Modified mRNA Formulation and Stability for Cardiac and Skeletal Muscle Delivery.

Directly injecting naked or lipid nanoparticle (LNP)-encapsulated modified mRNA (modRNA) allows rapid and efficient protein expression. This non-viral technology has been used successfully in modRNA vaccines against SARS-CoV-2. The main challenges in using modRNA vaccines were the initial requirement for an ultra-cold storage to preserve their integrity and concerns regarding unwanted side effects from this new technology. Here, we showed that naked modRNA maintains its integrity when stored up to 7 days at 4 °C, and LNP-encapsulated modRNA for up to 7 days at room temperature. Naked modRNA is predominantly expressed at the site of injection when delivered into cardiac or skeletal muscle. In comparison, LNP-encapsulated modRNA granted superior protein expression but also additional protein expression beyond the cardiac or skeletal muscle injection site. To overcome this challenge, we developed a skeletal-muscle-specific modRNA translation system (skeletal muscle SMRTs) for LNP-encapsulated modRNA. This system allows controlled protein translation predominantly at the site of injection to prevent potentially detrimental leakage and expression in major organs. Our study revealed the potential of the SMRTs platform for controlled expression of mRNA payload delivered intramuscularly. To conclude, our SMRTs platform for LNP-encapsulated modRNA can provide safe, stable, efficient and targeted gene expression at the site of injection.

直接注射裸的或脂质纳米颗粒（LNP）包裹的修饰信使核糖核酸（modRNA）可实现快速高效的蛋白质表达。这种非病毒技术已成功用于针对新冠病毒的modRNA疫苗。使用modRNA疫苗的主要挑战在于，最初需要超低温储存以保持其完整性，以及对这种新技术可能产生不良副作用的担忧。在此，我们表明裸的modRNA在4°C下储存长达7天能保持其完整性，而LNP包裹的modRNA在室温下储存长达7天也能保持其完整性。当裸的modRNA被递送至心肌或骨骼肌时，其主要在注射部位表达。相比之下，LNP包裹的modRNA可实现更优的蛋白质表达，但在心肌或骨骼肌注射部位之外也会有额外的蛋白质表达。为克服这一挑战，我们为LNP包裹的modRNA开发了一种骨骼肌特异性modRNA翻译系统（骨骼肌SMRTs）。该系统可主要在注射部位控制蛋白质翻译，以防止在主要器官中可能有害的泄漏和表达。我们的研究揭示了SMRTs平台用于肌肉内递送的信使核糖核酸有效载荷的可控表达的潜力。总之，我们用于LNP包裹的modRNA的SMRTs平台能够在注射部位提供安全、稳定、高效且靶向的基因表达。