Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland.
Nucleic Acids Res. 2024 Oct 14;52(18):10788-10809. doi: 10.1093/nar/gkae763.
The recent COVID-19 pandemics have demonstrated the great therapeutic potential of in vitro transcribed (IVT) mRNAs, but improvements in their biochemical properties, such as cellular stability, reactogenicity and translational activity, are critical for further practical applications in gene replacement therapy and anticancer immunotherapy. One of the strategies to overcome these limitations is the chemical modification of a unique mRNA 5'-end structure, the 5'-cap, which is responsible for regulating translation at multiple levels. This could be achieved by priming the in vitro transcription reaction with synthetic cap analogs. In this study, we combined a highly efficient trinucleotide IVT capping technology with several modifications of the 5' cap triphosphate bridge to synthesize a series of 16 new cap analogs. We also combined these modifications with epigenetic marks (2'-O-methylation and m6Am) characteristic of mRNA 5'-ends in higher eukaryotes, which was not possible with dinucleotide caps. All analogs were compared for their effect on the interactions with eIF4E protein, IVT priming, susceptibility to decapping, and mRNA translation efficiency in model cell lines. The most promising α-phosphorothiolate modification was also evaluated in an in vivo mouse model. Unexpected differences between some of the analogs were analyzed using a protein cell extract pull-down assay.
最近的 COVID-19 大流行展示了体外转录（IVT）mRNA 的巨大治疗潜力，但提高其生化特性，如细胞稳定性、反应原性和翻译活性，对于基因替代治疗和抗癌免疫治疗的进一步实际应用至关重要。克服这些限制的策略之一是对独特的 mRNA 5'端结构（5'-帽）进行化学修饰，该结构负责在多个水平上调节翻译。这可以通过用合成帽类似物启动体外转录反应来实现。在这项研究中，我们将高效的三核苷酸 IVT 封端技术与 5'帽三磷酸桥的几种修饰相结合，合成了一系列 16 种新的帽类似物。我们还将这些修饰与真核生物 mRNA 5'端特有的表观遗传标记（2'-O-甲基化和 m6Am）相结合，这是二核苷酸帽无法做到的。在模型细胞系中，比较了所有类似物对与 eIF4E 蛋白相互作用、IVT 引发、脱帽敏感性和 mRNA 翻译效率的影响。还在体内小鼠模型中评估了最有前途的α-硫代磷酸酯修饰。使用蛋白细胞提取物下拉测定分析了一些类似物之间的一些意外差异。
