PROJECT SUMMARY Sickle Cell Disease (SCD) affects millions of people around the world. Recently, stem cell gene therapy has emerged as a potentially curative option for SCD. Obtaining a sufficient dose of hematopoietic stem and progenitor cells (HSPCs) from peripheral blood is paramount to the success of these gene therapies. However, the higher numbers of RBCs in apheresis products can adversely impact the yield of valuable hematopoietic stem cells during purification (~46% loss). There is, therefore, an immediate unmet need to develop an isolation technology that can efficiently recover hematopoietic stem cells from apheresis products, irrespective of their hematocrits. To address this challenge, I will develop a microfluidic HSPC isolation chip (HSPC-iChip) capable of recovering >95% CD34+ cells from full apheresis products (~300 mL) in an hour (Aim 1). I will bring advancements (in microfluidic technologies) from the field of cancer diagnostics to the field of hematology to accomplish this. My central hypothesis is that the HSPC-iChip can isolate highly viable and functional hematopoietic stem cells. To test this hypothesis, I will genetically edit the isolated stem cells and analyze engraftment, disease correction, and human hematopoiesis in NBSGW mice (Aim 1). Additionally, under the influence of centrifugal forces, the hypercoagulable state of sickle cell patients can lead to the formation of cell clusters. These clusters have been observed to destabilize the cell collection interface, requiring highly skilled apheresis operators for stem cell collection from sickle cell patients. Once the apheresis product is collected, subsequent purification of ~1% HSPCs from the rest of the cells further necessitates specialized instruments and consumables. This restricts a broader implementation of sickle cell gene therapy as most patients reside in low-resource settings where skilled labor, bio-cleanrooms, and financial capabilities are restricted. To address this challenge, in Aim 2, I will test the feasibility of a Precision Apheresis technology that can directly separate HSPCs from peripheral circulation in a single step based on their surface epitopes (CD34). The training objective of this project is to provide Dr. Mishra—who has a strong background in microfluidics and cell sorting—with additional scientific training from leading pioneers in therapeutic gene editing for hemoglobinopathies (Dr. Bauer, Boston Children's Hospital/Harvard), stem cell apheresis and pathology (Dr. Manis, Boston Children's Hospital/Harvard), clinical hematology (Dr. Azar, MGH/Harvard) high-throughput microfluidics (Dr. Toner, lead mentor, MGH/Harvard), animal models and advanced tissue culture (Dr. Haber, co-mentor, MGH/Harvard), nanoparticle kinetics (Dr. Bhatia, MIT), computational modeling of blood cells (Dr. Koumoutsakos, Harvard), and closed-loop mouse-chip models (Dr. Manalis, MIT). This additional training will prepare Dr. Mishra to lead an independent transdis...