Genome-wide Analysis of Anticoagulant Heparin Sulfate for Bioengineering Heparan

项目摘要 肝素是世界上处方最广泛的抗凝血药，也是治疗和治疗肝病的常规药物 预防深静脉血栓形成和肺血栓形成。目前，治疗性肝素是一种分级的 一种来自动物来源的硫酸肝素，主要来自猪的结缔组织肥大细胞 粘膜来源：中国。虽然肝素是必需的，而且被广泛使用，但它也有严重的不良并发症。 在美国，每年约有60万名接受肝素治疗的患者患上危及生命的疾病 被称为肝素诱导的血小板减少症(HIT)，它是由肝素-血小板因子4的形成引起的 (PF4)免疫活性复合体。因此，迫切需要更安全的替代肝素来源。 重组肝素来源将更安全，避免供应链问题，并允许引入 生物修饰以防止命中。而参与肝素生物合成的酶与那些 对于硫酸乙酰肝素来说，关于引起 对肝素的抗凝血活性和生物合成的影响。肝素与高亲和力结合抑制凝血作用 与丝氨酸蛋白酶抑制剂抗凝血酶(AT)结合，增强其中和凝血酶和因子的活性 Xa在凝固级联中。此外，肝素与产生的趋化因子PF4具有高亲和力结合。 通过血小板，触发命中。由于PF4与肝素的结合取决于与AT相比不同的结合部位，因此我们 假设细胞可以被改造成产生抗凝剂硫酸乙酰肝素，但PF4亲和力降低 作为动物源性肝素更安全的替代品。我们之前的研究揭示了明显的监管 细胞中肝素生物合成的机制，表明存在调节抗凝剂的其他因素 肝素/HS的产生和修改AT和PF4的亲和力。这项建议的目标是利用我们的经验 在全基因组筛选试验中，确定控制肝素生物合成的遗传因素，并可利用 用于生物工程培养细胞中的抗凝血剂硫酸乙酰肝素。为了实现这一目标，我们的目标是(I)适应 全基因组筛选试验寻找调节AT和PF4与细胞表面结合的新因素 硫酸乙酰肝素，以及(Ii)验证来自筛查的优先命中，并利用这些来进行代谢工程 产生具有较低PF4亲和力的抗凝血剂硫酸乙酰肝素的哺乳动物细胞。成功者 这些目标的完成将提供一种生物工程细胞系，生产更安全的重组形式的肝素 还可能发现以前未知的与肝素的活性和组装相关的基因。重要的是 这个项目将导致未来详细的假设驱动的研究，将使我们更接近于找到替代方案 动物源性肝素用于通过预防HIT来改善患者护理和临床结果。

Project Summary Heparin is the most widely prescribed anticoagulant drug in the world and is used routinely for the treatment and prevention of deep vein thrombosis and pulmonary embolism. Currently, therapeutic heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa sourced from China. While essential and widely used, heparin has significant adverse complications. Approximately 600,000 patients per year treated with heparin in the USA develop a life-threatening condition known as heparin-induced thrombocytopenia (HIT), which results from the formation of heparin-platelet factor 4 (PF4) immunoreactive complexes. Therefore, there is an urgent need for safer, alternative sources of heparin. A recombinant source of heparin would be safer, avoid supply chain issues, and allow for the introduction of biological modifications to prevent HIT. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, there is a significant gap in knowledge regarding the regulatory mechanisms that give rise to the anticoagulant activity and biosynthesis of heparin. Heparin inhibits coagulation by binding with high affinity to the serine protease inhibitor, antithrombin (AT), and enhancing its activity to neutralize thrombin and Factor Xa in the coagulation cascade. Additionally, heparin exhibits high affinity binding to PF4, a chemokine produced by platelets, triggering HIT. Since PF4 binding to heparin depends on distinct binding sites compared to AT, we hypothesize that cells could be engineered to produce anticoagulant heparan sulfate with decreased PF4 affinity as a safer alternative to animal-derived heparin. Our previous studies have revealed distinct regulatory mechanisms for heparin biosynthesis in cells, suggesting that other factors exist that regulate anticoagulant heparin/HS production and modify AT and PF4 affinity. The goal of this proposal is to leverage our experience in genome-wide screening assays to identify genetic factors that control heparin biosynthesis and can be utilized for bioengineering anticoagulant heparan sulfate in cultured cells. To accomplish this goal, we aim to (i) adapt genome-wide screening assays to search for novel factors that regulate AT and PF4 binding to cell surface heparan sulfate, and (ii) validate prioritized hits from the screens and leverage these for metabolic engineering of mammalian cells to produce anticoagulant heparan sulfate with lowered PF4 affinity. The successful completion of these aims will provide a bioengineered cell line that produces a safer, recombinant form of heparin and may also uncover previously unknown genes associated with heparin’s activity and assembly. Importantly, this project will lead to future detailed hypothesis-driven studies that will bring us closer to finding an alternative to animal-derived heparin for improving patient care and clinical outcomes by prevention of HIT.