Journal: Molecular Therapy Advances

Article Title: Development of bicistronic plasmids and fusion proteins for clinical translation of tumor immune reprogramming

doi: 10.1016/j.omta.2026.201708

Figure Lengend Snippet: In vitro validation of delivery and function of bicistronic plasmids (A) Mouse Tnfsf9 (4-1BBL) and mouse IL12b-IL12a (IL-12) were assembled onto a single plasmid separated with 2A peptide. (B) Mouse 4-1BBL and mouse IL-12 were assembled onto a single plasmid separated by a (G4S)n linker. (C) B16-F10 cells were transfected with bicistronic plasmids as shown in the experimental timeline. (D) 4-1BBL expression levels after co-delivery of 4-1BBL and IL-12 as separate plasmids or bicistronic plasmids as determined by flow cytometry (one-way ANOVA, Dunnett’s test, compared to 4-1BBL/IL-12 as separate plasmids). (E) Surface IL-12 expression after co-delivery of 4-1BBL and IL-12 as separate plasmids or bicistronic plasmids as determined by flow cytometry (one-way ANOVA, Dunnett’s test, compared to 4-1BBL/IL-12 as separate plasmids). (F) IL-12 expression in cell culture supernatant after co-delivery of 4-1BBL and IL-12 as separate plasmids or bicistronic plasmids as determined by enzyme-linked immunosorbent assay (ELISA) (one-way ANOVA, Dunnett’s test, compared to 4-1BBL/IL-12 as separate plasmids). (G) B16-F10 cells were reprogrammed and co-cultured with splenocytes. (H) Comparison of IFNγ expression in cell culture supernatant between plasmids as determined by ELISA (one-way ANOVA, Dunnett’s test, compared to 4-1BBL/IL-12 as separate plasmids). (I) Comparison of IFNγ expression between plasmids with differing lengths of the (G4S)n tether as determined by ELISA (one-way ANOVA, Dunnett’s test, compared to 4G 5 12). Significance is represented by ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001. Each data bar represents mean ± standard error of the mean (SEM) with four biological replicates.

Article Snippet: Restriction enzymes NheI (New England Biolabs [NE Biolabs]; NEB, catalog no. R3131S) and SalI (NEB, catalog no. R0138S) were used to remove the inserts from Clonal Genes (Twist Biosciences) encoding mouse Tnfsf9 (4-1BBL) (residues 1–309 in 4G 5 12, 4T12, and residues 566–875 in 12T4) and mouse IL12b-IL12a (IL-12) (P40:P35) (residues 335–851 in 4G 5 12, residues 342–880, and residues 1–538 in 12T4) with the linkers (residues 310–334) and T2A sequences (residues 310–333 in 4T12 and 539–559 in 12T4) noted in .

Techniques: In Vitro, Biomarker Discovery, Plasmid Preparation, Transfection, Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecular Therapy Advances

Article Title: Development of bicistronic plasmids and fusion proteins for clinical translation of tumor immune reprogramming

doi: 10.1016/j.omta.2026.201708

Figure Lengend Snippet: B16-F10 tumor-bearing mice survived longer after intratumoral injection of 4-1BBL- and IL-12-loaded NPs (A) Experimental timeline of mice inoculated with B16-F10 tumors is shown. (B) Growth curves of B16-F10 tumors treated with systemic recombinant IL-12 (rIL-12), luciferase (fLuc) NPs, 4-1BBL/IL-12 NPs, 4G 5 12 NPs, or 4T12 NPs (two-way ANOVA, Dunnett’s test, compared to 4-1BBL/IL-12-treated mice). Each data bar represents mean ± SEM with eight biological replicates. (C) Survival curves for mice treated with rIL-12, luciferase NPs, or tAPC NPs (log rank test with Bonferroni correction for multiple comparisons). (D–I) Tumor growth curves for each treatment group. Data points represent mean ± SEM with eight biological replicates. Significance is represented by ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Restriction enzymes NheI (New England Biolabs [NE Biolabs]; NEB, catalog no. R3131S) and SalI (NEB, catalog no. R0138S) were used to remove the inserts from Clonal Genes (Twist Biosciences) encoding mouse Tnfsf9 (4-1BBL) (residues 1–309 in 4G 5 12, 4T12, and residues 566–875 in 12T4) and mouse IL12b-IL12a (IL-12) (P40:P35) (residues 335–851 in 4G 5 12, residues 342–880, and residues 1–538 in 12T4) with the linkers (residues 310–334) and T2A sequences (residues 310–333 in 4T12 and 539–559 in 12T4) noted in .

Techniques: Injection, Recombinant, Luciferase

Journal: Molecular Therapy Advances

Article Title: Development of bicistronic plasmids and fusion proteins for clinical translation of tumor immune reprogramming

doi: 10.1016/j.omta.2026.201708

Figure Lengend Snippet: Immunohistochemistry staining of harvested B16-F10 tumors reveals immune infiltration Tumor tissue excised from mice treated systemically with 3 ng/kg or 500 ng/kg of recombinant IL-12 or intratumorally with luciferase NPs, co-delivered 4-1BBL and IL12 plasmid NPs, 4G 5 12 NPs or 4T12 NPs. Melanin appears as beige brown. CD8 + cells are stained purple, CD4 + cells teal, and FOXP3 + cells dark brown. Scale bars, 250 μm.

Article Snippet: Restriction enzymes NheI (New England Biolabs [NE Biolabs]; NEB, catalog no. R3131S) and SalI (NEB, catalog no. R0138S) were used to remove the inserts from Clonal Genes (Twist Biosciences) encoding mouse Tnfsf9 (4-1BBL) (residues 1–309 in 4G 5 12, 4T12, and residues 566–875 in 12T4) and mouse IL12b-IL12a (IL-12) (P40:P35) (residues 335–851 in 4G 5 12, residues 342–880, and residues 1–538 in 12T4) with the linkers (residues 310–334) and T2A sequences (residues 310–333 in 4T12 and 539–559 in 12T4) noted in .

