Moradian Hanieh, Schwestka Marko, Roch Toralf, Gossen Manfred
Institute of Active Polymers, Helmholtz-Zentrum Hereon Teltow Germany.
Berlin Institute of Health Center for Regenerative Therapies (BCRT) Berlin Germany.
Bioeng Transl Med. 2023 Nov 28;9(4):e10622. doi: 10.1002/btm2.10622. eCollection 2024 Jul.
Recent technological advances in the production of in vitro transcribed messenger RNA (IVT-mRNA) facilitate its clinical use as well as its application in basic research. In this regard, numerous chemical modifications, which are not naturally observed in endogenous mRNA, have been implemented primarily to address the issue of immunogenicity and improve its biological performance. However, recent findings suggested pronounced differences between expression levels of IVT-mRNAs with different nucleoside modifications in transfected cells. Given the multistep process of IVT-mRNA delivery and subsequent intracellular expression, it is unclear which step is influenced by IVT-mRNA chemistry. Here, we deconvolute this process and show that the nucleoside modification does not interfere with complexation of carriers, their physicochemical properties, and extracellular stability, as exemplified by selected modifications. The immediate effect of mRNA chemistry on the efficiency of ribosomal protein synthesis as a contributor to differences in expression was quantified by in vitro cell-free translation. Our results demonstrate that for the nucleoside modifications tested, translatability was the decisive step in determining overall protein production. Also of special importance for future work on rational selection of tailored synthetic mRNA chemistries, our findings set a workflow to identify potentially limiting, modification-dependent steps in the complex delivery process.
近期体外转录信使核糖核酸（IVT-mRNA）生产技术的进步促进了其在临床及基础研究中的应用。在这方面，人们进行了多种内源性mRNA中未天然存在的化学修饰，主要是为了解决免疫原性问题并改善其生物学性能。然而，最近的研究结果表明，在转染细胞中，具有不同核苷修饰的IVT-mRNA的表达水平存在显著差异。鉴于IVT-mRNA递送及随后细胞内表达的多步骤过程，尚不清楚IVT-mRNA化学修饰影响的是哪个步骤。在此，我们对这一过程进行了剖析，结果表明，如所选修饰所示，核苷修饰不会干扰载体的复合、其理化性质及细胞外稳定性。通过体外无细胞翻译对作为表达差异原因的mRNA化学修饰对核糖体蛋白合成效率的直接影响进行了量化。我们的结果表明，对于所测试的核苷修饰，可翻译性是决定总体蛋白产量的决定性步骤。我们的研究结果还为合理选择定制化合成mRNA化学修饰的未来工作设定了一个工作流程，以识别复杂递送过程中潜在的、依赖修饰的限制步骤，这对未来工作也尤为重要。
