UCLA

Gene therapies that leverage engineered cells to produce a therapeutic effect via gene correction or modification are offering exciting treatment solutions for patients with cancer, genetic diseases, and infectious diseases. Accelerating the clinical translation of these interventions requires the development and application of new techniques for engineering target cell populations rapidly, efficiently, safely, and cost effectively. To address the limitations of traditional viral and non-viral gene delivery methods, we have designed an acoustofluidic intracellular delivery technology that enables precise sonoporation of cells as they pass through a microfluidic channel in the absence of contrast agents. A combination of acoustic radiation force and shearing force in the microfluidic system can transiently increase membrane permeability and promote cellular uptake. We demonstrated the delivery of a model expression plasmid encoding for non-integrating enhanced green fluorescent protein (eGFP) to Jurkat cells, peripheral blood mononuclear cells (PBMCs), and umbilical blood CD34+ hematopoietic stem and progenitor cells (CD34+ HSPCs) at a high throughput. We then extended the applications of this biophysical gene delivery platform to introduce different biomolecular cargoes to various cell types including both immortalized cells and human primary T cells. With optimized microfluidic designs, we generated functional chimeric antigen receptor (CAR) T cell populations via delivery of Sleeping Beauty plasmids designed to encode for the expression of a Glypican-3 (GPC3)-targeting CAR, which we packaged into supramolecular nanoparticle (SMNP) carriers. To understand the mechanisms of the acoustofluidic-mediated membrane disruption, we observed changes of cell membrane morphology post-acoustofluidic treatment and probed the dynamics of membrane deformation and repair that occur during/post acoustofluidic manipulation. Altogether, this study represents a promising step toward expanding the library of acoustofluidics-engineered cell products for advancing the generation of future cell-based gene therapeutics.