The use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications.

Exosomes are a subset of extracellular vesicles essential for cell-cell communication in health and disease with the ability to transport nucleic acids, functional proteins and other metabolites. Their clinical use as diagnostic biomarkers and therapeutic carriers has become a major field of research over recent years, generating rapidly expanding scientific interest and financial investment. Their reduced immunogenicity compared to liposomes or viral vectors and their ability to cross major physiological barriers like the blood-brain barrier make them an appealing and innovative option as biomarkers and therapeutic agents. Here, we review the latest clinical developments of exosome biotechnology for diagnostic and therapeutic purposes, including the most recent COVID-19-related exosome-based clinical trials. We present current exosome engineering strategies for optimal clinical safety and efficacy, and assess the technology developed for good manufacturing practice compliant scaling up and storage approaches along with their limitations in pharmaceutical industry.

Argininosuccinate lyase (ASL) is a key enzyme integral to the hepatic urea cycle which is required for ammonia detoxification, and the citrulline-nitric oxide (NO) cycle for NO production. ASL deficient patients present with argininosuccinic aciduria (ASA), an inherited metabolic disease with hyperammonaemia and a chronic systemic phenotype with neurocognitive impairment and chronic liver disease. ASL deficiency as an inherited model of systemic NO deficiency, shows enhanced nitrosative and oxidative stress. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics andin vivopositron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-L-glutamate ([18F]FSPG). Upregulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways.hASLmRNA encapsulated in lipid nanoparticles corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis and glutathione metabolism and ameliorating chronic liver disease. We further present [18F]FSPG PET as a novel non-invasive diagnostic tool to assess liver disease and therapeutic efficacy in ASA. These findings support clinical translation of mRNA therapy for ASA.

In academic research and the pharmaceutical industry, in vitro single cell line cultures and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have limitations, with incomplete reproduction of pathophysiological characteristics and absence of 3-dimensional architecture with cell lines or the use of live animals brings ethical considerations, limiting the experimental scale and design. The use of precision-cut tissue slices can bridge the gap between these mainstream models as this technique combines the advantages of studying all cell sub-types whilst preserving the tissue-matrix architecture, thereby closely mimicking a mini-organ. Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers. We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases.

Recently approved adeno-associated viral (AAV) vectors for liver monogenic diseases hemophilia A and B are exemplifying the success of liver-directed viral gene therapy. In parallel, additional strategies are rapidly emerging to overcome some inherent AAV limitations, such as non-persistence of episomal transgene in rapidly growing liver and immune response. Integrating lentiviral vectors and non-viral lipid nanoparticles encapsulating mRNA (LNP-mRNA) are rapidly being developed, currently at preclinical and clinical stages respectively. Macrophages are first effector cells of the innate immune response triggered by gene therapy vectors. Macrophage uptake and activation following administration of viral gene therapy and LNPs has been reported. In this study, we assessed the biodistribution of AAV, lentiviral and LNP-mRNA gene therapy following inhibition of tissue macrophages by clodronate liposomes in neonatal and juvenile mice. Juvenile clodronate-treated mice showed significant increase of lentiviral-transduced hepatocytes, and increasing trend of transduction was shown in neonatally-injected mice. In contrast, AAV- and LNP-mRNA-treated neonatal and juvenile animals did not show significant increase of liver biodistribution following clodronate administration. These findings will have translational application for liver-targeting gene therapy programmes.