Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
A novel intracellular delivery platform that works through mechanoporation that 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.
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.
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Technological Innovations in Immunology (TECH)
Current intracellular delivery methods face significant limitations, including a restricted range of compatible molecules and cell types, as well as unintended alterations to cell biology. CellPore™ Transfection System addresses these limitations by using mechanoporation technology to efficiently deliver mRNAs, CRISPR-Cas9 ribonucleoproteins (RNPs), siRNAs, proteins and other molecules to the cytosol.
Recent platform enhancements, including the introduction of new cartridge sizes, have expanded compatibility to a wider range of cell types. These include unactivated and activated primary immune cells (e.g. T, NK, B and myeloid cells), hematopoietic stem and progenitor cells and pluripotent stem cells, which traditionally have been challenging to reliably engineer via conventional methods.
In the context of immunology applications, we demonstrate efficient RNP-mediated knockout of surface MHC-I (74%) or TCRÉ’β (94%) in human T cells, and TIGIT (84%), CD16 (66%), NKG2A (81%) or NKp46 (87%) in human NK cells, with minimal impact to cell viability and function. We also demonstrate delivery of reporter mRNA and siRNA to monocytes and monocyte-derived dendritic cells for enabling transient gene expression and RNAi-mediated gene knockdown.
The CellPore™ system offers a gentle and efficient method to engineer a variety of cell types with a wide range of molecules. The simple and versatile workflow makes this a valuable tool for enabling new areas of research.
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Technological Innovations in Immunology (TECH)
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