引言

2023 年，Katalin Karikó 和 Drew Weissman 因在 mRNA 核苷堿基修飾方面重要的基礎性發現而榮獲諾貝爾生理學或醫學獎，這一發現使得開發出針對 COVID-19 的有效 mRNA 疫苗成為了可能^[1]。在 mRNA 體外轉錄 (In Vitro Transcription, IVT) 時摻入修飾的核苷, 如假尿苷 (Ψ)、N1-甲基假尿苷 (m1Ψ) 等, 可以降低其免疫原性、增強穩定性并提高產出。IVT mRNA 的核苷修飾已有廣泛研究, 如核苷修飾的 COVID-19 mRNA 疫苗在較早之前已得到緊急使用授權^[2]。近期，核苷修飾在 mRNA 體外轉錄工藝流程中的安全性和有效性引發了行業內外的高度關注。

理論基礎

2005 年，Katalin Karikó 等人首次發現將假尿苷 (Ψ) 引入 RNA 中能降低其免疫原性，并且 RNA 的免疫原性隨著假尿苷 (Ψ) 引入比例的增高而降低^[3]。2008 年，Katalin Karikó 等人再次發現用假尿苷 (Ψ) 完全替代尿苷 (U) 的 mRNA 不僅能極大地降低 mRNA 的免疫原性，還能提高 mRNA 的穩定性并增強其翻譯能力^[4]。2010 年，Anderson 等人進一步探究假尿苷 (Ψ) 增強 mRNA 翻譯能力的原因，結果顯示，含有尿苷的 mRNA 體外轉錄會激活 RNA 依賴性蛋白激酶 (PKR)，隨后磷酸化翻譯起始因子 (eIF-2α)并抑制翻譯，而含有假尿苷的 mRNA 在體外轉錄過程中較少的激活 PKR，其翻譯不會受到抑制^[5]。

前沿動態

2.2分離譜圖及數據

 

 

 

參考文獻

[1] The Nobel Prize in Physiology or Medicine 2023. https://www.nobelprize.org/prizes/medicine/2023/summary/

[2] 侯書婷, 于傳飛, 王蘭, 王軍志. 體外轉錄mRNA藥物的技術進展和應用前景[J]. 藥學學報, 2023, 58(8): 2047-2058. DOI: 10.16438/j.0513-4870.2023-0201

[3] Karikó, K., Buckstein, M., Ni, H. and Weissman, D. Suppression of RNA Recognition by Toll-like Receptors: The impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

[4] Karikó, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S. and Weissman, D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16, 1833–1840 (2008).

[5] Anderson, B.R., Muramatsu, H., Nallagatla, S.R., Bevilacqua, P.C., Sansing, L.H., Weissman, D. and Karikó, K. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38, 5884–5892 (2010).

[6] Overview of In Vitro Transcription. https://www.thermofisher.cn/cn/zh/home/industrial/pharma-biopharma/nucleic-acid-therapeutic-development-solutions/mrna-research/overview-in-vitro-transcription.html

[7] Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release. 2015 Nov 10;217:337-44. doi: 10.1016/j.jconrel.2015.08.051. Epub 2015 Sep 3. PMID: 26342664.

[8] Morais P, Adachi H, Yu YT. The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. Front Cell Dev Biol. 2021 Nov 4; 9:789427. doi: 10.3389/fcell.2021.789427. PMID: 34805188; PMCID: PMC8600071.

[9] Mulroney, T.E., Pöyry, T., Yam-Puc, J.C. et al. N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Nature 625, 189–194 (2024). https://doi.org/10.1038/s41586-023-06800-3

[10] Ziegenhals T, Frieling R, Wolf P, Göbel K, Koch S, Lohmann M, Baiersdörfer M, Fesser S, Sahin U, Kuhn AN. Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP. Front Mol Biosci. 2023 Dec 11;10:1291045. doi: 10.3389/fmolb.2023.1291045.

[11] Welbourne EN, Loveday KA, Nair A, Nourafkan E, Qu J, Cook K, Kis Z, Dickman MJ. Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development. Front Mol Biosci. 2024 Mar 7;11:1250833. doi: 10.3389/fmolb.2024.1250833. PMID: 38516194; PMCID: PMC10955092.

[12] Acclaim C30 column - Thermo ScientificTM AcclaimTM C30 columns provide unique selectivity and superior separations of hydrophobic, structurally related analytes.