This review provides an updated perspective on rapidly proliferating efforts to harness extracellular vesicles (EVs) for therapeutic applications. We summarize current knowledge, emerging strategies, and open questions pertaining to clinical potential and translation. Potentially useful EVs comprise diverse products of various cell types and species. EV components may also be combined with liposomes and nanoparticles to facilitate manufacturing as well as product safety and evaluation. Potential therapeutic cargoes include RNA, proteins, and drugs. Strategic issues considered herein include choice of therapeutic agent, means of loading cargoes into EVs, promotion of EV stability, tissue targeting, and functional delivery of cargo to recipient cells. Some applications may harness natural EV properties, such as immune modulation, regeneration promotion, and pathogen suppression. These properties can be enhanced or customized to enable a wide range of therapeutic applications, including vaccination, improvement of pregnancy outcome, and treatment of autoimmune disease, cancer, and tissue injury.

Background: Exosomes show great promise as targeted therapeutic delivery vehicles. Results: A strategy was identified for stabilizing targeting peptides on the surface of exosomes. Conclusion: Glycosylation protects targeting ligands displayed on the surface of exosomes from proteolytic degradation. Significance: Strategies for robust display of targeting peptides will enable targeted delivery of therapeutic exosomes.

Extracellular vesicles (EVs) mediate intercellular communication through transfer of RNA and protein between cells. Thus, understanding how cargo molecules are loaded and delivered by EVs is of central importance for elucidating the biological roles of EVs and developing EV-based therapeutics. While some motifs modulating the loading of biomolecular cargo into EVs have been elucidated, the general rules governing cargo loading and delivery remain poorly understood. To investigate how general biophysical properties impact loading and delivery of RNA by EVs, we developed a platform for actively loading engineered cargo RNAs into EVs. In our system, the MS2 bacteriophage coat protein was fused to EV-associated proteins, and the cognate MS2 stem loop was engineered into cargo RNAs. Using this Targeted and Modular EV Loading (TAMEL) approach, we identified a configuration that substantially enhanced cargo RNA loading (up to 6-fold) into EVs. When applied to vesicles expressing the vesicular stomatitis virus glycoprotein (VSVG) – gesicles – we observed a 40-fold enrichment in cargo RNA loading. While active loading of mRNA-length (>1.5 kb) cargo molecules was possible, active loading was much more efficient for smaller (~0.5 kb) RNA molecules. We next leveraged the TAMEL platform to elucidate the limiting steps in EV-mediated delivery of mRNA and protein to prostate cancer cells, as a model system. Overall, most cargo was rapidly degraded in recipient cells, despite high EV-loading efficiencies and substantial EV uptake by recipient cells. While gesicles were efficiently internalized via a VSVG-mediated mechanism, most cargo molecules were rapidly degraded. Thus, in this model system, inefficient endosomal fusion or escape likely represents a limiting barrier to EV-mediated transfer. Altogether, the TAMEL platform enabled a comparative analysis elucidating a key opportunity for enhancing EV-mediated delivery to prostate cancer cells, and this technology should be of general utility for investigations and applications of EV-mediated transfer in other systems.

Macrophages are ubiquitous innate immune cells that play a central role in health and disease by functionally “polarizing” to distinct phenotypes, which are broadly divided into classical inflammatory responses (M1) and alternative responses (M2) that promote immune suppression and wound healing. Although macrophages are attractive therapeutic targets, incomplete understanding of polarization limits clinical manipulation. While individual stimuli, pathways, and genes involved in polarization have been identified, how macrophages evaluate complex in vivo milieus comprising multiple divergent stimuli remains poorly understood. Here, we used combinations of “incoherent” stimuli – those that individually promote distinct macrophage phenotypes – to elucidate how the immunosuppressive, IL-10-driven macrophage phenotype is induced, maintained, and modulated under such combinatorial stimuli. The IL-10-induced immunosuppressive phenotype was largely dominant but required sustained IL-10 signaling to maintain this phenotype. Our data also implicate the intracellular protein, BCL3, as a key mediator of the IL-10-driven phenotype. IL-12 did not directly impact polarization of IL-10-treated macrophages, but IFNγ disrupted a positive feedback loop that may reinforce the IL-10-driven phenotype in vivo. This novel combinatorial perturbation approach thus generated new insights into macrophage decision making and local immune network function.

Our previously-developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26 and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm.