A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice

Abstract

Nonalcoholic steatohepatitis (NASH) is emerging as a major public health issue and is associated with significant liver-related morbidity and mortality. At present, there are no approved drug therapies for NASH. The transcriptional coactivator with PDZ-binding motif (TAZ; encoded by WW domain-containing transcription regulator 1 [WWTR1]) is up-regulated in hepatocytes in NASH liver from humans and has been shown to causally promote inflammation and fibrosis in mouse models of NASH. As a preclinical test of targeting hepatocyte TAZ to treat NASH, we injected stabilized TAZ small interfering RNA (siRNA) bearing the hepatocyte-specific ligand N-acetylgalactosamine (GalNAc-siTAZ) into mice with dietary-induced NASH. As a preventative regimen, GalNAc-siTAZ inhibited inflammation, hepatocellular injury, and the expression of profibrogenic mediators, accompanied by decreased progression from steatosis to NASH. When administered to mice with established NASH, GalNAc-siTAZ partially reversed hepatic inflammation, injury, and fibrosis. Conclusion: Hepatocyte-targeted siTAZ is potentially a novel and clinically feasible treatment for NASH.

Figure 1

Taz siRNA screen in mouse liver cells. (A) Forty‐eight siRNAs designed against mouse Taz (Wwtr1) were transfected into mouse Hepa1‐6 cells using an siRNA concentration of 10 nM; Wwtr1 mRNA was assayed 24 hours later. Data were normalized to Gapdh, and the values in the graph are shown as percentage of Wwtr1 expression relative to the mean value of three nonspecific control siRNAs (= 100%). The average of four biological replicas ± SD is shown. (B) Dose‐response curves of siRNA6 and siRNA8; the average of four biological replicas ± SD is shown. (C) Hepa1‐6 cells were left untreated (−) or incubated under mock conditions with siCtrl or with 5 or 50 nM siTAZ‐1 or siTAZ‐2, which are the GalNAc conjugates of siRNA6 and siRNA8, respectively. Taz protein was analyzed by immunoblot and quantified by densitometry. (D) Primary mouse hepatocytes were incubated with the indicated concentrations of siTAZ‐1 and siTAZ‐2 and then assayed for Wwtr1 mRNA. The average of four biological replicas ± SD is shown. (E) Primary mouse hepatocytes, bone marrow‐derived macrophages, and HSCs were incubated with 100 nM siControl or siTAZ‐2 (shown as 2) and then assayed for Wwtr1 mRNA. The average of four biological replicas ± SEM is shown. (F) Human PBMCs were left untreated (−); incubated under mock conditions; with a negative control or three positive controls, as defined in Materials and Methods; with siCtrl; or with 100 nM siTAZ‐2. After 24 hours, IFN‐α2a and IP‐10 were assayed. Data are shown for PBMCs from three healthy donors as mean average of two to six biological replicas ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: C, siControl; Ctrl, control; HC, primary hepatocyte; HSC, hepatic stellate cell; Mac, bone marrow‐derived macrophage.

Figure 2

TAZ silencing using GalNAc‐siTAZ in NASH mice. (A) Experimental scheme. Male C57BL/6J mice were fed the NASH diet for 9 weeks and injected with PBS, GalNAc‐siControl, or GalNAc‐siTAZ‐1 or GalNAc‐siTAZ‐2 at the indicated doses. (B) Immunoblots of YAP and TAZ in the livers of the treated mice. (C,D) Immunoblots of TAZ and YAP in primary hepatocytes and NPCs isolated from mice treated with GalNAc‐siControl or GalNAc‐siTAZ‐2. Abbreviations: Ctrl, control; HC, primary hepatocyte.

Figure 3

Mice treated with GalNAc‐siTAZ during steatosis to NASH progression have markedly lower liver TAZ without affecting metabolic endpoints, liver triglyceride, or plasma lipids. (A) Experimental scheme. Male C57BL/6J mice were fed the NASH diet for 8 weeks and then injected once weekly for 8 additional weeks with PBS, GalNAc‐siControl, GalNAc‐siTAZ‐1, or GalNAc‐siTAZ‐2 at 10 mg/kg. (B,C) Immunoblots of liver TAZ in mice from the various experimental groups. (D) Assay of plasma ALT. For panel D, n = 8 mice/group, and values shown are mean ± SEM; *P < 0.05 using one‐way ANOVA. Abbreviations: 1, siTAZ‐1; 2, siTAZ‐2; C, siControl; P, phosphate‐buffered saline.

Figure 4

Mice treated with GalNAc‐siTAZ during steatosis to NASH progression show improvements in liver inflammation and fibrosis and less liver cell death. (A) Livers of the mice described in Fig. 3 were stained with H&E (upper row of images) and sirius red (lower row of images) and then quantified for inflammatory cells per field and percentage sirius red area. Scale bar, 200 μm; * indicates difference from P. (B‐E) The following endpoints were assayed in the livers of these mice: (B) indicated mRNAs; (C) percent of F4/80⁺ cells and α‐SMA⁺ area; (D) MCP‐1 and TGF‐β1 concentration; and (E) percentage of liver cells that stained for TUNEL. For all panels, n = 8 mice per group; values shown are means ± SEM; *P < 0.05 using one‐way ANOVA. Abbreviations: 1, siTAZ‐1; 2, siTAZ‐2; C, siControl; P, phosphate‐buffered saline; Tnfa, tumor necrosis factor a mRNA.

Figure 5

GalNAc‐siTAZ treatment after the development of NASH reduces liver inflammation and fibrosis. (A) Experimental scheme. Male C57BL/6J mice were fed the NASH diet for 16 weeks and then injected once weekly for 12 additional weeks with PBS or GalNAc‐siTAZ‐2 at 10 mg/kg. (B) Livers were immunoblotted for TAZ and β‐actin and assayed for Wwtr1 mRNA. (C) Livers were stained with H&E (upper row of images) and sirius red (lower row of images) and then quantified for inflammatory cells per field and percentage sirius red area. Scale bars, 200 μm. (D) Livers were scored for fibrosis stage as described in Materials and Methods. P value was calculated from the average score for each group using the Wilcoxon rank‐sum test. (E‐K) The following endpoints were measured in the livers or plasma from these mice: (E) indicated mRNAs; (F) percentage of F4/80⁺ cells and the α‐SMA⁺ area; (G) MCP‐1 and TGF‐β1 concentrations; (H) percentage of liver cells that stained for TUNEL; (I) plasma ALT; (J) Ihh mRNA; and (K) MMP activity. For all graphs, n = 7 (PBS) and n = 8 (GalNAc‐siTAZ‐2) mice. Values shown for all graphs except panel D are means ± SEM; *P < 0.05 using the two‐tailed Student t test. Abbreviations: 2, siTAZ‐2; Acta2, smooth muscle aortic α‐actin; AU, arbitrary unit; Dpt, dermatopontin; Opn, osteopontin; P, phosphate‐buffered saline.