Mechanisms of resistance to Tmprss6 antisense oligonucleotides in a mouse model of β-thalassemia

Non-transfusion dependent β-thalassemia (NTDT) is characterized by ineffective erythropoiesis, suppression of hepcidin, and iron overload, which is a major cause of morbidity and mortality. Hepatic protease Tmprss6 regulates hepcidin by modulating BMP/Smad signaling. Suppression of Tmprss6 in the mouse model of NTDT (Th3 mice), increased hepcidin and improved disease outcomes, but similar benefits have not been reported in human trials. We hypothesized that the resistance to the effects of anti-TMPRSS6 treatment in patients with NTDT may be caused by high erythroferrone (ERFE) concentrations and more severe iron overload, both known to modulate BMP/Smad signaling in hepatocytes. We therefore examined how ERFE and iron overload modulate the effects of Tmprss6 antisense oligonucleotides (ASO) in mouse models.We treated ERFE-overexpressing Th3 mice (T-E(M)) and their littermates (wild-type, Th3, and Erfe-overexpressing E(M) mice) with Tmprss6 ASO or non-targeting ASO for 4 weeks. Tmprss6 ASO increased phospho-Smad1/5/8 and hepcidin levels, decreased splenomegaly, reduced extramedullary erythropoiesis and improved iron parameters in Th3 mice but not in T-E(M) mice, despite similar Tmprss6 decrease. To determine whether iron overload alone blunted the effects of Tmprss6 ASO, we assessed the responses of C57BL/6 mice after 10 mg of iron dextran or 5000 ppm high iron diet. Unlike iron-adequate mice, iron-overloaded mice already had high phospho-Smad and hepcidin, with no further increase after Tmprss6 ASO treatment, and no effect on erythroid or iron parameters.Higher ERFE concentrations and more severe iron overload may be responsible for diminished benefits of Tmprss6 ASO in human NTDT.