Methods for treating iron overload-related diseases

Abstract

In the present invention, we identified the hepatokine FGL1 as a previously unreported hepcidin suppressor that is highly induced in the liver in response to hypoxia during recovery from anemia and in thalassemia mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deficiency of Fgl1 in mice (Fgl1- / -) results in lower hepcidin repression after hemorrhage. Finally, we clearly demonstrated that FGL1 is a BMP antagonist that directly binds to BMP6 and impairs the canonical BMP-SMAD signaling cascade that controls hepcidin regulation. Therefore, the present invention relates to methods for preventing or treating iron overload-related diseases by targeting the hepatokine FGL1, a novel hepcidin repressor.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods for preventing or treating iron overload-related diseases, such as β-thalassemia, hemochromatosis, congenital dyserythroid anemia, and myelodysplastic syndromes, by targeting hepatokine FGL1 (fibrinogen-like protein 1), a newly identified hepcidin repressor.

[0002] Background of the Invention Anemia, defined as a reduced number of functional red blood cells, is a major cause of disease affecting one-third of the world's population. 1 Many conditions can cause anemia, including iron deficiency, bleeding, infection, and genetic disorders. Iron is an essential functional component of hemoglobin in red blood cells, and a continuous supply of iron to the bone marrow is necessary to produce red blood cells and ensure adequate oxygen delivery to tissues. 2,3 Iron is released from iron-recycling macrophages, enterocytes, and hepatocytes by ferroportin, a unique iron efflux protein. Hepcidin, a liver-derived hormone, regulates iron content in the body by binding to ferroportin and causing its sequestration and degradation. 4,5 .

[0003] Hepcidin synthesis is primarily regulated by the canonical BMP-SMAD signaling pathway and the bone morphogenetic proteins BMP2 and BMP6 6-8 Binding of BMP2 / 6 to the large receptor complex phosphorylates SMAD1, 5, and 8 effectors, which translocate to the nucleus and activate hepcidin transcription. 9 Hepcidin expression is rapidly repressed by the erythroid regulator erythroferon (ERFE) in conditions associated with increased erythropoiesis, such as anemia caused by hemorrhage or inflammation. 10,11 Conversely, excessive release of ERFE occurs in congenital conditions caused by genetic mutations (beta-thalassemia, congenital dyserythroid anemia, myelodysplastic syndromes). 12-14This leads to iron overload and severe clinical complications that threaten patient survival. In response to erythropoietin (EPO), ERFE is secreted by erythroid progenitor cells in the bone marrow and spleen and functions as a ligand trap that directly binds to BMP6 and inhibits the signaling cascade that induces hepcidin expression. 15 .

[0004] ERFE is essential for suppressing hepcidin within the first few hours after erythropoietic stress, and ERFE-deficient mice recover from anemia induced by hemorrhage and chronic inflammation. 10,11 Similarly, ERFE deletion or neutralization in thalassemia mice 16,17 This increases hepcidin levels and reduces whole-body iron content. However, restoration of physiological hepcidin levels is not sufficient to correct iron overload, and hepcidin synthesis remains inappropriately low relative to liver iron content. Together, these data indicate that hepcidin is repressed during anemia by an ERFE-independent mechanism.

[0005] Therefore, we examined hepcidin regulation during recovery from hemorrhage-induced anemia in WT and Erfe-deficient mice and confirmed ERFE-independent repression of hepcidin during anemia. Here, we report the identification of a novel hepcidin suppressor, fibrinogen-like 1 (FGL1), a hepatokine produced in the liver, that may contribute to hepcidin regulation during anemia.

[0006] Summary of the Invention A first object of the present invention relates to a fibrinogen-like protein 1 (FGL1) inhibitor for use in the treatment of subjects suffering from iron overload-related diseases.

[0007] A second object of the present invention relates to the combination of a fibrinogen-like protein 1 (FGL1) inhibitor and an erythroferron (ERFE) inhibitor for simultaneous or sequential use in preventing or treating iron overload related diseases.

[0008] A third object of the present invention relates to methods, particularly in vitro methods, for screening fibrinogen-like protein 1 (FGL1) inhibitors (or antagonists) for use in preventing or treating iron overload-related diseases.

[0009] In certain embodiments, the iron overload related disease is selected from the list consisting of hemochromatosis (adult and juvenile hereditary hemochromatosis), iron-loading anemia and chronic liver diseases, including alcoholic liver disease and chronic hepatitis B and C.

[0010] In certain embodiments, the iron-loading anemia is selected from the list consisting of alpha-thalassemia, beta-thalassemia, congenital dyserythroid anemia, myelodysplastic syndrome (MDS).

[0011] Detailed Description of the Invention In this study, we identified the hepatokine FGL1 as a previously undescribed hepcidin suppressor that is highly induced in the liver in response to hypoxia during recovery from anemia and in thalassemia mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deficiency of Fgl1 in mice (Fgl1- / -) blunts hepcidin suppression after hemorrhage. Finally, we clearly demonstrated that FGL1 is a BMP antagonist that directly binds to BMP6 and impairs the canonical BMP-SMAD signaling cascade that controls hepcidin regulation.

[0012] Finally, as demonstrated in Example 2, the present inventors developed antisense oligonucleotides specific for human and mouse FGL1 that were validated in hepatocytes. These antisense oligonucleotides have the potential to block FGL1 attraction and subsequently hepcidin suppression in a mouse model of anemia.

[0013] Thus, the present invention provides methods and compositions (eg, pharmaceutical compositions) for preventing or treating iron overload-related diseases.

[0014] In the context of the present invention, the terms "treatment" or "prophylaxis" refer to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such terms apply, or one or more symptoms of such disorder or condition. In particular, treatment of a disorder may consist of reducing iron overload resulting from low hepcidin expression in conditions such as β-thalassemia, hemochromatosis, congenital dyserythroid anemia, and myelodysplastic syndromes (MDS). Most preferably, such treatment completely eliminates the pathological features observed in iron overload-related diseases.

[0015] Preferably, the individual to be treated is a human or non-human mammal (e.g., a rodent, feline, canine, or primate) suffering from or likely to suffer from hepcidin dysfunction observed in iron overload-related diseases.

[0016] Preferably, the individual is a human.

[0017] Fibrinogen-like protein 1 (FGL1) inhibitor According to a first aspect, the present invention relates to a fibrinogen-like protein 1 (FGL1) inhibitor for use in the treatment of a subject suffering from an iron overload-related disease.

[0018] In the present invention, we demonstrated that fibrinogen-like protein 1 (FGL1) expressed in the liver is directly involved in modulating hepcidin expression, primarily by acting as a BMP6 antagonist.

[0019] In certain embodiments, the iron overload related disease is selected from the list consisting of hemochromatosis (adult and juvenile hereditary hemochromatosis), iron-loading anemia and chronic liver diseases, including alcoholic liver disease and chronic hepatitis B and C.

