Combination of Tmprss6-ASO and the Iron Chelator Deferiprone Improves Erythropoiesis and Reduces Iron Overload in a Mouse Model of Beta-Thalassemia

Patients affected by non-transfusion dependent thalassemia (NTDT) do not require chronic blood transfusion for survival. However, transfusion-independence in such patients is not without side effects. Ineffective erythropoiesis (IE), the hallmark of disease, leads to a variety of serious clinical morbidities. In NTDT the master regulator of iron homeostasis, hepcidin, is chronically repressed. Consequently, patients absorb abnormally high levels of iron, which eventually requires iron chelation to prevent the clinical sequelaes associated with iron overload. It has been shown that in mice affected by NTDT ( Hbbth3/+ ), a second-generation antisense oligonucleotide ( Tmprss6- ASO) can reduce expression of transmembrane serine protease Tmprss6, the major suppressor of hepcidin expression. This leads to reduction of hemichrome formation in erythroid cells, amelioration of IE and splenomegaly, and increased hemoglobin levels (Guo et al, JCI , 2013). Now we propose the use of Tmprss6 -ASO in combination with iron chelators for the treatment of NTDT using Hbbth3/+ mice as a preclinical model. Our hypothesis is that use of chelators will benefit from the positive effect of Tmprss6 -ASO on erythropoiesis and iron absorption, further ameliorating organ iron content. To this end, Hbbth3/+ animals were treated with Tmprss6 -ASO at 100 mg/kg/week for 6 weeks with or without the iron chelator deferiprone (DFP) at a dose of 1.25 mg/ml. Additional animals were treated with DFP alone. We fed the animals with a commercial or physiological diet, containing 200 or 35 ppm of iron, respectively. We did not observe major differences in the treated animals fed the commercial or physiological iron diet and, for this reason, the data were combined for simplicity. Administration of DFP alone was successful in decreasing organ iron content. Compared to untreated Hbbth3/+ animals, we observed a reduction of 30% and 33% in the liver and spleen, respectively, and no change in the kidney. However, erythropoiesis was not improved (looking at IE, splenomegaly, RBC production and total Hb levels). This was associated with increased serum iron levels (+25%). In Tmprss6- ASO treated Hbbth3/+ animals, we observed an improvement in liver iron content (36% reduction), amelioration of IE, and increased RBC and Hb synthesis (~2 g/dL). Compared to treatment with Tmprss6- ASO alone, combination of DFP with Tmprss6- ASO achieved the same level of suppression of Tmprss6 in the liver (~90%) and reduction of serum iron parameters. This was associated with improvement of IE, decreased reticulocyte counts and splenomegaly, and increased RBC and Hb synthesis (~2 g/dL). While we observed that both Tmprss6- ASO and DFP separately reduced liver iron content to the same extent (~30-36%), combination treatment further reduced iron concentrations in the liver and kidney (69% and 19%, respectively), with no changes in the spleen. Additional analyses are in progress to evaluate the amount of hepcidin in serum as well as expression of erythroferrone, the erythroid regulator of hepcidin. Our first conclusion is that administration of an iron chelator alone is not sufficient to improve erythropoiesis despite that organ iron content is reduced. We speculate that when iron is removed from the liver, hepcidin expression becomes more susceptible to the suppressive effect of IE rather than the enhancing effect of reduced liver organ iron concentration. In addition, the combined effect of iron mobilized from organs and unchanged (or even augmented) iron absorption leads to increased serum iron concentration. As we have shown previously, amelioration of IE in this model requires decreased erythroid iron intake and hemichrome formation. Therefore, iron chelation alone is likely insufficient to improve erythropoiesis. Additional experiments are in progress to further elucidate this mechanism. Our second conclusion is that use of Tmprss6- ASO together with DFP combines the best effects of these two drugs, in particular on erythropoiesis and organ iron content. In animals that received the combined treatment, kidney and liver iron concentrations were further decreased compared to the single treatments. This indicates that Tmprss6- ASO might be extremely helpful in the treatment of NTDT and it could further improve iron related-chelation therapies. Disclosures Casu: Merganser Biotech LLC: Employment; Isis Pharmaceuticals, Inc.: Employment. Aghajan: Isis Pharmaceuticals, Inc.: Employment. Guo: Isis Pharmaceuticals, Inc.: Employment. Monia: Isis Pharmaceuticals, Inc.: Employment. Rivella: bayer: Consultancy, Research Funding; isis Pharmaceuticals, Inc.: Consultancy, Research Funding; merganser Biotech LLC: Consultancy, Research Funding, Stock options , Stock options Other.