Volume 13, Issue 2, Pages 401-410 (February 2006) Overcoming Diabetes-Induced Hyperglycemia through Inhibition of Hepatic Phosphoenolpyruvate Carboxykinase (GTP) with RNAi  Alicia G. Gómez-Valadés, Anna Vidal-Alabró, Maria Molas, Jordi Boada, Jordi Bermúdez, Ramon Bartrons, José C. Perales  Molecular Therapy  Volume 13, Issue 2, Pages 401-410 (February 2006) DOI: 10.1016/j.ymthe.2005.08.026 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 pSHAG-664 induces PEPCK gene silencing in vitro. (A) pC/PEPCK-C (2.25 μg) was cotransfected with different molar ratios of pSHAG-664 (1:0 (striped), 1:6 (white), and 1:12 (gray)). As a control for nonspecific gene silencing pSHAG-Ff was cotransfected at a 1:24 ratio (black). pBluescript was used as stuffer. As an internal transfection control, 1 μg of pGL3 was added to all plates. PEPCK-C activity was measured 48 h after transfection and normalized for luciferase activity. Data are expressed as relative PEPCK activity (PEPCK activity/luciferase activity). (B) Silencing was confirmed at the protein level using Western blot. A representative blot is shown. Molecular Therapy 2006 13, 401-410DOI: (10.1016/j.ymthe.2005.08.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Liver-specific RNAi-induced gene silencing using hydrodynamic gene transfer. Luciferase expression vector pGL3 (10 μg) was co-injected with specific (pSHAG-Ff) or nonspecific (pSHAG-664) shRNA at a 1:6 molar ratio, using the hydrodynamic method. Luciferase activity was assayed 72 h later in liver extracts (n = 3). Molecular Therapy 2006 13, 401-410DOI: (10.1016/j.ymthe.2005.08.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 PEPCK-C gene silencing in healthy mice. Analysis of the silencing capacity of pSHAG-664 in nondiabetic mice assessed at the mRNA, enzyme activity, and protein levels. Mice were injected in the tail vein with 10% (w/v) physiological saline solution containing 100 μg of either pSHAG-664 or pSHAG-Ff plasmid. Silencing of PEPCK-C was assayed 24 h later. (A) Hepatic PEPCK mRNA content was analyzed from fed and 24-h fasted mice by Northern blot and is represented as the relative amount of PEPCK-C mRNA with respect to GAPDH mRNA. (B) Specific PEPCK activity in liver extracts from fasted animals is presented. Values are expressed as the means ± SE (n = 4, *P < 0.05). (C) Further confirmation of PEPCK silencing was obtained by analysis of the PEPCK-C protein content by Western blot. Representative blots are shown (n = 4). Molecular Therapy 2006 13, 401-410DOI: (10.1016/j.ymthe.2005.08.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Blood glucose and glucose tolerance after silencing PEPCK-C in the livers of diabetic animals. (A) Streptozotocin-induced diabetic mice received an intravenous injection as explained under Materials and methods with 100 μg of either pSHAG-Ff (solid bars) or pSHAG-664 (empty bars). Glycemia at 48 h (pSHAG-Ff, n = 26, and pSHAG-664, n = 25, ***P < 0.001) and 72 h (pSHAG-Ff, n = 12, and pSHAG-664, n = 10, P = 0.05) in 8-h fasted animals is presented. Results are expressed as relative glycemia. (B) A glucose tolerance test was performed at 48 h after hydrodynamic gene transfer by ip injection of a glucose bolus (1 mg/g) in pSHAG-Ff (filled squares), pSHAG-664 (empty squares), and healthy control animals (empty circles). Values are the means ± SE (n = 5, **P < 0.01). Molecular Therapy 2006 13, 401-410DOI: (10.1016/j.ymthe.2005.08.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 PEPCK-C silencing in the liver of diabetic mice. Mice received an intravenous injection of pSHAG-Ff or pSHAG-664 (100 μg). Liver extracts were prepared from animals at 48 and 72 h after injection as described under Materials and methods. PEPCK content was analyzed by Western blot and enzymatic activity. In addition, the level of a number of key proteins involved in the regulation of energy metabolism was analyzed. (A) Representative blots from independent experiments are shown. (B) PEPCK specific activity 72 h after the injection is shown. Values are the means ± SE (*P < 0.05). Molecular Therapy 2006 13, 401-410DOI: (10.1016/j.ymthe.2005.08.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Zonal expression in the liver after hydrodynamic gene delivery promotes partial PEPCK-C silencing throughout the liver parenchyma. Mice were injected with either (A to D) 10 or 20 μg of pEGFP or (E and F) 100 μg of pSHAG using the hydrodynamics procedure. Direct visualization of GFP (green) and indirect immunodetection of PEPCK (red) on fixed liver sections were analyzed using confocal microscopy. (A and B) correspond to representative fields of hepatic GFP distribution after hydrodynamic gene transfer at either 10 or 20 μg dose, respectively. Colocalization of GFP and PEPCK signals at the indicated doses (C, 10 μg, and D, 20 μg) in fixed liver sections is also shown. Representative PEPCK immunostaining after transfection of 100 μg pSHAG-Ff (E) or pSHAG-664 (F) is shown. Original magnification, 200×. Molecular Therapy 2006 13, 401-410DOI: (10.1016/j.ymthe.2005.08.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 7 PKR/interferon or apoptosis pathways are not activated upon pSHAG hydrodynamic injection. Positive control (C+) for PKR/interferon pathway activation was obtained from liver extracts of diabetic mice injected ip with 50 μg of a dsRNA analogue (poly(dI:dC)). Negative controls (C−) were saline-injected mice. eIF2α and eIF2α-P were detected by Western blot performed with liver extracts from the various groups. (A) Results are presented as the ratio of the phosphorylated form versus total eIF2α after densitometric analysis of the blots (n = 5). (B) Caspase 3 activity in liver extracts from healthy mice following hydrodynamic injection of with 100 μg of either pSHAG Ff or pSHAG 664 (n = 7). Positive control for hepatic apoptosis induction was obtained from liver extracts of mice injected ip with 700 mg/kg galactosamine and 100 mg/kg LPS (n = 3). Negative controls were ip saline-injected mice (n = 3). Molecular Therapy 2006 13, 401-410DOI: (10.1016/j.ymthe.2005.08.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions