Extracellular Vesicles & Nanoparticle Therapeutics Europe 2022 Agenda

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. 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.

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.

Networking Lunch

Investigating Extracellular Vesicle-based Therapeutic Options Using Alternatives for Animal Models
Bas WM van Balkom, Assistant Professor, University Medical Center Utrecht, Netherlands

Mesenchymal stromal cell (MSC)-derived small EVs (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, like elucidation of the mode of action (MoA) and formulation of safety and release criteria. sEVs tend to accumulate in the liver and at sites of injury. Biodistribution knowledge is crucial to assess MoA, efficacy and safety, and can be obtained using labelled sEVs in animal models, which come with ethical concerns, are time-consuming and expensive, and do not represent all human physiological processes equally well. Our models recapitulate the efficacy and biodistribution of MSC-sEVs as observed in animal models and provide alternatives for animal experiments. Their systemic or human background allows for in-depth analysis of the MoA and identification of potential side effects and accelerated development of EV-based therapeutics.

Mid-Afternoon Coffee and Tea Break and Networking

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.

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.

Beer and Wine + Networking

Morning Coffee, Tea and Networking

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.

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.

Networking Lunch