Incorporation of Heterologous Proteins in Engineered Exosomes

Engineering exosomes to upload heterologous proteins represents the last frontier in terms of nanoparticle-based technology. A limited number of methods suitable to associate proteins to exosome membrane has been described so far, and very little is known regarding the possibility to upload proteins inside exosomes. We optimized a method of protein incorporation in exosomes by exploiting the unique properties of a nonfunctional mutant of the HIV-1 Nef protein referred to as Nef(mut). It incorporates at high extents in exosomes meanwhile acting as carrier of protein antigens fused at its C-terminus. Manipulating Nef(mut) allows the incorporation into exosomes of high amounts of heterologous proteins which thus remain protected from external neutralization/degradation factors. These features, together with flexibility in terms of incorporation of foreign antigens and ease of production, make Nef(mut)-based exosomes a convenient vehicle for different applications (e.g., protein transduction, immunization) whose performances are comparable with those of alternative, more complex nanoparticle-based delivery systems.