Pandey Ekta, Harris Edward N
University of Nebraska, Department of Biochemistry, Beadle Center, 1901 Vine St., Lincoln, NE 68588, USA.
Mol Ther Nucleic Acids. 2023 Jul 19;33:430-443. doi: 10.1016/j.omtn.2023.07.019. eCollection 2023 Sep 12.
Non-DNA-binding Stabilin-2/HARE receptors expressed on liver sinusoidal endothelial cells specifically bind to and internalize several classes of phosphorothioate antisense oligonucleotides (PS-ASOs). After Stabilin-mediated uptake, PS-ASOs are trafficked within endosomes (>97%-99%), ultimately resulting in destruction in the lysosome. The ASO entrapment in endosomes lowers therapeutic efficacy, thereby increasing the overall dose for patients. Here, we use confocal microscopy to characterize the intracellular route transverse by PS-ASOs after Stabilin receptor-mediated uptake in stable recombinant Stabilin-1 and -2 cell lines. We found that PS-ASOs as well as the Stabilin-2 receptor transverse the classic path: clathrin-coated vesicle-early endosome-late endosome-lysosome. Chloroquine exposure facilitated endosomal escape of PS-ASOs leading to target knockdown by more than 50% as compared to untreated cells, resulting in increased PS-ASO efficacy. We also characterize cytosolic galectins as novel contributor for PS-ASO escape. Galectins knockdown enhances ASO efficacy by more than 60% by modulating EEA1, Rab5C, and Rab7A mRNA expression, leading to a delay in the endosomal vesicle maturation process. Collectively, our results provide additional insight for increasing PS-ASO efficacy by enhancing endosomal escape, which can further be utilized for other nucleic acid-based modalities.
肝血窦内皮细胞上表达的非DNA结合型Stabilin-2/HARE受体可特异性结合并内化几类硫代磷酸酯反义寡核苷酸（PS-ASO）。在Stabilin介导的摄取后，PS-ASO在内体中运输（>97%-99%），最终在溶酶体中被降解。内体中ASO的截留降低了治疗效果，从而增加了患者的总体用药剂量。在此，我们利用共聚焦显微镜来表征在稳定的重组Stabilin-1和-2细胞系中，PS-ASO在Stabilin受体介导的摄取后所穿过的细胞内途径。我们发现PS-ASO以及Stabilin-2受体遵循经典途径：网格蛋白包被囊泡-早期内体-晚期内体-溶酶体。与未处理的细胞相比，氯喹处理促进了PS-ASO的内体逃逸，导致靶点敲低超过50%，从而提高了PS-ASO的疗效。我们还将胞质半乳糖凝集素表征为PS-ASO逃逸的新因素。半乳糖凝集素的敲低通过调节EEA1、Rab5C和Rab7A的mRNA表达，使ASO疗效提高超过60%，导致内体囊泡成熟过程延迟。总体而言，我们的结果为通过增强内体逃逸来提高PS-ASO疗效提供了更多见解，这可进一步应用于其他基于核酸的治疗方式。
