The hallmarks of β-thalassemia (BT) include ineffective erythropoiesis (IE), splenomegaly and iron overload (IO). Recent studies have pointed to iron restriction (IR) to improve both anemia and IO in BT (Rivella, Blood). The decreased iron-uptake by early erythroid cells reduces hemichrome toxicity and prevents premature RBC hemolysis. One such IR therapy targets the matriptase-2 (Tmprss6) gene using antisense oligonucleotides (T-ASO). Our group has previously shown that treatment of Hbb th3/+(th3/+) mice (a mouse model for BT-intermedia) with T-ASO improved anemia, lengthened red blood cell (RBC) lifespan, reduced levels of erythroferrone (ERFE), hemichromes and reactive oxygen species, and ameliorated splenomegaly (Casu et al. Blood). Another novel therapeutic approach to improve anemia targets the Transforming Growth Factor (TGF)-β pathway to increase erythroid maturation. Luspatercept, a TGF-β trap-ligand, gained FDA approval in 2019 to treat transfusion dependent BT patients (Cappellini and Taher, Blood Adv). In mouse models of BT, its murine analog (RAP-536) was found to promote EPO-independent maturation of late-stage erythroid cells, and resulted in increased RBC parameters in a dose-dependent manner (Surgani, et al. Nat Med). In this work we treated th3/+ mice with an agent analogous to murine Luspatercept (RAP-GRL) in combination with the iron restriction (IR) drug T-ASO, (RAP-GRL+T-ASO) with the goal of targeting distinct morbidities associated with BT. To test our RAP-GRL construct, primary fibroblasts were transduced with an adenovirus containing the RAP-GRL sequence (FB Ad5RAP-GRL) and used to deliver RAP-GRL to mice. As a second strategy, RAP-GRL was expressed in a mammalian cell line and purified. Wild-type (WT) or th3/+ mice were subcutaneously (s.c.) implanted with 1x10 6 FB Ad5RAP-GRL or injected s.c. with 10mg/kg of RAP-GRL and monitored by complete blood counts. Implantation of FB Ad5RAP-GRL ortreatment with purified RAP-GRL increased RBC parameters in both WT and th3/+ mice (n=3-9, 2-4-month-old females and males). In the first combination therapy experiment we implanted FB Ad5RAP-GRL s.c. and delivered T-ASO via intraperitoneal (i.p.) injection in th3/+ mice. RBC parameters were increased in all treatment groups except controls after 6 weeks. The RAP-GRL+ T-ASO group displayed the most pronounced increase in RBC parameters with a mean increase in RBC of 3.067±0.73 10 6 cells/µL, Hb of 3.02±0.77 g/dL, and Hct of 5.88±2.36 % (Table 1). Additionally, we also treated th3/+ mice with two different doses of protein purified RAP-GRL in combination with T-ASO (Table 1). The best results using the protein purified RAP-GRL were achieved in the RAP-GRL+T-ASO group that was treated with two weekly 10mg/kg s.c. injections of RAP-GRL and two weekly 5mg/kg i.p. injections of T-ASO (Group 2) for 6 weeks. Flow cytometry analysis using CD71, TER119, and CD44 antibodies showed improvements in the bone marrow (BM) and spleen (SPL) of all treatment groups compared to controls. Additionally, ROS levels and splenomegaly were also greatly reduced in all T-ASO and RAP-GRL+T-ASO treated groups compared to controls. Serum assessment of T-ASO and RAP-GRL+T-ASO treated animals showed decreased levels of iron and transferrin saturations with a simultaneous increase in hepcidin levels. ERFE levels were decreased in all T-ASO and RAP-GRL+T-ASO groups, however, erythropoietin (EPO) levels were increased only in the RAP-GRL and RAP-GRL+T-ASO cohorts of Group 2. Additionally, although EPO was elevated in all RAP-GRL treated animals of Group 2, only the RAP-GRL+T-ASO group had reduced ERFE. This result is in agreements with our findings of decreased early (ERFE-producing) erythroid progenitors in the BM and SPL of RAP-GRL+T-ASO treated mice. This finding also suggests that higher doses of RAP-GRL may result in elevated EPO. Luspatercept, through heightened iron consumption, may increases EPO synthesis in the kidney via activation of the transcription factor HIF2-α, which can be stabilized not only by hypoxia, but also by iron deficiency. In conclusion our results provide pre-clinical support for combining IR and TFG-β trap-ligands in the treatment of BT. Our data shows that IR, in conjunction with the enhancing erythroid maturation action of Luspatercept (and potential activation of EPO), may offer an additive and more effective therapeutic strategy for BT patients. Figure 1 Figure 1. Disclosures Guo: Ionis Pharmaceuticals, Inc.: Current Employment. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.
Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe (monoferric N-TF), the C-lobe (monoferric-C), or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric. The iron-bound TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. Iron-bound TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and bone cells. Whereas TFR1 serves primarily as a cargo receptor, TFR2 serves primarily to influence cellular signaling events regulating hepcidin expression, erythropoiesis, and bone formation. We proposed that different transferrin forms provide differential signaling properties in this regulation. We thus generated TF mutant mice in which all iron-containing TF was either monoferric N (Tf monoN) or monoferric C (Tf monoC). Compared with Tf monoC mice, the Tf monoN mice demonstrated increased RBC production and increased hepcidin expression relative to iron status (Parrow et al. Blood). Based on observations in β-thalassemic mice treated with exogenous TF (Li et al. Nat Med), we hypothesized that β-thalassemic mice obligate for monoN TF would demonstrate improved erythropoietic and iron parameters compared with β-thalassemic mice obligate for monoC TF. To address this hypothesis, we crossed Hbb th3/+ mice (a mouse model of β-thalassemia intermedia) with Tf monoN and Tf monoC mice. Compared with Hbb th3Tf +/+mice, in Hbb th3/+Tf monoN mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology (Figure 1A-B), decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of ineffective erythropoiesis (as measured by the ratio of progenitors to RBC in the bone marrow). Additionally, serum ERFE was significantly reduced and hepcidin levels were increased in Hbb th3/+Tf monoN relative to Hbb th3/+Tf +/+controls. Conversely, hematological parameters from Hbb th3/+Tf monoC mice were comparable to Hbb th3/+Tf +/+ mice. Similarly, Hbb th3/+Tf monoCmice had no improvements in markers of ineffective erythropoiesis in the bone marrow compared with Hbb th3/+Tf +/+ mice. In summary, we demonstrate that the differential regulatory effects of monoN and monoC TF on erythropoiesis are relevant not only in steady-state, but also in the ineffective erythropoiesis that is characteristic of β-thalassemia. Because both monoN and monoC TF forms can deliver only one iron atom per TF-TFR1 binding event, our findings suggest that the improvements observed only in the Hbb th3/+Tf monoN mice were not due to iron restriction alone. We are now elucidating the mechanisms by which the two TF lobes exert their differential effects on ineffective erythropoiesis and exploring the translational potential of obligate monoN TF in the treatment of β-thalassemia. Figure 1 Figure 1. Disclosures Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.
The hallmarks of β-thalassemia (BT) include ineffective erythropoiesis (IE), splenomegaly and iron overload (IO). Recent studies have pointed to iron restriction (IR) to improve both anemia and IO in BT (Rivella, 2019). The decrease of iron-uptake by early erythroid cells results in reduced hemichrome toxicity and prevents premature red blood cell (RBC) hemolysis. One promising IR therapy strategy, which is currently in Phase II clinical trials (NCT04059406), targets the matriptase-2 (TMPRSS6) gene using antisense oligonucleotide technology (T-ASO). As previously shown, treatment of Hbbth3/+ (th3/+) mice (a mouse model for BT-intermedia) with T-ASO improves anemia, lengthens RBC lifespan, reduces levels of erythroferrone (ERFE), decrease hemichromes and lowers reactive oxygen species (ROS), and ameliorates splenomegaly (Casu et al., 2016(Casu et al., , 2020Guo et al., 2013). Another novel therapeutic approach to improve anemia in BT targets the TGF-β pathway. Luspatercept, a TGF-β ligand trap, gained FDA approval in 2019 to treat transfusion dependent BT patients (Cappellini and Taher, 2021). In mouse models of BT, its murine analog (RAP-536) was found to promote erythropoietin (EPO)-independent maturation of late-stage erythroid cells, resulting in increased RBC parameters in a dose-dependent manner (Suragani et al., 2014). In this work we generated a Luspaterceptlike protein (RAP-GRL) and used it to treat th3/+ mice in combination with T-ASO (RAP-GRL+T-ASO). Our goal was to investigate if this strategy would successfully target distinct morbidities associated with BT. We generated RAP-GRL against the mouse analog of Luspatercept (RAP-536). Wild-type (WT) and th3/+ mice were subcutaneously injected s.c. with 10mg/kg of purified RAP-GRL. Our results showed that treatment with RAP-GRL increased RBC levels in both WT and th3/+ mice. Next, we treated th3/+ mice with RAP-GRL (10mg/ kg), T-ASO (5mg/kg), or RAP-GRL+T-ASO. The RAP-GRL+T-ASO group displayed the most pronounced increase in RBC parameters and improvements in cell morphology. Flow cytometry analysis using CD71, TER119, and CD44 antibodies of the RAP-GRL+T-ASO groups showed the greatest improvements in IE in both the bone marrow (BM) and spleen (SPL). Additionally, splenomegaly was also greatly reduced in all T-ASO and RAP-GRL+T-ASO groups compared to RAP-GRL and control groups. In conclusion our results provide pre-clinical support for combining IR and TFG-β ligand-trap strategies for the treatment of BT. Our data provides evidence that IR, in conjunction with the erythroid maturation action of Luspatercept may offer additive and more effective therapeutic strategy for BT patients.
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