Techniques: Immunohistochemistry, Staining, Recombinant, Luciferase, Plasmid Preparation

Journal: Molecular Therapy Advances

Article Title: Development of bicistronic plasmids and fusion proteins for clinical translation of tumor immune reprogramming

doi: 10.1016/j.omta.2026.201708

Figure Lengend Snippet: Tumor rechallenge to assess durability of tAPC NP-induced anti-tumor effects (A) Long-term survivors were rechallenged with B16-F10 tumors on day 100 alongside age-matched naive mice (two-way ANOVA, Dunnett’s test, compared to naive mice). (B) Average size of new tumors in naive mice compared to 4-1BBL/IL-12-, 4G 5 12-, or 4T12-treated mice. Individual tumor growth in (C) naive mice ( n = 3), (D) 4-1BBL/IL-12 NP-treated mice ( n = 3), (E) 4G 5 12 NP-treated mice ( n = 2), and (F) 4T12 NP-treated mice ( n = 2). Data points represent mean ± SEM. Significance is represented by ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Restriction enzymes NheI (New England Biolabs [NE Biolabs]; NEB, catalog no. R3131S) and SalI (NEB, catalog no. R0138S) were used to remove the inserts from Clonal Genes (Twist Biosciences) encoding mouse Tnfsf9 (4-1BBL) (residues 1–309 in 4G 5 12, 4T12, and residues 566–875 in 12T4) and mouse IL12b-IL12a (IL-12) (P40:P35) (residues 335–851 in 4G 5 12, residues 342–880, and residues 1–538 in 12T4) with the linkers (residues 310–334) and T2A sequences (residues 310–333 in 4T12 and 539–559 in 12T4) noted in .

Techniques:

Journal: bioRxiv

Article Title: Activation-dependent lentiviruses enable antigen-specific T cell expansion and transduction

doi: 10.64898/2026.05.11.724165

Figure Lengend Snippet: (A) Pseudotyped lentiviral (LV) vectors designed to target 4-1BB. Co-display of a targeting ligand (e.g. 4-1BBL or anti-4-1BB scFv) alongside VSVGmut (K47Q + R354A) on the surface of an enveloped LV particle can enable cell-specific infection. Desired therapeutic cargo can be packaged inside LV vectors to achieve cell-specific gene delivery. (B) Transduction (% ZsGreen+) observed in h4-1BB+ (EGFR+) Jurkat T cells (top) or h4-1BB-(EGFR-) Jurkat T cells (bottom) by construct. Each bar represents triplicate data points per dose of virus added. Each virus was added in a dilution series with dextran, with each bar from left to right (per construct) representing a virus dose of: 1, 0.25, 0.0625, and 0.015625 µL. (C) Representative flow plots demonstrating transduction (ZsGreen+) observed in h4-1BB (EGFR+) Jurkat T cells with selected lead candidate constructs and pertinent controls at a single virus dose (0.0625 µL).

Article Snippet: Human 4-1BBL (Uniprot P41273) and murine 4-1BBL (Uniprot P41274) constructs were designed by us and obtained from Twist Biosciences and cloned into the pMD2 backbone.

Techniques: Infection, Transduction, Construct, Virus

Journal: bioRxiv

Article Title: Identification of Novel Extracellular Vesicles Scaffold Proteins for Versatile Cargo Engineering

doi: 10.64898/2026.01.04.697584

Figure Lengend Snippet: (A) Schematic diagram showing the EVs engineering of Cas9 mediated by RFTN1-N15. (B) Cas9 reporter assay analysis at 48 hours post EVs treatment. Scale bar: 20 μm. (C) Cas9 editing efficiency analysis as quantified by EGFP positive cells ratio from flow cytometry analysis. N=2. Values are plotted as mean ± SD. (D) Schematic diagram showing the EVs engineering of the minimal RISC comlpex mediated by RFTN1-N15. (E) EGFP silencing analysis at 48 hours post EVs treatment. Scale bar: 20 μm. (F) EGFP silencing efficiency analysis as quantified by EGFP mean fluorescence intensity from flow cytometry analysis. N=2. Values are plotted as mean ± SD. (G) Schematic diagram showing the EVs engineering of FCU1 mediated by RFTN1-N15. (H) Cellular toxicity assay analysis at 48 hours post EVs treatment. Scale bar: 100 μm. (I) Cell viability as quantified by CCK8 assay. N=2. Values are plotted as mean ± SD. (J) Schematic diagram showing the EVs engineering of type II transmembrane proteins mediated by RFTN1-N15. (K) Western blot analysis for engineered TMPRSS2 EVs. (L) Western blot analysis for engineered 4-1BBL EVs.

Article Snippet: The following antibodies were used: CD9 (abcam, AB263019), CD81 (Cell Signaling Technology, 56039S), Calnexin (abcam, ab22595), CD63 (Cell Signaling Technology, 2897), Cre recombinase (Cell Signaling Technology, 15036), TSG101 (abcam, ab125011), TMPRSS2 (Cell Signaling Technology, 39665), 4-1BBL (Cell Signaling Technology, 59127), Arginase1 (Cell Signaling Technology, 93668), iNOS (abcam, ab178945), HRP Goat Anti-Rabbit IgG (H+L) (ABclonal, AS014), HRP Goat Anti-Mouse IgG (H+L) (ABclonal, AS003).

Techniques: Reporter Assay, Flow Cytometry, Fluorescence, CCK-8 Assay, Western Blot