Divergent Delivery and Expression Kinetics of Lipid and Polymeric Nanoparticles across mRNA Modalities

Figures

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  Figure 1. Characterization of mRNA modalities and delivery modules. A) Illustration of the structural elements of the in vitro transcribed linear, circular and self‐amplifying mRNA. B) Lipid mixture composition formulated with mRNA modalities using microfluidic mixing to generate LNPs. C) Chemical structure of pABOL polymer complexed with mRNA modalities to generate PNPs. D) Dot blot image of double‐stranded RNA content in a serial dilution of mRNA modalities used.

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  Figure 2. Physicochemical properties of mRNA nanoparticles. A) Average zeta size in nanometers and polydispersity index of LNPs. B) Zeta potential in millivolts of LNPs. C) Zeta size in nanometers and polydispersity index of PNPs. D) Zeta potential in millivolts of PNPs. Data shown are mean values ± standard deviation from n = 3 technical replicates compared using One‐way ANOVA followed by Tukey's multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 3. Luciferase expression after LNP delivery of mRNA modalities. A) Bioluminescence images of mice after intramuscular injection of 5 µg of linRNATMV (black), linRNABNT (magenta), circRNA (turquoise) and VEEV‐saRNA (purple). B) Total flux detected on days indicated after administration of 5 µg or C) 0.5 µg of LNP‐mRNA. Data shown are mean values ± standard deviation from n = 3 biological replicates. D) Area under the curve of total flux curves for the duration of the study. Data shown are mean ± standard error of mean (SEM) compared using two‐way ANOVA followed by Tukey's multiple comparison test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 4. Luciferase expression after PNP delivery of mRNA modalities. A) Bioluminescence images of mice after intramuscular injection of 5 µg of linRNATMV (black), linRNABNT (magenta), circRNA (turquoise) and VEEV‐saRNA (purple). B) Total flux detected on days indicated after administration of 5 µg or C) 0.5 µg of pABOL‐mRNA. Data shown are mean values ± standard deviation from n = 3 biological replicates. D) Area under the curve of total flux curves for the duration of the study. Data shown are mean ± standard error of mean (SEM) compared using two‐way ANOVA followed by Tukey's multiple comparison test. **** p < 0.0001.

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  Figure 5. Phylogenetic analysis of alphavirus‐derived saRNA vectors evaluated. Phylogenetic tree generated using neighbor‐joining method based on the amino acid sequences of the non‐structural protein 4. Branch lengths represent evolutionary distances.

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  Figure 6. LNP and PNP delivery of genotypically distinct saRNA vectors. A) Total flux detected on days indicated after administration of LNPs containing 5 µg of New World and B) Old World alphaviral saRNA vectors. C) Area under the curve of total flux curves shown in A and B. D) Total expression relative to control VEEV‐saRNA after LNP administration. E) Total flux detected on days indicated after administration of pABOL complexed with 5 µg of New World and F) Old World alphaviral saRNA vectors. (C) Area under the curve of total flux curves shown in E and F. (D) Total expression relative to control VEEV‐saRNA after PNP administration. For A, B, E and F, data shown are mean values ± standard deviation from n = 3 biological replicates. For C, D, G, and H, data shown are mean ± standard error of mean (SEM) compared using one‐way ANOVA followed by Dunnett's multiple comparison test. * p < 0.05; **** p < 0.0001.