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Endocytosis Controls siRNA Efficiency: Implications for siRNA Delivery Vehicle Design and Cell-Specific Targeting.
Nucleic Acid Therapeutics ( IF 4 ) Pub Date : 2019-11-12 , DOI: 10.1089/nat.2019.0804
Daniel Vocelle 1 , Christina Chan 1, 2 , S Patrick Walton 1
Affiliation  

While small interfering RNAs (siRNAs) are commonly used for laboratory studies, development of siRNA therapeutics has been slower than expected, due, in part, to a still limited understanding of the endocytosis and intracellular trafficking of siRNA-containing complexes. With the recent characterization of multiple clathrin-/caveolin-independent endocytic pathways, that is, those mediated by Graf1, Arf6, and flotillin, it has become clear that the endocytic mechanism influences subsequent intracellular processing of the internalized cargo. To explore siRNA delivery in light of these findings, we developed a novel assay that differentiates uptake by each of the endocytic pathways and can be used to determine whether endocytosis by a pathway leads to the initiation of RNA interference (RNAi). Using Lipofectamine 2000 (LF2K), we determined the endocytosis pathway leading to active silencing (whether by clathrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types (HeLa, H1299, HEK293, and HepG2). We showed that LF2K is internalized by Graf1-, Arf6-, or flotillin-mediated endocytosis for the initiation of RNAi, depending on cell type. In addition, we found that a portion of siRNA-containing complexes is internalized by pathways that do not lead to initiation of silencing. Inhibition of these pathways enhanced intracellular levels of siRNAs with concomitant enhancement of silencing.

中文翻译:

内吞作用控制siRNA效率:对siRNA运载工具设计和细胞特异性靶向的意义。

尽管小干扰RNA(siRNA)通常用于实验室研究,但siRNA治疗剂的开发却比预期的要慢,部分原因是对含siRNA的复合物的内吞作用和细胞内运输的了解仍然有限。随着最近表征多个网格蛋白-/ caveolin独立的内吞途径,即那些由Graf1,Arf6和flotillin介导的途径,很明显,内吞机制影响内在货物的后续细胞内加工。为了根据这些发现探索siRNA的传递,我们开发了一种新颖的测定法,该测定法可区分每种内吞途径的摄取,并可用于确定通路的内吞作用是否导致RNA干扰(RNAi)的启动。使用Lipofectamine 2000(LF2K),我们确定了跨多种细胞类型(HeLa,H1299,HEK293和HepG2)导致主动沉默(无论是网格蛋白,小窝蛋白,Arf6,Graf1,弗洛蒂林还是大胞饮作用)的内吞途径。我们显示LF2K被Graf1,Arf6或弗洛特林介导的内吞作用内在化,以启动RNAi,具体取决于细胞类型。此外,我们发现一部分含siRNA的复合物被不导致沉默启动的途径内化。这些途径的抑制增强了siRNA的细胞内水平,同时伴随沉默的增强。或氟替林介导的内吞作用引发RNAi,具体取决于细胞类型。此外,我们发现一部分含siRNA的复合物被不导致沉默启动的途径内化。这些途径的抑制增强了siRNA的细胞内水平,同时伴随沉默的增强。或氟替林介导的内吞作用引发RNAi,具体取决于细胞类型。此外,我们发现一部分含siRNA的复合物被不导致沉默启动的途径内化。这些途径的抑制增强了siRNA的细胞内水平,同时伴随沉默的增强。
更新日期:2019-11-01
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