Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. We investigated restoration of hepatocyte functions by HNF4A mRNA transfection in vitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes in vitro. Repeated in vivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

The tightly controlled replication of hepatocytes in liver regeneration and uncontrolled proliferation of tumor cells in hepatocellular carcinoma (HCC) are often modulated by common regulatory pathways. Several microRNAs (miRNAs) are involved in HCC progression by modulating posttranscriptional expression of multiple target genes. miR-221, which is frequently up-regulated in HCCs, delays fulminant liver failure in mice by inhibiting apoptosis, indicating a pleiotropic role of miR-221 in hepatocytes. Here, we hypothesize that modulation of miR-221 targets in primary hepatocytes enhances proliferation, providing novel clues for enhanced liver regeneration. We demonstrate that miR-221 enhances proliferation of in vitro cultivated primary hepatocytes. Furthermore, applying two-thirds partial hepatectomy as a surgically induced liver regeneration model we show that adeno-associated virus-mediated overexpression of miR-221 in the mouse liver also accelerates hepatocyte proliferation in vivo. miR-221 overexpression leads to rapid S-phase entry of hepatocytes during liver regeneration. In addition to the known targets p27 and p57, we identify Aryl hydrocarbon nuclear translocator (Arnt) messenger RNA (mRNA) as a novel target of miR-221, which contributes to the pro-proliferative activity of miR-221. Conclusion: miR-221 overexpression accelerates hepatocyte proliferation. Pharmacological intervention targeting miR-221 may thus be therapeutically beneficial in liver failure by preventing apoptosis and by inducing liver regeneration. (Hepatology 2013;)

Death receptor-mediated apoptosis of hepatocytes contributes to hepatitis and fulminant liver failure. MicroRNAs (miRNAs), 19–25 nucleotide-long noncoding RNAs, have been implicated in the posttranscriptional regulation of the various apoptotic pathways. Here we report that global loss of miRNAs in hepatic cells leads to increased cell death in a model of FAS/CD95 receptor-induced apoptosis. miRNA profiling of murine liver identified 11 conserved miRNAs, which were up-regulated in response to FAS-induced fulminant liver failure. We show that ectopic expression of miR-221, one of the highly up-regulated miRNAs in response to apoptosis, protects primary hepatocytes and hepatoma cells from apoptosis. Importantly, in vivo overexpression of miR-221 by adeno-associated virus serotype 8 (AAV8) delays FAS-induced fulminant liver failure in mice. We additionally demonstrate that miR-221 regulates hepatic expression of p53 up-regulated modulator of apoptosis ( Puma ), a well-known proapoptotic member of the Bcl2 protein family. Conclusion: We identified miR-221 as a potent posttranscriptional regulator of FAS-induced apoptosis. miR-221 may serve as a potential therapeutic target for the treatment of hepatitis and liver failure. (HEPATOLOGY 2011;)

Objective: Liver fibrosis and cirrhosis resulting from chronic liver injury represent a major healthcare burden worldwide. Growth differentiation factor (GDF) 11 has been recently investigated for its role in rejuvenation of ageing organs, but its role in chronic liver diseases has remained unknown. Here, we investigated the expression and function of GDF11 in liver fibrosis, a common feature of most chronic liver diseases. Design: We analysed the expression of GDF11 in patients with liver fibrosis, in a mouse model of liver fibrosis and in hepatic stellate cells (HSCs) as well as in other liver cell types. The functional relevance of GDF11 in toxin-induced and cholestasis-induced mouse models of liver fibrosis was examined by in vivo modulation of Gdf11 expression using adeno-associated virus (AAV) vectors. The effect of GDF11 on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)+ liver progenitor cells was studied in mouse and human liver organoid culture. Furthermore, in vivo depletion of LGR5+ cells was induced by injecting AAV vectors expressing diptheria toxin A under the transcriptional control of Lgr5 promoter. Results: We showed that the expression of GDF11 is upregulated in patients with liver fibrosis and in experimentally induced murine liver fibrosis models. Furthermore, we found that therapeutic application of GDF11 mounts a protective response against fibrosis by increasing the number of LGR5+ progenitor cells in the liver. Conclusion: Collectively, our findings uncover a protective role of GDF11 during liver fibrosis and suggest a potential application of GDF11 for the treatment of chronic liver disease.

The lack of broad-spectrum anti-acute liver failure (ALF) therapeutic agents contributes to ALF-related mortality. MicroRNAs (miRNAs) are suggested to be potent serum biomarkers for ALF, but their functional and therapeutic relevance in ALF are unclear. Here we show an unbiased approach, using two complementary miRNA screens, to identify miRNAs that can attenuate ALF. We identify miR-125b-5p as a regulator of cell death that attenuates paracetamol-induced and FAS-induced toxicity in mouse and human hepatocytes. Importantly, administration of miR-125b-5p mimic in mouse liver prevents injury and improves survival in models of ALF. Functional studies show that miR-125b-5p ameliorates ALF by directly regulating kelch-like ECH-associated protein 1, in turn elevating expression of nuclear factor-E2-related factor 2, a known regulator in ALF. Collectively, our findings establish miR-125b-5p as an important regulator of paracetamol-induced and FAS-induced cell death. Thus, miR-125b-5p mimic may serve as a broad-spectrum therapeutic attenuator of cell death during ALF.