Browsing by Subject "Exosomes"
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Item Open Access 3D-MSCs A151 ODN-loaded exosomes are immunomodulatory and reveal a proteomic cargo that sustains wound resolution(Elsevier B.V., 2022-11) Camões, Sérgio P.; Bulut, Özlem; Yazar, Volkan; Gaspar, Maria M.; Simões, Sandra; Ferreira, Rita; Vitorino, Rui; Santos, Jorge M.; Gürsel, İhsan; Miranda, Joana P.Introduction: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. Objectives: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. Methods: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. Results: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-β, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-β, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. Conclusion: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines. © 2022Item Open Access The development of prophylactic and therapeutic vaccine using cell derived extracellular vesicles(Bilkent University, 2022-08) Yıldırım, MuzafferThe primary aim of this thesis is to extend the breadth of in vivo application of externally loaded cell-line derived and tumor derived exosomes as a prophylactic and therapeutic carrier against cancer treatment. Exosomes with a size between 30 to 150 nm are small extracellular vesicles secreted by all types of mammalian cells. They mediate a novel mode of intercellular communication through their bioactive cargos such as lipids, nucleic acids, metabolites, and proteins, which can be delivered to the target cells. Exosomes have successfully served as immunotherapeutic nanocarriers in cancer treatment using their natural delivery capabilities. Furthermore, they are attractive as a delivery system because of their stability in circulation, biocompatibility, and low toxicity. In the first part of this thesis, we used exosomes as a nanocarrier system to develop cancer vaccines in a therapeutic murine melanoma cancer treatment. We show that lyophilization of exosomes together with the CpG ODN, model antigen OVA and lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution is successfully accomplished. We analyzed the effect of the lyophilization on a cell line-derived exosomes and we characterized the exosomes by using bead-based technique via flow cytometry, qNano, Scaning electron microscopy, and western blotting. We showed that lyophilization does not harm exosomes’ vesicular integrity and fundamental biological features. Furthermore, we tested the biodistribution and activating capacity of encapsulated exosomes in mouse PEC, mesenteric lymph node, and spleen cells. We found out that loaded exosomes are mostly taken up by antigen-presenting cells. Also, we showed that loading CpG ODN into exosomes significantly improves APC activation markers in macrophages, B cells, and DCs and induced significantly higher IFNγ production from mouse mLN and splenocytes. Finally, we tested the therapeutic utility of the CpG ODN, OVA and αGC encapsulating exosome in the B16F10-OVA melanoma tumor-bearing mice. We found out that therapeutic vaccination with triple (CpG ODN, OVA, and αGC) ligand encapsulating exosomes suppressed the progression of established melanoma tumors in mice. Moreover, our triple ligand loaded exosomes triggered Th-1 biased anti-IgG OVA immunity and converted immune cells in tumor microenvironment to the tumor-suppressing phenotype. In the second part of this thesis, we used tumor-derived exosomes (TEXs) as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells, TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). We showed that engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic breast cancer model, we found out that administration of 4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regressed tumor growth in 4T1 tumor-bearing mice. As a result, this work implicated that an exosome based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. The last part of this thesis, we studied the therapeutic potential of cell line-derived exosomes loaded with superparamagnetic iron oxide nanoparticles(SPION) and immunostimulatory ligands. We showed that loading SPION enhanced the in vitro delivery of exosomes within immune cells. Also, we found out that spleen cells incubated with exosomes encapsulating with SPION and CpG ODN induced significantly higher levels of IL-12 and IFNγ. Finally, we tested our exosomal vaccine candidate in human hepatocellular carcinoma tumor model in athymic mice. We showed that TLR3 and TLR9 ligands encapsulated with SPION loaded exosomes induced pronounced innate immune activation and regressed tumors and improve survival rate of treated mice.Item Open Access Efficient production and enhanced tumor delivery of engineered extracellular vesicles(Elsevier Ltd, 2016) Watson, D. C.; Bayik D.; Srivatsan, A.; Bergamaschi, C.; Valentin, A.; Niu, G.; Bear, J.; Monninger, M.; Sun, M.; Morales-Kastresana, A.; Jones, J. C.; Felber, B. K.; Chen, X.; Gurse,l I.; Pavlakis, G. N.Extracellular vesicles (EV), including exosomes and microvesicles, are nano-sized intercellular communication vehicles that participate in a multitude of