Abstract

Modified messenger RNAs (mRNAs) hold great potential as therapeutics by using the body’s own processes for protein production. However, a key challenge is efficient delivery of therapeutic mRNA to the cell cytosol and productive protein translation. Lipid nanoparticles (LNPs) are the most clinically advanced system for nucleic acid delivery; however, a relatively narrow therapeutic index makes them unsuitable for many therapeutic applications. A key obstacle to the development of more potent LNPs is a limited mechanistic understanding of the interaction of LNPs with cells. To address this gap, we performed an arrayed CRISPR screen to identify novel pathways important for the functional delivery of MC3 lipid-based LNP encapsulated mRNA (LNP-mRNA). Here, we have developed and validated a robust, high-throughput screening–friendly phenotypic assay to identify novel targets that modulate productive LNP-mRNA delivery. We screened the druggable genome (7795 genes) and validated 44 genes that either increased (37 genes) or inhibited (14 genes) the productive delivery of LNP-mRNA. Many of these genes clustered into families involved with host cell transcription, protein ubiquitination, and intracellular trafficking. We show that both UDP-glucose ceramide glucosyltransferase and V-type proton ATPase can significantly modulate the productive delivery of LNP-mRNA, increasing and decreasing, respectively, with both genetic perturbation and by small-molecule inhibition. Taken together, these findings shed new light into the molecular machinery regulating the delivery of LNPs into cells and improve our mechanistic understanding of the cellular processes modulating the interaction of LNPs with cells.