Development of Antisense Oligonucleotides to Regulate Gamma' Fibrinogen Levels

开发反义寡核苷酸来调节γ纤维蛋白原水平

• 批准号: 10759950
• 负责人: DAVID Henry FARRELL
• 金额: $ 27.77万
• 依托单位: GAMMA THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-24 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10759950
• 关键词: Acute; Affinity; Age; Alternative Splicing; Animals; Antisense Oligonucleotides; Antisense Technology; Antithrombin III; Binding; Binding Sites; Biological Assay; Blood; Blood Coagulation Factor; Blood Vessels; Blood coagulation; Body mass index; C-reactive protein; COVID-19; COVID-19 patient; COVID-19 severity; Cardiovascular Diseases; Cell Line; Cells; Chest Pain; Clinic; Coagulation Process; Critical Illness; Development; Effectiveness; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Enzymes; Event; Exercise Tolerance; F8 gene; Fatigue; Ferritin; Fibrin; Fibrin fragment D; Fibrinogen; Fluorescein; Gender; Genes; Goals; Hemophilia A; HepG2; Heparin; Heparin Binding; Hepatocyte; Hospitalization; Hospitals; Human; India; Infection; Interleukin-6; Label; Lactate Dehydrogenase; Lead; Long COVID; Measures; Messenger RNA; Modeling; Myocardial Infarction; Normal Range; Oligonucleotides; Oxygen; Patients; Pharmaceutical Preparations; Phase; Primates; Protein Isoforms; Proteins; ROC Curve; Resistance; Resolution; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2 infection; Severity of illness; Shortness of Breath; Stroke; Symptoms; Syndrome; Thrombin; VWF gene; Variant; cardiovascular risk factor; comorbidity; coronavirus disease; gamma Fibrinogen; heparin cofactor; in vivo; indexing; inhibitor; medical schools; post-COVID-19; prevent; severe COVID-19; thrombotic; tissue culture; uptake

PROJECT SUMMARY/ABSTRACT g' (pronounced "gamma prime") fibrinogen (GPF) is an alternative splice isoform of the blood coagulation factor fibrinogen. This fibrinogen variant contains a high affinity binding site for the coagulation factor thrombin that localizes thrombin on the growing blood clot. Thrombin binds to GPF via thrombin's heparin binding site. This allows thrombin to continue forming the blood clot even in the presence of its inhibitor protein, antithrombin III, with the cofactor heparin. In addition, GPF forms clots that are resistant to breakdown by fibrinolytic enzymes, such that the clots persist in the blood vessel. Because of these activities, GPF is a risk factor for cardiovascular disease, including heart attacks and stroke. We have recently found that COVID-19 patients can develop extraordinarily high levels of GPF, which likely contributes to the thrombotic events that are seen in COVID-19 patients. In support of this hypothesis, critically ill COVID-19 patients are heparin resistant and do not benefit from heparin treatment. High GPF levels like these sequester thrombin in an active form that cannot be inhibited by antithrombin III/heparin. If the GPF levels could be lowered, many of these thrombotic events could likely be prevented. The Specific Aims of this application are therefore to: 1) Develop antisense morpholino oligos that bind their target g gene sequences and are taken up by the HepG2 human liver cell line. This will be accomplished using electrophoretic mobility shift assays to ensure that the antisense oligos bind to their target sequences. Cellular uptake into liver cells will be assayed using fluorescein-labeled oligos in a well- established tissue culture model of fibrinogen synthesis, the human HepG2 liver cell line. This cell line expresses both the gA and g' isoforms of the human g chain. 2) Optimize the morpholino oligomers for effectiveness in vivo using HepG2 cells. This will be accomplished by assaying the lead compounds developed in Aim 1 in HepG2 cells. The absolute levels and the relative expression ratios of the gA and g' mRNAs will be quantitated using RT-PCR. Total fibrinogen and GPF will be measured by ELISA. Milestones – The criteria for progress to animal studies in primates in Phase II are: 1. Development of antisense morpholino oligomers that bind their target g gene sequences. 2. Demonstration that the antisense morpholino oligomers modulate GPF levels in HepG2 liver cells.

项目摘要/摘要 g'（发音为“伽马素”）纤维蛋白原（GPF）是血液凝结因子的替代剪接同工型 纤维蛋白原。该纤维蛋白原变体包含一个高亲和力结合位点，用于凝血因子凝血酶，该凝血酶凝血酶 将凝血酶定位在生长的血凝块中。凝血酶通过凝血酶的肝素结合位点与GPF结合。这 即使在存在抑制剂蛋白抗凝血酶III的情况下，允许凝血酶仍可以继续形成血凝块 与辅因子肝素。另外，GPF形成云纤维蛋白酶抗崩溃的云，， 使血管持续存在的血管中。由于这些活动，GPF是 心血管疾病，包括心脏病发作和中风。我们最近发现Covid-19患者 可以发展出极高的GPF，这可能会导致血栓形成事件 在199位患者中。为了支持这一假设，病重于199例患者具有抗肝素的耐药性，DO 肝素治疗不受益受。像这样的高GPF水平，以这种活跃形式隔离凝血酶 被抗凝血酶III/肝素抑制。如果可以降低GPF水平，那么许多这些血栓事件 可能可以防止。因此，本应用程序的具体目的是： 1）开发反义的形态寡素，以结合其靶G基因序列并被 HEPG2人肝细胞系。 这将使用电泳移动性转移测定法实现，以确保反义寡聚结合到 他们的目标序列。细胞对肝细胞的摄取将使用荧光素标记的寡核酸分配 建立的纤维蛋白原合成组织组织培养模型，人类HEPG2肝细胞系。该单元线 同时表达人G链的GA和G'同工型。 2）优化使用HEPG2细胞在体内有效性的形态寡聚物。 这将通过测定HepG2细胞中AIM 1中开发的铅化合物来实现。绝对 使用RT-PCR将定量GA和G'mRNA的水平和相对表达比。全部的 纤维蛋白原和GPF将由ELISA测量。 里程碑 - 在第二阶段基本研究中进步的动物研究标准是： 1。结合其靶G基因序列的反义形态寡聚的发展。 2。证明反义的形态寡聚体调节HEPG2肝细胞中的GPF水平。