| 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.
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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
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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.
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