Edible Plant-Derived Extracellular Vesicles for Oral mRNA Vaccine Delivery.

Nucleic acid delivery through extracellular vesicles (EVs) is a well-preserved evolutionary mechanism in all life kingdoms including eukaryotes, prokaryotes, and plants. EVs naturally allow horizontal transfer of native as well as exogenous functional mRNAs, which once incorporated in EVs are protected from enzymatic degradation. This observation has prompted researchers to investigate whether EVs from different sources, including plants, could be used for vaccine delivery. Several studies using human or bacterial EVs expressing mRNA or recombinant SARS-CoV-2 proteins showed induction of a humoral and cell mediated immune response. Moreover, EV-based vaccines presenting the natural configuration of viral antigens have demonstrated advantages in conferring long-lasting immunization and lower toxicity than synthetic nanoparticles. Edible plant-derived EVs were shown to be an alternative to human EVs for vaccine delivery, especially via oral administration. EVs obtained from orange juice (oEVs) loaded with SARS-CoV-2 mRNAs protected their cargo from enzymatic degradation, were stable at room temperature for one year, and were able to trigger a SARS-CoV-2 immune response in mice. Lyophilized oEVs containing the S1 mRNA administered to rats via gavage induced a specific humoral immune response with generation of blocking antibodies, including IgA and Th1 lymphocyte activation. In conclusion, mRNA-containing oEVs could be used for developing new oral vaccines due to optimal mucosal absorption, resistance to stress conditions, and ability to stimulate a humoral and cellular immune response.

Oral Delivery of mRNA Vaccine by Plant-Derived Extracellular Vesicle Carriers.

mRNA-based vaccines were effective in contrasting SARS-CoV-2 infection. However, they presented several limitations of storage and supply chain, and their parenteral administration elicited a limited mucosal IgA immune response. Extracellular vesicles (EVs) have been recognized as a mechanism of cell-to-cell communication well-preserved in all life kingdoms, including plants. Their membrane confers protection from enzyme degradation to encapsulated nucleic acids favoring their transfer between cells. In the present study, EVs derived from the juice of an edible plant (Citrus sinensis) (oEVs) were investigated as carriers of an orally administered mRNA vaccine coding for the S1 protein subunit of SARS-CoV-2 with gastro-resistant oral capsule formulation. The mRNA loaded into oEVs was protected and was stable at room temperature for one year after lyophilization and encapsulation. Rats immunized via gavage administration developed a humoral immune response with the production of specific IgM, IgG, and IgA, which represent the first mucosal barrier in the adaptive immune response. The vaccination also triggered the generation of blocking antibodies and specific lymphocyte activation. In conclusion, the formulation of lyophilized mRNA-containing oEVs represents an efficient delivery strategy for oral vaccines due to their stability at room temperature, optimal mucosal absorption, and the ability to trigger an immune response.

Plant-Derived Extracellular Vesicles as a Delivery Platform for RNA-Based Vaccine: Feasibility Study of an Oral and Intranasal SARS-CoV-2 Vaccine.

Plant-derived extracellular vesicles (EVs) may represent a platform for the delivery of RNA-based vaccines, exploiting their natural membrane envelope to protect and deliver nucleic acids. Here, EVs extracted from orange (Citrus sinensis) juice (oEVs) were investigated as carriers for oral and intranasal SARS-CoV-2 mRNA vaccine. oEVs were efficiently loaded with different mRNA molecules (coding N, subunit 1 and full S proteins) and the mRNA was protected from degrading stress (including RNase and simulated gastric fluid), delivered to target cells and translated into protein. APC cells stimulated with oEVs loaded with mRNAs induced T lymphocyte activation in vitro. The immunization of mice with oEVs loaded with S1 mRNA via different routes of administration including intramuscular, oral and intranasal stimulated a humoral immune response with production of specific IgM and IgG blocking antibodies and a T cell immune response, as suggested by IFN-γ production by spleen lymphocytes stimulated with S peptide. Oral and intranasal administration also triggered the production of specific IgA, the mucosal barrier in the adaptive immune response. In conclusion, plant-derived EVs represent a useful platform for mRNA-based vaccines administered not only parentally but also orally and intranasally.

