Phosphorothioate-modified antisense oligonucleotide (PS-ASO) drugs are commonly used to modulate gene expression through RNase H1-mediated cleavage of target RNAs. Upon internalization through endocytic pathways into cells, PS-ASOs must be released from membraned endosomal organelles to act on target RNAs, a limiting step of PS-ASO activity. Here we report that Hsc70 protein mediates productive release of PS-ASOs from endosomes. Hsc70 protein was enriched in endosome fractions shortly after PS-ASO incubation with cells. Reduction of Hsc70 significantly decreased the activities of PS-ASOs in reducing target RNAs. PS-ASO uptake and transport from early endosomes to late endosomes (LEs) were not affected upon Hsc70 reduction; however, endosomal release of PS-ASOs was impaired. Reduction of Hsc70 led to more scattered mannose-6-phosphate receptor (M6PR) localization at LEs in the cytoplasm, in contrast to the perinuclear localization at trans-Golgi network (TGN) in control cells, suggesting that retrograde transport of M6PR from LEs to TGN was affected. Consistently, reduction of Hsc70 increased colocalization of M6PR and PS-ASOs at LEs, and also delayed M6PR antibody transport from LE to TGN. Together, these results suggest that Hsc70 protein is involved in M6PR vesicle escape from LEs and may thus enhance PS-ASO release from LEs.

中文翻译：

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Hsc70 促进甘露糖-6-磷酸受体介导的细胞内运输并增强硫代磷酸酯修饰的反义寡核苷酸的内体释放

硫代磷酸酯修饰的反义寡核苷酸 (PS-ASO) 药物通常用于通过 RNase H1 介导的靶 RNA 切割来调节基因表达。在通过内吞途径内化进入细胞后，PS-ASO 必须从膜内体细胞器中释放出来以作用于靶 RNA，这是 PS-ASO 活性的限制步骤。在这里，我们报告 Hsc70 蛋白介导 PS-ASO 从内涵体中的生产性释放。在 PS-ASO 与细胞孵育后不久，Hsc70 蛋白就在内涵体组分中富集。Hsc70 的减少显着降低了 PS-ASO 在减少靶 RNA 方面的活性。PS-ASO 从早期内体到晚期内体 (LEs) 的摄取和运输不受 Hsc70 减少的影响；然而，PS-ASO 的内体释放受损。对照细胞中的反式高尔基网络（TGN），表明 M6PR 从 LE 到 TGN 的逆行运输受到影响。一致地，Hsc70 的减少增加了 M6PR 和 PS-ASO 在 LE 的共定位，并且还延迟了 M6PR 抗体从 LE 到 TGN 的转运。总之，这些结果表明 Hsc70 蛋白参与了 M6PR 囊泡从 LEs 的逃逸，因此可能增强 PS-ASO 从 LEs 的释放。