physiological processes. Due to their biological properties, they are also promising candidates for the systemic delivery of therapeutic compounds, such as cytokines, chemotherapeutic drugs, siRNAs and viral vectors. However, low EV production yield and rapid clearance of administered EV by liver macrophages limit their potential use as therapeutic vehicles. We have used a hollow-fiber bioreactor for the efficient production of bioactive EV bearing the heterodimeric cytokine complex Interleukin-15:Interleukin-15 receptor alpha. Bioreactor culture yielded ∼40-fold more EV per mL conditioned medium, as compared to conventional cell culture. Biophysical analysis and comparative proteomics suggested a more diverse population of EV in the bioreactor preparations, while serum protein contaminants were detectable only in conventional culture EV preparations. We also identified the Scavenger Receptor Class A family (SR-A) as a novel monocyte/macrophage uptake receptor for EV. In vivo blockade of SR-A with dextran sulfate dramatically decreased EV liver clearance in mice, while enhancing tumor accumulation. These findings facilitate development of EV therapeutic methods. © 2016Item Open Access Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine(American Chemical Society, 2016-03) Fais, S.; O'Driscoll, L.; Borras, F. E.; Buzas, E.; Camussi, G.; Cappello, F.; Carvalho, J.; Cordeiro Da Silva, A.; Del Portillo, H.; El Andaloussi, S.; Ficko Trček, T.; Furlan, R.; Hendrix, A.; Gursel, I.; Kralj-Iglic, V.; Kaeffer, B.; Kosanovic, M.; Lekka, M. E.; Lipps, G.; Logozzi, M.; Marcilla, A.; Sammar, M.; Llorente, A.; Nazarenko, I.; Oliveira, C.; Pocsfalvi, G.; Rajendran, L.; Raposo, G.; Rohde, E.; Siljander, P.; Van, N. G.; Vasconcelos, M. H.; Yáñez-Mó, M.; Yliperttula, M. L.; Zarovni, N.; Zavec, A. B.; Giebel, B.Recent research has demonstrated that all body fluids assessed contain substantial amounts of vesicles that range in size from 30 to 1000 nm and that are surrounded by phospholipid membranes containing different membrane microdomains such as lipid rafts and caveolae. The most prominent representatives of these so-called extracellular vesicles (EVs) are nanosized exosomes (70-150 nm), which are derivatives of the endosomal system, and microvesicles (100-1000 nm), which are produced by outward budding of the plasma membrane. Nanosized EVs are released by almost all cell types and mediate targeted intercellular communication under physiological and pathophysiological conditions. Containing cell-type-specific signatures, EVs have been proposed as biomarkers in a variety of diseases. Furthermore, according to their physical functions, EVs of selected cell types have been used as therapeutic agents in immune therapy, vaccination trials, regenerative medicine, and drug delivery. Undoubtedly, the rapidly emerging field of basic and applied EV research will significantly influence the biomedicinal landscape in the future. In this Perspective, we, a network of European scientists from clinical, academic, and industry settings collaborating through the H2020 European Cooperation in Science and Technology (COST) program European Network on Microvesicles and Exosomes in Health and Disease (ME-HAD), demonstrate the high potential of nanosized EVs for both diagnostic and therapeutic (i.e., theranostic) areas of nanomedicine.Item Open Access Immunomodulatory effects of exosomes: promising candidates in immunotherapy(Bilkent University, 2018-08) Kılgöz, Havva ÖzgenExosomes are type of extracellular vesicles secreted from almost all cell types and carry numerous biological molecules such as nucleic acids, protein and lipids. They mediate many cellular processes including cellular communication and immune responses. Accumulating evidence suggests that these vesicles play a key role in the pathogenesis of inflammatory diseases, infectious diseases and many malignancies. The significant role of exosomes in cellular level also describe their intriguing potential in cancer therapeutics. The primary aim of this thesis is to identify the immunomodulatory roles of distinct exosome species and extend the knowledge of exosome utilization in immunotherapy. The exosomes purified from i) RAW264.7 (murine macrophage-like), ii) EG7 (murine T cell lymphoma), and iii) HUH7 (human hepatocellular carcinoma) had distinct characteristics as well as immunomodulatory features upon murine splenocyte stimulation either alone or in combination with poly(I:C) (a TLR3 ligand), R848(a TLR7/8 ligand), and CpG ODN(a TLR9 ligand). Strikingly, these cell line-derived exosomes displayed changing internalization kinetics by immune cells. Furthermore, the involvement of exosomes in the liver disease progression in the course of Hepatitis B virus (HBV) infection, cirrhosis and hepatocellular carcinoma (HCC) was investigated. As a result of patients’ exosome stimulation assays with healthy peripheral blood mononuclear cells (PBMCs) and murine splenocytes, patient plasma-derived exosomes had inflammatory effects correlated with the severity of liver damage suggesting that these exosomes might have a pathological role