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β-Thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly, and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (ie, using hepcidin activators like Tmprss6-antisense oligonucleotides [ASOs]) or increase erythropoiesis (by erythropoietin [EPO] administration or modulating the ability of transferrin receptor 2 [Tfr2] to control red blood cell [RBC] synthesis). Targeting Tmprss6 messenger RNA by Tmprss6-ASO was proven to be effective in improving IE and splenomegaly by inducing iron restriction. However, we postulated that combinatorial strategies might be superior to single therapies. Here, we combined Tmprss6-ASO with EPO administration or removal of a single Tfr2 allele in the bone marrow of animals affected by β-thalassemia intermedia (Hbbth3/+). EPO administration alone or removal of a single Tfr2 allele increased hemoglobin levels and RBCs. However, EPO or Tfr2 single-allele deletion alone, respectively, exacerbated or did not improve splenomegaly in β-thalassemic mice. To overcome this issue, we postulated that some level of iron restriction (by targeting Tmprss6) would improve splenomegaly while preserving the beneficial effects on RBC production mediated by EPO or Tfr2 deletion. While administration of Tmprss6-ASO alone improved the anemia, the combination of Tmprss6-ASO + EPO or Tmprss6-ASO + Tfr2 single-allele deletion produced significantly higher hemoglobin levels and reduced splenomegaly. In conclusion, our results clearly indicate that these combinatorial approaches are superior to single treatments in ameliorating IE and anemia in β-thalassemia and could provide guidance to translate some of these approaches into viable therapies.

Hepcidin agonists are a new class of compounds that regulate blood iron levels, limit iron absorption, and could improve the treatment of hemochromatosis, β-thalassemia, polycythemia vera, and other disorders in which disrupted iron homeostasis causes or contributes to disease. Hepcidin agonists also have the potential to prevent severe complications of siderophilic infections in patients with iron overload or chronic liver disease. This review highlights the preclinical studies that support the development of hepcidin agonists for the treatment of these disorders.

Nonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), Xenopus oocytes expressing wild-type or K8R Fpn, and mature human red blood cells. We conclude that nonclassical FD is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders.

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.

Anemia of inflammation (AI) is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine interleukin 6 (IL6) and the hormone hepcidin (HAMP). IL6 is an inflammatory cytokine which also limits iron absorption by inducing HAMP, which promotes the degradation of the iron exporter ferroportin. We hypothesized that knocking down both HAMP and IL6 simultaneously will help us to understand if their mode of action in AI is uniquely limited to iron absorption and erythroid iron intake or if they also have independent roles. Henceforth, we generated IL6/HampKO (DKO) mice and, unexpectedly, observed that IL6KO mice showed the best recovery in bone marrow (BM) erythropoiesis (using flow cytometry analysis and looking at the absolute number of erythroid progenitors) after BA administration when compared to wild type (WT), HampKO and DKO mice. The best differences were observed at 14 days post BA administration. In contrast, the extramedullary erythropoiesis in the spleen was more pronounced in HampKO and DKO mice compared to WT and IL6KO animals, indicating that the mechanism impairing erythropoiesis in the BM did not affect erythroid progenitors in the spleen. These observations suggest that HAMP and IL6 proteins contribute independently to AI, with IL6 having some effect on the erythropoiesis in the BM independent from the IL6-HAMP axis leading to iron restriction. Furthermore, these observations raised the question why both HampKO and DKO mice showed reduced BM erythropoiesis compared to IL6KO animals. We investigated inflammatory cytokines and altered iron parameters as potential mediators of impaired erythropoiesis. We compared several inflammatory cytokines, including IL6, TNFa and INFg following BA administration: cytokine levels were elevated 6 hrs, reduced 48hrs after BA administration and moderately increased again two weeks later. Interestingly, among all the cytokines the levels of IL1b were significantly attenuated in IL6KO mice at day 14 compared to WT and HampKO animals. Moreover, transferrin saturation and NTBI levels were higher in HampKO and DKO animals compared to IL6KO mice. These observations strongly suggested that BM erythropoiesis is more sensitive to inflammatory insult in presence of an excess of iron, while extramedullary erythropoiesis is mildly affected and can eventually thrive under supra-physiological transferrin saturation levels. To test if increased iron affects BM erythropoiesis in presence of inflammation, we treated both WT and IL6KO mice with combination of iron dextran and BA. Both WT and IL6KO mice were treated with a combination of BA and iron at day 0 followed by alternate day of iron injections showed the poorest erythropoiesis in the BM and became rapidly sick, although the effect was significantly more pronounced in WT animals, as suggested by their survival curve. Since mycobacterium infections lead to NLPR3 inflammasome activation and Caspase1 upregulation (Marim et al. Semin Immunopathology 2017), we investigated how erythroid progenitors were affected. By flow cytometry analyses, we observed a significantly higher upregulation of the Caspase1 protein in WT and DKO mice compared to IL6KO animals. This was also reproduced by culturing WT or IL6KO BM progenitor erythroid cells in presence of mouse serum derived from WT or IL6KO mice treated with BA. Most importantly, IL6KO mice treated with BA and iron showed the highest levels of Caspase1 compared to only BA treated IL6KO mice, indicating that excess of iron abrogates the beneficial effect of IL6 deficiency on erythropoiesis under conditions of AI. Furthermore, using flow cytometry, we observed in WT mice treated with BA or BA and iron a significant increase in mitochondrial mass, which is an indicator of mitochondrial stress. The mitochondrial mass was reduced in IL6KO mice treated with BA, but again increased in IL6KO mice treated with BA and iron. We have also observed an increase of mitochondrial superoxide by confocal microscopy in WT mice compared to IL6KO mice treated with BA. Altogether, these data support a model where inflammation in presence of an excess of iron impairs BM erythropoiesis through mechanisms at least in part mediated by Caspase1 and mitochondrial dysfunction, while iron excess itself is sufficient to boost extramedullary erythropoiesis to compensate and sustain RBC production. Disclosures Vinchi: PharmaNutra: Research Funding; Vifor Pharma: Research Funding; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Rivella: Ionis Pharmaceuticals: Consultancy; Meira GTx: Consultancy.

Anemia of Inflammation (AI) is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, or cancer. Induced expression of hepcidin by pro-inflammatory cytokines results in iron-restricted anemia. In particular, abnormally elevated levels of the cytokine Tumor Necrosis Factor-α (TNFα) is a hallmark of AI, however its contribution to the pathophysiology of AI is not well understood. In this study, we investigated the role of TNFα in the development of anemia in a TNFα knockout (TNFαKO) mouse model of AI, which is induced with a single intraperitoneal (i.p.) injection of heat-killed Brucella Abortus (BA) (Kim et al. Blood). We hypothesized that TNFαKO mice would show a less severe form of AI compared to WT animals when challenged with BA. Our results showed that WT-BA mice developed severe anemia within 2 weeks, which was resolved by 8 weeks, whereas the TNFαKO mice developed leukocytosis and an irreversible macrocytic, hyperchromic anemia. Serum analysis at 8 weeks showed that erythropoietin (EPO) and iron parameters were elevated in TNFαKO compared to WT mice, which ruled out iron-restriction as the cause for the persistent anemia. However, serum cytokine measurements of TNFαKO mice at 4 weeks showed continual elevation of interleukin (IL)-12p40 and Interferon-γ (IFNγ) compared to WT-BA controls. We hypothesized that TNFα served an anti-inflammatory role that restrained prolonged inflammation after BA, and in its absence, pro-inflammatory macrophages continuously secreted IL-12p40 levels and induced the proliferation of IFNγ secreting cells. To test if concurrent loss of IFNγ would correct the inflammatory phenotype, we crossed TNFαKO with IFNγKO mice (DKO). Indeed, IFNγKO and DKO mice challenged with BA showed complete reversal of the anemic phenotype present in WT-BA and TNFαKO-BA at 2 weeks. Additionally, serum levels of IL-12p40 were normalized by 4 weeks in IFNγKO-BA and DKO-BA compared to TNFαKO-BA mice. Flow cytometry analysis of the bone marrow (BM) and spleen (SPL) at 8 weeks showed T-lymphocytes and macrophages were markedly expanded, whereas erythrocytes and B-lymphocytes were reduced in TNFαKO-BA mice. However, only modest differences in erythrocytes, macrophage, T- and B-lymphocyte in WT-BA, IFNγKO-BA and DKO-BA in the BM and SPL were detected. Additionally, we performed immunohistochemistry using an anti-CD3 antibody in SPL and livers of BA treated mice sacrificed at 8 weeks. We found complete disorganization of the white pulp in the SPL and infiltration of T-Lymphocytes in livers of TNFαKO-BA but not in WT-BA, IFNγKO-BA or DKO-BA animals. These results led us to question if lack of TNFα skewed the BM towards T-Lymphocytes. We investigated the hematopoietic stem cell (HSC) LSK, multipotent myeloid progenitors (MPP) LK, and common lymphoid progenitor (CLP) compartments by flow cytometry. Shockingly, the TNFαKO had over a 30% increase of Lin-cKit+Sca1+ LSKs, compared to TNFαKO controls, while the Lin-cKit+Sca1- LK population was drastically reduced. By contrast, the Lin-CD217-cKit+Sca1+ CLP population was expanded by more than 40% in the TNFαKO-BA compared to TNFαKO controls. Considering this data, it is difficult to disentangle the effects of TNFα in AI in vivo from its role in regulating upstream stem and progenitor cell differentiation using a germline KO strategy. However, our observations reinforce the link between iron homeostasis and HSC self-renewal and provide a new model to study inflammation associated bone marrow failure. The TNFα-BA mice displayed severe anemia, which seems to result from persistent IFNγ elevation. A recent study identified TNFα as a major pro-survival and pro-regeneration factor for HSCs (Yamashita & Passegue, Cell Stem Cell). Other studies have shown that IFNγ restricts HSCs self-renewal (Chen et al. Blood). Our results in the TNFαKO-BA treated mice suggest that TNFα preserves balanced progenitor output by countering the action of IFNγ at the HSC level. Ongoing work aims to understand the relationship between TNFα and IFNγ in regulating HSC quiescence, self-renewal, and overall pool size. Disclosures Paulson: Forma Therapeutics: Consultancy. Rivella: Meira GTx: Consultancy; Ionis Pharmaceuticals: Consultancy.

Transferrin, the major plasma iron-binding molecule, interacts with cell-surface receptors to deliver iron, modulates hepcidin expression, and regulates erythropoiesis. Transferrin binds and releases iron via either or both of 2 homologous lobes (N and C). To test the hypothesis that the specificity of iron occupancy in the N vs C lobe influences transferrin function, we generated mice with mutations to abrogate iron binding in either lobe (Tf(N-bl) or Tf(C-bl)). Mice homozygous for either mutation had hepatocellular iron loading and decreased liver hepcidin expression (relative to iron concentration), although to different magnitudes. Both mouse models demonstrated some aspects of iron-restricted erythropoiesis, including increased zinc protoporphyrin levels, decreased hemoglobin levels, and microcytosis. Moreover, the Tf(N-bl/N-bl) mice demonstrated the anticipated effect of iron restriction on red cell production (ie, no increase in red blood cell [RBC] count despite elevated erythropoietin levels), along with a poor response to exogenous erythropoietin. In contrast, the Tf(C-bl/C-bl) mice had elevated RBC counts and an exaggerated response to exogenous erythropoietin sufficient to ameliorate the anemia. Observations in heterozygous mice further support a role for relative N vs C lobe iron occupancy in transferrin-mediated regulation of iron homeostasis and erythropoiesis.

In β-thalassemia and polycythemia vera (PV), disordered erythropoiesis triggers severe pathophysiological manifestations. β-Thalassemia is characterized by ineffective erythropoiesis, reduced production of erythrocytes, anemia, and iron overload and PV by erythrocytosis and thrombosis. Minihepcidins are hepcidin agonists that have been previously shown to prevent iron overload in murine models of hemochromatosis and induce iron-restricted erythropoiesis at higher doses. Here, we show that in young Hbb(th3/+) mice, which serve as a model of untransfused β-thalassemia, minihepcidin ameliorates ineffective erythropoiesis, anemia, and iron overload. In older mice with untransfused β-thalassemia, minihepcidin improves erythropoiesis and does not alter the beneficial effect of the iron chelator deferiprone on iron overload. In PV mice that express the orthologous JAK2 mutation causing human PV, administration of minihepcidin significantly reduces splenomegaly and normalizes hematocrit levels. These studies indicate that drug-like minihepcidins have a potential as future therapeutics for untransfused β-thalassemia and PV.

Biogenesis of mammalian red blood cells requires nuclear expulsion by orthochromatic erythoblasts late in terminal differentiation (enucleation), but the mechanism is largely unexplained. Here, we employed high-resolution confocal microscopy to analyze nuclear morphology and F-actin rearrangements during the initiation, progression, and completion of mouse and human erythroblast enucleation in vivo. Mouse erythroblast nuclei acquire a dumbbell-shaped morphology during enucleation, whereas human bone marrow erythroblast nuclei unexpectedly retain their spherical morphology. These morphological differences are linked to differential expression of Lamin isoforms, with primary mouse erythroblasts expressing only Lamin B and primary human erythroblasts only Lamin A/C. We did not consistently identify a continuous F-actin ring at the cell surface constriction in mouse erythroblasts, nor at the membrane protein-sorting boundary in human erythroblasts, which do not have a constriction, arguing against a contractile ring-based nuclear expulsion mechanism. However, both mouse and human erythroblasts contain an F-actin structure at the rear of the translocating nucleus, enriched in tropomodulin 1 (Tmod1) and nonmuscle myosin IIB. We investigated Tmod1 function in mouse and human erythroblasts both in vivo and in vitro and found that absence of Tmod1 leads to enucleation defects in mouse fetal liver erythroblasts, and in CD34+ hematopoietic stem and progenitor cells, with increased F-actin in the structure at the rear of the nucleus. This novel structure, the "enucleosome," may mediate common cytoskeletal mechanisms underlying erythroblast enucleation, notwithstanding the morphological heterogeneity of enucleation across species.

