Abstract
Β-thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload. Administration of agents that increase erythropoiesis (such as erythropoietin-Epo) can potentially improve anemia by enhancing the production of red blood cells (RBC). However, this improvement will likely result in exacerbation of splenomegaly and worsening of iron overload due to further suppression of hepcidin, the hormone that controls dietary iron absorption. However, we postulated that, following Epo administration, some level of iron restriction will limit the splenomegaly and iron overload without preventing the improvement of anemia.
To increase circulating Epo levels and increase erythropoiesis, we infused animals with primary dermal fibroblasts transduced with a helper dependent adenoviral vector carrying the Epo gene (TARGTEpo). Pharmacokinetic studies indicated that 1e6 cells were sufficient to significantly increase Epo synthesis (up to 47.6%in Hbbth3/+ and 90% in WT). This led to an increase in hemoglobin levels in both mouse models (average of 3-4 g/dL) through the end of the treatment (6 weeks). In Hbbth3/+mice, Hb levels rose to 10.7±1.0 g/dL in the first week and remained elevated through the end of the 6-weeks of treatment (compared to 7.7±0.7 g/dL in controls). As expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin.
To test our hypothesis, we then combined TARGTEpo with low iron diet or antisense oligonucleotides targeting Tmprss6 mRNA (Tmprss6-ASO; the last treatment only for Hbbth3/+animals). It has already been shown that inhibition of Tmprss6 increases hepcidin expression leading to decreased iron absorption and limited erythroid iron intake, improving anemia, IE, splenomegaly and iron overload in Hbbth3/+mice (Guo et al, JCI, 2013).
In WT animals, after three weeks, the combination of TARGTEpo with low iron diet significantly reduced Hb levels (-40%), RBC number (-38%) and reticulocytes (-80%), when compared to animals overexpressing Epo and receiving a normal iron diet. In contrast, Hbbth3/+animals on an iron-deficient diet or treated with Tmprss6-ASO in the presence of TARGTEpo showed decreased hemichrome formation and improved anemia through the end of treatment. In particular, Hb levels increased (on average 4 g/dL), reaching levels of 11-12g/dl, corresponding to +36% more than baseline levels in Hbbth3/+(7.7g/dL) and +25% more compared to Hbbth3/+mice treated with Tmprss6-ASO alone (9.0g/dL). Animals receiving TARGTEpo with Tmprss6-ASO showed a significant reduction in splenomegaly when compared to baseline levels (-35%) and to animals treated only with TARGTEpo (-52.4%). Preliminary data indicate that organ iron concentration improved or normalized compared to control Hbbth3/+mice when iron restriction was applied.
We then inquired how these two treatments combined improved erythropoiesis. We quantified the number of nucleated erythroid cells positive for the phosphorylated form of pAkt and pJak2, proteins activated by Epo but whose activity is potentially limited by iron restriction. For pAkt+ cells in the spleen, the number of proerythroblasts were increased in Hbbth3/+treated with TARGTEpo (+35% compared to control Hbbth3/+mice), while they were reduced in Hbbth3/+treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO (-72% and -43% respectively, compared to control Hbbth3/+mice). Other erythroid populations showed a comparable profile for both pAkt+ and pJak2+ nucleated erythroid cells. The total number of progenitor erythroid cells (i.e. BFUe) also followed a similar trend. Cell cycle analysis of splenic erythroid progenitor cells showed a decrease in the percentage of nucleated erythroid cells in mice treated with Tmprss6-ASO or TARGTEpo+Tmprss6-ASO compared to control or TARGTEpo-treated Hbbth3/+mice. Given that the number of RBC in circulation was highest in mice treated with TARGTEpo+Tmprss6-ASO, this data suggests that co-administration of these two drugs maximize the ability of each erythroid cell to develop into a functional RBC.
In conclusion, we propose that in β-thalassemia, combination of erythropoietic stimulating agents with iron restriction could maximize correction of anemia while improving splenomegaly and iron overload.
Disclosures
Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Aghajan:Ionis Pharmaceuticals, Inc: Employment. Neil:Aevi Genomic Medicine, Inc: Employment, Other: Officer and share holder. Guo:Ionis Pharmaceuticals, Inc: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; MeiraGTx: Other: SAB; Disc Medicine: Consultancy; Protagonist: Consultancy.
A mathematical model for the hemoglobin response to iron intake, based on iron absorption measurements from habitually consumed Indian meals