[0020] In a more particular embodiment, the iron-loading anemia is selected from the list consisting of β-thalassemia, congenital dyserythroid anemia, myelodysplastic syndrome (MDS).

[0021] As used herein, "fibrinogen-like protein 1" (abbreviated as FGL1) or "fibrinogen-like protein 1" (synonyms LFIRE-1, HFREPI, hepasocin, HP-041) refers to a protein of the fibrinogen protein family (which also includes fibrinogen-like protein 2 and blood clotting factors V, VIII, and XIII), which in humans is encoded by the FGL gene (Gene ID: 2267). The term "FGL1 expression" refers to both protein and mRNA expression in humans, unless otherwise specified. FGL1 contains its C-terminal portion that is homologous to fibrinogen, and this portion contains the four conserved cysteines common to all members of the fibrinogen family. However, FGL1 lacks the platelet-binding site, cross-linking region, and thrombin-sensitive site that enable other members of the fibrinogen family to promote fibrin clot formation. FGL1 is upregulated in the regenerating liver and is abundantly associated with the fibrin matrix after clot formation. Most FGL1 is found in plasma, but approximately 20% FGL1 remains in serum after blood clotting. The human FGL1 precursor has the amino acid sequence provided by the NCBI Reference Sequence Database under ID number NP004458 (see also Uniprot ref. Q08830), where amino acids 1-22 correspond to the signal peptide and amino acids 23-312 correspond to the mature protein. The N-terminal domain of fibrinogen-like protein 1 (FGL1) corresponds to amino acids 23-78. The C-terminal globular domain of fibrinogen-like protein 1 corresponds to amino acids 79-312.

[0022] FGL1 has also been observed to strongly bind and activate LAG-3, a regulatory protein expressed in T cells. Because LAG-3 plays an important role in regulating activated T cells, manipulating FGL1 binding to T cells has been proposed for both cancer immunotherapy and anti-inflammatory treatment (Wang J, et al. (2019). Cell. 176 (1-2): 334-347.e12).

[0023] A "fibrinogen-like protein 1 (FGL1) inhibitor" or "fibrinogen-like protein 1 (FGL1) antagonist" refers to a molecule (natural or synthetic) that can neutralize, block, inhibit, suppress, reduce, or interfere with the biological activity of fibrinogen-like protein 1 (FGL1) (including, for example, reducing or blocking the interaction between fibrinogen-like protein 1 (FGL1) and BMP6). Fibrinogen-like protein 1 (FGL1) inhibitors or antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, drugs and their metabolites, transcriptional and translational control sequences, and the like. Inhibitors or antagonists also include antagonist mutants of proteins, siRNA molecules against proteins, aptamers, which are antisense molecules against proteins, and ribozymes against proteins. For example, a fibrinogen-like protein 1 (FGL1) inhibitor or antagonist can be a molecule that binds to fibrinogen-like protein 1 (FGL1) and neutralizes, blocks, inhibits, abrogates, reduces, or prevents the biological activity of fibrinogen-like protein 1 (FGL1), which leads to the inhibition of hepcidin expression through direct inhibition of BMP6 in response to hypoxia during recovery from anemia and in thalassemia mice.

[0024] In some embodiments, the FGL1 inhibitor is a direct fibrinogen-like protein 1 (FGL1) inhibitor. Thus, a "direct fibrinogen-like protein 1 (FGL1) inhibitor" is a fibrinogen-like protein 1 (FGL1) inhibitor / antagonist that directly binds to fibrinogen-like protein 1 (FGL1) (protein or nucleic acid sequence (DNA or RNA)) and neutralizes, blocks, inhibits, abrogates, reduces or prevents the biological activity of fibrinogen-like protein 1 (FGL1).

[0025] In the context of the present invention, a direct fibrinogen-like protein 1 (FGL1) inhibitor directly binds to fibrinogen-like protein 1 (FGL1) (protein or nucleic acid sequence (DNA or RNA)) and (ii) inhibits the suppression of hepcidin expression (through blocking the fibrinogen-like protein 1 (FGL1)-induced BMP6 antagonism process).

[0026] In particular, the direct fibrinogen-like protein 1 (FGL1) inhibitors of the present invention include: 1) inhibitors of fibrinogen-like protein 1 (FGL1) activity (e.g., small organic molecules, antibodies, aptamers, polypeptides) and / or 2) Inhibitors of fibrinogen-like protein 1 (FGL1) gene expression (e.g., antisense oligonucleotides, nucleases, siRNA, etc.) is.

[0027] "Biological activity" of fibrinogen-like protein 1 (FGL1) in the context of the present invention means the inhibition of hepcidin expression (through the process of BMP6 antagonism).

[0028] Tests for determining the ability of a compound to be a fibrinogen-like protein 1 (FGL1) inhibitor are well known to those skilled in the art. In a preferred embodiment, the antagonist / inhibitor specifically binds to fibrinogen-like protein 1 (FGL1) (protein or nucleic acid sequence (DNA or mRNA)) sufficiently to inhibit the biological activity of fibrinogen-like protein 1 (FGL1). Binding to fibrinogen-like protein 1 (FGL1) and inhibition of the biological activity of fibrinogen-like protein 1 (FGL1) can be determined by any competitive assay known in the art. For example, the assay may consist of determining the ability of the agent being tested as a fibrinogen-like protein 1 (FGL1) inhibitor to bind to fibrinogen-like protein 1 (FGL1). Binding ability is reflected by Kd measurement. As used herein, the term "Kd" is intended to refer to the dissociation constant. The dissociation constant is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The Kd value for binding to a biological molecule can be determined using methods well established in the art. In a specific embodiment, an antagonist / inhibitor that "specifically binds to fibrinogen-like protein 1 (FGL1)" is intended to refer to an inhibitor that binds to human fibrinogen-like protein 1 (FGL1) polypeptide with a Kd of 1 μM or less, 100 mM or less, 10 nM or less, or 3 nM or less. A competitive assay can then be set up to determine the ability of an agent to inhibit the biological activity of fibrinogen-like protein 1 (FGL1) inhibition of hepcidin expression through blocking the fibrinogen-like protein 1 (FGL1)-induced BMP6 antagonism process.

[0029] "Inhibitor of fibrinogen-like protein 1 (FGL1) activity" as used herein refers to a compound capable of reducing or suppressing the inhibition of hepcidin expression. Given the teachings of the present disclosure, particularly the Examples, it is within the ability of one skilled in the art to assess whether a compound is an inhibitor of hepcidin suppression activity. For example, a suitable test involves assessing whether the compound blocks the inhibition of hepcidin expression in hepatocytes, i.e., restores hepcidin-induced expression via the BMP6 / SMAD2 pathway. Suitable tests for detecting hepcidin-induced expression are described in the Examples below.

