Microfluidic Technologies as Clinical Biomarker Platforms for Sickle Cell Gene Therapies

微流控技术作为镰状细胞基因治疗的临床生物标志物平台

• 批准号: 10001892
• 负责人: John D Roback
• 金额: $ 3.2万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2020-02-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001892
• 关键词: Address; Adhesions; Adhesiveness; Affect; Area; Award; Biological Assay; Biological Markers; Biomedical Engineering; Blood Cells; Blood Platelets; Blood Tests; Blood specimen; Cells; Characteristics; Child health care; Clinical; Clinical Pathology; Clinical Trials; Collaborations; Data; Devices; Diagnostic; Diagnostic tests; Disease; Doctor of Philosophy; Endothelial Cells; Endothelium; Evaluation; Flow Cytometry; Functional disorder; Funding; Genetic; Geometry; Goals; In Vitro; Institutes; Instruction; Laboratories; Laboratory Research; Malignant Neoplasms; Measures; Medical; Medicine; Methods; Microfluidic Microchips; Microfluidics; Minnesota; Modification; Monitor; Morbidity - disease rate; Pathologic; Pathologist; Pathology; Patients; Pediatrics; Performance; Physician Executives; Physiological; Positioning Attribute; Procedures; Process; Property; Publications; Quality Control; Reagent; Reproducibility; Research; Reticulocytes; Sampling; Shipping; Sickle Cell; Sickle Cell Anemia; Sickle Hemoglobin; Site; Testing; Translating; Universities; Validation; Veno-Occlusive Disease; Whole Blood; Wood material; assay development; biomarker validation; blood rheology; clinical biomarkers; design; direct application; drug discovery; gene correction; gene therapy; improved; in vivo; innovation; interest; microfluidic technology; mid-career faculty; next generation; novel; professor; prognostic; prospective; recruit; response; tool

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.

镰状细胞病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）替代一部分镰状血红蛋白（HbS）。 血红蛋白，以改变疾病的进程。为此，新的功能测定（在 除了HbS定量之外）对于评估是否达到HbS水平是非常有用的。 基因改造正在产生临床上有意义的影响。SCD的特点 是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/或血细胞与内皮之间的粘附。这些 相互作用是这种疾病中微血管病理生理学的关键决定因素， SCD发病率的贡献者。在SCD基因治疗的背景下，量化 这些细胞相互作用以可再现的、经验证的和质量受控的方式进行。换句 换句话说，开发提供充分验证的预后终点的测定将是有益的， 并且以实验室医学中的其他临床测试所期望的严格性来执行。 John Roback，医学博士，埃默里大学病理学和实验室医学教授， 埃默里医学实验室医学总监，Wilbur Lam，医学博士，副教授 儿科和生物医学工程（埃默里大学/格鲁吉亚理工学院），最近共同创立了 Emory Laboratory for Innovative Assay Development（ELIAD）ELIAD被设想为一个 实验室，在研究实验室中创建的新检测方法可以转化为临床应用 在使用临床病理学的标准方法进行彻底优化和验证之后。 这些检测试剂盒将代表适用的下一代实验室开发检测（LDT） 挑战性的临床情况。用于评估RBC粘附性、扩张性和 流动特性是ELIAD中实施的第一批测试，并直接适用于 定量血液样品中RBC和/或内皮细胞之间的相互作用，所述血液样品从 SCD患者，前瞻性或回顾性。 微流控测试，是这个建议的主题是在生物工程开发 Lam博士和大卫伍德博士（明尼苏达大学）的独立实验室， 协作林博士和伍德博士是这一领域的先驱，并开发了几种设备 其可以定量评估宽范围的镰状细胞病理活性，包括镰状细胞 体外血管闭塞，红细胞变形性，全血流变学，内皮细胞粘附， 不同的血细胞亚群这些测定可能具有显著的诊断实用性，因为 血液样本可被监测：在单细胞水平;高通量;可控 生理条件（例如，氧合/脱氧）;在内皮存在下;和 具有不同的微血管几何形状和体内流动条件。这些测定可用作 研究工具和药物发现平台。本应用程序旨在测试 这些测定作为基因治疗后疾病改变的量度或生物标志物的效用 在SCD患者中。请注意：根据最佳定义以及罗阿（OTA-19-007）， 红细胞的微流体流动特性有资格作为生物标志物，并且是高度优先的研究领域 对评估SCD患者对遗传疗法的反应感兴趣。 作为团队的一部分，我们还招募了大卫奥尔特，医学博士（病理学副教授 和实验室医学）和大卫阿彻博士（儿科副教授）。奥尔特博士是 委员会认证的临床病理学家和临床化学家，是埃默里核心主任 实验室，是验证LDT检测的专家。阿彻博士是流式细胞术专家 包括将应用于微流体测定的分析方法;他担任 亚特兰大儿童保健中心儿科/Winship流式细胞术中心主任， 埃默里大学温希普癌症研究所。集合起来的研究团队 制造本文所述的微流体装置，验证它们的性能， 特性，为这些设备开发可重现的试剂和质量控制规范， 并将其用于量化参与SCD患者的RBC功能特性变化 基因矫正试验