Antisense oligonucleotides ameliorate kidney dysfunction in podocyte-specific APOL1 risk variant mice.

Coding variants (named G1 and G2) in Apolipoprotein L1 (APOL1) can explain most excess risk of kidney disease observed in African American individuals. It has been proposed that risk variant APOL1 dose, such as increased risk variant APOL1 level serves as a trigger (second hit) for disease development. The goal of this study was to determine whether lowering risk variant APOL1 levels protects from disease development in a podocyte-specific transgenic mouse disease model. We administered antisense oligonucleotides (ASO) targeting APOL1 to podocyte-specific G2APOL1 mice and observed efficient reduction of APOL1 levels. APOL1 ASO1, which more efficiently lowered APOL1 transcript levels, protected mice from albuminuria, glomerulosclerosis, tubulointerstitial fibrosis, and renal failure. Administration of APOL1 ASO1 was effective even for established disease in the NEFTA-rtTA/TRE-G2APOL1 (NEFTA/G2APOL1) mice. We observed a strong correlation between APOL1 transcript level and disease severity. We concluded that APOL1 ASO1 may be an effective therapeutic approach for APOL1-associated glomerular disease.

Silencing of STE20-type kinase MST3 in mice with antisense oligonucleotide treatment ameliorates diet-induced nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is emerging as a leading cause of chronic liver disease worldwide. Despite intensive nonclinical and clinical research in this field, no specific pharmacological therapy is currently approved to treat NAFLD, which has been recognized as one of the major unmet medical needs of the 21st century. Our recent studies have identified STE20-type kinase MST3, which localizes to intracellular lipid droplets, as a critical regulator of ectopic fat accumulation in human hepatocytes. Here, we explored whether treatment with Mst3-targeting antisense oligonucleotides (ASOs) can promote hepatic lipid clearance and mitigate NAFLD progression in mice in the context of obesity. We found that administration of Mst3-targeting ASOs in mice effectively ameliorated the full spectrum of high-fat diet-induced NAFLD including liver steatosis, inflammation, fibrosis, and hepatocellular damage. Mechanistically, Mst3 ASOs suppressed lipogenic gene expression, as well as acetyl-CoA carboxylase (ACC) protein abundance, and substantially reduced lipotoxicity-mediated oxidative and endoplasmic reticulum stress in the livers of obese mice. Furthermore, we found that MST3 protein levels correlated positively with the severity of NAFLD in human liver biopsies. In summary, this study provides the first in vivo evidence that antagonizing MST3 signaling is sufficient to mitigate NAFLD progression in conditions of excess dietary fuels and warrants future investigations to assess whether MST3 inhibitors may provide a new strategy for the treatment of patients with NAFLD.

Antisense oligonucleotide treatment ameliorates IFN-γ-induced proteinuria in APOL1-transgenic mice.

African Americans develop end-stage renal disease at a higher rate compared with European Americans due to 2 polymorphisms (G1 and G2 risk variants) in the apolipoprotein L1 (APOL1) gene common in people of African ancestry. Although this compelling genetic evidence provides an exciting opportunity for personalized medicine in chronic kidney disease, drug discovery efforts have been greatly hindered by the fact that APOL1 expression is lacking in rodents. Here, we describe a potentially novel physiologically relevant genomic mouse model of APOL1-associated renal disease that expresses human APOL1 from the endogenous human promoter, resulting in expression in similar tissues and at similar relative levels as humans. While naive APOL1-transgenic mice did not exhibit a renal disease phenotype, administration of IFN-γ was sufficient to robustly induce proteinuria only in APOL1 G1 mice, despite inducing kidney APOL1 expression in both G0 and G1 mice, serving as a clinically relevant "second hit." Treatment of APOL1 G1 mice with IONIS-APOL1Rx, an antisense oligonucleotide (ASO) targeting APOL1 mRNA, prior to IFN-γ challenge robustly and dose-dependently inhibited kidney and liver APOL1 expression and protected against IFN-γ-induced proteinuria, indicating that the disease-relevant cell types are sensitive to ASO treatment. Therefore, IONIS-APOL1Rx may be an effective therapeutic for APOL1 nephropathies and warrants further development.