The ongoing clinical trials, based on the use of lenti-globin vectors for beta-globinopathies, indicate that current vectors require high number of integrations (~3-4 copies per genome) in a pancellular fashion to make a patient transfusion independent, regardless of the genotype. This increased VCN requirement could increase the risk of genome toxicity, limiting the application of these vectors and preventing their use in a reduced myeloablative regimen. To overcome this limitation we designed new vectors, using modification or inclusion of a variety of regulatory genomic elements aiming to increase expression of the beta-globin gene. We performed in vitro and in vivo studies to compare the ability of these constructs to express the therapeutic gene with a low number of integrations and reduced chimerism. We screened new constructs using a CRISPR-Cas9 modified clonal cell line, HUDEPM#13, which derives from the previously described erythroid HUDEP-2 cell line (Kurita, 2013). Upon differentiation, HUDEPM#13 cells produce a hemoglobin variant (HbMut) that can be discriminated by liquid chromatography from the adult hemoglobin (HbA) produced by the transgene in the lentiviruses. Among our candidates we identified a construct, indicated as ALS20, that synthesizes HbA at high level at a single integration (VCN). Moreover, ALS20 produced significantly more HbA per copy than constructs currently utilized in clinical trials, which were reproduced based on the literature (Negre, 2015; Miccio, 2008; and Boulad, 2014) and designated CV-1, CV-2, and CV-3, respectively. In erythroblasts differentiated in vitro from patients with SCD ALS20 produces, on average, 21% HbA at VCN=1(P In a parallel potency assay, we tested ALS20 using beta0/beta0 thalassemic specimens, which represent the most severe phenotype to correct, due to complete absence of HbA production. Statistical analyses indicate that ALS20 produces, on average, 32.5% HbA at VCN=1 (P Our ongoing bone marrow transplantation studies, using a semi-myeloablative conditioning based on busulfan administration on the beta thalassemia Hbbth3/+mouse model, indicate that ALS20 is curative at VCN lower than 1. In fact, mice with an average of 0.8 copies per genome and 65% transgenic chimerism, present Hb levels of 13.6g/dL, reiterating the potential of this new lentivirus in vivo. In immunocompromised NSG mice, injection of human CD34+cells transduced with ALS20 did not trigger tumor formation or meaningful pathological changes. The safety of our vector has been further assessed using an in vitro immortalization assay on primary mouse BM cells, in which no clonal survival has been observed. Finally, genome integration analyses on human CD34+cells infected with ALS20 showed that none of the examined samples had clones that exceeded the 20% abundance threshold, in accordance with the expected level of safety for gene therapy-based approaches. In summary, we have identified a powerful new lentiviral vector with an enhanced ability to synthesize hemoglobin with a low number of integrations. ALS20's performance has been demonstrated in specimens from patients with hemoglobinopathies as well as in mice affected by thalassemia. Treatment of thalassemia and SCD with ALS20 could reduce the risk of cytotoxic events due to high levels of integration, and also lessen the intensity of the myeloablative regimen to correct anemia in patients. This, in addition with its confirmed safety features, makes this construct an outstanding candidate for clinical trial. Disclosures Kwiatkowski: Imara: Consultancy; Apopharma: Research Funding; Terumo: Research Funding; Novartis: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Celgene: Consultancy. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

We have previously designed and investigated novel allosteric effectors of hemoglobin (AEHs), as potential targeted treatment of sickle cell disease (SCD). In general, AEHs transiently covalently bind to hemoglobin (Hb), increase its affinity for O2, increasing the fraction of oxygenated sickle Hb (HbS), thus reducing HbS polymerization and countering red blood cell (RBC) sickling. In the current study, we designed a novel class of AEH molecules, incorporating a secondary mechanism of action (MOA), which is independent of Hb O2-affinity by interacting with the F-helix of deoxygenated HbS to directly destabilize its polymerization. Here, we report current results from our in-vitro and in-vivo studies with a representative AEH compound (PP-14). First, we assessed the anti-sickling properties in-vitro by incubating 0.5, 1, and 2 mM of PP-14 with whole blood suspensions from a subject with homozygous SCD (SS, hematocrit: 20%) under hypoxic conditions, with subsequent RBC sickling assessment by microscopy. Next, we subjected the samples to anoxia (100% N2 gas) to demonstrate the O2-affinity-independent antisickling mechanism. Subsequently, we tested residual samples for the degree of Hb modification (i.e., HbS-AEH adduct formation) and O2-affinity (p50) shifts. In a second experiment to further assess the secondary MOA, we subjected SS blood samples treated with various concentrations of PP-14 to hypoxia in the Hemox analyzer, which permitted us to obtain aliquot samples at defined pO2 values to establish pO2-dependent sickling. Additionally, we conducted in silico and in-vitro ADME studies to evaluate possible metabolic inhibition of a panel of CYP enzymes. Finally, we conducted a preliminary in-vivo PK/PD study in wild-type mice administered single doses of PP-14 via the oral (P.O.: 100-200 mg/kg) and intraperitoneal (I.P.: 75 mg/kg) routes. Serial blood samples were collected for up to 52 h after P.O., and up to 30 h after I.P. administration, and samples were assayed to quantify PP-14 concentrations. Residual blood samples were assayed for in-vivo Hb-AEH adduct formation, and the corresponding change in O2-affinity (Δp50, %). Our in-vitro studies demonstrated concentration-dependent inhibition of cell sickling of 25.5±11%, 44.4±3.8% and 90.8±1%, at 0.5, 1 and 2 mM of PP-14, respectively. HbS was modified correspondingly (38.9±9%, 55.7±4.9 %, and 92.4±9.8%), and was correlated linearly with the left-shift in OEC (Δp50 values of 11.3±5.1%, 29.0±13.2%, and 67.5±8.2%). Importantly, the antisickling effect was sustained under anoxic conditions (100% N2), strongly supporting the notion of a secondary, O2-affinity-independent MOA. Furthermore, we observed a dose-dependent delay in sickling, with initiation of sickling recorded at a pO2 level of 40 mmHg in absence of PP-14; and at 30 and 20 mmHg at 0.5, and 1 mM PP-14 concentrations, respectively. Complete inhibition of sickling was observed at 2 mM PP-14 through the lowest recorded pO2 level of 1.5 mmHg, a unique, effect not previously observed in any analogous AEH. In-vitro partitioning studies demonstrated that >90% of PP-14 partitioned into the RBC compartment when whole blood was incubated with 100-300 µM concentrations. Metabolic studies using pooled human liver microsomes (HLM) and isozyme-specific probe substrates suggested that up to 100 µM PP-14 did not inhibit CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 or CYP2B6. Wild-type mice after I.P. administration of PP-14 (75 mg/kg, n=5) showed peak concentrations in blood at 7 hrs (416.3±81.2 µM), with corresponding PD effects (Δp50 of 41.6±13.5%; modified Hb levels of 43.6±8.0%). Orally-treated mice had peak drug concentrations after 10-24 hrs, (~150 µM at 200 mg/kg, n= 2), with corresponding PD effects (Δp50 of 36.5±7.0%; modified Hb levels of 28.8±4.9%), which declined by 52 hrs. Overall, our data confirm that PP-14 is novel antisickling AEH with a secondary, O2-independent MOA in addition to the primary O2-dependent effect, as demonstrated by the inhibition of sickling under anoxic conditions. Additionally, PP-14 showed: excellent partitioning into the RBC compartment; acceptable in-silico ADME properties and in-vivo oral bioavailability; PD effects; and low liability for metabolic drug-drug interactions. Further studies to investigate formal detailed pharmacokinetic properties, and biological activity after single- or repeat-doses in a SCD mouse model are ongoing. Disclosures Safo: Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Pagare:Virginia Commonwealth University: Patents & Royalties. Ghatge:Virginia Commonwealth University: Patents & Royalties. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy. Hines:Functional Fluidics: Equity Ownership. Liu:Functional Fluidics: Employment. Zhang:Virginia Commonwealth University: Patents & Royalties. Venitz:Virginia Commonwealth University: Patents & Royalties. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent.

Erythropoiesis normally occurs in the bone marrow within the pelvis and femur, and both erythropoiesis and bone metabolism are susceptible to changes in iron homeostasis. Thus, hematopoietic and osteoid systems require coordination of iron metabolism during stress or ineffective erythropoiesis. Recently, a more extensive understanding of the crosstalk between iron metabolism and erythropoiesis revealed that a bone marrow secreted protein, erythroferrone (ERFE), is a negative regulator of hepcidin [Kautz Nat Gen 2014]. Hepcidin in turn is the main negative regulator of iron absorption and recycling [Nemeth Science 2004] and its suppression enables an increase in iron availability during stress erythropoiesis. Diseases of ineffective erythropoiesis, such as β-thalassemia, with chronic erythroid expansion, are associated with thinning of cortical bone, leading to decreased bone mineral density [Haidar Bone 2011; Vogiatzi Bone 2006]. Mechanisms underlying coordination of erythropoiesis and bone metabolism are incompletely understood. However, because ERFE functions to suppress hepcidin by sequestering BMPs [Arezes Blood 2018], and because BMPs are crucially important for bone metabolism [Hogan Genes Dev 1996], we hypothesize that ERFE may be involved in coordinating iron metabolism, erythropoiesis, and bone homeostasis. Lastly, osteoblast expression of TfR2 was found to inhibit bone formation by activating BMP-p38MAPK signaling and expression of the Wnt inhibitor Sclerostin, protein product of the SOST gene [Rauner Nat Med 2019]. We thus propose to explore the role of ERFE in disordered bone metabolism in β-thalassemia. In vitro data demonstrates that osteoblasts from wild type (WT) mice express ERFE and this expression is enhanced by BMP2/6/7 (Figure 1a and 1b). Furthermore, osteoblasts from ERFE-/- mice exhibit enhanced bone mineralization (6.8-fold increased von Kossa staining, measured by image J) (Figure 1c), increased expression of osteoblast-specific markers (e.g. osterix (OSX))(Figure 1d), and higher SOST expression (Figure 1e) relative to WT osteoblasts. We anticipate that if TfR2 is central to bone metabolism, ERFE-/- osteoblasts may exhibit a decrease in TfR2; our results demonstrate only a trend toward decreased TfR2 in ERFE-/- osteoblasts (Figure 1f). In addition, we propose that ERFE is a negative regulator of osteoblast activity, predicting that ERFE loss in th3/+ mice would enhance bone mineral density. To this end, we analyzed bone mineral density and histomorphometry in WT, ERFE-/-, th3/+, and th3/+ERFE-/- mice. Surprisingly, although no differences are evident between WT, ERFE-/-, and th3/+ femora, th3/+ERFE-/- mice exhibit a decrease in bone mineral density and bone volume / total volume (BV/TV) (Figure 2a-2b) with a trend toward enhanced femoral mineral apposition rate (Figure 2c) relative to th3/+ mice. These results indicate enhanced osteoblast activity without increased bone formation. Because bone mineralization is a composite of the relative osteoblast and osteoclast activity, we hypothesize that osteoclast activity is further enhanced in th3/+ ERFE-/- mice. TRAP staining demonstrates a significantly increased number of osteoclasts in ERFE-/- relative to WT as well as th3/+ ERFE-/- relative to th3/+ femora (Figure 2d). Our studies demonstrate that ERFE, like other members of the TNFα superfamily [Lu J Bone Miner Res 2011], negatively regulates OSX which is critical for osteoblast function (Figure 3a). Thus, suppression of ERFE results in more OSX (Figure 1d), enhanced mineralization (Figure 1c), and higher SOST expression (Figure 1e) which results in the secretion of Sclerostin (Figure 3b). Sclerostin both feeds back to suppress Wnt signaling to decrease osteoblast function and increases RANKL production to stimulate osteoclast differentiation (Figure 3b). Taken together, ERFE functions as a negative regulator of both osteoblast and especially osteoclast activity such that its loss leads to more osteoclast activity and results in decreased bone mineral density in β-thalassemia. These findings provide novel insights into the complex interplay between regulation of iron metabolism and bone homeostasis in diseases of dysregulated erythropoiesis, when ERFE expression is increased, and support the rationale to further explore the role of ERFE and TfR2 in this crosstalk in β-thalassemia. Disclosures Fleming: Protagonist: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Consultancy; Ultragenyx: Consultancy. Rivella:Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy; Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees.

