Improvement ofIn VivoExpression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins

Abstract

Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.

Efficient Induction of T Cells against Conserved HIV-1 Regions by Mosaic Vaccines Delivered as Self-Amplifying mRNA

“… 42 For example, it was demonstrated for self-amplifying mRNA that potent initial induction of type I interferons by double-stranded RNA (dsRNA) was counterproductive to immunogen expression and subsequent immunogenicity. 83 , 84 Thus, any rational exploitation of the innate signals and/or immunomodulation will require careful evaluation, which will ultimately need to be confirmed in the target species, i.e., humans. 85 Nevertheless, new technologies can often precipitate the long-awaited breakthroughs in difficult challenges of public health, such as prevention and treatment of HIV-1, malaria, tuberculosis, cancer, and allergies, and the mRNA platform has the potential to be such a transforming technology.…”

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

“…116 The usage of modified nucleosides to silence self-amplifying mRNA recognition by innate immune sensors is not an option, because it could impair RDRP-mediated mRNA amplification in target cells, and the effect of modified nucleoside would also be lost after the first round of amplification. Beissert et al 117 have demonstrated that antigen expression from self-amplifying mRNA can be significantly improved in vitro and in vivo by co-transferring mRNAs encoding IFN and PKR inhibitory proteins, such as viral protein B18R from vaccinia virus. These data are consistent with previous results showing an increase in translation from an alphavirus self-amplifying mRNA co-expressing a dominant-negative PKR 118 or co-delivered with a recombinant vaccinia B18R protein.…”

Enlisting the mRNA Vaccine Platform to Combat Parasitic Infections

“…The innate immune response against exogenous mRNA can be minimized by avoiding the activation of TLR receptors, by using highly purified transcripts (complete removal of production byproducts, such as dsRNA and DNA template) and modified nucleosides (replacing uridine for pseudouridine) [25,62]. Another strategy is to suppress the receptors by co-expressing mRNA encoding immune evasion proteins, such as vaccinia virus proteins E3, K3, and B18 [64][65][66]. These proteins can locally and temporarily suppress PKR and IFN pathway activation and enhance expression of mRNA-encoded genes of interest.…”