Lin Cheng, Kuzmanović Adrian, Wang Nan, Liao Liangliang, Ernst Sabrina, Penners Christian, Jans Alexander, Hammoor Thomas, Stach Petra Bumnuri, Peltzer Mona, Volkert Ines, Zechendorf Elisabeth, Hassan Reham, Myllys Maiju, Liedtke Christian, Herrmann Andreas, Chakraborty Gurudas, Trautwein Christian, Hengstler Jan, Müller-Newen Gerhard, Wang Junqing, Ghallab Ahmed, Bartneck Matthias
Department of Internal Medicine III, University Hospital RWTH Aachen, Pauwelsstraße 30, 52074, Aachen, Germany.
Department of Rheumatology and Shanghai Institute of Rheumatology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200240, China.
Adv Sci (Weinh). 2025 Mar;12(9):e2409729. doi: 10.1002/advs.202409729. Epub 2025 Jan 10.
Most gene therapies exert their actions via manipulation of hepatocytes (parenchymal cells) and the reasons behind the suboptimal performance of synthetic mRNA in non-parenchymal cells (NPC) such as Kupffer cells (KC), and liver macrophages, remain unclear. Here, the spatio-temporal distribution of mRNA encoding enhanced green fluorescent protein (Egfp), siRNA, or both co-encapsulated into lipid nanoparticles (LNP) in the liver in vivo using real-time intravital imaging is investigated. Although both KC and hepatocytes demonstrate comparable high and rapid uptake of mRNA-LNP and siRNA-LNP in vivo, the translation of Egfp mRNA occurs exclusively in hepatocytes during intravital imaging. Despite attempts such as inhibiting intracellular ribonuclease, substituting uridine bases in mRNA with pseudouridine, and using a different ionizable lipid in the LNP mixture, no substantial increase in Egfp translation by NPC is possible. The investigation reveals that hepatocytes, which are distinct from other liver cells due to their polyploidy, exhibit significantly elevated levels of total RNA and protein, along with a higher proportion of ribosomal protein per individual cell. Consequently, fundamental cellular differences account for the low mRNA translation observed in NPC. The findings therefore suggest that cellular biology imposes a natural limitation on synthetic mRNA translation that is strongly influenced by cellular ploidy.
大多数基因疗法通过操纵肝细胞（实质细胞）发挥作用，而合成mRNA在非实质细胞（NPC）如库普弗细胞（KC）和肝巨噬细胞中的表现欠佳，其背后的原因仍不清楚。在此，利用实时活体成像研究了编码增强型绿色荧光蛋白（Egfp）、小干扰RNA（siRNA）或两者共封装于脂质纳米颗粒（LNP）中的mRNA在体内肝脏中的时空分布。尽管KC和肝细胞在体内对mRNA-LNP和siRNA-LNP均表现出相当高且快速的摄取，但在活体成像过程中，Egfp mRNA的翻译仅发生在肝细胞中。尽管尝试了诸如抑制细胞内核糖核酸酶、用假尿苷替代mRNA中的尿嘧啶碱基以及在LNP混合物中使用不同的可电离脂质等方法，但NPC对Egfp的翻译仍无法显著增加。该研究表明，由于其多倍体特性而与其他肝细胞不同的肝细胞，其总RNA和蛋白质水平显著升高，且每个细胞中核糖体蛋白的比例更高。因此，细胞的基本差异导致了NPC中观察到的低mRNA翻译。因此，这些发现表明细胞生物学对合成mRNA的翻译施加了自然限制，而这种限制受到细胞倍性的强烈影响。
