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Identifier

000444710

Title

Genomic engineering of adipocytes with CRISPR/Cas9 delivery particles for ex vivo therapeutic approach for type 2 diabetes

Alternative Title

Γενετική τροποποίηση λιποκυττάρων με σφαιρίδια μεταφοράς CRISPR/Cas9 για ex vivo θεραπευτική προσέγγιση για τον διαβήτη τύπου 2

Author

Τσαγκαράκη, Εμμανουέλα

Thesis advisor

Καρδάσης, Δημήτρης
Czech, Michael P.
Zannis, Vassilis

Abstract

Obesity and type 2 diabetes (T2D) are related with abnormalities in glucose and lipid homeostasis which is regulated by insulin. They can lead in severe complications including cardiovascular disease and steatohepatitis. Systemic glucose metabolism is controlled by distinct fat depots which include two main types: white adipose tissue and brown adipose tissue. White adipose tissue is composed by white adipocytes, and it stores energy in the form of lipids, whereas brown adipose tissue is composed by the brown adipocytes. These brown fat cells utilize the stored energy to produce heat and abundantly express uncoupling protein 1 (UCP1) and several secreted factors that altogether enhance metabolism. Brown adipose tissue or murine and human brown/beige adipocyte transplantations have been shown to improve glucose tolerance in obese mice. However, the application of such a therapeutic intervention in human has not been possible due to the limited availability of human brown/beige adipocytes. In this work, a large – scale expansion of human progenitor adipocytes from small samples of human adipose tissue has been used. Parallel to murine, in these human progenitor cells, efficient genome engineering with clustered regularly interspaced short palindromic repeats (CRISPR) – mediated was achieved. The purpose of this gene editing was to disrupt genes that physiologically prevent the conversion of white adipocytes into brown. Therefore, it was hypothesized that knocking out those genes would allow “browning” of the engineered white preadipocytes after differentiation and hence, favor glucose tolerance. Importantly, in order to make this approach translatable for therapy, a novel genome editing approach was developed that allowed to bypass CRISPR components immunogenicity, off – target and off – tissue effects. Particularly, for this project, an ex vivo delivery method of Streptococcus pyogenes Cas9 and guide RNA was developed and optimized to ensure their prompt degradation following the gene editing. For the 19 CRISPR-Cas9 delivery, electroporation was used with editing efficiency close to 100%. After screening multiple candidate genes identified in literature, nuclear repressor interacting protein 1 (Nrip1) showed the most promising results. The NRIP1 knock-out (NRIP1KO) adipocytes demonstrate a brown-like phenotype which includes UCP1 protein and several secreted factors. The engineered adipocytes were further characterized using a variety of tools including gene expression studies of thermogenic genes, genes related to mitochondrial respiration, fatty acid oxidation and secreted factors and oxygen consumption assay. Evaluation of UCP1 protein expression and transcriptome analysis by bulk RNA sequencing were also employed for the phenotypic characterization. Importantly, the CRISPR – enhanced murine and human adipocytes were implanted in recipient mice that were then placed on high – fat diet for the induction of type 2 diabetes. The engineered adipocyte implant recipients were found with lower levels of accumulated fat and triglycerides in the hepatic parenchyma and with improved glucose tolerance compared to age and gendered – matched control mice that received implants with unedited adipocytes. The CRISPR components were only transiently present in the edited cells as demonstrated by degradation of Cas9 protein after editing. These findings demonstrate a therapeutic approach for the improvement of metabolic homeostasis via CRISPR gene editing with human adipocytes bypassing the patient exposure to the immunogenic Cas9 and guide RNA as well as other vehicles for CRISPR-Cas delivery.

Language

English, Greek

Subject

Adipoce tissue

Glucose

Insulin

Ribonucleoprotein

Ήπαρ

Γλυκόζη

Εμφυτεύματα

Ηλεκτροδιάτρηση

Ινσουλίνη