Exosomes 101 – Exosome Engineering

by   |  September 28, 2018

The ultimate nanovehicle – engineer exosomes for custom cargo delivery shuttles into target cells. Cargoes include nucleic acid and even small molecules.

Engineering Exosomes – Custom surface and cargo designs
Used by cells to transport specific cargoes of active biomolecules, exosomes have the potential to be a powerful tool that scientists can use to package and traffic proteins and miRNAs. And SBI is ready to help. As the field transitions from observation and analysis of exosomes to custom exosome design for therapeutic and other uses, SBI is already offering the necessary tools for engineering exosomes – deliver miRNA for knock down studies, small molecules for biochemical or therapeutic studies – imagine the possibilities.

Load Exosomes with siRNA, microRNA, mRNA, plasmid DNA, and Small Molecules
Exo-Fect is a novel nucleic acid transfer agent that enables the transfection of nucleic acids and small molecules directly into isolated exosomes. The transfected siRNA, microRNA, mRNA, plasmid DNA, or small molecule can then be shuttled into target cells via the transfected exosome vesicles. Simply combine isolated exosomes with Exo-Fect and the nucleic acid or small molecule of your choice to generate exosome delivery vehicles. The protocol takes less than an hour and is highly efficient at placing nucleic acids into exosomes for transport, so you can create your very own “FedExosome”(1).

Tag miRNAs for Packaging into Exosomes In Vivo
The mechanism of how specific RNA sequences are selectively packaged into exosomes is an intensive area of investigation. SBI has identified an RNA sequence tag that targets a small RNA for exosome packaging, and used this to create a set of tools that enables researchers to generate exosomes enriched for a specific RNA. With the XMIR and AXMIR kits, simply fuse a sense (XMIR) or antisense (AXMIR) miRNA oligo to the XMIR/AXMIR RNA tag and transfect into the exosome-producing cells of your choice. The secreted exosomes will be enriched for your miRNA, which can then be delivered to target cells. Great for knocking down expression of a target in the recipient cells, or for studying other biological functions. You can also clone miRNA, anti-miRNA, or siRNA sequence into our XMIRXpress Lentivector System to create stable cellular exosome “factories” that are enriched for your RNA. Or SBI can design and build a custom XMIRXpress Lentivector for your studies.

Exo-Fect vs. XMIR/AXMIR
With two different options for generating exosomes with custom RNA cargo, which should you choose? As always, it depends on what your experimental goals are and what materials you’re working with. The Exo-Fect system can be used to deliver a wider variety of cargo to exosomes, such as plasmids, larger RNAs like mRNAs, and small molecules. The exosomes need to be either isolated or purchased first, and are then “exofected” (exosomes are “transfected”). In contrast, the XMIR/AXMIR systems enable cells to be transfected with tagged miRNA/anti-miRNA oligos, which then end up packaged into exosomes. For some studies, this may result in a larger supply of custom exosomes, and the XMIRXpress lentiviral system allows stable exosome packaging cell lines to be made for a constant supply of miRNA-loaded exosomes. Bottom line – the Exo-Fect system enables flexibility with cargo material and the ability to customize cargo when your exosomes are generated by difficult-to-transfect cell lines. The XMIR/AXMIR system enables generation of stable exosome packaging cell lines.

Use Exosomes as Transfection Vehicles
The EV Shuttle Kits are the “Next Generation” technology of transfecting cells that are typically resistant to most transfection methods (ie. Lipofectamine®) – utilizing exosomes! The EV shuttle kits enable the efficient transfer of RNA and DNA into cells that can only be nucleofected. The EV shuttles are more gentle and are successful in delivering the RNA or DNA cargo of your choice.

Label Exosome Cargo
What happens to exosome cargo once it’s delivered to a cell? You can now find out by fluorescently labeling RNA or protein in isolated exosomes, and then delivering the exosomes to target cells. With Exo-Glow the fate of exosomal RNA or proteins can be tracked once they’ve been delivered to the recipient cells.

Package Proteins inside Exosomes
XPack is the protein equivalent of XMIR/AXMIR, and tags proteins for incorporation into exosomes. We’ve optimized a peptide sequence that targets a protein to the interior exosomal membrane, allowing the fusion protein to be packaged into exosomes for secretion. You can use our convenient, pre-made XPack-GFP and XPack-Luciferase reporters for tracking exosomes, or design your own exosome-targeted protein for custom exosomes.

Exosome Targeting
Use exosomes to deliver protein, RNA, DNA, or small molecule cargo to specific tissues with XStamp. XStamp technology is an optimized, exosome surface display system that efficiently places protein sequences on exosomal surfaces. When a tissue-specific protein ligand is used, the engineered exosomes become targeted to the specified tissue, enabling directed cargo delivery.

Conclusions
Supporting researchers with a growing set of tools for engineering custom exosomes, SBI is truly putting the power of exosomes into your hands.

REFERENCE

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