A novel silicon-based intracellular delivery platform enables multiplexed delivery across cell types and cell states 2310229

Abstract

Immune cell engineering is a critical element of basic immunological research and translational applications. The ability to deliver modifying materials to cells without substantial phenotypic or survival impacts is a limiting factor for many immune cell types. To enable engineering in sensitive cell types or cell states, we have developed a novel intracellular delivery platform that works through mechanoporation. Cells are pushed through pores on a thin silicon membrane which creates temporary openings in the cell membrane that allow the payload of interest to diffuse into the cytoplasm before the membranes repair themselves. This method has been used to introduce CRISPR RNPs, mRNA, circRNA, siRNA and peptides and is compatible across cell types including naive and activated T cells, B cells, NK cells, monocytes, neutrophils, iPSC and HSC. Using our device, we simultaneously delivered a B2M targeting CRISPR RNP, GFP mRNA, and fluorescent dextran to primary unstimulated T cells and observed that 85% of the live population was B2M negative, GFP and dextran positive. The temporary membrane disruption is gentle enough to use with sensitive cells like neutrophils which allowed us to deliver dextran and GFP mRNA to neutrophils in whole blood. We measured 83% GFP positivity in the live, dextran positive population the next day. Similarly, we delivered a GFP-mRNA to PBMCs and observed expression in T cells, B cells, NK cells, and monocytes. This technology can also enable the use of viral vectors in challenging cell types. Using our method, we engineered B cells to express a circRNA ‘transduction enhancer’ (TE) and then infected them with a GFP lentivirus. After 3 days, the cells that received the TE had the highest GFP expression. Our technology unlocks scalable, gentle, and versatile intracellular delivery for sensitive immune cells, expanding the feasibility of complex genetic and molecular engineering for research and clinical cell therapies. n/a Technological Innovations in Immunology (TECH)