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）是具有化学修饰的寡核苷酸序列的短（~20 bp）寡核苷酸。 主链以抵抗细胞和生物流体中的核酸内切酶活性。ASO与靶向mRNA结合， 细胞，并作为催化剂的RNA酶H破坏mRNA，从而降低基因表达。最 常见的阿索修饰是磷酸二酯基团的非桥连氧原子被 硫原子以产生硫代磷酸酯（PS）部分，其迄今为止最常用于临床ASO。 进一步的稳定化和核酸酶抗性可以通过修饰寡核苷酸的2'位置来赋予。 阿索的核糖。众所周知，肝脏是PS-ASO的天然库，然而，PS-ASO的机制尚不清楚。 这项活动尚不清楚。我们已经发现稳定蛋白类（SR-H）清道夫受体（稳定蛋白-1 和稳定蛋白-2）是系统性PS-ASO清除的主要机制。稳定蛋白表达为 包括肝窦、淋巴结、脾、II型巨噬细胞、骨髓 等，这可能对PS-ASO向许多组织的递送有影响。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。我们的中央 假设是PS-ASO稳定蛋白介导的内吞作用沿沿着2条途径进行;其中一条途径是PS- 阿索穿梭于溶酶体（破坏），而PS-ASO在另一个溶酶体中被允许逃离溶酶体。 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 稳定蛋白-阿索结合复合物的生物相互作用，使用生物层干涉测量法， 在已知溶液和血浆中的竞争配体。第二，测定PS-ASO的动力学 在表达稳定蛋白的重组稳定细胞系和原代窦状核细胞系中的胞吞作用 肝内皮细胞。我们还将剖析Stabilin-2的内吞机制， 阐明PS-ASO活性（内体逃逸）所必需的相互作用分子。三是 测定PS-ASO在WT和Stabilin敲除小鼠中的全身清除率和生物活性，以评估 稳定蛋白依赖的生物分布和激活的组织而不仅仅是肝脏。我们将使用全球和 组织特异性稳定蛋白敲除小鼠用于这些研究。这将进一步描述两种可能的途径 （破坏与激活）在多种组织中的作用，以及稳定蛋白如何促进每种途径。预期 该项目的成果将有助于更好地了解结构-活性关系，功效， 临床级PS-ASO和Stabilin生物学/生物化学的总体代谢。