(623e) Engineering Exosomes to Probe and Program Loading of RNA Cargo Into Biological Nanoparticles

Exosomes are emerging as important new players in the landscape of intercellular communication. These extracellular vesicles serve as biological nanoparticles (~30-200 nm in diameter) that transfer proteins, mRNA, and microRNA from a “sender” cell to a “receiver” cell, wherein these cargo molecules functionally modulate cell state. This mechanism appears to play a particularly important role in cancer, and tumor-derived exosomes are a newly appreciated modality by which cancer cells modulate host function to promote tumor survival and metastasis. Given this capacity to functionally deliver biomolecules, exosomes are also potentially attractive vehicles for the therapeutic delivery of biomolecules [1], and recent reports have demonstrated the promise of this approach. To date, however, the mechanisms by which a subset of cellular proteins and RNAs are sorted into exosomes are poorly understood. Therefore, in order to better understand the roles of exosomes in normal and disease processes and to enable effective exosome-based therapeutics, we are developing novel biochemical tools for interrogating and directing the incorporation of specific RNA cargo molecules into exosomes. We have developed a system for engineering exosomal membrane proteins that bind to defined “packaging” sequences within engineered RNA cargo molecules. Using this system, we showed that by engineering defined protein-RNA interactions, we can direct the incorporation of specific proteins and RNA into exosomes. We also identified RNA motifs and features that may impact loading in a manner that cannot be explained by simple bulk flow or mass action driving forces. These results suggest that novel active sorting mechanisms and/or biophysical constraints dramatically modulate RNA loading into exosomes. We are using this platform to investigate the constraints that regulate packaging of RNAs into exosomes and the features of exosomal RNA cargo that affect function in receiver cells, in order to develop a quantitative framework for understanding and modulating exosome-mediated delivery. Together, these investigations serve to both improve our understanding of natural exosome biology and enable translation of this understanding into novel therapeutic capabilities.

[1] Marcus, M.E.; Leonard, J.N. FedExosomes: Engineering Therapeutic Biological Nanoparticles that Truly Deliver. Pharmaceuticals 2013, 6, 659-680.