Hashimoto Atsushi, Kunitomo Yuma, Kikuchi Ittoku, Yamada Hiroki, Kobayashi Keiko, Soshiroda Kazuhiro, Aman Hiromi, Kimura Yasuaki, Yamamoto Junichiro, Shiraishi Yasuhisa, Uchida Satoshi, Abe Hiroshi, Iwai Hiroto
Innovation Center, Research Division Kyowa Kirin Co., Ltd. 3-6-6 Asahi, Machida Tokyo 194-8533 Japan
Department of Chemistry, Graduate School of Science, Nagoya University Furo, Chikusa Nagoya 464-8602 Japan.
RSC Chem Biol. 2025 Aug 19. doi: 10.1039/d5cb00137d.
The poly(A) tail plays a crucial role in mRNA stability and translation efficiency. Chemical modification of the poly(A) tail is a promising approach for stabilizing mRNA against deadenylation. In this study, we investigated the effect of poly(A) chemical modifications using phosphorothioate (PS), 2'-fluoro (2'-F), 2'--methyl (2'-OMe), and 2'--methoxyethyl (2'-MOE) modifications. Notably, PS, 2'-OMe, and 2'-MOE modifications conferred resistance to CAF1, an enzyme responsible for deadenylation. Interestingly, only the PS modification retained the poly(A)-binding protein (PABP) binding activity, which is critical for translation, whereas 2'-F, 2'-OMe, and 2'-MOE modifications abolished this activity. Beyond the PS modification, the combination of 2'-F, 2'-OMe, and 2'-MOE modifications resulted in enhanced resistance to both CAF1 and other nucleases. Based on these results, a 12-nucleotide unmodified poly(A) sequence was inserted upstream of the modified poly(A) to confer both nuclease resistance and PABP-binding activity. Notably, the resulting poly(A) formulation significantly prolonged protein expression in cultured cells and mouse skin when applied to epidermal growth factor-encoding therapeutic mRNA. Collectively, this study presents a design concept for poly(A) chemical modifications to achieve durable protein expression from mRNA, offering a promising strategy for enhancing the function of mRNA-based therapeutics.
聚腺苷酸（poly(A)）尾巴在信使核糖核酸（mRNA）稳定性和翻译效率中起着关键作用。对聚腺苷酸尾巴进行化学修饰是一种稳定mRNA防止其去腺苷酸化的有前景的方法。在本研究中，我们使用硫代磷酸酯（PS）、2'-氟（2'-F）、2'-O-甲基（2'-OMe）和2'-O-甲氧基乙基（2'-MOE）修饰研究了聚腺苷酸化学修饰的效果。值得注意的是，PS、2'-OMe和2'-MOE修饰赋予了对CAF1（一种负责去腺苷酸化的酶）的抗性。有趣的是，只有PS修饰保留了对翻译至关重要的聚腺苷酸结合蛋白（PABP）结合活性，而2'-F、2'-OMe和2'-MOE修饰则消除了这种活性。除了PS修饰外，2'-F、2'-OMe和2'-MOE修饰的组合导致对CAF1和其他核酸酶的抗性增强。基于这些结果，在修饰的聚腺苷酸上游插入了一个12个核苷酸的未修饰聚腺苷酸序列，以赋予核酸酶抗性和PABP结合活性。值得注意的是，当应用于编码表皮生长因子的治疗性mRNA时，所得的聚腺苷酸制剂显著延长了培养细胞和小鼠皮肤中的蛋白质表达。总的来说，本研究提出了一种聚腺苷酸化学修饰的设计概念，以实现从mRNA持久表达蛋白质，为增强基于mRNA的治疗药物的功能提供了一种有前景的策略。
