Extracellular Vesicles & Nanoparticle-based Therapeutics Europe 2023 Agenda

Introduction to the EV Field -- circa 2023
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Contribution of Immuno-Cryo-Electron Microscopy to the EV Field
Alain Brisson, Professor Emeritus, University of Bordeaux, France

Extracellular vesicle (EV) samples isolated from body fluids or conditioned media are heterogeneous in cell origin, membrane compartment origin, size, molecular composition and functional properties. Given the diversity of biological functions and biomedical applications borne by EV, there is an urgent need for efficient and reliable methods of EV characterization and isolation, to improve our understanding on EV and fully exploit their potential. Our group has pioneered the use of cryo-transmission electron microscopy (cryo-EM), immuno-gold labeling and flow cytometry for imaging and quantifying EV. In this presentation, I will first summarize the main contribution of immuno-cryo-EM to the EV field, helping to answer the question “What do contain EV samples?”. Next, I will present recent results and discuss our attempts to identify EV-based biomarkers in sickle cell disease and preeclampsia.

Gene Editing and Targeted Delivery Using Engineered Extracellular Vesicles
Samir EL-Andaloussi, Associate Professor, Karolinska Institutet, Sweden

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication due to their ability to transfer bioactive lipids, proteins and different species of RNA into cells. As such, EVs can be harnessed for the delivery of macromolecular drugs. Adapting EVs for drug delivery requires cellular engineering that allows for selective loading of biotherapeutics inside or on the surface of EVs. This presentation will cover our recent developments in EV engineering, with focus on active loading and functional delivery of gene editing modalities. In addition to addressing PK, PD and functional delivery of EVs in normal and diseased mice, strategies to enhance tissue targeting and extending plasma half-life of circulating EVs will be discussed.

Targeting Hepatocytes with the Lipid Nanoparticular System in Fatty Liver Disease
Jiong-Wei Wang, Assistant Professor, National University of Singapore, Singapore

Non-alcoholic fatty liver disease (NAFLAD) is a spectrum of chronic liver disease caused by excessive fat accumulation in the liver, with a prevalence of up to 40% in the United States and in Singapore. NAFLAD can develop into a more severe form, non-alcoholic steatohepatitis (NASH), characterized with liver inflammation and fibrosis, and ultimately cirrhosis and liver cancer. Currently, efficacious drugs reversing the various forms of this disease are not yet available. Here in this talk, I will discuss our recent study that employs the lipid nanoparticles (LNP) for targeted inhibition of a clinically relevant sphingolipid metabolic pathway in the hepatocytes with siRNA. With this LNP-siRNA approach, we achieved effective lowering of both hepatic and circulating ceramides and improved therapeutic indexes in animal models of non-alcoholic fatty liver disease. Apart from biochemical and histological evidence, the therapeutic efficacy was also confirmed by our recently developed myeloperoxidase-responsive MRI imaging. This proof-of-concept study demonstrates the feasibility of LNP siRNA system for the treatment of metabolic diseases.

The EVs and the Secretome: Challenges and Obstacles with EV-based Therapeutics Development
Mario Gimona, Head of Manufacturing, GMP Unit, Paracelsus Medical University Salzburg, Austria

Vesicle-based therapy is increasingly being pursued as a safe, cell-free strategy to combat various immunological, musculoskeletal and neurodegenerative diseases. Extracellular vesicle (EV)-enriched preparations obtained from multipotent mesenchymal stromal cells (MSCs) are of particular interest for therapeutic use since they may convey anti-inflammatory, anti-scarring and cyto-protective activities to the recipient cells and tissues. MSCs secrete a variety of bioactive autocrine and paracrine factors including cytokines, chemokines, extracellular matrix proteases, EVs and growth factors. The MSC-derived secretome is thus a complex mixture of proteins, lipids, macromolecular assemblies and vesicular structures, including various types of EVs. The current knowledge on the dynamic structure of vesicular secretome and its interrelated functions is fragmented and the understanding of the nano- and mesoscale properties is limited. The heterogeneity of the vesicular secretome preparations hampers dose finding, the importance of EV uptake and natural tropism remains to be solved, and technologies to better investigate secretome dynamics and corona/halo formation must be developed. Importantly, activities must be initiated that attempt to quantitatively describe one or more biological activities present in/on EVs that are beneficial in elucidating the complex mode-of-action of the vesicular secretome and to assist in the required dose-finding for pre-clinical and clinical application.

