Miller Colton M, Donner Aaron J, Blank Emma E, Egger Andrew W, Kellar Brianna M, Østergaard Michael E, Seth Punit P, Harris Edward N
University of Nebraska-Lincoln, Dept. of Biochemistry, 1901 Vine Street Lincoln NE 68588, USA.
Ionis Pharmaceuticals, 2855 Gazelle Ct, Carlsbad, CA 92010, USA.
Nucleic Acids Res. 2016 Apr 7;44(6):2782-94. doi: 10.1093/nar/gkw112. Epub 2016 Feb 22.
Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro™, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.
在过去三十年中，硫代磷酸酯（PS）修饰的反义寡核苷酸（ASO）作为药物和治疗剂受到了广泛研究。第二代ASO药物Kynamro™最近已获美国食品药品监督管理局（FDA）批准，用于治疗纯合子家族性高胆固醇血症，超过35种第二代PS ASO正处于临床开发的不同阶段。在本报告中，我们发现清道夫受体中的Stabilin家族，此前认为其不与DNA结合，但确实能结合并内化PS ASO。利用来自小鼠和大鼠肝脏的原代细胞以及分别表达Stabilin-1和Stabilin-2各亚型（315-HARE和190-HARE）的重组细胞系，我们已确定PS ASO以高亲和力结合，且这些受体负责细胞内大量的网格蛋白介导的内吞作用。结合主要依赖于盐桥的形成以及完整蛋白质受体的正确折叠。在表达Stabilin的细胞系中，内化速率的提高也增强了ASO降低非编码RNA Malat-1表达的效力。更深入地了解ASO在细胞内内化的机制及其细胞内运输途径，将有助于设计具有更好治疗特性的下一代反义药物。