[0030] Functional assays based on hepcidin expression can be envisioned, such as assays that assess the ability to block the inhibition of the hepcidin expression process (in hepatocytes or in a mouse beta-thalassemia model) through blocking fibrinogen-like protein 1 (FGL1)-mediated BMP6 inhibition. Antagonism of FGL1 can be monitored by examining the restoration of hepcidin expression and p-SMAD5 expression by RT-qPCR and / or Western blot (see also Kautz L, et al. Nat. Genet. 2014;46(7):678-684). Other functional assays based on experimental mouse models of anemia (hemorrhage, chronic inflammation) or iron overload (i.e., beta-thalassemia models) can also be used, such as assays assessing the ability to increase hepcidin levels and decrease liver and serum iron levels (Kautz L, et al. Nat Genet. 2014;46(7):678-684; Kautz L, et al. Blood. 2014; Oct 16;124(16):2569-74; Kautz L, et al. Blood. 2015 Oct 22;126(17):2031-7). For example, serum iron levels can be assessed by a widely used chromogenic assay or transferrin saturation (Biolabo, Iron direct method & TIBC). Transferrin is the iron transport protein in plasma, and both parameters are currently used as diagnostic tests for iron deficiency anemia or iron overload disorders.

[0031] Those skilled in the art can readily determine whether a fibrinogen-like protein 1 (FGL1) antagonist / inhibitor neutralizes, blocks, inhibits, abrogates, reduces, or interferes with the biological activity of fibrinogen-like protein 1 (FGL1) to determine whether the FGL1 antagonist / inhibitor can bind to FGL1 and / or inhibit processes related to the inhibition of hepcidin expression (through the process of BMP6 antagonism) in the same manner as the first characterized inhibitors of FGL1. Binding assays and / or BMP6 activity assays can be performed for each antagonist.

[0032] Thus, a direct fibrinogen-like protein 1 (FGL1) inhibitor may be a molecule that binds to fibrinogen-like protein 1 (FGL1) selected from the group consisting of antibodies, aptamers.

[0033] One skilled in the art can readily determine whether a fibrinogen-like protein 1 (FGL1) inhibitor or antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of fibrinogen-like protein 1 (FGL1) by (i) binding to fibrinogen-like protein 1 (FGL1) (protein or nucleic acid sequence (DNA or mRNA)) and / or (ii) inhibiting the suppression of hepcidin expression or restoring hepcidin expression (through the process of BMP6 antagonism).

[0034] The term "iron overload-related disease" refers to a group of diseases and / or disorders commonly associated with abnormally low levels of hepcidin, including diseases in which abnormal iron metabolism directly causes the disease, or diseases in which blood iron levels are dysregulated by the disease, or diseases in which iron dysregulation is the result of another disease, or diseases that can be treated by modulating iron levels, etc. More specifically, iron metabolism diseases of the present disclosure include iron overload diseases, iron biodistribution disorders, other disorders of iron metabolism, and other disorders that may be related to iron metabolism, etc. Disorders of iron metabolism include hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemojuvelin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal hemochromatosis, hepcidin deficiency, transfusional iron overload, thalassemia, thalassemia intermedia, alpha thalassemia, beta thalassemia, African iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, and congenital dyserythroid anemia.

[0035] In certain embodiments, the iron overload related disease is selected from the list consisting of hemochromatosis (adult and juvenile hereditary hemochromatosis), iron-loading anemia and chronic liver diseases, including alcoholic liver disease and chronic hepatitis B and C.

[0036] The term "iron-loading anemia," also known as "secondary iron overload," refers to a disorder characterized by anemia, high serum iron, transferrin saturation, and ferritin levels, and hemosiderin deposition in parenchymal and reticuloendothelial tissues with or without organ dysfunction.

[0037] In a more particular embodiment, the iron-loading anemia is selected from the list consisting of β-thalassemia, congenital dyserythroid anemia, myelodysplastic syndrome (MDS).

[0038] In some embodiments, the iron overload disease is a myelodysplastic syndrome. In some cases, diseases and disorders included in the definition of "iron overload disease" are not typically identified as iron-related. For example, hepcidin is highly expressed in mouse pancreas. This suggests that diabetes (type I or type II), insulin resistance, impaired glucose tolerance, and other disorders can be alleviated by treating the underlying iron metabolism disorder. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, which is incorporated herein by reference. Thus, these diseases are encompassed by the broad definition. Those skilled in the art can readily determine whether a given disease is an "iron metabolism disease" of the present invention using methods known in the art, including the assays of WO 2004092405 (which is incorporated herein by reference) and assays that monitor hepcidin, hemojuvelin, or iron levels and expression (known in the art, as described in U.S. Pat. No. 7,534,764, which is incorporated herein by reference).

[0039] As used herein, diseases and disorders associated with hepcidin deficiency include adult and juvenile hereditary hemochromatosis, alpha-thalassemia, beta-thalassemia, and congenital dyserythroid anemia, as well as chronic liver diseases, including alcoholic liver disease and chronic hepatitis B and C.

[0040] According to certain embodiments of the present invention, the iron metabolism disorder is an iron overload disorder directly related to hepcidin deficiency, including secondary iron overload-related disorders also known as hemochromatosis (adult and juvenile hereditary hemochromatosis) or iron-loading anemia (including alpha-thalassemia, beta-thalassemia, congenital dyserythropoietic anemia, myelodysplastic syndromes), and chronic liver diseases including alcoholic liver disease, nonalcoholic steatohepatitis (NASH), and chronic hepatitis B and C.

[0041] In a more particular embodiment, the secondary iron overload related disease (or iron-loaded anemia) is selected from the list consisting of β-thalassemia, congenital dyserythroid anemia, myelodysplastic syndrome (MDS).

[0042] Preferably, the iron overload related disease is β-thalassemia.

[0043] Beta thalassemia (β-thalassemia) is a group of inherited blood disorders. Beta thalassemia is a form of thalassemia caused by reduced or absent synthesis of the hemoglobin beta chain. The resulting outcomes range from severe anemia to clinically asymptomatic individuals. The annual incidence worldwide is estimated at 1 / 100,000 (Galanello, et al (2010). Orphanet J Rare Dis. 5: 11). Beta thalassemia is caused by dysfunction of the hemoglobin subunit beta of HBB. The severity of the disease depends on the nature of the mutation. Over time, HBB blockade leads to decreased beta chain synthesis, accumulation of alpha chains, and premature apoptosis of erythroid precursors. Furthermore, the body's inability to construct new beta chains results in decreased production of HbA (adult hemoglobin). This in turn leads to an overall decrease in HbA available to fill red blood cells, resulting in microcytic anemia. With inadequate HbA protein for red blood cells to function properly, microcytic anemia eventually develops. This factor can lead to patients requiring blood transfusions to compensate for the beta chain blockage. Repeated blood transfusions can lead to serious problems related to iron overload (Galanello, et al (2010). Orphanet J Rare Dis. 5: 11).

[0044] Beta-thalassemia is medically incurable except through the use of blood transfusions, but frequent transfusions can lead to or increase iron overload (Greer, John P.; et al (2013). Wintrobe's Clinical Hematology. Lippincott Williams & Wilkins). Thus, there is a medical need to specifically treat subjects with beta-thalassemia with new therapeutic approaches.

