The Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
Faculty of Science, Charles University in Prague, Czech Republic.
J Gene Med. 2018 Jul;20(7-8):e3025. doi: 10.1002/jgm.3025. Epub 2018 Jun 25.
Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design.
The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone.
Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27% compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68% and 62%, respectively), which was comparable to nonpalmitoyl gapmers (68% and 82%, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68% and 66% in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67% and 66% in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent.
The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.
反义寡核苷酸药物需要递送和生物分布使能技术来提高体内疗效。一种有吸引力的方法是通过将脂肪酸纳入间隙设计来结合并由此介导内源性人血清白蛋白池的运输。
本研究调查了棕榈酰修饰及其位置对带硫代磷酸酯（PS）或磷酸二酯（PO）骨架的间隙物与白蛋白结合、细胞摄取和体外基因沉默的影响。
两个棕榈酰基仅位于 5'端，或一个棕榈酰基位于 3'和 5'位置，通过凝胶迁移分析显示与人血清白蛋白具有相似的结合。通过流式细胞术测定的细胞摄取减少（与非棕榈酰化间隙物相比为 27%）观察到棕榈酰化 Cy5.5 标记的间隙物。然而，用转染剂在 PC-3 和 Caco-2 细胞中显示出棕榈酰化间隙物的 HER3（人表皮生长因子受体 3）基因沉默（分别为 68%和 62%），与非棕榈酰化间隙物相当（分别为 68%和 82%）。重要的是，在没有转染剂的情况下，带有两个 3'和 5'位置的单个棕榈酰基的 PO 间隙物表现出高沉默效率（分别在 PC-3 和 Caco-2 细胞中为 68%和 66%），与 PS 非棕榈酰化间隙物相似（分别在 PC-3 和 Caco-2 细胞中为 67%和 66%）。
本研究定义了具有高基因沉默活性和白蛋白结合的磷酸二酯间隙物设计标准，可与可能与硫代磷酸酯间隙物设计相关的较少体内毒性一起使用。
