Developing a Galnac-Conjugated TMPRSS6 Antisense Therapy for the Treatment of β-Thalassemia

Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H1-mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previous mouse and human genetic data from multiple groups suggest that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with beta-thalassemia. We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment results in dose-dependent hepcidin upregulation, which leads to dramatic reductions in serum iron and transferrin saturation. This in turn ameliorated the anemia and iron overload phenotypes in a mouse model of beta-thalassemia (th3/+ mice), which recapitulates beta-thalassemia intermedia in humans (Guo et al. J Clin Invest. 2013; 123(4):1531-41). Moreover, this ASO can be combined efficiently with iron chelators for the management of iron overload and anemia in non-transfusion-dependent thalassemia (Casu et al. Haematologica. 2016; 101(1):e8-e11). TMPRSS6 is predominantly expressed in hepatocytes, for which we have developed a targeted delivery approach with triantennary N-acetyl galactosamine (GalNAc). With GalNAc-conjugated ASOs, a ~10-fold improvement in potency is observed for many liver targets (Prakash et al. Nucleic Acids Res. 2014; 42(13):8796-807). In order to characterize GalNAc-conjugated TMPRSS6 ASO, we treated normal mice with both parent ASO and its conjugated counterpart. As expected, the conjugated ASO demonstrated a ~10-fold improvement in ED50 (25 mg/kg/week versus 2.5 mg/kg/week for parent and conjugated ASOs, respectively). Next, we treated th3/+ mice for six weeks with 10 mg/kg/week GalNAc-conjugated TMPRSS6 ASO or a control ASO of the same chemistry. Compared to the control ASO treatment group, we observed >95% reduction of TMPRSS6 mRNA levels and >3-fold up-regulation of hepcidin mRNA levels in the liver. This resulted in a ~40% reduction in serum iron and ~50% reduction in transferrin saturation. In addition, anemia phenotypes were significantly improved as shown by a significant increase in hemoglobin and red blood cells (from 7.0 g/dL to 8.9 g/dL and from 5.8 to 7.5x10^6 cells/µl in the control ASO treatment group and in the TMPRSS6 ASO treatment group, respectively). Furthermore, there was an approximately 50% reduction in spleen weight. Improved erythroid maturation was indicated by a significant reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. A GalNAc-conjugated human TMPRSS6 clinical candidate was identified. Similar to mouse TMPRSS6, GalNAc-conjugated ASO demonstrated superior potency in human primary hepatocyte culture, in human TMPRSS6 transgenic mice and in cynomolgus monkey. Collectively, our data demonstrate that GalNAc-conjugated TMPRSS6 ASO could be an effective therapeutic for patients with beta-thalassemia and related disorders. A Phase 1 clinical trial is planned to initiate in 2017. Disclosures Aghajan:Ionis Pharmaceuticals: Employment, Equity Ownership. Booten:Ionis Pharmaceuticals: Employment, Equity Ownership. Monia:Ionis Pharmaceuticals: Employment, Equity Ownership. Guo:Ionis Pharmaceuticals: Employment, Equity Ownership.