Development of Antisense Oligonucleotides to Regulate Gamma' Fibrinogen Levels

项目总结/摘要 g'（读作“gamma prime”）纤维蛋白原（GPF）是凝血因子的一种选择性剪接异构体 纤维蛋白原。这种纤维蛋白原变体含有凝血因子凝血酶的高亲和力结合位点， 将凝血酶定位在正在生长的血块上凝血酶通过凝血酶的肝素结合位点与GPF结合。这 允许凝血酶继续形成血凝块，即使在其抑制蛋白，抗凝血酶III， 和辅助因子肝素此外，GPF形成对纤维蛋白溶解酶分解具有抗性的凝块， 使得凝块在血管中持续存在。由于这些活动，GPF是一个风险因素， 心血管疾病，包括心脏病发作和中风。我们最近发现， 可以发展出非常高水平的GPF，这可能有助于观察到的血栓形成事件。 在COVID-19患者中。为了支持这一假设，重症COVID-19患者对肝素具有耐药性， 不能从肝素治疗中获益高GPF水平像这些螯合凝血酶在一个活跃的形式，不能 被抗凝血酶III/肝素抑制。如果GPF水平可以降低，许多这些血栓事件 可能是可以避免的。因此，本申请的具体目的是： 1)开发反义吗啉代寡核苷酸，其结合其靶g基因序列并被 HepG 2人肝细胞系。 这将使用电泳迁移率变动测定来完成，以确保反义寡核苷酸结合至 它们的目标序列。将使用孔中的荧光素标记的寡核苷酸测定肝细胞的细胞摄取。 建立纤维蛋白原合成的组织培养模型，人HepG 2肝细胞系。该细胞系 表达人g链的gA和g'同种型。 2)使用HepG 2细胞优化吗啉代低聚物的体内有效性。 这将通过在HepG 2细胞中测定Aim 1中开发的先导化合物来实现。绝对 使用RT-PCR定量gA和g ′ mRNA的水平和相对表达比率。总 将通过ELISA测量纤维蛋白原和GPF。 Milestone-第II阶段灵长类动物研究进展的标准是： 1.反义吗啉寡聚体结合其靶基因序列的开发。 2.证明反义吗啉代寡聚体调节HepG 2肝细胞中的GPF水平。

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.