Chimeric antigen receptor T cell or CAR-T is a new type of immunotherapy that uses genetically modified T cells that express surface receptors which recognize an antigen on targeted cancer cells. CAR-T has been used to successfully treat several types of blood cancer, but carries the risk of severe side effects resulting from overactivation of therapeutic cells. One of the pathways for the development of cellular therapies is the use of synthetic biology to introduce molecular switches into CAR-T cells that can be controlled by small molecules to influence signaling pathways in the cell and subsequently regulate various cellular processes such as activation, proliferation or induction of apoptosis. We have prepared several transcription factors based on human protein STAT5b with added activator and/or repressor domains through which we may control its activity. We used two different approaches, the first was the utilization of protease TEV for the cleavage of covalently bound domains, the second was the utilization of dimerization domains DmrC and DmrA for induction of a noncovalent bond between the DNA binding domain of STAT5b and a repressor domain. We evaluated the activity of transcription factors in HEK293 cells by measuring the expression of the reporter gene. The selected transcription factor was additionally tested in Jurkat cells, where we also measured the expression of endogenous genes with qPCR. With the use of the DNA binding domain of STAT5b, covalently bound with an activation or repressor domain, we successfully controlled the expression of the reporter gene. However, there was no effect on the expression of the selected endogenous genes.
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