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.

中文翻译：

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实验设计自扩增RNA的体外转录产量优化

与质粒DNA疫苗相比，自扩增RNA（saRNA）疫苗能够以更高的成本效益和更快的生产过程诱导更高的抗原特异性免疫反应。saRNA是通过体外转录（IVT）合成的；此过程主要针对相对较短的mRNA进行了优化。在这里，我们通过设计实验（DoE）方法优化了大约9.4 kb的长saRNA的IVT工艺，以产生最大的RNA产量，并针对各种大小的RNA验证了最佳IVT方法。我们发现，镁对RNA的产量影响最大，乙酸根离子的收率高于氯离子。另外，镁与核苷三磷酸酯（NTPs）之间的相互作用对于IVT至关重要。在IVT期间进一步添加乙酸钠（NaOAc）不会增加RNA产量。此外，焦磷酸酶对于生产性IVT不是必需的。最佳的IVT方法可用于合成不同长度的RNA。这些发现强调了通过IVT合成高质量和大量saRNA的能力，并且每种成分的最佳量对于它们相互作用以产生高RNA产量至关重要。