Innate Inhibiting Proteins Enhance Expression and Immunogenicity of Self-Amplifying RNA

Abstract

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis -encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo , and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo . Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus ( RABV )-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC 50 ) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.

Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: Getting the message across

“…The in vitro results were however not confirmed in vivo , as neither of the proteins led to a substantial and significant increase in protein expression in both BALB/c and C57BL/6 mice. In contrast, both proteins were able to increase the percentage of transfected cells in human skin explants [4] . Nevertheless, this innate immune evasion strategy remains interesting to explore.…”

“…Nevertheless, this innate immune evasion strategy remains interesting to explore. However, future studies should include more in-depth research on the delivery system dependency, anti-vector immunity, possible combination of inhibitors and effects in humans [4] .…”

“…The recent FDA approval of the first messenger RNA (mRNA) vaccine formulations, developed by Pfizer/BioNTech and Moderna, to control the worldwide COVID-19 pandemic, created a new wave of attention for mRNA-based therapeutics [1] . Currently, several other mRNA-based COVID-19 vaccine formulations are still under investigation, containing either conventional non-amplifying mRNA or self-amplifying mRNA (saRNA) [2] , [3] , [4] , [5] . These successful outcomes are the result of a long and tedious journey that started in 1961 when conventional mRNA was first discovered, followed by successful pre-clinical experiments in 1990 [6] , [7] , [8] .…”

“…In contrast, the larger saRNA gained a lot of attention, mainly for vaccination purposes as first reported in 1994 due to is self-replicating ability [2] , [3] . The latter results in enhanced and prolonged protein expression which overcomes the often transient nature of conventional mRNA therapeutics [2] , [3] , [4] . Besides its use in vaccination against infectious diseases or cancer, mRNA is currently explored for many other non-immunotherapy applications, including protein replacement therapy, cell reprogramming and gene editing [7] , [9] , [10] .…”

Multifunctional Immunoadjuvants for Use in Minimalist Nucleic Acid Vaccines

“…This system provides not only prolonged expression of antigen, but also induces strong immunostimulatory effects owing to formation of the dsRNA structure during RNA self-amplification, which interacts with multiple PRRs. SAM RNA activates a type I IFN through endosomal sensing via TLRs 3/7/8, as well as cytosolic sensing via MDA5, RIG-I, and possibly unknown pathways [ 241 ]. Self-adjuvanted RNActive vaccine technology based on SAM RNA has emerged as a promising vaccine technology, inducing strong and balanced immune responses comprising humoral and cellular responses.…”