Virus-Free Method to Control and Enhance Extracellular Vesicle Cargo Loading and Delivery.

Extracellular vesicles (EVs)─including exosomes and microvesicles─are involved in cell-cell communication. EVs encapsulate different types of molecules such as proteins or nucleotides and are long-lasting contenders for the establishment of personalized drug delivery systems. Recent studies suggest that the intrinsic capacities for uptake and cargo delivery of basic EVs might be too limited to serve as a potent delivery system. Here, we develop two synergistic methods to, respectively, control EV cargo loading and enhance EV cargo delivery through fusion without requirement for any viral fusogenic protein. Briefly, cargo loading is enabled through a reversible drug-inducible system that triggers the interaction between a cargo of interest and CD63, a well-established transmembrane EV marker. Enhanced cargo delivery is promoted by overexpressing Syncytin-1, an endogenous retrovirus envelop protein with fusogenic properties encoded by the human genome. We validate our bioengineered EVs in a qualitative and quantitative manner. Finally, we utilize this method to develop highly potent killer EVs, which contain a lethal toxin responsible for protein translation arrest and acceptor cell death. These advanced methods and future downstream applications may open promising doors in the manufacture of virus-free and EV-based delivery systems.

Quantitative Measurement of Extracellular Vesicle Content Delivery Within Acceptor Cells.

Extracellular vesicles (EVs), including exosomes and microvesicles, are thought to transport bioactive molecules from donor to acceptor cells. Although EV uptake has been qualitatively assessed through subcellular imaging, EV content delivery has been rarely addressed due to a lack of adequate methods. Here we present a sensitive bulk assay to quantitatively measure EV uptake and content delivery in mammalian cell. In this assay, EVs containing a NanoLuc luciferase-tagged cargo are mixed with unlabeled acceptor cells. Cell fractionation separates membrane and cytosolic fractions, and luciferase activity is measured within each fraction to determine the percentage of cytosolic release. This assay can be used to further decipher cellular and molecular mechanisms that regulate the EV delivery process or to quantitatively test specific pairs of donor-acceptor cells.

Translocated Legionella pneumophila small RNAs mimic eukaryotic microRNAs targeting the host immune response.

Legionella pneumophila is an intracellular bacterial pathogen that can cause a severe form of pneumonia in humans, a phenotype evolved through interactions with aquatic protozoa in the environment. Here, we show that L. pneumophila uses extracellular vesicles to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways. The bacterial sRNA RsmY binds to the UTR of ddx58 (RIG-I encoding gene) and cRel, while tRNA-Phe binds ddx58 and irak1 collectively reducing expression of RIG-I, IRAK1 and cRel, with subsequent downregulation of IFN-ß. Thus, RsmY and tRNA-Phe are bacterial trans-kingdom regulatory RNAs downregulating selected sensor and regulator proteins of the host cell innate immune response. This miRNA-like regulation of the expression of key sensors and regulators of immunity is a feature of L. pneumophila host-pathogen communication and likely represents a general mechanism employed by bacteria that interact with eukaryotic hosts.