[0045] In another embodiment, the iron overload related disease is myelodysplastic syndrome.

[0046] Myelodysplastic syndrome (MDS) is a group of blood cancers in which immature blood cells in the bone marrow fail to mature and develop into healthy blood cells. Initially, there are typically no symptoms. Later symptoms may include fatigue, shortness of breath, bleeding disorders, anemia, or frequent infections. Some types can progress to acute myeloid leukemia. Risk factors include prior chemotherapy or radiation therapy, exposure to certain compounds such as cigarette smoke, pesticides, and benzene, and exposure to heavy metals such as mercury or lead. Problems with blood cell formation result in a combination of low red blood cell, platelet, and white blood cell counts. Some types have an increase in immature blood cells called blasts in the bone marrow and blood. Anemia is the most common cytopenia in MDS patients, but with inadequate use of blood transfusions, MDS patients rarely experience severe anemia-related injury. The two most serious complications in MDS subjects with cytopenia are bleeding (due to lack of platelets) or infection (due to lack of white blood cells). Chronic transfusion of packed red blood cells leads to iron overload (Rasel, Mohammad; Mahboobi, Sohail K. (2022), "Transfusion Iron Overload", StatPearls, Treasure Island (FL): StatPearls Publishing).

[0047] Treatment may include supportive care, drug therapy, and hematopoietic stem cell transplantation. Supportive care may include blood transfusions, medicines to increase red blood cell production, and antibiotics. Drug therapy may include the medicines lenalidomide, antithymocyte globulin, and azacitidine.

[0048] Typically, the direct fibrinogen-like protein 1 (FGL1) inhibitors of the present invention comprise: A) Inhibitors of fibrinogen-like protein 1 (FGL1) activity, such as anti-FGL1 antibodies and anti-FGL1 aptamers B) An inhibitor of fibrinogen-like protein 1 (FGL1) gene expression selected from the list consisting of an antisense oligonucleotide, a nuclease, an siRNA, an shRNA, or a ribozyme nucleic acid sequence. Including, but not limited to:

[0049] A) Inhibitors of fibrinogen-like protein 1 (FGL1) activity ·antibody In one embodiment, the inhibitor of fibrinogen-like protein 1 (FGL1) activity is an antibody (a term that includes fragments or portions of antibodies) that can directly or indirectly block the interaction between fibrinogen-like protein 1 (FGL1) and BMP6 protein.

[0050] In preferred embodiments, the fibrinogen-like protein 1 (FGL1) antagonist may consist of an antibody against fibrinogen-like protein 1 (FGL1) (neutralizing antibody), such that the antibody is capable of impairing the binding of fibrinogen-like protein 1 (FGL1) to BMP6 and neutralizing, blocking, inhibiting, abrogating, reducing or preventing the biological activity of fibrinogen-like protein 1 (FGL1). In some embodiments, the neutralizing antibody of the present invention is directed against the globular domain of FGL1 (SEQ ID NO: 11). In some embodiments, the neutralizing antibody of the present invention is directed against SEQ ID NO: 12. In some embodiments, the neutralizing antibody of the present invention is directed against SEQ ID NO: 13. In some embodiments, the neutralizing antibody of the present invention is directed against SEQ ID NO: 14.

[0051] For the present invention, then, a fibrinogen-like protein 1 (FGL1) neutralizing antibody is selected as described above for its ability to (i) bind to fibrinogen-like protein 1 (FGL1) (protein) and / or (ii) inhibit suppression of hepcidin expression or restore hepcidin expression in the liver (through the process of BMP6 antagonism).

[0052] In this study, we clearly demonstrated that 1) full-length FGL1 (in vitro and in vivo) and the globular domain of FGL1 (in vitro) repress hepcidin expression (Figures 5D-5E), 2) reduce its expression in serum in mice (Figures 5F-5G), and 3) FGL1 is a BMP antagonist that directly binds to BMP6 and impairs the canonical BMP-SMAD signaling cascade that controls hepcidin regulation (Figure 7). These results suggest that, similar to erythropoietin, FGL1 can function as a ligand trap for BMP6 to repress hepcidin transcription during hemorrhage recovery.

[0053] In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody portion comprises an antibody light chain. In one embodiment of the antibodies or portions thereof described herein, the antibody portion comprises an antibody heavy chain. In one embodiment of the antibodies or portions thereof described herein, the antibody portion comprises an Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody portion comprises an F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody portion comprises an Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody portion comprises an Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody portion comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

[0054] As used herein, "antibody" includes both natural and non-natural antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, as well as monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single-chain antibodies, and fragments thereof. Antibodies can be human or non-human. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans.

[0055] Antibodies are prepared according to conventional methods. Monoclonal antibodies can be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the present invention, mice or other suitable host animals are immunized with an antigenic form of fibrinogen-like protein 1 (FGL1) at appropriate intervals (e.g., twice weekly, once weekly, twice monthly, or once monthly). The animals can be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunoadjuvant during immunization. Suitable immunoadjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titemax, saponin adjuvants such as QS21 or Quil, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well known in the art. Animals may be immunized subcutaneously, intraperitoneally, intramuscularly, intravenously, intranasally, or by other routes. A given animal may be immunized with multiple forms of antigen and by multiple routes.

[0056] Briefly, recombinant fibrinogen-like protein 1 (FGL1) can be provided by recombinant cell lines or bacterial expression. Recombinant forms of fibrinogen-like protein 1 (FGL1) can be provided using any previously described method. Following an immunization regimen, lymphocytes are isolated from the animal's spleen, lymph nodes, or other organs and fused with an appropriate myeloma cell line using an agent such as polyethylene glycol to form hybridomas. After fusion, the cells are placed in a medium that allows the hybridoma to grow but not the fusion partner, using standard methods described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). After hybridoma cultivation, the cell supernatant is analyzed for the presence of antibodies of the desired specificity, i.e., antibodies that selectively bind to the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and Western blot. Other screening techniques are well known in the art. Preferred techniques are those that confirm the binding of antibodies to conformationally intact, natively folded antigens, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

[0057] In particular, as is well known in the art, a small portion of an antibody molecule, the paratope, is responsible for the binding of the antibody to its epitope (see generally Clark, WR (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). For example, the Fc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding. Antibodies from which the pFc' region has been enzymatically cleaved or which have been produced without the pFc' region, designated F(ab')2 fragments, retain both antigen-binding sites of an intact antibody. Similarly, antibodies from which the Fc region has been enzymatically cleaved or which have been produced without the Fc region, designated Fab fragments, retain one antigen-binding site of an intact antibody molecule. Furthermore, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, designated Fd. The Fd fragment is the primary determinant of antibody specificity (a single Fd fragment can associate with up to 10 different light chains without altering antibody specificity), and Fd fragments retain epitope binding ability upon isolation.