First pilot case-control interventional study using autologous extracellular vesicles to treat chronic venous ulcers unresponsive to conventional treatments.

Current therapeutic approaches for chronic venous ulcers (CVUs) still require evidence of effectiveness. Diverse sources of extracellular vesicles (EVs) have been proposed for tissue regeneration, however the lack of potency tests, to predict in-vivo effectiveness, and a reliable scalability have delayed their clinical application. This study aimed to investigate whether autologous serum-derived EVs (s-EVs), recovered from patients with CVUs, may be a proper therapeutic approach to improve the healing process. A pilot case-control interventional study (CS2/1095/0090491) has been designed and s-EVs recovered from patients. Patient eligibility included two or more distinct chronic lesions in the same limb with 11 months as median persistence of active ulcer before enrollment. Patients were treated three times a week, for 2 weeks. Qualitative CVU analysis demonstrated that s-EVs-treated lesions displayed a higher percentage of granulation tissue compared to the control group (Sham) (s-EVs 3 out of 5: 75-100 % vs Sham: none), further confirmed at day 30. s-EVs-treated lesions also displayed higher sloughy tissue reduction at the end of treatment even increased at day 30. Additionally, s-EV treatment led to a median surface reduction of 151 mm2 compared to 84 mm2 in the Sham group, difference even more evident at day 30 (s-EVs 385 mm2vs Sham 106 mm2p = 0.004). Consistent with the enrichment of transforming growth factor-ß1 in s-EVs, histological analyses showed a regenerative tissue with an increase in microvascular proliferation areas. This study first demonstrates the clinical effectiveness of autologous s-EVs in promoting the healing process of CVUs unresponsive to conventional treatments.

Stroma-derived miR-214 coordinates tumor dissemination.

BACKGROUND: Tumor progression is based on a close interaction between cancer cells and Tumor MicroEnvironment (TME). Here, we focus on the role that Cancer Associated Fibroblasts (CAFs), Mesenchymal Stem Cells (MSCs) and microRNAs (miRs) play in breast cancer and melanoma malignancy. METHODS: We used public databases to investigate miR-214 expression in the stroma compartment of primary human samples and evaluated tumor formation and dissemination following tumor cell injections in miR-214 overexpressing (miR-214over) and knock out (miR-214ko) mice. In addition, we dissected the impact of Conditioned Medium (CM) or Extracellular Vesicles (EVs) derived from miR-214-rich or depleted stroma cells on cell metastatic traits. RESULTS: We evidence that the expression of miR-214 in human cancer or metastasis samples mostly correlates with stroma components and, in particular, with CAFs and MSCs. We present data revealing that the injection of tumor cells in miR-214over mice leads to increased extravasation and metastasis formation. In line, treatment of cancer cells with CM or EVs derived from miR-214-enriched stroma cells potentiate cancer cell migration/invasion in vitro. Conversely, dissemination from tumors grown in miR-214ko mice is impaired and metastatic traits significantly decreased when CM or EVs from miR-214-depleted stroma cells are used to treat cells in culture. Instead, extravasation and metastasis formation are fully re-established when miR-214ko mice are pretreated with miR-214-rich EVs of stroma origin. Mechanistically, we also show that tumor cells are able to induce miR-214 production in stroma cells, following the activation of IL-6/STAT3 signaling, which is then released via EVs subsequently up-taken by cancer cells. Here, a miR-214-dependent pro-metastatic program becomes activated. CONCLUSIONS: Our findings highlight the relevance of stroma-derived miR-214 and its release in EVs for tumor dissemination, which paves the way for miR-214-based therapeutic interventions targeting not only tumor cells but also the TME.

Optimized Protocol for Plasma-Derived Extracellular Vesicles Loading with Synthetic miRNA Mimic Using Electroporation.

Extracellular vesicles (EVs) are a population of particles naturally released by cells to transport biological messages, including nucleic acids. Thus, EVs represent an ideal vehicle to deliver therapeutic miRNAs. The current challenge is the development of efficient protocols to load EVs with exogenous miRNAs. Human plasma is an abundant source of EVs which can be manipulated for therapeutic applications. Despite numerous techniques are currently available to load EVs, all of them present issues which limit their clinical application. Among all, electroporation was shown to be superior to other protocols and to efficiently load plasma-derived EVs with miRNAs. However, also the electroporation procedure presents issues that can reduce the miRNA delivery. In this chapter, we describe a protocol to isolate EVs from human plasma, to load synthetic miRNA mimics using electroporation, to evaluate EV integrity and miRNA loading into EVs. Finally, the analysis of EV functionality allows to investigate the ability of engineered EVs to transfer the miRNAs to target cells and to exploit their biological effects.

