Shuling Guo

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.

Clinical development of an antisense therapy for the treatment of transthyretin-associated polyneuropathy.

Transthyretin (TTR)-associated amyloidosis is a late-onset autosomal-dominant genetic disease. Over 100 amyloidogenic mutations have been identified in TTR which destabilize the TTR tetramer thereby inducing the formation of amyloid fibrils in tissues such as the heart and peripheral nerves. This disease mainly affects peripheral nerves, causing familial amyloid polyneuropathy (FAP) or heart, causing familial amyloid cardiomyopathy (FAC). Circulating TTR is predominantly produced by liver, and the only widely available clinical treatment for FAP is orthotopic liver transplantation (OLT), whereas no treatment currently exists for FAC. Using second-generation antisense technology, we identified an antisense oligonucleotide (ASO) targeting TTR, ISIS-TTRRx, for the treatment of TTR-associated amyloidosis. When tested in a human TTR transgenic mouse model (hTTR Ile84Ser), ISIS-TTRRx showed a dose-dependent reduction of human TTR (up to >80%) at both the mRNA and protein levels. In cynomolgus monkeys, ISIS-TTRRx treatment produced a time-dependent reduction in plasma TTR levels. After 12 weeks of treatment in monkey, liver TTR mRNA and plasma TTR protein levels were reduced by ~80%. As expected, treatment with ISIS-TTRRx also produced a significant decrease in plasma RBP4 levels that correlated with reductions in TTR levels. ISIS-TTRRx treatment was well tolerated in both rodents and monkeys and produced a PK/PD profile consistent with prior experiences using this chemistry platform. ISIS-TTRRx is currently under evaluation in a Phase 1 clinical trial in normal healthy volunteers, and interim results of this trial will be presented.

Reducing TMPRSS6 ameliorates hemochromatosis and β-thalassemia in mice

β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe(-/-)) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB(th3/+) mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.

Peripheral Androgen Receptor Gene Suppression Rescues Disease in Mouse Models of Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ-AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here, we tested this hypothesis using AR113Q knockin and human bacterial artificial chromosome/clone (BAC) transgenic mice that express the full-length polyQ-AR and display androgen-dependent weakness, muscle atrophy, and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery but not the CNS after subcutaneous administration. Suppression of polyQ-AR in the periphery rescued deficits in muscle weight, fiber size, and grip strength, reversed changes in muscle gene expression, and extended the lifespan of mutant males. We conclude that polyQ-AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

Inhibition of hepatic sulfatase‐2 In Vivo: A novel strategy to correct diabetic dyslipidemia

Type 2 diabetes mellitus (T2DM) impairs hepatic clearance of atherogenic postprandial triglyceride‐rich lipoproteins (TRLs). We recently reported that livers from T2DM db/db mice markedly overexpress the heparan sulfate glucosamine‐6‐O‐endosulfatase‐2 (SULF2), an enzyme that removes 6‐O sulfate groups from heparan sulfate proteoglycans (HSPGs) and suppresses uptake of TRLs by cultured hepatocytes. In the present study, we evaluated whether Sulf2 inhibition in T2DM mice in vivo could correct their postprandial dyslipidemia. Selective second‐generation antisense oligonucleotides (ASOs) targeting Sulf2 were identified. Db/db mice were treated for 5 weeks with Sulf2 ASO (20 or 50 mg/kg per week), nontarget (NT) ASO, or phosphate‐buffered saline (PBS). Administration of Sulf2 ASO to db/db mice suppressed hepatic Sulf2 messenger RNA expression by 70%‐80% (i.e., down to levels in nondiabetic db/m mice) and increased the ratio of tri‐ to disulfated disaccharides in hepatic HSPGs (P < 0.05). Hepatocytes isolated from db/db mice on NT ASO exhibited a significant impairment in very‐low‐density lipoprotein (VLDL) binding that was entirely corrected in db/db mice on Sulf2 ASO. Sulf2 ASO lowered the random, nonfasting plasma triglyceride (TG) levels by 50%, achieving nondiabetic values. Most important, Sulf2 ASO treatment flattened the plasma TG excursions in db/db mice after corn‐oil gavage (iAUC, 1,500 ± 470 mg/dL·h for NT ASO versus 160 ± 40 mg/dL·h for Sulf2 ASOP < 0.01). Conclusions: Despite extensive metabolic derangements in T2DM mice, inhibition of a single dys‐regulated molecule, SULF2, normalizes the VLDL‐binding capacity of their hepatocytes and abolishes postprandial hypertriglyceridemia. These findings provide a key proof of concept in vivo to support Sulf2 inhibition as an attractive strategy to improve metabolic dyslipidemia. (HEPATOLOGY 2012;55:1746–1753)

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.

Reducing glycosphingolipid biosynthesis in airway cells partially ameliorates disease manifestations in a mouse model of asthma

Lipid rafts reportedly play an important role in modulating the activation of mast cells and granulocytes, the primary effector cells of airway hyperresponsiveness and asthma. Activation is mediated through resident signaling molecules whose activity, in part, may be modulated by the composition of glycosphingolipids (GSLs) in membrane rafts. In this study, we evaluated the impact of inhibiting GSL biosynthesis in mast cells and in the ovalbumin (OVA)-induced mouse model of asthma using either a small molecule inhibitor or anti-sense oligonucleotides (ASOs) directed against specific enzymes in the GSL pathway. Lowering GSL levels in mast cells through inhibition of glucosylceramide synthase (GCS) reduced phosphorylation of Syk tyrosine kinase and phospholipase C gamma 2 (PLC-gamma2) as well as cytoplasmic Ca(2+) levels. Modulating these intracellular signaling events also resulted in a significant decrease in mast cell degranulation. Primary mast cells isolated from a GM3 synthase (GM3S) knockout mouse exhibited suppressed activation-induced degranulation activity further supporting a role of GSLs in this process. In previously OVA-sensitized mice, intra-nasal administration of ASOs to GCS, GM3S or lactosylceramide synthase (LCS) significantly suppressed metacholine-induced airway hyperresponsiveness and pulmonary inflammation to a subsequent local challenge with OVA. However, administration of the ASOs into mice that had been sensitized and locally challenged with the allergen did not abate the consequent pulmonary inflammatory sequelae. These results suggest that GSLs contribute to the initiation phase of the pathogenesis of airway hyperreactivity and asthma and lowering GSL levels may offer a novel strategy to modulate these manifestations.

Suppressing transthyretin production in mice, monkeys and humans using 2nd-Generation antisense oligonucleotides

Abstract Transthyretin amyloidosis (ATTR amyloidosis) is a rare disease that results from the deposition of misfolded transthyretin (TTR) protein from the plasma into tissues as amyloid fibrils, leading to polyneuropathy and cardiomyopathy. IONIS-TTRRx (ISIS 420915) is a 2nd-Generation 2′-O-(2-methoxyethyl) modified “2′-MOE” antisense oligonucleotide (ASO) that targets the TTR RNA transcript and reduces the levels of the TTR transcript through an RNaseH1 mechanism of action, leading to reductions in both mutant and wild-type TTR protein. The activity of IONIS-TTRRx to decrease TTR protein levels was studied in transgenic mice bearing the Ile84Ser human TTR mutant, in cynomolgus monkeys and in healthy human volunteers. Robust (>80%) reductions of plasma TTR protein were obtained in all three species treated with IONIS-TTRRx, which in mice and monkeys was associated with substantial reductions in hepatic TTR RNA levels. These effects were dose-dependent and lasted for weeks post-dosing. In a Phase 1 healthy volunteer study, treatment with IONIS-TTRRx for four weeks was well tolerated without any remarkable safety issues. TTR protein reductions up to 96% in plasma were observed. These nonclinical and clinical results support the ongoing Phase 3 development of IONIS-TTRRx in patients with ATTR amyloidosis.

Combination of Tmprss6-ASO and the iron chelator deferiprone improves erythropoiesis and reduces iron overload in a mouse model of beta-thalassemia intermedia

Beta-thalassemia is one of the most frequently inherited disorders caused by mutations in the beta globin gene or its promoter, leading to reduced or absent beta globin synthesis. Ineffective erythropoiesis (IE) and consequent extramedullary hematopoiesis, splenomegaly and systemic iron overload are major features of this disease. The disease course can be associated with severe anemia and need for lifelong transfusion therapy (thalassemia major, TM) or relatively less severe anemia (non-transfusion-dependent thalassemia, NTDT, or thalassemia inter-media, TI). Patients affected by beta-thalassemia intermedia do not require chronic blood transfusions for survival. However, transfusion-independence is still associated with a variety of serious clinical morbidities.1–3 In NTDT the master regulator of iron homeostasis, hepcidin (Hamp), is chronically repressed.4–7 Therefore, patients absorb abnormally high levels of iron, requiring iron chelation to prevent the clinical sequelae associated with iron overload. Iron homeostasis needs to be carefully regulated in order to avoid toxicity due to its excess. If untreated, iron overload leads to organ failure and death. For this reason, in beta-thalassemia and other iron-related disorders, the management of iron overload has become the main focus. Chelation therapy, however, does not target the mechanism responsible for abnormal iron absorption, which is low levels of Hamp expression and synthesis. It has been shown that in mice affected by NTDT (Hbbth3/+ or th3/+), second generation antisense oligonucleotides (Tmprss6-ASO) or lipid nanoparticle (LNP)-formulated siRNAs can reduce the expression of transmembrane serine protease Tmprss6, one of the major suppressors of hepcidin expression.8,9 Suppression of Tmprss6 led to an increase in hepcidin synthesis and hemoglobin levels. These observations were also associated with a net reduction in splenomegaly, iron overload, transferrin saturation (TfSat), formation of insoluble membrane-bound globins (hemichromes) and reactive oxygen species (ROS).9 Thus, we hypothesized that the simultaneous use of the iron chelator deferiprone (DFP) with Tmprss6-ASO (Tmprss6-ASO+DFP) could combine the positive effects of Tmprss6-ASO on erythropoiesis and iron absorption with the chelation benefit on organ iron content. In this study, 3- to 4-month-old Hbbth3/+ females were treated with 50 mg/kg of Tmprss6 antisense oligonucleotide (Tmprss6-ASO, twice a week for 6 weeks) or Tmprss6-ASO in combination with the oral iron chelator DFP dissolved in the drinking water at 1.25 mg/ml, using either a commercial diet (normally used in the facility where animals were housed) containing 200 ppm of iron, or a physiological diet containing 35 ppm of iron. The majority of the animals available were treated using the commercial diet and just a few animals per group received the physiological one. With both diets we obtained the same trend in behavior, but considering that the numbers were not comparable, we decided to show only the data obtained from the 200 ppm diet.

Transthyretin Antisense Oligonucleotides Lower Circulating RBP4 Levels and Improve Insulin Sensitivity in Obese Mice

Circulating transthyretin (TTR) is a critical determinant of plasma retinol-binding protein 4 (RBP4) levels. Elevated RBP4 levels cause insulin resistance, and the lowering of RBP4 levels improves glucose homeostasis. Since lowering TTR levels increases renal clearance of RBP4, we determined whether decreasing TTR levels with antisense oligonucleotides (ASOs) improves glucose metabolism and insulin sensitivity in obesity. TTR-ASO treatment of mice with genetic or diet-induced obesity resulted in an 80–95% decrease in circulating levels of TTR and RBP4. Treatment with TTR-ASOs, but not control ASOs, decreased insulin levels by 30–60% and improved insulin sensitivity in ob/ob mice and high-fat diet–fed mice as early as after 2 weeks of treatment. The reduced insulin levels were sustained for up to 9 weeks of treatment and were associated with reduced adipose tissue inflammation. Body weight was not changed. TTR-ASO treatment decreased LDL cholesterol in high-fat diet–fed mice. The glucose infusion rate during a hyperinsulinemic-euglycemic clamp was increased by 50% in high-fat diet–fed mice treated with TTR-ASOs, demonstrating improved insulin sensitivity. This was also demonstrated by 20% greater inhibition of hepatic glucose production, a 45–60% increase of glucose uptake into skeletal and cardiac muscle, and a twofold increase in insulin signaling in muscle. These data show that decreasing circulating TTR levels or altering TTR-RBP4 binding could be a potential therapeutic approach for the treatment of type 2 diabetes.