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.

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.

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.

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.