Administration of Minihepcidins to Animals Affected By β-Thalassemia Major Reduces Anemia and Splenomegaly

Both β-thalassemia intermedia and major are characterized by formation of hemichromes in erythroid cells, impairing their survival and the lifespan of red blood cells (RBC). Minihepcidins (MH) are novel compounds that function as hepcidin agonists and reduce iron absorption and transferrin saturation. Hbbth3/+ mice show features of β-thalassemia intermedia, such as ineffective erythropoiesis (IE), anemia and reduced hepcidin synthesis, but do not require blood transfusion for survival (non-transfusion dependent thalassemia or NTDT). As we have previously shown, the administration of MH in these animals decreased transferrin saturation, erythroid iron intake, heme synthesis and hemichrome formation, with a significant beneficial effect on RBC quality, lifespan and anemia (Casu et al, Blood 2016). In order to test if this approach could also benefit animals affected by β-thalassemia major we focused on generating a model that exhibited a low production of RBCs, severe anemia and a blood transfusion requirement for survival, as in patients affected by transfusion dependent thalassemia or TDT. We have previously shown that engraftment of Hbbth3/th3fetal liver cells (FLCs) into normal mice leads to a very severe anemia that requires blood transfusion for survival (Gardenghi et al, Blood 2007). However, Hbbth3/th3FLCs do not contain any adult or fetal-globin genes and are unable to make hemoglobin in the transplanted animals, in contrast to human β-thalassemia. Therefore, animals cannot benefit from therapies that decrease hemichrome formation and target IE such as MH. To overcome this limitation, we crossed Hbbth3/+mice with additional models of NTDT, indicated as Hbbth1/th1and Hbbth2/+. These mice harbor alternative mutations so that the synthesis of the mouse b-globin genes is different in each model. Hbbth1/th2and Hbbth1/th3pups were alive at birth, but unable to survive more than a couple of days even with the support of blood transfusion. However, recipient transgenic animals expressing GFP and engrafted with Hbbth1/th2andHbbth1/th3FLCs showed the desired phenotype 3 months post-transplant including production of GFP- RBCs (with less than 2% of host GFP+ RBC) and a different degree of anemia, respectively 5.6±0.5 g/dL and 3.1±1.5 g/dL. In the long term these animals require blood transfusion for survival. Therefore these models are useful to test drugs that have the potential to modify erythropoiesis and RBC production. Ten weeks following engraftment with Hbbth1/th2FLCs, mice were treated for six weeks with two different doses of MH (5.25 mg/kg and 2.625 mg/kg administered every other day) in absence of blood transfusion. Animals treated with vehicleshowed severe ineffective erythropoiesis and worsening anemia over 6 weeks (from 5.6±0.5 g/dL on D0 to 5.0±0.7 g/dL on D42 of treatment). In contrast, animals treated with MH showed reversal of anemia at 3 weeks (6.6±0.3 g/dL and 6.1±0.6 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 5.3±0.9 g/dL in controls), while at 6 weeks the differences were reduced compared to vehicle treated mice (6.0±0.4 g/dL and 5.7±0.5 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 4.9±0.7 g/dL in controls). The RBC number followed the same trend. Furthermore, the RBC morphology of animals treated with MH was improved compared to control animals. At 6 weeks, splenomegaly was also improved in the treatment groups (13.8±2.7 mg and 16.9±2.7 mg respectively in the 5.25 mg/Kg and 2.625 mg/Kg group compared to 26.9±3.5 mg in controls). Comparing the data at 3 versus 6 weeks, we speculate that, while the MH has a positive effect on RBC quality and production, this is insufficient, in the long term, to prevent the severe splenomegaly and the consequent entrapment of the RBC, which exacerbates the anemia over time. However, we hypothesized that administration of MH could have longer lasting beneficial effects in presence of blood transfusion, which would limit the splenomegaly. Presently, we are testing this hypothesis using both the Hbbth1/th2and Hbbth1/th3models. Complete characterization of these models and their parameters (CBC, erythropoiesis, iron metabolism and organ iron content) is in progress. In conclusion, these models can be utilized to characterize severe thalassemia phenotypes and new drugs that have the potential to ameliorate IE and improve RBCs generation. Disclosures MacDonald: Merganser: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.