Cracking the Code: Optimizing Ribonucleoprotein Delivery for Gene Editing in Human Myeloid Cells 4298

Abstract Description Several studies have shown that nucleofection can be used to edit human myeloid cells. However, nucleofection of myeloid cells has many disadvantages, including low viability and editing efficiency. The aim of this study is to develop a robust system to genetically engineer primary human monocyte-derived macrophages and dendritic cells (DCs), while maintaining their functional integrity. We tested several in vitro approaches to deliver ribonucleoproteins (RNPs). Using Beta-2 microglobulin (β2M) as a model gene target, nucleofection protocols to deliver RNPs yielded high editing efficiency (90-100%) at the genomic, transcriptional and protein levels. However, nucleofection severely compromised the functional integrity of the cells as they failed to secrete interleukin-1 beta in response to NLRP3 inflammasome activation. We tested alternative RNP delivery methods, including cell-penetrating peptides and lipid nanoparticles. However, the editing efficiencies only reached approximately 30% and resulted in a significant loss in viability. As a solution, we adapted protocols from the Doudna lab to use Enveloped Delivery Vehicles (EDVs) to deliver RNP complexes for gene editing. We reached up to 90% editing efficiency in both primary human macrophages and DCs. We are in the process of validating the functional integrity of EDV-edited myeloid cells and the transcriptional impact of EDV delivery. In conclusion, EDVs are robust and programmable gene editing tools for myeloid cells. Funding Sources NIH R01AI175614 Topic Categories Technological Innovations in Immunology (TECH)