A New Potent Secondary Amphipathic Cell-penetrating Peptide for siRNA Delivery Into Mammalian Cells
A New Potent Secondary Amphipathic Cell-penetrating Peptide for siRNA Delivery Into Mammalian Cells
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DOI：
10.1038/mt.2008.215
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发表时间：
2009-01-01
影响因子：
12.4
通讯作者：
Divita, Gilles
中科院分区：
文献类型：
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作者：
Crombez, Laurence;Aldrian-Herrada, Gudrun;Divita, Gilles
RNA干扰是生物学研究的有力工具，但也已成为最具挑战性的治疗策略之一。然而，基于小干扰RNA（siRNA）的策略遭受其差的递送和生物分布。细胞穿透肽（CPP）已显示出改善各种生物活性分子到活细胞中的细胞内递送，并且最近已应用于siRNA递送。为了改善siRNA进入挑战性细胞系的细胞摄取，我们设计了20个残基的二级两亲性肽（CADY），其结合了芳香族色氨酸和阳离子精氨酸残基。CADY在细胞膜内采用螺旋构象，从而在一侧暴露带电残基，而在另一侧暴露有利于细胞摄取的Trp基团。我们表明，CADY与siRNA形成稳定的复合物，从而增加其稳定性并改善其递送到多种细胞系中，包括悬浮细胞系和原代细胞系。CADY介导的递送亚纳摩尔浓度的siRNA导致靶基因在mRNA和蛋白质水平上的显著敲低。此外，我们证明CADY是无毒的，并通过一种机制，这是独立的主要内体途径进入细胞。鉴于其生物学特性，我们认为基于CADY的技术将对基于siRNA的基础和治疗应用的发展产生重大影响。
RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.