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588. Hydrodynamics-Based FGF21 Gene Transfer for Treatment and Prevention of High Fat Diet Induced Obesity and Insulin Resistance
CARDIOVASCULAR AND PULMONARY DISEASES II 588. Hydrodynamics-Based FGF21 Gene Transfer for Treatment and Prevention of High Fat Diet Induced Obesity and Insulin Resistance
Cardiovascular and Pulmonary Diseases II
Fibroblast growth factor 21 (FGF21) is a secreted protein that plays critical roles in regulating glucose and lipid metabolism. In this study, we evaluated the effects of FGF21 gene transfer on C57BL/6 mice fed a high fat diet (HFD). We demonstrate that hydrodynamic delivery of the FGF21 gene increased mRNA levels of FGF21 exclusively in the liver, subsequently generating a sustained high level of FGF21 protein in blood that peaked at 500 ng/ml one day after injection, and consequently leading to a variety of beneficial effects including blockade of HFD-induced obesity, alleviation of fatty liver and improvement in glucose homeostasis. These effects were associated with altered expression of Ucp1, Dio2, Pgc1α, Pparg2, Cd36, Mgat1, F4/80, Mcp1 and Tnfα, which are involved in thermogenesis, lipogensis and chronic inflammation in the liver and adipose tissues. Transfer of the FGF21 gene in HFD-induced obese mice greatly increased expression of thermogenic genes in adipose tissue, resulting in similar improvements in systemic metabolism including reduction of adiposity, alleviation of fatty liver and attenuation of insulin resistance. Mechanistic studies on the effects of FGF21 gene transfer in lean mice revealed that mice transferred with FGF21 gene displayed suppressed lipogenesis in the liver and enhanced thermogenesis in brown adipose tissue which was coincident with a significant improvement in glucose tolerance. Collectively, our results suggest transfer of the FGF21 gene could be considered a promising approach for preventing and treating obesity and its complications.
Hiroyuki Hirai,1 Nobuaki Kikyo.1 1 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN.
Mingming Gao,1 Yongjie Ma,1 Ran Cui,1 Dexi Liu.1 1 Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA.
589. Pancreas-Targeted Gene Delivery for Enhanced Beta-Cell Proliferation
Brian Lu,1 Jason M. Tonne,1 Kiran Kurmi,1 Miguel MunozGomez,1 Egon Jacobus,1 Yogish Kudva,2 Yasuhiro Ikeda.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN; 2 Endocrinology, Mayo Clinic, Rochester, MN. Aging and obesity are linked to an increased risk of type 2 diabetes (T2D), a chronic metabolic disorder resulting from relative insulin insufficiency. Increased β-cell proliferation would benefit patients with T2D. Previously, we have demonstrated that systemic delivery of AAV8 vectors with a mouse INS2 or ELS internal promoter resulted in β-cell- or acinar cell-specific transgene expression. When the β-cell-targeted vector system was used to over-express SV40 large T antigen, a 9-fold increase in β-cell proliferation was observed in aged mice (control, 1.7%; SV40T, 16.0%). In this study, we investigated the potential utility of glucokinase (GCK) and betatrophin (BTRP) for induced β-cell proliferation. When GCK was over-expressed in β-cells of young 6-week old mice, a 2-fold increase in β-cell proliferation was observed (control, 9.7%; GCK, 21.6%). This correlated with enhanced β-cell mass, enhanced glucose clearance and elevated circulating insulin in treated mice. When aged mice were treated with the same GCK vector, we observed a 6-fold increase in β-cell proliferation (control, 0.8%; GCK, 4.2%) and enhanced glucose clearance. GCK vector administration in aged, high-fat-diet mice resulted in a 3-fold increase in β-cell proliferation (control, 1.4%; GCK, 4.4%) but without notable effect on glucose clearance. When the acinar-cell-targeted AAV vector was used to over-express BTRP in aged mice, β-cell proliferation increased by 2-fold (control, 0.8%; BTRP 2.0%). BTRP vector showed no notable effect on circulating insulin levels or glucose tolerance. These data suggest the potential utility of GCK for enhance β-cell proliferation in young, aged and obese subjects with diabetes. S228
590. Small Molecule Inhibitors of Suppressive Histone Modification Promote Direct Reprogramming of Fibroblasts To CardiomyocyteLike Cells
