SELECTBIO invites you to its 2nd Annual Circulating Biomarkers Conference. This conference held in the city of Boston close to world-leading research institutions and hospitals seeks to bring together academic researchers, clinician-scientists, as well as researchers from companies all focusing on various classes of biomarkers.
Don’t miss the special session on exosomes – Monday, 24 March 2014
Session Title: Methodologies for Studying Exosomes, Microvesicles [Extracellular Vesicles, EVs]
|Session Chair: Sasha Vlassov, PhD|
|14:30||A Rapid and Non-destructive Magnetic Beads-based Exosome Isolation and Enrichment Method
Kenneth Henry, Senior Research Scientist, JSR Micro, Inc.In order to isolate exosomes from various body fluids and cell culture supernatants, we have successfully developed ExoCap, which utilizes a magnetic beads based isolation method. ExoCap consists of magnetic particles coupled with antibodies that recognize antigens on the exosome surface, an irrigation solution, and a reagent that releases the captured exosomes for analysis. The antibodies against CD9, CD63, CD81, and EpCAM were specifically selected for this kit. ExoCap can separate easily exosomes within 30 minutes, without ultracentrifuge or any special equipment. A sample amount of 0.1mL is sufficient. In addition, it is an animal free system which is superior to other methods for mass analysis. Moreover, this method enables non-destructive purification of exosomes. To confirm exosomal isolation from diverse body fluids (such as human serum, plasma, urine) and cell culture media, exosomes were examined by western blot, particle size distribution measurement, and scanning transmission electron microscopy (TEM). Exosomes isolated by ExoCap had a lipid bilayer membrane, showed a particle size distribution around 100nm, and expressed tetraspanin molecules.Authors:
Kenneth Henry, Ph.D, Senior Research Scientist, Life Sciences, JSR Micro, Inc.
Tetsuji Yamaguchi, Ph.D. JSR Life Sciences
|15:00||Heparin Affinity Purification of Extracellular Vesicles
Leonora Balaj, Research Fellow, Massachusetts General HospitalExtracellular vesicles (EVs) are membrane vesicles released by cells. They carry active biomolecules which can be transferred to recipient cells. Isolation and purification of EVs from in vitro conditioned culture media and in vivo biofluids is still a major challenge and the most widely used isolation method still remains ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs due to co-pelleting of proteins and lipids. Affinity purification of biomolecules is an efficient way to achieve high purity without requiring expensive equipment. Previously we have shown that heparin blocks EV uptake in mammalian cells in culture, suggesting a possible direct EV/heparin interaction. Here we show that EVs can be purified from conditioned media using heparin-coated agarose beads. We directly compared heparin-purified EVs to UC prepared and kit-isolated EVs and we show that we can efficiently isolate EVs a higher purity than UC, kit-isolated EVs and even sucrose gradient-purified EVs. Importantly these, heparin-purified EVs retained the RNA content, morphology, and uptake dynamics of UC-isolated EVs. In conclusion, we have discovered a simple and effective way to isolate a highly pure population of EVs using their affinity for heparin.
|15:30||Coffee Break and Networking with Exhibitors in the Exhibit Hall|
|16:00||New Biosensor Platform for the Isolation and Analyses of Microvesicles
Hakho Lee, Assistant Professor, Harvard Medical SchoolThis presentation will review new biosensor platforms developed in our laboratory for microvesicle analyses. Specifically, I will describe 1) microfluidic systems that can enrich microvesicles from native clinical specimen, and 2) microNMR sensors that can molecularly profile microvesicles. Clinical applications of these platforms on cancer detection will also be discussed.
|16:30||A Hybrid Extracellular vesicle/virus Vector System for Gene Therapy Applications
Casey Maguire, Assistant Professor of Neurology, Harvard Medical School/Mass General HospitalObtaining tissue-restricted transgene expression after intravenous (i.v.) injection of AAV vectors is a challenging task, especially for the brain, as the majority of vector is taken up by the liver. Although some vectors can cross the intact blood-brain barrier, improvements are still needed. Additionally, pre-existing antibodies against AAV can remove vector from the circulation. Other studies have shown that association of virus vectors with nanoparticles, such as microbubbles and cationic liposomes can alter the vector biodistribution to preferred sites. Another nanoparticle which may offer utility to the field of viral vector gene delivery are extracellular vesicles(EVs). EVs are small (50-200 nm in diameter) membrane limited structures naturally secreted by many cell types. We have recently shown that EV-associated AAV vectors (EV-AAV, a.k.a vexosomes) can deliver genes more efficiently on a genome copy per cell basis than AAV vectors alone using cultured cells. In the current study we are using the EV-AAV for targeted gene delivery to the brain after i.v. injection in mice. To investigate if EV-AAV can be targeted to the brain via over-expression of specific ligands on the EV surface we injected nude mice with EV-AAV9-Fluc or EV-AAV9-Fluc with a brain targeting peptide (RVG) fused to a trasmembrane domain.Enhanced transduction of the brain was observed with the RVG peptide compared to untargeted EV-AAV. The brain:peripheral organ transduction ratio was significantly higher for RVG-EV-AAV compared to standard AAV9. This work has provides the first evidence for the in vivo use of EV-AAV for gene therapy.
|17:00||Large Oncosomes as a Novel Source of Circulating DNA and miRNA in Cancer
Dolores Di Vizio, Associate Professor, Cedars-Sinai Medical Center
|17:30||Panel Discussion: Evolution of the Exosome Research Field
Johan Skog, Chief Scientific Officer, Exosome Diagnostics IncPanelists:Dominique de Kleijn, Professor, National University of Singapore
Xandra Breakefield, Professor, MGH
Shannon L. Stott, Professor, MGH
Pavan Kumar, Eisai