Metabolism of Antisense Oligonucleotides and other Polyanions in Liver

反义寡核苷酸(ASO)是一种经过化学修饰的短(约20个碱基)寡核苷酸 在细胞和生物体液中抵抗核酸内切酶活性的脊椎。ASO与靶向mRNAs结合 并充当RNaseH的催化剂，破坏mRNA，从而减少基因表达。最多的 常见的ASO修饰是用磷二酯基的未桥氧原子取代 一个硫原子来产生硫代磷酸(PS)部分，到目前为止，这种部分最常用于临床ASO。 进一步的稳定和核酸酶抗性可以通过修饰2‘位被赋予。 ASO的核糖。众所周知，肝脏是PS-ASOS的天然汇，然而，其机制 对于这一活动尚不清楚。我们发现Stablin类(SR-H)清道夫受体(Stablin-1 和Stablin-2)是系统性PS-ASO清除的主要机制。稳定蛋白在一种 组织数量包括肝、淋巴结、脾、II型巨噬细胞、骨髓、 等，这可能对PS-ASO在许多组织中的传递有影响。知识上的差距是如何 PS-ASO与稳定蛋白的相互作用增加了PS-ASO对靶向mRNAs的抑制作用。我们的中央 假说认为，他汀林介导的PS-ASO内吞作用沿着两条途径进行：一条是PS-ASO- 麻生被穿梭到溶酶体(破坏)，而在另一个中，PS-ASO被允许逃脱 内体与mRNAs相互作用(功效)。我们这个项目的主要目标是，首先，确定 生物膜干涉法研究斯坦比林-PS-ASO结合络合物的生物相互作用 已知溶液和血浆中的竞争配体。其次，确定了PS-ASO的动力学 表达稳定蛋白的重组稳定细胞株和原代肝窦中的内吞作用 肝内皮细胞。我们还将剖析Stablin-2的内吞机制，我们将在其中 阐明PS-ASO活性(内体逃逸)所必需的相互作用分子。第三，到 检测PS-ASOS在WT和Stablin基因敲除小鼠体内的清除能力和生物活性 他汀类药物在肝脏以外的其他组织中的生物分布和激活。我们将使用全局和 用于这些研究的组织特异性Stablin基因敲除小鼠。这将进一步勾勒出两条可能的路径 (破坏与激活)在多个组织中，以及稳定剂如何在每个途径中起作用。预期中的 该项目的成果将有助于更好地理解结构-活性关系、有效性和 临床级PS-ASOS和他汀类生物/生化的整体代谢。

Antisense oligonucleotides (ASOs) are short (~20 bp) oligonucleotides that have chemically-modified backbones to resist endonuclease activity in cells and biological fluids. ASOs bind to targeted mRNAs within cells and act as catalysts for RNAse H to destroy the mRNA, thus decreasing gene expression. The most common ASO modification is the substitution of the unbridging oxygen atom of the phosphodiester group with a sulfur atom to create the phosphorothioate (PS) moiety which is most often used in clinical ASOs to date. Further stabilization and nuclease resistance may be conferred through the modification of the 2’ position of ribose of the ASO. It is widely known that the liver is the natural sink for PS-ASOs, however, the mechanism for this activity is not clear. We have discovered that the Stabilin class (SR-H) scavenger receptors (Stabilin-1 and Stabilin-2) are the primary mechanism for systemic PS-ASO clearance. Stabilins are expressed in a number of tissues including the sinusoids of liver, lymph nodes, spleen, Type II macrophages, bone marrow, etc which may have implications for PS-ASO delivery to many tissues. The gap in knowledge is how interactions of PS-ASOs with the Stabilins increases PS-ASO knock-down of targeted mRNAs. Our central hypothesis is that Stabilin-mediated endocytosis of PS-ASO proceeds along 2 pathways; one in which the PS- ASO is shuttled to the lysosome (destruction) and the other in which the PS-ASO is allowed to escape the endosome to interact with mRNAs (efficacy). Our primary objectives for this project are first, to determine the biological interactions of Stabilin-PS-ASO binding complexes with the use of biolayer interferometry with competing ligands in both known solutions and in plasma. Second, to determine the kinetics of PS-ASO endocytosis in both recombinant stable cells lines expressing the Stabilins and in primary sinusoidal endothelial cells of liver. We will also dissect the endocytosis mechanisms of Stabilin-2 in which we will elucidate interacting molecules that are necessary for PS-ASO activity (endosomal escape). Third, to determine the systemic clearance and bioactivity of PS-ASOs in WT and Stabilin knock-out mice to assess Stabilin-dependent biodistribution and activation in tissues other than just the liver. We will use global and tissue-specific Stabilin knockout mice for these studies. This will further delineate the two possible pathways (destruction vs activation) in multiple tissues and how the Stabilins contribute to each pathway. The expected outcomes of this project will lend greater understanding for the structure-activity relationship, efficacy, and overall metabolism of clinical-grade PS-ASOs and Stabilin biology/biochemistry.