Targeted Delivery of Stk25 Antisense Oligonucleotides to Hepatocytes Protects Mice Against Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide. Currently, no specific pharmacologic therapy is available for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the 21st century. Our recent studies in genetic mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine protein kinase (STK)25 as a critical regulator of hepatic lipid partitioning and NAFLD/NASH. Here, we studied the metabolic benefit of liver-specific STK25 inhibitors on NAFLD development and progression in a mouse model of diet-induced obesity. METHODS: We developed a hepatocyte-specific triantennary N-acetylgalactosamine (GalNAc)-conjugated antisense oligonucleotide (ASO) targeting Stk25 and evaluated its effect on NAFLD features in mice after chronic exposure to dietary lipids. RESULTS: We found that systemic administration of hepatocyte-targeting GalNAc-Stk25 ASO in obese mice effectively ameliorated steatosis, inflammatory infiltration, hepatic stellate cell activation, nutritional fibrosis, and hepatocellular damage in the liver compared with mice treated with GalNAc-conjugated nontargeting ASO, without any systemic toxicity or local tolerability concerns. We also observed protection against high-fat-diet-induced hepatic oxidative stress and improved mitochondrial function with Stk25 ASO treatment in mice. Moreover, GalNAc-Stk25 ASO suppressed lipogenic gene expression and acetyl-CoA carboxylase protein abundance in the liver, providing insight into the molecular mechanisms underlying repression of hepatic steatosis. CONCLUSIONS: This study provides in vivo nonclinical proof-of-principle for the metabolic benefit of liver-specific inhibition of STK25 in the context of obesity and warrants future investigations to address the therapeutic potential of GalNAc-Stk25 ASO in the prevention and treatment of NAFLD.

Serine/threonine protein kinase 25 antisense oligonucleotide treatment reverses glucose intolerance, insulin resistance, and nonalcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD) contributes to the pathogenesis of type 2 diabetes and cardiovascular disease, and patients with nonalcoholic steatohepatitis (NASH) are also at risk of developing cirrhosis, liver failure, and hepatocellular carcinoma. To date, no specific therapy exists for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the twenty-first century. We recently identified serine/threonine protein kinase (STK)25 as a critical regulator of energy homeostasis and NAFLD progression. Here, we investigated the effect of antisense oligonucleotides (ASOs) targeting Stk25 on the metabolic and molecular phenotype of mice after chronic exposure to dietary lipids. We found that Stk25 ASOs efficiently reversed high-fat diet-induced systemic hyperglycemia and hyperinsulinemia, improved whole-body glucose tolerance and insulin sensitivity, and ameliorated liver steatosis, inflammatory infiltration, apoptosis, hepatic stellate cell activation, and nutritional fibrosis in obese mice. Moreover, Stk25 ASOs suppressed the abundance of liver acetyl-coenzyme A carboxylase (ACC) protein, a key regulator of both lipid oxidation and synthesis, revealing the likely mechanism underlying repression of hepatic fat accumulation by ASO treatment. We also found that STK25 protein levels correlate significantly and positively with NASH development in human liver biopsies, and several common nonlinked single-nucleotide polymorphisms in the human STK25 gene are associated with altered liver fat, supporting a critical role of STK25 in the pathogenesis of NAFLD in humans. Conclusion: Preclinical validation for the metabolic benefit of pharmacologically inhibiting STK25 in the context of obesity is provided. Therapeutic intervention aimed at reducing STK25 function may provide a new strategy for the treatment of patients with NAFLD, type 2 diabetes, and related complex metabolic diseases. (Hepatology Communications 2018;2:69-83).

Comprehensive Structure-Activity Relationship of Triantennary N-Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes.

The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.

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.

Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.

Triantennary N-acetyl galactosamine (GalNAc, GN3: ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, ∼ 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3: -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3: -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3: -ASO conjugate was detected in plasma suggesting that GN3: acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.

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.

Inhibition of protein tyrosine phosphatase-1B with antisense oligonucleotides improves insulin sensitivity and increases adiponectin concentrations in monkeys.

