Laboratoire Matière et Systèmes Complexes

There will be a live diffusion with Zoom :

Monday February 6th, 11h30 in room 454 A.

Please contact Jean-Baptiste Manneville to attend the visio-seminar.

Two talks of 30 min, Sheryl Bui then Anna Sebbagh.

Virus-free Method to Control and Enhance Extracellular Vesicle Cargo Loading and Delivery

Sheryl Bui

Co-authors, Julia Dancourt, Gregory Lavieu.

MSC-Med

Université Paris Cité, INSERM U1316, CNRS UMR7057, Paris, France.

Extracellular vesicles (EVs) – including exosomes and microvesicles – are involved in cell-cell communication. EVs encapsulate different types of molecules such as proteins or nucleotides and are long-lasting contenders for the establishment of personalized drug delivery systems. Recent studies suggest that the intrinsic capacities for uptake and cargo delivery of basic EVs might be too limited to serve as a potent delivery system. Here, we develop two synergistic methods to respectively control EV cargo loading and enhance EV cargo delivery through fusion, without requirement for any viral fusogenic protein. Briefly, cargo loading is enabled through a reversible drug-inducible system that triggers the interaction between a cargo of interest and CD63, a well-established transmembrane EV marker. Enhanced cargo delivery is promoted by overexpressing Syncytin-1, an endogenous retrovirus envelop protein with fusogenic properties encoded by the human genome. We validate our bioengineered EVs in a qualitative and quantitative manner. Finally, we utilize this method to develop highly potent Killer EVs, which contain a lethal toxin responsible for protein translation arrest and acceptor cell death. These advanced methods and future downstream applications may open promising doors in the manufacture of virus-free and EV-based delivery systems.

Characterization of the potential of turbulence-primed adipose stromal cells-derived extracellular vesicles for the treatment of radiation colitis and perianal fistulizing Crohn’s disease

Anna Sebbagh

MSC-Med

Université Paris Cité, CNRS UMR7057, Paris, France.

Crohn’s disease is a chronic inflammatory bowel disease with no curative treatment. Fistulae are devastating complications of Crohn’s disease where abnormal channels form between two organs or between one organ and the skin. Crohn’s fistulae are very hard to treat ; and recur often. Radiation colitis occurs as a complication of anti-cancer radiotherapy. There are no curative treatments to this day. Extracellular vesicles (EV) are membrane-bound particles with sizes ranging from a few tenths of nanometers to a few micrometers, which are secreted by cells and contain lipids, proteins and nucleic acids. In this PhD project, we aim to investigate whether EV produced from adipose-derived stromal cells (ADSC) could have a therapeutic potential in the context of perianal fistulizing Crohn’s disease and radiation colitis. EVs were produced from primary ADSCs by exposing the cells to a turbulent flow in stirred bioreactors, and concentrated by either tangential flow filtration, ultracentrifugation, size exclusion chromatography, or a combination thereof. The effect of EVs on cell survival and metabolic activity was investigated by Alamar blue assays on starved colon cells treated with either saline or EVs. We found that EVs concentrated by tangential flow filtration and/or ultracentrifugation significantly increased metabolic activity compared to saline at concentrations higher than 1010 particles/mL. This effect was not observed for EVs concentrated by tangential flow filtration plus size exclusion chromatography. To study whether EVs modulate inflammation, the secretion of pro-inflammatory cytokines by colon cells was measured after stimulation with TNF-alpha and treatment with either saline or EVs. Preliminary results tend to hint at a pro-inflammatory effect of EVs concentrated by tangential flow filtration alone, and immunomodulatory effects of EVs concentrated by tangential flow filtration plus ultracentrifugation or tangential flow filtration plus size exclusion chromatography. The therapeutic potential of EVs was then investigated in vivo in animal models of radiation colitis and perianal fistulizing Crohn’s disease. Radiation colitis was induced in Sprague-Dawley rats by fractionated irradiation (3 x 10 Gy). At days 7 and 11 after the end of the irradiation, the rats were treated by sub-mucosal endoscopic injections of either 2 x 106 ADSC ; 1011 EVs or saline. The rats were euthanized at day 13 for histological analysis. Assessment of the endoscopic score of injury at day 11 evidenced a tendency towards a decreased severity of the lesions in the EV group compared to the saline group. Preliminary histology results tend to indicate a decrease of the length of high-grade colon epithelium injury and a lower infiltration by macrophages in the EV group compared to the saline group. For the rat model of perianal fistulizing Crohn’s disease, inflammation of the colon was induced in Sprague-Dawley rats by rectal instillations of 2,4,6-trinitrobenzensulfonic acid, then perianal fistulae were created surgically by the insertion of iron wires between the rectum and the perianal skin. At day 24, the rats were treated by local injections of either 7 x 106 ADSC, 2 x 1011 EV, or saline. At day 29, the animals were sacrificed and the concentration of pro-inflammatory cytokines was assessed at the level of the external and internal fistula orifices, evidencing a tendency towards a lower expression of pro-inflammatory cytokines in the EV group compared to the saline group. Thus, in this work, we have shown that ADSC EV in vitro potency in terms of effects on cell survival, metabolism and immunomodulation may depend on their method of concentration. In the last semester of my PhD, I will continue the analysis of the histology results from the in vivo experiment in the rat model of radiation colitis ; and focus on comparing the impact of two methods of EV production (exposure to a turbulent flow versus starvation) on their in vitro potency.