The β thalassemia trait is associated with over 300 mutations in the β-globin gene that lead to reduced (β+ allele) or absent (β0 allele) synthesis of the β globin chain. A subset of these mutations affect the canonic splicing of the β globin mRNA. Such mutations activate aberrant splice sites, which lead to an altered splicing pathway and consequently affects protein synthesis. The (C>G) IVS-2-745 mutation is common in South Eastern Europe, Cyprus, Lebanon, India, Malaysia, and Indonesia. This mutation, located within intron 2 of the β-globin gene, creates an aberrant 5' splice site at nucleotide 745 of intron 2 and activates a cryptic 3' splice site within the same intron. Portions of the intronic sequence are incorrectly retained in the spliced mutant mRNA. The mutation results in a premature stop codon that prevents proper mRNA translation and causes a β‐globin deficiency, resulting in β‐thalassemia. The IVS-2-745 allele has the functional splice sites preserved, but produces a significantly reduced level of correctly spliced β-globin mRNA and results in only marginal synthesis of HbA. Therefore, the IVS-2-745 mutation in homozygosity leads to severe transfusion-dependent thalassemia major. Taking advantage of conserved canonical splice sites in defective β‐globin genes, such as IVS-2-745, recently developed approaches show that by targeting the aberrant splice sites it is possible to circumvent the aberrant splice site and restore the normal β-globin splicing pattern. We sought to use uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse aberrant splicing in the pre-mRNA for the IVS-2-745 mutation. Using these SSOs, we show effective aberrant-to-wild-type splice switching. This leads to an increase in adult hemoglobin (HbA) by up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a sickle/IVS-2-745-thalassemic genotype setting, we found that use of these oligos restored production of HbA and reduced HbS synthesis, which ultimately lessened cell sickling under hypoxic conditions. We confirmed increased WT β-globin expression in specimens treated with 2'-MOE-SSOs with semi- and quantitative methods (RT and Q-PCR), and further supported this evidence using a direct quantification method (ddPCR). Compared to treated specimens heterozygous for IVS-2-745 , homozygous specimens showed elevated WT HbA, reflecting the additive effect of targeting the aberrant splicing of both alleles as opposed to a single IVS-2-745 allele. In fact, while 2'-MOE-SSOs significantly reduced aberrant splicing, leading to a consequent 60% increase in HbA levels in specimens from patients with a β0/IVS-2-745 genotype, the same oligos produced a more robust effect in specimens with a homozygous IVS-2-745 genotype, resulting in an 80% increase in HbA levels. This level of increase could potentially be curative for patients with this particular genotype. Moreover, we compared the effect of 2'-MOE-SSOs treatment to a lentiviral vector carrying a WT β-globin gene. In this comparative assay, β0/IVS-2-745 cells treated with 2'-MOE-SSOs or the lentivector (with 1.13 copies integrated per genome) lead to a similar increase in HbA (50%). This suggests that the oligo-based technology is a competitive approach and a viable alternative to gene addition therapy to overcome anemia in IVS-2-745 β-thalassemia. In summary, 2'-MOE-SSOs are promising therapeutic tools for certain forms of β-thalassemia caused by aberrant splicing. Their ability to correct the underlying splicing defect offers a pharmacological treatment that is direct, specific, and accessible. In comparison, gene therapy approaches utilizing gene addition or editing are primarily available in advanced medical care environment resulting in an unfulfilled demand in regions where such conditions are not readily available. The restoration of target gene activity reported here suggests that this treatment strategy could be applicable to other forms of thalassemia resulting from mutations affecting splicing. This could have, with an effective method of delivery, potential clinical utility in helping patients reduce their transfusion dependence or even achieving transfusion independence. Disclosures Dong: Aruvant Sciences INC: Employment. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guo:Ionis Pharmaceutical, INC: Employment, Other: shareholders. Peralta:Ionis Pharmaceutical, Inc: Employment. Freier:Ionis Pharmaceuticals: Employment. Watt:Ionis Pharmaceuticals: Employment. Manwani:Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Cappellini:Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

Given that both Sickle Cell Disease (SCD) and beta-thalassemia (BT) are caused by mutations in the beta-globin gene, several lentivirus-based gene addition therapies have been developed. Results from recent trials indicate that the vectors used are safe; however, their efficacy inversely correlates with the severity of patients' hemoglobinopathy. The severity of the mutations (non-beta0 vs beta0) largely influences the outcome of the gene transfer. In fact, the data indicate that a relatively low number of integrations (in the range of 1-2 copies per genome) or vector copy number (VCN) is sufficient to cure patients whose mutations are categorized as non-beta0 and express relative high levels of endogenous hemoglobins (adult hemoglobin, HbA, and/or fetal hemoglobin, HbF). In contrast, the same level of VCN alleviates the transfusion regimen of patients with beta0 mutations, but it does not cure them. In addition, the lentiviruses currently used in clinical trials were engineered by different groups and to date no one has directly compared them side by side. In light of these limitations, here we describe a study that supplies a platform for rapid screening of lentiviral vectors expressing curative hemoglobin, based on the correlation between VCN and the increase in HbA levels. We also compared newly generated lentiviral vectors to vectors currently used in clinical trials. Our ultimate goal is to generate a new vector that can increase the yield of beta globin expressed per VCN in patients' cells. Using CRISPR-Cas9 we modified the erythroid Hudep-2 cell line (Kurita et al, 2013) to generate a clonal cell line, named Hudep #M13, which, upon differentiation, produces a hemoglobin variant (HbMut) that can be discriminated from that produced by the lentiviruses (HbA). In parallel, we immortalized erythroid progenitor cells isolated from a SCD donor (SCD #13), using the HPV16-E6/E7 expression system, which was introduced into the cells by lentiviral transduction. Using Hudep #M13, we compared the correlation between gene transfer and the production of HbA for 5 novel lentiviral vectors, indicated as ALS16-20. Our new vectors include the Ankyrin insulator in the 3' LTR (Breda et al 2012), the full beta-globin gene (including the native introns), the full 3' enhancer region, a combination of different portions of the beta-globin promoter, as well as modifications and inclusion of novel genomic elements from the locus control region (LCR). Our ALS- constructs were then compared to lentiviral vectors currently utilized in clinical trials. These constructs were reproduced based on information available from the literature (Negre et al, 2015; Miccio et al, 2008; and Boulad et al, 2014) and indicated as CV-1, CV-2, and CV-3, respectively. All these vectors contain the beta-globin gene with deletions in intron 2, different portions of the beta-globin promoter and/or 3' enhancer region, and different elements and sizes of the hypersensitive sites (HS) of the LCR. In Hudep #M13, linear regression analysis of the ratio of HbA to vector copy number (VCN) for each treatment, indicates that ALS17 and ALS20 yield roughly 40, 157 and 84% more HbA per copy than CV-1, CV-2 and CV-3, respectively. Similar increment in HbA% were confirmed on primary and immortalized (SCD #13) SCD erythroblasts derived CD34+ cells isolated from patients' blood. In these specimens, ALS20 maintained a 40% HbA increase compared to CV-1, when exploring a range of VCN from 0 to 3 with a linear mixed effects model. To assess the ability of these constructs to increase hemoglobin content in vivo, we are performing murine bone marrow transplants using thalassemic hematopoietic stem cells treated with CV1 and our two most powerful vectors. Based on most recently reported data (Thompson et al, 2018), 1 copy of the vector we reproduced as CV-1, makes on average 6.8g/dL of HbA. Hence, 1 copy of our best vector has the potential to make up to 9.5g/dL HbA. This could lead to a much greater clinical impact for patient with hemoglobinopathies, especially those who require higher Hb production to become transfusion independent, like patients with the beta0 genotype. The completion of these studies will provide not only a comparative analysis of our new best vector to those already in clinical trial, but also a way to predict how much therapeutic hemoglobin per vector copy number will be produced in the clinical setting. Disclosures Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Kwiatkowski:bluebird bio: Consultancy, Honoraria, Research Funding; Agios Pharmaceuticals: Consultancy, Research Funding; Novartis: Research Funding; Apopharma: Research Funding; Terumo: Research Funding. Rivella:Disc Medicine: Consultancy; Protagonist: Consultancy; Ionis: Consultancy; Meira GTX: Membership on an entity's Board of Directors or advisory committees.

Mutations in the HBB gene causes β-thalassemia (BT). Treatment for BT presents a major clinical challenge in the United States, as patients require chronic and expensive treatment for survival. A new drug in Phase III clinical trials, Luspatercept (ACE-536), has been shown to improve BT symptoms via an erythropoietin (EPO) -independent pathway. ACE-536 is a peptide drug identical to the extracellular domain of activin receptor IIB (ACVR2B). Upon administration, it competes with ACVR2B to bind members of the transforming growth factor (TGF) β superfamily. Growth differentiation factor 11 (GDF11) has been pinpointed as the primary target by which the trap ligand exerts its therapeutic efforts. Studies in murine models of BT using RAP-536 (the mouse analog of ACE-536), have suggested that Gdf11 is overexpressed in erythroblasts and that overexpression functions to inhibit erythroid differentiation. Interestingly, however, ACE-536 and RAP-536 have been shown to stimulate RBC synthesis in healthy humans and mice, where GDF11/Gdf11 overexpression has not been reported. Additionally, the expression data in mice has been questioned because of the unavailability of antibodies that can discriminate between Gdf11 and other TGF-β ligands. Due to the novelty of RAP-536 promoting erythropoiesis through an Epo-independent pathway and the lack of specific antibodies to distinguish between TGF-β ligands, we resorted to genetic tools to investigate the role of Gdf11 in erythropoiesis. For our study, we generated Hbb+/+Gdf11flox/floxand Hbbth3/+Gdf11flox/floxmice and crossed them with EpoRCre and VavCre transgenic lines, resulting in offspring harboring the Gdf11 deletion in erythroid cells and the complete hematopoietic compartment. If Gdf11 is secreted by erythroid cells, and it plays a role in inhibiting erythroid differentiation, then mice lacking Gdf11 in either erythroid cells or all hematopoietic cell lineages should show some increase in red blood cell (RBC) production, hemoglobin (Hb) and hematocrit (Hb). Furthermore, in Hbbth3/+mice, where Gdf11 has been proposed to be overexpressed, improvements in erythroid cell differentiation should be most apparent. Surprisingly, we did not detect any differences in RBC number, Hb or Hct levels of Gdf11 deficient Hbb+/+or Hbbth3/+mice compared to their Gdf11 containing controls. The discrepancy between our results and published data could be explained if Gdf11 is produced by non-hematopoietic tissues and indirectly influences erythropoiesis. Since Gdf11-/-are embryonic-lethal, we crossed Hbb+/+ Gdf11flox/floxand Hbbth3/+ Gdf11flox/flox mice with a tamoxifen (TAM) inducible Cre recombinase under the global Rosa26 promoter (RosaCre) to assess the effect of a pancellular deletion of Gdf11. No detectable differences were found in RBC, Hb or Hct levels of flthese animals after TAM treatment either acutely nor up to 5-6 months post deletion of Gdf11. Administration of RAP-536 significantly improved and increased hematopoietic parameters in the peripheral blood in all six models lacking Gdf11. In the RAP-536-treated Hbbth3/+models, amelioration of anemia was noted by a decrease in spleen size and improved ineffective erythropoiesis indicated by an increased hematological parameters and increased ratio of mature to immature erythroblasts in spleen analyzed by FACS. Therefore, lack of Gdf11 at the erythroid, hematopoietic and pancellular level did not prevent a response to the drug. Next, we investigated the effects of RAP-536 directly on erythroid cells. Since the drug causes increases in RBC and Hb of normal patients, we challenged CD34+ cells with RAP-536 at various concentrations. Results showed no increases in cell numbers, erythroid viability, hemoglobin content nor differentiation. Currently we are investigating the mRNA expression of activin receptors IIA and IIb along with TGF-β ligands in healthy and BT CD34+ cells as well as in erythroid specific progenitors of the Hbbth3/+ mouse model. Our findings suggest that Gdf11 is not the sole target of RAP-536, nor that Gdf11 is required to promote improvement of erythropoiesis. Most importantly, we show that in the absence of Gdf11, RAP-536 is effective at increasing hematological parameters in both Hbb+/+ and Hbbth3/+ mice. The results of this study demonstrate potential alternative target(s) for the action of RAP-536. Future work will focus on identifying the unknown targets of RAP-536. Disclosures Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Martinez:Acceleron Pharma: Employment. Suragani:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; Protagonist: Consultancy; Disc Medicine: Consultancy; MeiraGTx: Other: SAB.

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.

Allosteric effectors of hemoglobin (AEH) remain promising as a viable therapeutic approach for the management of sickle cell disease (SCD). AEH bind to hemoglobin (Hb) in a transiently covalently manner, increasing Hb affinity for oxygen (O2), with concomitant inhibition of polymerization of deoxygenated sickle Hb (HbS) and erythrocyte sickling. As part of our ongoing efforts to surmount well-known AEH druggability challenges, we have designed and synthesized VZHE-039, a novel substituted benzaldehyde with potent in-vitro anti-polymerization activity, as well as more favorable in-vivopharmacokinetic and pharmacodynamic (PK/PD) properties. Here, we report the results of our early findings. The anti-sickling properties of VZHE-039 were tested in-vitro by incubating 0.5, 1, and 2 mM concentrations with blood suspensions from a subject with homozygous SCD (hematocrit: 20%) under hypoxic conditions (4% O2/96% N2) at 37°C for 2 h. At conclusion, aliquots were drawn into a fixative (2% glutaraldehyde solution), and sickling was assessed by microscopy. Aliquot samples were also subjected to cation-exchange HPLC analyses to measure the degree of Hb modification (Hb adduct formation), as well as standard O2 equilibrium curves (OEC) to assess p50 shifts. Subsequently, we conducted in-vivo PK/PD studies in wild-type mice that received single doses of VZHE-039 via the intravenous (I.V.: 25 and 50 mg/kg), intraperitoneal (I.P.: 100-150 mg/kg) and oral (P.O.: 100-200 mg/kg) routes. Serial blood samples were collected up to 24 h after I.V. and P.O., and up to 6 h after I.P. administration, respectively; aliquots were hemolyzed, de-proteinized and subjected to reverse-phase HPLC-UV assay to quantify VZH-039 blood concentrations. Residual blood samples were also hemolyzed, and clarified lysates were assayed for in-vivoHb adduct formation, and the corresponding change in Hb oxygen affinity (Δp50, %). The results of our in-vitrostudies demonstrated a concentration-dependent inhibition of SS cell sickling: 39±1.4%, 68.6±4.9%, and 89.9±5.2%, at 0.5, 1 and 2 mM of VZHE-039, respectively. HbS was modified correspondingly (46±7.4%, 83.6±6.4%, and 96.4±6.2%), and this adduct formation was correlated linearly with the left shift in OEC (Δp50 values of 34.3±8.7%, 63.6±2.4%, and 76.6±2.5%). Noncompartmental PK analysis showed that, after I.V. administration, systemic PK was dose-independent with a CLtot of 0.5 mL/min/kg, resulting in a terminal half-life of 9 h. After I.P. administration, AUCtrap increased supra-proportionally with dose; the limited sampling schedule suggested an I.P. bioavailability of >20%. After P.O. administration, AUCtrap increased supra-proportionally with dose as well due to an increase in oral bioavailability to ~ 10%, with peak blood concentrations up to ~ 0.25 mM; the terminal half-lives were prolonged (relative to I.V.) to 14 h. The PD effect-time course of change in %Hb adduct paralleled (and was reasonably linearly related to) blood concentrations for all routes. Baseline-corrected peak PD effects after I.V. administration were 15.7% and 31.9%, after 25 and 50 mg/kg respectively; after P.O. administration of 200 mg/kg, the peak change was 17.6%. Overall, VZHE-039 exhibited a low (metabolic) clearance, presumably reflecting high-affinity and sustained Hb (and possibly albumin) binding, leading to the observed extended terminal half-life and duration of action. While its oral bioavailability is low (likely due to limited GI solubility) and there is evidence of saturable first-pass effects and possibly enterohepatic recycling, oral in-vivo exposures were associated with PD effects that were consistent with the results from the in-vitrostudies. In conclusion, the results of our current studies establish VZHE-039 as a novel, potent anti-sickling agent, confirm its proposed mechanism of action in-vitro and in-vivo, and suggest that therapeutically-relevant blood concentrations may be achieved after daily oral doses. Therefore, VZHE-039 is our new lead drug candidate that may require further modifications and studies to (a) improve its GI solubility and oral bioavailability; (b) elucidate atomic level structural interactions and kinetics of its Hb binding; and (c) formally investigate its biological activity after repeat-doses in a SCD mouse model. Disclosures No relevant conflicts of interest to declare.

