Humanized extracellular vesicles for efficient RNA delivery

Engineered extracellular vesicles (EVs) are a class of nonviral delivery vectors for RNA-based vaccines and gene therapies. A specialized form of engineered EVs, known as enveloped protein nanocages (EPNs), has been developed to enhance cargo loading and delivery. When EPNs are equipped with a viral fusogen, such as vesicular stomatitis virus glycoprotein (VSV-G), they have been shown to deliver proteins or RNA efficiently into recipient cells. Comparisons across different EPN types and optimization of their different features have been difficult, as assays for their activity have not been reported for single, active units. As we were interested in optimizing EVs, we first developed a biological titration assay inspired by the methods used for infectious viral particles. With this assay, we optimized EVs using a modular platform, creating EVs composed predominantly of human-derived protein components. This system achieved efficient RNA delivery, with functional titers comparable to those of lentiviral vectors. The optimized chimeric proteins comprising the EV particles integrate domains from human epsin 1, human citramalyl-CoA lyase beta-like protein (CLYBL), and human CEP55. The constructs also include a short 21-amino-acid peptide from a nonhuman source for RNA packaging, resulting in an EV-based RNA delivery system with reduced immunogenicity compared with EPNs and retroviral virus-like particles (VLPs).