Characterization of Circulating Extracellular Vesicle Surface Antigens in Patients With Primary Aldosteronism.

[Figure: see text].

Serum Derived Extracellular Vesicles Mediated Delivery of Synthetic miRNAs in Human Endothelial Cells.

Extracellular vesicles (EVs) have emerged in the last decades as a cell-to-cell communication mechanism. One of their mechanism of action is the direct delivery of their cargo, composed of bioactive molecules to target cells. Different methods (direct electroporation, cell transfection, chemical transfection) were developed to vehicle therapeutic molecules through EVs. However, most of these techniques presented some limitations such as EV disruption and aggregation. In the present study, we demonstrated that a direct temperature-controlled co-incubation of EVs with defined miRNAs is a stable method to deliver information to target cells without affecting EV constitutive content. We chose serum as an easy and abundant source of EVs applicable to autologous treatment after EV modification. Exogenous cel-miR-39 loaded on serum EVs (SEVs) was taken up by human endothelial cells, demonstrating an adequate miRNA loading efficacy based on the co-incubation method. Moreover, SEVs co-incubation with the angiomiRNA-126 (miR-126) enhanced their angiogenic properties in vitro and in vivo by increasing the capacity to induce capillary-like structure formation of human endothelial cells. MiR-126 loaded EVs were also shown to stimulate mouse endothelial cells to invade Matrigel plugs and create more vessels with respect to the EV naive counterpart. When SEVs were loaded with miR-19b, an anti-angiogenic miRNA, they were able to reduce Vascular endothelial growth factors (VEGF) pro-angiogenic capacity, supporting the selective biological effect mediated by the carried miRNA. Lastly, we identified Annexin A2 (ANXA2) as one of the molecules involved in the exogenous RNA binding to serum EV surface, favoring miRNA delivery to target endothelial cells for potential therapeutic application.

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes.

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.

Targeting IL-3Rα on tumor-derived endothelial cells blunts metastatic spread of triple-negative breast cancer via extracellular vesicle reprogramming.

The lack of approved targeted therapies highlights the need for new treatments for triple-negative breast cancer (TNBC) patients. Interleukin-3 (IL-3) acts as an autocrine factor for tumor-endothelial cells (TEC), and exerts pro-angiogenic paracrine action via extracellular vesicles (EVs). IL-3Rα blockade on TEC changes TEC-EV (anti-IL-3R-EV) microRNA (miR) content and promotes the regression of established vessels. As TEC is the doorway for "drug" entry into tumors, we aimed to assess whether IL-3R blockade on TEC impacts tumor progression via its unique EV cargo. First, the expression of IL-3Rα was evaluated in 27 human TNBC samples. It was noticed that, besides TEC and inflammatory cells, tumor cells from 55.5% of the human TNBC samples expressed IL-3Rα. Using human TNBC cell lines for in vitro studies, we found that, unlike native TEC-EVs (nEVs), anti-IL-3R-EVs increase apoptosis and reduced cell viability and migration. In vivo, anti-IL-3R-EV treatment induced vessel regression in established tumors formed of MDA-MB-231 cells, decreased Vimentin, ß-catenin, and TWIST1 expression, almost abolished liver and lung metastases from primary tumors, and reduced lung metastasis generated via the intravenous injection of MDA-MB-231 cells. nEVs depleted of miR-24-3p (antago-miR-24-3p-EVs) were effective as anti-IL-3R-EVs in downregulating TWIST1 and reducing metastatic lesions in vivo. Consistent with network analyses of miR-24-3p gene targeting, anti-IL-3R-EVs and antago-miR-24-3p-EVs upregulate SPRY2 in MDA-MB-231 cells. Finally, SPRY2 silencing prevented anti-IL-3R-EV and antago-miR-24-3p-EV-mediated apoptotic cues.Overall, these data provide the first evidence that IL-3Rα is highly expressed in TNBC cells, TEC, and inflammatory cells, and that IL-3Rα blockade on TEC impacts tumor progression.