in liver disease progression and/or pathogenesis. In the final part, we used a dehydration-rehydration technique enabling external loading of desired Toll-like receptors (TLR) ligand within exosome and liposome. We developed a robust therapeutic vaccine delivery system in which injection of therapeutic vaccine to tumor burden animals enhanced anti-tumor activity and prolonged survival of HCC xenografts. In summary, this approach could broaden the immunotherapeutic utility of exosomes in the clinic.Item Open Access Immunomodulatory potential of human umbilical cord tissue-derived mesenchymal stromal cell (UCX®) exosomes in combination with immunosuppressive “A151” oligodeoxynucleotide(Bilkent University, 2019-07) Bulut, ÖzlemMesenchymal stromal or stem cells (MSCs) modulate immune responses apart from their regenerative capacities. Accumulating evidence suggests that MSCs exert their paracrine effects through extracellular vesicles known as exosomes. In this study, we utilized a particular human umbilical cord tissue-derived MSC type termed as UCX®. UCX® is superior to the gold-standard bone marrow-derived MSCs in terms of immunosuppressive properties. We aimed to characterize and employ UCX® exosomes as cell-free immunosuppressive therapeutic agents. Another aim was to compare the functionalities of exosomes either isolated from 2-dimensional (2D) cultures or isolated from 3-dimensional (3D) spheroid cultures. 3D culture provides better cell-to-cell and cell-to-matrix interactions thereby mimics the in vivo environment better. A synthetic oligodeoxynucleotide called A151 ODN, which consists of 4 repeats of the mammalian telomeric TTAGGG motif, has broad immunosuppressive effects. Delivery of A151 ODN within liposomes or exosomes protects it from degradation by nucleases and improve the desired outcome. We also aimed to combine the immunomodulatory potentials of UCX® exosomes and A151 ODN through direct loading of A151 ODN into exosomes with ~95% efficiency via a dehydration-rehydration-based lyophilization method. First, we determined the binding and internalization kinetics of exosomes with different immune cells. 3D-exosomes interacted with the target cells much faster and more efficiently. Next, we investigated how UCX® exosomes influence Toll-like receptor (TLR) signaling in mouse splenocytes and bone marrow-derived macrophages (BMDMs). 3D-exosomes compared to 2D-exosomes were more potent to suppress IFNγ, IL6, IL12, and to a lesser extent TNFα production mediated by TLR1/2, TLR4, TLR7/8 and TLR9 but not by TLR3 triggering. A151 ODN-loading to either 2D- or 3D-exosomes improved the inhibition of all the above mentioned pro-inflammatory cytokines. Especially 3D-exosomes downregulated co-stimulatory molecules CD80 and CD86 along with MHC-II on BMDMs following TLR stimulation. Macrophage polarization experiments revealed that UCX® exosomes reprogram BMDMs to produce high amounts of nitric oxide and arginase-1 which are the key immunomodulatory factors induced by myeloid-derived suppressor cells (MDSCs) to inhibit T- and NK-cell activity. Besides shifting macrophages to an MDSC-like suppressive phenotype, exosomes also supported expansion of MDSC populations in vivo upon intraperitoneal injection. Next, we tested the therapeutic efficiency of UCX® exosomes with or without A151 ODN in zymosan-induced peritonitis and dextran sodium sulfate (DSS)-induced colitis models in mice. Exosomes could not alleviate zymosan-induced peritonitis which is an acute and severe inflammation. However, 3D-exosomes and A151 ODN-loaded versions of both 2D- and 3D-exosomes remarkably prevented DSS-induced colitis progression. A151 ODN itself was also therapeutic, albeit to a lesser degree. Standalone 3D-exosomes and A151 ODN-loaded exosomes prevented weight loss and colon shortening. All treatments except for 2D-exosomes could restore DSS-induced loss of T-cell numbers and cytokine-producing capacities in mesenteric lymph nodes and spleen. All treatments except for A151 ODN prevented DSS-induced macrophage accumulation in the lymph nodes. 3D-exosomes and A151 ODN-loaded versions of both exosomes normalized serum IL6 levels while only A151 ODN-loaded 3D-exosomes could impact the cytokine production capacities of macrophages. Finally, we tested the effects of UCX® exosomes with or without A151 ODN on wound healing. In vitro, 2D- and 3D-exosomes differentially upregulated the productions of wound healing-related cytokines and growth factors such as IL1α, TGFβ and VEGFα from fibroblast and keratinocyte cell lines. In vivo, in an excisional wound healing model, free or A151 ODN-loaded exosomes did not accelerate wound closure. However, they caused systemic immunosuppression at the late stages of wound healing. Systemic outcomes include reduced inflammatory capacity of macrophages and higher granulocytic MDSC numbers in spleen. A151 ODN-loaded 3D-exosomes also reduced T-cell numbers in spleen and pro-inflammatory cytokine levels in circulation. Taken together, this study revealed that 2D- but more importantly 3D-culturing of umbilical cord MSCs result in functionally different