SARS-CoV-2 inhibition through mRNA delivery using engineered extracellular vesicles displaying the spike protein

Coronavirus disease (COVID-19) caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has been the biggest pandemic in recent years, and there is a growing demand for the development of new modalities to treat emerging infections rapidly. Extracellular vesicles (EVs) are promising new biocompatible drug carriers; however, their specificity and delivery efficiency remain challenging. In this study, we aimed to develop EVs displaying the SARS-CoV-2 Spike (S) protein as a new modality to inhibit SARS-CoV-2 infection. The S protein-displaying EVs which were the pellet fractions of centrifugation at 10,000×g (S-EVs^10K) were found to selectively bind to cells expressing angiotensin-converting enzyme 2 (ACE2). Cleavage of the S protein using proteases such as transmembrane serine protease 2 (TMPRSS2) and cathepsins led to successful membrane fusion of the EVs^10K with the target cell. Experiments using viral infection inhibitors in TMPRSS2-expressing Vero E6 cells further confirmed the membrane fusion of S-EVs^10K in an ACE2-and TMPRSS2-dependent manner. Additionally, we demonstrated the potential of S-EVs^10K as novel mRNA carriers to inhibit SARS-CoV-2 infection by encapsulating the mRNA encoding HAI-2, a TMPRSS2-inhibiting membrane protein. Marked suppression of SARS-CoV-2 entry into TMPRSS2-expressing Vero E6 cells was confirmed using pseudotyped virus-like particles. These findings suggest the potential of S-EVs^10K for selective mRNA delivery to target cells via membrane fusion, serving as a new modality for inhibiting SARS-CoV-2 infection by delivering mRNA encoding inhibitory proteins.