Extracellular vesicles in onco-nephrology.

Extracellular vesicles (EVs) are important mediators of intercellular communication in cancer and in normal tissues. EVs transfer biologically active molecules from the cell of origin to recipient cells. This review summarizes the studies on EVs derived from renal cell carcinoma and from a subpopulation of CD105-positive renal cancer stem cells. While EVs from renal cell carcinoma show mild biological activity, EVs from renal cancer stem cells enhance tumor angiogenesis and metastasis formation. The effect is probably due to the transfer of proangiogenic RNA cargo to endothelial cells, which acquire an activated angiogenic phenotype. In vivo, treatment with EVs favors the formation of a premetastatic niche in the lungs. Moreover, EVs derived from renal cancer stem cells modify gene expression in mesenchymal stromal cells, enhancing the expression of genes involved in matrix remodeling, cell migration, and tumor growth. Mesenchymal stromal cells preconditioned with tumor EVs and then coinjected in vivo with renal cancer cells support tumor growth and vessel formation. Finally, tumor EVs promote tumor immune escape by inhibiting the differentiation process of dendritic cells and the activation of T cells. Thus, tumor-derived EVs act on the microenvironment favoring tumor aggressiveness, may contribute to angiogenesis through both direct and indirect mechanisms and are involved in tumor immune escape.

Improved Loading of Plasma-Derived Extracellular Vesicles to Encapsulate Antitumor miRNAs.

Extracellular vesicles (EVs) carry various molecules involved in intercellular communication and have raised great interest as drug delivery systems. Several engineering methods have been investigated for vesicle loading. Here, we studied the electroporation of EVs isolated from plasma to load antitumor microRNAs (miRNAs). First, we optimized the transfection protocol using miRNA cel-39 by evaluating different parameters (voltage and pulse) for their effect on vesicle morphology, loading capacity, and miRNA transfer to target cells. When compared with direct incubation of EVs with miRNA, mild electroporation allowed more efficient loading and better protection of miRNA from RNase degradation. Moreover, electroporation preserved the naive vesicle cargo, including RNAs and proteins, and their ability to be taken up by target cells, supporting the absence of vesicle damage. EVs engineered with antitumor miRNAs (miR-31 and miR-451a) successfully promoted apoptosis of the HepG2 hepatocellular carcinoma cell line, silencing target genes involved in anti-apoptotic pathways. Our findings indicate an efficient and functional miRNA encapsulation in plasma-derived EVs following an electroporation protocol that preserves EV integrity.

Noncoding RNAs Carried by Extracellular Vesicles in Endocrine Diseases.

RNA molecules are essential and fine regulators of important biological processes. Their role is well documented also in the endocrine system, both in physiological and pathological conditions. Increasing interest is arising about the function and the importance of noncoding RNAs shuttled by extracellular vesicles (EVs). In fact, EV membrane protects nucleic acids from enzyme degradation. Nowadays, the research on EVs and their cargoes, as well as their biological functions, faces the lack of standardization in EV purification. Here, the main techniques for EV isolation are discussed and compared for their advantages and vulnerabilities. Despite the possible discrepancy due to methodological variability, EVs and their RNA content are reported to be key mediators of intercellular communication in pathologies of main endocrine organs, including the pancreas, thyroid, and reproductive system. In particular, the present work describes the role of RNAs contained in EVs in pathogenesis and progression of several metabolic dysfunctions, including obesity and diabetes, and their related manifestations. Their importance in the establishment and progression of thyroid autoimmunity disorders and complicated pregnancy is also discussed. Preliminary studies highlight the attractive possibility to use RNAs contained in EVs as biomarkers suggesting their exploitation for new diagnostic approaches in endocrinology.

Extracellular Vesicles in Renal Pathophysiology.

