Associate editor: B. RodriguezExosomes: Therapy delivery tools and biomarkers of diseases
Introduction
First described as small vesicles by which maturating sheep reticulocytes discard obsolete cellular components (Johnstone et al., 1987, Pan and Johnstone, 1983, Trams et al., 1981), exosomes and other secreted extracellular vesicles (EVs) are now considered a prominent and universal form of cell–cell communication. Fundamentally all cells in the organism release EVs that are taken up by surrounding cells or circulate in the blood and eventually are taken up by cells at a distance. EVs transport biologically active molecules including proteins and nucleic acids that regulate gene expression and cellular function in target cells. As such, EVs mediate autocrine, paracrine, and endocrine effects that can be exploited therapeutically (Andaloussi, Mager, Breakefield, & Wood, 2013). For example, mesenchymal stem cells (MSCs) and other progenitor cells used in cell therapy studies mediate cytoprotective, angiogenic, and regenerative effects that are recapitulated by the EVs they release (Baglio et al., 2012, Barile et al., 2014). This observation raises the exciting prospect of “cell therapy without the cells”. Efforts to harness EVs as carriers of signaling molecules for therapeutic applications have been focused on exosomes, nanosized EVs of endosomal origin. Another area of intense investigation is the use of EVs as biomarkers of disease. Healthy subjects and patients with different diseases secrete EVs with different contents into the circulation and bodily fluids, which can be measured for diagnostic purposes.
To contextualise exosomes as potential biotherapeutics and drug delivery vectors within the broader field of vesicle biology, current knowledge of their biogenesis, composition, and functional roles in health and disease is summarized in the initial part of this communication. Potential roles of exosomes as indicators of diseases and novel exosome-based drug delivery systems will then be addressed. More comprehensive information on the classification, composition, and functions of EVs can be found at http://www.isev.org (International Society for Extracellular Vesicles), http://www.asemv.org (American Society for Exosomes and Microvesicles), http://microvesicles.org (Vesiclepedia, a compendium for EVs with continuous community annotation) (Kalra et al., 2012), http://www.exocarta.org (ExoCarta, a web-based compendium of exosomal cargo) (Keerthikumar et al., 2016), and http://exrna.org (Extracellular RNA communication program).
Section snippets
Vesicle classes
The classification of EVs as a heterogeneous mixture of membrane particles has been inconsistent and somewhat confusing. Three major populations have been distinguished: (i) exosomes, initially defined as 50–100 nm lipid bilayer particles released from cells; the size range was then increased to include particles as small as 20 nm in diameter and those as large as 150 nm in diameter, although a size range of 30–100 nm was used in most studies; (ii) microvesicles, also referred to as shedding
Biogenesis of exosomes
Endosomes arise from invaginations of the plasma membrane and fuse with molecular payloads sorted in the endoplasmic reticulum and processed in the Golgi complex, forming multivesicular endosomes (also referred to as multivesicular bodies; MVBs). When MVBs mature and eventually merge with the plasma membrane, their content is released into the extracellular space as exosomes (Fig. 1). Rab GTPase proteins regulate fusion of MVBs with the cell membrane (Pfeffer, 2010) and the spatio-temporal
Lipid and protein composition of exosomes
An in-depth discussion of the lipidomics and proteinomics of exosomes is beyond the scope of this article (for reviews, see Choi et al., 2013, Simpson et al., 2009). Briefly, exosomes released from different cell types contain different lipids and proteins. However, the lipid composition of exosomes differs from that of the plasma membrane of the parent cell, in part because exosomes also contain lipids from the Golgi. Exosome membranes are enriched in glycosphingolipids, cholesterol (Llorente
RNA sorting into exosomes
