In-vitro-transcribed messenger RNA (mRNA) has recently shown increasing significance in the development of vaccines and therapeutics. Immunogenic double-stranded RNA (dsRNA) is an undesired byproduct formed during in vitro transcription (IVT), and it is challenging to reduce dsRNA byproduct from mRNA due to their similar sizes and intrinsic characteristics. Removal of dsRNA relies heavily on post-IVT chromatography purifications, such as reverse-phase high-pressure liquid chromatography, which increase manufacturing costs, reduce yield, and often decrease integrity, especially for long mRNA. Thus, it would be ideal to reduce and control the level of dsRNA during IVT. We herein present a simple, scalable, and controllable method to reduce the formation of dsRNA byproducts during IVT. Selected chaotropic agents at optimized concentrations are included during IVT to create a mild denaturing environment to prevent the undesired intermolecular or intramolecular base-pairing that is thought to promote RNA-templated dsRNA formation by RNA polymerase. Compared with regular IVT, our improved method produces mRNA with significantly less dsRNA, much lower immuno-stimulation, and more efficient protein expression. Therefore, this method potentially eliminates dsRNA removal purification steps and does not require reduced magnesium concentration, elevated temperature, or custom reagents, enabling a straightforward, high-yield, and cost-effective scale-up approach for mRNA manufacturing.

体外转录的信使 RNA (mRNA) 最近在疫苗和治疗剂的开发中显示出越来越重要的意义。免疫原性双链 RNA (dsRNA) 是在体外形成的不需要的副产物转录（IVT），并且由于它们相似的大小和内在特征，减少来自 mRNA 的 dsRNA 副产物具有挑战性。dsRNA 的去除在很大程度上依赖于 IVT 后色谱纯化，例如反相高压液相色谱，这会增加制造成本、降低产量并经常降低完整性，尤其是对于长 mRNA。因此，在 IVT 期间降低和控制 dsRNA 水平将是理想的。我们在此提出了一种简单、可扩展且可控的方法，以减少 IVT 期间 dsRNA 副产物的形成。在 IVT 过程中包括选定浓度的离液剂，以创建温和的变性环境，以防止不希望的分子间或分子内碱基配对，这种碱基配对被认为会促进 RNA 模板化的 dsRNA 通过 RNA 聚合酶形成。与常规 IVT 相比，我们改进的方法产生的 mRNA 具有明显更少的 dsRNA、更低的免疫刺激和更有效的蛋白质表达。因此，该方法潜在地消除了 dsRNA 去除纯化步骤，并且不需要降低镁浓度、升高温度或定制试剂，从而为 mRNA 制造提供了一种简单、高产且具有成本效益的放大方法。