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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.

Schmidt PJ, Racie T, Westerman M, Fitzgerald K, Butler JS, Fleming MD - Am. J. Hematol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts.

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Iron parameters in Hbbth3/+ 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.]
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fig01: Iron parameters in Hbbth3/+ 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.]

Mentions: We determined whether modulating systemic iron stores by dietary restriction or pharmacological chelation could add to the beneficial effects of Tmprss6 siRNA therapy on Hbbth3/+ anemia and iron phenotypes. To do so, we treated wild type and Hbbth3/+ animals with biweekly LNP-Tmprss6 siRNA injections in conjunction with an iron replete synthetic control diet (50 ppm iron), a low iron diet (3–5 ppm iron), or an iron replete diet containing the orally bioavailable chelator deferiprone (50 ppm iron, 0.125% wt/wt chelator). In wild-type animals, we observed qualitatively similar effects on gene expression and iron metabolism, including Tmprss6 suppression and the induction of systemic iron deficiency, as we did previously (Supporting Information Fig. 1) [14]. As expected, LNP-Tmprss6 treatment of Hbbth3/+ animals on all diets suppressed Tmprss6 mRNA expression and up-regulated serum hepcidin (Fig. 1A,B). Furthermore, treated animals had decreased transferrin saturation and serum iron (Fig. 1C,D), with the largest and most significant changes seen in animals on the diet containing deferiprone. When compared to control siRNA-treated animals on the same diet, nonheme liver iron was diminished only in animals treated with deferiprone (Fig. 1E), possibly due to the relatively low iron (50 ppm), which is only approximately two times the recommended adult murine daily requirement, in the formulated test diets compared to conventional mouse chow (380 ppm iron) that we employed previously [14]. In comparison to the control diet, LNP-Tmprss6 treated animals on either an iron deficient diet or diet with added chelator had lower liver iron levels, with the latter having a greater effect. In Hbbth3/+ animals, this change in liver iron can be visualized on DAB-enhanced Perls' stained tissue sections as decreased periportal hepatocellular iron (Fig. 1F). Total spleen iron (Fig. 1G) decreased significantly in treated Hbbth3/+ animals on the iron replete and low iron diets, but only trended downward in animals fed chlelator-enhanced chow, possibly due to effects on erythroid iron utilization or RBC turnover.


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.

Schmidt PJ, Racie T, Westerman M, Fitzgerald K, Butler JS, Fleming MD - Am. J. Hematol. (2015)

Iron parameters in Hbbth3/+ 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.]
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fig01: Iron parameters in Hbbth3/+ 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.]
Mentions: We determined whether modulating systemic iron stores by dietary restriction or pharmacological chelation could add to the beneficial effects of Tmprss6 siRNA therapy on Hbbth3/+ anemia and iron phenotypes. To do so, we treated wild type and Hbbth3/+ animals with biweekly LNP-Tmprss6 siRNA injections in conjunction with an iron replete synthetic control diet (50 ppm iron), a low iron diet (3–5 ppm iron), or an iron replete diet containing the orally bioavailable chelator deferiprone (50 ppm iron, 0.125% wt/wt chelator). In wild-type animals, we observed qualitatively similar effects on gene expression and iron metabolism, including Tmprss6 suppression and the induction of systemic iron deficiency, as we did previously (Supporting Information Fig. 1) [14]. As expected, LNP-Tmprss6 treatment of Hbbth3/+ animals on all diets suppressed Tmprss6 mRNA expression and up-regulated serum hepcidin (Fig. 1A,B). Furthermore, treated animals had decreased transferrin saturation and serum iron (Fig. 1C,D), with the largest and most significant changes seen in animals on the diet containing deferiprone. When compared to control siRNA-treated animals on the same diet, nonheme liver iron was diminished only in animals treated with deferiprone (Fig. 1E), possibly due to the relatively low iron (50 ppm), which is only approximately two times the recommended adult murine daily requirement, in the formulated test diets compared to conventional mouse chow (380 ppm iron) that we employed previously [14]. In comparison to the control diet, LNP-Tmprss6 treated animals on either an iron deficient diet or diet with added chelator had lower liver iron levels, with the latter having a greater effect. In Hbbth3/+ animals, this change in liver iron can be visualized on DAB-enhanced Perls' stained tissue sections as decreased periportal hepatocellular iron (Fig. 1F). Total spleen iron (Fig. 1G) decreased significantly in treated Hbbth3/+ animals on the iron replete and low iron diets, but only trended downward in animals fed chlelator-enhanced chow, possibly due to effects on erythroid iron utilization or RBC turnover.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts.

Show MeSH
Related in: MedlinePlus