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:
- 金额:$ 22.65万
- 依托单位:
- 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:
- 关键词:AffectAffinityAnabolismAnimal SourcesAnimalsAntibodiesAnticoagulantsAntidotesBindingBinding SitesBiologicalBiological AssayBiological ProductsBiomedical EngineeringBlood PlateletsCRISPR screenCandidate Disease GeneCategoriesCell LineCell physiologyCell surfaceCellsCellular biologyChinaClinicalClustered Regularly Interspaced Short Palindromic RepeatsCoagulation ProcessComplexComplicationConnective TissueCultured CellsDeep Vein ThrombosisDevelopmentEngineeringEnzymesEpitopesExhibitsFactor XaFamily suidaeFlow CytometryFutureGenesGeneticGoalsGuide RNAHemorrhageHeparan Sulfate BiosynthesisHeparinHeparitin SulfateImmuneIncidenceIndividualIntestinesKnock-outKnowledgeLifeLigand BindingMammalian CellMass Spectrum AnalysisMediatingMethodsModificationMolecularMucous MembraneNational Heart, Lung, and Blood InstituteOligosaccharidesOperative Surgical ProceduresOralOutcomePF4 GenePathway interactionsPatient CarePatientsPharmaceutical PreparationsPlasma ProteinsPreventionProcessProductionPropertyPublic HealthPulmonary EmbolismRNA libraryRecombinantsRegulationReportingResearchRiskSerine Proteinase InhibitorsSiteSortingSourceSulfateTherapeuticThrombinTranscription RepressorWorkacute carecandidate validationchemokineenhancing factorexperiencefunctional genomicsgenetic testinggenome wide screengenome-widegenome-wide analysisheparin-induced thrombocytopeniaimmunoreactivityimprovedinhibitorinnovationmast cellmetabolic engineeringnoveloverexpressionpreventscreeningstable cell linesupply chainwhole 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
肝素是世界上最广泛使用的抗凝药物,通常用于治疗和
预防深静脉血栓和肺栓塞。目前,治疗性肝素是一种分级的
一种硫酸乙酰肝素,来源于动物,主要来源于猪的结缔组织肥大细胞
来源于中国。虽然肝素是必需的且广泛使用,但其具有显著的不良并发症。
在美国,每年约有600,000名接受肝素治疗的患者发生危及生命的疾病
称为肝素诱导的血小板减少症(HIT),其由肝素-血小板因子4的形成引起
(PF4)免疫反应复合物。因此,迫切需要更安全的肝素替代来源。
肝素的重组来源将更安全,避免供应链问题,并允许引入
生物学修饰以防止HIT。虽然参与肝素生物合成的酶与那些
对于硫酸乙酰肝素,关于引起
与肝素的抗凝活性和生物合成有关。肝素通过高亲和力结合抑制凝血
丝氨酸蛋白酶抑制剂,抗凝血酶(AT),并增强其活性,以中和凝血酶和因子
凝血级联中的Xa。此外,肝素表现出与PF 4的高亲和力结合,PF 4是一种产生的趋化因子,
引发HIT由于与AT相比,PF 4与肝素的结合依赖于不同的结合位点,我们
假设细胞可以被工程化以产生具有降低的PF 4亲和力的抗凝血硫酸乙酰肝素
作为动物源性肝素的更安全的替代品。我们之前的研究表明,
细胞中肝素生物合成的机制,表明存在其他调节抗凝剂的因子
肝素/HS产生和改变AT和PF 4亲和力。本提案的目的是利用我们的经验
在全基因组筛选试验中鉴定控制肝素生物合成的遗传因子
用于培养细胞中的生物工程抗凝剂硫酸乙酰肝素。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,以寻找调节AT和PF 4与细胞表面结合的新因子
硫酸乙酰肝素,和(ii)验证筛选的优先命中,并利用这些代谢工程
的哺乳动物细胞,以产生具有降低的PF 4亲和力的抗凝剂硫酸乙酰肝素。成功
这些目标的完成将提供一种生物工程细胞系,其产生更安全的重组形式的肝素
也可能揭示以前未知的与肝素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,这将使我们更接近于找到一种替代方案
涉及动物源性肝素,用于通过预防HIT改善患者护理和临床结果。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Ryan Joseph Weiss其他文献
Ryan Joseph Weiss的其他文献
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{{ truncateString('Ryan Joseph Weiss', 18)}}的其他基金
Investigating the molecular mechanisms of glycosaminoglycan assembly
研究糖胺聚糖组装的分子机制
- 批准号:
10715380 - 财政年份:2023
- 资助金额:
$ 22.65万 - 项目类别:
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