High-Throughput CRISPR/Cas9 Mediated Gene Editing of Primary Human T Cells in a Microfluidic Device for Cellular Therapy Manufacturing

Autologous cellular therapies have been highly successful in treating hematological cancers and have the potential to be used for a variety of indications. Manufacturing these therapies rapidly and at low cost remains a major challenge. A key bottleneck in cellular therapy manufacturing is genetic modification of target cells, which is often done using viral vectors. Because vectors are expensive to develop and produce, non-viral gene transfer using electroporation is emerging as a preferred transfection method for next-generation therapies. However, most commercial electroporation systems are built for research use rather than large-scale clinical manufacturing. The microfluidic, continuous-flow electroporation device presented here offers several advantages including large-scale and high throughput processing, high performance, and the potential for automation. It transfects primary human T cells with Cas9-guide ribonucleic acid (RNA) ribonucleoprotein complexes (RNP) and messenger RNA (mRNA) with up to 99-100% efficiency and minimal impact on viability. In addition, this device transfects 3.5 kbp plasmid deoxyribonucleic acid with up to 86% efficiency after preliminary optimization studies. A single microchannel can deliver a total cellular processing throughput of up to 9.6 billion per hour. The combination of high throughput and high performance enables the scale of processing required for future off-the-shelf allogeneic cellular therapies.

Automated summary

Autologous cellular therapies have shown great success in treating hematological cancers and have potential for various indications. However, manufacturing these therapies at a rapid pace and low cost remains challenging, especially due to the genetic modification of target cells using expensive viral vectors. This study presents a microfluidic continuous-flow electroporation device that offers high throughput processing, high performance, and potential for automation, making it suitable for large-scale clinical manufacturing.