07:30 | Conference Registration and Materials Pick-Up |
08:15 |  | Conference Chair Welcome and Introduction to the Conference by Conference Senior Co-Chairperson
Michael Graner, Professor, Dept of Neurosurgery, University of Colorado Anschutz School of Medicine, United States of America
|
|
|
Session Title: Conference Opening Session - Extracellular Vesicles 2024 |
| |
08:30 |  | Conference Chair Extracellular Vesicle-Based Gene Therapy
Mei He, Associate Professor, University of Florida, United States of America
Genome editing is an emerging and powerful therapeutic tool for treating diverse diseases. However, clinical translation has been challenging, due to tremendous limitations in current delivery vehicles such as traditional viral vectors for carrying CRISPR Cas9 systems. Alternatively, extracellular vesicles (EV) based gene delivery is emerging as a safe and highly biocompatible approach for addressing current challenges in gene therapy. We developed a novel Microfluidic Droplet-based EV Electroporation System (µDES), which can handle various cargos loaded into EVs in large throughput and high efficiency. We achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading CRISPR RNP complexes into EVs compared with conventional bulk electroporation. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery for future clincial translation. |
|
09:00 |  | Keynote Presentation Integrating Microphysiological Systems and Extracellular Vesicle-Based Technologies to Advance Regenerative Medicine
Danilo Tagle, Director, Office of Special Initiatives, National Center for Advancing Translational Sciences at the NIH (NCATS), United States of America
Microphysiological systems are microfluidic cell culture chips capable of recapitulating key functional aspects of physiological human tissue and organ response. MPS have many contexts of use including evaluation of toxicity/safety, and efficacy of promising therapeutic compounds, disease modeling of both rare and common diseases, as well as within the regenerative and precision medicine space. Extracellular vesicles (EVs) are nano-sized, membrane-enclosed carriers of bioactive lipids, protein, and nucleic acids that are used for intracellular communication. Extracellular vesicles (EVs), membrane-bound particles containing a variety of RNA types, DNA, proteins and other macromolecules, are now appreciated as an important means of communication between cells and tissues, both in normal cellular physiology and as a potential indicator of cellular stress and other environmental exposures and early disease pathogenesis. EVs have pleiotropic actions in physiological and pathological conditions. EVs are commonly heterogeneous in size, ranging from 20 to 1,000 nm in diameter depending on their origin and mechanism of release, direct shedding or budding from the plasma membrane. Exosomes are vesicles with a diameter of 20–100 nm formed by the inward budding of endosomal membranes to form large multivesicular bodies (MVBs) and released extracellularly when MVBs fuse with the plasma membrane. Exosomes have recently been studied for their potential use in therapy as a 1) targeted and non-immunogenic delivery system for drugs or biological molecules, and 2) in the maintenance of tissue homeostasis and their contribution to tissue repair and regeneration. For the past few years, MPS and EV-based technologies have been combined within the regenerative medicine space to find safer, more efficacious patient therapies, as well as to probe for non-invasive diagnostic biomarkers. Combination of these technologies could potentially help address a key drug development challenge, i.e., on-target delivery without off-tissue toxicity by delivering therapeutics (small molecules, macromolecules, nucleic acids, etc.) via EVs that only act at the diseased tissue, regardless of whether a target is expressed elsewhere. This presentation will summarize NIH-funded activities in exploring the therapeutic applications of exosomes along with application of new experimental models, including organ-on-chip (OOC) systems and in vitro approaches to extend findings. |
|
09:30 |  | Keynote Presentation Plasma Extracellular Vesicles for Cardiovascular Disease
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands
Cardiovascular Disease (CVD) is with the cardiovascular events of Ischemic Heart Disease and Stroke, the number 1 and 2 cause of death in the world and expect to increase especially in Asia. Ischemic heart disease (IHD) comprises 2 entities: Chronic Coronary Syndrome (CCS) and Acute Coronary Syndrome (ACS). Because CCS is associated with 6-8 times increased risk of adverse cardiovascular events like myocardial infarction and death, early recognition of CCS. Blood markers for CCS do not exist, resulting in that 80-90% of chest pain patients undergoing costly imaging do not have CCS. We use plasma extracellular vesicle protein content of vesicles from plasma subfractions as an accurate source for early diagnosis of CCS better than a clinical risk model. Using automated plasma EV fraction analysis and CD9 as an internal marker, we hope with the plasma EV test to reduce the numbers of patients without CCS that are referred for costly imaging. |
