PhD Scientific Days 2022

Budapest, 6-7 July 2022

Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)

Generation of cell lines that produce fluorescent vesicles containing a coronavirus antigen protein on their surface

Orsolya Mózner1,2, Edit I. Buzás3,4,5, Balázs Sarkadi2,6
1 Doctoral School of Molecular Medicine, Semmelweis University, 1094 Budapest, Hungary
2 Institute of Enzymology, ELKH Research Centre for Natural Sciences, 1117 Budapest, Hungary
3 Department of Genetics, Cell- and Immunobiology, Semmelweis University, 1089 Budapest, Hungary
4 HCEMM-SU Extracellular Vesicle Research Group, 1089 Budapest, Hungary
5 ELKH-SE Immune-Protegenomics Extracellular Vesicle Research Group, 1089 Budapest, Hungary
6 Department of Biophysics and Radiation Biology, Semmelweis University, 1094 Budapest, Hungary

Text of the abstract

The best-known forms of extracellular vesicles (EVs) are the small vesicles arising from multivesicular bodies (MVBs), the exosomes, produced by eukaryotic cells. The use of exosomes for human therapeutic purposes is a promising and widely researched area. EVs are known to have many advantages over artificial liposomes, drug molecules conjugated to polymers or gold nanoparticle carriers, but it is challenging to control the components present on the surface of the EVs produced by mammalian cells. CD63 is a marker protein of exosomes that shows a steady state accumulation in multivesicular bodies. It has also been shown that by tagging the CD63 protein, one can selectively express proteins in EVs bound either to CD63 N or to C termini. In both cases, the labeling appears on the inner surface of EVs. According to results published in 2020, one of the four transmembrane helices of CD63 alone is enough to induce the production of EVs, and it has also been shown that this transmembrane domain can be labelled at both the N and C termini. Thus, the originating vesicles can contain a peptide or even a protein of choice both on their surface and inside the EVs, bound to the membrane.
The aim of this project was to test and use the above-mentioned method in a new application to produce EVs that contain the receptor binding domain (RBD) of the SARS-CoV2 Spike protein. First, the DNA constructs were created that coded the CD63 transmembrane helix 3, tagged with blue fluorescent protein on one end and the RBD on the other. To create stable HEK293 cell lines that express the engineered CD63 protein, the Sleeping Beauty transposon-transposase system was used.
Cell lines created by this method express the blue fluorescent protein bound to the EV membrane and the green fluorescent protein in the cytoplasm. The sorted cells show green and blue fluorescence even after long-term culturing. Sequencing results show the presence of the modified CD63 protein-coding DNA sequence incorporated in the genome of the cell lines. Our aim is to further characterize the generated cell lines, separate and analyze the extracellular vesicles produced by the HEK293 cells.

SUPPORTED BY THE ÚNKP-21-3-I NEW NATIONAL EXCELLENCE PROGRAM OF THE MINISTRY FOR INNOVATION AND TECHNOLOGY FROM THE SOURCE OF THE NATIONAL RESEARCH, DEVELOPMENT AND INNOVATION FUND.