[0058] As is well known in the art, the antigen-binding portion of an antibody contains complementarity-determining regions (CDRs), which directly interact with the antigen epitope, and framework regions (FRs), which maintain the tertiary structure of the paratope (see generally Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of an IgG immunoglobulin, there are four framework regions (FR1 to FR4), each separated by three complementarity-determining regions (CDR1 to CDRS). The CDRs, particularly the CDRS regions, especially the heavy chain CDRS, are responsible for most of the antibody specificity.

[0059] It is now well established in the art that the non-CDR regions of a mammalian antibody can be replaced with the analogous regions of a homologous or heterologous antibody while retaining the epitope specificity of the original antibody. This is most clearly demonstrated in the development and use of "humanized" antibodies, in which non-human CDRs are covalently linked to human FR and / or Fc / Fc' regions to produce a functional antibody.

[0060] In certain embodiments, the present invention provides compositions and methods comprising humanized forms of antibodies. As used herein, "humanized" refers to antibodies in which some, most, or all of the amino acids outside the CDR regions have been replaced with corresponding amino acids derived from human immunoglobulin molecules. Humanization methods include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference. U.S. Pat. Nos. 5,585,089 and 5,693,761, as well as WO 90 / 07861, also propose four potential criteria that can be used to design humanized antibodies. The first proposal was to use a framework from a specific human immunoglobulin that is typically homologous to the donor immunoglobulin being humanized, or to use a consensus framework from many human antibodies, for the acceptor. The second proposal was to select a donor amino acid rather than an acceptor amino acid if an amino acid in the human immunoglobulin framework is unavailable and the donor amino acid at that position is typical of the human sequence. The third proposal was to select a donor amino acid rather than an acceptor amino acid at positions immediately adjacent to the three CDRs in the humanized immunoglobulin chain. The fourth proposal was to use donor amino acid residues at framework positions where, in a three-dimensional model of the antibody, amino acids within 3A of the CDRs are predicted to have side chain atoms and be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that can be used by those skilled in the art to construct humanized antibodies. Those skilled in the art will be familiar with other methods for humanizing antibodies.

[0061] In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR regions are replaced with amino acids derived from a human immunoglobulin molecule, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are possible, as long as they do not abrogate the antibody's ability to bind to a given antigen. Suitable human immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. A "humanized antibody" retains antigen specificity similar to that of the original antibody. However, certain humanization methods can be used to improve the binding affinity and / or specificity of the antibody using "directed evolution" methods, as described in Wu et al., Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

[0062] Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, and 6,150,584, and the references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified to be functionally deficient in endogenous (e.g., murine) antibody production. The animals are further modified to contain all or part of the human germline immunoglobulin gene loci, such that immunization of these animals will result in the production of fully human antibodies against the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and therefore will not elicit a human anti-mouse antibody (KAMA) response when administered to humans.

[0063] In vitro methods for producing human antibodies also exist. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0064] Thus, as will be apparent to those skilled in the art, the present invention also provides F(ab')2, Fab, Fv, and Fd fragments; chimeric antibodies in which the Fc and / or FR and / or CRD1 and / or CDR2 and / or light chain CDR3 regions are replaced with homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced with homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced with homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions are replaced with homologous human or non-human sequences. The present invention also includes so-called single-chain antibodies.

[0065] The various antibody molecules and fragments may be derived from any of the commonly known immunoglobulin classes, including, but not limited to, IgA, secretory IgA, IgE, IgG, and IgM. IgG subclasses are also known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4.

[0066] In another embodiment, the antibody of the present invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to a single heavy chain variable domain of a type of antibody that can be found in camelids and that is essentially devoid of light chains. Such VHHs are also called "nanobodies®". According to the present invention, the sdAb can in particular be a llama sdAb.

[0067] Those skilled in the art can employ routine techniques to generate humanized antibodies for treating iron overload-related diseases (e.g., B-thalassemia as disclosed herein) using the antigen-binding sequences (e.g., CDRs) of these antibodies.

[0068] Examples of neutralizing monoclonal antibodies against fibrinogen-like protein 1 (FGL1) that can be used in the present invention are disclosed in Qian et al. J Hematol Oncol (2021) 14:147; WO 2019241098; WO 2022007543. Examples of such anti-FGL1 antibodies include FGL1 antibody (Proteintech) (Chiu CF, et al. Biomolecules. 2021;11 and Son Y,. Int J Mol Sci. 2021;22), ab197357 (Abcam) (Sun C et al. Respir Res. 2020;21:210), and anti-FGL1 (clone 177R4) (Wang Jet et al. Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3. Cell. 2019;176:334-47).

[0069] Those skilled in the art can employ routine techniques to generate humanized antibodies for treating iron overload-related diseases, such as beta-thalassemia, using the antigen-binding sequences (e.g., CDRs) of these antibodies.

[0070] Aptamers In another embodiment, the fibrinogen-like protein 1 (FGL1) antagonist is an aptamer against fibrinogen-like protein 1 (FGL1). Aptamers are a class of molecules that are alternatives to antibodies for molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences capable of recognizing virtually any class of target molecule with high affinity and specificity. Such ligands can be isolated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) of random sequence libraries, as described in Tuerk C. and Gold L., 1990. Random sequence libraries can be obtained by combinatorial chemical synthesis of DNA, in which each member is a linear oligomer of a unique sequence, ultimately chemically modified. The possible modifications, uses, and advantages of this class of molecules are reviewed in Jayasena SD, 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by platform proteins, e.g., R. coli thioredoxin A, selected from combinatorial libraries by the two-hybrid method (Colas et al., 1996).

[0071] Then, for the present invention, a neutralizing aptamer of fibrinogen-like protein 1 (FGL1) is selected as described above for its ability to (i) bind to fibrinogen-like protein 1 (FGL1) and / or (ii) inhibit the suppression of hepcidin expression or restore hepcidin expression (through the process of BMP6 antagonism).

[0072] B) Inhibitors of fibrinogen-like protein 1 (FGL1) gene expression In yet another embodiment, the fibrinogen-like protein 1 (FGL1) antagonist is an inhibitor of fibrinogen-like protein 1 (FGL1) gene expression. An "inhibitor of expression" refers to a natural or synthetic compound that has the biological effect of inhibiting gene expression. Thus, an "inhibitor of fibrinogen-like protein 1 (FGL1) gene expression" refers to a natural or synthetic compound that has the biological effect of inhibiting the expression of the fibrinogen-like protein 1 (FGL1) gene (or gene transcript: RNA).

[0073] In a preferred embodiment of the present invention, the inhibitor of fibrinogen-like protein 1 (FGL1) gene expression is an antisense oligonucleotide, a nuclease, an siRNA, an shRNA or a ribozyme nucleic acid sequence.

