Improving the Repeatability and Efficacy of Intradermal Electroporated Self-Replicating mRNA

Abstract

Local administration of naked self-replicating mRNA (sr-mRNA) in the skin or muscle using electroporation is effective but hampered by low repeatability. In this manuscript, we demonstrated that intradermal electroporation of sr-mRNA in combination with a protein-based RNase inhibitor increased the expression efficiency, success rate, and repeatability of the data. The RNase inhibitor should be added just before administration because storage of the inhibitor together with the sr-mRNA at -80°C resulted in a partial loss of the beneficial effect. Furthermore, the location of intradermal electroporation also had a major effect on the expression of the sr-mRNA, with the highest and longest expression observed at the tail base of the mice. In contrast with previous work, we did not observe a beneficial effect of calcium ions on the efficacy of naked sr-mRNA after intradermal injection. Finally, another important finding was that the traditional representation of in vivo bioluminescence data as means in logarithmic graphs can mask highly variable data. A more truthful representation can be obtained by showing the individual data points or by displaying median values in combination with interquartile ranges. In conclusion, intradermal sr-mRNA electroporation can be improved by adding an RNase inhibitor and injecting at the tail base.

Keywords: Ca(2+); RNase inhibitors; electroporation; intradermal injection; mice; self-replicating mRNA.

Graphical abstract

Figure 1

Schematic Representation of the sr-mRNA and Its Replication The sr-mRNA starts at its 5′ end with a cap1, a 5′ UTR, and the sequences of the non-structural proteins (nsP1–4) of Venezuelan equine encephalitis virus (VEEV). These non-structural proteins are translated as a polyprotein that forms a replicase (brown). The non-structural proteins are followed by a subgenomic promoter (SGP, red), which starts in nsP4. The sequence of the protein(s) of interest (blue) can be found behind the SGP. In this work, the protein of interest was firefly luciferase. At the 3′ end, an UTR and a polyA tail are present. When the sr-mRNA arrives in the cytosol, the nsP1–4 polyprotein is translated and cleaved by nsP2 to generate the early replication complex (replicase), which consists of nsP1–3 and associated nsP4. In a later phase, nsP1–3 is also cleaved, and the individual nsPs join together to form the cleaved replicase. Three promoter elements (PEs) trigger the replicase and cleaved replicase to generate, respectively, complementary minus-RNA strands and new copies of the original genomic RNA starting from the minus-RNA strands. In addition, the SGP triggers the cleaved replicase to produce an excessive amount of subgenomic RNAs.

Figure 2

Effect of the RNase Inhibitor on Luciferase Expression after Intradermal Electroporation of sr-mRNA in the Flank of Mice (A–D) Five micrograms of luciferase encoding sr-mRNA dissolved in 50 μL PBS was supplemented with either 0 (A), 0.33 (B), or 1.0 U/μL (C and D) of RNase inhibitor. In (B) and (C), the RNase inhibitor was added to the sr-mRNA just before administration, whereas in (D), the RNase inhibitor (1.0 U/μL) was added to the sr-mRNA before storage at −80°C. The graphs show the individual expression profiles in each of the six injection spots (n = 6).

Figure 3

The Use of Median Values and Interquartile Ranges Is More Appropriate for Presenting Highly Variable Bioluminescence Data (A and B) The mean (± SEM) (A) and median (± interquartile range) (B) bioluminescence signals obtained after intradermal electroporation of luciferase encoding sr-mRNA in the absence or presence of RNase inhibitor. Five micrograms of luciferase encoding sr-mRNA dissolved in 50 μL PBS was injected in the flanks of mice and immediately electroporated. The green curves show the luciferase expression of the sr-mRNA without supplementation with RNase inhibitor. The blue and red curves depict the luciferase expression after adding, respectively, 0.33 and 1.0 U/μL RNase inhibitor to the sr-mRNA just before injection. The black curves represent the luciferase expression after adding 1.0 U/μL RNase inhibitor before storage at −80°C. (n = 6). Due to the high variation in luciferase expression, no significant differences were observed between the groups.

Figure 4

Comparison of the Luciferase Expression Kinetics after Intradermal Electroporation of sr-mRNA at the Flank or at the Tail Base (A–D) Five micrograms of sr-mRNA-encoding luciferase was just before injection supplemented with 1.0 U/μL RNase inhibitor and after intradermal injection immediately electroporated. Each curve in (A) and (B) represents the evolution of the individual bioluminescence signal in the separate injection spots of the flank (A) or the tail base (B) (n = 6). (C) The median bioluminescence signal over time after intradermal electroporation in the flank (blue) and in the tail base (green) is shown (error bars represent interquartile range; n = 6). (D) The area under the curve (AUC) of the curves in (C) and the median AUC (line) are plotted. Note the differences in the scales of the x axes in the graphs. **p = 0.0022.

Figure 5

The Effect of Calcium Ions on the Expression of Naked sr-mRNA after Intradermal Administration without Electroporation (A) Median luciferase expression with interquartile range after intradermal injection of sr-mRNA (5 μg) dissolved in PBS (Ca/Mg free) without (blue curve) or with 3 mEq/L CaCl₂ (red curve). The naked sr-mRNA was supplemented with 1.0 U/μL RNasin just before intradermal injection (six mice per group). The luciferase expression after intradermal electroporation of 5 μg sr-mRNA (dissolved in Ca/Mg-free PBS, green curve) in the tail base is also shown as a reference. The gray zones display significant differences between intradermally electroporated sr-mRNA and intradermally injected naked sr-mRNA with or without calcium. *p < 0.05. (B and C) Luciferase expression in the individual intradermal injection spots of 5 μg sr-mRNA dissolved in PBS (B) or in PBS supplemented with 3 mEq/L CaCl₂ (C) (6 mice per group). All intradermal injections in these graphs were performed at the tail base.