The recently described direct reprogramming approach is faster than an iPSCs approach by which first mature cells be reprogrammed to iPSCs and then differentiated into cardiomyocytes. Acceleration of direct reprogramming of cardiomyocytes are specifically important for cardiac gene therapy. Small molecules have great impact in combination with direct reprogramming approach, because they have ability to easily penetrate through multiple cell layers of wound tissues to regulate cellular signaling, yielding more consistent results compared to transduction of peptides or proteins that may not be able to access faster. Fibroblasts can be directly reprogrammed to cardiomyocyte-like cells by introducing defined genes. However, the reprogramming efficiency remains extremely low, delaying clinical application of this strategy to regenerative cardiology. MyoD is a master regulator for skeletal muscle differentiation and is an exceptionally potent transcription activator. Recently we found that the fusion of the highly potent transactivation domain isolated from the MyoD protein to the pluripotency factor Oct4 can improve the efficiency of converting skin fibroblasts to induced pluripotent stem cells (iPSCs) more than 10-100 fold. We examined whether fusion of the MyoD transactivation domain to cardiac transcription factors would facilitate reprogramming. We fused the MyoD transactivation domain to Mef2c, Gata4, Hand2, Tbx5 and other genes and transduced these genes in various combinations into non-cardiac mouse embryonic fibroblasts. Transduction of some of the fusion genes produced much larger beating clusters of cardiomyocyte-like cells faster than the combination of the 4 wild-type genes, with more than 18-fold greater efficiency of 3.5%. Then we examined two histone methyltransferase (HMTase) inhibitors, GSK126 and UNC0638 with cardiac reprogramming factors. GSK126 inhibits Enhancer of Zeste Homolog 2 (EZH2), which induces di- and trimethylation of Lys27 on histone H3 (H3K27me2 and H3K27me3, respectively) as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). H3K27me3 is typically associated with suppressed genes, although the underlying mechanisms have not been fully characterized. GSK126 or UNC0638 treated cells exhibited decreased H3K27 trimethylation or H3K9 dimethylation without changes of other histone H3 methylation marks. Using these inhibitors, we showed that inhibition of EZH2 in beating iCM cells formation results in increased cardiac reprogramming, making 23% of the cells spontaneously beat. Thus, inhibition of EZH2 enzymatic activity by GSK126 may provide a therapeutic option for the direct reprogramming treatment of cardiac repear.
591. Hyperactive AAVLDLR Variants Efficiently Reduce Cholesterol in Humanized Mouse Models of Familial Hypercholesterolemia Stably Expressing hPCSK9 or hIDOL
Suryanarayan Somanathan,1 Frank Jscobs,1 Qiang Wang,1 Daniel J. Rader,2 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Division of Translational Medicine and Human Genetics, University of Pennsylvania, Philadelphia, PA. Familial hypercholesterolemia (FH) is a metabolic disorder that arises due to loss-of-function mutations in the low-density lipoprotein receptor (LDLR). The disorder is characterized by high Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
Cardiovascular and Pulmonary Diseases II
Fibroblast growth factor 21 (FGF21) is a secreted protein that plays critical roles in regulating glucose and lipid metabolism. In this study, we evaluated the effects of FGF21 gene transfer on C57BL/6 mice fed a high fat diet (HFD). We demonstrate that hydrodynamic delivery of the FGF21 gene increased mRNA levels of FGF21 exclusively in the liver, subsequently generating a sustained high level of FGF21 protein in blood that peaked at 500 ng/ml one day after injection, and consequently leading to a variety of beneficial effects including blockade of HFD-induced obesity, alleviation of fatty liver and improvement in glucose homeostasis. These effects were associated with altered expression of Ucp1, Dio2, Pgc1α, Pparg2, Cd36, Mgat1, F4/80, Mcp1 and Tnfα, which are involved in thermogenesis, lipogensis and chronic inflammation in the liver and adipose tissues. Transfer of the FGF21 gene in HFD-induced obese mice greatly increased expression of thermogenic genes in adipose tissue, resulting in similar improvements in systemic metabolism including reduction of adiposity, alleviation of fatty liver and attenuation of insulin resistance. Mechanistic studies on the effects of FGF21 gene transfer in lean mice revealed that mice transferred with FGF21 gene displayed suppressed lipogenesis in the liver and enhanced thermogenesis in brown adipose tissue which was coincident with a significant improvement in glucose tolerance. Collectively, our results suggest transfer of the FGF21 gene could be considered a promising approach for preventing and treating obesity and its complications.
Hiroyuki Hirai,1 Nobuaki Kikyo.1 1 Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN.
Mingming Gao,1 Yongjie Ma,1 Ran Cui,1 Dexi Liu.1 1 Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA.
