Conference Registration, Materials Pick-Up, Morning Coffee and Tea

Morning Coffee and Tea Break and Networking

Extracellular Vesicle-mediated RNA Delivery: From Mechanistic Insights Towards Therapeutic Applications
Pieter Vader, Associate Professor, University Medical Center Utrecht, Netherlands

Extracellular vesicles (EVs) form an endogenous system for information transfer between cells. Since the recent discovery that EVs are also capable of functionally transferring RNA molecules, they are increasingly being considered as therapeutic RNA delivery systems. Despite extensive research into the engineering of EVs for RNA delivery, our understanding of the pathways and mechanisms regulating EV-mediated RNA delivery and processing is limited. Moreover, little is known about how their intrinsic RNA delivery efficiency compares to current synthetic RNA delivery systems. Here, we developed a novel CRISPR/Cas9-based reporter system in which eGFP expression is activated upon functional delivery of targeting single guide RNAs (sgRNAs) that allows study of EV-mediated RNA transfer at single-cell resolution. We employed this system to compare the delivery efficiency of EVs to clinically approved state-of-the-art DLin-MC3-DMA lipid nanoparticles and several in vitro transfection reagents. We found that EVs delivered RNA several orders of magnitude more efficiently than these synthetic systems. In addition, we prepared EV-liposome hybrid nanoparticles and evaluated them as siRNA delivery systems in terms of cellular uptake, toxicity, and gene-silencing efficacy [4]. We show that hybrids combine benefits of both synthetic and biological drug delivery systems and might serve as future therapeutic carriers of siRNA. Our data underline the potential of EVs as RNA delivery vehicles and highlight the need to study the mechanisms by which EVs achieve their efficiency. This may in turn contribute to the development of more efficient EV-based RNA delivery systems and accelerate clinical adoption of therapeutic EVs.

Extracellular Vesicles from Skin Selected Amniotic Fluid MSCs Improve Wound Healing in vitro
Mattis Holma, R&D Scientist, Amniotics, Sweden

Neonatal mesenchymal stem cells (MSC) derived from term amniotic fluid (TAF) were sorted with a skin specific marker identified with RNASeq data from TAF-MSC clones. One of these markers was used for cell-sorting using Tyto MACSQuant cell sorter and the positive cell population was expanded to final product stage (CutiStem). CutiStem cells were starved and secretome was collected. Extracellular vesicles were purified by ultracentrifugation and characterized. A THP-1 assay showed that EV derived from CutiStem reduce inflammation by downregulation of the NK- ?B. Furthermore, a scratch assay with human dermal fibroblasts revealed that EV derived from Cutistem also increase wound healing in vitro.

Networking Lunch

Production and Biological Activity of RBC-derived Extracellular Vesicles Containing STING Agonist (ADU-S100) Using ERYCEV Platform
Karine Senechal, R&D Project Manager, Erytech pharma, France

RBC-derived extracellular vesicles (RBCEVs) represent a new valuable Drug Delivery System (DDS), due to their intrinsic properties (small size, natural targeting to immune cells, biocompatibility…) and ease of production[1]. This study evaluates the feasibility of producing functional cargo-loaded RBCEVs from ERYCAPS pre-loaded RBCs[2]. To this end, RBCs encapsulating ADU-S100 or empty RBCs processed with ERYCAPS (hypotonic dialysis encapsulation technology) were subjected to physical vesiculation to produce ERYCEV-STINGa and ERYCEV respectively. The RBCEVs produced and purified, analyzed by NTA, revealed a uniform vesicles population of 95 nm mean size and 32 µg/mL mean ADU-S100 concentration in ERYCEV-STINGa. Luminal EVs markers TSG101 and ALIX were successfully detected by western-blot. Additionally, FACS analysis of surface markers showed moderate CD81 and CD235a levels and low CD47 and PS levels. Functionality of produced RBCEVs were successfully demonstrated in vitro. First, uptake of PKH67-labelled ERYCEV was confirmed in both THP-1 monocytes and EMT6 murine tumor cells. Moreover, THP1-derived macrophages phagocytosed pHrodo-labelled ERYCEV-STINGa, which successfully activated STING pathway, demonstrated by luciferase activity in THP1-Dual™ cells and type I interferon (IFN-beta) production. This first proof of concept to produce functional ERYCEV-STINGa supports the development of ERYCEV platform by further investigating pharmacologic properties in vivo and other cargo loading.

