PT  - JOURNAL ARTICLE
AU  - Karnyart Samnuan
AU  - Anna K. Blakney
AU  - Paul F. McKay
AU  - Robin J. Shattock
TI  - Design-of-Experiments In Vitro Transcription Yield Optimization of Self-Amplifying RNA
AID  - 10.1101/2021.01.08.425833
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.01.08.425833
4099  - http://biorxiv.org/content/early/2021/01/08/2021.01.08.425833.short
4100  - http://biorxiv.org/content/early/2021/01/08/2021.01.08.425833.full
AB  - Self-amplifying RNA (saRNA) vaccines are able to induce a higher antigen-specific immune response with a more cost-effective and rapid production process compared to plasmid DNA vaccines. saRNAs are synthesized through in vitro transcription (IVT) however; this process has mainly been optimized for relatively short mRNAs. Here, we optimized the IVT process for long saRNAs, approximately 9.4 kb through a design of experiment (DoE) approach to produce a maximal RNA yield and validated the optimal IVT method on various sizes of RNA. We found that magnesium has the highest impact on RNA yield with acetate ions enabling a higher yield than chloride ions. In addition, the interaction between magnesium and nucleoside triphosphates (NTPs) is highly essential for IVT. Further addition of sodium acetate (NaOAc) during IVT provided no added benefit in RNA yield. Moreover, pyrophosphatase was not essential for productive IVT. The optimal IVT method can be used to synthesize different lengths of RNA. These findings emphasize the ability to synthesize high quality and quantity of saRNA through IVT and that the optimal amount of each component is essential for their interactions to produce a high RNA yield.Competing Interest StatementThe authors have declared no competing interest.Abbreviations usedsaRNAself-amplifying RNA;IVTin vitro transcription;DoEdesign of experiment;pDNAplasmid DNA;GOIgene of interest;NTPnucleoside triphosphate;VEEVVenezuelan Equine Encephalitis Virus;firefly luciferasefLuc