Modulação da bioactividade e da capacidade de direccionamento de vesículas extracelulares para o tratamento da diabetes

EVOLUTION will engineer EVs with miRNAs and peptides to improve their bioactivity and targeting capacity, respectively. To accomplish this, we devised the following objectives: Objective 1: engineering EVs with GLT-protective miRNAs and ß cell targeting peptides: In brief, we will modify EVs with miRNAs known to rescue ß cells from GLT-induced cell death in vitro (bioactivity) and with a ß cell targeting peptide (GLP1-RA) using methods established by the team. We will engineer the EVs using the top three miRNAs identified in our screening (SI 1) and two GLP1-RA-targeting peptides (one that activates the receptor (Exendin 4) and one that does not (Exendin-(9-39)) (Göke et al., 1993). The engineered EVs will be characterized using methods previously reported by us (e.g. NTA analysis, presence of classical EV markers and absence of contaminants, surface charge before and after modification, stability in solution and upon freezing) (de Abreu et al., 2021). Objective 2: in vitro evaluation of engineered EVs: we will evaluate the capacity of the engineered EVs to rescue human ß cells from GLT-induced cell death (GICD). To accomplish this, we will test the EVs engineered in objective 1 in human ß cells (EndocBH3 and iPSC-derived ß cells) and control cells (e.g., fibroblasts). In brief, human ß cells will be exposed to engineered EVs and their uptake, internalization and intracellular trafficking analyzed using techniques reported by us (fluorescently-labelled EVs, markers for the different intracellular compartments involved in trafficking and confocal microscopy) (Fernandes et al., 2022). In addition, we will also evaluate their capacity to rescue human ß cells from GICD. Based on the results of these experiments, we will rank and select the best formulations for further in vivo studies. Objective 3: in vivo evaluation of engineered EVs in the survival of ß beta cells upon transplantation: we will use human islets or islet-like iPS-derived cells and study their survival in vivo. In brief, obese immune-compromised mice will be treated with low, medium and high dose of alloxan to mimic different levels of ß cell failure due to the destruction of native ß cells. Fourteen days post alloxan treatment, different concentrations of human islets will be transplanted intramuscularly and their survival and function evaluated 14 and 28 days later. Basal (before islet transplantation) glucose levels will be measured to determine hyperglycemic state and intraperitoneal glucose tolerance test (IPGTT) will be the readout used to assess ß cell function. After confirming the limited survival of the transplanted human islets, we will test the therapeutic effect of our engineered EVs. Three different doses of engineered EVs will be delivered intramuscularly (peri-implant) and cell survival will be analysed in animals treated with our engineered EVs (scramble-engineered EVs will be used as control). EVOLUTION will deliver a highly modular system where each component has a major role in restoring ß cell function. More importantly, the proposed platform can be tailored using different EV sources, miRNAs and/or targeting moieties.