Both β-thalassemia intermedia and major are characterized by formation of hemichromes in erythroid cells, impairing their survival and the lifespan of red blood cells (RBC). Minihepcidins (MH) are novel compounds that function as hepcidin agonists and reduce iron absorption and transferrin saturation. Hbbth3/+ mice show features of β-thalassemia intermedia, such as ineffective erythropoiesis (IE), anemia and reduced hepcidin synthesis, but do not require blood transfusion for survival (non-transfusion dependent thalassemia or NTDT). As we have previously shown, the administration of MH in these animals decreased transferrin saturation, erythroid iron intake, heme synthesis and hemichrome formation, with a significant beneficial effect on RBC quality, lifespan and anemia (Casu et al, Blood 2016). In order to test if this approach could also benefit animals affected by β-thalassemia major we focused on generating a model that exhibited a low production of RBCs, severe anemia and a blood transfusion requirement for survival, as in patients affected by transfusion dependent thalassemia or TDT. We have previously shown that engraftment of Hbbth3/th3fetal liver cells (FLCs) into normal mice leads to a very severe anemia that requires blood transfusion for survival (Gardenghi et al, Blood 2007). However, Hbbth3/th3FLCs do not contain any adult or fetal-globin genes and are unable to make hemoglobin in the transplanted animals, in contrast to human β-thalassemia. Therefore, animals cannot benefit from therapies that decrease hemichrome formation and target IE such as MH. To overcome this limitation, we crossed Hbbth3/+mice with additional models of NTDT, indicated as Hbbth1/th1and Hbbth2/+. These mice harbor alternative mutations so that the synthesis of the mouse b-globin genes is different in each model. Hbbth1/th2and Hbbth1/th3pups were alive at birth, but unable to survive more than a couple of days even with the support of blood transfusion. However, recipient transgenic animals expressing GFP and engrafted with Hbbth1/th2andHbbth1/th3FLCs showed the desired phenotype 3 months post-transplant including production of GFP- RBCs (with less than 2% of host GFP+ RBC) and a different degree of anemia, respectively 5.6±0.5 g/dL and 3.1±1.5 g/dL. In the long term these animals require blood transfusion for survival. Therefore these models are useful to test drugs that have the potential to modify erythropoiesis and RBC production. Ten weeks following engraftment with Hbbth1/th2FLCs, mice were treated for six weeks with two different doses of MH (5.25 mg/kg and 2.625 mg/kg administered every other day) in absence of blood transfusion. Animals treated with vehicleshowed severe ineffective erythropoiesis and worsening anemia over 6 weeks (from 5.6±0.5 g/dL on D0 to 5.0±0.7 g/dL on D42 of treatment). In contrast, animals treated with MH showed reversal of anemia at 3 weeks (6.6±0.3 g/dL and 6.1±0.6 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 5.3±0.9 g/dL in controls), while at 6 weeks the differences were reduced compared to vehicle treated mice (6.0±0.4 g/dL and 5.7±0.5 g/dL in the 5.25 mg/kg and 2.625 mg/kg group, respectively, compared to 4.9±0.7 g/dL in controls). The RBC number followed the same trend. Furthermore, the RBC morphology of animals treated with MH was improved compared to control animals. At 6 weeks, splenomegaly was also improved in the treatment groups (13.8±2.7 mg and 16.9±2.7 mg respectively in the 5.25 mg/Kg and 2.625 mg/Kg group compared to 26.9±3.5 mg in controls). Comparing the data at 3 versus 6 weeks, we speculate that, while the MH has a positive effect on RBC quality and production, this is insufficient, in the long term, to prevent the severe splenomegaly and the consequent entrapment of the RBC, which exacerbates the anemia over time. However, we hypothesized that administration of MH could have longer lasting beneficial effects in presence of blood transfusion, which would limit the splenomegaly. Presently, we are testing this hypothesis using both the Hbbth1/th2and Hbbth1/th3models. Complete characterization of these models and their parameters (CBC, erythropoiesis, iron metabolism and organ iron content) is in progress. In conclusion, these models can be utilized to characterize severe thalassemia phenotypes and new drugs that have the potential to ameliorate IE and improve RBCs generation. Disclosures MacDonald: Merganser: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

β-Thalassemia intermedia or non-transfusion dependent thalassemia (NTDT) is a genetic blood disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly and systemic iron overload mediated by down-regulation of hepcidin. Using Hbbth3/+animals affected by NTDT, we have shown that iron restriction improves anemia and reduces splenomegaly by reducing the formation of hemichromes and improving the lifespan of red blood cells (RBC; Gardenghi et al, JCI, 2010; Guo et al, JCI, 2013; Casu et all Blood 2016). These findings emphasize the strong correlation between erythropoiesis, RBC synthesis and iron metabolism. In theory, agents that can stimulate erythropoiesis (such as erythropoietin or EPO) could improve anemia by increasing the production of RBC and, under conditions of reduced iron absorption, correct the iron overload by consuming the excess of iron stored in organs. Here we used a new ex vivo technology called TARGT (Transduced Autologous Restorative Gene Therapy) for prolonged production and secretion of therapeutic agents. Primary dermal fibroblasts transduced with helper dependent adenovirus expressing mouse Epo were embedded in Matrigel and implanted in the dorsal area of 2-3 months old Hbbth3/+and wild-type (WT) C57BL/6 mice. Three groups were generated for each genotype using different amounts of genetically modified fibroblasts (10e5, 5x10e5 and 1x10e6 transduced cells), while one group received no transduced fibroblasts as control. Complete blood count and serum harvest for Epo levels measurement were performed one week after implantation and then every ten days until the end of the experiment, at six weeks. In WT mice, already one week after fibroblasts implantation, hemoglobin (Hb) levels raised 2/3 g/dL, showing erythrocytosis. At the end of the treatment animals showed significantly increased Hb levels (18.5±1.7 g/dL, 19.4±0.5 g/dL and 19.8±0.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 13.5±0.8 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 27% to 39%. In Hbbth3/+mice Hb levels rose about 3 g/dl already one week after fibroblasts implantation and remained elevated until the end experiment (8.7±0.3 g/dL, 10.7±1.0 g/dL and 10.6±1.8 g/dL in the groups implanted with 10e5, 5x10e5 and 1x10e6 fibroblasts, respectively, compared to 7.7±0.7 g/dL in controls). RBC numbers and HTC follow the same trend, showing increases in the range of 7% to 27%. At the end of the treatment, serum Epo levels were markedly increased at all doses both in Hbbth3/+and WT animals (more than 50% and up to 90%) when compared with their respective controls. However, as expected, stimulation of erythropoiesis led to worsening of splenomegaly and suppression of hepcidin, likely preventing the beneficial effect of erythroid-mediated consumption of stored iron in Hbbth3/+animals. Here we postulated that, in presence of agents that increase erythropoiesis such as Epo, some level of iron restriction is required to improve the anemia, prevent exacerbation of the splenomegaly and, concurrently, decreased iron overload. As a proof of principle of the ability of this technology to improve anemia and decrease iron overload, TARGTEPO was combined with low iron diet or iron chelation. We expect that decreased iron intake will improve the quality of the RBC, while the larger number of RBC produced should utilize more stored iron, reversing the iron overload in Hbbth3/+animals. Preliminary results at three weeks from the beginning of the experiment indicated that in WT implanted-animals the combination of TARGTEPO with low iron diet significantly reduced Hb levels (-40%), HTC (-42%), RBC number (-38%) and reticulocytes (-80%) when compared to animals overexpressing Epo and receiving normal iron diet or iron chelation. This indicates that in WT animals, after 3 weeks, the stored iron is insufficient to support the increased erythropoiesis. In contrast, in Hbbth3/+animals this drop in Hb was not seen, suggesting that this effect is delayed due to the highest level of stored iron. Complete characterization of these models and their parameters is in progress. These preliminary results suggest that the TARGTplatformcould be utilized, in combination with drugs that limit iron intake, to improve anemia and decrease iron overload in NTDT. Additional experiments are in progress to test this hypothesis. Disclosures Casu: Ionis Pharmaceuticals: Research Funding; Medgenics LLC: Research Funding; Merganser Biotech: Research Funding. Neil:Medgenics: Employment. Miari:Medgenics: Employment. Shapir:Medgenics: Employment.

The current treatment of β-thalassemia only partially mitigates the phenotype of the disease, making the need for novel therapeutic agents imperative. The investigational drug Luspatercept (ACE-536) is a ligand trap that contains the modified extracellular domain of activin receptor IIB (ACVR2B) and induces red blood cell production in an erythropoietin independent fashion. ACE-536 binds with high affinity to members of the transforming growth factor (TGF) β superfamily and therefore alters activin/GDF signaling through the intracellular SMAD complex. In search of the specific ligands, recent studies in a mouse model of β thalassemia intermedia identified growth differentiation factor 11 (GDF11) as a possible target of the drug. It has been proposed that GDF11 is overexpressed in thalassemic erythroblasts and inhibits terminal erythroid maturation via SMAD complex phosphorylation. A negative role of GDF11 in erythropoiesis has been postulated also in myelodysplastic syndrome (MDS). We recapitulate, by a genetic approach, the phenotype of thalassemic and MDS mice treated with RAP-536, the murine counterpart of ACE-536. We generated and analyzed animals with GDF11 deletion in erythroid cells (Hbbth3/+ Gdf11fl/flEpoR-Cre and NUP98-HOXD13 Gdf11fl/flEpoR-Cre) and in all hematopoietic tissues (Hbbth3/+Gdf11fl/flVav-Cre and NUP98-HOXD13 Gdf11fl/flVav-Cre). We did not detect any changes in red blood cell number, reticulocyte count, hemoglobin or hematocrit levels compared to thalassemic or MDS mice in absence of the floxed gene. Focusing on thalassemic mice, administration of RAP-536 significantly improved the anemia and other hematopoietic parameters in the peripheral blood, decreased spleen size and ameliorated ineffective erythropoiesis as indicated by an increased ratio of mature to immature splenic erythroblasts analyzed by flow cytometry. Similar endpoints were seen comparing floxed and non-floxed animals treated with RAP-536. Therefore, the lack of GDF11 in erythroid or bone marrow derived cells did not prevent a response to the drug. To assess the effect of a pancellular absence of GDF11, we are currently in process of generating a model of β-thalassemia with total Gdf11 deletion. To investigate the possible effects of RAP-536, we treated erythroid cells derived from normal or thalassemic patients with the drug. Erythroid cell viability, number, differentiation and cell cycle remained unvaried. Of note, we did not detect significant expression of GDF11/Gdf11 in human and mouse erythroid cells. To investigate the effect of an exogenous source of GDF11 production on erythroid cells, we treated murine erythroleukemia (MEL) cells with recombinant GDF11. Upon treatment we observed phosphorylation of the SMAD2/3 complex by western blot. This effect was hindered by co-treatment of GDF11 with RAP-536. We further assessed the effects of RAP-536 on the mouse erythroid transcriptome, using RNA seq analysis in splenic erythroid populations. After administration of a single dose of RAP-536, thalassemic mice were euthanized. We used flow cytometry to identify possible alterations on differentiating erythroid populations in the spleen. Notably, between 60 and 72h we observed reduced numbers of basophilic and increased numbers of polychromatophilic erythroblasts. Analysis at 60h revealed that signal transducer and activator of transcription 5a (Stat5a), cyclin-dependent kinase 6 (Cdk6) and other cell cycle-related and metabolic genes were increased in the basophilic erythroid progenitors treated with RAP-536. This effect suggests that RAP-536 promotes proliferation and/or differentiation of erythroblasts. Thus, our genetic analyses suggest that lack of GDF11 may be required but not sufficient to improve erythropoiesis. Furthermore, erythroid cells do not produce but can respond to exogenous GDF11, likely synthesized by non-erythroid cells and under conditions of ineffective erythropoiesis. Even though we detected in vitro effects, these may not mimic physiological effects, as experimental conditions may not correlate with GDF11 concentrations in vivo. As additional ligands have been proposed (such as GDF8 and Activin B), our future studies will focus on the potential role of these molecules. Altogether, these results reveal a potential alternative target of action for ACE-536 and may lead to the discovery of new therapeutic molecules. Disclosures Suragani: Acceleron Pharma: Employment, Equity Ownership. Kumar:Acceleron Pharma: Employment, Equity Ownership.

Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H1-mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previous mouse and human genetic data from multiple groups suggest that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with beta-thalassemia. We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment results in dose-dependent hepcidin upregulation, which leads to dramatic reductions in serum iron and transferrin saturation. This in turn ameliorated the anemia and iron overload phenotypes in a mouse model of beta-thalassemia (th3/+ mice), which recapitulates beta-thalassemia intermedia in humans (Guo et al. J Clin Invest. 2013; 123(4):1531-41). Moreover, this ASO can be combined efficiently with iron chelators for the management of iron overload and anemia in non-transfusion-dependent thalassemia (Casu et al. Haematologica. 2016; 101(1):e8-e11). TMPRSS6 is predominantly expressed in hepatocytes, for which we have developed a targeted delivery approach with triantennary N-acetyl galactosamine (GalNAc). With GalNAc-conjugated ASOs, a ~10-fold improvement in potency is observed for many liver targets (Prakash et al. Nucleic Acids Res. 2014; 42(13):8796-807). In order to characterize GalNAc-conjugated TMPRSS6 ASO, we treated normal mice with both parent ASO and its conjugated counterpart. As expected, the conjugated ASO demonstrated a ~10-fold improvement in ED50 (25 mg/kg/week versus 2.5 mg/kg/week for parent and conjugated ASOs, respectively). Next, we treated th3/+ mice for six weeks with 10 mg/kg/week GalNAc-conjugated TMPRSS6 ASO or a control ASO of the same chemistry. Compared to the control ASO treatment group, we observed >95% reduction of TMPRSS6 mRNA levels and >3-fold up-regulation of hepcidin mRNA levels in the liver. This resulted in a ~40% reduction in serum iron and ~50% reduction in transferrin saturation. In addition, anemia phenotypes were significantly improved as shown by a significant increase in hemoglobin and red blood cells (from 7.0 g/dL to 8.9 g/dL and from 5.8 to 7.5x10^6 cells/µl in the control ASO treatment group and in the TMPRSS6 ASO treatment group, respectively). Furthermore, there was an approximately 50% reduction in spleen weight. Improved erythroid maturation was indicated by a significant reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. A GalNAc-conjugated human TMPRSS6 clinical candidate was identified. Similar to mouse TMPRSS6, GalNAc-conjugated ASO demonstrated superior potency in human primary hepatocyte culture, in human TMPRSS6 transgenic mice and in cynomolgus monkey. Collectively, our data demonstrate that GalNAc-conjugated TMPRSS6 ASO could be an effective therapeutic for patients with beta-thalassemia and related disorders. A Phase 1 clinical trial is planned to initiate in 2017. Disclosures Aghajan: Ionis Pharmaceuticals: Employment, Equity Ownership. Booten:Ionis Pharmaceuticals: Employment, Equity Ownership. Monia:Ionis Pharmaceuticals: Employment, Equity Ownership. Guo:Ionis Pharmaceuticals: Employment, Equity Ownership.

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β−thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interrupting the interaction between macrophages and erythroblasts (MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. We speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta−1 (Itgβ1) and also intracellular molecules such as focal adhesion kinase (Fak1), Talin−1 and Sharpin might play a role in stress erythropoiesis. Furthermore, there is increased interaction between Itgb1 and Fak1 in erythroblasts co−cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgβ1 or Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgβ1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. Enucleation of erythroblasts was accompanied by a marked loss of Itgβ1 expression, indicating that Itgβ1 may be involved in erythroblast enucleation and differentiation. We crossed Hbbth3/+ mice with animals in which Itgβ1 or Fak1 were floxed and carrying an inducible Cre−recombinase (Mx1−Cre). From these animals, we investigated three different models; two obtained from breeding (Hbbth3/+−Itgβ1fl/fl−Mx1−Cre and Hbbth3/+−Fak1fl/fl−Mx1−Cre) and one by bone marrow transplant (BMT) of hematopoietic stem cells (HSCs) of Hbbth3/+−Itgβ1fl/fl −Mx1−Cre animals into wt mice to generate thalassemic animals that expressed the floxed Itgβ1 only in hematopoietic cells. After serial administration of Poly(I)−Poly(C) [poly(I:C)] the animals were analyzed for their erythropoiesis in the bone marrow and spleen. All the animals treated with poly(I:C) showed populations of Itgβ1 or Fak1 negative cells in the bone marrow and spleen. This indicated that all the HSCs were successfully depleted of the Itgβ1 or Fak1 gene. Interestingly, the spleen weight of all the treated animals was reduced, on average, 50% compared to untreated thalassemic mice. Similar results were seen also in Hbbth3/+−Itgβ1fl/fl−Mx1−Cre animals generated through BMT. Therefore, Itgβ1 and Fak1 might contribute to the pathophysiology of thalassemia and their removal might result in reduced stress erythropoiesis, erythroid proliferation and, as a consequence, amelioration of splenomegaly. Iron analysis and quantification of Erythroferrone (ERFE) are in progress to evaluate the impact of depleting Itgβ1 and Fak1 on these mechanisms. We are now in the process of identifying compounds that target MEI and, in particular, Itgβ1. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. Disclosures Feldman: Bayer ealthCare Phamaceuticals Inc.: Employment. Rivella:isis Pharmaceuticals: Consultancy; Merganser Biotech: Other: Stock options; Novartis Pharmaceuticals: Consultancy; Medgenics Pharmaceuticals: Consultancy; Bayer Healthcare: Consultancy, Research Funding.

Antisense technology is a powerful drug discovery approach for identifying oligonucleotide analogs that can specifically modify RNA expression through multiple mechanisms including RNase H-mediated degradation of RNA and modulation of RNA splicing. We have successfully applied this technology towards targeting a number of transcripts in a wide-range of therapeutic areas. Beta-thalassemia, one of the most common genetic disorders worldwide, is characterized by reductions in beta-globin and ineffective erythropoiesis. This in turn leads to suppression of hepcidin, a peptide hormone that serves as the master regulator of iron homeostasis. Inappropriately low levels of hepcidin trigger increased dietary iron absorption resulting in iron overload, which is the major cause of morbidity and mortality in beta-thalassemia patients. TMPRSS6 is a transmembrane serine protease mainly produced by hepatocytes that negatively regulates hepcidin expression. Previous mouse and human genetic data from multiple groups suggest that lowering TMPRSS6 expression could up-regulate hepcidin and ameliorate many of the disease symptoms associated with β-thalassemia. We identified potent antisense oligonucleotides (ASOs) against mouse TMPRSS6. Downregulation of TMPRSS6 with ASO treatment resulted in dose-dependent hepcidin upregulation and reduction in serum iron and transferrin saturation in normal mice. In a mouse model of beta-thalassemia (th3/+ mice), which effectively recapitulates beta-thalassemia intermedia in humans, TMPRSS6 reduction resulted in induction of hepcidin and dramatic reductions of serum transferrin saturation (from 55-63% in control group down to 20-26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40-50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (~30-40%), hemoglobin (~2 g/dl), and hematocrit (~20%); reduction of splenomegaly; decreases in serum erythropoietin levels; improved erythroid maturation as indicated by a strong reduction in reticulocyte number and a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Encouraged by the strong pharmacology of TMPRSS6 suppression in animal models, we initiated an effort to identify a human TMPRSS6 clinical candidate with a liver-targeted delivery strategy. Over 2000 ASOs were screened in cell lines and the most active compounds were evaluated in rodent tolerability studies. A human TMPRSS6 transgenic mouse model was established enabling evaluation of ASO activity toward human TMPRSS6 transcript in vivo. Furthermore, lead compounds were tested in a 3-month study in normal monkeys. With repeated dosing, TMPRSS6 mRNA levels in monkey liver were reduced by >90%, accompanied by time-dependent reductions of serum iron (from ~100-120ug/dl to Disclosures Guo: Isis Pharmaceuticals: Employment, Other: Shareholder. Aghajan:Isis Pharmaceuticals: Employment, Other: Shareholder. Booten:Isis Pharmaceuticals: Employment, Other: Shareholder. Monia:Isis Pharmaceuticals: Employment, Other: Shareholder.

Macrophages have been implicated in erythropoiesis historically as a mediator of iron recycling and a key component of the erythroblastic island-consisting of a central macrophage surrounded by erythroid cells in different stages of differentiation. Recently we and others have shown that macrophages contribute to stress erythropoiesis, and such contributions extend beyond the known macrophage function of iron recycling. This finding necessitates the investigation of processes within a macrophage itself that might facilitate stress erythropoiesis and the characterization of macrophage transcriptome signatures associated with the same. Macrophages in the bone marrow have been identified previously as CD115LowGR1LowF4/80High cells. Extending this to the spleen, we sorted and sequenced singlet macrophages in the bone marrow and spleen of two mice models of stress erythropoiesis: acute stress induced by retro orbital phlebotomy and chronic stress by bone marrow transplanted Polycythemia Vera. We found that splenic macrophages show more significant changes upon induction of stress (both acute and chronic) in comparison of macrophages in the bone marrow. The mean of normalized count of each gene when plotted against fold change (Figure 1) show that the acute and chronically stressed splenic macrophages differ significantly over non stressed macrophages but not over each other. Fig 1: Acute over non stressed Chronic over non stressed Acute over non stressed Fig 1:. Acute over non stressed Chronic over non stressed Acute over non stressed We further found that stressed splenic macrophages have a characteristic gene expression profile associated with erythropoietic stress, common to both models of induced and chronic stress. There were a total of 2602 genes showing significant changes in expression upon induction of acute stress and a total of 2196 genes showing significant changes in expression upon chronic stress. Out of these, 1529 genes common to both acute and chronically stressed splenic macrophages showed an expression profile common to both acute and induced stress. (Fig 2) Fig 2. Fig 2. We used the open bioinformatics resource DAVID and Ingenuity Pathway Analysis (IPA) to look at biological processes encompassing our significantly up regulated candidates (FDR 10%), upstream analysis of our candidates and further network analysis. Some biological processes of highest significance were cellular growth and proliferation, hematological system development and function, inflammatory response, response to infection, cellular adhesion, cellular movement and immune cell trafficking. We validated some of our key candidates under these biological processes by flow cytometry, such as the iron exporter ferroportin, the receptor for the anti inflammatory cytokine, IL-10 and the cellular adhesion molecule ICAM-1. Some transcription factors significantly upregulated were Hes1, Nfkb1A, RelB, Bcl3 to mention a few. Upstream analysis using IPA predicted activation of transcription factors such as Notch1, Jun, RelA etc. Along with further validation of our key candidates, we are currently investigating macrophage gene expression in another model of chronic stress, beta thalassemia and how this profile associated with the physiological condition of erythropoietic stress compares to those under in vivo models of classical and alternative macrophage activation. Disclosures Casu: Merganser Biotech: Research Funding; Isis Pharmaceuticals Inc.: Research Funding. Rivella:Merganser Biotech: Consultancy, Research Funding, Stock options, Stock options Other; Bayer: Consultancy, Research Funding; Isis Pharmaceuticals. Inc.: Consultancy, Research Funding.

Polycythemia Vera (PV) is a myeloproliferative disorder in which the rate of erythropoiesis is massively increased. Most of the health concerns associated with PV are caused by increased red cell production, leading to increased risk of thrombosis due to red cell sludging in small vessels. PV is caused by JAK2 tyrosine kinase mutations that cause unregulated activation of the erythropoietin signaling pathway and massive overproduction of red cells. PV shows characteristic features of stress erythropoiesis, which is highly dependent on iron absorption and erythroid iron intake. The mainstay of therapy is therapeutic phlebotomy, the goal of which is to reduce the hematocrit (Hct) to 45% or less to minimize the risk of thrombosis. Since phlebotomy has no effect on bone marrow production of red cells the effect is transient and dependent on the development of an iron-deficient state for control of Hct. Poor compliance with phlebotomy and the intermittent nature of the intervention frequently leads to treatment failure. Iron deficiency or erythroid iron restriction may be effective in the treatment of PV by causing inhibition of erythropoietin signaling downstream of JAK2, mediated by inhibition of aconitase. We have previously shown that increased hepcidin level reduces transferrin saturation and erythroid iron intake and has a beneficial effect on the number of erythroid progenitors in a mouse model of Thalassemia Intermedia. Hepcidin mimetic peptides (minihepcidins (MH)) which replicate the biological effects of hepcidin may be beneficial in the treatment of PV by limiting iron availability to the developing erythrocyte, thereby overriding JAK2 stimulated erythropoiesis and reducing erythrocyte production. A mouse model of PV was generated by crossing a floxed heterozygous Jak2V617Ffl/fl (V617F is the most common mutation causing human PV) mouse with a mouse hemizygous for the Vav-iCre transgene expressed in hematopoietic cells. We generated a cohort of mice with the disease by engrafting Jak2V617Ffl/fl-Vav-iCre bone marrow into wild type (WT) mice. M009, which is a potent MH that decreases ferroportin expression in vitro and serum iron in vivo was administered subcutaneously at a supramaximal dose of 400 µg/mouse twice weekly. A high dose of M009 was used to ensure that a significant level of iron restriction was present throughout the study. In PK/PD studies in the rat equivalent doses of M009 had previously been shown to reduce serum iron by >80% for 48-72h post-dose. MH treatment for six weeks or vehicle control was initiated 2 months after bone marrow transplant, when the phenotype was fully established. Hct was 62.5 ± 4.3% (mean ± SD) in controls (n=5) at baseline, rising to 85.0 ± 4.0% at the end of study. In contrast in M009 treated animals Hct dropped from 69.3 ± 4.3% (n=6) to 38.8 ± 7.4%. Interim evaluation at 3 weeks indicated that the effect of M009 was evident early in the treatment period (Hct reduction of 36% in M009 treated animals versus 17% increase in vehicle controls). The effect of M009 on Hct was predominantly caused by a decrease in RBC (-34%) rather than MCV (-14%). These data indicate that MH-induced iron restriction can rapidly reduce the rate of erythropoiesis in this mouse model of human PV, leading to large reductions in Hct that would be highly clinically relevant if replicated in clinical trials. This effect was observed despite the fact that Hct continued to increase in control animals, indicative of even greater underlying disease activity during the treatment period. The dose of M009 that was used was high and probably supratherapeutic since circulating Hb was reduced below the level observed in WT mice. Nevertheless these results suggest that MH may be considered for development as a “medical phlebotomy” to provide continuous and improved control of accelerated erythropoiesis in the treatment of PV. Disclosures Casu: Merganser Biotech LLC: Employment; Isis Pharmaceuticals, Inc.: Employment. Nemeth:Merganser Biotech LLC: Stockholders Other. Ganz:Merganser Biotech LLC: Stockholders Other. MacDonald:Merganser Biotech LLC: Employment, Equity Ownership. Rivella:merganser Biotech LLC: Consultancy, Research Funding, Stock options, Stock options Other; bayer: Consultancy, Research Funding; isis Pharmaceuticals, Inc.: Consultancy, Research Funding.

