PLT+ Characterization and IND-Enabling Studies

项目摘要/摘要 血小板生物发生已经开发出一种千分流生物反应器，它再现了成人的关键特征 骨髓微环境使临床规模生产非供者依赖的血小板成为可能 (PLT+)从人诱导的多能干细胞(HiPSCs)。基于美国食品和药物管理局的指导 美国药品监督管理局(FDA)，这项提案的目标是进行安全性和有效性研究 遵循良好实验室操作规范(GLP)原则，以支持将允许 对我们的产品进行临床测试。血小板是导致血栓形成的重要血液成分 形成和血管修复。血小板计数低是癌症的危险后果 治疗、移植、手术和血小板是预防的关键一线治疗。 失控出血。输血单位完全来自人类志愿捐赠者和 必须在≥22°C下存储，以避免不可逆的活化/聚集。细菌生长受限的风险 保质期为5天，其中3天是通过筛选和运输消耗的。正因为如此 限制，血液中心通常只有1.5天的库存，很快就会用完 紧急情况[1，2]。我们已经开发了一种两步法体外生产平台，在其中hPSCs (可冷冻多年的可补充祖细胞)经历分化为前 巨核细胞(前MK+)被储存并用于在我们的 生物反应器。在我们的第二阶段项目中，我们成功地生成并表征了功能性preMKs+ 和PLTS+从合格的HiPSC系[3，4]使用建立的无血清/饲养器、可伸缩电流 符合良好制造规范(CGMP)的流程[5]。在确认PLT+质量和功能后， 我们在体内验证了止血和血栓形成的潜力。在这些成果的基础上，本阶段IIB SBIR提案概述了执行GLP研究的三个具体目标：1)建立PLT+版本 标准和存储配置文件，2)评估体内畸胎瘤风险，以及3)测量循环和 体内清除时间。就所有目标而言，人类捐献的血小板将作为生理基准 以供比较。完成这些目标将有助于提交调查新的 药物(IND)申请。 目的1.建立PLT+释放质量和储存概况。我们将评估PLT+生物标记物， 在FDA批准的储存条件下，功能、代谢活性和无菌。 目的2.评估小鼠的畸胎瘤风险。遵循GLP指南，我们将确定是否存在任何风险 免疫低下(NSG)小鼠的肿瘤生长与PLT+产物有关。 目的3.评估小鼠PLT+循环和清除时间。我们将评估发行量 免疫低下(NSG)小鼠对PLT+的清除。

PROJECT SUMMARY/ABSTRACT Platelet BioGenesis has developed a millifluidic bioreactor that reproduces key features of the adult bone marrow microenvironment to enable clinical-scale production of donor-independent platelets (PLT+) from human induced pluripotent stem cells (hiPSCs). Based on guidance from the U.S. Food & Drug Administration (FDA), the goal of this proposal is to perform safety and efficacy studies following good laboratory practice (GLP) principles to support a regulatory package that will permit clinical testing of our product. Platelets are essential blood components responsible for clot formation and blood vessel repair. Low platelet count is a dangerous consequence of cancer treatment, transplant, and surgery, and platelets are a critical first-line therapy to prevent uncontrolled bleeding. Transfusion units are derived exclusively from human volunteer donors and must be stored at ≥22°C to avoid irreversible activation/aggregation. Risk of bacterial growth limits shelf life to 5 days, 3 of which are consumed by screening and transport. Because of these limitations, blood centers often only have a 1.5-day inventory that is quickly depleted by emergencies [1,2]. We have developed a 2-step ex vivo production platform in which hiPSCs (replenishable progenitors that can be frozen for years) undergo differentiation into pre- megakaryocytes (preMK+) that are banked and used for on-demand PLT+ generation in our bioreactor. In our Phase II project, we successfully generated and characterized functional preMKs+ and PLTs+ from a qualified hiPSC line [3,4] using an established serum/feeder-free, scalable current good manufacturing practice (cGMP)-compliant process [5]. After confirming PLT+ quality and function, we verified hemostatic and thrombogenic potentials in vivo. Building on these results, this Phase IIB SBIR proposal outlines three specific aims to perform GLP studies to 1) establish PLT+ release criteria and storage profile, 2) assess teratoma risk in vivo, and 3) measure circulation and clearance time in vivo. For all aims, human donor platelets will serve as a physiological benchmark for comparison. Completion of these aims will facilitate submission of an Investigational New Drug (IND) application. Aim 1. Establish PLT+ release quality and storage profile. We will evaluate PLT+ biomarkers, function, metabolic activity, and sterility under FDA-approved storage conditions. Aim 2. Assess teratoma risk in mice. Following GLP guidelines, we will determine whether any risk of tumorigenic growth is associated with PLT+ product in immunocompromised (NSG) mice. Aim 3. Assess PLT+ circulation and clearance times in mice. We will assess the circulation and clearance of PLT+ in immunocompromised (NSG) mice.