Cellular Reprogramming via Epi-CRISPRs-lnduced Targeted DNA Methylation

Abstract

Diabetes is the perfect candidate for cell replacement therapy, since it is caused by either an absolute (type 1 diabetes) or relative (type 2 diabetes) defect of insulin-producing pancreatic beta cells. Our research is focused on applying a novel synthetic epigenetic tool (Epi-CRISPRs) for straightfonward, one-step mouse pancreatic alpha (a-cells) to beta cell transdifferentiation by targeted DNA methylation and suppression of genes essential for maintaining pancreatic cell identity (homeobox Arx gene (Arx)). Up to now, we succeeded to transiently transfect a-cells with Epi-CRISPR constructs and 275 gRNA or mix gRNA. The suppression of Arx in a-cells was confirmed on day 5 and 8 posttransfection. The reduction of glucagon synthesis and beginning of insulin production in transfected a-cell was confirmed and visualised by immunostaining. DNA methylation-mediated suppression of Ao( in a-cells leads to their transdifferentiation to insulin-producing beta cells will be confirmed by bisulfite sequencing (undergoing experiments). Furthermore, we are also investigating an epithelial-mesenchymal transition (ETM), the mechanism which underlies the progressive decline in organ functioning in diabetes, such as the development of kidney and liver fibrosis. ETM is a process of reprogramming epithelial cells from a fully differentiated epithelial state to a more mesenchymal state. Our aim is to analyse the DNA methylation profile and gene expression of either epithelial (E-cadherin) or mesenchymal markers (a-smooth muscle actin and fibronectin) whose differential methylation and gene suppression could lead to more epithelial-like or mesenchyme-like phenotype. This will be accomplished using an in vitro model system based on epithelial cells treated with TGF-pi and 2. The obtained data should enable ETM reversal and stop fibrosis in diabetes and other pathologies using different compounds that act as DNA methylating/demethylating agents or using Epi-CRISPRs-based targeted DNA methylation/demethylation in future. We are on the way to develop a clear-cut technology able to provide a perfect delivery system for increase of insulin-producing cells in vitro. This system will allow for targeted gene silencing via increased DNA methylation of gene of interest. In addition, we are able to test if any compound used for treatment in different pathological conditions affects the DNA methylation profile of the examined cells. On the other hand, there is a great need for chemical compounds able to act as DNA hypo or hypermethylated agents