Targeting TMPRSS6 Using Antisense Technology for the Treatment of Beta-Thalassemia

Abstract Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H-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 β-thalassemia. We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment resulted in dose-dependent hepcidin upregulation and reduction in serum iron and transferrin saturation in normal mice. In a mouse model of beta-thalassemia (th3/+ mice), which effectively recapitulates beta-thalassemia intermedia in humans, TMPRSS6 reduction resulted in induction of hepcidin and 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; decreases in serum erythropoietin levels; improved erythroid maturation as indicated by a strong reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Encouraged by the strong pharmacology of TMPRSS6 suppression in animal models, we initiated an effort to identify a human TMPRSS6 clinical candidate with a liver-targeted delivery strategy. Over 2000 ASOs were screened in cell lines and the most active compounds were evaluated in rodent tolerability studies. A human TMPRSS6 transgenic mouse model was established enabling evaluation of ASO activity toward human TMPRSS6 transcript in vivo. Furthermore, lead compounds were tested in a 3-month study in normal monkeys. With repeated dosing, TMPRSS6 mRNA levels in monkey liver were reduced by >90%, accompanied by time-dependent reductions of serum iron (from ~100-120ug/dl to Disclosures Guo: Isis Pharmaceuticals: Employment, Other: Shareholder. Aghajan: Isis Pharmaceuticals: Employment, Other: Shareholder. Booten: Isis Pharmaceuticals: Employment, Other: Shareholder. Monia: Isis Pharmaceuticals: Employment, Other: Shareholder.