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.  View our data in Nature Communications. 

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

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplantation, with no approved therapeutics available. Despite years of research, the exact molecular mechanism of NASH progression is not well understood, but fat accumulation is believed to be the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long term gene silencing with single subcutaneous administration. Here we identified a hyper-functional, fully chemically stabilized GalNAc conjugated siRNA targeting DGAT2 (Dgat2-1473) that upon injection elicits up to three months of DGAT2 silencing (>80-90%, p<0.0001) in wild-type and NSG-PiZ “humanized” mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (> 50%, p:0.0008), resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat didn’t translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH. Our laboratory is currently exploring additional targets that may synergize with DGAT2 silencing to alleviate NASH. View our data in Molecular Therapy 

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.