O4 - Enfermedades humanas

Engineered extracellular vesicles with immunomodulatory and targeting functions

Grosso, Tomás Martín ¹ - Russo, Constanza ¹ - Schatcher, Julieta ² - Álvarez, Cora ² - Pozner, Roberto ³ - Witwer, Kenneth ⁴ - Ostrowski, Matías ¹ - Pérez, Paula Soledad ¹

1) Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (INBIRS), Universidad de Buenos Aires (UBA), CONICET
2) Instituto de Química y Físico-Química Biológicas “Prof. Alejandro C. Paladini”, Universidad de Buenos Aires (UBA), CONICET
3) Instituto de Medicina Experimental (IMEX), Academia Nacional de Medicina (ANM), CONICET
4) Department of Comparative and Molecular Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Contacto: grossotomas@gmail.com

Extracellular vesicles (EVs) are emerging as versatile platforms for immunotherapy due to their natural biocompatibility and capacity for molecular engineering. Here, we report the generation of multifunctional EVs derived from HEK293T cells engineered to express human ectonucleotidases (EV-ADO), with or without a selectin ligand (EV-ADO-P). Ectonucleotidases sequentially catalyze the hydrolysis of pro-inflammatory ATP/ADP into immunosuppressive adenosine, while the adhesion molecule facilitates targeting to inflamed or thrombotic tissuesvia selectin binding.

HEK293T cells were genetically modified using lentiviral vectors to stably express human ectonucleotidases and the selectin ligand. EVs were purified from cell culture supernatants by differential ultracentrifugation. We validated the expression and enzymatic activity of ectonucleotidases in both cells and EVs and confirmed selectin ligand enrichment in EV-ADO-P. Functional assays demonstrated that EV-ADO and EV-ADO-P significantly suppressed inflammatory responses in vitro, including reduced TNF secretion and enhanced IL-10 production in macrophages, inhibition of oxidative burst in neutrophils, and decreased T cell proliferation. Platelet aggregation assays revealed that EV-ADO hydrolyzed ADP and prevented platelet activation, suggesting potential antithrombotic effects.

Preliminary in vivo biodistribution studies in a murine model of TPA-induced ear inflammation showed that EV-ADO-P accumulated more efficiently in inflamed tissue, as compared to EV-ADO, which was predominantly sequestered in the liver. These findings suggest that the incorporated adhesion molecule enhances EV targeting to sites of inflammation.

Altogether, our results demonstrate the feasibility of engineering EVs with dual functionality: enzymatic immunomodulation and selective tissue targeting. This strategy holds promise for the development of next-generation EV-based therapeutics for inflammatory and thromboinflammatory diseases.

Palabras clave: ENGINEERED EXTRACELLULAR VESICLES, ECTONUCLEOTIDASES, INFLAMMATION, IMMUNOMODULATION, TARGETED THERAPY, ADENOSINE SIGNALING