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β-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for β-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects β-globin synthesis in β-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the β-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits β-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) 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 toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of β-thalassemia caused by mutations leading to aberrant splicing.

Minihepcidins are hepcidin agonists that have been previously shown to reverse iron overload and improve erythropoiesis in mice affected by non-transfusion-dependent thalassemia. Given the extreme anemia that occurred with the previous model of transfusion-dependent thalassemia, that model was inadequate for investigating whether minihepcidins can improve red blood cell quality, lifespan and ineffective erythropoiesis. To overcome this limitation, we generated a new murine model of transfusion-dependent thalassemia with severe anemia and splenomegaly, but sufficient red cells and hemoglobin production to test the effect of minihepcidins. Furthermore, this new model demonstrates cardiac iron overload for the first time. In the absence of transfusions, minihepcidins improved red blood cell morphology and lifespan as well as ineffective erythropoiesis. Administration of a minihepcidin in combination with chronic red blood cell transfusion further improved the ineffective erythropoiesis and splenomegaly and reversed cardiac iron overload. These studies indicate that drugs such as minihepcidins have therapeutic potential for patients with transfusion-dependent thalassemia.

There is a Blood Commentary on this article in this issue.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2—a STAT5-dependent lipid binding protein downstream of erythropoietin—as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis., Maria Feola et al., elucidate the compensatory role of pleckstrin-2 in ineffective erythropoiesis in β-thalassemic mice by reducing cofilin-mediated apoptosis and enhancing enucleation by activating RacGTPases. These findings could support future therapeutic interventions.

Background:Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis.Methods:To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as β–thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone.Results:We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss.Conclusions:Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia.Funding:YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.

Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low–bone–mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP–mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in β–thalassemic (Hbbth3/+) mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β–thalassemia.

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34(+) cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.

Patients with thalassemia exhibit an increased risk of thrombotic events that is augmented after splenectomy. Heparanase protein enhances cancer progression, angiogenesis, and inflammation; it also activates the coagulation system through direct interaction with tissue factor (TF). Additionally, erythropoietin, which is elevated in anemic patients, up-regulates heparanase expression via the Janus kinase 2 (JAK-2) pathway. This study aimed was to explore the heparanase profile in thalassemia. Coagulation factors were analyzed via immunostaining, enzyme-linked immunosorbent assay, and heparanase procoagulant activity assay. In spleen specimens of thalassemia major patients, a higher level of heparanase staining was observed compared with control spleens resected after trauma (P

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.

Iron is one of the most abundant elements in the Earth and a fundamental component of enzymes and other proteins that participate in a wide range of biological processes. As the human body has no mechanisms to eliminate the excess of iron, its metabolism needs to be tightly controlled in order to avoid all the sequelae associated with high iron levels. Iron overload is the main cause of morbidity and mortality in beta thalassemia. The master regulator of iron homeostasis, hepcidin, is chronically repressed in this disorder, leading to increased intestinal iron absorption and consequent iron overload. Many groups have focused on obtaining a better understanding of the pathways involved in iron regulation. New molecules have recently been synthesized and used in animal models of dysregulated iron metabolism, demonstrating their ability to target and reduce iron load. Antisense oligonucleotides, as well as lipid nanoparticle-formulated small interfering RNAs and minihepcidins peptides, are novel agents that have already proved to be efficient in modulating iron metabolism in mouse models and are therefore promising candidates for the treatment of patients affected by iron disorders.

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.

Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23(-/-)) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.

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.

β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe(-/-)) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB(th3/+) mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.

Regulation of erythropoiesis is achieved by integration of distinct signals. Among these, macrophages are emerging as erythropoietin-complementary regulators of erythroid development, particularly under stress conditions. We investigated the contribution of macrophages for physiological and pathological conditions of enhanced erythropoiesis. We utilized mouse models of induced anemia, Polycythemia vera and β-thalassemia in which macrophages were chemically depleted. Our data indicate that macrophages contribute decisively for recovery from induced anemia as well as the pathological progression of Polycythemia vera and β-thalassemia by modulating erythroid proliferation and differentiation. We validated these observations in primary human cultures, showing a critical direct impact of macrophages on proliferation and enucleation of erythroblasts from healthy individuals and Polycythemia vera or β-thalassemic patients. In summary, we identify a new mechanism that we named “Stress Erythropoiesis Macrophage-supporting Activity” (SEMA) that contributes to the pathophysiology of these disorders and will have critical scientific and therapeutic implications in the near future.

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss.

Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO). INTRODUCTION: PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO. METHODS: Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO. RESULTS: Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P

In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.

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.

Lentiviral-mediated beta-globin gene transfer successfully treated beta-thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different beta-globin mutations found in patients. Most mutations can be classified as beta(0) or beta(+), based on the amount of beta-globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two-phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human beta-globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooley's anemia patients.

Iron is one of the most abundant elements in the Earth and a fundamental component of enzymes and other proteins that participate in a wide range of biological processes. As the human body has no mechanisms to eliminate the excess of iron, its metabolism needs to be tightly controlled in order to avoid all the sequelae associated with high iron levels. Iron overload is the main cause of morbidity and mortality in beta thalassemia. The master regulator of iron homeostasis, hepcidin, is chronically repressed in this disorder, leading to increased intestinal iron absorption and consequent iron overload. Many groups have focused on obtaining a better understanding of the pathways involved in iron regulation. New molecules have recently been synthesized and used in animal models of dysregulated iron metabolism, demonstrating their ability to target and reduce iron load. Antisense oligonucleotides, as well as lipid nanoparticle-formulated small interfering RNAs and minihepcidins peptides, are novel agents that have already proved to be efficient in modulating iron metabolism in mouse models and are therefore promising candidates for the treatment of patients affected by iron disorders.