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The Extracellular Vesicle Foundry

Periodic Reporting for period 1 - evFOUNDRY (The Extracellular Vesicle Foundry)

Reporting period: 2018-09-01 to 2019-08-31

Extracellular vesicles (EVs) are biological messenger nanoparticles involved in cell-to cell communication. To study their structure and content can be exploited to improve fundamental aspects of everyday life, from diseases diagnosis and therapy to environment and public health. However, EV understanding and manipulation ability are immature. evFOUNDRY aims at advancing EV field by developing a technology that streamlines the production of high-grade therapeutic EVs from sustainable sources (milk and parasitic worms), leading to EV bioprocessing. This is necessary for effective EV medical translation (large clinical trials and regulatory initiatives) and opens to new EV applications (nanotechnology, nutraceuticals, cosmeceuticals, veterinary).

evFOUNDRY specific objectives are:
1) to produce pure, stable and reproducible sets of nanosized EVs from bovine milk and Ascaris suum incubation medium;
2) to unveil the interaction of EVs with surfaces and exploit it to develop innovative, high-performance materials for separation of EVs;
3) to design and fabricate a multi-modular device for automatic separation and grading of EVs.
All the planned activities are within three established main actions:

- Action 1. Setting EV sources and EV quality standards. This action featured: (a) separation of high-grade nanosized EVs from bovine milk and Ascaris suum incubation media by the highest state-of-the-art batch low-volume methods (these fluids are very attractive “infinite” sources of EVs because they are cheap, widely available, sustainable and ethically compliant and their EVs have biological activity and medical potentiality); (b) determination of compositional, structural and physical-chemical properties of the separated EVs; (c) determination immunomodulatory properties of the separated EVs.

- Action 2. Development of enabling knowledge, materials and methods for reproducible separation of nanosized EVs (from the featured fluids) based on EV size and/or membrane properties and suitable to be operated under continuous or pulsed flow.
This action featured:
(a1) Identification of architectures to separate EV populations by size that provide the best characteristics in terms of cost/benefits, integrability into microfluidic platforms, reproducibility, robustness and scalability. (a2) films made of lipid bilayers of planar structure and of bended cubic structure were successfully prepared and characterized. (b) Studies on how EVs and EV fluids interact with surfaces have started. (c) Assessment of methods for EV grading which are quick, accessible, robust and suitable for in-line sampling quality control.

- Action 3. Realization of a modular system for robust separation and grading of immune modulatory EVs under continuous or pulsed flow. The first prototype system was designed in three modules (1) a loading module, (2) separation module based on size, and (3) multi-characterization module. Analysis of operational and design challenges has been determined for each module as well as module specification and the evaluation of the hardware setting that will be part of the integrated device.

It is to note that each partner involved in evFOUNDRY hired young researchers (nine persons, from Ph.D students to Post-Docs, see picture in the attachment). Their skills and enthusiasm fundamentally contributed to the first successful year of activity.
"The above technical actions were constantly complemented by the planned actions for achieving impact and supported by proper management. This to exploit evFOUNDRY results and to obtain the highest impact both in the scientific field and on society. Made progresses that go beyond the state of the art include:
- setting of quality standards and Standard Operating Procedures (SOPs) for immunomodulatory nanosized bovine milk EVs and Ascaris EVs;
- augmented and innovative methods for EV physicochemical characterization and grading;
- innovative tools based on Deterministic Lateral Displacement (DLD) devices to separate EVs depending on their size.

In the first reporting period:
- two papers have been published on peer review scientific journals (https://www.mdpi.com/2073-4409/7/12/273 Repository link: http://hdl.handle.net/11379/515026; one featured in the journal inside cover https://onlinelibrary.wiley.com/doi/10.1002/admt.201970048 Repository link: https://zenodo.org/record/3352780#.XZHEh1Uzapo) - more than 5 papers are under review.
This dissemination activity has been complemented with:
-the participation to the foundation of the Italian Society of Extracellular Vesicle (EVIta https://www.evitasociety.org) and of the Danish Society of Extracellular Vesicles (DSEV) https://www.dsev.dk/)
- the organization of the first ""FET open projects cluster event"" (https://www.evitasociety.org/congressi/convegno)
- 17 presentations at international meetings.
evFOUNDRY members organized and participated also to several communication events to the general public and to stakeholders and organized several training workshops on the (or inspired by) the project featured science and technology, directed to both internal and external members of the consortium.

evFOUNDRY is expected to set the seminal concepts, science and technology for EV bioprocess and product development. This new possibility will consolidate on a mid-term existing EV research initiatives (EV structure and biology, EV-based therapeutics for personalized medicine, regenerative medicine and immunotherapies)) and foster new radical ones (cosmeceuticals, nutraceuticals and veterinary), unlocking the EV translational cycle and the pace of its adoption in the next 5-10 years.
In particular, evFOUNDRY technology will be decisive towards establishing principles for EV large-scale production. Results will be applied to achieve good manufacturing practice (GMP), a fundamental step to translate EV based therapeutics from “lab bench” to clinical application in the next future.
In addition, dairy science, animal healthcare and parasitology, will benefit a direct impact. By evFOUNDRY, bovine milk is likely to become the first source of EV-based pharmacological effectors (dairy market > 60 billion Euro/year in EU, worth yearly, Eurostat). Parasitic worms result in major economic penalty in livestock production in Europe and have by now developed multi-drug resistance. When largely available in a controlled manner, parasitic EVs will hold the potential to be the needed novel approach for sustainable parasite control.
As parasitic worms are a major public health issue in less developed areas of the world the results of evFOUNDRY will also contribute in fighting such neglected diseases affecting a billion people.
More generally, by enabling large availability of standard EV samples, evFOUNDRY will address a key feasibility step in future research towards deciphering intercellular and inter-organism communication. This will impact next studies on microbiome and superorganism biology – which will be crucial to address global issues, such as water pollution and decontamination, antibiotic resistance, non-communicable diseases (NCDs) or food safety – contributing to reshape our perspective on life science, the environment and public health."
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