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Identifier 000417029
Title Genetic editing of adipocytes by CRISPR/Cas9 delivery particles for ex vivo therapeutic approaches to type 2 diabetes
Alternative Title Γενετική τροποποίηση λιποκυττάρων με σωματίδια μεταφοράς CRISPR/Cas9 για ex vivo θεραπευτικές προσεγγίσεις για τον διαβήτη τύπου 2
Author Τσαγκαράκη Εμμανουέλα
Thesis advisor Καρδάσης, Δημήτριος
Reviewer Czech, Michael
Ζαννής, Βασίλειος
Abstract Obesity and type 2 diabetes are major health threats with increasing prevalence affecting productive age groups in the developed countries. So far, there are no therapeutic approaches that target the causative mechanisms for type 2 diabetes in routine clinical practice. All pharmaceutical components that are administered daily throughout life, aim to lower blood glucose levels targeting different mechanisms of glucose synthesis, cell uptake and secretion. However, high blood glucose levels that cause the devastating cardiovascular complications of diabetes, seem to be the end-result of still unknown pathogenic mechanisms, occurring in patients with type 2 diabetes. Yet, remarkable progress has been made towards the understanding of insulin resistance pathogenesis, which is an important aspect of the efforts towards the development of more efficient therapeutic approaches. Our group has recently demonstrated that suppression of de novo lipogenesis by deletion of the enzymatic complex fatty acid synthase (FASN) in white adipose tissue results in “browning”, improvement of cold adaptation and whole-body metabolism. Furthermore, white adipose tissue derived from FASN knock-out mice has been shown to have a beneficial impact on glucose homeostasis, when transplanted into metabolically healthy, chow-fed wild type mice. 6 While exploring the role of the DNL pathway, and glucose metabolism in our lab, we have also made a conscious effort to continue to develop novel therapies to target adipocytes and diabetes in vitro and in vivo. To address this, we developed a powerful technology for gene deletion utilizing CRISPR/Cas9 genome editing called CRISPR delivery particles (CriPs), composed of Cas9, sgRNA and the amphipathic peptide Endoporter. The advantage to the CriPs is the possibility to be systemically administered in vivo or in cell cultures ex vivo to introduce gene deletions with a simple non-viral, plasmid-free system. This system is not cell type-specific, thus, we can efficiently introduce Cas9 endonuclease and single guide RNA to delete a target gene in many cell types such as macrophages and primary pre-adipocytes, without the adverse effects of plasmid or virus – mediated Cas9 and sgRNA engineered cells. Results presented here show that significant depletion of the FASN protein was obtained upon treating isolated adipocytes with CriPs containing sgRNA targeting the Fasn gene. Up to 50% loss of FASN protein was obtained in these experiments. The purpose of this research project is to exploit new data supporting the beneficial effects of de novo lipogenesis suppression and improvement in glucose tolerance by using the CriPs technology available to advance a novel therapeutic approach for type 2 diabetes with a significant curative impact. The steps required to achieve this goal consist of improving the genomic editing efficiency of CriPs to target mature adipocytes, deleting FASN in these cells, followed by transplanting 7 the genetically modified cells back into mice, in order to improve glucose tolerance and reverse insulin resistance. Results presented here show successful transplantation of brown adipose tissue or immortalized brown adipocytes into the visceral region of recipient mice to achieve improvement in glucose tolerance. Such transplants will serve as controls for future experiments designed to test whether FASN depletion in primary white adipocytes will promote glucose tolerance when similarly transplanted into mice. If successful, this would be a promising novel and long-lasting therapeutic approach to improve energy homeostasis in patients with type 2 diabetes.
Language English
Subject translational
Issue date 2018-07-18
Collection   School/Department--School of Medicine--Department of Medicine--Post-graduate theses
  Type of Work--Post-graduate theses
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