Combination therapy with a Tmprss6 RNAi-therapeutic and the oral iron chelator deferiprone additively diminishes secondary iron overload in a mouse model of β-thalassemia intermedia

Abstract

β-thalassemias result from diminished β-globin synthesis and are associated with ineffective erythropoiesis and secondary iron overload caused by inappropriately low levels of the iron regulatory hormone hepcidin. The serine protease TMPRSS6 attenuates hepcidin production in response to iron stores. Hepcidin induction reduces iron overload and mitigates anemia in murine models of β-thalassemia intermedia. To further interrogate the efficacy of an RNAi-therapeutic downregulating Tmprss6, β-thalassemic Hbb(th3/+) animals on an iron replete, an iron deficient, or an iron replete diet also containing the iron chelator deferiprone were treated with Tmprss6 siRNA. We demonstrate that the total body iron burden is markedly improved in Hbb(th3/+) animals treated with siRNA and chelated with oral deferiprone, representing a significant improvement compared to either compound alone. These data indicate that siRNA suppression of Tmprss6, in conjunction with oral iron chelation therapy, may prove superior for treatment of anemia and secondary iron loading seen in β-thalassemia intermedia.

Figure 1

Iron parameters in Hbb^th3/+ treated with LNP-Tmprss6 siRNA and dietary iron deficiency or chelation. Six-week old female animals were injected with LNP-siRNA formulations (1 mg/kg) every other week for 6 weeks and then killed 14 days after the final injection. On the day of the first injection, animals were placed on a control (50 ppm iron), iron deficient (3–5 ppm iron), or control diet supplemented with deferiprone (50 ppm iron, 0.125% deferiprone). A: Liver Tmprss6 mRNA assessed by quantitative real-time PCR and normalized to β-actin (Actb). B: ELISA serum hepcidin analysis (ng/ml). C: Serum transferrin saturation (%). D: Serum iron (μg/dl). E: Nonheme liver iron (μg/g). F: 3,3′-diaminobenzidine (DAB)-enhanced iron stain of liver sections. G: Total spleen iron (μg). Error bars are ±SEM. (n = 4−6 for each group) Student's t-test P-values: ****P < 0.001, ***P < 0.005, **P <0.01, and *P < 0.05. [Color figure can be viewed in the online issue, which is available at http://wileyonlinelibrary.com.]

Figure 2

Erythropoietic parameters in Hbb^th3/+ animals treated with LNP-Tmprss6 siRNA and dietary iron deficiency or chelation. Animals were treated as in Figure 1. A: Hemoglobin (g/dl). B: Red blood cell count. C: Mean red blood cell volume (fl). D: RBC morphology on Wright's stained peripheral blood smears. E: Fraction of reticulocytes (%). F: Absolute reticulocyte count. G: Serum erythropoietin levels (pg/ml). H: Spleen weight (g). Error bars are ±SEM. (n = 4−6 for each group) Student's t-test P-values: ****P < 0.001, ***P < 0.005, **P <0.01, and *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]