[0074] Inhibitors of fibrinogen-like protein 1 (FGL1) gene expression for use in the present invention can be based on antisense oligonucleotide constructs. Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, will act to directly block translation of fibrinogen-like protein 1 (FGL1) mRNA by binding to it, thereby preventing protein translation or increasing mRNA degradation, thereby reducing the level of fibrinogen-like protein 1 (FGL1) and therefore its activity in cells. For example, antisense oligonucleotides of at least about 15 bases complementary to unique regions of the mRNA transcript sequence encoding fibrinogen-like protein 1 (FGL1) can be synthesized, for example, by conventional phosphodiester technology, and can be administered, for example, by intravenous injection or infusion. Methods for using antisense technology to specifically inhibit gene expression of genes whose sequences are known are well known in the art (see, e.g., U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

[0075] Small inhibitory RNAs (siRNAs) can also function as inhibitors of fibrinogen-like protein 1 (FGL1) gene expression for use in the present invention. Fibrinogen-like protein 1 (FGL1) gene expression can be reduced by using small double-stranded RNA (dsRNA) or vectors or constructs that produce small double-stranded RNA, such that fibrinogen-like protein 1 (FGL1) gene expression is specifically inhibited (i.e., RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequences are known (see, e.g., Tuschi, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, ​​TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559, and WO 01 / 36646, 99 / 32619, and 01 / 68836).

[0076] Examples of siRNAs against fibrinogen-like protein 1 (FGL1) that can be used in the present invention are disclosed in Pei X, et al. Chem Eng J. 2021;421: 129774; Gong C, et al. J Nanobiotechnology. 2021;19:58; Sun C. et al Respir Res. 2020;21: 210; Son Y. et al Int. J. Mol. Sci. 2021, 22, 5330; Tang XY. Et al Transl Lung Cancer Res 2022;11(3):404-419.

[0077] Examples of commercial siRNAs against fibrinogen-like protein 1 (FGL1) include, but are not limited to, FGL1 siRNA(h) sc-62453 from Santa Cruz Biotechnology.

[0078] Inhibitors of fibrinogen-like protein 1 (FGL1) gene expression for use in the present invention may be based on nuclease therapy (e.g., Talen or Crispr).

[0079] The term "nuclease" or "endonuclease" refers to a synthetic nuclease that consists of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease and is used in gene targeting efforts. The synthetic nucleases of the present invention exhibit enhanced selectivity and specificity for bisected or trisected DNA target sites (i.e., TALEN or CRISPR recognition sites) that contain DNA binding and restriction endonuclease target sites, while preventing cleavage at off-target sites that contain only the restriction endonuclease target site.

[0080] The guide RNA (gRNA) sequence directs a nuclease (i.e., Cas9 protein) to induce a site-specific double-strand break (DSB) in genomic DNA at the target sequence.

[0081] The restriction endonucleases (also called restriction enzymes) of the present invention referred to herein are capable of recognizing and cleaving DNA molecules at specific DNA cleavage sites between defined nucleotides. In contrast, some endonucleases, such as Fokl, contain a cleavage domain that nonspecifically cleaves DNA at a particular position, regardless of whether a nucleotide is present at that position. Therefore, preferably, the specific DNA cleavage site and the DNA recognition site of a restriction endonuclease are identical. Furthermore, preferably, the cleavage domain of the chimeric nuclease is derived from a restriction endonuclease that has reduced DNA binding and / or reduced catalytic activity compared to the wild-type restriction endonuclease.

[0082] In accordance with the knowledge that restriction endonucleases, particularly type II restriction endonucleases, bind to DNA in an ordered manner as homodimers, the chimeric nucleases referred to herein may involve the homodimerization of two restriction endonuclease subunits. Preferably, according to the present invention, the cleavage module referred to herein has the ability to form reduced homodimers in the absence of a DNA recognition site, thereby preventing nonspecific DNA binding. Thus, functional homodimers are formed only upon recruitment of the chimeric nuclease monomer to a specific DNA recognition site. Preferably, the restriction endonuclease from which the cleavage module of the chimeric nuclease is derived is a type IIP restriction endonuclease. Preferably, the palindromic DNA recognition site of these restriction endonucleases consists of at least four or at most eight consecutive nucleotides. Preferably, type IIP restriction endonucleases cleave DNA within a recognition site or a site immediately adjacent to the recognition site that occurs more frequently in the genome. The IIIP-type restriction endonucleases referred to herein are preferably Pvull, EcoRV, BamHl, Bcnl, BfaSORF1835P, BfiI, Bgll, Bglll, BpuJl, Bse6341, BsoBl, BspD6I, BstYl, Cfr101, Ecl18kl, EcoO109l, EcoRI, EcoRI, EcoRV, EcoR124l, EcoR124ll, Selected from the group consisting of HinP11, Hincll, Hindlll, Hpy99l, Hpy188l, Mspl, Munl, Mval, Nael, NgoMIV, Notl, OkrAl, Pabl, Pacl, PspGl, Sau3Al, Sdal, Sfil, SgrAl, Thal, VvuYORF266P, Ddel, Eco57l, Haelll, Hall, Hindll and Ndel.

[0083] Examples of commercial gRNAs for fibrinogen-like protein 1 (FGL1) include, but are not limited to, FGL1 sgRNA CRISPR Lentivirus Set (human: CAT. No. 205331110101) from Applied Biological Materials, Human FGL1 Activation Kit with CRISPRa (CAT#: GA101589) from Origene, and Hepacosin CRISPR / Cas9 KO Plasmid (human ref. No. sc-409612) from SANTA CRUZ BIOTECHNOLOGY, INC.

[0084] Other nucleases for use in the present invention are disclosed in WO 2010 / 079430, WO 2011072246, WO 2013045480, Mussolino C, et al. (Curr Opin Biotechnol. 2012 Oct;23(5):644-50), and Papaioannou I. et al. (Expert Opinion on Biological Therapy, March 2012, Vol. 12, No. 3: 329-342), all of which are incorporated herein by reference.

[0085] Ribozymes may also function as inhibitors of fibrinogen-like protein 1 (FGL1) gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA, followed by endonucleolytic cleavage. Therefore, engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of the fibrinogen-like protein 1 (FGL1) mRNA sequence are useful within the scope of the present invention. First, specific ribozyme cleavage sites within any potential RNA target are identified by scanning the target molecule for ribozyme cleavage sites. Target molecules typically contain the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of approximately 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure. This characteristic may render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by examining their accessibility to hybridization with complementary oligonucleotides, using, for example, ribonuclease protection assays.

[0086] Antisense oligonucleotides, siRNAs, and ribozymes useful as inhibitors of fibrinogen-like protein 1 (FGL1) gene expression can be prepared by known methods. These include techniques for chemical synthesis, such as solid-phase phosphoramidite chemical synthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a variety of vectors incorporating appropriate RNA polymerase promoters, such as the T7 or SP6 polymerase promoter. Various modifications to the oligonucleotides of the present invention can be introduced as a means of improving intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiester linkages within the oligonucleotide backbone.

