Everton Elissa, Rizvi Fatima, Smith Anna R, Beattie Mitchell, Tam Ying, Pardi Norbert, Weissman Drew, Gouon-Evans Valerie
Center for Regenerative Medicine and the Section of Gastroenterology, Boston University and Boston Medical Center, Boston, USA.
Acuitas Therapeutics, Vancouver, Canada.
Bio Protoc. 2021 Oct 5;11(19):e4184. doi: 10.21769/BioProtoc.4184.
With the recent availability of the SARS-CoV-2 mRNA-based vaccines, public attention has been drawn to this new technology and how it may be applied to other indications. Temporal activation of key hepatic regenerative pathways can induce liver regeneration, overcoming the lack of donor organs for liver transplantation and ineffectiveness of alternative treatments. Recombinant protein therapies and genetic therapies that target these pathways require frequent and repeated injections or, when integrated into the genome, may lead to deleterious effects. In contrast, nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) are non-integrative and induce transient yet robust expression of proteins that could serve as an ideal therapeutic tool to treat specific liver diseases. For instance, our recent publication in used mRNA-LNP to express hepatic mitogens, hepatocyte growth factor, and epidermal growth factor to induce liver regeneration following both acute and chronic liver injuries. Initial testing with firefly luciferase mRNA-LNP transfection and imaging confirmed specific hepatotropic delivery. In this protocol, we describe in detail the necessary steps to deliver mRNA-LNP to the murine liver and, following intravenous injection of eGFP mRNA-LNP, verify transfection efficiency using flow cytometry and liver cell specificity using immunofluorescence analyses. This procedure presents an unprecedented tool that can be customized with mRNA-LNP encoding any protein of interest to be expressed by virtually all hepatocytes, ~70% endothelial cells, and ~40% Kupffer cells for promoting liver function and/or regeneration. Graphic abstract: (Created with BioRender.com).
随着基于严重急性呼吸综合征冠状病毒2（SARS-CoV-2）信使核糖核酸（mRNA）的疫苗近期问世，这项新技术及其可能如何应用于其他适应症已引起公众关注。关键肝脏再生途径的短暂激活可诱导肝脏再生，克服肝移植供体器官短缺以及替代治疗无效的问题。针对这些途径的重组蛋白疗法和基因疗法需要频繁重复注射，或者整合到基因组中时可能会产生有害影响。相比之下，包裹在脂质纳米颗粒中的核苷修饰mRNA（mRNA-LNP）不会整合到基因组中，能诱导蛋白质短暂而强劲的表达，可作为治疗特定肝脏疾病的理想治疗工具。例如，我们最近在[具体文献]中发表的研究使用mRNA-LNP来表达肝脏促细胞分裂剂、肝细胞生长因子和表皮生长因子，以诱导急性和慢性肝损伤后的肝脏再生。使用萤火虫荧光素酶mRNA-LNP转染和成像进行的初步测试证实了其对肝脏的特异性递送。在本方案中，我们详细描述了将mRNA-LNP递送至小鼠肝脏的必要步骤，并且在静脉注射增强型绿色荧光蛋白（eGFP）mRNA-LNP后，使用流式细胞术验证转染效率，并使用免疫荧光分析验证肝细胞特异性。该程序提供了一种前所未有的工具，可通过编码几乎所有肝细胞、约70%的内皮细胞和约40%的库普弗细胞中任何感兴趣蛋白质的mRNA-LNP进行定制，以促进肝功能和/或肝脏再生。图形摘要：（由BioRender.com创建）
