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.

Expression Kinetics and Innate Immune Response after Electroporation and LNP-Mediated Delivery of a Self-Amplifying mRNA in the Skin

“…By evaluating the success rate as a function of the dose, we found that the percentage of successful injections gradually dropped when the dose of sa-RNA was lowered. We recently showed that this observation may be attributed to the presence of RNases and that the repeatability and efficacy of intradermal electroporated sa-RNA can be increased by adding a protein-based RNase inhibitor to the sa-RNA just before injection 42 . RNases are abundantly present on the skin, 43 and these RNases can contaminate the tip of the needle during injection.…”

A new generation of vaccines based on alphavirus self-amplifying RNA

“… Plurifunctional CD4+ and CD8+ T cells [ 12 ] ta-SFV Nak. RNA ID HA Protection from influenza virus challenge [ 41 •• ] VEEV EP & LNP Luc Expression: EP, days 3−10; LNP, peak 24h [ 16 ] EP Pig High expression for 12 days [ 17 • ] Mouse Optimal expression in tail base versus flank [ 19 • ] EP+RNase inh. RNase inh.…”

“… RNase inh. increased reproducibility [ 19 • ] LNP IM HIV ag.-lumaz. High HIV gp120-antibodies [ 37 ] SARS-2 spike SARS-2 neutralizing antibodies/cell response [ 45 •• ] LION Mouse/NHP SARS-2 neutralizing antibodies/cell response [ 46 •• ] LNPout HIV gp140 Mouse Equivalent antibody versus LNP with RNA inside [ 25 ] Exp Luc/GFP Hum skin exp Cephalin LNPs increased expression sevenfold [ 35 ] Cationic NLP IM Luc Mouse Enhanced complexation & delivery [ 34 ] NLC SEAP/ZIKV ag.…”

“…On one hand, it is a very large and heterologous organ, hence caution should be taken when choosing the immunization site. In mice, EP of a VEEV-luc saRNA at the tail base resulted in a significantly higher and longer luc expression compared with the same administration at the flank [ 19 • ]. This observation emphasizes the fact that location of the ID immunization should be chosen with thoughtfulness in human clinical trials.…”

“…Preclinical studies delivering saRNA ID in combination with EP had shown a high variability in expression levels, which could be due to RNA degradation. The addition of placental RNase inhibitor to saRNA before ID injection was shown to increase efficacy and reproducibility of expression in mice [ 19 • ].…”