Sioud Mouldy
Division of Cancer Medicine, Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway.
Methods Mol Biol. 2025;2965:127-149. doi: 10.1007/978-1-0716-4742-4_4.
The success of messenger RNA (mRNA)-based vaccines against SARS-CoV-2 during the COVID-19 pandemic has driven significant advancements in mRNA-based therapeutics. Key innovations include the use of modified nucleosides and ionizable lipid nanoparticles. Modified mRNA is produced through in vitro transcription, typically employing phage T7 RNA polymerase along with a DNA template and ribonucleoside triphosphates. Chemical modifications at the 5-position of pyrimidines, (cytosine and uridine) are generally well tolerated by T7 RNA polymerase and can reduce innate immune activation, though their effects on translation vary considerably. Therefore, the nature and codon-positions of these modifications, as well as the mRNA sequence, should be considered when designing mRNA therapeutics. This chapter details the production, purification, and evaluation of translation in human cells for modified mRNA containing 5-methyluridine, 5-methoxyuridine, 5-hydroxymethyluridine, pseudouridine, N1-methylpseudouridine, or N1-ethylpseudouridine.
在新冠疫情期间，基于信使核糖核酸（mRNA）的抗严重急性呼吸综合征冠状病毒2（SARS-CoV-2）疫苗的成功推动了基于mRNA的治疗方法取得重大进展。关键创新包括使用修饰核苷和可电离脂质纳米颗粒。修饰的mRNA通过体外转录产生，通常使用噬菌体T7 RNA聚合酶以及DNA模板和核糖核苷三磷酸。嘧啶（胞嘧啶和尿苷）5位的化学修饰通常能被T7 RNA聚合酶很好地耐受，并且可以减少先天免疫激活，尽管它们对翻译的影响差异很大。因此，在设计mRNA治疗方法时，应考虑这些修饰的性质和密码子位置以及mRNA序列。本章详细介绍了含有5-甲基尿苷、5-甲氧基尿苷、5-羟甲基尿苷、假尿苷、N1-甲基假尿苷或N1-乙基假尿苷的修饰mRNA在人细胞中的生产、纯化及翻译评估。
