The objective of this project is to develop a treatment of heart failure (HF) based on intravenous injections of the extracellular vesicle (EV)-enriched secretome of cardiovascular progenitor cells. The rationale is that the primary mechanism of action of the cells is the paracrine activation of endogenous repair pathways, largely contributed by the EV that they secrete. The intravenous route has been selected primarily because of its lack of invasiveness which opens the possibility of repeating injections. However, EV delivered by this approach are primarily sequestered in lungs, spleen and liver with few of them reaching the heart. Even though this peripheral trapping can induce indirect cardioprotective effects through a modulation of systemic immune-inflammatory responses, experimental studies suggest that this benefit could be optimized by increasing directly the presence of EV in the target myocardial tissue. So far, this cardiac targeting of EV has been achieved by chemical or genetic manipulations of the EV parental cells or of EV directly but although these approaches have been efficacious, their complexity may hamper their clinical implementation. Our objectives are thus (1) to leverage the presence, at the surface of the EV, of glycans amenable to a chemical modification (exofucosylation) allowing them to express a ligand of E-selectin (sialyl Lewis X). This approach has a dual rationale: (i) E-selectin is an adhesion receptor activated on the endothelium in an inflammatory environment, which is a hallmark of HF, and as such it looks a logical target for enhancing homing of EV towards the heart, and (ii) exofucosylation is a technique the feasibility, safety and efficacy of which have already been established and validated in the clinics; (2) to document the biodistribution of these glycan-engineered EV following their direct radio-labeling by gallium-oxine and tracking of their fate by positron emission tomography, and (3) to assess the functional and histological consequences of this cardiac targeting of glycan-engineered EV following their intravenous injection in a murine model of postinfarction left ventricular dysfunction.

The main drawback of the coral-algae model is that the microalgae Symbiodinium are refractory to molecular biology techniques, which seriously restrict any functional exploration. In this proposal, this obstacle is circumvented by the use of a new emerging model, the photosymbiotic worm Symsagittifera roscoffensis. This flatworm establishes a sustainable photosymbiosis with a green alga Tetraselmis convolutae. This symbiotic association has many major assets (i) a complete life cycle of worms amenable in laboratory conditions (ii) a functional approach on the green algae The aim of the ANIMALGA project is therefore to combine a functional approach on the photosymbiont T. convolutae and the use of free-algae juvenile worms as high throughput screenings to isolate Tetraselmis mutants impaired in photosymbiotic relationship. This proposal offers a major breakthrough for exploring marine photosymbioses in particular in the light of oxygen as a key component driving photosymbiosis.

Glycogen and starch define the two most widespread forms of storage polysaccharides with opposing physicochemical properties respectively amorphous and hydrosoluble or solid and semicrystalline. They are nevertheless both composed solely of glucose with the same basic glucosidic linkages, yielding alpha-1,4-linked glucan chains associated through alpha-1,6 branches. The aggregation of polysaccharides within huge semicrystalline granules defines a specific property of the algae and plants that diverged following primary plastid endosymbiosis, as well as of two eukaryotic lines resulting from secondary plastid endosymbioses: the cryptophytes and alveolates (including ancestral ciliates, dinoflagellates, chromerids and apicomplexa parasites). Years ago, the coordinator of this application proposed that insolubilization of starch resulted from the action of isoamylase, a direct debranching enzyme of prokaryotic ancestry absent from the glycogen metabolism enzyme network of eukaryotes. Isoamylase is believed to act by debranching selectively those glucans that prevent crystallisation and aggregation into solid starch during synthesis of otherwise glycogen polysaccharides. This model known as the glucan trimming model is now generally accepted. However, both cryptophytes and alveolates are now proven to lack isoamylase or other forms of direct debranching enzyme, begging the question of how these starch-storing organisms manage to render their storage polysaccharide microgranular solid and crystalline. Hence, we now propose to reveal how alveolates have converged from the synthesis of glycogen to the accumulation of crystalline starch through the use of a completely different molecular toolkit from that operating in plants, but which is more related to those active in fungi and animals for glycogen synthesis. The originality of our approach consists of a novel strategy allowing for previously impossible mutant selection and characterization in the chromerid alveolate alga Chromera velia, an organism previously thought to be genetically untractable. If successful, the analysis of these mutants would define the first case where non-model single cell eukaryotes would have been subjected to a full detailed forward genetic analysis, complete with molecular and biochemical characterization. This would pave the way for comparative biochemistry in the whole eukaryotic tree of life enlightening the evolution of important but ancient biochemical pathways and illuminating mechanistic convergences to get a deeper understanding of eukaryotic metabolism. Knowledge on the mechanism of starch crystallization in alveolates is likely to impact several very different fields of activities such as the breeding of valuable starch polymers for food and non-food end-uses in crops or the use of novel alveolate enzymes for postharvest modification of starch-derived products. With respect to health issues, it is expected to bring novel insights into the pathological aggregation of glycogen into starch in the brains and tissues of humans suffering from Lafora’s disease and to provide novel targets for antiparasitic drugs targeted against starch-storing apicomplexa parasites. Finally, the identification of novel starch binding proteins from Chromera could be of use for the targeting of protein vaccinal epitopes to starch granules.

Polysialic acids (polySia) are natural carbohydrate polymers of growing interest, since they interact with important players of the innate immune system, such as antimicrobial peptides and extracellular histones. Whereas in mammals only homopolymers of N-Acetylneuraminic acid are unambiguously detected, in fish a huge diversity of differently structured polySia chains is described. We want to decipher the fish polysialylation machinery and elucidate the molecular basis underlying this biochemical diversity. Based on our findings, a range of recombinant fish polysialyltransferases will be applied to generate various polysialylated proteins and surfaces with different interacting properties. The resulting sugar chains will be used as molecular anchors for immunomodulatory substances thereby regulating their activity or mediating an accumulation of bioactive polySia-carriers to hot spot of inflammation. Thus, the project opens new avenues to target key mechanisms of the immune system.

Monoclonal antibodies (mAb) used to treat cancers, immune and infectious diseases represent a multibillion dollar industry. Chinese Hamster Ovary cells are currently the gold standard. However, high expense, potential virus contamination and mAb heterogeneity represent major drawbacks that drive scientists to seek alternatives. Among these, the diatom P. tricornutum represent an option because of (i) its robustness in industrial process, (ii) its ability to be cultivated in mixotrophy, (iii) its genome availability and possibility to be genetically engineered. The proof of concept in P. tricornutum has already been exemplified through the production of a glycosylated mAb. A human-like N-glycosylation is now required for mAb optimal safety and efficacy when used in human therapies. Therefore, we propose to engineer the N-glycosylation pathway of P. tricornutum by gene silencing and expressing heterologous enzymes, thus allowing galactosylated algae-made mAb production.