Mechanical Stimulation after Centrifuge-Free Nano-Electroporative Transfection Is Efficient and Maintains Long-Term T Cell Functionalities.

Abstract

Transfection is an essential step in genetic engineering and cell therapies. While a number of non-viral micro- and nano-technologies have been developed to deliver DNA plasmids into the cell cytoplasm, one of the most challenging and least efficient steps is DNA transport to and expression in the nucleus. Here, the magnetic nano-electro-injection (MagNEI) platform is described which makes use of oscillatory mechanical stimulation after cytoplasmic delivery with high aspect-ratio nano-structures to achieve stable (>2 weeks) net transfection efficiency (efficiency×viability) of 50% in primary human T cells. This is, to the best of the authors' knowledge, the highest net efficiency reported for primary T cells using a centrifuge-free, non-viral transfection method, in the absence of cell selection, and with a clinically relevant cargo size (>12kbp). Wireless mechanical stimulation downregulates the expression of microtubule motor protein gene, KIF2A, which increases local DNA concentration near the nuclei, resulting in enhanced DNA transfection. Magnetic forces also accelerate membrane repair by promoting actin cytoskeletal remodeling which preserves key biological attributes including cell proliferation and gene expressions. These results demonstrate MagNEI as a powerful non-viral transfection technique for progress toward fully closed, end-to-end T cell manufacturing with less human labor, lower production cost, and shorter delay.