Microfluidic Technologies as Clinical Biomarker Platforms for Sickle Cell Gene Therapies

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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.