Archives

  • 2026-10
  • 2026-09
  • 2026-08
  • 2026-07
  • 2026-06
  • 2026-05
  • 2025-12
  • 2025-11
  • 2025-10

    • Serial LNP–mRNA Delivery in Human T Cells

    2026-09-23

    Research Question

    The study asked whether a non-viral lipid nanoparticle system could deliver mRNA repeatedly to primary human T cells while preserving more viable, usable cells than electroporation. The central formulation comparison was between an SM102 ionizable-lipid LNP containing DSPC and related formulations in which DSPC was replaced or supplemented with the cationic lipid DOTAP and the fusogenic helper lipid DOPE. The authors also examined whether delivery-associated signals represented labeled-RNA uptake, translated protein production, or functional activity from a therapeutic mRNA payload.

    The reported system is SM102-based rather than ALC-0315-based. Its relevance to ALC-0315 and mRNA-delivery research is therefore comparative: it illustrates how ionizable-lipid, cationic-lipid, helper-lipid, cholesterol, and PEG-lipid composition can influence ex vivo mRNA delivery without providing direct evidence about ALC-0315.

    Study Design and Methods

    Formulation comparison. Primary human CD3+ T cells were isolated from healthy donors, activated for 48 hours, and cultured in serum-free ImmunoCult-XF medium supplemented with IL-2. The benchmark SM102-DSPC LNP contained SM102, DSPC, cholesterol, and DMG-PEG2000 at 50%, 10%, 38.5%, and 1.5% molar ratios, respectively. SM102-DOTAP5 replaced 5% DSPC with DOTAP, and SM102-DD contained 5% DOTAP plus 5% DOPE, with the remaining formulation components adjusted as described by the authors. DOTAP was titrated from 5% to 40% in the initial screen.

    The LNPs were prepared by vortex mixing an aqueous mRNA phase with an ethanol lipid phase at a 1.8:1 aqueous-to-ethanol ratio. Reporter transcripts encoded eGFP or firefly luciferase and were chemically modified with an M6 cap and N1-methyl-pseudouridine. Particle size and polydispersity were measured by dynamic light scattering, and mRNA encapsulation was measured with a RiboGreen assay. The tested formulations had hydrodynamic dimensions below 300 nm, polydispersity indices below 0.3, and at least 90% encapsulation efficiency in the reported formulation screens.

    Delivery and expression readouts. Reporter delivery was assessed after 24 hours using eGFP flow cytometry and luciferase activity. GhostRed780 staining was used to identify membrane-compromised cells; it was a viability readout rather than evidence of mRNA uptake or translation. To separate delivery from functional expression, Cy5-labeled eGFP mRNA was encapsulated in SM102-DSPC, SM102-DOTAP5, or SM102-DD LNPs. Flow cytometry measured Cy5 signal as a labeled-RNA-associated uptake signal and eGFP fluorescence as translated protein. The authors therefore could identify Cy5-positive/eGFP-negative cells in which labeled RNA was detected without corresponding reporter protein output.

    Cell entry and trafficking. Pharmacological inhibitors were used to probe macropinocytosis, clathrin-mediated endocytosis, dynamin-dependent processes, caveolae-associated uptake, and cholesterol-dependent membrane organization. Separately, DiI-labeled LNPs were imaged with LysoTracker and Hoechst staining by confocal microscopy. DiI reported nanoparticle-associated lipid signal, not translated protein. Partial DiI/LysoTracker colocalization was used to assess intracellular trafficking, but the experiment did not directly quantify cytosolic mRNA release.

    Serial delivery and functional payload. Reporter mRNAs were delivered to primary T cells four times at 24-hour intervals using SM102-DD LNPs, SM102-DSPC LNPs, or electroporation with the EO115 or CM138 pulse codes. Total cells, viable cells, transfected cells, and dead transfected cells were followed over time. In a second series, mRNA encoding a FLAG-tagged, second-generation uPAR-targeting CAR was delivered four times at 24-hour intervals. CAR surface expression, cell survival, uPAR-positive target-cell depletion, activation markers, exhaustion markers, and cytotoxicity against uPAR-expressing HEK293 target cells were measured. IFN-γ secretion was measured after co-culture.

    Main Findings

    Paper finding: formulation composition changed reporter expression. Adding DOTAP to the SM102-DSPC formulation increased eGFP and firefly-luciferase expression in primary T cells, although higher DOTAP levels also reduced viability. SM102-DOTAP5 was selected as the lead DOTAP formulation because it showed the lowest cytotoxicity among the tested DOTAP-containing formulations. Adding DOPE to create SM102-DD further increased reporter delivery at lower mRNA doses compared with SM102-DOTAP5. In the discussion, the authors report that replacing DSPC with 5% DOTAP produced a 50-fold increase in transfected-cell yield and that adding DOPE produced a further 1.8-fold improvement; these values are the authors’ reported formulation-level comparisons.

    Paper finding: culture medium strongly influenced delivery. SM102-DD-mediated eGFP and luciferase expression was higher in serum-free ImmunoCult-XF than in RPMI containing 10% fetal bovine serum. The formulations also showed medium-dependent changes in zeta potential, consistent with serum-protein adsorption altering the particle surface. This result means that LNP performance in a primary T-cell culture cannot be interpreted independently of the surrounding medium or inferred directly from Jurkat-cell experiments performed under different culture conditions.