exosomes, 3D culture-derived exosomes display higher immunosuppressive potential, A151 ODN-loading into these exosomes improves immunosuppressive capacity and A151 ODN-loaded UCX® exosomes could be a valuable therapeutic agent for inflammatory and autoimmune disorders.Item Open Access Immunotherapeutic action of extracellular vesicles and effects of TLR signaling to immune dysfunction of SCI patients(Bilkent University, 2017-03) Güçlüler, GözdeThe primary aim of this thesis is to extend the breadth of in vivo application of externally loaded exosomes as prophylactic or therapeutic carriers against disease treatment. Exosomes are secreted from all cells and could be purified from all bodily fluids; however, engineering of exosomes to carry specific ligands post-purification is a daunting task. Herein, we show that lyophilization of exosomes together with the biological cargo alone or in combination of CpG ODN motifs, model protein antigen ovalbumin or lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution successfully internalizes these cargos within exosomes. Furthermore, the bioactivity of the loaded ligand(s) surpasses the unloaded free ligand activities. When tested in vivo, exosome incorporated ligand(s) proved to be significantly effective against model tumors such as E.G7 thymoma or established melanoma models. The mechanism behind this elevated immune activity is the ability of exosomes to be delivered to target cells and boost immune antigen dependent immune activation. Our in vitro findings revealed that encapsulation of CpG ODN into exosomes enhances immunostimulatory activity of CpG ODN than free form as evidenced by superior levels of cytokines like IL6, IL12 and Type-I and II interferons. This magnified immune activity might be partly due the increased APC activation observed as elevated CD86/MHCII surface marker expression. Immunization of C57/Bl6 mice with exosomal CpG ODN plus OVA induced strong Th1-biased anti-OVA response. Following thymoma induction in naive and OVAimmunized animals, >85% of exosomal vaccine treated mice cleared tumors whereas almost all naive animals were positive for tumor. This data suggests that CpG ODN encapsulation into exosomes improve immunostimulatory activity, provide better anti-OVA immunity thereby contribute effective tumor clearance in mice. A second aim of this thesis was to establish that it is feasible to load exosomes with more than two ligands. Next, invariant natural killer T (iNKT) cell ligand αGC was included within exosomes as the third element next to OVA and CpG ODN. Initial in vivo studies revealed that exosomes containing αGC were significantly more potent in inducing antigen dependent immune responses in comparison to free form of CpG ODN, OVA and αGC. In therapeutic tumor vaccine model, two exosome injections (@d: 9 and d: 15) were done to B16-OVA tumor bearing animals and tumor regression was followed. Mice that had triple exosomal ligands significantly reduced tumors compared to mice treated with non-exosomal ligands. This study confirmed that exosomes with triple ligands could be effectively control established tumor development. In this thesis, the elucidation of the involvement of extracellular vesicles (EVs) on the pathogenesis of autoimmune/autoinflammatory diseases was studied. The underlying mechanism in BD pathogenesis is still unclear. We found that one of the human cathelicidin group members, antimicrobial peptide LL37 along with EVs were elevated in active BD patients` plasmas. Strikingly, majority of plasma LL37 was associated with circulating EVs. We found that there was a strong correlation between i) LL37 level, ii) EV #/ml plasma and iii) cytokine production. In the last part of this thesis, one of the possible mechanisms of immune dysfunction contributing to severe neurological deterioration of chronic spinal cord injured (SCI) patients was unearthed. We aimed to investigate whether there is a correlation between susceptibility to infections of chronic SCI patients within the context of impaired innate recognition of pathogen associated molecular patterns (PAMPs). Our data implicated that although there was no dysfunction of B cell, or CD4+ Treg activity, but sensing TLR7 and TLR9 ligands by monocytes and pDCs were ablated in patients with SCI, leading to lower IFNγ and IP10 production along with costimulatory molecule expression, that could explain the immunological dysfunction in patient with SCI contributing to persistent complications.Item Open Access Mesenchymal stem cell derived extracellular vesicles: promising immunomodulators against autoimmune, autoinflammatory disorders and SARS-CoV-2 infection(Scientific and Technical Research Council of Turkey, 2020) Bulut, Özlem; Gürsel, İhsanDiscovery of novel and broad-acting immunomodulators is of critical importance for the prevention and treatment of disorders occurring due to overexuberant immune responseincluding SARS-CoV-2 triggered cytokine storm leading to lung pathology and mortality during the ongoing viral pandemic. Mesenchymal stem/stromal cells (MSCs), highly regarded for their regenerative capacities, also