Tfr2 Genetic Deletion Makes Transfusion-Independent a Murine Model of Transfusion-Dependent β-Thalassemia
Authors: Di Modica Simona Maria , Olivari Violante , Tanzi Emanuele , Pettinato Mariateresa , Lidonnici Maria Rosa , Pagani Alessia , Tiboni Francesca , Ferrari Giuliana , Silvestri Laura , Rivella Stefano , Nai Antonella 
Published: November 2021

Abstract
β-thalassemia is an autosomal recessive disorder due to mutations in the β-globin gene, that leads to defective production of hemoglobin (Hb) and red blood cells (RBC). The main features of the disease are anemia, ineffective erythropoiesis and iron overload. Patients affected by the most severe form of β-thalassemia are transfusion-dependent (TDT) and require lifelong blood transfusions and iron chelation, symptomatic treatments that affect the quality of life. The only curative option, unfortunately limited by the insufficient number of HLA-matched donors, is allogenic bone marrow (BM) transplantation. Other recently approved treatments (i.e. Luspatercept and gene therapy) are only partially effective and/or suitable for selected patients. Thus, the identification of novel therapeutic approaches is a clinical need.
Transferrin receptor 2 (TFR2) contributes to the transcriptional activation of hepcidin, the master regulator of iron homeostasis, in the liver and is a brake of Erythropoietin signaling in erythroid cells, thus balancing RBC production and iron availability. We have recently proved that BM Tfr2 deletion enhances erythropoiesis in wild-type mice (Nai et al., Blood 2015) and ameliorates anemia in non-TDT mice models, both alone (Artuso et al., Blood 2018) and in combination with iron-restricting agents (Casu, Pettinato et al., Blood 2020).
Here we aim at investigating whether Tfr2 targeting might be beneficial also for TDT. To this purpose we generated TDT mice (Hbb th1/th2; Casu et al., Haematologica 2020) with heterozygous (Tfr2 BMhetero/Hbb th1/th2) and homozygous (Tfr2 BMKO/Hbb th1/th2) BM Tfr2 deletion by transplantation of Hbb th1/th2, Tfr2-hetero/Hbb th1/th2 and Tfr2-ko/Hbb th1/th2 fetal liver cells (FLT) from day E14.5 embryos into lethally irradiated wild-type mice. BM Tfr2 deficient mice have increased RBC count and Hb levels and decreased percentage of reticulocytes with a gene dosage effect 8 weeks after FLT, before the onset of transfusion-dependance. At this time-point, complete BM Tfr2 deletion ameliorates ineffective erythropoiesis, decreasing the percentage of polychromatic erythroblasts and increasing orthochromatic erythroblasts and mature RBCs both in the BM and in the spleen. The improved anemia was also accompanied by a partial correction of two debilitating TDT complications, iron overload and cardiomegaly.
The beneficial effect of Tfr2 deletion was maintained over time. Indeed, Hbb th1/th2 mice became transfusion-dependent 14 weeks after FLT, when Hb levels drop below 5.5 g/dL, requiring transfusions of an average 108.75±56.87μL of blood/animal/week. On the contrary, animals with both heterozygous and homozygous BM Tfr2 deletion are still non-transfusion-dependent at 20 weeks, maintaining Hb levels above 7 and 9 g/dL respectively.
Overall, our results prove that, despite the persistence of the genetic defect, hematopoietic Tfr2 deletion ameliorates anemia, ineffective erythropoiesis and secondary complications also in the most severe form of β-thalassemia. This improvement is associated to a substantial increase in transfusion-free survival of both Tfr2 BMhetero/Hbb th1/th2 and Tfr2 BMKO/Hbb th1/th2 mice, which are transfusion-independent 6 weeks after the time of blood transfusion requirement in Hbb th1/th2 animals. The difference of blood transfusions needs will be evaluated over time for at least 10 additional weeks.
Our findings demonstrate that TFR2 targeting represents a new promising therapeutic opportunity for the management of β-thalassemia, worth to be tested both as a monotherapy and in association with available treatments.

Disclosures
Rivella: Incyte: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.