Structural components for amplification of positive and negative strand VEEV splitzicons
Open accessAbstract
RNA is a promising nucleic acid technology for both vaccines and therapeutics, and replicon RNA has gained traction as a next-generation RNA modality. Replicon RNA self-amplifies using a replicase complex derived from alphaviral non-structural proteins and yields higher protein expression than a similar dose of messenger RNA. Here, we debut RNA splitzicons; a split replicon system wherein the non-structural proteins (NSPs) and the gene of interest are encoded on separate RNA molecules, but still exhibit the self-amplification properties of replicon RNA. We designed both positive and negative strand splitzicons encoding firefly luciferase as a reporter protein to determine which structural components, including the 5' untranslated region (UTR), a 51-nucleotide conserved sequence element (CSE) from the first nonstructural protein, the subgenomic promoter (SGP) and corresponding untranslated region, and an internal ribosomal entry site (IRES) affect amplification. When paired with a NSP construct derived from the whole, wild type replicon, both the positive and negative strand splitzicons were amplified. The combination of the 51nt CSE, subgenomic promoter and untranslated region were imperative for the positive strand splitzicon, while the negative strand was amplified simply with inclusion of the subgenomic promoter. The splitzicons were amplified by NSPs in multiple cell types and show increasing protein expression with increasing doses of NSP. Furthermore, both the positive and negative strand splitzicons continued to amplify over the course of 72 h, up to >100,000-fold. This work demonstrates a system for screening the components required for amplification from the positive and negative strand intermediates of RNA replicons and presents a new approach to RNA replicon technology.
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From COVID-19 mRNA vaccines: Platforms and current developments (2022) · cites this paper as [157]
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- Authors
- Blakney, A. K., McKay, P. F., & Shattock, R. J.
- Affiliations
- Imperial College London (GB)
- Corresponding author
- Shattock, Robin J.
- Venue
- Frontiers in Molecular Biosciences
- Publisher
- Frontiers Media SA
- Published
- 2018-07-26
- Volume
- 5
- Pages
- 71-71
- ISSN
- 2296-889X (ISSN-L)
- PMC
- PMC6070733
- OpenAlex
- W2883754781
- Cited by
- 47
- References
- 30
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 9 · 2023: 9 · 2022: 9 · 2021: 7 · 2020: 3 · 2019: 2
- FWCI
- 1.07
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Frontiers in Molecular Biosciences (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Spiral (Imperial College London) (submittedVersion, cc-by)
- Funders
- Imperial College London
- Engineering and Physical Sciences Research Council (EP/R013764/1)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Mosquito-borne diseases and control
- Keywords
- vesicular stomatitis virus, self-amplification, non-structural proteins, conserved sequences, subgenomic promoter, internal ribosome entry site, 5' untranslated region, firefly luciferase reporter, RNA vaccines, RNA therapeutics
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