|
10:00 |  | Keynote Presentation Moving Neural Stem Cell Derived Exosome into Clinical Trials: Manufacturing and Mechanistic Considerations
Steven Stice, Co-Founder and Chief Scientific Officer, Aruna Bio; DW Brook Distinguished Professor and Director of the Regenerative Bioscience Center, Georgia Research Alliance Eminent Scholar, University of Georgia, United States of America
Neural stem cell EVs (NSC EVs) derived in bioreactors have therapeutic potential for treating neurological disease and acute ischemic stroke (AIS). New FDA Investigational New Drug (IND) applications are being filed and specifically, we have an open IND for AIS. As the field grows, new INDs will be filed for various other therapeutic indications. In order for EV therapeutics to move efficiently through the regulatory process to approval, there is a need for more emphasis on and development of analytical assays directly related to complex, and likely multimodal, mechanisms of action. Research focused on this area will lead to new disease-specific potency assays and identification of critical quality attributes. Beyond AIS, one of the most promising applications of NSC EVs is in the treatment of amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease that affects the motor neurons in the brain and spinal cord. In a preliminary study, we have shown that NSC EVs significantly preserved motor function, decreased serum neurofilament light chain, and prolonged survival in ALS mice. NSC EVs also reduced inflammatory mediators TNFa, IL-1ß, IL-6, RIPK1, and NLRP3 in the lumbar spinal. These results suggest that NSC EVs have the potential to be developed as a therapeutic for ALS. The complex pathogenesis in the central nervous system during ALS suggests the need to develop drugs with multimodal therapeutic action and will likely require the development of multiple potency assays relevant to the active agents in and on the surface of the NSC EVs. |
|
10:30 | Mid-Morning Coffee Break and Networking in the Exhibit Hall |
11:00 | EVs in Treatment of Lung Diseases and Other Targets
Marcin Jurga, Chief Scientific Officer, EXO Biologics SA, Belgium
|
11:30 |  | Keynote Presentation High-Resolution Analysis of Single Extracellular Vesicles and Particles with Digital Flow Cytometry and Super-Resolution Imaging
Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America
We have developed a multi-parametric high-throughput flow-based method for the analysis of individual extracelluar vesicles and particles (EVPs), and a super-resolution method for sizing individual EVPs in a high-throughput fashion. EVPs are highly heterogeneous and comprise a diverse set of surface protein markers as well as intra-vesicular cargoes. Yet, current approaches to the study of EVPs lack the necessary sensitivity and precision to fully characterize and understand the make-up and the distribution of various EV subpopulations that may be present. Digital flow cytometry (dFC) provides single-fluorophore sensitivity and enables multiparameter characterization of EVPs, including single-EVP phenotyping, the absolute quantitation of EVP concentrations, and biomarker copy numbers. dFC has a broad range of applications, from analysis of single EVPs such as exosomes or RNA-binding proteins to characterization of therapeutic lipid nanoparticles, viruses, and proteins. dFC also provides absolute quantitation of non-EVP samples such as dyes, beads, and Ab-dye conjugates. |
|
12:00 |  Characterization of Extracellular Vesicles and Other Biological Nanoparticles Using Nanoparticle Tracking Analysis (NTA)
Sven Rudolf Kreutel, Chief Executive Officer, Particle Metrix GmbH and CEO, Particle Metrix Inc., USA
Nanoparticle Tracking Analysis (NTA) has emerged as a fast and vital characterization technology for Extracellular Vesicles (EVs), Exosomes and other biological material in the size range from 30 nm to 1 µm. While classic NTA scatter operation feeds back the size and total particle concentration, the user typically cannot discriminate whether the particle is a vesicle, protein aggregate, cellular trash or an inorganic precipitate. The fluorescence detection capabilities of f-NTA however enables the user to gain specific biochemical information for phenotyping of all kinds of vesicles and viruses. Alignment-free switching between excitation wavelengths and measurement modes (scatter and fluorescence) allow quantification of biomarker ratios such as the tetraspanins (CD63, CD81 and CD9) within minutes. Furthermore, specific colocalization studies using c-NTA gives a deeper understanding of the composition of biomarker on single particle.
