Project Summary Stem cell-based gene therapies that leverage gene-editing approaches to address disease-causing mutations are emerging as viable medical interventions across a variety of pathologies. Current viral-vector-based and non-viral gene-transfer methods of delivering gene editing machinery, which involve either chemical or energetic disruption of cell membranes, are used routinely in laboratory settings, but fall short when scaled up for clinically relevant applications targetting the manufacture of therapeutic cell products. New methods that enable efficient, rapid, safe, and economical delivery of gene editing packages are needed to support the infrastructures that will be required to translate these gene therapies broadly for application in patient care. Our solution to this critical unmet need leverages innovations in gene editing and nanotechnology to render cell membranes transiently porous, enabling intracellular delivery of biomolecular cargoes. We will design and apply new methods that use acoustic waves generated within microfluidic systems (i.e., acoustofluidics) to mechanically disrupt cell membranes, facilitating the rapid and efficient delivery of CRISPR/Cas9 gene-editing components that are packaged into supramolecular nanocarriers. We use sickle cell disease, one of the most common hemoglobinopathies worldwide, as an initial clinical target for evaluating the proposed platform as it arises from a well-defined genetic mutation that can be targetted for site-specific correction in hematopoietic stem cells with gene editing systems such as CRISPR/Cas9. Successful execution of this research will pave the way for technologies that enable rapid and sustainable processing of stem cell-based gene therapies at clinically- applicable doses ? effectively establishing scalable, good manufacturing practice-compatible assembly lines for manufacturing gene modified therapeutic cell products to treat a wide variety of disesases and will streamline the clinical deployment of future cellular therapies.