Mid-Afternoon Coffee and Tea Break and Networking

The Importance of Suitable Media for Exosome Production
Joo Youn Lee, CTO, Xcell Therapeutics Inc.

Mesenchymal stem cells (MSCs) are commonly used as a source of regenerative medicine due to their strong immunosuppressive and regenerative effects. Paracrine effect is one of the key mechanisms of MSC. Recently, it has been shown that the extracellular vesicles (EVs), which include exosomes and microvesicles, orchestrate the principle mechanisms of action of MSCs after infusion. These vesicles are involved in cell-to-cell communication, cell signaling, and altering cell or tissue metabolism at short or long distances in the body. Exosomes are nanoscale-specific lipid bilayer vesicles secreted by cells, with a diameter of 30-150 nm. The use of MSC-derived exosomes may provide several advantages over their counterpart live cells (MSC), potentially reducing undesirable side effects including immune reaction and low stability. However, for effective pharmaceutical research, only the target cell-derived exosomes must be produced and isolated, but contamination of animal/human component-derived exosomes already contained in cell culture media has reduced the purity of exosomes. Therefore, this study aims to introduce the ideal culture environment for the production of exosomes by cultivating MSCs in a serum-free chemically defined media (CDM) and confirming the characteristics of secreted exosomes. MSC derived exosome productivity was compared using several media including CDM. Wound healing assay and angiogenesis assay were conducted to evaluate the characteristics of the produced exosomes.

RNA Printer to Answer Increasing Demands for GMP-Grade mRNA Material
Martin Winter, Senior Director Strategic Marketing, CureVac RNA Printer GmbH, Germany

New approaches for personalized cancer therapies and fast responses to pandemics require rapid manufacturing of mRNA material. GMP-grade mRNA manufacturing normally is time consuming, expensive and requires extensive and specialized laboratories as well as highly qualified staff. To answer this demand, CureVac has developed an mRNA manufacturing platform, the mRNA Printer, for automated and integrated manufacturing of GMP-grade vaccines and therapeutics, engineered in collaboration with Tesla Automation. The system covers all steps for standardized and fast production of mRNA material and herewith facilitates broad access to mRNA technology and speeds up the process from research to therapy. The RNA Printer is capable to produce several grams of LNP-formulated mRNA within a few weeks and can be switched from one mRNA product to another in a short time. Due to its modular design, the platform is mobile and will therefore enable local manufacturing of mRNA medicines.

Networking Reception with Dutch Beer Sponsored by Emulate: Engage and Network with Colleagues

Close of Day 1 of the Conference

Morning Coffee, Tea and Networking

Cost-Effective GMP Production of Extracellular Vesicles
Marcin Jurga, Chief Scientific Officer, EXO Biologics SA, Belgium

The therapeutic effect of Extracellular Vesicles (EVs) has been demonstrated in numerous non-clinical studies. The efficacy and safety of EVs have been proven in different animal models including treatment of Bronchopulmonary Dysplasia (BPD), among other indications. Currently, the most advanced projects are being translated into the clinic to confirm the safety and efficacy of EVs in humans. This requires pharmaceutical grade EVs, manufactured to be compliant with Good Manufacturing Practice (GMP). In the emerging EV field, there are various methods and instruments qualified for the production of GMP-grade EVs and most of the manufacturers use their own proprietary techniques. It is important for a biotech company to develop a robust and cost-effective process, capable of producing EVs on different scales, starting from low-volume, which can be used in the non-clinical phase, and moving on to a large-scale production for clinical trials and ultimately commercial drug manufacture. Considering that EVs as biological drug products are defined by their production process, any major changes in the manufacturing method can have an impact on the drug product identity and activity. EXO Biologics has developed a GMP-approved custom platform for EV manufacturing, based on a closed and scalable bioreactor system. The production process is flexible and allows for a manual production with a low-volume bioreactor during the early phases of drug development. Subsequently, the process can be scaled up to a fully automated large-volume system for commercial manufacturing, without changing the characteristics of the product. The platform can be used for production of multiple EV types, including the 1st generation of non-modified EVs, which are currently being manufactured for clinical tests in BPD, and also engineered EVs designed for enhanced activity and drug delivery.

