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.

中文翻译：

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阵列式 CRISPR 筛选确定了通过基于 MC3 的脂质纳米颗粒提高 mRNA 高效递送的新靶点。

通过使用人体自身的蛋白质生产过程，修饰的信使 RNA (mRNA) 具有作为治疗剂的巨大潜力。然而，一个关键的挑战是有效地将治疗性 mRNA 传递到细胞质和生产性蛋白质翻译。脂质纳米颗粒 (LNP) 是临床上最先进的核酸递送系统；然而，相对狭窄的治疗指数使其不适用于许多治疗应用。开发更有效的 LNP 的一个关键障碍是对 LNP 与细胞相互作用的有限机制理解。为了解决这一差距，我们进行了阵列式 CRISPR 筛选，以确定对基于 MC3 脂质的 LNP 封装的 mRNA (LNP-mRNA) 的功能传递很重要的新途径。在这里，我们开发并验证了一个强大的、高通量筛选友好的表型测定，以确定调节生产性 LNP-mRNA 递送的新靶标。我们筛选了可药用基因组（7795 个基因）并验证了 44 个增加（37 个基因）或抑制（14 个基因）LNP-mRNA 生产性传递的基因。许多这些基因聚集成与宿主细胞转录、蛋白质泛素化和细胞内运输有关的家族。我们表明，UDP-葡萄糖神经酰胺葡萄糖基转移酶和 V 型质子 ATP 酶均可显着调节 LNP-mRNA 的生产性传递，分别通过遗传扰动和小分子抑制增加和减少。综合起来，