Elucidate exosome transfer between cells and determine the role of caspases in the regulation of exosome assembly and secretion

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Establishing a novel biological model to elucidate exosome transfer between cells and determine the role of caspases in the regulation of exosome assembly and secretion under physiological conditions.Exosomes are a subtype of extracellular vesicles that efficiently transport proteins, lipids and RNA, between cells. Although their instrumental role during cell communication is broadly accepted, the basic molecular mechanisms responsible for their loading and cellular release remain largely unknown. We are developing a Cre-LoxP reporter system suitable for investigating exosome loading and transfer between Drosophila S2 cells and fly tissues. Preliminary results using this system in cell culture based assays suggest that Cre mRNAs can be potentially packaged within extracellular vesicles and transferred between cells. Interestingly, specific exosome-loading and subsequent transfer of mRNAs can be virtually enhanced, by adding a 25-nucleotide extracellular vesicle 'zipcode'. Our tool is currently in the process of adaptation within complex Drosophila tissues, aiming to investigate further the biology of exosome-mediated cell communication. In this regard, we will focus our scientific efforts on uncovering the molecular features of mRNAs that facilitate the loading within exosomes using RNA-Seq analysis. In addition, we will investigate the involvement of the apoptotic factors known as caspases in exosome release. Finally, we aim to exploit this knowledge to change cellular properties remotely, by using exosomes loaded with customised cargos. Our project holds the potential to improve our fundamental understanding of exosome loading/transfer, whilst paving the way to the development of biological tools to manipulate intercellular communication via exosomes.BBSRC Priorities CoveredThe replacement, refinement and reduction (3Rs) in research using animals - Adapting the Cre-LoxP system for studying exosome loading and transfer to Drosophila cell culture and in vivo models, reducing the need to utilise a comparable system established in mice.Technology development for the biosciences - Drosophila cell based and in vivo models to investigate the molecular mechanisms of exosome loading and their potential as molecule carriers, providing a novel resource to the EV and biological research communities.World-class bioscience - Using a collaborative approach and varied biological/bioinformatic techniques to answer fundamental biological questions.Bioscience for health - Exosomes have been implicated in a number of diseases and thus, understanding their fundamental biology improves our understanding of their role in disease. Our work paves the way to harnessing the ability to utilise exosomes for the loading, release and directed transfer of therapeutic molecules for drug delivery.WUB, ENWW