Development of Antisense Oligonucleotides to Regulate Gamma' Fibrinogen Levels

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.