[0087] The antisense oligonucleotides, siRNAs, and ribozymes of the present invention can be introduced in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the introduction of an antisense oligonucleotide, siRNA, or ribozyme nucleic acid into a cell, preferably a cell expressing fibrinogen-like protein 1 (FGL1). Preferably, the vector transports the nucleic acid into the cell with reduced degradation relative to the level that would occur in the absence of the vector. Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources that have been engineered with the insertion or incorporation of an antisense oligonucleotide, siRNA, gRNA, or ribozyme nucleic acid sequence. Viral vectors are a preferred type of vector, including, but not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV-40 type viruses; polyoma viruses; Epstein-Barr virus; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses, such as retroviruses. Other vectors not named but known in the art can readily be utilized.

[0088] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentiviruses), whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by proviral integration into host cell DNA. Retroviruses have been approved for human gene therapy trials. The most useful retroviruses are those that are replication-deficient (i.e., capable of directing desired protein synthesis but unable to produce infectious particles). Such genetically modified retroviral expression vectors have general utility for highly efficient gene transduction in vivo. Standard protocols for producing replication-defective retrovirus (including the steps of incorporating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, producing recombinant retrovirus by the packaging cell line, harvesting the viral particles from tissue culture medium, and infecting target cells with the viral particles) are provided by KRIEGLER ("A Laboratory Manual," W.H. Freeman CO, New York, 1990) and MURRY ("Methods in Molecular Biology," vol. 7, Humana Press, Inc., Cliffton, NJ, 1991).

[0089] Preferred viruses for certain applications are adenoviruses and adeno-associated viruses. These viruses are double-stranded DNA viruses already approved for human use in gene therapy. Adeno-associated viruses can be engineered to be replication-deficient and are capable of infecting a wide range of cell types and species. Adeno-associated viruses have additional advantages, such as heat and aliphatic solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and the lack of superinfection inhibition, allowing for multiple transductions. Reportedly, adeno-associated viruses can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and alterations in the expression characteristics of inserted genes due to retroviral infection. In addition, wild-type adeno-associated virus infection has been demonstrated in tissue culture for over 100 generations in the absence of selective pressure. This indicates that adeno-associated virus gene integration is a relatively stable event. Adeno-associated viruses may also function extrachromosomally.

[0090] Other vectors include plasmid vectors. Plasmid vectors have been widely described in the art and are well known to those skilled in the art. See, for example, SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. For several years, plasmid vectors have been used as DNA vaccines to transfer antigen-encoding genes into cells in vivo. Plasmid vectors are particularly advantageous for this purpose because they do not have the same safety concerns as many viral vectors. However, these plasmids, which contain promoters compatible with the host cell, can express peptides from genes operably encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. Furthermore, plasmids can be custom-designed using restriction enzymes and ligation reactions to remove and add specific DNA fragments. Plasmids can be introduced via a variety of parenteral, mucosal, and topical routes. For example, DNA plasmids can be injected intramuscularly, intradermally, subcutaneously, or by other routes. They can also be administered by nasal spray or drops, rectal suppositories, and orally. They can also be administered to the epidermis or mucosal surfaces using a gene gun. Plasmids can be provided in aqueous solution, dried onto gold particles, or associated with other DNA delivery systems, including (but not limited to) liposomes, dendrimers, coclate, and microcapsules.

[0091] In a preferred embodiment, the antisense oligonucleotide, nuclease (i.e., CrispR containing gRNA), siRNA, shRNA, or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter, which may be specific for hepatocytes.

[0092] Methods for preventing or treating pathological conditions The present invention further contemplates a method of preventing or treating an iron overload-related disease in a subject, comprising administering to the subject a therapeutically effective amount of a fibrinogen-like protein 1 (FGL1) inhibitor.

[0093] In specific embodiments, the iron overload related disease is selected from the list consisting of hemochromatosis (adult and juvenile hereditary hemochromatosis), iron-loading anemia and chronic liver diseases, including alcoholic and non-alcoholic liver disease and chronic hepatitis B and C.

[0094] In a preferred embodiment, the iron-loading anemia is selected from the list consisting of β-thalassemia, congenital dyserythroid anemia, myelodysplastic syndrome (MDS).

[0095] Preferably, the iron overload related disease is beta-thalassemia.

[0096] In one aspect, the present invention provides a method of inhibiting an iron overload-related disease in a subject, the method comprising administering a therapeutically effective amount of a direct fibrinogen-like protein 1 (FGL1) inhibitor.

[0097] The "therapeutically effective amount" of the direct fibrinogen-like protein 1 (FGL1) inhibitor described above refers to an amount of the antagonist sufficient to prevent or treat iron overload-related disorders. However, it will be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective amount level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, weight, health, sex, and diet of the subject; the time of administration, route of administration, and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination with or simultaneously with the specific inhibitor employed, and similar factors well known in the medical field. For example, it is well known to those skilled in the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product can vary over a wide range, from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of active ingredient, with the dosage adjusted depending on the condition of the subject being treated. Pharmaceuticals typically contain about 0.01 mg to about 500 mg of active ingredient, preferably 1 mg to about 100 mg. Effective amounts of the drug are usually supplied at dosage levels of 0.0002 mg to about 20 mg per kg of body weight per day, and particularly about 0.001 mg to 7 mg per kg of body weight per day.

[0098] The present invention also relates to methods of treating iron overload-related disorders in subjects exhibiting low hepcidin levels in a biological sample (e.g., a blood sample or a urine sample) compared to a predetermined reference value with a direct fibrinogen-like protein 1 (FGL1) inhibitor.

[0099] The present invention also relates to direct fibrinogen-like protein 1 (FGL1) inhibitors for use in treating iron overload-related diseases in subjects exhibiting low hepcidin levels in a biological sample compared to a predetermined reference value.

[0100] The methods and uses include measuring the level of hepcidin protein expression (protein or nucleic acid sequence (DNA or mRNA)) in a biological sample obtained from the subject and comparing the measured level with a reference control value.

[0101] Low hepcidin levels predict a high risk of having or developing iron overload-related diseases (e.g., β-thalassemia, hemochromatosis, congenital dyserythroid anemia, and myelodysplastic syndromes), meaning that direct fibrinogen-like protein 1 (FGL1) inhibitors can be used.

[0102] Typically, a biological sample is obtained from a subject, and hepcidin levels are measured in this blood sample. Indeed, reducing fibrinogen-like protein 1 (FGL1) levels may be particularly beneficial for subjects with low levels of hepcidin.

[0103] Pharmaceutical compositions of the present invention The above-described inhibitors of fibrinogen-like protein 1 (FGL1) activity / inhibitors of fibrinogen-like protein 1 (FGL1) gene expression can be combined with pharmaceutically acceptable excipients, and optionally with sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0104] Thus, the present invention relates to a pharmaceutical composition comprising a direct fibrinogen-like protein 1 (FGL1) inhibitor of the present invention and a pharmaceutically acceptable carrier.

[0105] The present invention also relates to a pharmaceutical composition for use in preventing or treating iron overload-related diseases (e.g., β-thalassemia, hemochromatosis, congenital dyserythroid anemia, and myelodysplastic syndromes), comprising a direct fibrinogen-like protein 1 (FGL1) inhibitor of the present invention and a pharmaceutically acceptable carrier.

