![]() ![]() The applied high-voltage pulses create transient pores in the cell membrane that allow molecules to diffuse into the cells 19, 20. ![]() In the standard, static electroporation method that has been used for many years, an electric field is created by applying high-voltage electrical pulses to cell suspensions within a cuvette 18. Similarly, electroporation was recently used to generate CAR-T cells through the use of Sleeping Beauty transposon system for the first clinical trial with virus-free CAR-T cells in Europe 16 as well as with the piggyBac system 17. For example, Bozza et al., demonstrated the ability to use electroporation to generate recombinant T cells using nonintegrating DNA nanovectors that bypass many of the drawbacks associated with viral approaches 8. Among non-viral transfection methods, electroporation is a well-studied approach commonly used to deliver DNA, RNA and proteins into cells that is recognized as a leading contender for the replacement of viral vectors. To circumvent these limitations, research efforts have increasingly focused on non-viral transfection methods to replace viral delivery 9, 14, 15. ![]() Furthermore, the field is trending towards more complex reprogramming methods, such as multiple gene edits via CRISPR/Cas9 technology and gene insertion by transposon elements, that are not compatible with typical packaging limits associated with viral approaches 9, 10, 11, 12, 13. Viral vectors have enabled high efficiency transduction of difficult-to-transfect primary human immune cells but have several drawbacks related to their complex and costly manufacturing processes, immunogenicity, and potential for insertional mutagenesis 6, 7, 8. However, the first generation of approved CAR-T cell therapies rely on viral vectors such as lentivirus or adeno-associated virus (AAV) for cellular reprogramming 1, 2, 3, 4, 5. Research is being directed toward treating other cancers such as solid tumors. In particular, immunotherapies that utilize autologous T cells modified to express chimeric antigen receptors (CARs) have achieved remarkable rates of complete response with durable, long-lasting remissions for certain hematological cancers. This study displays the capabilities of our system to address unmet needs for efficient, non-viral engineering of T cells for cell manufacturing.Ĭellular therapies have generated enthusiasm for their potential to treat a variety of inherited and acquired diseases. Finally, we demonstrate a therapeutically relevant modification of primary T cells using CRISPR/Cas9 to knockdown T cell receptor (TCR) expression. We present methods for scaling delivery that achieve an experimental throughput of 256 million cells/min. We demonstrate delivery of plasmid DNA and mRNA to primary human T cells with high efficiency and viability, such as > 95% transfection efficiency for mRNA delivery with < 2% loss of cell viability compared to control cells. Here, we present a novel electroporation platform capable of rapid and reproducible electroporation that can efficiently transfect small volumes of cells for research and process optimization and scale to volumes required for applications in cellular therapy. Electroporation has emerged as an approach for non-viral transfection of primary cells, but standard cuvette-based approaches suffer from low throughput, difficult optimization, and incompatibility with large-scale cell manufacturing. Viral vectors represent a bottleneck in the manufacturing of cellular therapies. ![]()
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