Cationic LNP-formulated mRNA expressing Tie2-agonist in the lung endothelium prevents pulmonary vascular leakage

Figures

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  Figure 1. cLNP-mediated delivery leads to mRNA uptake predominantly in murine pulmonary capillary ECs (A) Cryoelectron microscopy, (B) dynamic light scattering analysis of particle size (Z-Ave), PDI, and (C) zeta potential of cLNP. (D) Luciferase assay of indicated tissue samples from mice 6 h after i.v. injection of 1.5 mg/kg of cLNP containing mRNA encoding for nonsecreted Firefly luciferase (mRNAFLUC) shown by relative light units (RLUs) per milligram of tissue. Data are represented as mean ± SEM; p < 0.0001. (E) Experimental workflow of single-cell RNA sequencing (scRNA seq) analysis of murine lung cells and results of loupe-based t-SNE clustering of murine lung cell types clustered by single-cell transcriptional analysis, showing a total of 13 distinct cell-type clusters as indicated by numbers. (F) t-SNE plots showing Log2 levels for Luciferase-encoding mRNAFLUC. NK, natural killer.

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  Figure 2. Expression and Tie2 activation by mRNA-directed expression of human COMP-Ang-1 in vitro (A) General structure of mRNA with signal peptide (SP) sequence flanked by designated 5′ and 3′ UTR regions plus defined polyA-tail. Schematic representation of protein domain structure of the natural protein Ang1 and the derived engineered humanized fusion proteins COMP-Ang1 and CMP-Ang1. Protein domains of human Ang1: SCD, super-clustering domain; CCOD, coiled-coil domain; L, linker; FLD, Fibrinogen-related domain; the multimerization domains of COMP (orange) and of CMP protein (red) are indicated. (B) Expression, (C) secretion, and (D) multimerization of full-length Ang1 and Ang1 derivates in HeLa cells. Cells were transfected for 2 h with mRNA-52 encoding Ang1, mRNA-76 encoding COMP-Ang1, or mRNA-59 encoding CMP-Ang1. Cell lysates (B) and supernatants (C, D) were harvested at indicated time points posttransfection (p.t.) and analyzed by Western blotting using Ang1-specific antibody. (E) Immunoblot of Tie2 expression and Tie2 phosphorylation from mRNATie2–transfected HeLa cells after 15 or 30 min incubation with supernatant from HeLa cells expressing secreted Nano-Luciferase (N-LUC), Ang1,…

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  Figure 3. cLNP-mediated delivery of COMP-Ang1-encoding mRNA-76 stabilizes endothelial barrier in vitro (A) Effect of COMP-Ang1 on F-actin remodeling and VE-cadherin distribution in isolated HPMECs. HPMECs grown on glass coverslips were transfected for 2 h using mRNA-cLNP formulations with control mRNANLUC or with mRNA-76 in serum-free media; cells were then kept in serum-containing media for a further 16 h before addition of TNF-α (10 ng/mL) for another 6 h followed by immunofluorescence staining with VE-cadherin-specific antibody and with Alexa Fluor 594 phalloidin to detect actin filaments (F-actin). Arrows indicate disrupted VE-Cadherin complexes. (B) TER measurements of HPMEC monolayers. HPMEC were incubated for 60 min with supernatant from mRNA-76 (encoding COMP-Ang1) or mRNANLUC (Nano-Luciferase) transfected HeLa cells and stimulated with the indicated amount of PLY (0.25, 0.5 μg/mL). PLY stimulation decreased TER of HPMEC monolayers, displaying loss of endothelial integrity when incubated with control luciferase supernatant (solid curves). Preincubation with COMP-Ang1 containing supernatant attenuated the PLY-induced TER decrease (dashed curves). After PLY stimulation the AUC was…

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  Figure 4. Localized expression of COMP-Ang1 in murine pulmonary ECs by cLNP-mediated delivery of mRNA-76 (A) Immunoblot of lung tissue lysates from individual animals 6 and 24 h after administration of 1.5 mg/kg mRNA-76-cLNP (+) or saline control (−). (B) Immunoblot of different tissue lysates from individual animals 6 h postadministration of 1.5 mg/kg mRNA-76-cLNP (+) or saline control (−). (C) Immunoprecipitation (IP) (top) and signal quantification (bottom) of COMP-Ang1 protein using Tie2-Fc-coupled magnetic beads from indicated tissue samples of individual animals 4 h postadministration of 1.5 mg mRNA-76-cLNP (+) or saline control (−). Quantification data are represented as mean ± SEM. (D) Loupe-based t-SNE clustering of murine lung cell types clustered by single-cell transcriptional analysis, showing a total of 13 distinct cell-type clusters as indicated by numbers. (E) t-SNE plots showing levels for COMP-Ang1-encoding mRNA-76. (F, G) t-SNE plots showing levels for canonical markers of ECs Tie2 (F) and VE-cadherin (G). (H) Cells expressing Ang1 (pericytes class 1 and 2, fibroblasts class 1). The intensity of expression (Log2) is indicated by red coloring. ∗, unspecific band. NK,…

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  Figure 5. cLNP-mediated delivery of mRNA-76 leads to time-dependent Tie2 pathway activation in vivo (A) Immunoblot (top) and signal quantification (bottom) after IP of murine Tie2 from lung samples from individual animals analyzed for Tie2 protein levels, Tie2 phosphorylation, and levels of coimmunoprecipitated COMP-Ang1 at indicated time points after i.v. administration of 1.5 mg/kg mRNA-76-cLNP. Quantification data are represented as mean ± SEM. (B) Immunoblot (top) and signal quantification (bottom) of murine lunge tissue lysates from individual animals analyzing COMP-Ang1 expression level and Akt phosphorylation status at indicated time points after i.v. administration of 1.5 mg/kg mRNA-76-cLNP or saline. Quantification data are represented as mean ± SEM. ∗, unspecific band.

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  Figure 6. Systemic mRNA-76 treatment stabilized endothelial barrier lung function in vivo (A) Study outline of ex vivo perfused and ventilated mouse lung model. At 6 and 15 h postadministration of mRNA-76-cLNP (1, 1.5, and 2 mg/kg) or control mRNAFLUC-cLNP (2 mg/kg) lungs were isolated and stimulated with PLY (1.4 μg/mL) for 1 min. After 30 min, lung vascular permeability was assessed by quantifying respective concentrations of continuously infused HSA in the BALF. (B) Treatment with mRNA-76 significantly decreases PLY-induced hyperpermeability of mouse lungs as compared to treatment with control mRNAFLUC-cLNP 6 h (top) and 15 h (bottom) as shown by HSA ELISA. Quantification data are represented as mean ± SEM (n = 10, ∗∗p < 0.01 between indicated groups) concentration of HSA in BALF. HSA concentration of individual mice are indicated as dots. (C) Systemic mRNA-76 treatment prevents neutrophilia transmigration in LPS-induced pulmonary inflammation in vivo. Study outline: animals (n = 10) were challenged intratracheally with 0.9 % w/v saline or LPS (3 mg/kg). A fixed volume of 50 μL, equal to an approximate intratracheal dose volume of 2.5 mL/kg, based on a 20 g mouse, was applied…