Steps to Translate EV-Based Therapeutics into Clinics
Eva Rohde, Head of Department for Transfusion Medicine, Director of GMP Laboratory, Paracelsus Medical University Salzburg, Austria

Extracellular vesicles from mesenchymal stromal cells (MSC-EVs) are being investigated as novel promising biologic drug candidates. In her talk, Eva Rohde will highlight the pharmaceutical and clinical development of naïve umbilical cord-derived MSC-EVs as candidate therapeutics since about 10 years. Therapeutic concepts for selected target diseases are based on observed anti-inflammatory, anti-fibrotic, and neuroprotective biological activities of MSC-EVs in various in vitro and in vivo models. Non-clinical safety and efficacy data required for clinical evaluation include pharmacodynamic, pharmacokinetic and toxicological results for the selected routes of administration and specific indications. Pilot clinical experiments to confirm the EV-associated prevention of secondary tissue damage following acute traumatic injury or chronic (neuro)degeneration and the developmental road for first-in-human clinical trials are presented.

Clinical Potential of MSC-EVs and Translational Challenges
Bernd Giebel, Group Leader, Institute for Transfusion Medicine, University Hospital Duisburg-Essen, Germany

Synthetic Lipid Nanocarriers: From Liposomes to Lipid Nanoparticles
Raymond Schiffelers, Professor of Nanomedicine, University Medical Center Utrecht, Netherlands

2023 marks the sixtieth birthday of ‘liposomes’. These structures were shown to spontaneously form when amphiphilic lipids were brought in an aqueous environment. In the next decades, many lessons were learned on the biomedical applications of liposomes on the effects of liposome size and surface charge, on steric stabilization and lipid composition, on targeting and immune evasion. Tens of liposome-based products are currently marketed or in advanced stages of clinical development. The most recent addition to the clinically used nanocarriers are the lipid nanoparticles for the delivery of nucleic acids. Building on the foundations of liposomes, these newest type of particles have been successfully used in the siRNA-product patisiran. This recipe also formed the basis for the mRNA COVID-vaccines. These successes paved the way for many new applications where synthetic lipid nanoparticles enable new nucleic acid modalities for advanced therapeutic interventions.

LNP 2.0+: Gene Medicine Beyond Covid
Fred Campbell, Scientific Director, NanoVation Therapeutics UK, United Kingdom

mRNA vaccines against SARS-Cov2 have emphatically shown lipid nanoparticles (LNPs) can deliver safe and effective gene therapies in humans, can be developed at unprecedented speed, manufactured at scale and all at reasonable cost. A genetic healthcare revolution is now upon us. How quickly these new, diverse and life changing treatments come to market, however, will require many outstanding challenges to be met (both scientifically and commercially). Here, I will briefly outline NTx’s strategic and scientific efforts to accelerate the development of new LNP-based gene medicines and democratize healthcare for all.

Screening and Scale-Up Lipid Nanoparticles Using a High-Throughput Microfluidic System
Nan Zhang, Assistant Professor, University College Dublin, Ireland

