Seed-derived Therapeutic Protein for Acute Ischemic Stroke

种子源治疗性蛋白质治疗急性缺血性中风

• 批准号: 9149036
• 负责人: Kevin Yueju Wang
• 金额: $ 6.45万
• 依托单位: NORTHEASTERN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9149036
• 关键词: Acute; Adopted; Affinity Chromatography; Age; Alteplase; Biological Assay; Blood; Blood Vessels; Blood coagulation; Brain; C-terminal; Cause of Death; Cell Line; Cells; Cessation of life; Chinese Hamster Ovary Cell; Chiroptera; Coagulation Process; Codon Nucleotides; Cytolysis; Cytosol; Data; Developing Countries; Disease; Dose; Ensure; Evaluation; Fibrin; Food; Food Contamination; Frequencies; Future; Gene Expression; Goals; Harvest; Health Services Accessibility; Human; In Vitro; Ischemic Stroke; Laboratories; Methods; Nickel; Nicotiana tabacum; Patients; Peptide Hydrolases; Peptide Signal Sequences; Pharmaceutical Preparations; Plant Leaves; Plant Roots; Plants; Plasminogen Activator; Policies; Principal Investigator; Production; Proteins; Publishing; Recombinant Proteins; Recombinants; Reporting; Research; Seeds; Serine Protease; Source; Stroke; System; Testing; Tissues; Tobacco; Transgenes; Transgenic Organisms; Transgenic Plants; United States; Vial device; base; cost; disability; expression vector; his6 tag; human tissue; improved; in vitro activity; large scale production; overexpression; prevent; programs; promoter; protein purification; public health relevance; stroke survivor; stroke treatment; therapeutic protein; vector

Program Director/Principal Investigator (Last, first, middle): Wang, Kevin, Yueju A stroke occurs when a blood clot or a narrowing of blood vessels prevents blood from getting to the brain. Globally, stroke is the second leading cause of death among people over the age of 60 years. It is also a major source of serious, long-term disability. The only clot- busting drug approved for the treatment of acute ischemic stroke (AIS) disease is a recombinant human tissue plasminogen activator (rt-PA) obtained from Chinese Hamster Ovary cell (CHO) lines. However, yields of rt-PA produced in CHO cells are relatively low, while the dose of rt-PA required for treatment is high, making this therapy very expensive for developing countries. Therefore, a more efficient method of providing rt-PA would greatly reduce the cost and beof great benefit to patients suffering from acute ischemic stroke disease. The objective of the proposed project is to develop a low-cost, plant seed-based platform for the large scale production of functional t-PA for treatment of acute ischemic stroke patients. Our preliminary data revealed that overexpression of several blood-clot dissolving serine protease genes in plant leaves resulted in death of the leaf tissue. In contrast, our preliminary data also indicated that a recombinant vampire bat plasminogen activator (DSPA1) could be reliably produced in seeds with sufficient activity to dissolve human blood clots. In this project, appropriate plant expression vectors will be developed to target t-PA gene expression and production to seeds in order to obtain high levels of expression and increased stability of the end product. Transgenic rt-PA plant lines will be generated and in vitro evaluation of seed- derived rt-PA, such as fibrin degradatin and human clot-dissolving assay, will be performed. The overall goal is to develop a seed-based platform in plants for potential large scale, low- cost production of functional rt-PA for the treatment of AIS in humans. 1 Research Strategy

项目负责人/主要研究者（末位、首位、中间）：Wang、Kevin、Yueju 当血凝块或血管狭窄阻止血液从血管中流出时， 到达大脑。在全球范围内，中风是老年人死亡的第二大原因。 60岁的年龄。它也是严重、长期残疾的一个主要原因。唯一的血块- 一种被批准用于治疗急性缺血性中风（AIS）的药物是一种 重组人组织型纤溶酶原激活剂（rt-PA） 卵巢细胞（CHO）系。然而，在CHO细胞中产生的rt-PA的产率相对较低， 虽然治疗所需的rt-PA剂量很高，使得这种治疗对于 发展中国家因此，提供rt-PA的更有效的方法将极大地 降低了成本，对急性缺血性脑卒中患者有很大的益处， 疾病 拟议项目的目标是开发一个低成本的植物种子平台 用于大规模生产治疗急性缺血性中风患者的功能性t-PA。 我们的初步数据显示，几种溶解血凝块的丝氨酸蛋白酶的过度表达， 植物叶片中的蛋白酶基因导致叶片组织死亡。相比之下，我们的初步 数据还表明，重组吸血蝙蝠纤溶酶原激活剂（DSPA β 1）可以被 在种子中可靠地产生，具有足够的活性来溶解人体血块。在这个项目中， 将开发合适的植物表达载体以靶向t-PA基因表达， 为了获得高水平的表达和增加的稳定性， 最终产品将产生转基因rt-PA植物系，并对种子进行体外评价。 将进行衍生的rt-PA，如纤维蛋白降解和人凝块溶解测定。 总体目标是在植物中开发一个基于种子的平台，用于潜在的大规模，低成本， 用于治疗人类AIS的功能性rt-PA的成本生产。 1 研究策略