Fazirsiran for Liver Disease Associated with Alpha1-Antitrypsin Deficiency.

BACKGROUND: Alpha1-antitrypsin (AAT) deficiency results from carriage of a homozygous SERPINA1 "Z" mutation (proteinase inhibitor [PI] ZZ). The Z allele produces a mutant AAT protein called Z-AAT, which accumulates in hepatocytes and can lead to progressive liver disease and fibrosis. This open-label, phase 2 trial investigated the safety and efficacy of fazirsiran, an RNA interference therapeutic, in patients with liver disease associated with AAT deficiency. METHODS: We assigned adults with the PI ZZ genotype and liver fibrosis to receive fazirsiran at a dose of 200 mg (cohorts 1 [4 patients] and 2 [8 patients]) or 100 mg (cohort 1b [4 patients]) subcutaneously on day 1 and week 4 and then every 12 weeks. The primary end point was the change from baseline to week 24 (cohorts 1 and 1b) or week 48 (cohort 2) in liver Z-AAT concentrations, which were measured by means of liquid chromatography-mass spectrometry. RESULTS: All the patients had reduced accumulation of Z-AAT in the liver (median reduction, 83% at week 24 or 48). The nadir in serum was a reduction of approximately 90%, and treatment was also associated with a reduction in histologic globule burden (from a mean score of 7.4 [scores range from 0 to 9, with higher scores indicating a greater globule burden] at baseline to 2.3 at week 24 or 48). All cohorts had reductions in liver enzyme concentrations. Fibrosis regression was observed in 7 of 15 patients and fibrosis progression in 2 of 15 patients after 24 or 48 weeks. There were no adverse events leading to trial or drug discontinuation. Four serious adverse events (viral myocarditis, diverticulitis, dyspnea, and vestibular neuronitis) resolved. CONCLUSIONS: In this small trial, fazirsiran was associated with a strong reduction of Z-AAT concentrations in the serum and liver and concurrent improvements in liver enzyme concentrations. (Funded by Arrowhead Pharmaceuticals; AROAAT-2002 ClinicalTrials.gov number, NCT03946449.).

The Inflammatory Cytokine Profile Associated With Liver Damage Is Broader and Stronger in Patients With Chronic Hepatitis B Compared to Patients With Acute Hepatitis B.

Liver damage in hepatitis B is immune driven and correlates with inflammatory markers in patient serum. There is no comparison of these markers to determine if inflammatory profiles are distinct to different types of liver damage across patients at different stages of disease. We measured 25 inflammatory markers in patients with acute hepatitis B and chronic hepatitis B with hepatitis B e antigen seroconversion and chronic patients stopping nucleoside analogue therapy. Myeloid markers dominated the inflammatory profile in all stages of hepatitis B. More inflammatory markers were detectable in chronic patients, including elevated concentrations of cytotoxic effectors Fas ligand, TRAIL, and TNF-α.

Hepatic-targeted RNA interference provides robust and persistent knockdown of alpha-1 antitrypsin levels in ZZ patients.

BACKGROUND & AIMS: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder causing pulmonary and liver disease. The PiZ mutation in AAT (SERPINA1) results in mis-folded AAT protein (Z-AAT) accumulating in hepatocytes, leading to fibrosis and cirrhosis. RNAi-based therapeutics silencing production of hepatic Z-AAT might benefit patients with AATD-associated liver disease. This study evaluated an RNAi therapeutic to silence production of AAT. METHODS: Part A of this double-blind first-in-human study randomized 54 healthy volunteers (HVs) into single dose cohorts (two placebo: four active), receiving escalating doses of the investigational agent ARC-AAT from 0.38 to 8.0 mg/kg or placebo. Part B randomized 11 patients with PiZZ (homozygous for Z-AAT) genotype AATD, who received up to 4.0 mg/kg of ARC-AAT or placebo. Patients with baseline FibroScan® >11 kPa or forced expiratory volume in one second (FEV1) <60% were excluded. Assessments included safety, pharmacokinetics, and change in serum AAT concentrations. RESULTS: A total of 36 HVs received ARC-AAT and 18 received placebo (part A). Seven PiZZ individuals received ARC-AAT and four received placebo (part B). A dose response in serum AAT reduction was observed at doses ≥4 mg/kg with similar relative reductions in PiZZ patients and HVs at 4 mg/kg and a maximum reduction of 76.1% (HVs) vs. 78.8% (PiZZ) at this dose. The time it took for serum AAT to return to baseline was similar for HV and PiZZ. There were no notable differences between HV and PiZZ safety parameters. The study was terminated early because of toxicity findings related to the delivery vehicle (ARC-EX1) seen in a non-human primate study. CONCLUSION: PiZZ patients and HVs responded similarly to ARC-AAT. Deep and durable knockdown of hepatic AAT production based on observed reduction in serum AAT concentrations was demonstrated. LAY SUMMARY: Accumulation of abnormal proteins in the livers of patients with alpha-1 antitrypsin deficiency may lead to decreased liver function and potentially liver failure. Therapeutics targeting the production of these abnormal proteins may be used to prevent or treat liver disease in patients with alpha-1 antitrypsin deficiency. CLINICAL TRIAL REGISTRATION NUMBER: NCT02363946.

Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

Alpha-1-Antitrypsin Deficiency.

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Knockdown of Alpha-1 Antitrypsin with antisense oligonucleotide does not exacerbate smoke induced lung injury.

Alpha-1 Antitrypsin (AAT) is a serum protease inhibitor that regulates increased lung protease production induced by cigarette smoking. Mutations in the Serpina1 gene cause AAT to form hepatoxic polymers, which can lead to reduced availability for the protein's primary function and severe liver disease. An AAT antisense oligonucleotide (ASO) was previously identified to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knockdown of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups each for the smoking and smoke-flu injury models. The ASO and control (No-ASO) were injected subcutaneously starting with smoking or four days prior to influenza infection and then injected weekly at 50 mg/kg body weight. ASO treatment during a 3-month smoke exposure significantly decreased the serum and lung AAT expression, resulting in increased Cela1 expression and elastase activity. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly worsened by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure, but did not observe an ASO treatment effect. Similarly, with the smoke-flu model, differences were only observed between smoke-flu and room air controls, but not as a result of ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment, while sufficient to protect from liver injury, may not be robust enough to lead to lung injury. The results also suggest that previously described AAT ASO treatment for AAT mutation related liver disease may attenuate hepatic injury without being detrimental to the lungs. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.

Preserved Gut Microbial Diversity Accompanies Upregulation of TGR5 and Hepatobiliary Transporters in Bile Acid-Treated Animals Receiving Parenteral Nutrition.

BACKGROUND: Parenteral nutrition (PN) is a lifesaving therapy but is associated with gut atrophy and cholestasis. While bile acids (BAs) can modulate intestinal growth via gut receptors, the gut microbiome likely influences gut proliferation and inflammation. BAs also regulate the bile salt export pump (BSEP) involved in cholestasis. We hypothesized that the BA receptor agonist oleanolic acid (OA) regulates gut TGR5 receptor and modulates gut microbiota to prevent PN-associated injury. MATERIALS AND METHODS: Neonatal piglets were randomized to approximately 2 weeks of isocaloric enteral nutrition (EN), PN, or PN + enteral OA. Serum alanine aminotransferase, bilirubin, BAs, hepatic BSEP, gut TGR5, gut, liver morphology, and fecal microbiome utilizing 16S rRNA sequencing were evaluated. Kruskal-Wallis test, pairwise Mann-Whitney U test, and multilevel logistic regression analysis were performed. RESULTS: PN support resulted in gut atrophy substantially prevented by OA. The median (interquartile range) for villous/crypt ratio was as follows: EN, 3.37 (2.82-3.80); PN, 1.73 (1.54-2.27); and OA, 2.89 (2.17-3.34; P = .006). Pairwise comparisons yielded P = .002 (EN vs PN), P = .180 (EN vs OA), P = .026 (PN vs OA). OA upregulated TGR5 and BSEP without significant improvement in serum bilirubin ( P = .095). A decreased microbial diversity and shift toward proinflammatory phylum Bacteroidetes were seen with PN, which was prevented by OA. CONCLUSIONS: OA prevented PN-associated gut mucosal injury, Bacterioides expansion, and the decreased microbial diversity noted with PN. This study demonstrates a novel relationship among PN-associated gut dysfunction, BA treatment, and gut microbial changes.

Oleanolic Acid Improves Gut Atrophy Induced by Parenteral Nutrition.

BACKGROUND: Nutrition support with parenteral nutrition (PN) is associated with gut atrophy. Prior studies have shown improvement with enteral chenodeoxycholic acid, a dual agonist for the farnesoid X receptor (FXR) and bile acid receptor TGR5. We hypothesized that gut growth is induced by TGR5 activation, and gut atrophy during PN administration could be prevented with the TGR5-specific agonist oleanolic acid (OA). METHODS: Neonatal pigs were implanted with duodenal and jugular vein catheters. Animals were provided equi-nutritious PN or enteral swine milk. A PN subgroup received enteral OA at 50 mg/kg/d. RESULTS: PN caused marked gut atrophy compared with enterally fed (EN) control animals. OA treatment led to preservation of gut mass demonstrated grossly and histologically. The mean ± SD gut weight as a percentage of body weight was 4.30 ± 0.26 for EN, 1.92 ± 0.06 for PN (P < .05, EN vs PN), and 3.39 ± 0.79 for PN+OA (P < .05, PN+OA vs PN). Mean ± SD gut density (g/cm) was 0.31 ± 0.03 for EN, 0.18 ± 0.03 for PN (P < .05 EN vs PN), and 0.27 ± 0.01 for PN+OA (P < .05 PN+OA vs PN). Histologically, a markedly decreased villous to crypt ratio was noted with PN, and OA significantly prevented this decrease. The mean ± SD v/c ratio was 3.51 ± 0.59 for EN, 1.69 ± 0.10 for PN (P < .05, EN vs PN), and 2.90 ± 0.23 for PN+OA (P < .05, PN+OA vs PN). Gut TGR5 messenger RNA expression was significantly elevated with OA treatment compared with both PN and EN. CONCLUSION: The bile acid-activated G protein-coupled receptor TGR5 agonist OA prevented gut atrophy associated with PN.