Mesenchymal Stromal Cell-derived Extracellular Vesicles as Therapeutic Tool in Neonatology: The Case of Bronchopulmonary Dysplasia
Maurizio Muraca, Professor, Università degli Studi di Padova, Italy

Extreme premature newborns are a fragile population because of immaturity of various organs, requiring variable stays in the ICU. Bronchopulmonary Dysplasia is the most common respiratory disease in babies, resulting in high mortality and high incidence of long-lasting complications and for which no definite cure exists. Treatment with mesenchymal stromal cells (MSCs) and more recently with MSC-derived extracellular vesicles has provided proof-of-principle in animal models and is now entering the clinical phase.   In addition to providing preclinical evidence and to complying with regulatory requirements, the therapeutic approach to this peculiar patient population requires a delicate balance between efficacy and potential risks.

Mid-Morning Coffee, Tea and Networking

From Small Molecules to Proteins and Nanoparticles: Exploring Novel Tools For Intracellular Delivery
Koen Raemdonck, Professor, Ghent University, Belgium

Intracellular delivery of membrane-impermeable cargo (e.g. nucleic acid- or protein-based drugs) provides unique opportunities for cell biology and biomedical applications. A wide variety of intracellular delivery technologies is available to date, such as carrier- and membrane disruption-based approaches. However, existing tools are often suboptimal and alternative technologies that merge delivery efficiency, biocompatibility and applicability remain highly sought after. This presentation will first describe the repurposing of two distinct cationic amphiphiles, i.e. both low molecular weight cationic amphiphilic drugs (CADs) as well as lung surfactant-inspired proteins and peptides, to improve cellular delivery of RNA therapeutics. Both approaches significantly promote cytosolic RNA delivery, albeit by adopting a different mode-of-action for permeabilization of the endosomal and/or lysosomal compartments. In contrast to carrier-based protocols, physical delivery approaches do not rely on the endosomal pathway and allow the direct cytosolic access of cell-impermeable agents. Our group recently developed two innovative physical delivery technologies based on non-viral nanoparticles that can be implemented in cell engineering protocols. One platform (Hydrogel-enabled nanoPoration or HyPore) exploits cationic hydrogel nanoparticles to disrupt the plasma membrane of cells, enabling cytosolic delivery of nanobodies and enzymes. Of note, HyPore-mediated delivery of the neutral MRI contrast agent gadobutrol significantly improved T1-weighted MRI signal intensities in primary human T cells, outperforming state-of-the-art nucleofection. A second platform technology is nanoparticle-sensitized photoporation. By attaching photothermal nanoparticles to the cell surface followed by pulsed laser illumination, transient membrane pores can be generated that allow delivery of biologics into cells with high efficiency. To avoid direct contact of the nanoparticles with cells and the associated regulatory concerns, light-sensitive iron oxide nanoparticles were embedded in electrospun nanofibers. The resulting photothermal electrospun nanofibers (PENs) successfully delivered CRISPR-Cas9 ribonucleoprotein complexes and siRNAs into embryonic stem cells and T cells, while maintaining cellular fitness and phenotype (6). In conclusion, the above described approaches are considered promising concepts towards improved cellular delivery of a wide variety of cargo for biomedical applications.

SPARTA® - Enabling Nanoformulations For Next-Generation Therapeutics
Jelle Penders, CEO & CTO, SPARTA Biodiscovery Ltd., United Kingdom

SPARTA^® (Single Particle Automated Raman Trapping Analysis) is a cutting-edge benchtop instrument that chemically analyses and characterizes single nanoparticles in a fully automated, high-throughput, non-destructive label-free process. Dr. Penders will talk through the benefits of this technique for nanoformulation research as well as industry applications and show the breath of data that can be acquired and analyzed.

Networking Lunch

Periphery-Brain Communication via Extracellular Vesicles
Roosmarijn Vandenbroucke, Associate Professor, Ghent University, Group Leader at VIB, Belgium

Our brain is protected from the periphery via the presence of different brain barriers, including the blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier. While this blocks access to the brain for most molecules, recent research has shown that some extracellular vesicles (EVs) are able to cross these barriers. In this talk, I will focus on the consequences of this phenomenon, including the impact of bacterial-derived EVs on the brain and the use of EVs to deliver therapeutics to the brain.