Comparison of the Expression Kinetics and Immunostimulatory Activity of Replicating mRNA, Nonreplicating mRNA, and pDNA after Intradermal Electroporation in Pigs

Abstract

Synthetic mRNA is becoming increasingly popular as an alternative to pDNA-based gene therapy. Currently, multiple synthetic mRNA platforms have been developed. In this study we investigated the expression kinetics and the changes in mRNA encoding cytokine and chemokine levels following intradermal electroporation in pigs of pDNA, self-replicating mRNA, and modified and unmodified mRNA. The self-replicating mRNA tended to induce the highest protein expression, followed by pDNA, modified mRNA, and unmodified mRNA. Interestingly, the self-replicating mRNA was able to maintain its high expression levels during at least 12 days. In contrast, the expression of pDNA and the nonreplicating mRNAs dropped after respectively one and two days. Six days after intradermal electroporation a dose-dependent expression was observed for all vectors. Again, also at lower doses, the self-replicating mRNA tended to show the highest expression. All the mRNA vectors, including the modified mRNA, induced elevated levels of mRNA encoding cytokines and chemokines in the porcine skin after intradermal electroporation, while no such response was noticed after intradermal electroporation of the pDNA vector.

mRNA as a Transformative Technology for Vaccine Development to Control Infectious Diseases

“…Similar superior kinetics and potency have been previously described for CNE-formulated self-amplifying mRNA. 108 Leyman et al 109 recently compared luciferase expression of self-amplifying mRNA, nucleoside-modified conventional mRNA, and unmodified conventional mRNA after i.d. electroporation in pigs.…”

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

“…Synthetic self-amplifying mRNAs are known for their high in vivo translational efficiency [21,22]. However, in vivo administration of sa-mRNAs may induce a strong type I IFN response [5,11,23]. While this can be considered advantageous when the sa-mRNA is used for vaccination purposes [11,24], several studies have demonstrated that triggering type I IFN production can negatively impact the intended adaptive immune response of intramuscularly and intradermally administered mRNA vaccines [9,11,15].…”

“…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.…”

Expression kinetics and innate immune response after electroporation and lipid nanoparticle mediated delivery of a self-amplifying mRNA in the skin of mice

“…Indeed, if the innate immune response has not faded away at the moment of the next mRNA injection a chronical inflammatory condition will be established. The duration of the innate immune response after in vivo delivery of mRNA is not well studied and most information comes from studies that used RT-PCR or ELISA to quantify key innate immune proteins [22][23][24]. Although RT-PCR is a very sensitive technique, the correlation between mRNA levels and protein expression are not always linear [25].…”