Extracellular vesicles are a heterogeneous population of microparticles released by virtually all living cells which have been recently widely investigated in different biological fields. They are typically composed of two primary types (exosomes and microvesicles) and are recently commanding increasing attention as mediators of cellular signaling. Indeed, these vesicles can affect recipient cells by carrying and delivering complex cargos of biomolecules (including proteins, lipids and nucleic acids), protected from enzymatic degradation in the environment. Their importance has been demonstrated in the pathophysiology of several organs, in particular in kidney, where different cell types secrete extracellular vesicles that mediate their communication with downstream urinary tract cells. Over the past few years, evidence has been shown that vesicles participate in kidney development and normal physiology. Moreover, EVs are widely demonstrated to be implicated in cellular signaling during renal regenerative and pathological processes. Although many EV mechanisms are still poorly understood, in particular in kidney, the discovery of their role could help to shed light on renal biological processes which are so far elusive. Lastly, extracellular vesicles secreted by renal cells gather in urine, thus becoming a great resource for disease or recovery markers and a promising non-invasive diagnostic instrument for renal disease. In the present review, we discuss the most recent findings on the role of extracellular vesicles in renal physiopathology and their potential implication in diagnosis and therapy.

Exosome and Microvesicle-Enriched Fractions Isolated from Mesenchymal Stem Cells by Gradient Separation Showed Different Molecular Signatures and Functions on Renal Tubular Epithelial Cells.

Several studies have suggested that extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) may mediate MSC paracrine action on kidney regeneration. This activity has been, at least in part, ascribed to the transfer of proteins/transcription factors and different RNA species. Information on the RNA/protein content of different MSC EV subpopulations and the correlation with their biological activity is currently incomplete. The aim of this study was to evaluate the molecular composition and the functional properties on renal target cells of MSC EV sub-populations separated by gradient floatation. The results demonstrated heterogeneity in quantity and composition of MSC EVs. Two peaks of diameter were observed (90-110 and 170-190 nm). The distribution of exosomal markers and miRNAs evaluated in the twelve gradient fractions showed an enrichment in fractions with a flotation density of 1.08-1.14 g/mL. Based on this observation, we evaluated the biological activity on renal cell proliferation and apoptosis resistance of low (CF1), medium (CF2) and high (CF3) floatation density fractions. EVs derived from all fractions, were internalized by renal cells, CF1 and CF2 but not CF3 fraction stimulated significant cell proliferation. CF2 also inhibited apoptosis on renal tubular cells submitted to ischemia-reperfusion injury. Comparative miRNomic and proteomic profiles reveal a cluster of miRNAs and proteins common to all three fractions and an enrichment of selected molecules related to renal regeneration in CF2 fraction. In conclusion, the CF2 fraction enriched in exosomal markers was the most active on renal tubular cell proliferation and protection from apoptosis.

AKI Recovery Induced by Mesenchymal Stromal Cell-Derived Extracellular Vesicles Carrying MicroRNAs.

Phenotypic changes induced by extracellular vesicles have been implicated in mesenchymal stromal cell-promoted recovery of AKI. MicroRNAs are potential candidates for cell reprogramming toward a proregenerative phenotype. The aim of this study was to evaluate whether microRNA deregulation inhibits the regenerative potential of mesenchymal stromal cells and derived extracellular vesicles in a model of glycerol-induced AKI in severe combined immunodeficient mice. We generated mesenchymal stromal cells depleted of Drosha to alter microRNA expression. Drosha-knockdown cells produced extracellular vesicles that did not differ from those of wild-type cells in quantity, surface molecule expression, and internalization within renal tubular epithelial cells. However, these vesicles showed global downregulation of microRNAs. Whereas wild-type mesenchymal stromal cells and derived vesicles administered intravenously induced morphologic and functional recovery in AKI, the Drosha-knockdown counterparts were ineffective. RNA sequencing analysis showed that kidney genes deregulated after injury were restored by treatment with mesenchymal stromal cells and derived vesicles but not with Drosha-knockdown cells and vesicles. Gene ontology analysis showed in AKI an association of downregulated genes with fatty acid metabolism and upregulated genes with inflammation, matrix-receptor interaction, and cell adhesion molecules. These alterations reverted after treatment with wild-type mesenchymal stromal cells and extracellular vesicles but not after treatment with the Drosha-knockdown counterparts. In conclusion, microRNA depletion in mesenchymal stromal cells and extracellular vesicles significantly reduced their intrinsic regenerative potential in AKI, suggesting a critical role of microRNAs in recovery after AKI.