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

基本信息

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
项目类别：
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

项目摘要

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产生人类血小板 (PLT) 的骨髓（生理微环境）是“带-”。血液中的辅助物质，负责凝块形成和血管修复，PLT 计数低是一个重要因素。辐射事故或蓄意核攻击的后果，PLT 是重要的一线疗法以防止因失控出血而导致的死亡。PLT 单位包含约 1.5x109 PLT/mL（总共 3x1011 PLT）。目前仅来自人类捐赠者，并且必须储存在 22oC 或以上以避免室温下存在不可逆的温度相关激活/聚集风险。储存会导致保质期较短（5 天），血液中心的 PLT 通常不超过 1.5 天。非紧急情况下可用于输血的库存 (1, 2)，目前没有获得许可的血小板减少症 PLT 疗法可治疗辐射暴露(3)。为了满足未满足的需求，我们将验证我们的生物反应器，以在临床规模上从巨核细胞祖细胞生成功能性 PLT，以治疗辐射引起的血小板减少症 (RIT) 迄今为止，我们已经证明：(1) 是可行的。从人类诱导多能干细胞（iPSC，一种潜在的祖细胞来源无限，可以冷冻保存多年）（4）和（2）我们可以提高比率人类 iPSC 衍生巨核细胞的 PLT 生产程度高于已建立的静态培养方法 (5)。在我们当前的生物反应器中，PLT 生产开始的时间从 6 小时减少到立即，产生 PLT 的祖细胞的百分比从 10% 增加。 90%以上，完成时间 PLT 产量（每 300 µL 输血单位约 1.5x106 个 PLT）从 18 小时减少到 8 小时，并且 PLT 形态、超微结构和体外功能与血液PLT 相当且一致(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 1x108 鼠 PLT 原代巨核细胞，并在体外评估 PLT 功能。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激活条件下的血液 PLT 进行比较。目标 2. 将 bdPLT 注入亚致死性照射的小鼠中，以测量 bdPLT 的免疫原性、清除率、循环时间、止血活性，并测定bdPLT在体内的功能。 bdPLT 在体内具有功能，我们将评估 bdPLT 清除率和循环时间（相对于血液 PLT 对照），并在遗传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确定 bdPLT 止血活性和血栓形成 RIT（非 GLP 试点研究）为了确定 bdPLT 是否可以应用于治疗 RIT，我们将评估 bdPLT。亚致死 γ 辐射（~6.25 Gy）后成年小鼠的功能。