Tmprss6-ASO treatment, together with overexpression of Epo, significantly improves anemia and reduces splenomegaly in Hbb^th3/+ animals. Treatment with Tmprss6-ASO induced increased Hb levels (A) and RBC count (B) in Hbb^th3/+ mice. Tmprss6-ASO also induced a reduction of MCV (C) and had a minimal effect on MCH (D). Improvement in anemia was also associated with a significant reduction in reticulocyte (Retics) number (E) and splenomegaly (F) when compared with animals receiving empty fibroblasts. When EPO was coadministered with Tmprss6-ASO, animals displayed normalization of Hb levels and RBC number when compared with controls (A-B), as well as normalization of reticulocyte number and splenomegaly, when compared with animals receiving fibroblasts overexpressing Epo (E-F). This was also confirmed by flow cytometry analysis of the erythroid compartment in the spleen (expressed in Abs#). (G). Coadministration of EPO with Tmprss6-ASO led to higher levels of serum EPO (H) as well as serum ERFE (I). Suppression of endogenous Epo expression was observed in the kidney of animals receiving fibroblasts overexpressing Epo (L). Dotted red line indicates mean values for untreated WT mice. Dashed black lines indicates mean values for untreated Hbb^th3/+ mice. Bars represent SD. Asterisks refer to statistically significant differences (****P ≤ .001; ***P ≤ .005; **P ≤ .01; *P ≤ .05). E, erythroblasts.