Our project's main objective is to set up a standardized biological system for the production of engineered exosomes. For that, we have designed several composite parts that, when introduced in a cell line, produce the boosting of exosome biogenesis, facilitate shRNA cargo loading and exosome purification and provide cell type tropism for those exosomes. With that, we present our Part Collection:
- Exosome boosters: BBa_K4501011 and BBa_K4501012
These constructs contain an array of genes implicated in the biogenesis of exosomes, inducing the boosting of exosome production. The STEAP3, SDC4, NadB and nSMase genes have been correlated with exosome biogenesis. They contain, respectively, a puromycin and hygromycin resistance gene to facilitate transfection.
- Marker and loader. His-tag CD63-L7Ae: BBa_K4501014
This construct contains the expression vector for CD63, a tetraspannin present in exosomes, with some modifications. On the one hand, it contains a His-tag on the extracellular domain to facilitate purification by affinity chromatography. On the other hand, it contains the archaeal gene L7Ae, that interacts with the C/D-box motif to load the labeled shRNAs. It is coupled to an RFP gene to facilitate the selection of the transfected cells.
- Cell type ligand: BBa_K4501017
In our case, we use a ligand for CD19 to target B cells. By substituting the CD19-L for a ligand of choice, the tropism of the exosomes can be modified. The ligand is located in the extracellular domain of Lamp2B, a constitutive protein of exosomes. It is coupled to a BFP reporter.
- Cytosolic delivery helper: BBa_K4501021
This expression cassette codes for a mutated version of Connexin 43, a mutation that constitutively activates the channel. With that, the delivery of shRNAs into the exosomes at the Multivesicular Body is increased. It is coupled to a YFP reporter.
- shRNA expression cassette: BBa_K4501019
With this expression cassette for shRNA generation (in our case, the shRNA is designed against myc), the cell expresses the shRNA by itself eliminating the need of artificially loading the shRNA into the exosomes. Moreover, the shRNA is labelled with the C/D-box sequence that allows its loading to the exosomes. It is coupled to a GFP reporter.