|
12:30 | Networking Lunch Break in the Atrium -- Network with Colleagues, Visit Exhibitors and View Posters |
|
Session Title: Extracellular Vesicles for Drug Delivery and Therapeutics Applications |
| |
13:30 | Large Scale Manufacturing, Cargo Profiling, and Functional Effects of hTERT MSC EVs
Heather Branscome, Senior Scientist, ATCC and Research Assistant, George Mason University, United States of America
Ocular diseases are a major cause of visual impairment and morbidity. Furthermore, exposure to ionizing radiation (IR) can cause direct damage to the eye. Therefore, there is an urgent need to explore novel ocular therapeutics. Extracellular Vesicles (EVs) from Mesenchymal Stem Cells (MSCs) have demonstrated widespread regenerative properties across multiple pathologies. However, the reparative effects of MSC EVs against ocular damage remains relatively unexplored. Here, we report a large-scale platform for manufacturing of EVs from hTERT-immortalized MSCs and evaluate their reparative properties on retinal cells before and after exposure to IR. Additionally, we evaluate the efficacy of a novel EV lyophilization buffer for improved EV storage, transport, and stability. Physical and biochemical properties of EVs were assayed using various techniques including NTA, western blot, mass spectrometry, RNAseq, and multiplex immunoassays. EV functionality was evaluated in vitro using a combination of assays to assess cell viability, cell migration, cell cycle, and apoptosis before and after exposure to IR. Collectively, our data suggests that hTERT MSC EVs are enriched with unique cargos and that these EVs exert reparative properties on retinal cells in vitro against irradiation-induced damage. Importantly, lyophilization of EVs further extended their shelf life without impacting their function. |
14:00 |  | Keynote Presentation Therapeutic Development and Use of Exosomes in Neurological Disease and Injury
Damien Pearse, Professor, Department of Neurological Surgery; The John M. and Jocelyn H.K. Watkins Distinguished Chair in Cell Therapies, University of Miami Miller School of Medicine, United States of America
Exosomal vesicles (EVs) derived from neural and non-neural sources have shown potential in limiting damage to the CNS as well as promoting neurorepair. Our laboratory is interested in the development and utility of glial cell-derived exosomes as therapeutic agents in models of spinal cord injury, multiple sclerosis, and amyotrophic lateral sclerosis, among other neurological conditions. Herein I will discuss our advances in characterizing EVs from different cell sources, examining their ability to alter cellular responses in vitro in neural cell assays as well as investigating their capacity to alter pathological processes in neurological injury and disease paradigms in rodents with the goal of moving this approach towards clinical evaluation in humans. |
|
14:30 |  Step up your EV Characterization with Leprechaun
Alex Shephard, Market Manager, Unchained Labs
Accurately characterizing extracellular vesicles (EVs) can be challenging, even in highly purified cell culture samples. Interference from lipoproteins, cell debris and protein aggregates make it hard to be confident that you're counting the right stuff. Add in complex biofluids, limited sample volumes, and rare EV subpopulations and the task gets even tougher. Leprechaun lightens the load by isolating EVs on its Luni consumable, before measuring particle size, concentration, and analyzing EV phenotype for up to 4 surface or cargo markers simultaneously, from <25 uL of sample. Leprechaun jives with a range of materials, from crude cell culture media to murine cerebral spinal fluid, without the need for sample purification. With sensitivity down to 5x10^5 particles/mL, single particle analysis and the ability to size EVs as small as 35 nm, Leprechaun is ready to help you step up your EV characterization no matter how rare or small.
|
15:00 | Engineering EVs for Cardiovascular Diseases
Eun Ji Chung, Dr. Karl Jacob Jr. and Karl Jacob III Early Career Chair, Associate Professor of Biomedical Engineering, Chemical Engineering and Materials Science, Surgery, and Medicine, University of Southern California, United States of America
Extracellular vesicles derived from healthy sources contain natural homing properties and therapeutic cargo and can be leveraged as biomimetic carriers for targeted delivery. Additionally, extracellular vesicles represent an endogenous source of nanoparticles and can offer enhanced safety as a nanoparticle platform technology. In this presentation, EVs derived from healthy cells of the vasculature and their ability to be engineered as agents that inhibit vascular calcification and inflammation in atherosclerosis will be included. Strategies to enable EV surface modification and enhance cargo loading will be presented, and the potential of engineered EVs towards clinical applications will be discussed. |
15:30 | Mid-Afternoon Coffee Break and Networking in the Exhibit Hall |
16:00 |  Single vs Multi-Parameter EV Isolation Methods
Amber Murray, Senior VP, Application, Exokeryx
The success of extracellular vesicles (EVs) in diagnostics and therapeutics depends on scalable isolation methods that produce high recovery of highly pure EVs. Current methods for EV isolation exploit one physical property at a time—density for ultracentrifugation, size for size exclusion chromatography, presence of a given surface marker for immunoprecipitation, etc. As such, isolated EVs exhibit high recovery at the expense of purity for the cruder techniques or high purity (and bias) at the expense of recovery for the more tailored techniques. In contrast, we introduce a new EV isolation technique called dielectrophoresis that exploits two physical properties at once—particle diameter and particle composition (dipole moment) in the presence of a radio frequency electric field.
