Project Summary Sickle cell disease (SCD) is among the most common monogenetic inherited disorders. Clinical management of SCD is primarily supportive. However, in the most severe cases, the only definitive curative option for patients suffering from SCD is an allogeneically matched hematopoietic stem cell transplant. This hemoglobinopathy directly affects the structure and function of hemoglobin, leading to deficiencies of β-globin chains in the development of functional adult hemoglobin. Furthermore, the lack of fully matched donors for patients to receive a stem cell transplant runs the risk of adverse immunogenic reactions, such as auto-immune disorders or graft-versus-host disease. Recent efforts to address this disease and its clinical sequela have focused on gene therapies based on the transplantation of autologous gene-modified hematopoietic stem & progenitor cells (HSPC), where a patient's own cells are corrected and reinfused to enable production of fully functioning erythrocytes. However, non-viral strategies for the batch processing of stem cell gene therapies are known to be inefficient and are unable to meet clinical demands. We hypothesize that the optimization of an acoustofluidic therapeutic platform that physically permeabilizes cells for the delivery of CRISPR-Cas9 biomolecules will address this technologic gap. This high-throughput gene-delivery strategy will enable our long-term goal to generate gene-modified stem cell therapies quickly and efficiently for curing sickle cell disease. This physical permeabilization process renders target cells transiently permeable, enabling vector uptake while minimizing damage to the cell membrane and maintaining high levels of viability. In order to achieve our clinical target, our proposed specific aims include: 1) optimize acoustofluidic gene delivery in model cell lines harboring the sickle cell mutation and 2) evaluate site-specific correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. Given the utility of this acoustofluidic technology, there is a wide range of heart, lung, and blood disorders that can be addressed, overcoming the state of the art for gene delivery. We expect the generation of rapid and safe gene-modified stem cell therapies using our acoustofludic technology will greatly improve access to these medical interventions and the quality of life for patients with the most severe cases of SCD.