Individuals affected by non-transfusion-dependent thalassemia (NTDT) develop severe ineffective erythropoiesis that causes a number of serious clinical morbidities, such as chronic anemia, splenomegaly and systemic iron overload requiring chelation therapy. In NTDT, low hepcidin levels caused by ineffective erythropoiesis result in increased iron absorption which may in turn exacerbate erythroid cell damage, apoptosis and ineffective erythropoiesis. Minihepcidins (MH) are short peptide mimetics that reproduce the iron restrictive effects of hepcidin. Using a mouse model of NTDT (Hbbth3/+), we previously showed that MH-induced iron restriction significantly reduced erythroid cell damage, leading to reduced ineffective erythropoiesis and improved anemia. Accumulation of tissue iron was significantly diminished in MH treated animals. In clinical settings, use of MH for the treatment of NTDT would likely be concomitant with oral chelation therapy. Therefore we conducted studies in the (Hbbth3/+) mouse to evaluate whether the concurrent use of the oral iron chelator deferiprone (DFP) and MH would alter a) the hematological benefit of MH or b) the tissue iron reduction benefit of DFP. MH may improve the efficacy of DFP by reducing dietary iron absorption, thus increasing net reduction in tissue iron. Studies were performed in six week old mice using MH M004, which reduces cell surface expression of ferroportin with an EC50of 9.7 nM and causes >80% reduction in serum iron at a dose of 7.5 mg/kg in the rat. M004 (2.65 mg/kg) or vehicle control was administered twice weekly by subcutaneous injection for six weeks. Half of the mice in each group had free access to water containing DFP (1.25 mg/mL). Treatment with MH alone produced a profile of hematological changes resulting in a significant increase in circulating hemoglobin of 1.6 g/dL. Flow cytometry studies of bone marrow erythroid populations from MH-treated animals demonstrated an increase in the relative proportion of mature erythroid cells, reduced apoptosis and reduced levels of ROS. Parameters of erythrocyte damage (red cell distribution width, red cell morphology, red cell survival time) were improved and an increase in peripheral red cell number was observed. Reticulocyte count and spleen size were both reduced, reflecting improved erythropoietic efficiency. In separate studies in Hbbth3/+ mice that also expressed a hypoxia induced luciferase-reporter gene, treatment with MH resulted in reduction of tissue hypoxia. Mice treated with DFP alone showed no hematologic improvement compared to vehicle control whereas mice treated with MH and DFP together showed similar hematologic benefit as mice treated with MH alone (Hb increase of 2.3 g/dL versus control). Both DFP and MH administered separately caused a reduction in total liver iron compared to vehicle controls but the different was only statistically significant in MH treated animals. When administered concurrently, the combination of DFP and MH caused a further non-significant reduction compared to either agent alone. Although spleen iron/g wet weight was increased by MH, reflecting the iron-sequestering effect of hepcidin, total spleen iron was not significantly increased because spleen size was also reduced. DFP was equally effective in reducing spleen iron/g wet weight with or without concurrent MH treatment. However total spleen iron was non-significantly reduced (-35%) in DPF-treated mice compared to vehicle controls (1,435 ± 627 µg (mean ± SEM) vs 2,210 ± 213 µg) but a larger, statistically significant reduction in spleen iron was observed in MH + DFP treated mice compared to MH alone (-50%, 1,258 ± 414 vs 2,531 ± 246, p In conclusion, the beneficial hematological effects of MH treatment in this model of NTDT remained even during concurrent treatment with DFP. DFP had no hematological benefits when used as a single agent, reflecting the inability of iron chelators to cause clinically meaningful iron restriction. Reduction in tissue iron by DFP was either unaffected or enhanced by concurrent treatment with MH. The improved effect of DFP on spleen iron burden in the presence of MH may reflect the complementary effects of these agents to increase iron excretion and reduce dietary iron absorption respectively. Enhancement of the clinical efficacy of iron chelators may be an important benefit of MH therapy in addition to the observed increase in circulating hemoglobin. Disclosures Casu: Isis Pharmaceuticals, Inc.: Employment; Merganser Biotech LLC: Employment. Nemeth:Merganser Biotech LLC: Stockholders Other. Ganz:Merganser Biotech LLC: Stockholders Other. MacDonald:Merganser Biotech LLC: Employment, Equity Ownership. Rivella:bayer: Consultancy, Research Funding; merganser Biotech LLC: Consultancy, Research Funding, Stock options, Stock options Other; isis Pharmaceuticals, Inc.: Consultancy, Research Funding.

After blood loss, the production of red cells must be increased by stress erythropoiesis. This phenomenon is associated with increased proliferation and reduced differentiation of the erythroblasts, leading to a net increase in the number of progenitor erythroid cells and red cells (erythron). In normal conditions, after expansion of the pool of erythroblasts, these cells eventually differentiate to erythrocytes and the anemia resolves. However, in diseases such as β-thalassemia, production of healthy mature erythrocytes is impaired, resulting in anemia. Over time, the expansion, rather than the differentiation, of the erythron further exacerbates the ineffective erythropoiesis (IE), reducing the ability of the erythroid progenitors to generate erythrocytes. Interupting the interaction between macrophages and erythroblasts (macrophage-erythroblast interaction, MEI) in thalassemia models is efficacious in reducing IE and alleviating the disease phenotype. Targeting MEI, using a number of approaches, caused a significant improvement in blood parameters in β-thalassemia intermedia (BTI) mouse models (Hbbth3/+) and a rapid and dramatic improvement in splenomegaly, an outcome that is relevant for clinical practice. Importantly, MEI is not critical for hematopoiesis under non-stress conditions, and ablation of this interaction in normal mice showed minimal effects on blood parameters. As our initial observations indicate that MEI is essential to support stress erythropoiesis, we investigated adhesion molecules that might activate downstream pathways in erythroblasts that regulate cell proliferation. We also speculate that these molecules are also responsible for the homing of erythroid progenitor cells to extramedullary organs, such as the spleen and liver. Our studies in erythroblasts indicate that integrin beta 1 (Itgb1) and also intracellular molecules such as Fak1, Talin1 and Sharpin might play a role in stress erythropoiesis. There is increased interaction between Itgb1 and Fak1 in erythroblasts co-cultured with macrophages as demonstrated by immunocytochemistry and in vitro proximity ligation assays. In addition, targeting either Itgb1 and Fak1 prevents expansion of erythroid cells when cultured in the presence of macrophages. Strikingly, using Itgb1 together with Ter119 as selection parameters in flow cytometry, a distinct subset of erythroblasts, not discernable using CD44 or CD71, was observable, which we found to be part of the mixed orthochromatic erythroblast/reticulocyte population as determined with CD44 expression. More specifically, when measuring the content of DNA, we were able to demonstrate that enucleation of erythroblasts was accompanied by a marked loss of Itgb1 expression, indicating that there may be an important role for Itgb1 in erythroblast enucleation, and differentiation in general. Lack of Itgb1 in thalassemic mice prevents erythroid cells from homing to and expanding in the spleen, the major source of chronic stress erythopoiesis in this disorder. In particular, such a role of Itgb1 is supported by our analysis of thalassemic mice in which this molecule was partially depleted by induction of the Cre recombinase. These animals were generated by crossing th3/+ mice with animals in which Itgb1 was floxed and carrying an inducible Cre-recombinase (Mx1-CRE). We utilized the BM of these animals (Hbbth3/+, Itgb1fl/fl, Mx1-CRE) to generate thalassemic animals that expressed the floxed Itgb1 only in hematopietic cells. After serial administration of polyI:C the animals were analyzed for their erythropoiesis in the bone marrow and spleen. Interestingly, all the animals analyzed show chimeric populations of Itgb1 positive and negative erythroid cells in the bone marrow. This indicated that not all the HSCs were successfully depleted of the Itgb1 gene. However, when we investigated Itgb1 in the spleen, we observed only erythroid cells positive for the expression of this adhesion molecule. This last observation strongly suggests that depletion of Itgb1 prevents homing and expansion of erythroid cells in the spleen and drugs that by inhibit Itgb1 could reduce erythroid spleen colonization, splenomegaly and limit erythropoiesis. We are now in the process of identifying compounds that target MEI. Such molecules might be utilized for development of new treatments for thalassemia or additional disorders of aberrant erythropoiesis. Disclosures Casu: Merganser Biotech : Research Funding; Isis Pharmaceuticals, Inc.: Research Funding.

Patients affected by non-transfusion dependent thalassemia (NTDT) do not require chronic blood transfusion for survival. However, transfusion-independence in such patients is not without side effects. Ineffective erythropoiesis (IE), the hallmark of disease, leads to a variety of serious clinical morbidities. In NTDT the master regulator of iron homeostasis, hepcidin, is chronically repressed. Consequently, patients absorb abnormally high levels of iron, which eventually requires iron chelation to prevent the clinical sequelaes associated with iron overload. It has been shown that in mice affected by NTDT (Hbbth3/+), a second-generation antisense oligonucleotide (Tmprss6-ASO) can reduce expression of transmembrane serine protease Tmprss6, the major suppressor of hepcidin expression. This leads to reduction of hemichrome formation in erythroid cells, amelioration of IE and splenomegaly, and increased hemoglobin levels (Guo et al, JCI, 2013). Now we propose the use of Tmprss6-ASO in combination with iron chelators for the treatment of NTDT using Hbbth3/+ mice as a preclinical model. Our hypothesis is that use of chelators will benefit from the positive effect of Tmprss6-ASO on erythropoiesis and iron absorption, further ameliorating organ iron content. To this end, Hbbth3/+ animals were treated with Tmprss6-ASO at 100 mg/kg/week for 6 weeks with or without the iron chelator deferiprone (DFP) at a dose of 1.25 mg/ml. Additional animals were treated with DFP alone. We fed the animals with a commercial or physiological diet, containing 200 or 35 ppm of iron, respectively. We did not observe major differences in the treated animals fed the commercial or physiological iron diet and, for this reason, the data were combined for simplicity. Administration of DFP alone was successful in decreasing organ iron content. Compared to untreated Hbbth3/+ animals, we observed a reduction of 30% and 33% in the liver and spleen, respectively, and no change in the kidney. However, erythropoiesis was not improved (looking at IE, splenomegaly, RBC production and total Hb levels). This was associated with increased serum iron levels (+25%). In Tmprss6-ASO treated Hbbth3/+ animals, we observed an improvement in liver iron content (36% reduction), amelioration of IE, and increased RBC and Hb synthesis (~2 g/dL). Compared to treatment with Tmprss6-ASO alone, combination of DFP with Tmprss6-ASO achieved the same level of suppression of Tmprss6 in the liver (~90%) and reduction of serum iron parameters. This was associated with improvement of IE, decreased reticulocyte counts and splenomegaly, and increased RBC and Hb synthesis (~2 g/dL). While we observed that both Tmprss6-ASO and DFP separately reduced liver iron content to the same extent (~30-36%), combination treatment further reduced iron concentrations in the liver and kidney (69% and 19%, respectively), with no changes in the spleen. Additional analyses are in progress to evaluate the amount of hepcidin in serum as well as expression of erythroferrone, the erythroid regulator of hepcidin. Our first conclusion is that administration of an iron chelator alone is not sufficient to improve erythropoiesis despite that organ iron content is reduced. We speculate that when iron is removed from the liver, hepcidin expression becomes more susceptible to the suppressive effect of IE rather than the enhancing effect of reduced liver organ iron concentration. In addition, the combined effect of iron mobilized from organs and unchanged (or even augmented) iron absorption leads to increased serum iron concentration. As we have shown previously, amelioration of IE in this model requires decreased erythroid iron intake and hemichrome formation. Therefore, iron chelation alone is likely insufficient to improve erythropoiesis. Additional experiments are in progress to further elucidate this mechanism. Our second conclusion is that use of Tmprss6-ASO together with DFP combines the best effects of these two drugs, in particular on erythropoiesis and organ iron content. In animals that received the combined treatment, kidney and liver iron concentrations were further decreased compared to the single treatments. This indicates that Tmprss6-ASO might be extremely helpful in the treatment of NTDT and it could further improve iron related-chelation therapies. Disclosures Casu: Merganser Biotech LLC: Employment; Isis Pharmaceuticals, Inc.: Employment. Aghajan:Isis Pharmaceuticals, Inc.: Employment. Guo:Isis Pharmaceuticals, Inc.: Employment. Monia:Isis Pharmaceuticals, Inc.: Employment. Rivella:bayer: Consultancy, Research Funding; isis Pharmaceuticals, Inc.: Consultancy, Research Funding; merganser Biotech LLC: Consultancy, Research Funding, Stock options , Stock options Other.

