Abstract
RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.
Highlights
RNA-based therapies have great potential to treat many undruggable human diseases
In this work, we have demonstrated a comprehensive system for the monitoring of nanoparticle trafficking from uptake, to endosomal escape, and to protein translation within a single microscopy assay and established a screening platform by integrating the assay with robotic formulation and high-throughput biophysical analysis of lipid nanoparticles (LNPs)
This approach can highlight the general effect of component substitution but reduces the reliance on using a single LNP to make the decision of efficacy
Summary
RNA-based therapies have great potential to treat many undruggable human diseases Their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. This assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. The undeniable importance of other components such as the sterol or the PEG-lipid for modulating uptake, improving pharmacokinetics or modifying the particle’s corona composition is being increasingly appreciated[11,12,13,14]
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