Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered mRNA backbones for gene expression in human T cells

doi: 10.1016/j.omtn.2026.102913

Figure Lengend Snippet: T cell-specific UTRs for optimized CAR expression, reactivity, and tonic signaling (A). Representative flow cytometry analysis of PBMC-derived T cells electroporated with CD19-CAR-encoding constructs using various 5′ UTRs. (B) Quantification of percentages of CD19-CAR + cells in (A). Data are normalized to the HBA1 UTR condition and presented as mean ± SEM from four independent experiments ( n = 4). Kruskal-Wallis test revealed no statistically significant difference ( p = 0.29). (C) MFI of CD19-CAR + cells in (A) normalized to the HBA1 UTR. Data represent four independent experiments ( n = 4). Kruskal-Wallis test indicated a significant difference among groups ( p < 0.005); Dunn’s post hoc test revealed significant reduction in the TNF-UTR group compared to HBA1 ( p = 0.0022). (D) Secreted IFN-γ levels in co-culture supernatants of PBMC-derived T cells electroporated with CD19-CAR mRNA using different 5′ UTRs and CD19 + NALM6 target cells. Data from two healthy donors (D29 and D40) are shown at various effector-to-target (E:T) ratios. (E) Left: interferon gamma ELISA in media taken from co-cultures of PBMC-derived T cells electroporated with CD19-CAR mRNA using different UTRs, either together with CD19 + (filled) or CD19 − (NALM6 KO, empty) at an E:T ratio of 4:1. Right: delta of interferon gamma secretion of E between the co-culture of electroporated T cells with CD19 + vs. CD19 − NALM6 cells. Bars represent mean ± SEM, n = 5 (CD19 + co-cultures) and n = 2 (CD19 − co-cultures) per construct. (F) Flow cytometry analysis of virus-specific T cells (VSTs) electroporated with mRNA constructs encoding for CD19-CAR using various UTRs. Note that cells are grown without target cells to demonstrate tonic signaling. (G) Pie charts of PD-1/TIM-3 population distribution of (F). Data are representative of two independent experiments.

Article Snippet: Cells were harvested 24–48 h post-electroporation, washed in FACS buffer (PBS with 2% FBS), and stained with the following fluorochrome-conjugated antibodies: CD19 CAR Detection Reagent, Biotin (Miltenyi Biotec, #130-129-550), followed by secondary staining with Anti-Biotin-APC (Miltenyi Biotec, REAfinity #130-113-854); TIM-3 APC-Cy7 (BioLegend, #345025); 4-1BB PE-Cy7 (BioLegend, #309820); Viability Dye eFluor 506/AmCyan (Thermo Fisher Scientific, #65-0866-14).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Construct, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Virus