Extracellular-vesicle-based cell-to-cell communication is conserved across all kingdoms of life. There is compelling evidence that extracellular vesicles are involved in major (patho)physiological processes, including cellular homoeostasis, infection propagation, cancer development and cardiovascular diseases. Various studies suggest that extracellular vesicles have several advantages over conventional synthetic carriers, opening new frontiers for modern drug delivery. Despite extensive research, clinical translation of extracellular-vesicle-based therapies remains challenging. Here, we discuss the uniqueness of extracellular vesicles along with critical design and development steps required to utilize their full potential as drug carriers, including loading methods, in-depth characterization and large-scale manufacturing. We compare the prospects of extracellular vesicles with those of the well established liposomes and provide guidelines to direct the process of developing vesicle-based drug delivery systems. In this Review the authors discuss the biological role of extracellular vesicles and how they can be applied as drug carriers, focusing on the current state of their manufacturing and existing challenges.

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Extracellular vesicles as a next-generation drug delivery platform.

Cancer-derived extracellular vesicles (EVs) are regarded as having promising potential to be used as therapeutics and disease biomarkers. Mechanistically, EVs have been shown to function in most, if not all, steps of cancer progression. Cancer EVs, including small EVs (sEVs), contain unique biomolecular cargo, consisting of protein, nucleic acid and lipids. Through progress in the identification of this specific cargo, cancer biomarkers have been identified and developed, opening up novel and interesting opportunities for cancer diagnosis and prognosis. Intriguingly, we still lack a comprehensive understanding of the cancer-specific pathways that govern EV biogenesis in cancer cells. Filling this knowledge gap will rapidly improve cancer EV biomarkers, as it will also allow discrimination of the procancer and anticancer actions of those EVs. Even more promising is uncovering therapeutically targetable, tumour-specific EV pathways and content, which will generate novel classes of cancer therapies. This Review highlights the progress the cancer sEV field has made in the areas of biomarker discovery and validation as well as sEV-based therapeutics, highlights the challenges we are facing and identifies gaps in our knowledge, which currently prevent us from developing the full potential of sEVs in cancer diagnostic and therapy.

The evolving translational potential of small extracellular vesicles in cancer.

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these. Extracellular vesicles (EVs) mediate cell–cell communication in physiology and pathology but many questions remain about the mechanisms governing their delivery to recipient cells. This Expert Recommendation article highlights areas of progress and challenges in establishing the importance of EV-mediated communication in vivo. 

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Challenges and directions in studying cell–cell communication by extracellular vesicles

https://orca.cardiff.ac.uk/id/eprint/149367/1/CLAYTON%2C%20ALED%20-%20Challenges%20and%20directions%20in%20studying%20cell%3Fcell%20communication%20by%20extracellular%20vesicles.pdf

Extracellular vesicles (EVs) are biocompatible, nano-sized secreted vesicles containing many types of biomolecules, including proteins, RNAs, DNAs, lipids, and metabolites. Their low immunogenicity and ability to functionally modify recipient cells by transferring diverse bioactive constituents make them an excellent candidate for a next-generation drug delivery system. Here, the recent advances in EV biology and emerging strategies of EV bioengineering are summarized, and the prospects for clinical translation of bioengineered EVs and the challenges to be overcome are discussed.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/advs.202003505

Shedding Light on Extracellular Vesicle Biogenesis and Bioengineering

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications.

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RNA delivery by extracellular vesicles in mammalian cells and its applications.

https://doi.org/10.1016/j.celrep.2022.110651

Uptake, functionality, and re-release of extracellular vesicle-encapsulated cargo

https://doi.org/10.1016/j.biomaterials.2022.121366

Using genetically modified extracellular vesicles as a non-invasive strategy to evaluate brain-specific cargo

Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise extracellular vesicles, virus-like particles, and lentiviral vectors. Here, we introduce a non-enzymatic assembly of purified BVs, supercharged proteins, and plasmid DNA called pDNA-scBVs. This multicomponent vehicle results from the interaction of negative sugar moieties on BVs and supercharged proteins that contain positively charged amino acids on their surface to enhance their affinity for pDNA. pDNA-scBVs were demonstrated to mediate floxed reporter activation in culture by delivering a Cre transgene. We introduced pDNA-scBVs containing both a CRE-encoding plasmid and a BV-packaged floxed reporter into the brains of Ai9 mice. Successful delivery of both payloads by pDNA-scBVs was confirmed with reporter signal in the striatal brain region. Overall, we developed a more efficient method to load isolated BVs with cargo that functionally modified recipient cells. Augmenting the natural properties of BVs opens avenues for adoptive extracellular interventions using therapeutic loaded cargo. 

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Exogenous loading of extracellular vesicles, virus-like particles, and lentiviral vectors with supercharged proteins

Aberrant activation of the MET receptor in cancer is sustained by genetic alterations or, more frequently, by transcriptional upregulations. A fraction of MET-amplified or mutated tumors are sensible to MET targeting agents, but their responsiveness is typically short-lasting, as secondary resistance eventually occurs. Since in the absence of genetic alterations MET is usually not a tumor driver, MET overexpressing tumors are not/poorly responsive to MET targeted therapies. Consequently, the vast majority of tumors exhibiting MET activation still represent an unmet medical need.Here we propose an immunotherapy strategy based on T lymphocytes expressing a Chimeric Antigen Receptor (CAR) targeting MET overexpressing tumors of different histotypes. We engineered two different MET-CAR constructs and tested MET-CAR-T cell cytotoxic activity against different MET overexpressing models, including tumor cell lines, primary cancer cells, organoids, and xenografts in immune-deficient mice.We proved that MET-CAR-T exerted a specific cytotoxic activity against MET expressing cells. Cell killing was proportional to the level of MET expressed on the cell surface. While CAR-T cytotoxicity was minimal versus cells carrying MET at physiological levels, essentially sparing normal cells, the activity versus MET overexpressing tumors was robust, significantly controlling tumor cell growth in vitro and in vivo. Notably, MET-CAR-T cells were also able to brake acquired resistance to MET targeting agents in MET amplified cancer cells carrying secondary mutations in downstream signal transducers.We set and validated at the pre-clinical level a MET-CAR immunotherapy strategy potentially beneficial for cancers not eligible for MET targeted therapy with inhibitory molecules, including those exhibiting primary or secondary resistance. 

/pdf/efficacy-of-car-t-immunotherapy-in-met-overexpressing-tumors-febgbc06.pdf

Stefano Ughetto

Papers

RNA delivery by extracellular vesicles in mammalian cells and its applications.

Uptake, functionality, and re-release of extracellular vesicle-encapsulated cargo

Using genetically modified extracellular vesicles as a non-invasive strategy to evaluate brain-specific cargo

Exogenous loading of extracellular vesicles, virus-like particles, and lentiviral vectors with supercharged proteins

Efficacy of CAR-T immunotherapy in MET overexpressing tumors not eligible for anti-MET targeted therapy