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Pettinato, M.1, 2; Aghajan, M.3; Lidonnici, M. R.4; Olivari, V.2; Silvestri, L.1, 2; Guo, S.3; Ferrari, G.1, 4; Camaschella, C.2; Nai, A.1, 2

doi: 10.1097/01.HS9.0000558812.83696.45
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1Vita-Salute San Raffaele University

2Division of Genetics and Cell Biology, Ospedale San Raffaele, Milan, Italy

3Ionis Pharmaceuticals, Inc., Carlsbad, United States

4SR-Tiget, Ospedale San Raffaele, Milan, Italy

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Transferrin receptor 2 (TFR2) is a type II transmembrane protein mainly expressed in the liver and in the erythroid compartment. In the liver it acts as an activator of the iron hormone hepcidin and, when mutated, is responsible for type 3 hereditary hemochromatosis. In erythroid cells TFR2 binds the erythropoietin (EPO) receptor and serves as a brake of the EPO-EPOR signaling pathway. We have recently demonstrated that the complete loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity and improves anemia, ineffective erythropoiesis and iron-overload in a β-thalassemia intermedia murine model (Hbbth3/+), without reducing splenomegaly. On the contrary, approaches based on the induction of iron restriction were proven to be effective in ameliorating splenomegaly and anemia in thalassemic mice. Among these, targeting the hepcidin inhibitor TMPRSS6 is one of the most promising strategies.

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The main aim of the present work is to investigate whether the combination of Tfr2-haploinsufficiency in the BM and TMPRSS6 antisense oligonucleotide (ASOs) administration will reduce splenomegaly and provide a synergistic benefit on thalassemic erythropoiesis and anemia.

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We generated thalassemic mice with both or a single Tfr2 allele in the BM by transplanting BM cells from Hbbth3/+ or Tfr2+/-/Hbbth3/+ mutants into lethally irradiated wild-type mice. Mice were treated with TMPRSS6-ASO (or control ASO) twice a week for 6 weeks starting 9 weeks after BM transplantation. Complete blood count was determined at the beginning of the treatment and after 3 and 6 weeks. At sacrifice, blood and organs were collected for full phenotypic analysis.

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Deletion of a single Tfr2 allele in Hbbth3/+ mice BM mildly increased Red Blood Cells (RBC) count and Hemoglobin (Hb) levels and improved erythropoiesis, with a concomitant decrease in EPO levels, but no reduction of spleen size. A similar phenotype was induced by TMPRSS6-ASO treatment of Hbbth3/+ animals, with additional partial correction of splenomegaly.

The combination of BM Tfr2 haploinsufficiency and TMPRSS6-ASO had a striking effect on the thalassemic phenotype: indeed, an impressive increase in RBC count (up to 11.2 ± 1.2*106/μl) and Hb (˜3 g/L) compared with Hbbth3/+ was attained, accompanied by the normalization of reticulocyte and EPO levels, improved erythropoiesis and spleen size reduction. Mean corpuscular volume and hemoglobin were strongly reduced. This indicates a disproportionate erythropoietic expansion relative to the available iron, whose circulating levels are decreased relative to untreated thalassemic controls.

Summary/Conclusion: While Tfr2 haploinsufficiency does not impair systemic iron homeostasis, here we demonstrate that deletion of a single Tfr2 allele in the BM is sufficient to improve the hematological parameters of thalassemic mice. This finding paves the way toward a potential targeted therapy based on partial Tfr2 inhibition that might be beneficial for thalassemia (and potentially for other forms of anemia) without inducing defective hepcidin activation and iron overload. In addition, our results prove that targeting Tfr2 in combination with an anti-TMPRSS6 therapy may be a valuable therapeutic approach that strongly improves the thalassemic phenotype. However, Tmprss6 inhibition must be tightly controlled to avoid excessive iron restriction. Further experiments will allow setting up the optimal conditions.

Copyright © 2019 The Authors. Published by Wolters Kluwer Health Inc., on behalf of the European Hematology Association.