Fig 2: Organ toxicity of OX40L mRNA-LNP. Mice were euthanized at the end of treatment and their organs were removed. Heart, liver, spleen, lung, kidney and brain sections were stained with H&E to evaluate organ toxicity (scale bars: 100μm).

Fig 3: In vivo efficacy of intratumoral therapy with OX40L mRNA. (A) Timeline for treatment of H22 tumor-bearing mice. (B) Kaplan-Meier analysis of mouse survival after treatment with OX40L-mRNA and non-coding mRNA; log-rank test, p =0.0183. (C) Growth of subcutaneous tumors growth curves (median ± interquartile range [IQR]) of each group treated with OX40L-mRNA and the control. Two-way repeated-measures ANOVA using the Sidak multiple-comparisons test, **p≤ 0.01, ***p≤ 0.001, compared to non-coding mRNA. (D) Images of mice from the two groups at day 37. (E) Tumors; white circle, no tumor. (F) Tumor volume at day 37. Statistical significance was calculated via t test. ***p ≤ 0.001. (G) Tumor weight at day 37. Statistical significance was calculated via t test. ***p≤0.001.

Fig 4: OX40L mRNA elicited lymphocyte activation in the H22 tumor bearing mice. (A, C) T-cell activation by flow cytometry. CD4+ T cells and CD8+ T cells in the spleen of the H22 tumor-bearing BALB/c mice. (B, D) Quantitative analysis of the proportions in (A, C). Statistical significance was calculated via t test. **P ≤ 0.01, ***P ≤ 0.001. (E, F) Representative FACS profiles of CD69+ CD8+ and CD69+ CD4+ activated T cells. (G) Results of quantitative analysis of the proportions in (E, F). Statistical significance was calculated via t test. *P ≤0.05. (H) Immunohistochemistry of the mechanisms of action of OX40L mRNA in subcutaneous H22 tumors (scale bars: 100μm).

Fig 5: Optimization and characterization of encapsulated OX40L mRNA. (A) Structure of the optimized mRNA. Codons in red are optimized. (B) Identification of recombinant plasmid and restriction-digested plasmid by gel electrophoresis 1) 15000 bp marker, 2) 600 ng OX40L-pcDNA3.1 and 3) 200 ng Xba I -digested OX40L-pcDNA3.1. (C) Electrophoretic profile of mRNA 4) 2000 bp marker; 5) 2 ug OX40L-mRNA. (D) Size distribution of OX40L mRNA-LNP. (E) TEM image of OX40L mRNA-LNP (scale bar: 200 nm).