Zebrafish for exploring Exosome-like Nanovesicle THerapeutic potential – ZENiTh

Extracellular vesicles (EVs) like exosomes generate high expectations from the biomedical world as to their future use as non-invasive diagnostic and prognostic biomarkers and natural therapeutic vectors as illustrated by the numerous EV-based biotechs founded and by an expected global EV market size of $ 2,28 B by 2030. While the methods of isolation, production and functionalization of EVs seem mature enough to reach a GMP grade, clinical application of EVs comes up against a gap of knowledge of the characteristics of EVs in vivo. The origin and half-life of EVs, their ability to cross biological barriers, their extremely rapid clearance or their effectiveness in reaching a cellular target are all functional parameters which are not clearly established in vivo. However, these parameters are crucial in pre-clinic studies to exploit and improve the therapeutic potential of EVs. This lack of data was primarily due to the impossibility of tracking EVs in real time and at the scale of the single vesicle of 100nm in murine models. Most studies therefore relied on in vitro approaches which only partially reflect the behavior of EVs in vivo.
The goal of our project is to establish the zebrafish as a relevant vertebrate model organism to study in preclinic and in vivo the therapeutic potential of Evs. Our project is based on the already published and functional zebrafish model that we have developed. This is the first model organism allowing the in vivo investigation of the biology of endogenous EVs and functionalized EVs at the scale of the single vesicle and in real time (Verweij et al Dev Cell 2019). To achieve the following three objectives, our consortium brings together experts of EVs from different tissues (vascular endothelium, brain and muscle) and a company developing functionalized EVs for clinical applications. In order to establish the relevance of our model, we propose the following objectives.
1) We will generate a catalog of the main characteristics of the sub-populations of endogenous EVs secreted in zebrafish by the different cell types mentioned above. And we will compare them with the data obtained in murine and human in vitro models by the different partners.
2) We will map the spatiotemporal bio-distribution of endogenous VEs and we will compare it in parallel with those of exogenous EVs produced by the different partners and injected into the corresponding zebrafish models.
3) Through close collaboration with the industrial partner, we will first establish the proof of concept of the use of zebrafish for preclinical tests of functionalized EVs. Then we will develop together new functionalities to improve EV targeting and assess in real time drug delivery by EVs. This part may in particular take the form of co-supervision of PhD student with the company (CIFRE).

Completion of this project will establish the zebrafish as a pre-clinical reference model to refine the use of EVs as biomarkers and to assess their effectiveness as therapeutic vectors. IN this model we will compare the features and functions of EVs of zebrafish and murin and human origin to lift the species barrier and to map precisely the sites of accumulation and the targets EVs in vivo. On this groundwork, we will assess the features and potency of functionalized EVs to stengthen the relevance of this model to test therapeutical EVs in vivo. Ultimately, this model will serve as a basis to establish a service platform that would propose to new partners to test their functionalized EVs and in parallel to identify by drug-screening patentables molecules able to improve the therapeutic use of functionalized EVs.