The field of gene therapy has evolved over the past two decades after the first introduction of nucleic acid drugs, such as plasmid DNA (pDNA). With the development of in vitro transcription (IVT) methods, synthetic mRNA has become an emerging class of gene therapy. IVT mRNA has several advantages over conventional pDNA for the expression of target proteins. mRNA does not require nuclear localization to mediate protein translation. The intracellular process for protein expression is much simpler and there is no potential risk of insertion mutagenesis. Having these advantages, the level of protein expression is far enhanced as comparable to that of viral expression systems. This makes IVT mRNA a powerful alternative gene expression system for various applications in regenerative medicine. In this review, we highlight the synthesis and preparation of IVT mRNA and its therapeutic applications. The article includes the design and preparation of IVT mRNA, chemical modification of IVT mRNA, and therapeutic applications of IVT mRNA in cellular reprogramming, stem cell engineering, and protein replacement therapy. Finally, future perspectives and challenges of IVT mRNA are discussed.

在首次引入核酸药物（例如质粒DNA（pDNA））后的过去二十年中，基因治疗领域得到了发展。随着体外的发展转录（IVT）方法，合成的mRNA已成为一种新兴的基因治疗方法。IVT mRNA与常规pDNA相比在靶蛋白表达方面具有多个优势。mRNA不需要核定位即可介导蛋白质翻译。蛋白表达的细胞内过程非常简单，并且没有潜在的插入诱变风险。具有这些优点，与病毒表达系统相比，蛋白质表达水平大大提高。这使IVT mRNA成为强大的替代基因表达系统，可用于再生医学的各种应用。在这篇综述中，我们重点介绍了IVT mRNA的合成，制备及其治疗应用。本文包括IVT mRNA的设计和制备，IVT mRNA的化学修饰，IVT mRNA在细胞重编程，干细胞工程和蛋白质替代疗法中的应用和治疗应用。最后，讨论了IVT mRNA的未来观点和挑战。