Genome-wide Analysis of Anticoagulant Heparin Sulfate for Bioengineering Heparan

用于生物工程类乙酰肝素的抗凝剂硫酸肝素的全基因组分析

基本信息

批准号：
10742641
负责人：
Ryan Joseph Weiss
金额：
$ 22.65万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10742641
关键词：
Affect Affinity Anabolism Animal Sources Animals Antibodies Anticoagulants Antidotes Binding Binding Sites Biological Biological Assay Biological Products Biomedical Engineering Blood Platelets CRISPR screen Candidate Disease Gene Categories Cell Line Cell physiology Cell surface Cells Cellular biology China Clinical Clustered Regularly Interspaced Short Palindromic Repeats Coagulation Process Complex Complication Connective Tissue Cultured Cells Deep Vein Thrombosis Development Engineering Enzymes Epitopes Exhibits Factor Xa Family suidae Flow Cytometry Future Genes Genetic Goals Guide RNA Hemorrhage Heparan Sulfate Biosynthesis Heparin Heparitin Sulfate Immune Incidence Individual Intestines Knock-out Knowledge Life Ligand Binding Mammalian Cell Mass Spectrum Analysis Mediating Methods Modification Molecular Mucous Membrane National Heart, Lung, and Blood Institute Oligosaccharides Operative Surgical Procedures Oral Outcome PF4 Gene Pathway interactions Patient Care Patients Pharmaceutical Preparations Plasma Proteins Prevention Process Production Property Public Health Pulmonary Embolism RNA library Recombinants Regulation Reporting Research Risk Serine Proteinase Inhibitors Site Sorting Source Sulfate Therapeutic Thrombin Transcription Repressor Work acute care candidate validation chemokine enhancing factor experience functional genomics genetic testing genome wide screen genome-wide genome-wide analysis heparin-induced thrombocytopenia immunoreactivity improved inhibitor innovation mast cell metabolic engineering novel overexpression prevent screening stable cell line supply chain whole genome

项目摘要

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.

项目摘要肝素是世界上规定最广泛的抗凝药物，通常用于治疗和预防深静脉血栓形成和肺栓塞。目前，治疗性肝素是分数源自动物来源的乙par硫酸盐的形式，主要来自猪的结缔组织肥大细胞粘膜来自中国。肝素虽然必不可少且广泛使用，但却具有重大的广告并发症。在美国，每年大约有600,000名患有肝素治疗的患者会发展生命。被称为肝素诱导的血小板减少症（HIT），这是由肝素 - 静脉 - 静脉因子因子4的形成而引起的（PF4）免疫反应性复合物。因此，迫切需要更安全的肝素替代来源。肝素的重组来源是安全的，避免供应链问题，并允许引入生物修饰以防止受到打击。而参与肝素生物合成的酶与那些相同对于硫酸乙酰肝素，关于引起的调节机制的知识存在很大的差距肝素的抗凝活性和生物合成。肝素通过与高亲和力结合来抑制凝血到丝氨酸蛋白抑制剂抗凝血酶（AT），并增强其活性以中和凝血酶和因子 Xa在凝血级联中。此外，肝素表现出与PF4的高亲和力结合，PF4是趋化因子产生的通过血小板，触发命中。由于PF4与肝素的结合取决于与AT相比的不同结合位点假设可以设计细胞以产生硫酸乙酰肝素，并具有降低的PF4亲和力作为动物来源肝素的更安全替代品。我们以前的研究揭示了明显的调节细胞中肝素生物合成的机制，表明存在其他因素来调节抗凝剂肝素/HS的生产和修改和PF4亲和力。该提议的目的是利用我们的经验在全基因组筛查测定中，以识别控制肝素生物合成的遗传因素，并且可以使用用于培养细胞中硫酸乙酰肝素的生物工程抗凝剂。为了实现这一目标，我们的目标是（i）适应全基因组筛选测定法以搜索调节和PF4结合细胞表面的新型因素硫酸乙酰肝素，（ii）验证屏幕中的优先命中率，并利用它们用于代谢工程哺乳动物细胞的产生抗凝剂硫酸乙酰肝素具有降低的PF4亲和力。成功这些目标的完成将提供生物工程的细胞系，从而产生更安全的重组形式的肝素并且可能还可以发现以前未知的基因与肝素的活性和组装相关。重要的是，该项目将导致未来的详细假设驱动的研究，这将使我们更加接近寻找替代方案通过预防命中率来改善动物衍生的肝素，以改善患者护理和临床结果。