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The new chromatin opening models as a strong potential tool for recombinant production and gene therapy applications

O F Anakok(1) A GOSCHI(2) P Kose(2) K Bayindirli(2)

1:Bolu Abant Izzet Baysal University; 2:Ataturk University

Our new chromatin opening models we recently developed which free from potential mutation sites that also considerably smaller (500bp) than the current UCOE models used in gene therapy and recombinant protein biotechnology studies,were tested on various cell groups, including mouse embryonic stem cells and human İPS cells.

It has been demonstrated in terms of replacing existing UCOE models that these new UCOE candidates we have developed are more efficient than the previous ones and that they are also a safer profile model for clinical gene therapy studies since they are free from additional enhancing vector cassette areas. These new UCOE models ubiquitously shows a powerfull resistance to DNA methylation- mediated silencing and also provides a higher and stable transfection profile.

To understand the potentiality of our new generation UCOE designs in gene therapy studies, it was tested whether the universal chromatin opening abilities will be retained stable of activation on human induced pluripotent stem cells by differentiating them into different tissue cell types as done before on mouse embryonic stem cells.

In the light of the obtained results, the new UCOE designs that we developed, have maintained their expression levels stably on human iPS cells before and after differentiation into three different tissue type cells. And additionally, they also showed their potential on monoclonal antibody production with CHO cells as producing mg and gr level of recombinant antibodies into two months of period in another parallel study we conducted.

Moreover, by the urgent need of vaccine development for COVID-19 during the pandemic, we aimed to produce a potential recombinant vaccine by using this new chromatin opening models of our own design. For this purpose a part of the spike and nucleocapsid gene sequences of COVID-19 were firstly cloned into our UCOE models. These UCOEs plasmids were then transferred into 293T cells along with plasmids carrying the genes that will form the lentivirus vectors (LVs). After harvesting and calculation of LV vector titers, the cloned vectors were then transfected into the CHO cells which the targeted recombinant production of the antigen proteins will be carried out. Antigenic structures were then isolated from the culture medium of CHO cells in following days for confirmation. Using HPLC and qTOF mass spectrometer methods, these structures in the medium were confirmed to be the units of spike and nucleocapsid proteins of the COVID-19 virus. In order to produce large amount of the recombinant antigens, the culture was then carried out with bioreactors in liters. At the final stage, these recombinantly produced antigen proteins were tested on rats to measure their immunogenic responses, and the study recently been completed successfully as a potential recombinant vaccine against COVID-19. 

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