Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.

中文翻译：

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表皮生长因子受体介导的细胞摄取促进了硫代磷酸酯修饰的反义寡核苷酸的细胞内活性

具有硫代磷酸酯（PS）键的化学修饰的反义寡核苷酸（ASO）已被广泛研究用作研究和治疗药物。PS-ASO甚至可以在不存在转染试剂的情况下进入细胞，并通过RNase H1触发互补RNA的切割。已经鉴定出许多结合PS-ASO并介导其细胞摄取的细胞表面蛋白。但是，导致PS-ASO生产性内在化的机制尚不十分清楚。在这里，我们表征了PS-ASO与表皮生长因子受体（EGFR）之间的相互作用。我们发现PS-ASO与EGF和EGFR一起进入网格蛋白包被的凹坑结构。在早期的内体和在扩大的晚期内体内部也观察到它们的共定位。EGFR的减少会降低PS-ASO活性，而不会影响EGF介导的信号通路，而EGFR的过表达会增加细胞中PS-ASO的活性。此外，EGFR的降低延迟了PS-ASO从早期到晚期的内体运输。因此，EGFR通过其货物依赖性运输过程将PS-ASO从早期内体迁移到晚期内体，在细胞表面与PS-ASO结合并介导PS-ASO主动（生产性）细胞摄取的基本步骤。此EGFR介导的过程还可以用作额外的模型，以更好地了解PS-ASO的细胞内摄取和内体释放机制。EGFR在细胞表面与PS-ASO结合，并通过其货物依赖性运输过程介导PS-ASO主动（生产性）细胞摄取（从早期内体向晚期内体迁移PS-ASO的重要步骤）。此EGFR介导的过程还可以用作额外的模型，以更好地了解PS-ASO的细胞内摄取和内体释放机制。EGFR在细胞表面与PS-ASO结合，并通过其货物依赖性运输过程介导PS-ASO主动（生产性）细胞摄取（从早期内体向晚期内体迁移PS-ASO的重要步骤）。此EGFR介导的过程还可以用作额外的模型，以更好地了解PS-ASO的细胞内摄取和内体释放机制。