Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

Profiling of Extracellular Small RNAs Highlights a Strong Bias towards Non-Vesicular Secretion.

The extracellular environment consists of a plethora of molecules, including extracellular miRNA that can be secreted in association with extracellular vesicles (EVs) or soluble protein complexes (non-EVs). Yet, interest in therapeutic short RNA carriers lies mainly in EVs, the vehicles conveying the great majority of the biological activity. Here, by overexpressing miRNA and shRNA sequences in parent cells and using size exclusion liquid chromatography (SEC) to separate the secretome into EV and non-EV fractions, we saw that >98% of overexpressed miRNA was secreted within the non-EV fraction. Furthermore, small RNA sequencing studies of native miRNA transcripts revealed that although the abundance of miRNAs in EVs, non-EVs and parent cells correlated well (R2 = 0.69-0.87), quantitatively an outstanding 96.2-99.9% of total miRNA was secreted in the non-EV fraction. Nevertheless, though EVs contained only a fraction of secreted miRNAs, these molecules were stable at 37 °C in a serum-containing environment, indicating that if sufficient miRNA loading is achieved, EVs can remain delivery-competent for a prolonged period of time. This study suggests that the passive endogenous EV loading strategy might be a relatively wasteful way of loading miRNA to EVs, and active miRNA loading approaches are needed for developing advanced EV miRNA therapies in the future.

Considerations and Implications in the Purification of Extracellular Vesicles - A Cautionary Tale.

Extracellular vesicles (EVs) are nano-sized particles constitutively released from cells into all biological fluids. Interestingly, these vesicles contain genetic cargoes including proteins, RNA and bioactive lipids that can be functionally delivered and affect recipient cells. As a result, there is growing interest in studying EVs in pathological conditions, including central nervous system (CNS)-related diseases, as EVs may be used for diagnostic purposes or as therapeutic agents. However, one major bottleneck is the need for better EV purification strategies when considering complex biological sources such as serum/protein-rich media or plasma. In this study, we have performed a systematic comparison study between the current gold-standard method: ultracentrifugation, to an alternative: size-exclusion chromatography (LC), using induced pluripotent stem cell (iPSC) derived complex media as a model system. We demonstrate that LC allows for derivation of purer EVs from iPSCs, which was previously impossible with the original UC method. Importantly, our study further highlights the various drawbacks when using the conventional UC approach that lead to misinterpretation of EV data. Lastly, we describe novel data on our iPSC-EVs; how they could relate to stem cell biology and discuss their potential use as EV therapeutics for CNS diseases.

Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome.

Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding- and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.