Arrayed CRISPR Screening Identifies Novel Targets That Enhance the Productive Delivery of mRNA by MC3-Based Lipid Nanoparticles

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.

Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

“…of NTC; Supplementary table 5). This validation rate (66%) is comparable to that of a previous arrayed CRISPR screen that used a subset of the same crRNA library to analyse delivery of lipid nanoparticleencapsulated mRNA (50%) 31 .…”

Report on Webinar Series Cell and Gene Therapy: From Concept to Clinical Use

“…Findings from this work has revealed rapid trafficking of LNPs is one of the hallmarks of high transfection cell lines. Detailed assessment of functional delivery of LNPs was performed in the H358 cell line known for a high rate of endosomal entrapment [18]. Targets that were found to contribute to dramatic improvements in functional gene delivery included phosphatidylinositol at the plasma membrane, early endosomes and the cytoskeleton.…”

“…Targets that were found to contribute to dramatic improvements in functional gene delivery included phosphatidylinositol at the plasma membrane, early endosomes and the cytoskeleton. Such phenotypic screens can enable high throughput initial evaluation of prototypes for mechanistic characterization during early discovery stages [16,18]. The talk by Dr Desai concluded with the design of a modified LNP formulation process which could evade the clathrin-mediated endocytosis and utilize micropinocytosis for cellular internalization [15].…”

Lipid-Based Nanocarrier Systems for Drug Delivery: Advances and Applications

“…Under physiological conditions, neutral LNP can avoid the toxicity and side effects caused by cationic lipids. 73 Delivery of RNAs to target cells by clinically translational LNPs provides vast opportunities to tackle a series of life-threatening diseases including the novel coronavirus disease 2019 (COVID-19). 74 In 2020, two mRNA vaccines, BNT162b2 and mRNA-1273, received emergency authorization for use from the FDA and European Medicines Agency as the vaccines for the prevention of COVID-19.…”