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

• 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
• 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

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）是具有化学修饰的短（约20 bp）寡核苷酸 骨架可抵抗细胞和生物液中的核酸内切酶活性。 ASO与靶向的mRNA结合 细胞并充当RNase H破坏mRNA的催化剂，从而降低基因表达。最多 常见的ASO修饰是用磷酸酯基的脱氧氧原子取代 硫原子可产生磷酸盐剂（PS）部分，该部分最常用于迄今为止的临床ASO。 可以通过修饰2'的位置来赋予进一步的稳定和抗核酸酶抗性 ASO的核糖。众所周知，肝脏是PS-ASO的天然水槽，但是机构 因为这项活动尚不清楚。我们已经发现稳定蛋白类（SR-H）清道夫接收器（稳定蛋白1 稳定蛋白2）是全身性PS-ASO清除率的主要机制。稳定蛋白在 组织数量，包括肝脏的正弦，淋巴结，脾脏，II型巨噬细胞，骨髓， ETC可能对PS-ASO向许多组织产生影响。知识的差距是如何 PS-ASO与稳定蛋白的相互作用增加了靶向mRNA的PS-ASO敲低。我们的中心 假设是稳定蛋白介导的PS-ASO的内吞作用沿2个途径进行。一个PS- ASO被穿梭到溶酶体（破坏），另一个允许PS-ASO逃脱 内体与mRNA相互作用（功效）。我们对该项目的主要对象首先确定 使用Biolayer干扰与 在已知溶液和等离子体中竞争配体。第二，确定PS-ASO的动力学 表达稳定蛋白和原发性正弦的两个重组稳定细胞系的内吞作用 肝内皮细胞。我们还将剖析稳定蛋白-2的内吞作用机制 阐明PS-ASO活性所需的相互作用分子（内体逃逸）。第三，到 确定WT和稳定蛋白敲除小鼠的PS-ASO的全身清除率和生物活性以评估 稳定蛋白依赖性生物分布和在肝脏以外的组织中的激活。我们将使用全球和 这些研究的组织特异性稳定敲除小鼠。这将进一步描述两个可能的途径 （破坏与激活）在多个时机中以及稳定蛋白如何对每种途径贡献。预期 该项目的结果将对结构活动关系，效率和 临床级PS-ASO和稳定蛋白生物学/生物化学的总体代谢。