    Paper finding: labeled-RNA uptake and translation were not equivalent. SM102-DD produced the highest combined Cy5-positive/eGFP-positive population among the compared formulations. However, Cy5-positive/eGFP-negative cells were detected with every formulation. Thus, the study directly demonstrates that detecting labeled mRNA-associated signal is not sufficient to establish cytosolic release, translation, or functional protein production. The higher SM102-DD signal reflected an improvement in both the measured uptake-associated and translation-associated populations, but the assay did not establish that every internalized transcript reached the cytosol.

    Paper finding with interpretation: uptake was consistent with macropinocytosis. EIPA and methyl-β-cyclodextrin reduced eGFP expression and the yield of transfected live cells, whereas inhibitors of clathrin-mediated, dynamin-dependent, and caveolae-associated pathways did not significantly reduce transfected-cell yield. Confocal imaging showed robust intracellular DiI signal for SM102-DD and SM102-DOTAP5 but minimal signal for SM102-DSPC. The authors interpret these findings as evidence that SM102-DD enters primary T cells predominantly through macropinocytosis and traffics through the endolysosomal system. The partial separation of DiI from LysoTracker signal is compatible with material outside lysosomes or in earlier compartments, but it is not a direct measurement of endosomal escape.

    Paper finding: repeated LNP delivery preserved viable-cell output. SM102-DD maintained reporter expression across four daily transfections and produced consistently greater cumulative expression than SM102-DSPC under the same dosing schedule. Electroporation produced a higher absolute number of live transfected cells at some early time points, but serial electroporation caused greater cell loss over the full course. By the later time points, SM102-DD cultures contained significantly more total viable cells and preserved a larger population of viable untransfected bystander cells. The abstract reports a 100% higher live-cell yield for DOTAP/DOPE-LNP delivery compared with electroporation, while the detailed results emphasize the cumulative nature of this advantage rather than superiority in every single-round transfection metric.

    Paper finding: serial CAR mRNA delivery generated functional cells. SM102-DD delivered uPAR CAR mRNA to the cell surface after each dose, and CAR surface expression increased with the single-dose mRNA amount tested. During serial delivery, LNP-treated cultures maintained higher total and live-cell counts than electroporated cultures at several time points, although electroporation temporarily produced more live CAR-positive cells through 72 hours and slightly more at 96 hours. Both approaches generated CAR-positive cells capable of depleting endogenous uPAR-positive cells in culture. In co-culture with engineered uPAR293 targets, SM102-DD-derived CAR-T cells showed CAR-dependent cytotoxicity, increased activation, and IFN-γ secretion, with functional activity comparable to the electroporation-derived CAR-T cells under the reported assay conditions.

    Interpretation. The study supports a delivery tradeoff: electroporation can provide strong early transfection, whereas SM102-DD LNPs can reduce cumulative cell loss during repeated exposure. The useful endpoint for a serial manufacturing workflow may therefore be the final number of viable, functionally modified cells rather than the percentage or absolute number of transfected cells after only one delivery event.

    Limitations

    • The experiments were performed ex vivo with activated primary human T cells and did not evaluate in vivo biodistribution, organ targeting, or tissue-specific delivery.
    • The study tested SM102 and DMG-PEG2000 formulations, not ALC-0315, and did not provide a systematic comparison across alternative ionizable lipids or PEG-lipid chemistries.
    • Particle size, polydispersity, encapsulation efficiency, and medium-dependent zeta potential were measured, but the supplied study text does not report a formal storage-stability or long-term physicochemical stability study.
    • Macropinocytosis and endosomal trafficking were inferred from inhibitor sensitivity and DiI/LysoTracker imaging. Direct cytosolic mRNA release or a quantitative endosomal-escape assay was not reported.
    • The Cy5 assay distinguished labeled-RNA-associated signal from eGFP protein output, but Cy5 signal can include labeled RNA that remains intracellular without being translated. Likewise, DiI signal tracked lipid-associated material rather than mRNA translation.
    • Reporter mRNAs were appropriate tools for comparing delivery and viability, but they do not establish the therapeutic performance of a CAR, transcription factor, cytokine, or genome-editing payload. The CAR experiments addressed function more directly, yet they remained in vitro.
    • The study used different donor numbers for different experiments, and donor-to-donor variation in absolute yield was observed. The authors also note that the donor cohort was not age- or sex-matched.
    • CAR-associated cell death was not fully separable from electroporation-associated cell death in some assays. Activation and exhaustion measurements were mainly endpoint assessments, and inflammatory responses such as IL-6 were not evaluated after repeated LNP exposure.
    • The supplied article is a journal pre-proof rather than the definitive Version of Record; the paper notes that copyediting, typesetting, and further production checks could still identify errors.

    Source

    Ramamurthy A, Scimonelli G, Murphy WL, Saha K. Human T Cell Engineering via Serial Delivery of mRNA Encapsulated within Lipid Nanoparticles. Molecular Therapy – Nucleic Acids (2026), journal pre-proof. https://doi.org/10.1016/j.omtn.2026.103081

    Research Materials

    The local material record identifies APExBIO as the supplier for EZ Cap™ Cy5 EGFP mRNA (5-moUTP), SKU R1011, linked at https://www.apexbt.com/ez-captm-cy5-egfp-mrna-5-moutp.html. This provenance statement is limited to the Cy5-labeled reporter-mRNA material used for the study’s uptake-versus-translation assay and does not make the product the subject of the paper.