Protein tyrosine phosphatase (PTP)-1B antagonizes insulin signaling and is a potential therapeutic target for insulin resistance associated with obesity and type 2 diabetes. To date, studies of PTP-1B have been limited by the availability of specific antagonists; however, treatment of rodents with antisense oligonucleotides (ASOs) directed against PTP-1B improves insulin sensitivity, inhibits lipogenic gene expression, and reduces triglyceride accumulation in liver and adipose tissue. Here we investigated ASO-mediated PTP-1B inhibition in primates. First, PTP-1B ASO (ISIS 113715) dose-dependently inhibited PTP-1B mRNA and protein expression in cultured monkey hepatocytes. Subcutaneous administration of ISIS 113715 reduced PTP-1B mRNA expression in liver and adipose tissue of normal-weight monkeys by 40-50% and improved insulin sensitivity during an iv glucose tolerance test (IVGTT). In obese, insulin-resistant rhesus monkeys, treatment with 20 mg/kg ISIS 113715 for 4 wk reduced fasting concentrations of insulin and glucose and reduced insulin responses during an IVGTT. In these animals, adiponectin concentrations were also increased by 70%, most of which was an increase of high-molecular-weight oligomers. These effects were not observed in monkeys on a lower, dose-escalation regimen (1-10 mg/kg over 9 wk). Overall, the increase of adiponectin concentrations during ISIS 113715 treatment was correlated with the lowering of insulin responses during IVGTT (r = -0.47, P = 0.042). These results indicate that inhibition of PTP-1B with ASOs such as ISIS 113715 may be a viable approach for the treatment and prevention of obesity-associated insulin resistance and type 2 diabetes because they potently increase adiponectin concentrations in addition to improving insulin sensitivity.

Reduction of JNK1 expression with antisense oligonucleotide improves adiposity in obese mice.

To investigate the role of JNK1 in metabolism, male ob/ob and diet-induced obese mice were treated with a JNK1-specific antisense oligonucleotide (ASO) or control ASO at 25 mg/kg or saline twice/wk for 6 and 7 wk, respectively. JNK1 ASO reduced JNK1 mRNA and activity by 65-95% in liver and fat tissues in both models. Compared with controls, treatment with JNK1 ASO did not change food intake but lowered body weight, fat pad weight, and whole body fat content. The treatment increased metabolic rate. In addition, the treatment markedly reduced plasma cholesterol levels and improved liver steatosis and insulin sensitivity. These positive observations were accompanied by the following changes: 1) increased mRNA levels of AR-beta(3) and UCP1 by >60% in BAT, 2) reduced mRNA levels of ACC1, ACC2, FAS, SCD1, DGAT1, DGAT2, and RBP4 by 30-60% in WAT, and 3) reduced mRNA levels of ACC1, FAS, G-6-Pase, and PKCepsilon by 40-70% and increased levels of UCP2 and PPARalpha by more than twofold in liver. JNK1 ASO-treated mice demonstrated reduced levels of pIRS-1 Ser(302) and pIRS-1 Ser(307) and increased levels of pAkt Ser(473) in liver and fat in response to insulin. JNK1 ASO-transfected mouse hepatocytes showed decreased rates of de novo sterol and fatty acid synthesis and an increased rate of fatty acid oxidation. These results indicate that inhibition of JNK1 expression in major peripheral tissues can improve adiposity via increasing fuel combustion and decreasing lipogenesis and could therefore provide clinical benefit for the treatment of obesity and related metabolic abnormalities.

Reduced adiposity and improved insulin sensitivity in obese mice with antisense suppression of 4E-BP2 expression.

