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

Abstract

In this work, we studied the expression kinetics and innate immune response of a self-amplifying mRNA (sa-RNA) after electroporation and lipid-nanoparticle (LNP)-mediated delivery in the skin of mice. Intradermal electroporation of the sa-RNA resulted in a plateau-shaped expression, with the plateau between day 3 and day 10. The overall protein expression of sa-RNA was significantly higher than that obtained after electroporation of plasmid DNA (pDNA) or non-replication mRNAs. Moreover, using IFN-β reporter mice, we elucidated that intradermal electroporation of sa-RNA induced a short-lived moderate innate immune response, which did not affect the expression of the sa-RNA. A completely different expression profile and innate immune response were observed when LNPs were used. The expression peaked 24 h after intradermal injection of sa-RNA-LNPs and subsequently showed a sharp drop. This drop might be explained by a translational blockage caused by the strong innate immune response that we observed in IFN-β reporter mice shortly (4 h) after intradermal injection of sa-RNA-LNPs. A final interesting observation was the capacity of sa-RNA-LNPs to transfect the draining lymph nodes after intradermal 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.…”

Corticosteroids and cellulose purification improve respectively the in vivo translation and vaccination efficacy of self-amplifying mRNAs

“…Subsequently, an additional cellulose-based purification was performed to remove dsRNA by-products as previously described [18]. Briefly, cellulose fibers (Sigma- The procedure of electroporation involved two short high-voltage pulses of 450 V with a duration of 0.05 ms and an interval of 300 ms followed by eight long low-voltage pulses of 100 V with a duration of 10 ms and an interval of 300 ms [4,15]. Innate immune inhibitors (Fig.…”

“…Furthermore, synthetic mRNAs allow cell-free production and exert a transient and more predictable expression [1,2]. In recent years, synthetic self-amplifying mRNAs (sa-mRNAs) have also been gaining interest because of their higher and longer-lasting expression compared to non-amplifying mRNAs [4,5]. Self-amplifying RNAs encode an RNAdependent RNA polymerase (replicase) that gives them the capacity to trigger a temporal amplification of their backbone.…”

“…We recently demonstrated that the innate immune response triggered by the selfamplifying mRNA platform [4,5,11] reduced the cellular and humoral responses of an sa-mRNA vaccine against Zika virus [15]. Therefore, strategies that can reduce the innate immunity of sa-mRNAs may improve the efficacy of sa-mRNA vaccines and the acceptance of sa-mRNA therapeutics in general.…”

“…Therefore, strategies that can reduce the innate immunity of sa-mRNAs may improve the efficacy of sa-mRNA vaccines and the acceptance of sa-mRNA therapeutics in general. Tempering the innate immunity of sa-mRNA by inclusion of modified nucleosides is expected to have a negative impact on the replication of the sa-RNA [4,16]. Moreover, the large size (> 10 kb) of sa-RNAs makes them more prone to shearforces, which complicates their purification by reversed-phase HPLC.…”