Platelet Bioreactor for Treatment of Radiation-Induced Thrombocytopenia

用于治疗辐射引起的血小板减少症的血小板生物反应器

• 批准号: 9253542
• 负责人: Lea M Beaulieu
• 金额: $ 30万
• 依托单位: PLATELET BIOGENESIS, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-05 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9253542
• 关键词: Achievement; Address; Adult; Agreement; American; Animals; Biogenesis; Biological; Biological Markers; Bioreactors; Bleeding time procedure; Blood; Blood Circulation; Blood Platelets; Blood Vessels; Bone Marrow; Clinical; Clot retraction; Coagulation Process; Collection; Cytoplasmic Granules; Cytoskeletal Modeling; Data; Drug Approval; Drug Kinetics; Embryo; Emergency Situation; Engineering; Equipment and supply inventories; Fetal Liver; Freezing; Grant; Growth; Half-Life; Hemorrhage; Hemostatic Agents; Hour; Human; Human Volunteers; Immunocompromised Host; Immunologics; In Vitro; Infusion procedures; Investigational Drugs; Label; Letters; Licensing; Life; Measures; Mediation; Medical; Megakaryocytes; Microfluidics; Morphology; Mouse Strains; Mus; Nuclear; Operative Surgical Procedures; Perfusion; Phase; Physiological; Pilot Projects; Platelet Count measurement; Platelet Transfusion; Production; Radiation; Radiation Accidents; Radiation exposure; Radiation therapy; Recovery; Research; Rest; Risk; Safety; Small Business Innovation Research Grant; Source; Stem cells; Survival Rate; Tail; Temperature; Testing; Therapeutic; Thrombocytopenia; Thrombus; Time; Transfusion; Transplantation; Vendor; Viral; cancer therapy; immunogenicity; improved; in vivo; induced pluripotent stem cell; irradiation; mortality; mouse model; prevent; progenitor; repaired; shear stress; standard of care; volunteer

Platelet Biogenesis is developing a microfluidic bioreactor that reproduces key features of adult bone marrow (physiological microenvironment) to produce human platelets (PLTs). PLTs are the 'band- aids' of the bloodstream, responsible for clot formation and blood vessel repair. Low PLT count is a significant consequence of a radiation accident or deliberate nuclear attack, for which PLTs are a critical first-line therapy to prevent mortality due to uncontrolled bleeding. PLT units comprising ~1.5x109 PLTs/mL (3x1011 PLTs total) are currently derived exclusively from human volunteer donors, and must be stored at or above 22oC to avoid irreversible temperature-related activation/aggregation. Risk of bacterial/viral growth during room temperature storage results in short shelf life (5 days). Blood centers typically do not have more than a 1.5-day PLT inventory available for transfusion under non-emergency situations (1, 2), and there are currently no licensed PLT therapeutics for thrombocytopenia to treat radiation exposures(3). To address this major unmet need we will validate our bioreactor to generate functional PLTs from megakaryocyte progenitors at clinical scale to treat radiation-induced thrombocytopenia (RIT). To date we have shown that: (1) It is feasible to generate functional megakaryocytes and PLTs from human induced pluripotent stem cells (iPSCs, a potentially unlimited source of progenitor cells which can be stored frozen for years)(4) and (2) we can improve the rate and extent of PLT production from human iPSC-derived megakaryocytes above established static culture approaches(5). In our current bioreactor time to initiation of PLT production is reduced from 6 hours to immediately, the percent of PLT-producing progenitors is increased from 10% to more than 90%, the time to completion of PLT production (~1.5x106 PLTs per 300 µL transfusion unit) is reduced from 18 hours to 8 hours, and PLT morphology, ultrastructure and in vitro function is comparable to and consistent with blood PLTs(6). Aim 1. Apply our existing bioreactor to produce 1x108 murine PLTs per 300 µL transfusion unit from primary megakaryocytes, and assess PLT function in vitro. Morphology, cytoskeletal organization, granule content, ultrastructure, biomarker expression, aggregation, and clot retraction of bioreactor-derived PLTs will be compared to blood PLTs under resting and activated conditions. Aim 2. Infuse bdPLTs into sub-lethally irradiated mice to measure bdPLT immunogenicity, clearance, circulation time, hemostatic activity, and determine bdPLT function in vivo. To determine whether bdPLTs are functional in vivo, we will assess bdPLT clearance and circulation time (relative to blood PLT controls), and test their immunogenicity in genetically-distinct and isogenic mouse strains. Aim 3. Determine bdPLT hemostatic activity and thrombus formation in an isogenic adult mouse model of RIT. (non-GLP pilot study) To determine whether bdPLTs can be applied to treat RIT, we will assess bdPLT function in adult mice following sub-lethal γ-irradiation (~6.25 Gy).

血小板生物发生正在开发一种微流体生物反应器，该生物反应器重现了成人的关键特征 骨髓（生理微环境）产生人血小板（PLT）。 plt是'band- 血液的辅助，负责凝块形成和血管修复。低PLT计数是重要的 辐射事故或故意核攻击的结果，PLT是一线疗法的关键 为了防止因不受控制的出血而导致死亡率。 PLT单元完成〜1.5x109 PLT/ml（总计3x1011 PLT） 目前仅来自人类的志愿捐助者，必须在22oC或以上存储以避免 不可逆的温度相关激活/聚集。室温期间细菌/病毒生长的风险 存储会导致短暂的保质期（5天）。血液中心通常不超过1.5天 在非紧急情况下可用于输血的库存（1，2），目前没有 血小板减少症治疗辐射暴露的许可PLT治疗（3）。解决这个专业 未满足的需求我们将验证生物反应器以在临床规模上从巨核细胞祖细胞中产生功能性PLT，以治疗辐射诱导的血小板细胞减少症（RIT）。迄今为止，我们已经表明：（1） 从人类诱导的多能干细胞（IPSC，一种潜在的潜在 祖细胞的无限来源可以冷冻多年）（4）和（2）我们可以提高速率 与固定静态培养方法高于人类IPSC衍生的巨核细胞的PLT生产程度（5）。在我们目前的生物反应器开始开始PLT生产的时间从6小时降低到立即，生产PLT的祖细胞百分比从10％增加到90％以上，这是完成的时间 PLT生产（每300 µL输血单元）的PLT生产（〜1.5x106 PLT）从18小时减少到8小时，PLT 形态，超微结构和体外功能与血液PLT相媲美，并一致（6）。 AIM 1。应用我们现有的生物反应器，每300 µL输血单元产生1x108鼠PLT 原发性巨核细胞并在体外评估PLT功能。形态学，细胞骨骼组织，颗粒 含量，超微结构，生物标志物表达，聚集和生物反应衍生的PLT的凝结缩回将 在静止和活化条件下与血液PLT进行比较。 AIM 2。将BDPLT注入亚致死的小鼠中，以测量BDPLT免疫原性，清除率， 循环时间，止血活性并确定体内BDPLT功能。确定是否 BDPLT在体内具有功能性，我们将评估BDPLT清除率和循环时间（相对于血液PLT对照），并测试其在遗传差异和同源性小鼠菌株中的免疫原性。 AIM 3。在等源成年小鼠模型中确定BDPLT止血活性和血栓形成 RIT。 （非GLP初步研究）为了确定是否可以应用BDPLT来治疗RIT，我们将评估BDPLT 亚致死性γ-辐照后成年小鼠的功能（〜6.25 Gy）。