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
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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.
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