Exosomes contain nucleic acids including mRNA, microRNA (miRNA), ribosomal RNA, long noncoding RNA (lncRNA), and variably some DNA. miRNA is a class of small noncoding RNAs that regulate gene expression at the post-transcriptional level (Yates, Norbury, & Gilbert, 2013). The miRNA repertoire of exosomes varies as a function of the parent cell and its physiological state (Gibbings, Ciaudo, Erhardt, & Voinnet, 2009). The composition of exosomes is a tightly regulated process that is modulated by
Exosome uptake
Exosomes interact with target cells through multiple mechanisms (de Curtis and Meldolesi, 2012, Mittelbrunn and Sánchez-Madrid, 2010). Receptor binding can initiate exosome uptake (Miyanishi et al., 2007, Nolte-'t Hoen et al., 2009). Fusion (Prada & Meldolesi, 2016) and endocytotic processes including clathrin-coated pits, pinocytosis, caveolae, macropinocytosis (Fitzner et al., 2011), and phagocytosis may participate in the internalization of exosomes by different cell types (Morelli et al.,
Exosomes regulate cell functions
A variety of cell types including DCs and other APCs (Thery et al., 2002), mast cells (Skokos, Goubran-Botros, Roa, & Mecheri, 2002), B and T cells (Clayton et al., 2005, Taylor and Gercel-Taylor, 2005), intestinal epithelial cells (van Niel et al., 2001), neurons (Faure et al., 2006), cardiac myocytes (Gupta & Knowlton, 2007), MSCs (Lai et al., 2010), endothelial cells (de Jong et al., 2012), and cancer cells (Taylor and Gercel-Taylor, 2005, Zhao et al., 2015) secrete EVs. Early studies
EVs as carriers of genetic information
By delivering nucleic acids to target cells, EVs exchange genetic information between cells. Exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively) and from primary bone marrow-derived mouse mast cells were shown to contain RNA from ∼ 1300 genes (Valadi et al., 2007). In vitro translation showed that exosomal mRNAs were functional. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred
EVs and pathogens
EVs share biological features with enveloped viruses, such as HSV-1, EBV, and vaccinia virus regarding biogenesis, biophysical properties, cell entry, and functional protein and RNA delivery to target cells (Meckes & Raab-Traub, 2011). Several enveloped viruses exploit the cellular vesiculation machinery for budding and assembly (Rossman & Lamb, 2011). EVs secreted by cells infected by enveloped viruses carry viral envelope factors that mediate EV attachment and possibly fusion with target
Exosomes as biomarkers of diseases
Exosomes released by cells into the circulation and bodily fluids display different protein and RNA contents in healthy subjects and patients with different diseases, which can be measured as potential diagnostic markers (Clayton et al., 2003, Pant et al., 2012, Revenfeld et al., 2014). Tumour-derived exosomes are rich in miRNAs that may serve as tumour markers (Kumar et al., 2015, Mishra, 2014, Schwarzenbach, 2015). Patients with glioblastoma multiforme differed from healthy subjects in their
Therapeutic potential of naturally secreted EVs
EVs naturally released from certain cell types exhibit therapeutic potential. MSC-derived EVs recapitulate immunomodulatory and cytoprotective activities of their parent cells (Baglio et al., 2012, Yeo et al., 2013). Bone marrow MSC-derived exosomes were protective in models of myocardial ischemia/reperfusion injury (Lai et al., 2010), hypoxia-induced pulmonary hypertension (Lee et al., 2012), and brain injury (Doeppner et al., 2015, Zhang et al., 2015). These exosomes likewise stimulated
Biodistribution of systemically delivered exosomes
After systemic administration, the biodistribution of EVs influences the therapeutic efficacy and toxicity (György et al., 2015). Like any other nanotherapeutics, unmodified exosomes delivered systemically in animals accumulated preferentially in liver, kidney, and spleen, and were rapidly eliminated by bile excretion, renal filtration, or phagocytosis in the reticuloendothelial system, respectively. Exogenous exosome concentrations in target tissues were very low. The fate of delivered EVs in
Loading exosomes with exogenous cargo molecules
As natural carriers of signaling molecules, exosomes offer appealing features for therapeutic delivery, including biocompatibility, stability in the circulation, biological barrier permeability, low immunogenicity, and low toxicity. Expression of CD55 and CD59 may contribute to EV stability in the blood by preventing complement-mediated lysis (Clayton et al., 2003). Being nano-sized, exosomes may evade fast clearance by the mononuclear phagocyte system (van den Boorn et al., 2011).
