Lipid nanoparticle-encapsulated mRNA therapy corrects serum total bilirubin level in Crigler-Najjar syndrome mouse model.

Crigler-Najjar syndrome is a rare disorder of bilirubin metabolism caused by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) mutations characterized by hyperbilirubinemia and jaundice. No cure currently exists; treatment options are limited to phototherapy, whose effectiveness diminishes over time, and liver transplantation. Here, we evaluated the therapeutic potential of systemically administered, lipid nanoparticle-encapsulated human UGT1A1 (hUGT1A1) mRNA therapy in a Crigler-Najjar mouse model. Ugt1 knockout mice were rescued from lethal post-natal hyperbilirubinemia by phototherapy. These adult Ugt1 knockout mice were then administered a single lipid nanoparticle-encapsulated hUGT1A1 mRNA dose. Within 24 h, serum total bilirubin levels decreased from 15 mg/dL (256 µmol/L) to <0.5 mg/dL (9 µmol/L), i.e., slightly above wild-type levels. This reduction was sustained for 2 weeks before bilirubin levels rose and returned to pre-treatment levels by day 42 post-administration. Sustained reductions in total bilirubin levels were achieved by repeated administration of the mRNA product in a frequency-dependent manner. We were also able to rescue the neonatal lethality phenotype seen in Ugt1 knockout mice with a single lipid nanoparticle dose, which suggests that this may be a treatment modality appropriate for metabolic crisis situations. Therefore, lipid nanoparticle-encapsulated hUGT1A1 mRNA may represent a potential treatment for Crigler-Najjar syndrome.

Altered ethanol metabolism and increased oxidative stress enhance alcohol-associated liver injury in farnesoid X receptor-deficient mice.

BACKGROUND & AIMS: Pharmacological activation of farnesoid X receptor (FXR) ameliorates liver injury, steatosis and inflammation in mouse models of alcoholic liver disease (ALD), but the underlying mechanisms of the protective effect of FXR against ALD remain unclear. METHODS: To investigate the role of FXR in ALD, we used the NIAAA model of chronic plus binge ethanol feeding in FXR-deficient knockout (FXR KO) mice. RESULTS: Ethanol-mediated liver injury and steatosis were increased in FXR KO mice, while both WT and FXR KO mice consumed the same amount of alcohol. Ethanol feeding induced liver inflammation and neutrophil infiltration that were further increased in FXR KO mice. In addition, collagen accumulation and expression of profibrotic genes were markedly elevated in the liver of alcohol-fed FXR KO compared to wild-type mice, suggesting that ethanol-induced liver fibrosis is enhanced in the absence of FXR. Surprisingly, FXR KO mice showed reduced blood alcohol levels post-binge, while CYP2E1 and ALDH1A1 were upregulated compared to WT mice, suggesting that alcohol metabolism is altered in FXR KO mice. Notably, exacerbated liver injury in FXR KO mice was associated with increased oxidative stress. ALDH1A1 activity was upregulated in FXR-deficient mouse primary hepatocytes, contributing to reactive oxygen species (ROS) generation, in vitro. Finally, using an ALDH1A1 inhibitor, we showed that ALDH1A1 activity is a key contributor to alcohol-induced ROS generation in FXR-deficient hepatocytes, in vitro. CONCLUSION: ALD pathogenesis in FXR KO mice correlates with altered ethanol metabolism and increased oxidative stress, providing new insights into the protective function of FXR in ALD.

Evaluation of cytosine base editing and adenine base editing as a potential treatment for alpha-1 antitrypsin deficiency.

Alpha-1 antitrypsin deficiency (AATD) is a rare autosomal codominant disease caused by mutations within the SERPINA1 gene. The most prevalent variant in patients is PiZ SERPINA1, containing a single G > A transition mutation. PiZ alpha-1 antitrypsin (AAT) is prone to misfolding, leading to the accumulation of toxic aggregates within hepatocytes. In addition, the abnormally low level of AAT secreted into circulation provides insufficient inhibition of neutrophil elastase within the lungs, eventually causing emphysema. Cytosine and adenine base editors enable the programmable conversion of C⋅G to T⋅A and A⋅T to G⋅C base pairs, respectively. In this study, two different base editing approaches were developed: use of a cytosine base editor to install a compensatory mutation (p.Met374Ile) and use of an adenine base editor to mediate the correction of the pathogenic PiZ mutation. After treatment with lipid nanoparticles formulated with base editing reagents, PiZ-transgenic mice exhibited durable editing of SERPINA1 in the liver, increased serum AAT, and improved liver histology. These results indicate that base editing has the potential to address both lung and liver disease in AATD.

