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).

Platelet Biogenesis正在开发一种微流体生物反应器，可重现成人的关键特征 骨髓（生理微环境）产生人血小板（PLT）。血小板是“带- 血液循环的“艾滋病”，负责凝块形成和血管修复。血小板计数低是一个显著的 辐射事故或蓄意核攻击的后果，其中PLT是关键的一线治疗 以防止由于不受控制的出血而导致的死亡。PLT单位包含约1.5x109 PLT/mL（总计3x 1011 PLT） 目前仅来自人类志愿者捐赠者，必须储存在22 ℃或以上，以避免 不可逆的温度相关活化/聚集。室温下细菌/病毒生长的风险 储存导致短的保存期（5天）。血液中心通常不会有超过1.5天的PLT 库存可用于非紧急情况下的输血（1，2），目前没有 获得许可的血小板减少症治疗药物，用于治疗辐射暴露（3）。为了解决这一重大问题， 为了满足未满足的需求，我们将验证我们的生物反应器，以临床规模从巨核祖细胞产生功能性PLT，以治疗辐射诱导的血小板减少症（RIT）。到目前为止，我们已经表明：（1）它是可行的， 从人诱导多能干细胞（iPSC）产生功能性巨核细胞和PLT， 无限的祖细胞来源，可以冷冻储存多年）（4）和（2）我们可以提高 以及人iPSC衍生的巨核细胞的PLT产生程度高于已建立的静态培养方法（5）。在我们目前的生物反应器中，开始PLT生产的时间从6小时减少到立即，产生PLT的祖细胞的百分比从10%增加到90%以上，完成PLT生产的时间从10%增加到90%以上。 PLT生产（约1.5x106 PLT/300 µL输血单位）从18小时缩短至8小时， 形态、超微结构和体外功能与血液PLT相当并一致（6）。 目标1。应用我们现有的生物反应器，每300 µL输血单位生产1x 108个小鼠PLT， 原代巨核细胞，并在体外评估PLT功能。形态学、细胞骨架组织、颗粒 生物反应器衍生的PLT的含量、超微结构、生物标志物表达、聚集和凝块收缩将 与静息和活化条件下的血液PLT进行比较。 目标2.将bdPLT输注到亚致死剂量照射的小鼠中以测量bdPLT免疫原性、清除率， 循环时间、止血活性，并测定体内bdPLT功能。以确定是否 由于bdPLT在体内具有功能性，我们将评估bdPLT清除和循环时间（相对于血液PLT对照），并在遗传上不同和同基因小鼠品系中测试其免疫原性。 目标3。在同基因成年小鼠模型中测定bdPLT止血活性和血栓形成 的RIT。（非GLP初步研究）为了确定bdPLT是否可用于治疗RIT，我们将评估bdPLT 亚致死剂量γ辐射（~6.25戈伊）后成年小鼠的功能。