Unfortunately, we did not have enough time to fulfill all of our aims. However, after the competition we will keep working on the project, thanks to our research group host, Dr. Gaël Roué’s group, that will hire one of our team members to allow expand the progress on this promising drug delivery system. In the following months, we expect to perform the following:
For that, lentiviral vectors are to be produced by using our pJUMP-based transfer plasmid system. For the constructs containing fluorescent reporters, cells that display high intensity of fluorescence are to be sorted by FACS. For Exosome booster constructs containing a antibiotic resistance selector, the selection will be performed by one-week treatment with the antibiotic.
We will transfect the following constructs to generate different cell lines: CD63_His-tag, CD63_His-tag L7Ae (x1), CD63_His-tag L7Ae (x2) and CD63_His-tag L7Ae (x3). After verification of the transfection, we will assess if the constructions are easily purified by affinity chromatography (using Ni-NTA resin, see Protocols). The amount of exosomes obtained will be quantified by NTA.
Once all of the CD63 constructs are stably transfected into our cell line, we will assess wether the shRNA is loaded propperly into the exosomes that are afterwards secreted. For that, we will purify the exosomes and perform RT-qPCR to determine the amount of shRNA inside the exosomes. With that, we will determine which one of the CD63 constructs works better for shRNA loading.
We will also test this with our without the cytosolyc delivery helper construct to determine its effect on shRNA loading into exosomes.
All the three myc shRNA constructions will be tested wether they are able to induce myc sylencing. For that, the plasmids will be transfected by lentiviral vectoring into Burkitt Lymphoma cell lines that highly express myc. Expression of myc will be assessed at different time points by Wester Blot.
This experiment will serve to confirm the specificity of the CD19-L-lamp2b bearing-exosomes towards CD19+ cells.
Eventually, we will use our purified shRNA-loaded exosomes with an in vivo model: the CAM (see Proof of concept). For that, a tumour is induced in the CAM by injecting a Burkitt lymphoma cell line prior to the immune system development, and afterwards is treated with our exosomes. In that , we expect to verifiy the therapeutic effect of our exosomes.