Extracellular Vesicles 2023: Drug Delivery, Biologics & Therapeutics Agenda

Morning Coffee and Networking in the Exhibit Hall

Mid-Morning Coffee Break and Networking in the Exhibit Hall

The Aethlon Hemopurifier for the Removal of Enveloped Viruses and Injurious Extracellular Vesicles
Steven LaRosa, Chief Medical Officer, Aethlon Medical

The construction and mechanism of action for removal of enveloped viruses, virus particles, and extracellular vesicles will be discussed. Next, the data demonstrating the in vitro removal of viruses and extracellular vesicles from biologic and non-biologic fluids will be presented. Finally, the clinical experience with the Aethlon Hemopurifier to date and clinical plan will be discussed.

Enhancing Therapeutic Extracellular Vesicle Biomanufacturing via Mechanoregulation
Steven Jay, Associate Professor, University of Maryland, United States of America

This presentation will recap progress on enhancing production of mammalian vesicles via 3D-printed perfusion bioreactors. Additional studies on the effects of various mechanical stimuli (substrate stiffness, cellular confinement) will also be discussed.

X-Cell Therapeutics Technology Spotlight Presentation

EVs and the Inner Ear: Diagnostic and Treatment Opportunities
Hinrich Staecker, David and Mary Zamierowsky Professor, Department of Otolaryngology, University of Kansas Health System, United States of America

Inner ear diseases are common, yet no good diagnostic or therapeutic interventions exist. EVs provide an opportunity to diagnose inner ear disease and with custom manufacturing approaches can be applied to treat inner ear disease.

Networking Lunch - Network with Colleagues and Meet Exhibitors

Title to be Confirmed.
Heather Branscome, Research Assistant, ATCC and George Mason University, United States of America

The AlphaV-Beta3 Integrin/NgR2 Complex is Up-regulated in Prostate Cancer Cell-derived EVs and Patient-derived EVs and Represents a Promising Therapeutic Target
Anna Testa, Medical Resident, Thomas Jefferson University; Internal Medicine Fellow, University of Tur, United States of America

The AlphaV-Beta3 integrin, known to promote growth and metastasis of several types of cancer, is highly expressed in neuroendocrine prostate cancer (NEPrCa). PrCa cell-derived small extracellular vesicles (sEVs) expressing AlphaV-Beta3 contribute to NEPrCa differentiation in recipient cells. AlphaV-Beta3 integrin upregulates NgR2, a glycosylphosphatidylinositol-anchored receptor, in NEPrCa cells. By investigating the impact of AlphaV-Beta3 expression in sEVs on downstream protein expression via proteomic analysis, we demonstrate that AlphaV-Beta3+ sEVs also show an up-regulation in NgR2 expression; furthermore, a down-regulation of typical effectors involved in apoptosis and necrosis and an up-regulation of tumor cell survival factors emerge, as compared to control sEVs. We also show that EVs isolated from metastatic castrate-resistant prostate cancer (mCRPC) patients plasma are enriched in AlphaV-Beta3 and NgR2. sEVs used in these experiments were isolated by density gradient and characterized by nanoparticle tracking analysis and immunoblotting. Additionally, by testing the effect of AlphaV-Beta3 inhibition in NEPrCa-patient derived xenografts (PDXs) using LM609, a monoclonal antibody (mAb) specific for AlphaV-Beta3, we demonstrate a significant reduction of tumor volume and weight in NE-PDXs carrying mice upon treatment with LM609. Our findings suggest that AlphaV-Beta3 integrin and NgR2 are key components of NEPrCa progression and represent a promising therapeutic target both in vitro and in vivo.

An Effective Peptide-based Platform for Efficient Exosomal Loading and Cellular Delivery of a microRNA
Zucai Suo, Eminent Professor & Dorian and John Blackmon Chair in Biomedical Science, Florida State University College of Medicine, United States of America

Exosomes, membrane-bound nanosized vesicles of biologic origin, are known to contain various molecules, e.g., proteins, lipids, and nucleic acids, which contribute to the exosomes’ ability to mediate cell-to-cell communication. Recent impediments of artificial nanoparticles in drug delivery, including low cellular uptake, activation of the immune system, and tissue obstacles, have led scientists to engineer exosomes as drug delivery vehicles. Though exosomes possess inherent properties of stability, biocompatibility, low immunogenicity, and capability to cross biological barriers, there is a need to develop technologies that allow the efficient loading of therapeutic materials into exosomes. Here, we introduced a simple peptide-equipped technology that can enhance the cargo-loading potential of exosomes in a mild loading environment. Specifically, a known cell-penetrating peptide, YARA, derived from human immunodeficiency virus-1 trans-activator of transcription, was covalently conjugated with miR21-5p, a mammalian microRNA. The conjugate YARA-miR-21-5p was then incubated with exosomes, isolated from either mesenchymal stem cells or cancer cells, for loading. Exosomal loading of YARA-miR-21-5p was time-dependent and demonstrated an impressive 18.6-fold increase in efficiency over exosomal loading of miR-21-5p through incubation. After effective cellular uptake, the loaded exosomes rapidly delivered YARA-miR-21-5p into mammalian cells. Relative to unloaded exosomes and free YARA-miR21-5p, the loaded exosomes significantly enhanced the proliferation, migration, and invasion of human and mouse fibroblasts, which are vital steps in wound healing. This study lays the groundwork for using cell-penetrating peptides as an innovative approach to efficiently load therapeutic cargos, e.g., microRNAs, into exosomes, which can then be employed to deliver the cargos into cells to yield biological effects.