Anemia of inflammation (AI) is a complex condition commonly observed in chronic inflammatory states and associated with poor health outcomes and low quality of life. Increased hepcidin (Hamp) expression triggered by interleukin-6 (IL6) is a hallmark of AI and is responsible for iron-restricted erythropoiesis and anemia. Several translational studies have been aimed at neutralizing Hamp overexpression as a therapeutic strategy against AI. However, additional Hamp-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In our study, we investigated the distinct roles of iron metabolism and inflammation triggered by Hamp and IL6 in AI. We utilized Hamp-knockout (Hamp-KO), IL6-KO, and wild-type (WT) mice in which we induced inflammation with an intraperitoneal injection of heat-killed Brucella abortus (HKBA). In these mice, we characterized erythropoiesis with weekly CBC, FACS, and red blood cell (RBC) lifespan analysis. In addition, we measured cytokine expression, iron and transferrin (Tf) saturation in sera as indicators of inflammation and iron availability reflecting of iron stores. CBC analysis showed that hemoglobin (Hb) reached a nadir 2 weeks after HKBA injection in WT mice (6.0±1.1 g/dl), and 1 week after HKBA injection in IL6-KO (8.8±1.8 g/dl), and Hamp-KO mice (11.3±1.0 g/dl) (N=10/group), demonstrating that knockout mice were partially protected against HKBA-induced anemia. Additionally, knockout mice fully recovered within 3-4 weeks, whereas WT mice required more than 6 weeks. Mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and reticulocyte hemoglobin content (CHr) were decreased in both WT and IL6-KO mice after HKBA, although values were higher in the latter. Interestingly, Hamp-KO mice showed the highest levels of serum iron, Tf-saturation, MCH, MCV and CHr, indicating that the mild anemia induced by HKBA in these mice was a consequence of superior hemoglobinization of their reticulocytes and RBCs. Iron-depleted Hamp-KO mice were still less sensitive to HKBA administration, suggesting that lack of Hamp expression was contributing to protect these mice from anemia, rather than merely iron overload. FACS analysis performed 3 days after HKBA injection showed that BM erythropoiesis was disrupted in all treated mice. After 1 week, only IL6-KO mice were recovering, although their marrow erythroid progenitors were still reduced compared to PBS-treated mice. The faster recovery in IL6-KO mice was supported by the increased production of reticulocytes compared to Hamp-KO and WT mice. After 1 week, we observed stress erythropoiesis in the spleen of all mice, which was most significant in Hamp-KO, who also showed the highest levels of erythropoiesis in the spleen at steady state. Further investigation of stress erythropoiesis in phlebotomized WT mice injected with HKBA demonstrated that spleen erythropoiesis was partially spared from the inflammatory insult. We showed that a shortened RBC lifespan was contributing to anemia in HKBA-treated mice, and that impaired production of RBCs was accompanied by an increased rate of elimination. In fact, by FACS analysis we characterized macrophages (Gr1-/CD115-/SSClow/F4/80hi) engulfing erythroid cells (Ter119+) by discriminating multiplets vs. single cells (SSC-H vs. SSC-A), and observed that hemophagocytosis was occurring in HKBA-treated mice. Finally, we analyzed 58 inflammatory cytokines (RodentMap, Myriad RBM) from mouse sera and showed that HKBA administration changed the level of expression of inflammatory molecules more substantially in Hamp-KO than in IL6-KO compared to WT mice (26 cytokines changed in Hamp-KO mice, with fold change > or In conclusion, we showed that both IL6 and Hamp ablation offered protection against AI and that IL6-KO and Hamp-KO mice exhibited different patterns in the development and resolution of anemia. We believe that our data support the notion that not only IL6 and Hamp play distinct roles in modulating erythropoiesis in AI, but also that cytokines could be targeted as a therapeutic approach to improve marrow erythropoiesis in AI. Disclosures: Sasu: Amgen Inc.: Employment. Cooke:Amgen Inc.: Employment. Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Introduction Beta-thalassemia is a inherited disorder characterized by mutations in the beta-globin gene that lead to reduced or absent beta-globin synthesis. As a consequence, in erythroid cells there is a relative excess of alpha globin chains and heme forming toxic hemichromes, which lead to oxidative damage, impaired erythroid cell maturation, premature cell death and anemia. Under these conditions, the anemia and the elevated erythropoietin levels increase the proliferation of the erythroid progenitors, suppressing the expression of the iron master regulator hepcidin. Hepcidin targets the iron exporter ferroportin present on enterocytes and macrophages. High levels of hepcidin limit iron absorption and iron recycling but low levels of hepcidin increase iron absorption. Chronic low levels of hepcidin, as in β-thalassemia, eventually lead to iron overload. The th3/+ mouse model of thalassemia intermedia (Non-Transfusion Dependent Thalassemia or NTDT), reproduces the main features of the disease including anemia, increased erythropoiesis, splenomegaly and iron overload due to low levels of hepcidin. It has been previously demonstrated that modestly increasing hepcidin levels in these mice ameliorates both anemia and iron overload (Gardenghi et al, JCI, 2010). Methods We used minihepcidin M004, a modified peptide analog of the 9 N-terminal amino acids of hepcidin, to reproduce the biological effects of hepcidin. In a dose-ranging study, we treated th3/+ animals with daily intraperitoneal (ip) injections of M004 at doses of 6.25-100 µg/day for 2-6 weeks or with subcutaneous (sc) dosing of 52.5 μg twice a week for 6 weeks. Results In animals treated with the lower ip doses of M004 (6.25-25 μg/day for 2-4 weeks), mild iron restriction was observed (Tf sat ≥15%). Flow cytometry studies using the markers CD44 and Ter119 demonstrated an increase in the proportion of mature erythroid cells. Consistent with the flow cytometry findings, a 30% increase in peripheral red cells (rbc) was observed associated with a reduction in reticulocyte count (>62%) and a reduction in spleen size (>40%), reflecting improved erythropoietic efficiency. At the 12.5 and 25 µg doses, Hb level increased by 1.8-2.5 g/dL. Longer treatment (6 weeks) with 12.5-25 µg/day led to greater iron restriction and the loss of therapeutic effect at 6 weeks. In the same animals, measurement of total organ iron concentrations demonstrated no changes in the liver and kidney, but a 4-fold iron increase in the spleen. Prussian blue staining confirmed increased sequestration of iron in splenic macrophages and Kupffer cells. At high ip doses of M004 (100 μg/day for 2 weeks) the mice developed severe iron restriction (Tf sat = 6%) and exacerbation of anemia (Hb reduction of 4.5 g/dL). Accumulation of immature erythroid precursors was observed by flow cytometry studies, reflecting the erythroid maturation block caused by lack of iron for hemoglobin generation. Subsequent pharmacokinetic studies in rats indicated that plasma levels of M004 increased gradually with sc dosing and were still increasing 24h after dosing, indicating accumulation of drug levels and iron restriction with daily administration. We therefore explored whether less frequent dosing may be preferable to achieve sustained improvement in anemia and normalization of tissue iron. Sc dosing of M004 at 52.5 µg twice weekly was associated with an increase in rbc at 6 weeks to levels observed in WT animals, and an Hb increase of almost 3 g/dL. Reduced spleen weight (-51%) and reticulocyte count (-61%) reflected improved erythropoietic efficiency, further corroborated by flow cytometry studies. Reduction in hemichrome and ROS formation coupled with improvement in rbc morphology and reduced RDW indicated that hematological improvement was likely caused by improved erythroid cell survival as a result of reduced oxidative damage secondary to decreased erythroid iron uptake. Evaluation of total organ iron concentrations showed a reduction of 77% and 54% in the liver and kidney, respectively. Remarkably, the liver iron values were normalized compared to WT animals. Splenic iron increased only ∼1.5 times or 148%. However, when the total weight of the spleen was taken in account, the total splenic iron was reduced (-39%). Conclusion Minihepcidin peptides may be beneficial for the treatment of beta thalassemia intermedia by improving anemia and reducing tissue iron burden. Disclosures: Casu: Merganser: Research Funding. Goldberg:Merganser: Research Funding. Nemeth:Merganser: Equity Ownership. Ganz:Merganser: Equity Ownership. MacDonald:Merganser : Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Rivella:Alexion: Consultancy; Biomarin: Consultancy; Merganser: Equity Ownership, Research Funding; Isis: Consultancy, Research Funding; Bayer: Consultancy; Novartis: Consultancy; Imago: Consultancy.

Macrophages, strategically positioned in the center of erythroblastic islands while surrounded by developing erythroblasts (EB), are important for both steady state and stress erythropoiesis. In addition to their function in iron recycling and disposal of nuclei expelled by the maturing red cells during differentiation, macrophages are able to drive erythropoietic activity directly, making them, along with erythropoietin and iron, key regulators of erythropoiesis. This stress erythropoiesis-supporting macrophage activity (SEMA) has been demonstrated recently; there is, however, only limited understanding with regard to the exact cellular mechanism by which the macrophage activity is conveyed to the proliferating erythroid cells. Erythroblastic signaling through integrin β1 (Itgb1) and, further downstream, focal adhesion kinase-1 (Fak1), has previously been linked to stress erythropoiesis. Therefore, the current study explored the role of the Itgb1/Fak1 pathway in the macrophage-EB interplay, as well as its therapeutic potential in disorders marked by chronic stress erythropoiesis (CSE). Mice with beta-thalassemia intermedia (Hbbth3/+ or BTI), which present with ineffective erythropoiesis, characterized by high proliferation but limited differentiation of (CD71+) erythroblasts, anemia, and splenomegaly, were used as a model of CSE. In BTI mice, the depletion of macrophages by intravenous administration of clodronate-loaded liposomes (Clod) resulted in an augmentation of thalassemic erythroid maturation, characteristically marked by a decrease of immature (CD71+Ter119+) and increase of mature (CD71-Ter119+) erythroid cells in bone marrow; a reduction in actively cycling erythroblasts in the spleen, i.e. percentage of cells in S-phase (Clod (20%) compared to PBS (40%) PBS, p To focus on the macrophage-EB interaction in specific, human erythroid islands were generated in vitro after isolation of macrophages and CD34+ EBs from the peripheral blood of volunteers, allowing the evaluation of proliferation, expansion and differentiation of EBs upon co-culture with macrophages. Cycling and proliferation of EBs were significantly increased in the presence of autologous macrophages (60-fold increase in cell number after 8 days; versus 10-fold increase for EBs cultured alone, p To investigate the role of Itgb1-Fak1 signaling in the macrophage-EB crosstalk, the expression of both proteins was studied further in vitro. Flow cytometry analysis demonstrated higher surface expression of Itgb1 by EBs co-cultured with macrophages, as compared to EBs cultured alone, even though the expression of other differentiation markers was unchanged; and Fak1 expression was increased in macrophage-cultured EBs isolated from healthy volunteers, as well as from patients with beta-thalassemia major or Polycythemia Vera. Finally, a Fak1 inhibitor (FAK inhibitor 14) was used to test whether inhibition of Fak1 (Fak1i) could reverse the effect of macrophages on EB proliferation and maturation, thereby suggesting a link between erythroblastic Fak1 and the regulating effect of macrophages on erythroid development. In human CD34+ cells in vitro, Fak1i abolished the effect macrophages on the proliferation of EB, resulting in proliferation similar to that of EBs cultured alone, whereas the direct effect of Fak1i on EB proliferation was limited. In vivo, Fak1i administration to mice with BTI, rapidly reverted splenomegaly (0.2 g FAK1i vs 0.3 g PBS after 2 days, p In conclusion, in the present studies Itgb1 and Fak1 signaling were correlated to the mechanism by which macrophages contribute to SE and CSE, which may have critical scientific and therapeutic implications in the future. Disclosures: Rivella: Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Abstract 481 Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia. Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes. In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures: Guo: Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

Abstract 2055 β-thalassemia and sickle cell disease (SCD) are two of the most common genetic red cell blood disorders, affecting millions. Although both conditions originate from genetic defects that reside within the β-globin gene, β-thalassemia is characterized by limited or absent synthesis of β-globin chains, whereas SCD by production of an aberrant β-globin molecule. The only definitive cure for these disorders requires allogeneic bone-marrow (BM) transplant, a procedure whose success is limited by the availability of suitable donors and the occurrence of graft versus host disease. Therefore, the modification of a patient's own BM cells by insertion of the correct β-globin gene might offer a relatively safe alternative therapy. Recently, a patient affected by βE/β0-thalassemia received an autologous bone marrow transplant of hematopoietic stem cells treated with a lentiviral vector carrying the β-globin gene (Cavazzano-Calvo, Nature, 2010). This patient no longer requires transfusion therapy raising great hope that this disease can be cured in this way. However, only one-third of the total hemoglobin content in the patient is derived from the vector, the remainder being the endogenous hemoglobin, half adult and half fetal. To date, no study has focused on the correlation between gene transfer and increased hemoglobin levels in patients carrying different β-globin mutations and exhibiting phenotypic differences. Therefore, it would be extremely helpful if one could anticipate a patient response to gene transfer before undergoing myeloablation. For this purpose we devised a novel method to analyze patient derived erythroid cells in vitro following gene transfer. We generated lentiviral vectors carrying the human β-globin gene, large elements of the locus control region (LCR) with (AnkT9W) and without (T9W) an ankyrin insulator inserted in the 3' self-inactivating long terminal repeat. Analysis of Murine Erythroleukemia (MEL) cells single-integrant-clones indicated that the presence of the ankyrin insulator increased the synthesis of chimeric α-mouse/β-human hemoglobin by 47% (p=0.0023). This was further validated by comparing the amelioration of hematological parameters of thalassemic animals (Hbbth3/+) transplanted with thalassemic hematopoietic stem cells transduced with T9W or AnkT9W. To better understand the mechanism for increased globin expression in the AnkT9W-bearing MEL cells, we performed a time-course real-time PCR analysis on the human β-globin messenger, chromatin immunoprecipitation (ChIP) and polysomal analyses. Our results suggest a novel mechanism triggered by the presence of the ankyrin element, which increases the rate of transcription and confers temporal advantage of the transgenic β-globin mRNA during erythroid differentiation, facilitating ribosomal loading and efficient translation. We also established a preclinical assay to assess in vitro the response to gene transfer with AnkT9W of hematopoietic cells, isolated from twentytwo patients with β-thalassemia and SCD. Among β-thalassemic individuals, we found that in specimens carrying one or two β+ alleles the integration of 0.6 copies of the vector achieved hemoglobin production comparable to specimens from healthy individuals and 35% higher compared to erythroid cells from patients harboring two β0 mutations (p From our results we conclude that the ankyrin element is particularly effective for the purpose of expressing the β-globin gene not only in a quantitative but also in a qualitative fashion. Furthermore, this approach could provide vital information to select the best gene therapy tools for patients before undergoing myeloablation and bone marrow transplant. Further experiments are in progress to increase the number of SCD specimens and to analyze whether the integration pattern is different in cells infected with T9W versus AnkT9W. Disclosures: No relevant conflicts of interest to declare.

