Exosomes
Exosomes, a class of small vesicles continuously released from all cells of the body, may serve as a most interesting target for in vitro diagnostics of a liquid biopsy. Exosomes (30-150 nm in size) are the smallest type of extracellular vesicle, produced through inward budding of multivesicular bodies, resulting in intra-luminal vesicles, which are released into the extracellular space by exocytosis. Exosome-associated proteins are currently used to isolate EVs from biological fluids such as blood, urine, or cerebral fluid and identify the cellular source of their origin. Examples of such proteins are tetraspanins (CD9, CD63, CD81), HLA molecules, membrane transport and fusion proteins (Rab 5, Rab 11). Exosomes contain DNA, RNA, microRNA, proteins as well as lipids and, therefore, have the potential to be used as “liquid biopsy” (van Niel et al. 2018, Nature Reviews in Molecular Cell Biology). Since exosomes contain DNA, RNA, lipids and proteins from the cell they are released from, they provide the opportunity to gain a more comprehensive view of the functional state of cells also within tissues.
The molecular composition of exosomes is assumed to reflect (patho-) physiological changes of their cells or tissues of origin. Thus, exosomes secreted from virus-infected cells have the potential to aid in the identification of virus-infected organs and cell populations and the changes associated with infection in cells.Similarly, exosomes released from cancer cells shall mirror the phenotype of parental as well as metastatic cancer cells. Exosomes released from immune cells are supposed to reflect the phenotype and functional properties of immune cells during ongoing immune responses.
therawis develops innovative technologies to use exosomes for diagnosis and treatment monitoring in viral infection, oncology and immune-mediated diseases. Diagnostic exosomes will add a novel dimension to in vitro diagnostics.
ELAEXIA® - Elective Labelling of Exosomes for Identification and Analysis
Currently applied methods for exosome purification are ultracentrifugation, precipitation using PEG, immunocapture with antibodies against CD9, CD63, CD81, TSG, Rab5, HLA-ABC, the use of microfluidic isolation, filtration, and size exclusion chromatography. However, methods such as filtration, size exclusion chromatography and precipitation lead to the isolation of a mixture of microvesicles, lipoproteins and exosomes and also bear the risk of damaging or destroying exosomes. The use of antibodies to CD9, CD81 and CD63 for immunocapturing and purification of exosomes will yield subpopulations of exosomes because oft he heterogenous loading of protein cargo into exosomes depending on the state of the cell. There is a lack of a standardised and reliable detection of exosome hampers (cross)validation of methods that are required to validate diagnostic workflows. To solve this problem, an identification and detection method is required, which is based on a ubiquitously present characteristic/element for all exosomes independent of their origin and protein cargo.
We developed ELAEXIA® as a diagnostic tool that binds specifically to the exosome lipid bilayer membrane, thus identifying all exosomes. Exosome biogenesis occurs inside multi-vesicular bodies, where the donor membrane for the budding exosome undergoes inward budding. This process leads to lipids located in the inner leaflet of membranes in cells, such as phosphatidylethanolamine (PE) or phosphatidylserine (PS) lipids such as phosphatidylserine (PS), being exposed on the outer leaflet of the exosome membrane (see Figure).
ELAEXIA® is a technology using a biopeptide binding with high specificity and affinity to phosphatidylethanolamine (PE) present on the outer leaflet of exosome membranes.
ELAEXIA® technology combines high specificity for exosomes with high sensitivity allowing thorough standardization of exosome-based workflows.
therawis diagnostics develops and delivers to themarket life science products based on this proprietary exosome platform as prime liquid biopsy diagnostics in oncology, virology and immunology enabling easier biomarker discovery and diagnostic performance through its unprecedented specificity and sensitivity.