EVs can be
Targeted exosomes
The biodistribution profiles of naturally secreted EVs with no modifications to their composition prohibit their systemic administration. However, EVs secreted by a few cell types exhibit target selection. DC-derived EVs are recruited by activated T cells through interactions between ICAM-1 expressed on DC-EVs and lymphocyte function-associated antigen-1 (LFA-1) expressed on T cells (Nolte-'t Hoen et al., 2009). B-cell-derived exosomes are recruited by hepatic and splenic macrophages through
Artificial exosome mimetics
All strategies outlined above relay on naturally secreted EVs with no or limited modifications to their composition. However, exosomes exhibit complex structures that are difficult to characterize pharmaceutically. Their composition varies with the producer cell type and its physiological state, as well as with manufacturing protocols. The purification of single populations of EVs is a complex procedure. While pure populations of exosomes can be isolated from exosome-secreting cell lines, these
Immune responses to exosomes
Unlike viral gene transfer vectors or liposomes (Seow & Wood, 2009), autologous exosomes show negligible immunogenicity. Repeated i.v. injections of autologous exosomes from immature DCs did not trigger significant immune responses in mice (Alvarez-Erviti et al., 2011). However, therapeutic applications for acute clinical events (e.g., myocardial infarction) would require ready-to-use exosome preparations in storage. Frozen exosome preparations can be stored for extended periods of time without
Experience in clinical trials
The safety of EV-based anti-tumour and anti-bacterial vaccines was tested in several phase-I clinical trials and one phase-II trial. In a phase-I trial, DCs of patients with advanced melanoma were isolated and pulsed with tumour antigen. Exosomes presenting the tumour antigen were purified and injected intradermally and subcutaneously to the patients. Exosome administration was tolerated for up to 21 months. A mild inflammatory reaction at the site of injection was observed in some patients. One
Conclusion
As mediators of intercellular communication, EVs can be harnessed for both diagnostic and therapeutic purposes. While naturally secreted exosomes may mediate beneficial effects in certain disease conditions, targeted exosomes loaded with therapeutic molecules may optimize efficacy while also reducing off-target delivery. Therapeutic molecules may include biological molecules that are difficult to deliver intracellularly without the use of a carrier, such as miRNA, siRNA and recombinant
Conflict of interest
Authors declare that there are no conflicts of interest.
On behalf of my co-author, Giuseppe Vassalli, I declare that authors do not have any actual or potential conflict of interest including any financial, personal or other relationships with individuals or organizations within three years of initiating the work that could inappropriately influence, or be perceived to influence, the study design or data interpretation.
Acknowledgments
G.V. and L.B. are the recipients of a research grant by the Swiss National Science Foundation (31003A_169306), Swiss Heart Foundation n° 4433; Cecilia-Augusta Foundation, Lugano (Switzerland); SHK Stiftung für Herz- und Kreislaufkrankheiten Foundation.
References (264)
- et al.
Microvesicles derived from endothelial progenitor cells protect the kidney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells
Kidney International
(2012) - et al.
Exosomes from ovarian cancer cells induce adipose tissue-derived mesenchymal stem cells to acquire the physical and functional characteristics of tumor-supporting myofibroblasts
Gynecologic Oncology
(2011) - et al.
Apoptosis, subcellular particles, and autoimmunity
Clinical Immunology
(2004) - et al.
Extracellular vesicles—Their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases
Seminars in Cell & Developmental Biology
(2015) - et al.
Intravenous phage display identifies peptide sequences that target the burn-injured intestine
Peptides
(2012) - et al.
Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer
Molecular Therapy
(2008) - et al.
Artificial exosomes as tools for basic and clinical immunology
Journal of Immunological Methods
(2009) - et al.
Exosome display technology: Applications to the development of new diagnostics and therapeutics
Blood Cells, Molecules & Diseases
(2005) - et al.
Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA
Blood
(2007) - et al.
Exosomes are released by cultured cortical neurones
Molecular and Cellular Neurosciences
(2006)