Fig 1: Sequence-engineered TPO mRNA yielded high TPO expression in vitro(A) Expression of HA-tagged mouse TPO mRNA with different nucleotide modifications in AML12 cells. Unmod, uridine without any modification; m5C/Ψ, 5-methylcytosine and pseudouridine; Ψ, pseudouridine; mo5U, 5-methoxyuridine; m1Ψ, N1-methylpseudouridine. IP-IB: HA, HA-tagged proteins in cell culture supernatant were immunoprecipitated using anti-HA magnetic beads followed by western blot with anti-HA antibody. (B) Expression of HA-tagged human TPO mRNA with different nucleotide modifications in HEK293T cells. (C) Molecular weight of mouse TPO secreted by different types of cells. (D) Molecular weight of N-glycans removed from mouse TPO secreted by AML12 and C2C12 cells. PNGase treatment could remove N-glycans on protein. (E) Molecular weight of N-glycans removed from human TPO secreted by HEK293T and HepG2 cells. (F) Molecular weight of human TPO-HA after removal of N-glycans by PNGase F treatment in the whole cell lysates of HEK293T.
Fig 2: Zyx−/− mice display macrothrombocytopenia.A, B Platelet count (A) and volume (B) were analyzed in whole blood from WT (n = 21) and Zyx−/− (n = 17) mice. Means are indicated by horizontal lines. ***P < 0.001 compared with WT mice by Mann–Whitney test in (A) and unpaired Student’s t-test with Welch correction in (B). C Representative images of Wright–Giemsa stained blood smears from WT and Zyx−/− mice (original magnification × 1,000). Scale bar: 10 μm. Platelets were indicated with arrows. D Representative transmission electron microscopy (TEM) images of WT and Zyx−/− platelets (original magnification ×13,500). Scale bar: 1 μm. Images were obtained from five mice in each genotype in (C) and (D). E WT and Zyx−/− mice were intravenously injected with NHS-biotin, and peripheral blood was taken at the indicated time points after injection. The percentage of biotinylated platelets was determined by flow cytometry; n = 10 mice per genotype. Data are expressed as means ± SD. F Serum TPO levels in WT and Zyx−/− mice; n = 7 mice per genotype. Means are indicated by horizontal lines.
Fig 3: In vivo safety profile of TPO mRNA-LNPs(A) Hematoxylin and eosin (H&E) staining of heart, liver, spleen, or kidney sections from the PBS and TPO mRNA-LNP treatment groups in the repeated injection experiment in Figure 4E. Scale bar, 100 µm. No obvious changes were found in (B) ALT and (C) AST levels after different doses of TPO mRNA-LNPs administration. Error bars represent the SD (n = 3). (D) H&E and reticulin staining of bone marrow after four weekly repeated injections of romiplostim and TPO mRNA-LNPs. Scale bar, 50 µm. Idiopathic pulmonary fibrosis (IPF) model sections were shown to be a positive control of reticulin staining.
Fig 4: Expression and biodistribution of lipid nanoparticle-packaged mRNA(A) Scheme of LNPs packaging. Percentages in brackets show molar ratios of lipids, and concentration of mRNA represents molar concentration of nucleotides. DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; PEG, polyethylene glycol. (B) Cryo-TEM images of TPO mRNA-LNPs. mRNA was encapsulated as arrows indicated. (C) Particle size distribution and polydispersity index (PDI) of LNPs. d.nm, diameter (nm). Error bas represent the SD (n = 3). (D) Whole cell lysates were prepared from HEK293T cells treated with TPO mRNA-LNP or transfected with HA-tagged EGFP and TPO mRNA using lipofectamine 2000. HA-tagged proteins in cell culture supernatant were immunoprecipitated using anti-HA magnetic beads, followed by western blot with anti-HA antibody. (E) In vivo imaging at different time points after 0.5 mg/kg luc mRNA-LNPs were administered in mice. Duration and biodistribution of mRNA-LNPs in mice administered through intravenous and intramuscular injection were recorded. Different scale of luminescence is indicated for different groups. (F) Bioluminescence intensity quantification after luciferase (luc) mRNA-LNPs were delivered through intravenous and intramuscular injection. Error bars represent the SEM (n = 3).
Fig 5: Therapeutic efficacy of TPO mRNA-LNPs in mouse thrombocytopenia models(A) Platelet count of acute thrombocytopenia model generated by intravenous injection of 0.25 mg/kg R300. Error bars represent the SEM (n = 4). (B) TPO levels increased on the first day after injection in the acute thrombocytopenia model. Error bars represent the SEM (n = 4). (C) TPO mRNA-LNPs promoted thrombopoiesis in the acute thrombocytopenia model. R300 was injected to deplete platelets at day 0, and LNPs were injected 1–2 days in advance. Error bars represent the SEM (n = 4). (D) Platelet count of prolonged thrombocytopenia model generated by repeated subcutaneous injection of low-dose R300. Each single dose was 0.08 mg/kg, and red arrows indicate the date of injection. Error bars represent the SEM (n = 5). (E) TPO levels in the prolonged thrombocytopenia model were measured with ELISA. Error bars represent the SD (n = 4). In the prolonged thrombocytopenia model, the therapeutic effects of TPO mRNA-LNPs, romiplostim, and recombinant TPO protein were compared at the dosages of 0.1 mg/kg (F) and 0.03 mg/kg (G). Red arrows indicate the date of injection. TPO mRNA-LNPs were injected through tail vein. Romiplostim was injected subcutaneously, and recombinant TPO was injected intraperitoneally. Dose and injection date of R300 was the same in (D). Error bars represent the SEM (n = 5).
Supplier Page from Abcam for Mouse Thrombopoietin ELISA Kit