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Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a range of clinical applications. Forin vivofunctionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, we developed a bioengineering platform for efficient mRNA loading and functional delivery using naturally-derived nanoparticles, Extracellular Vesicles (EVs). By expressing the highly specific PUFe RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA, our system exceeds mRNA loading efficiencies by up to 4.5-fold over previously reported EV-based approaches. By combining with an mRNA-stabilizing protein, PABPc, and a fusogenic endosomal escape moiety, VSVg, functional mRNA deliveryin vivois achieved at doses substantially lower than clinically used lipid nanoparticles. Our technology solves substantial drawbacks currently associated with EV-based nucleic acid delivery systems and could enable new applications for mRNA therapeutics.

Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration., Graphical abstract

PMCID: PMC1276709, Phage ø29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that 29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host-encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected ø29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses ø29 development by repressing the main early ø29 promoters via different and novel mechanisms and also by preventing activation of the single late ø29 promoter., This investigation was supported by grants 2RO1 GM27242-24 from the National Institutes of Health and BMC2002-03818 from the Spanish Ministry of Science and Technology to MS and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular 'Severo Ochoa'. VC is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology, which also supported WJJM by means of the 'Ramón y Cajal' program.

RNA granules have been observed in different organisms, cell types and under different conditions, and their formation is crucial for the mRNA life cycle. However, very little is known about the molecular mechanisms governing their assembly and disassembly. The aggregation-prone LSCRs (low-sequence-complexity regions), and in particular, the polyQ/N-rich regions, have been extensively studied under pathological conditions due to their role in neurodegenerative diseases. In the present review, we discuss recent in vitro, in vivo and computational data that, globally, suggest a role for polyQ/N regions in RNA granule assembly.

Members of groups 1 (e.g. ϕ29) and 2 (e.g. Nf) of the ϕ29 family of phages infect the spore forming bacterium Bacillus subtilis. Although classified as lytic phages, the lytic cycle of ϕ29 can be suppressed and its genome can become entrapped into the B. subtilis spore. This constitutes an alternative infection strategy that depends on the presence of binding sites for the host-encoded protein Spo0A in the ϕ29 genome. Binding of Spo0A to these sites represses ϕ29 transcription and prevents initiation of DNA replication. Although the Nf genome can also become trapped into B. subtilis spores, in vivo studies showed that its lytic cycle is less susceptible to spo0A-mediated suppression than that of ϕ29. Here we have analysed the molecular mechanism underlying this difference showing that Spo0A differently affects transcription and replication initiation of the genomes of these phages. Thus, whereas Spo0A represses all three main early promoters of ϕ29, it only represses one out of the three equivalent early promoters of Nf. In addition, contrary to ϕ29, Spo0A does not prevent the in vitro initiation of Nf DNA replication. Altogether, the differences in Spo0A-mediated regulation of transcription and replication between ϕ29 and Nf explain their different behaviours in vivo., Spanish Ministry of Education and Science (BFU2005-00733 to M.S., BFU2005-01878 to W.J.J.M.); Spanish Ministry of Science and Innovation (BFU2008-00215/BMC to M.S.); institutional grant—Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’; predoctoral fellowship from the Spanish Ministry of Education and Science (to V.C.L.L.). Funding for open access charge: BFU2008-00215/BMC. Conflict of interest statement. None declared.

The host of the lytic bacteriophage φ29 is the spore-forming bacterium Bacillus subtilis. When infection occurs during early stages of sporulation, however, φ29 development is suppressed and the infecting phage genome becomes trapped into the developing spore. Recently, we have shown that Spo0A, the key transcriptional regulator for entry into sporulation, is directly responsible for suppression of the lytic φ29 cycle in cells having initiated sporulation. Surprisingly, we found that φ29 development is suppressed in a subpopulation of logarithmically growing culture and that spo0A is heterogeneously expressed during this growth stage. Furthermore, we showed that kinC and, to a minor extent, kinD, are responsible for heterogeneous expression levels of spo0A during logarithmical growth that are below the threshold to activate sporulation, but sufficient for suppression of the lytic cycle of φ29. Whereas spo0A was known to be heterogeneously expressed during the early stages of sporulation, our findings show that this also occurs during logarithmical growth. These insights are likely to have important consequences, not only for the life cycle of φ29, but also for B. subtilis developmental processes., This investigation was supported by Grants BFU2005-00733 and BFU2005-01878 from the Spanish Ministry of Education and Science to M.S. and W.J.J.M. respectively, and an Institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular ‘Severo Ochoa’. V.C. is holder of a predoctoral fellowship from the Spanish Ministry of Science and Technology.