Mechanoporation-based Intracellular Delivery of Functional Molecules for Primary Immune Cell Engineering 2252000

Introduction 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. Methods 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. Results 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. Conclusion 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.