Therapeutic effects of Tmprss6 suppression are negated by iron overload in a mouse model of beta-thalassemia Free

Beta-thalassemia, caused by genetic deficiency of beta-globin, is characterized by ineffective erythropoiesis and anemia. In addition to iron overload generated by episodic or regular erythrocyte transfusions, the erythroid hormone erythroferrone (ERFE) is pathologically overproduced in beta-thalassemia, suppressing the hepatic production of the master iron-regulatory hormone hepcidin, and thereby causing excessive iron absorption and iron overload. Iron overload is a major cause of morbidity and mortality in beta-thalassemia, and is therefore an important therapeutic target.

One approach to ameliorating iron overload is to increase hepcidin levels by suppressing the hepatic transmembrane serine protease Tmprss6. Tmprss6 is a negative regulator of hepatic hepcidin transcription, and acts by dampening iron-related BMP signaling in hepatocytes. Inhibition of Tmprss6 with antisense oligonucleotides (ASO) or neutralizing antibodies in mouse models of thalassemia not only decreased serum and liver iron, but also ameliorated ineffective erythropoiesis as reflected by increased hemoglobin and RBC count and reduced splenomegaly. However, there have been no reports to date of similar beneficial outcomes in several relevant human trials. One possible explanation is that the commonly used Hbb^Th3/+ (Th3) thalassemic mouse model does not replicate the severity of iron overload seen in patients, in part because ERFE expression in mice may be lower, and in part because the mouse model does not undergo blood transfusions. Tmprss6 protein levels have been shown to be downregulated under high iron conditions (at least in rodents), which may render therapeutic Tmprss6 suppression less effective in beta-thalassemia as iron overload increases. Thus, the goal of this study was to determine how iron overload in beta-thalassemia modulates the effectiveness of Tmprss6 ASOs.

Our lab previously generated ERFE-overexpressing transgenic mice E(M), (Coffey et al, Blood 2022), here referred to as E and showed that they developed dose-dependent iron overload and relative hepcidin deficiency. When E mice were crossed with Th3 mice, Th3 mice overexpressing ERFE (T-E) developed greater iron loading and worse ineffective erythropoiesis compared to Th3-only mice (Olivera, Zhang et al, Blood Adv. 2023). In the current study, we generated T-E mice as well as wildtype (WT), E, and Th3 littermate controls. From 6 weeks of age, these mice received a weekly i.p. injection of 5 ug/g Tmprss6 ASO or non-targeting ASO for 4 weeks prior to harvest. We observed a 4-8-fold decrease in liver Tmprss6 mRNA levels after Tmprss6-ASO treatment in all groups along with increased liver Hamp levels and decreased serum iron, indicating the effectiveness of the Tmprss6 ASO in increasing hepcidin concentrations. However, Tmprss6 ASO treatment was considerably less effective in increasing liver Hamp mRNA and decreasing serum iron in T-E than in Th3 groups. Additionally, the Tmprss6 ASO treatment improved splenomegaly and increased RBC counts in the Th3 group, but not the T-E group.

To determine whether iron overload itself was sufficient to interfere with the effect of decreased Tmprss6 expression, we utilized two different mouse models of iron overload. C57BL/6 mice were either treated with 10 mg of i.p. iron dextran prior to the previously described Tmprss6 ASO treatment or placed on 5000 ppm high iron diet throughout the duration of the Tmprss6 ASO treatment. Another group of mice was placed on 100 ppm iron diet as iron-replete controls. As expected, liver Tmprss6 mRNA expression was lowered by Tmprss6 ASO compared to the non-targeting ASO in all three groups. However, both the iron dextran and high iron diet already maximally elevated liver Hamp mRNA levels, and treatment with Tmprss6 ASO did not further increase it. Neither serum and tissue iron, nor CBCs were altered in these high iron groups after Tmprss6 inhibition.

Overall, we have demonstrated that while Tmprss6 inhibition is effective in increasing hepcidin and improving thalassemia under usual iron-replete conditions, its effect is blunted in the presence of more severe iron overload caused by either high ERFE concentrations or by the administration of exogenous iron. Similar factors may make patients with beta-thalassemia resistant to treatments targeting Tmprss6.