Role of Noncoding RNAs in Acetaminophen-Induced Liver Injury.

Genomic and transcriptomic analyses have well established that the major fraction of the mammalian genome is transcribed into different classes of RNAs ranging in size from a few nucleotides to hundreds of thousands of nucleotides, which do not encode any protein. Some of these noncoding RNAs (ncRNAs) are directly or indirectly linked to the regulation of expression or functions of 25,000 proteins coded by <2% of the human genome. Among these regulatory RNAs, microRNAs are small (2125 nucleotides) RNAs that are processed from precursor RNAs that have stemloop structure, whereas noncoding RNAs >200 nucleotides are termed long noncoding RNAs (lncRNAs). Circular RNAs (circRNAs) are newly identified lncRNA members that are generated by back-splicing of primary transcripts. The functions of ncRNAs in modulating liver toxicity of xenobiotics are emerging only recently. Acetaminophen (N-acetyl-para-aminophenol, paracetamol or APAP) is a safe analgesic and antipyretic drug at the therapeutic dose. However, it can cause severe liver toxicity that may lead to liver failure if overdosed or combined with alcohol, herbs, or other xenobiotics. This review discusses the role of ncRNAs in acetaminophen metabolism, toxicity, and liver regeneration after APAP-induced liver injury (AILI).

Regulation of hepatic glutamine metabolism by miR-122.

OBJECTIVE: It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine (Gln) is a critical energy and anaplerotic source for mammalian cells, we assessed Gln metabolism in control wild type (WT) mice and miR-122 knockout (KO) mice by stable isotope resolved metabolomics (SIRM) studies. METHODS: Six-to eight-week-old WT and KO mice and 12- to 15-month-old liver tumor-bearing mice were injected with [U-13C5,15N2]-L-Gln, and polar metabolites from the liver tissues were analyzed by nuclear magnetic resonance (NMR) imaging and ion chromatography-mass spectrometry (IC-MS). Gln-metabolism was also assessed in a Gln-dependent hepatocellular carcinoma (HCC) cell line (EC4). Expressions of glutaminases (Gls and Gls2) were analyzed in mouse livers and human primary HCC samples. RESULTS: The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of 13C- and/or 15N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney-type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3'-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased 13C-Gln, 13C-á-ketoglutarate, 13C-isocitrate, and 13C-citrate levels. In contrast, 13C-phosphoenolpyruvate and 13C-G6P levels were elevated in cells expressing ectopic miR-122, suggesting enhanced gluconeogenesis. Finally, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database analysis showed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared with those expressing lower levels of these proteins. CONCLUSIONS: Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating the therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.

miRNA-122 Protects Mice and Human Hepatocytes from Acetaminophen Toxicity by Regulating Cytochrome P450 Family 1 Subfamily A Member 2 and Family 2 Subfamily E Member 1 Expression.

Acetaminophen toxicity is a leading cause of acute liver failure (ALF). We found that miRNA-122 (miR-122) is down-regulated in liver biopsy specimens of patients with ALF and in acetaminophen-treated mice. A marked decrease in the primary miR-122 expression occurs in mice on acetaminophen overdose because of suppression of its key transactivators, hepatocyte nuclear factor (HNF)-4α and HNF6. More importantly, the mortality rates of male and female liver-specific miR-122 knockout (LKO) mice were significantly higher than control mice when injected i.p. with an acetaminophen dose not lethal to the control. LKO livers exhibited higher basal expression of cytochrome P450 family 2 subfamily E member 1 (CYP2E1) and cytochrome P450 family 1 subfamily A member 2 (CYP1A2) that convert acetaminophen to highly reactive N-acetyl-p-benzoquinone imine. Upregulation of Cyp1a2 primary transcript and mRNA in LKO mice correlated with the elevation of aryl hydrocarbon receptor (AHR) and mediator 1 (MED1), two transactivators of Cyp1a2. Analysis of ChIP-seq data in the ENCODE (Encyclopedia of DNA Element) database identified association of CCCTC-binding factor (CTCF) with Ahr promoter in mouse livers. Both MED1 and CTCF are validated conserved miR-122 targets. Furthermore, depletion of Ahr, Med1, or Ctcf in Mir122-/- hepatocytes reduced Cyp1a2 expression. Pulse-chase studies found that CYP2E1 protein level is upregulated in LKO hepatocytes. Notably, miR-122 depletion sensitized differentiated human HepaRG cells to acetaminophen toxicity that correlated with upregulation of AHR, MED1, and CYP1A2 expression. Collectively, these results reveal a critical role of miR-122 in acetaminophen detoxification and implicate its therapeutic potential in patients with ALF.