To investigate the possible role of eukaryotic initiation factor 4E-binding protein-2 (4E-BP2) in metabolism and energy homeostasis, high-fat diet-induced obese mice were treated with a 4E-BP2-specific antisense oligonucleotide (ASO) or a control 4E-BP2 ASO at a dose of 25 mg/kg body wt or with saline twice a week for 6 wk. 4E-BP2 ASO treatment reduced 4E-BP2 levels by >75% in liver and white (WAT) and brown adipose (BAT) tissues. Treatment did not change food intake but lowered body weight by approximately 7% and body fat content by approximately 18%. Treatment decreased liver triglyceride (TG) content by >50%, normalized plasma glucose and insulin levels, and reduced glucose excursion during glucose tolerance test. 4E-BP2 ASO-treated mice showed >8.5% increase in metabolic rate, >40% increase in UCP1 levels in BAT, >45% increase in beta(3)-adrenoceptor mRNA, and 40-55% decrease in mitochondrial dicarboxylate carrier, fatty acid synthase, and diacylglycerol acyltransferase 2 mRNA levels in WAT. 4E-BP2 ASO-transfected mouse hepatocytes showed an increased fatty acid oxidation rate and a decreased TG synthesis rate. In addition, 4E-BP2 ASO-treated mice demonstrated approximately 60 and 29% decreases in hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinase mRNA, respectively, implying decreased hepatic glucose output. Furthermore, increased phosphorylation of Akt(Ser473) in both liver and fat of 4E-BP2 ASO-treated mice and increased GLUT4 levels in plasma membrane in WAT of the ASO-treated mice were observed, indicating enhanced insulin signaling and increased glucose uptake as a consequence of reduced 4E-BP2 expression. These data demonstrate for the first time that peripheral 4E-BP2 plays an important role in metabolism and energy homeostasis.

Specific reduction of hepatic glucose 6-phosphate transporter-1 ameliorates diabetes while avoiding complications of glycogen storage disease.

D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.

miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.

Current understanding of microRNA (miRNA) biology is limited, and antisense oligonucleotide (ASO) inhibition of miRNAs is a powerful technique for their functionalization. To uncover the role of the liver-specific miR-122 in the adult liver, we inhibited it in mice with a 2'-O-methoxyethyl phosphorothioate ASO. miR-122 inhibition in normal mice resulted in reduced plasma cholesterol levels, increased hepatic fatty-acid oxidation, and a decrease in hepatic fatty-acid and cholesterol synthesis rates. Activation of the central metabolic sensor AMPK was also increased. miR-122 inhibition in a diet-induced obesity mouse model resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis, accompanied by reductions in several lipogenic genes. These results implicate miR-122 as a key regulator of cholesterol and fatty-acid metabolism in the adult liver and suggest that miR-122 may be an attractive therapeutic target for metabolic disease.

Antisense oligonucleotide reduction of DGAT2 expression improves hepatic steatosis and hyperlipidemia in obese mice.

In this study, we investigated the role of acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) in glucose and lipid metabolism in obese mice by reducing its expression in liver and fat with an optimized antisense oligonucleotide (ASO). High-fat diet-induced obese (DIO) C57BL/6J mice and ob/ob mice were treated with DGAT2 ASO, control ASO, or saline. DGAT2 ASO treatment reduced DGAT2 messenger RNA (mRNA) levels by more than 75% in both liver and fat but did not change DGAT1 mRNA levels in either of these tissues, which resulted in decreased DGAT activity in liver but not in fat. DGAT2 ASO treatment did not cause significant changes in body weight, adiposity, metabolic rate, insulin sensitivity, or skin microstructure. However, DGAT2 ASO treatment caused a marked reduction in hepatic triglyceride content and improved hepatic steatosis in both models, which was consistent with a dramatic decrease in triglyceride synthesis and an increase in fatty acid oxidation observed in primary mouse hepatocytes treated with DGAT2 ASO. In addition, the treatment lowered hepatic triglyceride secretion rate and plasma triglyceride levels, and improved plasma lipoprotein profile in DIO mice. The positive effects of the DGAT2 ASO were accompanied by a reduction in the mRNA levels of several hepatic lipogenic genes, including SCD1, FAS, ACC1, ACC2, ATP-citrate lyase, glycerol kinase, and HMG-CoA reductase. In conclusion, reduction of DGAT2 expression in obese animals can reduce hepatic lipogenesis and hepatic steatosis as well as attenuate hyperlipidemia, thereby leading to an improvement in metabolic syndrome.