Autoimmune diseases, such as type 1 diabetes, often arrise as a result of a defect in one or mechanisms of immune tolerance. An important part of immune tolerance are a subpopulation of T cells, called regulatory T cells (Treg), which prevent the autoimmune activity of aggresive immune cells, and whose depletion or inactivity can lead to severe autoimmune reactions. Stable expression of the master regulator transcription factor FOXP3 is key for their development from thymocytes. Stable induction of FOXP3 expression could potentially drive the differentiation of other T cell population, as well as other cells, towards a Treg lineage and an acquisition of an immosupresive phenotype. In my masters thesis, we aimed at developing and testing transcription activators, bound to modified Cas9 proteins, for the specific induction of FOXP3 expression. We have designed novel short guide RNA (sgRNA), that direct the Cas9 to specific sites on key regulatory regions of the FOXP3 gene. We have achieved high in vivo expression induction of FOXP3 with the use of a tripartite transcription activator VPR, bound to a nuclease-null mutant of Cas9 protein (dCas9:VPR). The highest induction of expression was obtained when we used dCas9:VPR in combination with sgRNA which target the core promotor of FOXP3 and a regulatory region, which we termed Cage1. After the success in achieving transcription activation, we wanted to check the expression profile of key FOXP3 target genes in cells, which normally do not express FOXP3. Initial experiments in HEK293T cell line showed a certain degree of correlation of gene expression to that of Treg cells, after the activation of FOXP3 expression. With our work we have presented a powerfull tool for targeted gene expression, as well as the imporatance of the Cage1 region for the activation of FOXP3 expression. Our tool could be used in further experiments which aim at the generation of Treg cells, which could potentially be used to treat different autoimmune diseases.
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