Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, California.
Department of Medicinal Chemistry, Ionis Pharmaceuticals, Inc., Carlsbad, California.
Nucleic Acid Ther. 2019 Oct;29(5):245-255. doi: 10.1089/nat.2019.0794. Epub 2019 Jun 3.
Antisense oligonucleotides modified with phosphorothioate linkages (PS-ASOs) can enter cells via endocytic pathways and must escape from membraned organelles to reach target RNAs. We recently found that membrane destabilization induced by different lipid species contributes to PS-ASO release from late endosomes (LEs). In this study, we characterized intracellular uptake, trafficking, and activities of PS-ASOs conjugated with different lipid species. We found that palmitic acid-, tocopherol-, and cholesterol-conjugated PS-ASOs have increased protein binding and enhanced intracellular uptake compared to unconjugated PS-ASOs. Similar to the parental PS-ASO, the lipid-conjugated PS-ASOs traffic from early to LEs without incorporation into lipid droplets. Unlike parental PS-ASOs, the lipid-conjugated PS-ASOs tend to remain associated with plasma or endosomal membranes, and this appears to influence their release from endosomes. The lipid-conjugated PS-ASOs were released more rapidly than parental PS-ASO. These results suggest that lipid conjugation enhances the interactions of PS-ASOs with proteins or membranes, in turn facilitating intracellular trafficking and endosomal release.
经硫代磷酸酯键修饰的反义寡核苷酸（PS-ASO）可通过内吞途径进入细胞，并且必须从膜细胞器中逃逸才能到达靶 RNA。我们最近发现，不同脂质种类诱导的膜不稳定有助于 PS-ASO 从晚期内体（LE）中释放。在这项研究中，我们对与不同脂质种类缀合的 PS-ASO 的细胞内摄取、转运和活性进行了表征。我们发现，与未缀合的 PS-ASO 相比，棕榈酸酯、生育酚和胆固醇缀合的 PS-ASO 具有增加的蛋白结合和增强的细胞内摄取。与亲本 PS-ASO 相似，脂质缀合的 PS-ASO 从早期到 LE 的转运而不整合到脂滴中。与亲本 PS-ASO 不同，脂质缀合的 PS-ASO 往往与质膜或内体膜保持关联，这似乎影响它们从内体中的释放。脂质缀合的 PS-ASO 比亲本 PS-ASO 更快地释放。这些结果表明，脂质缀合增强了 PS-ASO 与蛋白质或膜的相互作用，从而促进细胞内转运和内体释放。
