Metabolism of Antisense Oligonucleotides and other Polyanions in Liver

反义寡核苷酸和其他聚阴离子在肝脏中的代谢

基本信息

批准号：
10689248
负责人：
EDWARD N HARRIS
金额：
$ 30.01万
依托单位：
UNIVERSITY OF NEBRASKA LINCOLN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10689248
关键词：
Address Affect Affinity Animal Model Antisense Oligonucleotides Binding Biochemistry Biodistribution Biological Biology Bone Marrow Cancer cell line Cells Chemicals Chemistry Chondroitin Sulfates Circulation Clinic Clinical Code Complex Custom Data Dedications Discontinuous Capillary Down-Regulation Drug Kinetics Endocytosis Endosomes Endothelial Cells Excipients Fluorescence Microscopy Future Gene Expression Genes Genetic Genetic Diseases Goals Heparin Heparitin Sulfate Hepatocyte Hyaluronan Injections Intelligence Interferometry Kidney Kinetics Knock-out Knockout Mice Knowledge Ligands Liquid substance Liver Lysosomes Macrophage Mediating Messenger RNA Metabolic Metabolic Clearance Rate Metabolic Diseases Metabolism Methods Modification Oligonucleotides Organ Outcome Oxygen Paper Pathway interactions Pharmaceutical Preparations Physiological Plasma Polymers Positioning Attribute Productivity Property RNA RNA Interference Reaction Recombinants Research Research Project Grants Resistance Ribonuclease H Ribose Specificity Spleen Structure-Activity Relationship Sulfur System Therapeutic Therapeutic Uses Tissues Urine Vertebral column base catalyst cell type combat design endonuclease in vivo insight intraperitoneal knock-down lymph nodes mouse model next generation nuclease phosphodiester phosphorothioate polyanion receptor receptor internalization receptor recycling recruit scavenger receptor stable cell line subcutaneous trafficking transcytosis uptake

项目摘要

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.

反义寡核苷酸 (ASO) 是经过化学修饰的短 (~20 bp) 寡核苷酸抗拒细胞和生物体液中核酸内切酶活性的骨架，ASO 与目标 mRNA 结合。细胞并充当 RNAse H 的催化剂来破坏 mRNA，从而降低基因表达。常见的 ASO 修饰是将磷酸二酯基团的非桥氧原子替换为硫原子生成硫代磷酸酯 (PS) 部分，迄今为止，该部分最常用于临床 ASO。通过修饰 2' 位置可以赋予进一步的稳定性和核酸酶抗性众所周知，肝脏是 PS-ASO 的天然库，但其机制却存在争议。我们发现了Stabilin类(SR-H)清道夫受体(Stabilin-1)的这种活性尚不清楚。和 Stabilin-2) 是系统性 PS-ASO 清除的主要机制，以稳定蛋白的形式表达。组织数量，包括肝窦、淋巴结、脾脏、II 型巨噬细胞、骨髓、等，这可能会对 PS-ASO 递送到许多组织产生影响。知识差距在于如何递送。 PS-ASO 与稳定素的相互作用增加了 PS-ASO 对目标 mRNA 的敲低。假设稳定蛋白介导的 PS-ASO 内吞作用沿着 2 条途径进行；其中一条途径是 PS-ASO。 ASO 被运送到溶酶体（破坏），而另一个则允许 PS-ASO 逃脱内体与 mRNA 相互作用（功效）我们这个项目的主要目标是首先确定使用生物层干涉测量法研究 Stabilin-PS-ASO 结合复合物的生物相互作用其次，确定 PS-ASO 的动力学。表达稳定素的重组稳定细胞系和原代正弦细胞中的内吞作用我们还将剖析 Stabilin-2 的内吞机制。第三，阐明 PS-ASO 活性（内体逃逸）所必需的相互作用分子。确定 WT 和 Stabilin 敲除小鼠中 PS-ASO 的全身清除率和生物活性，以评估除肝脏以外的组织中的稳定素依赖性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稳定蛋白敲除小鼠这将进一步描绘出两种可能的途径。（破坏与激活）在多个组织中的作用以及稳定素如何对每个途径做出贡献。该项目的成果将有助于人们更好地理解结构-活动关系、功效和临床级 PS-ASO 和 Stabilin 生物学/生物化学的整体代谢。