Abstract 901 β-Thalassemia is a genetic disorder characterized by reduced or absent β-globin synthesis. Complications include anemia, ineffective erythropoiesis (IE), splenomegaly and iron overload. Homeostatic iron levels are maintained by the hepatic hormone hepcidin (HAMP), which degrades the iron exporter ferroportin. Iron-responsive transcriptional control of HAMP is normally mediated by the bone morphogenetic protein (BMP)/SMAD pathway. Despite the high organ iron load associated with β-thalassemia, low Hamp levels have been reported in patients with this disorder. HAMP levels are also low relative to organ iron load in mouse models of β-thalassemia. Although iron overload is a central component of the pathogenesis of this disease, the factors contributing to inadequate HAMP expression have not been fully described. We previously showed that moderate transgenic overexpression of HAMP increased hemoglobin levels, improved erythropoiesis, and decreased both splenomegaly and iron overload in a mouse model of β-thalassemia intermedia (th3/+). In order to define the pathways controlling HAMP transcription in β-thalassemia, initial studies examined HAMP expression in the context of BMP/SMAD pathway members and targets genes. qRT-PCR analyses confirm decreased HAMP expression in 2 month old th3/+ mice and indicate a significantly larger decrease in mice with β-thalassemia major (th3/th3) relative to wild-type (+/+) controls. A significant decrease in the common SMAD pathway member, SMAD4, is also evident in both strains. TMPRSS6, the transcript encoding the protease matriptase-2, is also decreased in th3/th3 mice. In contrast, at 5 months of age the transcriptional profile of these genes in th3/+ mice is indistinguishable from that of wild-type mice. By 12 months of age, there is a trend toward increased HAMP transcription corresponding to increases in the BMP/SMAD targets ID1 and ATOH8. Examination of the effect of a low-iron diet on the transcriptional levels of these genes over time in th3/+ mice indicates no significant differences from +/+ mice after the data were normalized to liver iron concentrations. In contrast, comparison of the transcriptional profile of these genes with +/+ mice fed a high-iron diet indicates that HAMP expression is blunted in thalassemic mice. Preliminary Western blot analyses indicate that phosphorylation of SMAD 1/5/8 in th3/+ and th3/th3 liver samples, although comparable to that in +/+ samples, is lower than the levels observed in mice fed a high-iron diet. These data indicate that iron sensing through the BMP/SMAD pathway is intact, but somewhat blunted in thalassemic mice. Thus an alternative mechanism, possibly mediated by the IE that is characteristic of this disease, is likely responsible for the suppression of HAMP. In order to gain support for this hypothesis, qRT-PCR analyses were performed on mice overexpressing transgenic erythropoietin (TgEpo) to examine the effect of erythropoiesis on HAMP expression. The data indicate suppression of HAMP transcription despite clear activation of the BMP/SMAD pathway, as evidenced by increases in BMP6 and ID1. Of note, SMAD7 transcription was not detected in these animals. Compared to +/+ mice, phlebotomized mice also show decreased transcription of HAMP and SMAD4 without corresponding decreases in the BMP/SMAD pathway targets ID1 and ATOH8. TMPRSS6 transcription was also decreased, but FURIN expression was increased. FURIN, a known hypoxia target gene, was also transcribed at a significantly higher rate in th3/th3 mice than in either th3/+ or +/+ mice. qRT-PCR data indicate that this is mediated by HIF-2α, as evidenced by decreased transcription of the HIF-1α target, pyruvate dehydrogenase kinase 1, and increased transcription of the HIF-2α target, erythropoietin (EPO), in th3/th3 liver samples. HIF2-α expression in th3/th3 liver lysates was confirmed by Western blot analysis. Collectively, these data indicate that HAMP expression in thalassemia is suppressed through a mechanism distinct from a BMP/SMAD feedback loop and provide support for the existence of an ‘erythroid factor'. Results also suggest the increased suppression of HAMP observed in th3/th3 mice is attributable to the activation of HIF2-α, which in turn would be expected to aggravate the production of an erythroid factor through the upregulation of EPO. Disclosures: No relevant conflicts of interest to declare.

Abstract 3187 ß-Thalassemia and sickle cell disease (SCD) are the most common genetic red blood cell (RBC) disorders characterized respectively by limited production of aberrant ß-globin chains. In both cases, chronic transfusions and iron chelation are required to treat the anemia and/or formation of abnormal RBC. In ß-thalassemia, anemia stimulates erythropoietin (Epo) synthesis, which in turn leads to increased erythropoiesis and development of hepatosplenomegaly, often resulting in the need for splenectomy. Recently, we demonstrated that erythroid cells from ß-thalassemic mice have a hyper-activation of Jak2, a kinase that mediates the signaling triggered by the binding of Epo to the Epo receptor. This led us to hypothesize that Jak2 inhibitors could be utilized to minimize erythroid expansion in this disorder, limiting splenomegaly. A Jak2 inhibitor (Tg101209 or Tg) was first tested in mice affected by ß-thalassemia intermedia (th3/+). Two doses of Tg (150 and 100 mg/Kg/day) were given orally for 10 days. Tg administration induced a mild decrease of hemoglobin levels (8.8±0.2, 8±0.2 and 7.8±0.2g/dL for placebo, Tg 100 mg/Kg and Tg 150 mg/Kg treated mice, respectively. p We then evaluated the outcome of combining Tg with blood transfusion, a common therapy in b-thalassemia and SCD. In b-thalassemia, massively enlarged spleens are believed to sequester a significant proportion of circulating RBC, thereby limiting their lifespan and the efficacy of transfusion regimens. We hypothesize that decreasing splenomegaly by administration of Jak2 inhibitors could increase the efficacy of transfusion. This was first tested in th3/+ animals. In this case, transfusion alone was sufficient to increase the hemoglobin (Hb) levels approximately 3 g/dL and reduce the spleen size to 65% of that seen in non-transfused controls. In this model, the combined effect of transfusion and administration of Tg was more effective, the spleen size been 50% of non-transfused controls (p Lastly, we tested combination therapy in a mouse model of SCD. Mice treated with Tg and transfusion exhibited slightly lower levels of Hb than transfused controls (Hb=9.7±0.2g/dL versus Hb=10.9±0.2g/dL). However, compared to the control, mice receiving combination therapy exhibited a larger percentage of donor RBCs, while endogenous erythropoiesis was markedly suppressed along with the production of sickle RBCs (1.3±0.3×106 RBC/ul compared to transfused-controls exhibiting 2.7±0.3×106 RBC/ul). In summary, administration of Jak2 inhibitors might reduce the production of pathological cells that, together with preservation of the splenic architecture, could minimize the propensity of patients to thrombotic events. Furthermore, suppression of endogenous erythropoiesis and reduction of the transfusion regimen would be expected to also reduce iron accumulation, making it easier to prevent its toxic effects through chelation therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1035 β-Thalassemia is a disorder associated with abnormal β-globin production, leading to anemia, extramedullary hematopoiesis (EMH), a decreased lifespan of the red cells and iron overload. In this disorder erythropoiesis is ineffective due to increased erythroid apoptosis and erythroblast proliferation, as well as deficient differentiation. Recent evidence suggests that erythroid development, especially under conditions of anemia (stress erythropoiesis), is highly dependent on microenvironmental factors within the erythroid niche, potentially mediated by the interaction of erythroblasts with macrophages. However, little is known about the function of these cells in pathological anemias associated with abnormal erythropoiesis. Our goal was to study the role of macrophages in normal, stress and ineffective erythropoiesis (IE). Macrophages were eliminated by intravenous administration of clodronate-containing liposomes. Treatment was carried out for up to 12 weeks, serial measurements being made of erythropoietic and pathological parameters. As a model of stress erythropoiesis, phlebotomized wt mice were used. To study IE we utilized th3/+ mice, a model of β-thalassemia intermedia (TI). Clodronate treatment effectively depleted splenic and bone marrow (BM) macrophages as shown by FACS and immunohistochemical analyses. Depletion of macrophages in wt mice had little effect on steady state erythropoiesis. In contrast, clodronate treatment drastically impaired the response to stress erythropoiesis in these mice, as shown by the slow recovery from phlebotomy-induced anemia. This was associated with a very slow rate of RBC and reticulocyte production, suggesting that erythroid activity was markedly impaired. Accordingly, mice depleted of macrophages were unable to expand their pool of erythroid progenitors in the BM and spleen in response to anemia, suggesting that macrophages play a critical role in this process. A similar defect was observed in response to Epo stimulation, suggesting that an intact erythroid niche is essential for normal activity of Epo in promoting erythroid expansion. Interestingly, TI mice treated with clodronate exhibited an improvement of the thalassemic phenotype. Within 40 hours of clodronate treatment, mice showed an increase in hemoglobin (Hb), RBC and reticulocyte counts in the peripheral blood, and a reduction of extra-medullary hematopoiesis (increased ratio of mature to immature erythroid cells) and splenomegaly (P In conclusion, our data suggests that macrophages have two major roles in β-thalassemia: 1) to modulate iron availability for erythroid cells; 2) to impair erythroid development, as suggested by the amelioration of splenomegaly and EMH observed after clodronate treatment. We hypothesize that the macrophages within erythroblastic islands control erythropoiesis, acting as modulators of this process. Under conditions of stress erythropoiesis they positively influence erythroid development, promoting proliferation to increase the pool of erythroid cells. However, under conditions of chronic stress such as in TI, macrophages limit differentiation and promote excessive expansion of the erythron, contributing to IE. Disclosures: No relevant conflicts of interest to declare.

Abstract 1046 Anemia of inflammation (AI) is a widespread multi-factorial form of anemia characterized by hepcidin-induced iron restricted erythropoiesis as well as direct cytokine effects on the bone marrow, blunted erythropoietin production and efficacy, and shortened red blood cell (RBC) lifespan. Our aim is to perform an in depth study of AI, identifying the components and mechanisms associated with its pathophysiology. We generated a mouse model of AI using a single intraperitoneal injection of heat-killed Brucella abortus (HKBA). In this model we explored the role played by interleukin-6 and hepcidin in the onset of anemia. We utilized wild-type (WT), interleukin-6 knockout (IL-6 KO) and hepcidin knockout (Hamp KO) mice (n ≥ 6/group) injected with HKBA, and conducted weekly CBC's for 7 weeks to follow the progression and resolution of anemia. Anemia started developing one week after HKBA administration and reached a nadir after 2 weeks in all mice. Hemoglobin values from WT mice were lowest 2 weeks after injection (6.4 ± 1.2 g/dl) but slowly recovered over 7 weeks. Initially, IL-6 KO mice were equally affected with similar hemoglobin values at 2 weeks (6.9 ± 1.3 g/dl). However, these mice recovered after 3 weeks. Hamp KO mice were less anemic throughout the course of the study, with hemoglobin values of 10.3 ± 0. 9 g/dl at 2 weeks and resolution after 4 weeks. These data demonstrate that while both interleukin-6 and hepcidin contribute to AI, lack of either molecule alone is not sufficient to prevent AI. Therefore, additional factors likely play an important role in the etiology of AI. In order to rule out the effect of iron overload on the reduced severity of anemia observed in Hamp KO mice injected with HKBA, 1 week-old mice were fed an iron-deficient diet in order to first deplete their iron stores, and then returned to the normal diet before HKBA injection. We observed that iron-depleted Hamp KO mice were still less sensitive to HKBA administration, suggesting that this effect was independent of iron overload and dependent on the intrinsic lack of hepcidin expression. We further investigated the erythropoiesis in WT, IL-6 KO, and Hamp KO mice one week after HKBA injection. We performed FACS analyses of BM and spleen using CD44 and Ter119 antibodies. Both the mature RBCs (CD44−/Ter119+) and erythroid progenitor cells (CD44+/Ter119+) were dramatically reduced in the BM of HKBA-treated WT mice compared to controls (CD44+/Ter119+ cells diminished from 35.5 ± 0.2% to 2.8 ± 0.8%; CD44−/Ter119+ cells from 17.2 ± 0.2% to 8.2 ± 0.8%). The reduction of erythroid cells was attenuated in HKBA-treated IL-6 KO mice (CD44+/Ter119+ cells diminished from 32.8 ± 0.1% to 7.5 ± 6.0%; CD44−/Ter119+ cells from 22.1 ± 0.5% to 10.4 ± 3.8%). Hamp KO mice, on the other hand, showed a dramatic reduction of the CD44+/Ter119+ population in their BM (from 24.1 ± 2.5% to 1.8 ± 0.3%), while mature CD44−/Ter119+ cells were less affected (from 15.4 ± 2.3% to 14.1 ± 2.6%). Erythropoiesis was altered in the spleen as well. However, while the CD44+/Ter119+ cells were reduced in all the mice strains, the CD44−/Ter119+ population was increased one week after HKBA injection. This profile was more similar to ineffective erythropoiesis than iron-restricted erythropoiesis. Splenomegaly was also observed in all HKBA-treated mice. In addition, we measured increased apoptosis and production of reactive oxygen species (ROS) in the reticulocytes and orthochromatic erythroblasts of the spleen and BM of all mice. Overall, these data suggest that, in addition to iron restricted-erythropoiesis, an acute inflammatory effect on erythropoiesis is occurring in the HKBA model of AI, affecting erythroid cell survival and/or proliferation. Further analyses aimed at determining the RBC life span and survival in these mice are in progress. Moreover, we are analyzing iron-related gene expression in all groups of mice, along with measurement of their serum iron levels, iron stores, and serum cytokine levels, at different time points. Preliminary data indicate that numerous cytokine mRNAs (including IL-1α, IL-1β, TNF-α, INF-γ) are elevated in the spleen of WT mice 6 hours after HKBA injection. We are investigating the role that these cytokines might have on erythropoiesis, and the anemia observed in IL-6 KO and Hamp KO mice after injection of HKBA. Disclosures: Cooke: Amgen: Employment. Sasu:Amgen: Employment.