5′ terminal nucleotide determines the immunogenicity of IVT RNAs

In vitro transcription (IVT) is a technology of vital importance that facilitated the production of mRNA therapeutics and drove numerous breakthroughs in RNA biology. T7 polymerase-produced RNAs can begin with either 5′-triphosphate guanosine (5′-pppG) or 5′-triphosphate adenosine (5′-pppA), generating potential agonists for the RIG-I/type I interferon response. While it is established that IVT can yield highly immunogenic double-stranded RNA (dsRNA) via promoterless transcription, the specific contribution of initiating nucleosides to this process has not been previously reported. Our study shows that IVT-derived RNAs containing 5′-pppA are significantly more immunogenic compared with their 5′-pppG counterparts. We observed heightened levels of dsRNAs triggered by IVT with 5′-pppA RNA, activating the RIG-I signaling pathway in cultured cells, as well as in ex vivo and in vivo mouse models where the IFN-β gene was substituted with the mKate2+ reporter. Elevated levels of dsRNA were found in both short 5′-pppA RNAs and full-length mRNAs, including those of COVID-19 vaccines. These findings reveal the unexpected source of IVT RNA immunogenicity, offering valuable insights for both academic research and medical applications of this technology.

体外转录（In vitro transcription, IVT）是一项至关重要的技术，为mRNA疗法的生产提供了关键支撑，并推动了RNA生物学领域的诸多突破性进展。由T7聚合酶转录得到的RNA，其5′端可起始于5′-三磷酸鸟苷（5′-triphosphate guanosine，5′-pppG）或5′-三磷酸腺苷（5′-triphosphate adenosine，5′-pppA），进而可能成为RIG-I/Ⅰ型干扰素应答的潜在激动剂。已知体外转录可通过无启动子转录过程生成具有高度免疫原性的双链RNA（double-stranded RNA，dsRNA），但此前尚未有研究阐明起始核苷在该过程中的具体作用。本研究发现，携带5′-pppA的体外转录源RNA，其免疫原性显著高于以5′-pppG为起始的同类RNA。我们观察到，由5′-pppA起始的体外转录所触发的双链RNA水平更高，可在培养细胞、离体及体内小鼠模型中激活RIG-I信号通路；其中小鼠模型的IFN-β基因已被替换为mKate2+报告基因。无论是短链5′-pppA RNA还是包括新冠疫苗mRNA在内的全长mRNA，均检测到了升高的双链RNA水平。本研究揭示了体外转录源RNA免疫原性的意外来源，为该技术的学术研究与医学应用提供了极具价值的见解。