possessesremarkable immunoregulatory functions affecting all types of innate and adaptive immune cells. Owing to that, MSCs have been heavily investigated in clinic for the treatment of autoimmune and inflammatory diseases along with transplant rejection. Extensive research in the last decaderevealed that MSCs carry out most of their functions through paracrine factors which are soluble mediators and extracellular vesicles (EVs). EVs, including exosomes and microvesicles, are an efficient way of intercellular communication due to their unique ability to carry biological messages such as transcription factors, growth factors, cytokines, mRNAs and miRNAs over long distances. EVs originate through direct budding of the cell membrane or the endosomal secretion pathway and they consist of the cytosolic and membrane components of their parent cell. Therefore, they are able to mimic the characteristics of the parent cell, affecting the target cells upon binding or internalization. EVs secreted by MSCs are emerging as a cell-free alternative to MSC-based therapies. MSC EVs are being tested in preclinical and clinical settings where they exhibit exceptional immunosuppressivecapacity. They regulate the migration, proliferation, activation and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory response. Being as effective immunomodulators as their parent cells, MSC EVs are also preferable over MSC-based therapies due to their lower risk of immunogenicity, tumorigenicity and overall superior safety. In this review, we present the outcomes of preclinical and clinical studies utilizing MSC EVs as therapeutic agents for the treatment of a wide variety of immunological disorders.Item Open Access Therapeutic and diagnostic applications of extracellular vesicles(Bilkent University, 2016-03) Kahraman, TamerExtracellular vesicles (EV), consisting of exosomes and microvesicles, are secreted biological nanovesicles and assumed plethora of physiological functions ranging from transport of cargo, regulating distant cell communication, and altering immune response. Accumulating evidence suggests that extracellular vesicles may participate in disease pathogenesis of inflammatory diseases. Moreover, accumulating evidence suggests that EVs are promising nanocarrier capable of modulating immune response. This thesis aims to harness EVs in immunotherapeutic and diagnostic applications. Behçet’s Disease (BD) activity is manifested with sustained, over exuberant immune activation, yet the underlying mechanisms leading to active BD state is poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 and EVs are elevated in BD plasma. Our data suggested that majority of LL37 is associated with EVs. This association drives plasma EVs to immune cells, enhancing pathologic and sustained immune response, thereby leading to aggravating BD pathology. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. Findings of this study could be of clinical interest in the management of BD, implicating that LL37/EV association as one of the major contributors of BD pathogenesis and might be used as a diagnostic readout to stratify the severity of BD patients. EVs, more specifically exosomes, suggested as new tools for biomedical applications such as drug/vaccine carrier vesicles. However, efforts to engineer cells to express desired cargo in/on these secreted exosomes or induce physical complexation with candidate bioactive agents or even use of membrane-breaching techniques such as electroporation to load exosomes with desirable cargo showed limited in vivo performance. Here we developed a mild and simple technique enabling external loading of any type of desired bioactive molecule within exosomes at high yield. Using this approach, we exploited therapeutic potential of exosomes encapsulating CpG ODN together with a protein antigen as a vaccine cancer for preventive tumor therapy. CpG ODN loaded within exosomes displayed pronounced in-vitro activity as evidenced by up to 6-fold higher IL6 and IL12 secretion from splenocytes as well as increased IFNα secretion from pDCs. Exosomes protected CpG ODN from digestion by DNase-I up to 90%. In order to demonstrate improved in-vivo activity, exosomes co-encapsulating CpG ODN and ovalbumin were tested as a potential vaccine vector against EG-7 thymoma. Animals that received Exo(CpG ODN+OVA) vaccine led to a magnified and persistent Th1-biased antiOVA IgG responses that was sufficient to fully protect mice from EG-7 derived tumor challenge even after 24 weeks post-booster injection as opposed to free vaccine combination. Our results suggest that EVs could be of clinical interest in both prognosis and management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. In addition, our studies related with exosomes present a platform that opens a new avenue to personalized cell-free therapeutic intervention and could be developed to harbor other therapeutically important molecules ranging from plasmid to mRNA or si/miRNA for more effective therapeutic modality development in the clinic against debilitating diseases ranging from cancer to infectious diseases