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

Abstract

Haematologica 2015 [Epub ahead of print] Citation: Casu C, Aghajan M, Rea Oikonomidou P, Guo S, Monia BP, and Rivella S. Combination of Tmprss6-ASO and the iron chelator deferiprone improves erythropoiesis and reduces iron overload in a mouse model of beta-thalassemia intermedia. Haematologica. 2015; 100:xxx doi:10.3324/haematol.2015.133348 Publisher's Disclaimer. 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 intermedia-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. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print 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 (hemichrome) 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 Hbb th3/+ females were treated with 50 mg/kg of Tmprss6 antisense 2 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 35ppm 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 the data obtained from the 200ppm diet only. As expected, Tmprss6-ASO treatment, alone or in combination with DFP, suppressed Tmprss6 expression in the liver reaching an 82% decrease (P<1.8E-08) ( 11 Amelioration of erythropoiesis in this model of NTDT requires decreased erythroid iron intake and hemichrome formation. 9 Since DFP did not decrease Tfsat, we postulated that hemichrome formation was not decreased in this setting. In fact, hemichrome levels were unchanged in DFP-treated animals compared to Hbb th3/+ controls, while they were reduced in animals that received Tmprss6-ASO alone or Tmprss6-ASO+DFP 12 In 3 this study only upon hemichrome reduction there was a correlation with improvement of IE, observed as reduced proportions of immature erythroid cells. Using Ter119 and CD44 antibodies on BM and spleen cells we were able to perform FACS analysis which allowed us to discriminate different stages of erythroid differentiatio