapeutic persistence in solid tumor microenvironments. Even so, the work establishes a rationally designed, nonviral platform that overcomes the insertional mutagenesis risks, cellular damage, and prohibitive costs associated with viral vectors and electroporation, and it points toward a future in which CAR T cells and other engineered immune cell therapies can be manufactured more safely, more scalably, and more affordably.

Subject of Research: Lipid nanoparticle-mediated mRNA delivery for nonviral CAR T cell engineering

Article Title: Lipid-based mRNA chimeric antigen receptor T cells for enhanced in vitro antitumor activity

Article References: Gao, H., Fang, Y., Tian, Z., Guo, C., Wang, P., Liu, T., Sun, Y., Zhang, P., & Qian, Q. (2026). Lipid-based mRNA chimeric antigen receptor T cells for enhanced in vitro antitumor activity. iScience, 29(10), Article 117390. https://doi.org/10.1016/j.isci.2026.117390

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117390

Keywords: CAR T cells, lipid nanoparticles, mRNA delivery, ionizable lipids, immunotherapy, nonviral gene delivery, MUC1, T cells, endosomal escape, cancer therapy, transient expression, ex vivo engineering