Stimulating Production of Osteoclast-derived Regulatory Extracellular Vesicles to Therapeutically-control Bone Remodeling
Shannon Holliday, Associate Professor of Orthodontics and Anatomy & Cell Biology, University of Florida, United States of America

Osteoclasts produce extracellular vesicles (EVs) that stimulate bone formation by osteoblasts. Our recent data suggest that the production of these EVs can be increased experimentally.  We are now seeking to dissect the regulatory pathways that are involved. The goal is to therapeutically-stimulate osteoclasts to produce EVs that promote bone formation. Such agents could prove more useful than current therapeutics for treating bone diseases, or repairing bone defects.

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.

Mid-Afternoon Coffee Break and Networking

sEV Sub-populations Derived from Cancer Cells that Express the AlphaVBeta3 Integrin
Cecilia Verrillo, Research Student, Thomas Jefferson University, United States of America

Small extracellular vesicles (sEVs) released from prostate cancer (PrCa) cells contain pro-tumorigenic cargo and are crucial components of cellular crosstalk.  The AlphaVBeta3 integrin is expressed in PrCa sEVs.  The primary goal of this project was to identify the impact of AlphaVBeta3 expression and associated proteins in sEVs derived from PrCa cells.  sEVs were isolated by differential ultracentrifugation followed by density gradient separation. After separation, sEVs were characterized by NTA, western blot analysis, and single vesicle analysis by Exoview.  While there are no differences in tetraspanin compositions of sEVs isolated from C4-2B AlphaVBeta3+ compared to C4-2B Mock cells, the expression of AlphaVBeta3 in C4-2B PrCa cells results in a distinct shift to low density sEVs.  These sEVs contain novel pro-tumorigenic cargo molecules: NgR2 and PD-L1, an effector of AlphaVBeta3 and a key player in immune evasion, respectively.  AlphaVBeta3/NgR2+ sEVs increase NgR2 expression upon transfer to recipient cells.  sEVs isolated from PrCa cell lines and mouse models of PrCa also specifically express six-transmembrane epithelial antigen of the prostate 1 (STEAP1), a metalloreductase upregulated in PrCa and not its family members STEAP2 or STEAP3.  Overall, our results show that AlphaVBeta3 expression is a key regulator of sEV density and pro-tumorigenic cargo loading.

Extracellular Vesicle-Mediated Amyloid Transfer and Intercellular Communication within the Neurovascular Unit
Michal Toborek, Professor and Vice-Chair for Research, University of Miami School of Medicine, United States of America

It is widely accepted that elevated brain deposits of amyloid beta (A-beta) contribute to neuropathology in Alzheimer’s disease. Additionally, A-beta deposition was demonstrated to be elevated in the brains of HIV-infected patients and associated with neuro-cognitive decline; however, the mechanisms of these processes are poorly understood. The role of the blood-brain barrier (BBB) as an interface for the transfer of A-beta from the periphery into the brain and the cells of neurovascular unit is poorly characterized. Indeed, substantial population of neural progenitor cells (NPC) reside near brain capillaries that form the BBB. The purpose of this study is to understand the impact of brain endothelium-derived extracellular vesicles containing A-beta (A-beta-EVs) on metabolic functions and differentiation of NPCs. Mechanistically, we focused on the role of mitochondria, the receptor for advanced glycation end products (RAGE), and activation of the inflammasome on these events. We demonstrate that physiological concentrations of A-beta-40 can transfer and accumulate in NPCs via endothelial EVs. This transfer results in mitochondrial dysfunction, disrupting cristae morphology, metabolic rates, fusion and fission dynamics of NPCs, as well as their neurite development. Moreover, our results show that A-beta partly co-localized with the inflammasome markers ASC and NLRP3 in the recipient NPCs. This co-localization was affected by HIV and RAGE inhibition by a high-affinity specific inhibitor, FPS-ZM1. Interestingly, both A-beta-EVs and RAGE inhibition altered NPC differentiation. Overall, our data indicates that intercellular transfer of A-beta-40 is carried out by brain endothelium derived EVs that can induce mitochondrial dysfunction and alter cellular signaling, leading to aberrant neurogenesis of NPCs. These events may modulate EV-mediated amyloid pathology in the HIV-infected brain and contribute to the development of HIV-associated neuro-cognitive disorders.

Genetic Engineering of Nanoscale Exosomes for Targeted Drug Delivery and Therapy
Bill Lu, Associate Professor, Santa Clara University, United States of America

Human cell-derived nanoscale exosomes are the fast-growing class of biologics with potential treatments of many difficult-to-manage human diseases such as cancer, hereditary and genetic defect, viral infection and inflammation, as well as cardiovascular and neurological disease.  Owning to their intrinsic biocompatibility, high tissue-penetrating ability, and large capacity in delivery of various biological cargos, exosomes have overtaken the traditional small-molecular therapeutic paradigm and opened new horizons in advanced therapeutic applications.  This seminal talk focuses on novel techniques for exosome surface engineering on functional moiety installation, drug loading and intracellular delivery. Furthermore, specific examples of how exosomes are engineered to display targeting moieties on their outer surface or to load therapeutic agents in living human cells are also discussed.

Title to be Confirmed.
Natalia Higuita-Castro, Assistant Professor, The Ohio State University, United States of America

Title to be Confirmed.
Daniel Gallego-Perez, Associate Professor, The Ohio State University, United States of America

Close of Day 2 Conference Programming