Introduction
Since different devices are incorporated into our system design, there are several proofs of concept that have to be considered: one for each step of the process, and a final one encompassing all of them as the complete system.
shRNA Validation
Burkitt leukemia is characterized by chromosomal rearrangements of the c-myc oncogene, which leads to the overexpression of MYC transcription factor. As MYC is a potent proto-oncogene and transcriptional regulator, when its chromosomal translocation occurs, this gene is dysregulated, and crucial oncogenic events initiate. This contributes to lymphomagenesis by altering the cell cycle regulation, cell differentiation, apoptosis, adhesion, and metabolism (1). MYC downregulation was studied through qPCR analysis after transfecting Ramos cell lines with the shRNA expression plasmid through electroporation. Real time quantitative PCR is an efficient, simple and low cost technique that is frequently used to quantify gene expression (5), so it perfectly fits the aim of the shRNA validation test.
Results showed that by the efficient incorporation of our customized shRNA plasmid, the oncogene expression presented a significant variance compared to the normal levels of this transcription factor. The almost 2 fold difference between the samples exemplifies the clear influence of our artificial RNA molecule on the c-myc oncogene silencing and so on, the potential of it as a downregulator.
Purification of exosomes device Validation
In order to assess the success of the exosome purification, the supernatant of the cultured HEK293T CD63_His-tag-transfected cells was purified by His-tag Ni-NTA chromatography (see Results webpage). The analytical NTA analysis that was done for the unbound and the eluted fractions of the column, indicated the presence of exosomes in both. The signaling obtained on the elution fractions indicate that we were able to correctly purify exosomes through the specific interaction between the CD63_His-tag-L7Ae receptor and the nickel ions of the affinity column. However the certain presence of exosomes in other fractions and for instance the protein recovery yield obtained, meant that the system is not fully optimized yet.
Final Proof of Concept
To ensure that our project is viable, not only in theory but is also applicable to a real setting, we propose to test the optimized vesicles (previously synthetized and purified) on both in vitro and in vivo models.
As it has been already demonstrated that inhibiting the expression of oncogenic c-myc produced high anti-cancer effects, it is remarkable the necessity of an appropriate silencing agent for a more effective cancer treatment. However, the development of a viable anti-c-myc-RNAi-based platform is still largely dependent on the design of an appropriate carrier (2).
Being our project focused on the treatment of Burkitt lymphoma, a highly aggressive B cell non-Hodgkin lymphoma, our proof of concept would be based on the usage of the exosome carrying short hairpin RNA (shRNA) expression plasmids in Ramos cell line. These are lymphoblast-like cells used in immunology research. Its hematopoietic origin, more concretely derived from human Burkitt’s lymphoma (BL), make them the optimal target for proving the effectiveness of our experiment. By analyzing both the growing rate and the cell concentration, before and after the treatment with the optimized vesicles, we would be able to evaluate how the effector nucleic acid acts against c-myc oncogene.
Furthermore, to complement the research, we would also base our in vivo assays on the choriallantoic membrane (CAM) model. Not only its tissue composition and accessibility makes it a robust experimental platform, but also several CAM-tumor model experiments have described the successful inoculation and treatment of BL cell lines on the chick chorioallantoic membrane. These facts reaffirm the potential usage of the CAM model to verify the usefulness of the optimized exosomes as growth inhibitors of human BL xenograft tumors (3,4).