Drug delivery systems in forms of nanoparticles have a broad spectrum of applications in area of gene therapy, cell therapy, and vaccine development. Lipid nanoparticles (LNPs) or liposomes that consist of a lipid bilayer and have a hollow structure are the most widely used drug carriers for nanomedicines. It is possible to encapsulate a variety of low molecular compounds (e.g., drugs, peptides, antibody, plasmid-DNA, mRNA, siRNA etc.) in the lipid bilayer membrane or to encapsulate them into the hollow structure. However, significant concerns remain on efficacy, safety, consistency, scale-up of manufacturing and stability when translating LNPs from formulation into clinical application. LNPs’ properties, e.g., size, size distribution, charge, drug encapsulation, transfection efficiency etc., can significantly affect the bio-distribution and pharmacokinetics of the drug to be delivered. In order to obtain LNPs with expected characteristics, microfluidics that exploit fluidic control to synthesize LNPs have grown in acceptability and applications in laboratory and larger scales due to offering a reproducible and robust manufacturing through the precise control of flowing conditions. In the present talk, the author will share the newly developed high throughput microfluidic system for laboratory screening and formulation of nanoparticles based on a novel aerofoil microfluidic mixing system. The formulation based on low, medium and high flow rates will be demonstrated. The automation of the system will be introduced for industrial scale formulation. The device has demonstrate the strong potential for low volume screening, medium volume scale up and high volume GMP production for nanoparticle development.

Gene Silencing in HSPCs with an Apolipoprotein A1-based Nanodelivery Platform
Roy van der Meel, Assistant Professor, Department of Biomedical Engineering, , Eindhoven University of Technology, Netherlands

Nucleic acid therapeutics are revolutionizing healthcare via gene inhibition, addition, replacement or editing. However, nucleic acid-based drugs require chemical modifications and sophisticated nanotechnology to prevent their degradation, reduce immunostimulatory effects, and ensure intracellular delivery. Lipid nanoparticle (LNP) technology is the current gold standard platform that enabled the clinical translation of the first siRNA drug Onpattro and the COVID-19 mRNA vaccines. Nevertheless, current LNP systems are mostly suited for vaccine purposes following local administration or hepatic delivery following intravenous administration. To unleash RNA’s full therapeutic potential, we introduce modular nanoplatform technology for systemic nucleic acid delivery to immune cells in hematopoietic organs using apolipoproteins.

Process Analytics for Nanotherapeutics Manufacturing, Enabled by Novel Inline Nanoparticle Sizing Technology
Rut Besseling, Scientific Director, InProcess Instruments BV, Netherlands

The rise of nanotherapeutics -and nano based products in general- has increased  the need for better characterization, understanding and control of nano-particle (NP) suspensions in synthesis and processing. In Pharma this is emphasized by regulatory drives for Quality by Design (QbD) and process monitoring via Process Analytical Technology (PAT). But continuous measurement of NPs like LNPs or liposomes using PAT integrated in the production process, has so far hardly been possible. Specifically, NP size characteristics -critical for endproduct quality- could previously not be measured inline due to the challenges regarding e.g. suspension turbidity and ‘agitation’ for standard methods (e.g. standard Dynamic Light Scattering, DLS). This has been an important barrier in successful development, scale-up and manufacturing of nanotherapeutics. Here I will describe applications of a new non-invasive PAT instrument – the NanoFlowSizer (NFS)- providing continuous, real-time, in-process size characterization of NP suspensions, over a vast range of suspension turbidities and flow conditions. The NFS resolves scattered light signals as function of depth in the suspension (‘Spatially Resolved Dynamic Light Scattering’), which provides the key solution to the turbidity/flow challenges mentioned for inline NP sizing. After a brief technology intro, several examples are described highlighting the NFS’ novel process monitoring capabilities. These include monitoring of solid NP syntheses (SiO2/TiO2/Ibuprofen), pharmaceutical nano-emulsion homogenization, processing of protein solutions and, lastly, continuous manufacturing of liposome formulations, including unique PAT-based automated process control for ‘on target’ production.

Anti-Aging MSC-EVs
Sai Kiang Lim, Research Director, Institute of Medical Biology, A*STAR, Singapore

Aging is believed to be, in part, a result of the decline in functional adult stem cells that are crucial for maintaining tissue homeostasis. We had previously postulated that MSC EVs could help preserve the balance of cell numbers by activating survival signaling pathways. Indeed, our research showed that EVs derived from MSCs effectively reduced senescence both in cell cultures and in live organisms, leading to an improvement in the natural lifespan of aging mice.