Microfluidic Technologies as Clinical Biomarker Platforms for Sickle Cell Gene Therapies

镰状细胞病治疗性基因治疗的主要目标 (SCD)至少部分是用正常的非镰状血红蛋白(HBS)取代一部分镰状血红蛋白 以此来修饰疾病的进程。为此，新的功能分析(in 除HBS量化外)将非常有助于评估是否实现了 基因改造正在产生临床上有意义的影响。SCD的特征性特征 血细胞之间的血管闭塞相互作用(例如，镰状红细胞，白细胞亚群， 网织红细胞和血小板)和/或血细胞和内皮细胞之间的粘连。这些 相互作用是本病微血管病理生理学的关键决定因素，也是 导致SCD发病率的因素。在SCD基因治疗的背景下，量化将是有用的 这些细胞间的相互作用是可重现的、经过验证的和质量可控的。在其他 换句话说，开发提供经过充分验证的预后终点的分析方法将是有益的， 并以实验室医学中其他临床测试预期的严格性进行。 约翰·罗巴克，医学博士，埃默里大学病理学和实验室医学教授 Emory医学实验室医学主任和副教授Wilbur Lam医学博士 儿科和生物医学工程(埃默里大学/佐治亚理工学院)，最近共同创立 埃默里创新分析开发实验室(EILAD)。伊莱德被设想为一个 实验室，在研究实验室创造的新的分析可以转化为临床使用 在使用临床病理学的标准方法进行彻底优化和验证之后。 这些测试将代表适用的下一代实验室开发的测试(LDT) 到具有挑战性的临床情况。微流控分析评估红细胞粘附性、扩张性和 流特性是Eliad中实现的第一个测试，并且直接适用于 量化血样中红细胞和/或内皮细胞之间的相互作用 SCD患者，无论是前瞻性的还是回顾性的。 作为这项建议的主题的微流控测试是在生物工程中开发的。 林博士和大卫·伍德博士(明尼苏达大学)独立和 协作。林博士和伍德博士是这一领域的先驱，他们已经开发了几种设备 这可以定量评估广泛的镰状细胞的病理活动，包括镰状细胞 体外血管闭塞、红细胞变形性、全血流变学和内皮细胞黏附 各种血细胞亚群。这些化验可能具有重要的诊断实用价值，因为 可以监测血液样本：单细胞水平；高通量；可控性 生理条件(如氧合/除氧)；在内皮存在的情况下； 具有不同的微血管几何形状和体内流动条件。这些化验结果可以作为 研究使能工具和药物发现平台。本应用程序旨在测试 这些检测作为基因治疗后疾病修饰的手段或生物标记物的应用 在SCD患者中。请注意：根据最佳定义，以及ROA(OTA-19-007)， 红细胞的微流体流动特性可作为生物标志物，是优先研究的领域 对评估SCD患者对基因治疗的反应感兴趣。 作为团队的一部分，我们还聘请了David Alter医学博士(病理学副教授 和David Archer博士(儿科学副教授)。奥尔特博士是 董事会认证的临床病理学家和临床化学家，是Emory Core的主任 实验室，是验证低密度脂蛋白检测的专家。阿彻博士是流式细胞术的专家， 包括将应用于微流控分析的分析方法；他担任 亚特兰大儿童保健儿科/Winship流式细胞术中心主任 埃默里大学温希普癌症研究所。总而言之，聚集在一起的研究团队很好 定位于：制造本文所述的微流控设备，验证其性能 特性，为这些设备开发可重复使用的试剂和质量控制规范， 并将其用于量化参与SCD的患者的红细胞功能特性的变化 基因修正试验。

The primary goal of curative genetic therapies for sickle cell disease (SCD), at least in part, is to replace a fraction of sickle hemoglobin (HbS) with normal non-sickling hemoglobin in order to modify the disease process. To this end, novel functional assays (in addition to HbS quantitation) would be highly useful for assessing whether the achieved levels of genetic modification are having a clinically meaningful impact. Characteristic hallmarks of SCD are the vaso-occlusive interactions between blood cells (e.g., sickle RBCs, WBC subpopulations, reticulocytes, and platelets) and/or adhesion between blood cells and endothelium. These interactions are critical determinants of microvascular pathophysiology in this disease, and key contributors to SCD morbidity. In the setting of SCD genetic therapy, it would be useful to quantify these cellular interactions in a reproducible, validated, and quality-controlled manner. In other words, it would be beneficial to develop assays that provide well-validated prognostic endpoints, and which are performed with the rigor expected for other clinical tests in laboratory medicine. John Roback, MD, PhD, Professor of Pathology and Laboratory Medicine at Emory as well as Medical Director of Emory Medical Laboratories, and Wilbur Lam, MD PhD, Associate Professor of Pediatrics and Biomedical Engineering (Emory University/Georgia Tech), recently co-founded the Emory Laboratory for Innovative Assay Development (ELIAD). ELIAD is envisioned as a laboratory where novel assays created in research laboratories can be translated to clinical use after being thoroughly optimized and validated using standard approaches of clinical pathology. These assays will represent the next generation of laboratory-developed tests (LDT) applicable to challenging clinical situations. Microfluidic assays to assess RBC adhesion, distensibility, and flow properties are the first tests being implemented in ELIAD, and are directly applicable to quantifying interactions between RBCs and/or endothelial cells in blood samples obtained from patients with SCD, either prospectively or retrospectively. The microfluidic tests that are the subject of this proposal were developed in the bioengineering laboratories of Dr. Lam and David Wood, PhD (University of Minnesota) independently and in collaboration. Drs. Lam and Wood are pioneers in this area, and have developed several devices that can quantitatively assess a broad range of sickle cell pathologic activities including sickle cell vaso-occlusion in vitro, RBC deformability, whole blood rheology, and endothelial adhesion to various blood cell subpopulations. These assays likely have significant diagnostic utility because blood samples can be monitored: at the single-cell level; with high-throughput; under controllable physiologic conditions (e.g., oxygenation/deoxygenation); in the presence of endothelium; and with varying microvascular geometries and in vivo flow conditions. These assays can function as research enabling tools and drug discovery platforms. The present application is designed to test the utility of these assays as measures or biomarkers of disease modification after gene therapy in SCD patients. Please note: per the BEST definition, and also the ROA (OTA-19-007), microfluidic flow properties of RBCs qualify as biomarkers and are high priority areas of research interest for assessing responses of SCD patients to genetic therapies. As part of the team, we have also recruited David Alter, MD (Associate Professor of Pathology and Laboratory Medicine) and David Archer, PhD (Associate Professor of Pediatrics). Dr. Alter is a Board-certified Clinical Pathologist and Clinical Chemist, is the Director of the Emory Core Laboratory, and is an expert at validating LDT assays. Dr. Archer is an expert in flow cytometry, including the analysis methods which will be applied to microfluidic assays; he serves as the Director of the Pediatrics/Winship Flow Cytometry Core at Children’s Healthcare of Atlanta and Winship Cancer Institute of Emory University. Together, the assembled research team is well positioned to: manufacture the microfluidic devices described herein, validate their performance characteristics, develop reproducible reagent and quality control specifications for these devices, and deploy them to quantify changes in RBC functional properties in patients participating in SCD gene correction trials.