Potential Therapeutic Applications of Jak2 Inhibitors and Hif2a-ASO for the Treatment of β-Thalassemia Intermedia and Major
Authors: Casu Carla , Oikonomidou Paraskevi Rea , Lo Presti Vania , Aghajan Mariam , Guo Shuling , Osheiza Abdulmalik , Melchiori Luca , Ramos Pedro , Rivella Stefano 
Published: November 2019

Abstract
β-Thalassemia is one of the most common genetic red blood cell (RBC) disorders characterized by reduced (such as in non-transfusion dependent thalassemia or NTDT) or absent (such as in transfusion dependent thalassemia or TDT) production of β-globin chains. Ineffective erythropoiesis (IE) with consequent anemia leads to extra-medullary hematopoiesis (EMH), splenomegaly and iron overload mediated by low levels of hepcidin. We previously demonstrated that IE in β-thalassemia is associated with increased proliferation and reduced differentiation of erythroid progenitors (Libani et al, 2008, Blood). This is mediated by increased production of erythropoietin (EPO), which activates the downstream JAK2 kinase in erythroid progenitors. As a consequence, hepatosplenomegaly may result, often requiring splenectomy to prevent serious morbidities and mortality. The increased synthesis of EPO in thalassemia is the result of hypoxia. Hypoxia Inducible Factor-2a (HIF2a) is a central mediator of cellular adaptation to hypoxia and stimulates renal and hepatic EPO synthesis. Furthermore, splenomegaly in thalassemia also exacerbates the anemia, as a large proportion of the circulating RBC are engulfed and eliminated by an enlarged spleen.
Therefore, we hypothesized that targeting the EPO/JAK2 pathway limits the number of erythroid progenitor cells and reduces the splenomegaly, and it could be utilized as an alternative to splenectomy. We utilized JAK2 inhibitors or HIF2a antisense oligonucleotides (HIF2a-ASO) to target the EPO/JAK2 pathway. Moreover, we hypothesized that combination of these drugs with blood transfusion therapy will further reduce the splenomegaly and, eventually, improve the blood transfusion regimen.
We tested two commercially available JAK2 inhibitors in mice affected by NTDT (Hbbth3/+): INCB018424 (Ruxolitinib) and TG101348 (Fedratinib, SAR302503). Both drugs were administered for 10 days, twice daily by oral gavage at a dose of 180 and 120 mg/Kg respectively. A mild reduction in hemoglobin (Hb) levels, (in the range of 9%), was observed in animals treated with both inhibitors when compared to vehicle- treated mice. Splenomegaly was significantly reduced with both inhibitors (up to 56% in reduction). Flow cytometry studies on spleen cells revealed that animals receiving the inhibitors exhibited a reduction in the number of erythroid progenitors compared to the placebo-treated animals . In parallel, we performed pharmacokinetic[BM1] studies using Hif2a-ASO. Animals received Hif2a-ASO at a dose of 25 mg/kg twice weekly for 10 days or 3 weeks by IP injections. After 10 days of treatment the spleen weight was reduced 58%, while the Hb level was reduced in the range of 27%%. After 3 weeks the effect observed on the anemia was more pronounced, with a reduction of more that 40% in Hb levels, while the spleen reduction was 36%. We then compared and combined these drugs with blood transfusion using Hbbth3/+ mice. Blood transfusion reduced splenomegaly 49% and 53% when compared, respectively, to non-transfused controls and animals treated with JAK2 inhibitors alone. When transfusion was combined with the administration of JAK2 inhibitors for 10 days, the spleen size was further reduced (72% when compared with non-transfused controls). Combination of Hif2a-ASO and blood transfusion is in progress. We then tested the JAK2 inhibitors in mice affected by TDT. TDT animals are generated by engrafting WT mice with Hbbth3/th3 fetal liver cells. Following engraftment, these mice show a large splenomegaly and rapidly become dependent on chronic blood transfusion for survival (Gardenghi et al, 2007, Blood). Preliminary studies suggest that administration of JAK2 inhibitors for 10 days, together with blood transfusion, further reduces spleen weight by 71% compared to transfusion alone. Combination of Hif2a-ASO and blood transfusion in these animals is also in progress. In summary, JAK2 inhibitors and Hif2a-ASO reduce splenomegaly by targeting the EPO/JAK2 pathway and limiting the excessive proliferation of erythroid cells. Therefore, these drugs could be effective in reversing the splenomegaly and may offer an important approach to splenectomy. Additional studies are in progress to evaluate the outcome of combination therapy on the efficacy of transfusions.
[BM1]These aren't pharmacokinetic studies. Suggest just striking the word

Disclosures
Casu: Medgenics LLC: Research Funding; Ionis Pharmaceuticals: Research Funding; Merganser Biotech: Research Funding. Aghajan:Ionis Pharmaceuticals: Employment, Equity Ownership. Guo:Ionis Pharmaceuticals: Employment, Equity Ownership. Melchiori:Adaptimmune Ltd: Employment. Ramos:Novartis: Employment.