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Current Research Projects

Our lab offers a variety of Graduate Student Rotation Projects exemplified by the following abstracts and the lab colleagues involved in the studies:

An Ex Vivo Therapeutic Approach for Type 2 Diabetes with Genome-engineered Adipocytes by CRISPR/Cas9

Emmanouela Tsagkaraki, Sarah Nicoloro, Yuefei Shen and Mark Kelly
Molecular Medicine, UMass Chan Medical School, Worcester, MA     

Type 2 diabetes, a disruption of glucose homeostasis, is a major global health problem in search of therapeutics that can fully prevent or cure the disease. One approach is to exploit distinct adipose tissue depots that are known to differentially regulate systemic glucose metabolism. White adipose tissue (WAT) is abundant in humans and is mainly lipogenic, whereas much less abundant thermogenic brown adipose tissue (BAT) is associated with a lean, insulin-sensitive phenotype possibly achieved through beneficial secreted factors. Interventions that cause “browning” of WAT to produce “beige” adipocytes appear to be beneficial, and heterologous transplantation of BAT or beige cells in mice significantly improves glucose homeostasis. We aim to convert abundant white adipocytes into beige cells by CRISPR genome-editing with gRNA-Cas9 ribonucleoprotein complexes (RNP). CRISPR-based methods in our lab avoid uncontrolled integration of foreign DNA segments into the adipocyte genome, adverse immune responses and off-target effects that may disrupt favorable metabolic actions of therapeutic, gene modified brown or beige adipocytes.

We previously achieved genome-editing via CRISPR delivery Particles (CriPs), achieving indel percentage up to 43.8% (Shen et al, JBC, 2008) in the Nrip1 gene identified as a suppressor of adipocyte “browning” (Powelka et al, JCI, 2006). In our present studies we developed a new method to deliver RNPs to pre-adipocytes and achieved frameshift indel percentages for Nrip1 up to 97%. With this optimized technology, a marked increase in adipocyte browning is achieved, while residual Cas9 protein and sgRNA are rapidly degraded. In collaboration with the Silvia Corvera laboratory, implantation of the CRISPR-enhanced human brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure to immunogenic Cas9 or delivery vectors. See our data in BioRxiv.

An RNAi therapeutics strategy for Nonalcoholic Steatohepatitis (NASH) in type 2 diabetes

Batuhan Yenilmez, Kyounghee Min and Mark Kelly in collaboration with the Anastasia Khvorova laboratory

Liver steatosis and inflammation, causing the disease condition denoted as Non-alcoholic steatohepatitis (NASH), is a highly prevalent problem worldwide. NASH just became the number one reason for liver transplantation and there are no clear diagnostics and no drugs approved to treat it. The mechanisms behind how the reversible state of hepatic steatosis often seen in obesity progress to NASH are not fully understood. One proposed pathway that drives NASH reported recently by the Ira Tabas laboratory involves TAZ, a transcription regulator downstream of the Hippo pathway, which initiates crosstalk between hepatocytes and stellate cells. An increase in hepatocyte TAZ protein levels in NASH induces stellate cell activation through Hedgehog signaling and promotes fibrosis formation. To advance a potential therapeutic strategy in this disease state, the Anastasia Khvorova laboratory developed siRNAs that are chemically modified to increase their stability as well as their cellular uptake targeting TAZ in vivo. These chemically modified siRNAs (cdRNAs) give robust knockdown of target proteins in liver after a simple subcutaneous injection without inflammation or obvious toxicity. In collaboration with the Khvorova laboratory, we screened 48 cdRNA sequences against TAZ that are specifically targeting TAZ mRNA in vitro to identify the most active and potent cdRNA compounds. Bi-weekly subcutaneous injection of the most active cdRNA compound targeting TAZ in a methionine/choline deficient diet NASH mouse model alleviated the increase in plasma ALT and in some mice liver inflammation markers and collagen type 1 protein levels. These data indicate that the cdRNAs used here are a viable approach to develop therapeutics for NASH and perhaps other liver maladies.  We are currently targeting other gene products with cdRNA that are known to enhance fatty liver and NASH in animal models and our goal is ultimately to advance this technology to human subjects in clinical trials.

Molecular mechanisms of insulin resistance

Felipe Henriques, Alex Bedard, Leslie Rowland, Emmanouela Tsagkaraki, Sarah Nicoloro, Batuhan Yenilmez, Mark Kelly and Adilson Guilherme
Program in Molecular Medicine, UMass Chan Medical School

Obesity attenuates the effect of insulin to lower blood glucose, and this “insulin resistance” is associated with glucose intolerance, type 2 diabetes and other serious maladies. Despite tens of thousands of publications suggesting dozens of hypotheses on how insulin resistance develops, there is still deep uncertainty on the mechanisms underlying this condition. A unifying concept is that multiple adipose tissue subtypes are central regulators of systemic glucose homeostasis through modulation of hepatic glucose output and skeletal muscle glucose disposal, and this regulation is disrupted in obesity. In seeking metabolic pathways within adipocytes that may control whole body glucose homeostasis and that are severely disrupted in obesity, de novo lipogenesis (DNL) catalyzed by the enzymes ACLY, ACC and FASN stands out. Consistent with this pathway’s importance, adipose-selective FASN KO or FASN & ACLY double KO in HFD mice caused the strong appearance of beige adipocytes within subcutaneous white adipose tissue (WAT) and reversed the systemic glucose intolerance. Insulin sensitivities of liver, muscle and adipocytes are all increased in these adipose-selective FASN KO mice in hyperinsulinemic clamp studies, and energy expenditure is elevated without decreases in HFD food intake. The beige adipose tissue in FASN KO mice displayed increased sympathetic nerve fibers, but denervation studies showed WAT innervation was not required for the beiging process as it is during cold exposure. Single cell sequencing of the non-adipocytes from WAT of FASN KO mice combined with data from FACS analysis revealed an increase in macrophage polarization towards M2-type macrophages. Clodronate-mediated macrophage depletion of adipose tissues completely eliminated the beiging of WAT in adipose-selective FASN KO mice. Taken together, these studies suggest a powerful immune cell signaling axis is responsive to changes in adipocyte DNL activity and can contribute to WAT browning as well as enhanced systemic insulin sensitivity and glucose tolerance. We are actively exploring the molecular basis for immune cell signaling to adipocytes that can drive adipocyte “browning”.

This work was supported by NIDDK grants DK030898 and DK103047 from the National Institutes of Health.

Czech Lab News

emmanouela-tsagkaraki-keystone-symposiaEmmanouela Tsagkaraki receives Keystone Symposia Scholarship and presented her data at the conference on Engineering the Genome in Banff, Canada. Award through: National Center for Advancing Translational Sciences, Grant #1R13TR003025-01

felipe-diabetes-association-award.jpgAmerican Diabetes Association Postoctoral Fellowship awarded to Felipe Henriques. Learn more about Felipe's work.

adlison-associate-professor.jpgAdilson Guilherme promoted to Associate Professor at UMass Medical School. Congrats Adilson!

batu-heart-association-award.jpgAmerican Heart Association Predoctoral Fellowship awarded to Batu Yenilmez. Congrats Batu!

felipe-henriques-keystone-symposiumFelipe Henriques receives Keystone Symposia Scholarship and presented his data at the conference on Obesity and Adipose Tissue Biology, 2019, in Banff, Canada. Awarded through: National Institute of Diabetes and Digestive and Kidney Diseases, Grant # 5R13DK104611-05

czech-corvera-grant.jpgCzech and Corvera Labs awarded $2.5M partnering Department of Defense grant to advance potential therapy for type 2 diabetes. Learn more