Endothelialized microfluidic assays for emerging therapies in sickle cell disease

项目总结 镰状细胞病(SCD)困扰着10万美国人和全世界数百万人，其特点是 贫血、痛苦的血管闭塞危象、缺血、炎症、显著发病率和早期死亡率。在SCD中， 脱氧镰状血红蛋白(Deoxy-HBs)聚合，使红细胞(RBC)变形，改变 膜的性质，并引发炎症、血栓形成和血管病变。传统治疗 方法包括常规输血和羟基尿素治疗，而这两种选择都是固有的 挑战和限制。最近在SCD中开发新的治疗方法的努力侧重于靶向 旨在防止低氧介导的镰状血红蛋白聚合和异常粘连的治疗 血液和内皮细胞之间的相互作用。因此，美国食品和药物管理局(FDA)已经 最近批准了两种治疗SCD的新疗法：ADAKVEO(诺华公司的Crizanlizumab)和Oxbryta(Voxelotor， 全球血液治疗公司)，以防止异常的红细胞黏附和镰状。除了这些，还有许多其他的 针对特定黏附途径的候选药物目前正处于开发阶段。因为SCD 病理生理学是极其复杂和异质性的，预测患者将如何/是否受益是至关重要的 一种特定的靶向治疗。抗粘连疗法可能不会使特定的患者群体受益，或者可能需要 并辅之以额外的治疗方法(即抗病毒药)。此外，特定于患者的 对这种疗法的反应可能会受到临床变量(例如乳酸)的影响或与之相关 脱氢酶水平、网织红细胞计数、血红蛋白水平等)。能够预测患者对 使用仿生体外工具进行特定的靶向治疗或多种治疗的组合将大大帮助 整个SCD患者群体。因此，迫切需要一种新型的仿生体外检测方法。 作为药物筛选平台，了解新出现的疗法对单个患者的影响 在筛选可能缓解SCD病理生理学的候选药物方面具有重要意义。 我们在此STTRI/II阶段提案中的目标是开发标准化和经验证的内皮化 微流控细胞黏附试验在体外预测靶向治疗患者特异性反应的研究 通过探测血细胞与激活的内皮细胞的黏附。在第一阶段，我们建议标准化和 验证内皮细胞的培养和激活以及内皮细胞内的血细胞黏附 A型芯片在生理相关的流动条件下。第二阶段的目标和里程碑侧重于相关活动 确定基线细胞黏附水平，建立概念验证的测试方法，以及 临床验证芯片上的内皮细胞。我们的目标是证明内皮的效用- A芯片在预测SCD患者对单一和联合靶向治疗的特异性反应中的作用。

PROJECT SUMMARY Sickle Cell Disease (SCD) afflicts 100,000 Americans and millions of people worldwide, and is characterized by anemia, painful vaso-occlusive crises, ischemia, inflammation, significant morbidity, and early mortality. In SCD, deoxygenated sickle hemoglobin (deoxy-HbS) polymerizes, deforms red blood cells (RBCs), changes membrane properties, and triggers inflammation, thrombophilia, and vasculopathy. Traditional treatment approaches include routine blood transfusions and hydroxyurea therapy while both options exhibit inherent challenges and limitations. Recent efforts in developing new treatment approaches in SCD focused on targeted therapies, aiming to prevent hypoxia-mediated polymerization of sickle hemoglobin and the abnormal adhesive interactions between blood and endothelial cells. Accordingly, the Food and Drug Administration (FDA) has recently approved two new treatments for SCD: ADAKVEO (Crizanlizumab, Novartis) and Oxbryta (Voxelotor, Global Blood Therapeutics) to prevent abnormal RBC adhesion and sickling. In addition to those, many other drug candidates targeting a specific adhesion pathway are currently in the development stage. Because SCD pathophysiology is extremely complex and heterogenous, it is crucial to predict how/if a patient will benefit from a specific targeted therapy. An anti-adhesive therapy may not benefit a certain patient population or may need to be supplemented with additional therapeutic approaches (i.e., anti-sickling drugs). Further, patient-specific responses to such therapies may be impacted by or associated with clinical variables (e.g., lactate dehydrogenase level, reticulocyte count, hemoglobin levels, etc.). Being able to predict a patient's response to a certain targeted therapy, or combination of multiple, using biomimetic in vitro tools will substantially help the entire SCD patient population. Therefore, there is an urgent need for novel biomimetic in vitro assays that can serve as a drug screening platform, both in understanding the effect of emerging therapies on a single patient level and in screening drug candidates for potential mitigation of SCD pathophysiology. Our objective in this STTR Phase I/II proposal is to develop a standardized and validated endothelialized microfluidic cellular adhesion assay to predict in vitro patient-specific responses to a targeted therapy by probing blood cell adhesion to activated endothelial cells. In Phase I, we propose to standardize and validate the culture and activation of endothelial cells as well as blood cell adhesion within the Endothelium-on- a-chip under physiologically relevant flow conditions. Phase II aims and milestones focus on activities related to determining baseline cellular adhesion levels, establishing the testing methodologies for proof-of-concept, and clinically validating the Endothelium-on-a-chip. Our goal is to demonstrate the utility of the Endothelium-on- a-chip in predicting patient-specific responses to single and combined targeted therapies in SCD.