A modified CD9 tag for efficient protein delivery via extracellular vesicles

Extracellular vesicles (EVs) are attracting growing attention for therapeutic use and as diagnostic markers, particularly for cancer. Although therapies based on small interfering RNAs are under intensive research, other therapeutic molecules, especially proteins, have not been sufficiently investigated. One of the major method for loading proteins into EVs is electroporation; however, it damages membrane integrity and requires repeated purification, precluding clinical applications. Thus, natural and efficient protein transfer is a prerequisite for the clinical application of protein-based EV therapy. Another prerequisite is an efficient endosomal escape, as most EVs incorporated into receptor cells result in endosomal degradation. Therefore, we generated a short CD9 (sCD9)-INF/TAT tag for efficiently transfers fused proteins to the EV and enhances endosomal escape to address the abovementioned problems. Interestingly, protein transfer via EVs drastically improved when the EV producer and receptor cells were cocultured, strongly indicating bystander effects of cells producing therapeutic proteins fused with a sCD9-INF/TAT tag. This method can be applied to a wide range of therapeutic technologies, including cellular transplantation or viral therapy.