|
16:30 | Delivery of Mitochondria-Containing Extracellular Vesicles to the BBB
Devika Manickam, Associate Professor, Duquesne University, United States of America
Extracellular vesicles (EVs) are natural, cell-secreted nanoparticles that have known roles in intercellular communication. Our work has demonstrated that the innate mitochondrial cargo in EVs can be transferred to recipient cells and tissues resulting in increased mitochondrial function. My talk will describe how delivery of innate mitochondrial cargo can be exploited for BBB protection in ischemic stroke. |
17:00 |  | Keynote Presentation Determining Critical Quality Attributes (CQAs) of Adeno-Associated Virus Gene Therapies using Resistive Pulse Sensing
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America
Adeno-associated virus (AAV) vectors have been used to successfully introduce therapeutic gene fragments (i.e., gene therapy) into host cells and thus offer a significant tool for combating diseases that are unaffected by conventional drug therapy. This has led to a significant number of new clinical trials involving AAVs. However, broad application of AAV gene therapy across potential disease targets is hampered by a lack in definition of critical quality attributes (CQAs), analytics to measure CQAs, development of universal customizable vector cassettes, and affordable manufacturing methods. Presently, one of the most significant production and quality control issues facing AAV manufacturing is the presence of non-transducing viral particles (including empty particles) in the final vector preparation. Not only does this introduce errors and inconsistencies in the identification of actual titer delivered to the patient, but it also results in decreased infectivity of the dose due to increased host immune response to the defective virions. The issue of empty capsids is considered one of the top five major concerns in the production of AAVs today. The establishment of quantifiable traits at various points in the production process along with suitable analytical techniques are needed. Techniques to determine the full-to-empty capsid ratio include transmission electron microscopy (TEM) and analytical ultracentrifugation. Unfortunately, these techniques are fraught with challenges. There have also been a number of different chromatographic techniques to determine the full-to-empty ratio, but are challenged by inter-laboratory variability. All of the aforementioned techniques are batch-type processes and thus, cannot do real-time reporting to optimize the manufacturing process in-line. In this presentation, we will discuss the use of synthetic nanopore-based sensors capable of detecting and characterizing AAVs. Specifically, we will discuss the use of nanopore technology to detect capsids, characterize capsids as either full or empty, and to analyze capsids to determine if they contain full-length or foreshortened gene fragments. The sensors consist of dual in-plane nanopores that flank either end of a nano-column from which one can deduce the electrophoretic mobility of the target nanoparticle. We will show that empty and full capsids possess different mobilities and with the use of resistive pulse sensing (RPS) and machine learning, we can classify the particles as either full or empty quantitatively in near real-time. |
|
17:30 |  | Keynote Presentation Nucleic Acid Delivery with Biological and Synthetic Lipid Nanoparticles
Raymond Schiffelers, Professor of Nanomedicine, University Medical Center Utrecht, Netherlands
Nucleic acid nanomedicines gain momentum. Starting with Onpattro, delivering siRNA to the liver and followed by the local injection of COVID mRNA vaccines, we currently witness an avalanche of new therapeutic applications. Following the same lipid nanoparticle recipe as Onpattro and the vaccines, enzyme replacement therapy for rare metabolic disorders in the liver and CRISPR mRNA/sgRNA combinations for hepatic gene editing are being explored clinically, amongst many others. For other applications in new tissues, this recipe needs to be tweaked. In this lecture, applications to spleen targeting, heart delivery and immune cell targeting will be discussed, and biological and synthetic systems are compared. |
|
18:00 | Networking Prosecco, Beer and Wine Reception - Meet Exhibitors, View Posters, Network with Colleagues |
19:30 | Close of Day 1 of the Conference |
