Detzer Anke, Overhoff Marita, Wünsche Winfried, Rompf Maria, Turner John J, Ivanova Gabriela D, Gait Michael J, Sczakiel Georg
Institut für Molekulare Medizin, Universität zu Lübeck and Schleswig-Holstein, Germany.
RNA. 2009 Apr;15(4):627-36. doi: 10.1261/rna.1305209. Epub 2009 Feb 18.
In the last decade short interfering RNA (siRNA) became an important means for functional genomics and the development of gene-specific drugs. However, major technical hurdles in the application of siRNA include its cellular delivery followed by its intracellular trafficking and its release in order to enter the RNA interference (RNAi) machinery. The novel phosphorothioate-stimulated cellular uptake of siRNA contrasts other known delivery systems because it involves a caveosomal pathway in which large amounts of siRNA are delivered to the perinuclear environment, leading to measurable though moderate target suppression. Limited efficacy seems to be related to intracellular trapping of siRNA. To study the role of intracellular trafficking of siRNA for biological effectiveness we studied whether a signal peptide for trans-membrane transport of bacterial protein toxins, which is covalently attached to siRNA, can promote its release from the perinuclear space into the cytoplasm and thereby enhance its biological effectiveness. We show that attachment of the peptide TQIENLKEKG to lamin A/C-directed siRNA improves target inhibition after its PS-stimulated delivery. This is related to increased efflux of the siRNA-peptide conjugate from the ER-specific perinuclear sites. In summary, this study strongly suggests that intracellular release of siRNA leads to increased biological effectiveness. Thus covalent peptide-siRNA conjugates are proposed as new tools to study the relationship between intracellular transport and efficacy of siRNA.
在过去十年中，短干扰RNA（siRNA）成为功能基因组学和基因特异性药物研发的重要手段。然而，siRNA应用中的主要技术障碍包括其细胞递送，随后是细胞内运输及其释放，以便进入RNA干扰（RNAi）机制。新型硫代磷酸酯刺激的siRNA细胞摄取与其他已知递送系统不同，因为它涉及一种小窝体途径，大量siRNA被递送至核周环境，导致可测量但适度的靶标抑制。有限的疗效似乎与siRNA的细胞内滞留有关。为了研究siRNA的细胞内运输对生物学有效性的作用，我们研究了与siRNA共价连接的细菌蛋白毒素跨膜运输信号肽是否能促进其从核周空间释放到细胞质中，从而增强其生物学有效性。我们表明，将肽TQIENLKEKG连接到针对核纤层蛋白A/C的siRNA上，在PS刺激递送后可改善靶标抑制。这与siRNA-肽缀合物从内质网特异性核周位点的流出增加有关。总之，这项研究强烈表明，siRNA的细胞内释放会导致生物学有效性增加。因此，共价肽-siRNA缀合物被提议作为研究siRNA细胞内运输与疗效之间关系的新工具。