Controversies in the Mechanism of Total Parenteral Nutrition Induced Pathology.

Over 30,000 patients are permanently dependent on Total Parenteral Nutrition (TPN) for survival with several folds higher requiring TPN for a prolonged duration. Unfortunately, it can cause potentially fatal complications. TPN infusion results in impairment of gut mucosal integrity, enhanced inflammation, increased cytokine expression and trans-mucosal bacterial permeation. It also causes endotoxin associated down regulation of bile acid transporters and Parenteral Nutrition Associated Liver Disease (PNALD), which includes steatosis, disrupted glucose metabolism, disrupted lipid metabolism, cholestasis and liver failure. Despite multiple theories, its etiology and pathophysiology remains elusive and is likely multifactorial. An important cause for TPN related pathologies appears to be a disruption in the normal enterohepatic circulation due to a lack of feeding during such therapy. This is further validated by the fact that in clinical settings, once cholestasis sets in, its reversal occurs when a patient is receiving a major portion of calories enterally. There are several other postulated mechanisms including gut bacterial permeation predisposing to endotoxin associated down regulation of bile acid transporters. An additional potential mechanism includes toxicity of the TPN solution itself, such as lipid mediated hepatic toxicity. Prematurity, leading to a poor development of bile acid regulating nuclear receptors and transporters has also been implicated as a causative factor. This review presents the current controversies and research into mechanisms of TPN associated injury.

Validating hyperbilirubinemia and gut mucosal atrophy with a novel ultramobile ambulatory total parenteral nutrition piglet model.

Total parenteral nutrition (TPN) provides all nutrition intravenously. Although TPN therapy has grown enormously, it causes significant complications, including gut and hepatic dysfunction. Current models use animal tethering which is unlike ambulatory human TPN delivery and is cost prohibitive. We hypothesize that using ultramobile infusion pumps, TPN can be delivered cost-effectively, resulting in classical gut and hepatic injury, and we thus aim to establish a new model system. Neonatal pigs (n=8) were implanted with jugular vein and duodenal catheters. Animals were fitted in dual-pocket jackets. An ultramobile ambulatory pump was placed in one pocket and connected to the jugular vein or duodenal catheter. Isocaloric TPN or swine formula was placed in the other pocket. Rigorous Wifi-based video and scheduled monitoring was performed. After 14days, the animals were euthanized. The mean (±SD) daily weight gain (in grams) for enteral-fed control (EN) vs TPN animals was 102.4±10.8 and 91.03±12.1 respectively (P<.05). Total parenteral nutrition resulted in significant conjugated bilirubin elevation and hepatomegaly. Mean (±SD) serum conjugated bilirubin (in µmol/L) was 1.5±0.7 for EN and 6.3±2.8 for TPN (P<.05). Marked gut atrophy was noted with TPN. The mean (±SD) gut weight as a percent of body weight was 4.30±0.26 for EN and 2.62±0.48 for TPN (P<.05). Surgical sites healed well. All animals remained completely mobile. We thus established that TPN can be successfully delivered using ultramobile pumps and believe that this remains the first such description of an ambulatory piglet TPN model system. In addition to cholestasis and gut atrophy, classical TPN-induced injury was documented.

Using antisense technology to develop a novel therapy for α-1 antitrypsin deficient (AATD) liver disease and to model AATD lung disease.

Alpha-1 antitrypsin (AAT) is a serum protease inhibitor that belongs to the serpin superfamily. Mutations in AAT are associated with α-1 antitrypsin deficiency (AATD), a rare genetic disease with two distinct manifestations: AATD lung disease and AATD liver disease. AATD lung disease is caused by loss-of-function of AAT and can be treated with plasma-derived AAT. AATD liver disease is due to the aggregation and retention of mutant AAT protein in the liver; the only treatment available for AATD liver disease is liver transplantation. Here we demonstrate that antisense oligonucleotides (ASOs) targeting human AAT efficiently reduce levels of both short and long human AAT transcript in vitro and in transgenic mice, providing a novel therapy for AATD liver disease. In addition, ASO-mediated depletion of mouse AAT may offer a useful animal model for the investigation of AATD lung disease.

Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin-related liver disease in mice.

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease.