Genetically engineering cells to produce therapeutically boosted extracellular vesicles for cardiovascular calcification

Abstract

Calcification associated with atherosclerosis is a major driver of morbidity globally. Despite the correlation of calcification with plaque rupture and sudden death, there are no clinically approved therapies that treat vascular calcification. Notably, vascular smooth muscle cells (VSMCs) represent a promising target to inhibit vascular calcification, as VSMCs are the primary source of calcification deposition in the vasculature. To that end, we report a novel approach using extracellular vesicles (EVs) to deliver anti-osteogenic miR-133 to osteochondrogenic VSMCs in atherosclerosis. Traditionally, loading miRs into EVs is marred by low loading efficiency, inefficient EV modification, miR degradation, or loss of EV structural integrity. To address these challenges, VSMCs were transduced to create cell lines expressing miR-133 modified with ExoMotifs, or 4–8 nucleotide motifs which enable binding to proteins involved in miR sorting, resulting in VSMCs that secrete EVs highly loaded with miR-133. Additionally, EVs were surface functionalized with a hydroxyapatite binding peptide (HABP) to enable targeting to areas of vascular calcification. Our results show that HABP-miR-133-EVs can inhibit osteochondrogenic VSMCs, promote contractile VSMCs genes, and inhibit vascular calcification both in vitro and in vivo in murine atherosclerosis models. More broadly, we demonstrate a platform strategy to develop cellular factories for miR-loaded, therapeutic EVs that can be tailored for a variety of diseases.