Future Work

FUTURE WORK



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:



Stable transfection of HEK293T cell line with our constructs

  • Exosome booster 1 and 2
  • CD63_His-tag and CD63_His-tag L7Ae (x1), CD63_His-tag L7Ae (x2) and CD63_His-tag L7Ae (x3
  • Cytosolic delivery helper
  • CD19-L-Lamp2b fusion protein
  • myc shRNAs

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.



Optimization of the purification of exosomes via His-tag


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.



Assessment of loading of exosomes in the different L7Ae configurations


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.



Ability of the shRNA to downregulate myc expression


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.



Docking of CD19-L-Lamp2b with CD19+ positive cells


This experiment will serve to confirm the specificity of the CD19-L-lamp2b bearing-exosomes towards CD19+ cells.



In vivo proof of concept of shRNA-laoded exosomes efficacy


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.



Bibliography

  1. Zhu X, Badawi M, Pomeroy S, Sutaria DS, Xie Z, Baek A, Jiang J, Elgamal OA, Mo X, Perle K, Chalmers J, Schmittgen TD, Phelps MA. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. J Extracell Vesicles. 2017 Jun 6;6(1):1324730. doi: 10.1080/20013078.2017.1324730. PMID: 28717420; PMCID: PMC5505007
  2. Vilanova-Perez T, Jones C, Balint S, Dragovic R, L Dustin M, Yeste M, Coward K. Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro. Nanomedicine (Lond). 2020 Aug;15(20):1965-1980. doi: 10.2217/nnm-2020-0056 . Epub 2020 Aug 14. PMID: 32794431.