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Abstract

In multicellular organisms, cells communicate with each other in order to form tissues and act collectively. Extracellular vesicles (EVs) are lipid membrane-lined compartments in the nanometre to micrometre range that have recently been identified as important mediators of intercellular communication. EVs transport a variety of cargo, such as membrane and soluble proteins, metabolites, nucleic acids and organelles. Depending on their composition and the target cell type, they elicit a range of cellular responses in health and disease, some of which could be exploited for medical applications. However, EVs are conventionally isolated from cell culture supernatants or body fluids. This leads to heterogeneity and batch-to-batch variation of EV preparations, challenges in scaled production, limited control over EV composition and thus obstacles in understanding their mechanisms of action. A bottom-up synthetic biology approach to engineer fully synthetic EVs (synEVs) could overcome these limitations and enable clinical translation. EVs from human mesenchymal stem cells (MSCs) are of particular medical interest due to their inherent immunomodulatory and pro-regenerative functions. For example, they ameliorate symptoms of atopic dermatitis (AD) in mice. AD is the most common chronic inflammatory skin disease and is characterised by a damaged skin barrier and an over-active, Th2-dominated immune response. Due to the prevalence of AD, its high impact on life quality and currently limited treatment options with numerous side effects, new therapeutic approaches are needed. In this thesis, I have designed, assembled and tested bioinspired fully synthetic EVs for 3D AD skin tissue treatment. Towards this end, I have isolated EVs from human adipose tissue MSCs (hASCs) and have analysed their biophysical and biochemical characteristics. The analysis results were used to assemble synEVs that mimic the lipid composition, size, surface charge and proteins of hASC EVs and could include identified miRNAs as well. For efficient formation of high-purity large synEVs, I have adapted and optimised a charge-mediated method that has previously been designed for giant unilamellar vesicles. I have characterised the interactions of small and large synEVs with keratinocytes in 2D and have found internalisation, which was enhanced by a specific set of surface proteins. The designed small synEVs penetrated deeply into the 3D human organotypic skin models of AD and remarkably improved the phenotype of 3D AD tissues, particularly epidermal histology. To date, this thesis presents the closest mimicry of a specific EV type for a targeted therapeutic application and highlights the therapeutic potential of synEVs. Additionally, the demonstration of immune-cell independent and protein-mediated therapeutic effects of synEVs on skin cells could provide hypotheses about natural EVs for further research. Ongoing and planned experiments focus on optimisation of synEV composition and mechanistic analysis of the observed treatment effects, in particular whole-transcriptome expression analysis of the tissues. In the future, translational studies in vivo or transferring the analysis and assembly pipeline to other EV types and diseases could be feasible. Overall, I hope this research will contribute to the future development of synEV-based therapeutics as well as an improved understanding of natural EV biology.