589. Pancreas-Targeted Gene Delivery for Enhanced Beta-Cell Proliferation
Brian Lu,1 Jason M. Tonne,1 Kiran Kurmi,1 Miguel MunozGomez,1 Egon Jacobus,1 Yogish Kudva,2 Yasuhiro Ikeda.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN; 2 Endocrinology, Mayo Clinic, Rochester, MN. Aging and obesity are linked to an increased risk of type 2 diabetes (T2D), a chronic metabolic disorder resulting from relative insulin insufficiency. Increased β-cell proliferation would benefit patients with T2D. Previously, we have demonstrated that systemic delivery of AAV8 vectors with a mouse INS2 or ELS internal promoter resulted in β-cell- or acinar cell-specific transgene expression. When the β-cell-targeted vector system was used to over-express SV40 large T antigen, a 9-fold increase in β-cell proliferation was observed in aged mice (control, 1.7%; SV40T, 16.0%). In this study, we investigated the potential utility of glucokinase (GCK) and betatrophin (BTRP) for induced β-cell proliferation. When GCK was over-expressed in β-cells of young 6-week old mice, a 2-fold increase in β-cell proliferation was observed (control, 9.7%; GCK, 21.6%). This correlated with enhanced β-cell mass, enhanced glucose clearance and elevated circulating insulin in treated mice. When aged mice were treated with the same GCK vector, we observed a 6-fold increase in β-cell proliferation (control, 0.8%; GCK, 4.2%) and enhanced glucose clearance. GCK vector administration in aged, high-fat-diet mice resulted in a 3-fold increase in β-cell proliferation (control, 1.4%; GCK, 4.4%) but without notable effect on glucose clearance. When the acinar-cell-targeted AAV vector was used to over-express BTRP in aged mice, β-cell proliferation increased by 2-fold (control, 0.8%; BTRP 2.0%). BTRP vector showed no notable effect on circulating insulin levels or glucose tolerance. These data suggest the potential utility of GCK for enhance β-cell proliferation in young, aged and obese subjects with diabetes. S228
590. Small Molecule Inhibitors of Suppressive Histone Modification Promote Direct Reprogramming of Fibroblasts To CardiomyocyteLike Cells
The recently described direct reprogramming approach is faster than an iPSCs approach by which first mature cells be reprogrammed to iPSCs and then differentiated into cardiomyocytes. Acceleration of direct reprogramming of cardiomyocytes are specifically important for cardiac gene therapy. Small molecules have great impact in combination with direct reprogramming approach, because they have ability to easily penetrate through multiple cell layers of wound tissues to regulate cellular signaling, yielding more consistent results compared to transduction of peptides or proteins that may not be able to access faster. Fibroblasts can be directly reprogrammed to cardiomyocyte-like cells by introducing defined genes. However, the reprogramming efficiency remains extremely low, delaying clinical application of this strategy to regenerative cardiology. MyoD is a master regulator for skeletal muscle differentiation and is an exceptionally potent transcription activator. Recently we found that the fusion of the highly potent transactivation domain isolated from the MyoD protein to the pluripotency factor Oct4 can improve the efficiency of converting skin fibroblasts to induced pluripotent stem cells (iPSCs) more than 10-100 fold. We examined whether fusion of the MyoD transactivation domain to cardiac transcription factors would facilitate reprogramming. We fused the MyoD transactivation domain to Mef2c, Gata4, Hand2, Tbx5 and other genes and transduced these genes in various combinations into non-cardiac mouse embryonic fibroblasts. Transduction of some of the fusion genes produced much larger beating clusters of cardiomyocyte-like cells faster than the combination of the 4 wild-type genes, with more than 18-fold greater efficiency of 3.5%. Then we examined two histone methyltransferase (HMTase) inhibitors, GSK126 and UNC0638 with cardiac reprogramming factors. GSK126 inhibits Enhancer of Zeste Homolog 2 (EZH2), which induces di- and trimethylation of Lys27 on histone H3 (H3K27me2 and H3K27me3, respectively) as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2). H3K27me3 is typically associated with suppressed genes, although the underlying mechanisms have not been fully characterized. GSK126 or UNC0638 treated cells exhibited decreased H3K27 trimethylation or H3K9 dimethylation without changes of other histone H3 methylation marks. Using these inhibitors, we showed that inhibition of EZH2 in beating iCM cells formation results in increased cardiac reprogramming, making 23% of the cells spontaneously beat. Thus, inhibition of EZH2 enzymatic activity by GSK126 may provide a therapeutic option for the direct reprogramming treatment of cardiac repear.
591. Hyperactive AAVLDLR Variants Efficiently Reduce Cholesterol in Humanized Mouse Models of Familial Hypercholesterolemia Stably Expressing hPCSK9 or hIDOL
Suryanarayan Somanathan,1 Frank Jscobs,1 Qiang Wang,1 Daniel J. Rader,2 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Division of Translational Medicine and Human Genetics, University of Pennsylvania, Philadelphia, PA. Familial hypercholesterolemia (FH) is a metabolic disorder that arises due to loss-of-function mutations in the low-density lipoprotein receptor (LDLR). The disorder is characterized by high Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy