Target TMPRSS6 Using Antisense Technology for the Treatment of Hereditary Hemochromatosis and β-Thalassemia

Abstract Abstract 481 Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia. Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes. In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures: Guo: Isis Pharmaceuticals: Employment. Booten: Isis Pharmaceuticals: Employment. Watt: Isis Pharmaceuticals: Employment. Freier: Isis Pharmaceuticals: Employment. Rivella: Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia: Isis Pharmaceuticals: Employment.