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Improvement of In Vivo Expression 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)-? 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.

SUBMITTER: Beissert T

PROVIDER: S-EPMC5737720 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

Beissert Tim T Koste Lars L Perkovic Mario M Walzer Kerstin C KC Erbar Stephanie S Selmi Abderraouf A Diken Mustafa M Kreiter Sebastian S Türeci Özlem Ö Sahin Ugur U

Human gene therapy 20170906 12

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, patte ...[more]

PMID: 28877647

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Project description:The optimal vaccination strategy to boost responses in the context of pre-existing immune memory to the SARS-CoV-2 spike (S) glycoprotein is an important question for global public health. To address this, we explored the SARS-CoV-2-specific humoral and cellular immune responses to a novel self-amplifying RNA (saRNA) vaccine followed by a UK authorised mRNA vaccine (BNT162b2) in individuals with and without previous COVID-19, and compared these responses with those who received an authorised vaccine alone. 35 subjects receiving saRNA (saRNA group) as part of the COVAC1 clinical trial and an additional 40 participants receiving an authorised SARS-CoV-2 vaccine only (non-saRNA group) were recruited. Antibody responses were measured by ELISA and a pseudoneutralisation assay for wildtype, Delta and Omicron variants. Cellular responses were measured by IFN-ƴ ELISpot and an activation induced marker (AIM) assay. Approximately 50% in each group had previous COVID-19 prior to vaccination, confirmed by PCR or antibody positivity on ELISA. All of those who received saRNA subsequently received a full course of an authorised vaccine. The majority (83%) of those receiving saRNA who were COVID-19 naïve at baseline seroconverted following the second dose, and those with previous COVID-19 had an increase in antibody titres two weeks following saRNA vaccination (median 27-fold), however titres were lower when compared to mRNA vaccination. Two weeks following the 2nd authorised mRNA vaccine dose, binding and neutralising antibody titres were significantly higher in the saRNA participants with previous COVID-19, compared to non-saRNA, or COVID-19 naive saRNA participants. Cellular responses were again highest in this group, with a higher proportion of spike specific CD8+ than CD4+ T cells when compared to those receiving the mRNA vaccine only. These findings suggest an immunological benefit of increased antigen exposure, both from natural infection and vaccination, particularly evident in those receiving heterologous vaccination with saRNA and mRNA.

Project description:RNA-baffsed vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part due to the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an antigen, formulated with a synthetic delivery system, and induce broad-based immune responses in preclinical animal models. BALB/c mice (three biological replicates per group) were injected bilaterally intramuscularly (i.m.) with 50 Î¼l per quadriceps of Phosphate Buffered Saline alone (Control group), Ribonucleic Acid Expressing Respiratory Syncytial Virus F Protein (RNA) (500 ng), lipid nanoparticle (LNP) alone, or LNPâformulated RNA (LNP_RNA). A single quadriceps from each mouse was homogenized using QIAzol reagent and the FastPrepâ24 instrument (MP Bio). Total RNA was purified using RNeasy columns according to the manufacturerâs instructions (Qiagen). RNA labeling, hybridization, and scanning were performed using methods, reagents, software, and hardware purchased from Agilent Technologies. Briefly, 100 ng RNA was retro transcribed, labeled using Cy3, and column purified Qiagen. The efficiency of Cy3 dye incorporation was assessed on a NanoDrop 1000 instrument. Reactions with specific activities >6 pmol dye/Î¼g cRNA were used for hybridization. cRNA (1.65 Î¼g) was hybridized onto a 4 X 44 Whole Mouse Genome Microarray (G4122F). After scanning, images were analyzed using Feature Extraction 10.7.3.1 software. Microarray data was analyzed using the R/Bioconductor Limma openâsource package. Briefly, each spot was background corrected and spot intensities between arrays normalized using the quantile method. The average normalized spot intensity from experimental replicates was determined and differentially expressed genes (compared to the PBS control group) calculated using a moderated tâstatistic (Empirical Bayes method) followed by BenjaminiâHochberg false discovery rate (FDR) correction. Genes with an FDR adjusted pâvalue â¤ 0.05 and a foldâchange greater than or less than 2 (log2 = 1) were considered significantly changed. Functional clustering was performed using the DAVID online database. Heat maps were generated using Spotfire 4.0.3 software.

Dataset Information

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

Publications

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

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