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: Generation of 5′ UTR library for T cell-specific gene expression (A) General scheme of mRNA constructs used throughout the research. The constructs are composed of replaceable 5′ UTRs, KOZAK sequence, a gene of interest (GOI), a conserved 3′ UTR of HBA1 which is shared in all constructs, and a poly-A sequence. (B) GC/AU contents for each 5′ UTR used in the study. (C) Centroid secondary structure and free energy for EGFP-encoding constructs using the 5′ UTR of TOX , HBA1 , and CD39 ( ENTPD1 ). (D) Psuedouridine-corrected centroid secondary structure and free energy for EGFP-encoding constructs using the 5′ UTR of IL 2 , HBA1 , and CD247 . (E) Centroid free-energy calculation for all 14 constructs using either native uridine or pseudouridine. The blue-dashed line represents the mean of the native uridine calculation, and the red line represents the mean of the psuedouridine calculation. ∗ p < 0.05, paired t test. (F) Distribution of centroid free energy of all 14 EGFP encoding constructs including HBA1 5′ UTR, corrected for the usage of pseudouridine instead of native uridine. Constructs with energy lower than HBA1 5′ UTR are predicted to be more stable and thus enhance expression, while constructs with energy higher than HBA1 5′ UTR are predicated to be less stable and impair expression.

Article Snippet: IVT was performed using the HiScribe T7 ARCA mRNA Kit (NEB #E2065S) enriched with pseudouridine-UTP (Ψ-UTP).

Techniques: Gene Expression, Construct, Sequencing, Expressing

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: Modulation of gene reporter expression using T cell-specific UTRs (A) Representative flow cytometry analysis of PBMC-derived T cells mock-electroporated or electroporated with EGFP-encoding constructs using either α-globin or IFN G 5′ UTR. Data are collected 24 h post-electroporation. Plots are representative of 3 independent donors. (B) Full analysis of PBMC-derived T cell EGFP MFI across days from electroporation. Data are relative to EGFP expression using HBA1 5′ UTR. (C) Plot of centroid minimal energy, corrected for pseudouridine usage as in F, by normalized EGFP expression at day 2, compared with expression of HBA1 5′-UTR- EGFP. Correlation bar is in red. Note the low correlation. (D) Luciferase activity in PBMC-derived T cells electroporated with mRNA constructs harboring various 5′ UTRs, 24 h post-electroporation. Data are shown relative to the HBA1 control and represent mean ± SEM ( n = 3). (E) Luciferase activity in melanoma-derived tumor-infiltrating lymphocytes electroporated with mRNA constructs harboring various 5′ UTRs. Data are shown relative to the HBA1 control and represent mean ± SEM ( n = 3). (F) Representative flow cytometry analysis of HEK293 cells electroporated with mRNA constructs harboring various 5′ UTRs. (G) Full analysis of HEK293 cell EGFP MFI 24 h post-electroporation. Data are relative to EGFP expression using HBA1 5′ UTR. Data represent mean ± SEM ( n = 2). (H) Full analysis of HEK293 cell luciferase 24 h post-electroporation. Data are relative to luciferase activity using HBA1 5′ UTR. Data are normalized to HBA1 and presented as mean ± SEM ( n = 2).

Article Snippet: IVT was performed using the HiScribe T7 ARCA mRNA Kit (NEB #E2065S) enriched with pseudouridine-UTP (Ψ-UTP).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Construct, Electroporation, Luciferase, Activity Assay, Control

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: IVT was performed using the HiScribe T7 ARCA mRNA Kit (NEB #E2065S) enriched with pseudouridine-UTP (Ψ-UTP).

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