Messenger RNA (mRNA) and self-amplifying RNA (saRNA) vaccines against SARS-CoV-2 produced using transcription (IVT) were clinically approved in 2020 and 2022, respectively. While the industrial production of mRNA using IVT has been extensively optimized, the optimal conditions for saRNA have been explored to a lesser extent. Most T7 polymerase IVT protocols have been specifically optimized for mRNA which is ∼5–10-fold smaller than saRNA and may have profound effects on both the quality and yield of longer transcripts. Here, we optimized IVT conditions for simultaneously increasing the yield of full-length transcripts and reducing dsRNA formation through Design of Experiments. Using a definitive screening approach, we found that the key parameters are temperature and magnesium in the outcome of RNA quality (% full length transcript) and yield in small scale synthesis. The most important parameter for reducing dsRNA formation for both mRNA and saRNA was Mg concentration (10 mM). We observed that a lower temperature was vital for production of high quality saRNA transcripts. mRNA quality was optimal at higher Mg concentration (>40 mM). High quality transcripts correspond to significantly reduced product yield for saRNA, but not for mRNA. The differences between mRNA and saRNA requirements for high quality product and the relationship between high quality large saRNA molecules and low temperature synthesis have not been reported previously. These findings are key for informing future IVT parameters design and optimization for smaller and larger RNA transcripts.

中文翻译：

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体外转录条件对高质量信使和自扩增RNA产量的影响

使用转录 (IVT) 生产的针对 SARS-CoV-2 的信使 RNA (mRNA) 和自扩增 RNA (saRNA) 疫苗分别于 2020 年和 2022 年获得临床批准。虽然使用 IVT 的 mRNA 工业生产已得到广泛优化，但 saRNA 的最佳条件的探索程度较低。大多数 T7 聚合酶 IVT 方案都针对 mRNA 进行了专门优化，该 mRNA 比 saRNA 小约 5-10 倍，可能对较长转录本的质量和产量产生深远影响。在这里，我们通过实验设计优化了 IVT 条件，以同时提高全长转录本的产量并减少 dsRNA 的形成。使用明确的筛选方法，我们发现小规模合成中 RNA 质量（全长转录物百分比）和产量结果中的关键参数是温度和镁。减少 mRNA 和 saRNA dsRNA 形成的最重要参数是 Mg 浓度 (10 mM)。我们观察到较低的温度对于生产高质量 saRNA 转录本至关重要。 mRNA 质量在较高的 Mg 浓度 (>40 mM) 下达到最佳。高质量转录本对应于 saRNA 产物产量的显着降低，但 mRNA 则不然。高质量产品的mRNA和saRNA要求之间的差异以及高质量大saRNA分子与低温合成之间的关系尚未有报道。这些发现对于未来较小和较大 RNA 转录本的 IVT 参数设计和优化至关重要。