Optimising the new ucoe models as higher direct transgene expression profile for effective gene therapy applications

Abstract

Background & Aim: Our new chromatin opening models which free from potential mutation sites that reduce the size of the current UCOE models used in gene therapy and recombinant protein biotechnology studies, tested on various cell groups, including mouse stem cell groups. As a result, 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 cassette areas. To see 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. Methods, Results & Conclusion: After transfection of induced human pluripotent stem cells with Lentiviral vectors carrying the UCOE [Figure presented] [Figure presented] candidates, the activation of UCOE elements which carry a reporter gene, were measured by flowytometry, immunofluorescence staining and DNA methylation analyses for two months without differentiation and also after tissue type differentiation into nerve, hepatocyte and cardiomycet type cells. In the light of the obtained results, it has been observed that the new UCOE designs (1.2kb and 1.7kb UCOE chromatin elements) 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. In addition, our new 0.5kb design (0.5kb UCOE), produced from another CpG density region of HNRPA2B1 gene, has been observed to display a stable expression level compared to our other designs and even a more stable profile. The new 0.5kb, 1.2kb 1.7kb (without expression enhancing cassette regions, thus eliminating the potential mutation problem) with a length of 10, 6 and 3 times shorter than the current standard A2UCOE models showed to be more advantageous for gene therapy and recombinant protein production studies as well. Recently, we have been using these new models now on producing the two of the target antigens of COVID-19 to provide a potential recombinant vaccine candidate.