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.

A vaccine antigen, when launched as DNA or RNA, can be presented in various forms, including intracellular, secreted, membrane‐bound, or on extracellular vesicles (EVs). Whether an antigen in one or more of these forms is superior in immune induction remains unclear. In this study, we used GFP as a model antigen and first compared the EV‐loading efficiency of transmembrane domains (TMs) from various viral glycoproteins, and then investigated whether EV‐bound GFP (EV‐GFP) would enhance immune induction. Our data showed that GFP fused to viral TMs was successfully loaded onto the surface of EVs. In addition, GFP‐bound EVs were predominantly associated with the exosome marker CD81. Immunogenicity study with EV‐GFP‐producing plasmids in mice demonstrated that antigen‐specific IgG and IgA were significantly increased in EV‐GFP groups, compared to soluble and intracellular GFP groups. Similarly, GFP‐specific T cell response‐related cytokines produced by antigen‐stimulated splenocytes were also enhanced in mice immunized with EV‐GFP constructs. Immunogenicity study with purified soluble GFP and GFP EVs further confirmed the immune enhancement property of EV‐GFP in mice. In vitro uptake assays indicated that EV‐GFP was more efficiently taken up than soluble GFP by mouse splenocytes and such uptake was B cell preferential. Taken together, our data indicate that viral TMs can efficiently load antigens onto the EV surface, and that EV‐bound antigen enhances both humoral and cell‐mediated antigen‐specific responses.

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. Advances in the development and rational optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by the limited availability of screening methods that 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. This assay has, furthermore, been integrated with a screening platform for nanoparticle formulation to optimise LNPs. 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 also identify nanoparticles with superior escape properties and demonstrate significant cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.