Efficiency and immunogenicity of lipid nanoparticle-mediated cardiac mRNA delivery are lipid composition-dependent

Figures

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  Figure 1. A robust local immune response is apparent 5 days after local intramyocardial administration of modRNA-LNPs H&E staining of transversal heart sections at 1 (A) and 5 days (B) after intramyocardial administration of C12-200 LNPs (C12-200 40%/Chol. 43.5%/DOPE 15%/DMG-PEG 1.5%) containing 4 μg of LNP-encapsulated modRNA. The histological sections of the hearts show marked differences in the local immune response to mRNA-LNP administration. Representative areas of the treated myocardium are presented by squares in ai and bi and magnified in aii and bii, respectively. Scale bars, 100 μm.

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  Figure 2. LNP size, polydispersity, surface charge, and modRNA encapsulation efficiency are similar among the tested LNP formulations (A) Overview of the lipid molar compositions of the four tested LNP formulations. LNP characterization consisted of LNP particle size (B), polydispersity index (PDI) (C), particle surface ζ-potential (ZP) (D), and modRNA encapsulation efficiency (E). Data represent the mean ± SD of three measurements. (F) Chemical structures of ionizable lipids used in this study.

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  Figure 3. The tested LNP formulations are effective for cardiac modRNA delivery, with SM-102 LNPs presenting the most favorable profile Cardiac delivery efficiency of luciferase-encoding modRNA was measured by luminescence imaging of hearts (A) and other organs (B) 24 h after intramyocardial administration of modRNA LNPs. Luminescence quantification was also performed in organ lysates (C and D). Data represent the mean ± SD of n = 4 for the treatment groups and n = 1 for the PBS group. ∗∗∗∗, p-value <0.0001; ∗∗∗, p-value <0.001; ∗∗, p-value <0.01; ∗, p-value <0.05; blank: no significant difference.

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  Figure 4. LNPs induce different degrees of cardiac inflammatory responses, characterized by neutrophil, macrophage, and T cell infiltration at the injection site 5 days after modRNA-LNP intramyocardial administration (A) Representative images of H&E, Ly6G, MAC-3, and CD3 stainings of mouse heart sections collected 5 days after local injection of 4 μg modRNA encapsulated in LNPs or PBS into the left ventricular wall. (B) Quantification of maximum cell infiltration in H&E-stained heart sections. (C) Quantification of the area of Ly6G+ infiltrating cells in heart sections, corresponding to neutrophils. (D) Quantification of the area of MAC-3+ infiltrating cells in heart sections, corresponding to macrophages. (E) Quantification of the number of CD3+ infiltrating cells in heart sections, corresponding to T cells. At least three histological sections per staining and per animal were analyzed. For each staining, the maximum infiltrated area (B, C, D) or maximum number of positive cells (E) per animal was plotted. All scale bars in (A) represent 1 mm, except for the zoomed-in CD3 images in the bottom row, which represent 100 μm. Data represent the mean ± SD of n = 1–3 animals (B, C, D, E).

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  Figure 5. ALC-0315 LNPs and SM-102 LNPs do not induce upregulation of pro-inflammatory cytokines in hearts or serum 24 h post local administration Quantification of pro-inflammatory cytokines in hearts (A) and in serum (B) 24 h after local LNP administration in the heart. (C) Quantification of liver transaminases ALT (Ci) and AST (Cii) in serum. Abbreviations are as follows: GM-CSF, granulocyte macrophage colony stimulating factor; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; ALT, alanine transaminase; AST, aspartate aminotransferase. Data represent the mean ± SD of n = 4 for treatment groups and n = 3 for PBS groups. ∗∗∗∗, p-value <0.0001; ∗∗∗, p-value <0.001; ∗∗, p-value <0.01; ∗, p-value <0.05; blank: no significant difference.