UC Irvine

Intracellular delivery is an important yet challenging step in gene and cell-based therapies, especially Chimeric antigen receptor T (CAR-T) cell therapy. Viral-based transduction is the most common method for cell delivery in clinical application because of its high efficiency and specificity. However, there’re safety concern remain unsolved such as insertional toxicity, immunogenicity, and cytotoxicity. Other non-viral transfection, such as cationic lipids and electroporation also have challenges such as low delivery efficiency for suspension cells, potential cell toxicity, and high cell mortality. In addition, due to their bulk nature, cationic lipids and electroporation do not offer uniform and dosage-controlled delivery across cell populations. Furthermore, part of the CAR-T therapy efficacy, in which the CAR gene was delivered into primary T cells and expression receptor on the cell membrane to recognize tumor cells, heavily relies on the method of gene delivery. Besides the side effect mentioned above of both viral and non-viral transfection, it’s also crucial that the intracellular delivery method can control the dosage deliver into the cell population. Recent clinical studies have recognized that the heterogeneity of CAR expression level in cell population determines therapy efficacy and safety. Specifically, CAR-T cells with lower CAR expression cause less therapeutic efficacy while high CAR expression lead cytokine release syndrome and tonic signaling. Hence, a precise-controlled method that can not only manufacture CAR-T cells efficiently but also titer the CAR expression density in an optimal range is desirable. To address the challenges from viral or non-viral gene delivery methods and enable to control the dosage of cargoes intracellular delivery, we developed an Acoustic-Electric Shear Orbiting Portation (AESOP) platform for intracellular delivery. By applying acoustic induced shear and relatively low electric field to initiate pores on cell membrane and expand the size of pores, a wide range of cargos (include dextran, mRNA and plasmid) can be delivered into multiple cells, such as HeLa, K562, Jurkat cells, and primary T cells without compromising cell viability. In addition, with assistant of micro-vortices induced by acoustic energy, the cargoes can be mixed uniformly with the targeted cells, thus each cell can uptake approximately the same dosage of cargo, which lead to similar protein expression on the cell membrane in cell population after transfection. Finally, we demonstrated that AESOP can delivered mRNA encoding anti CD19 CAR gene into primary T cells uniformly and can control the CAR expression in the cell population via adjusting the input mRNA concentration.