RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA; Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA.
Mol Ther. 2018 Aug 1;26(8):1973-1982. doi: 10.1016/j.ymthe.2018.05.024. Epub 2018 Jun 21.
Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5'-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5'-(E)-vinylphosphonate and alternating 2'-fluoro and 2'-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at ∼3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles.
细胞外囊泡是治疗性 RNA 的有前途的递药载体。胆固醇偶联的小干扰 RNA（siRNA）能够有效地将治疗性货物装载到细胞外囊泡中，且重现性好。siRNA 通常经过化学修饰以适应特定应用。然而，siRNA 的化学修饰模式尚未针对细胞外囊泡介导的递送来进行专门优化。在这里，我们使用胆固醇偶联的、疏水性修饰的不对称 siRNA（hsiRNA）来评估骨架、5' - 磷酸酯和连接子化学修饰对有效负载 hsiRNA 到细胞外囊泡上的影响。具有 5' -（E） - 乙烯膦酸酯和交替 2'- 氟和 2'-O- 甲基骨架修饰的 hsiRNA 在神经元中进行亨廷顿基因沉默的细胞外囊泡介导方面优于以前使用的部分修饰的 siRNA。在两种商业上可获得的连接子（三甘醇[TEG]和 2-氨基丁基-1-3-丙二醇[C7]）中，用于将胆固醇连接到 siRNA 的连接子中，TEG 比 C7 更有利于有效地进行外泌体加载。连接子的不稳定完全消除了负载细胞外囊泡的沉默活性。胆固醇偶联 siRNA 的装载量在每个细胞外囊泡约 3000 个 siRNA 拷贝时达到饱和。超载会损害细胞外囊泡的沉默活性。本文报道的数据提供了一种优化方案，用于成功地使用疏水性修饰作为将 RNA 货物有效装载到细胞外囊泡中的策略。
