在过去的十年中，基于 mRNA 的疗法在广泛的临床应用中显示出了巨大的前景。 mRNA 技术飞跃发展的最引人注目的例子是大流行期间针对 COVID-19 的大规模疫苗接种。 mRNA免疫大规模技术和积极经验的出现，引发了抗病毒和抗癌mRNA疫苗以及遗传性疾病和其他疾病治疗性mRNA药物的发展。为了促进 mRNA 递送，脂质纳米粒子 (LNP) 已被成功使用。然而，mRNA 治疗方法的多样化使用需要开发适应性强的 LNP 递送系统，以控制靶细胞中 mRNA 摄取和表达的动力学。在这里，我们报告了通过含有 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11、 16,20-四氮杂六烷四盐酸盐(2X3)或新应用的1,30-双(cholest-5-en-3β-基氧基羰基氨基)-9,13,18,22-四氮杂-3,6,25,28-四氧三十二烷四盐酸盐(2X7) 阳离子脂质。通过对 Fluc mRNA 表达的终点和实时监测，我们发现这些 LNP 表现出异常延迟（在基于 2X7 的系统中超过 10 小时），但在细胞中具有高效且持久的报告活性。因此，用 1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺-N-[氨基（聚乙二醇）-2000] (DSPE-PEG2000) 修饰的两种 LNP 制剂都能在小鼠体内提供有效的荧光素酶产生，并在肌内注射后第 3 天达到峰值。注射。值得注意的是，仅在大腿尾部肌肉的注射部位观察到生物发光，从而证明了感兴趣的模型基因的局部表达。所开发的 mRNA 递送系统有望用于需要持续合成防御蛋白的预防性应用，并为基于 mRNA 的疗法打开了新的可能性之门。

Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3β-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.