Therapeutic and diagnostic applications of extracellular vesicles

Abstract

Extracellular 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