Iron overload is a potentially lethal complication of β-thalassemia, affecting the structure and function of many organs. Hepcidin (Hamp) is a peptide of hepatic origin, which regulates iron metabolism by triggering the degradation of ferroportin (Fpn), an iron-transport protein localized on absorptive enterocytes, hepatocytes and macrophages. We showed that while iron overload increases with time in mice with thalassemia intermedia (th3/+), Hamp1 is expressed at a low level relative to the amount of body iron (Gardenghi et al. Blood. 2007 109:5027–35). Thalassemic patients exhibit a similar picture, having low levels of urinary HAMP. We hypothesize that th3/+ mice absorb more iron than they need for erythropoiesis and that reducing their iron intake has no effect on anemia. Accordingly, we postulated that selectively increasing the Hamp concentration might be therapeutic, limiting iron overload by reducing the amount of Fpn. However, increasing Hamp might also impair erythropoiesis by preventing release of iron from macrophages. We utilized different strategies to investigate the effects of increased Hamp on iron overload and erythropoiesis. First, we generated animals over expressing Hamp1, both wt (Tg-Hamp1; N=8) (Roy et al. Blood. 2007 109:4038) and th3/+ mice (Tg-Hamp1/th3; N=8). The animals were fed a defined iron sufficient diet (35 ppm) for 1 and 5 mo and compared with wt and th3/+ mice fed diets containing 2.5, 35 and 200 ppm of iron; N>5 per group). The 200-ppm diet was standard rodent chow containing approximately 10 times more iron than physiologically required. At 5 mo, mice fed the 35- and 200-ppm iron diets had similar organ iron contents and erythropoietic parameters. The 2.5-ppm diet induced a progressive anemia in wt mice (Hgb: 14.6 ± 0.7 g/dL and 8.7 ± 3.0 g/dL at 1 and 5 mo, respectively). In contrast, the same diet did not worsen the anemia in th3/+ animals, confirming our first hypothesis (Hgb: 8.2 ± 1.2 g/dL vs 8.2 ± 1.7 g/dL at 1 and 5 mo, respectively). In th3/+ mice, the combined amount of iron in the liver and spleen decreased over time and after 5 mo was 10 times less than at the beginning of the 2.5-ppm diet. The relative amount of iron in the liver was reduced and that in the spleen increased, indicating mobilization of iron from storage to erythropoietic tissues. At 1 mo, Tg-Hamp1 mice showed mild anemia (Hgb: 10.6 ± 2.2 g/dL vs 13.6 ± 1.2 g/dL), and a greater number of immature erythroid progenitor cells in the spleen, compared to wt control mice. However, Tg-Hamp1/th3 mice did not have significant changes in hematological values compared to control th3/+ animals. The total iron content of the liver was decreased in both Tg-Hamp1 (59 ± 18 ug vs 145 ± 25 ug in wt controls) and Tg-Hamp1/th3 mice (156 ± 91 ug vs 264 ± 18 ug in th3/+ controls), while no significant changes in iron content were detected in the spleen. These data showed that over expression of Hamp1 in Tg- Hamp1/th3 mice could reduce tissue iron overload with no effect on anemia. A second strategy to increase the Hamp level involved direct i.v. injection of synthetic Hamp25 peptide (50 ug) into wt and th3/+ mice (N=6) for 14 days. Our data indicate that Hamp administration decreases the iron level in the serum, compared to animals injected with PBS, without any effect on anemia. Quantitative real-time PCR analysis on the liver of injected mice also revealed down-regulation of endogenous Hamp1 expression after Hamp25 administration. Organ iron analysis is in progress. Third, we generated lentiviral vectors where a synthetic murine transthyretin (TTR) promoter specifically drives Hamp1 cDNA expression in the hepatocytes of 3-day-old …