[0106] "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0107] In therapeutic applications, the compositions are administered to a patient already suffering from the described disease in an amount sufficient to cure or at least partially halt the symptoms of the disease and its complications. Appropriate doses of pharmaceutical compositions can be easily determined according to any one of several well-established protocols. For example, animal studies (e.g., in mice or rats) are commonly used to determine the maximum tolerated dose per kilogram of body weight of a bioactive agent. Typically, at least one of the animal species tested is a mammal. The results of animal studies can be extrapolated to determine dosages for use in other species, such as humans. What constitutes an effective dose also depends on the nature and severity of the disease or condition and the patient's health status.

[0108] In therapeutic treatment, the antagonist contained in the pharmaceutical composition can be administered in multiple doses or in a single dose until the desired response is achieved. Treatment is typically monitored, and repeated doses can be administered as needed. The compounds of the present invention can be administered according to established dosing regimens when inactivation of fibrinogen-like protein 1 (FGL1) is required.

[0109] The daily dosage of the product can vary over a wide range, from 0.01 to 1,000 mg per adult per day. Preferably, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient, with the dosage adjusted depending on the condition of the patient being treated. The pharmaceutical typically contains about 0.01 mg to about 500 mg of the active ingredient, preferably 1 mg to about 100 mg. An effective amount of the drug is usually supplied at a dosage level of 0.0002 mg to about 20 mg per kg of body weight per day, particularly about 0.001 mg to 10 mg per kg of body weight per day. It will be understood, however, that the specific dose level and frequency of administration for any particular subject may vary and will depend upon a variety of factors, including the activity of the particular compound employed, the metabolic stability and duration of action of that compound, age, body weight, health, sex, diet, method and time of administration, rate of excretion, drug combination, the severity of the particular condition and the host being treated.

[0110] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active principle can be administered alone or in combination with another active principle to animals and humans in unit dosage forms, in admixture with conventional pharmaceutical carriers. Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms.

[0111] Suitable unit dosage forms include oral dosage forms, such as tablets, gelatin capsules, powders, granules and orally ingested solutions or suspensions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, intramuscular, intravenous, intranasal or intraocular dosage forms and rectal dosage forms.

[0112] In the pharmaceutical composition of the present invention, the active principle is generally formulated as a dosage unit containing 0.5 to 1000 mg, preferably 1 to 500 mg, more preferably 2 to 200 mg of the active principle per dosage unit per day.

[0113] When preparing solid compositions in the form of tablets, a wetting agent, such as sodium lauryl sulfate, can be added to the active principle, which can optionally be finely divided, which is then mixed with a pharmaceutical vehicle, such as silica, gelatin, starch, lactose, magnesium stearate, talc, gum arabic, etc. The tablets can be coated with sucrose, various polymers or other suitable substances, or they can be treated in other ways to have a sustained or delayed release, so that a predetermined amount of the active principle is continuously released.

[0114] Preparations in the form of gelatin capsules are obtained by mixing the active principle with a diluent, for example a glycol or glycerol ester, and filling the mixture obtained into soft or hard gelatin capsules.

[0115] A preparation in the form of a syrup or elixir may contain the active principle together with a sweetener (preferably calorie-free), methylparaben and propylparaben as preservatives, a flavoring and an appropriate coloring.

[0116] The water-dispersible powders or granules may contain the active principle mixed with a dispersing or wetting agent or suspending agent, for example polyvinylpyrrolidone, and also with sweeteners or taste-modifying agents.

[0117] The active principle may also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives.

[0118] Among the sustained-release forms useful for chronic treatment, implants can be used, which can be prepared in the form of an oily suspension or a suspension of microspheres in an isotonic medium.

[0119] The direct fibrinogen-like protein 1 (FGL1) inhibitors of the present invention can be administered by any suitable route of administration. For example, the direct fibrinogen-like protein 1 (FGL1) inhibitors of the present invention can be administered orally (including buccal and sublingual), rectally, nasally, topically (intracolonically), pulmonary, vaginally, or parenterally (including intramuscular, intraarterial, intrathecal, subcutaneous, and intravenous) via administration.

[0120] For direct fibrinogen-like protein 1 (FGL1) inhibitors of the present invention, in preferred embodiments, the FGL1 inhibitors can be administered orally (including buccal and sublingually), rectally, or topically (intracolonic) by administration.

[0121] The direct fibrinogen-like protein 1 (FGL1) inhibitors of the present invention can be formulated into a variety of oral dosage forms.

[0122] The term "preparation" is intended to include formulating an active compound with an encapsulating material as a carrier, providing a capsule in which the active ingredient (with or without a carrier) is surrounded by the carrier in association with it. Cachets and lozenges are also included. Tablets, powders, capsules, pills, cachets, and lozenges may be solid preparations suitable for oral administration. Other forms suitable for oral administration include liquid preparations, including emulsions, syrups, elixirs, aqueous solutions, and aqueous suspensions, or solid preparations intended to be converted to liquid preparations immediately before use. Emulsions can be prepared in solutions, for example, in aqueous propylene glycol solutions, or can contain emulsifying agents, such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickeners. Aqueous suspensions can be prepared by dispersing the finely divided active ingredient in water with viscous substances, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. Solid preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0123] Combination of FGL1 inhibitor and ERFE inhibitor It has been previously reported that hepcidin expression is rapidly suppressed by the erythropoiesis regulator erythroferon (ERFE) in conditions associated with increased erythropoiesis, such as anemia caused by bleeding or inflammation. 10,11 Conversely, excessive release of ERFE occurs in congenital conditions caused by genetic mutations (beta-thalassemia, congenital dyserythropoietic anemia, myelodysplastic syndromes). 12-14 This leads to iron overload and severe clinical complications that threaten patient survival. In response to erythropoietin (EPO), ERFE is secreted by erythroid progenitor cells in the bone marrow and spleen and functions as a ligand trap that directly binds to BMP6 and inhibits the signaling cascade that induces hepcidin expression.15 The present invention relates to the identification of a novel hepcidin suppressor, fibrinogen-like 1 (FGL1), a hepatokine produced in the liver, that contributes to hepcidin regulation during anemia. Briefly, similar to erythroferon, FGL1 functions as a ligand trap for BMP6 to repress hepcidin transcription during recovery from hemorrhage.

[0124] This means that direct fibrinogen-like protein 1 (FGL1) inhibitors can be used in combination with erythroferon (ERFE) inhibitors to treat iron disorders associated with low hepcidin expression (e.g., β-thalassemia, hemochromatosis, congenital dyserythropoietic anemia, and myelodysplastic syndromes).

[0125] In certain embodiments, the iron overload related disease is selected from the list consisting of hemochromatosis (adult and juvenile hereditary hemochromatosis), iron-loading anemia and chronic liver diseases, including alcoholic liver disease and chronic hepatitis B and C.

[0126] In a more particular embodiment, the iron-loading anemia is selected from the list consisting of β-thalassemia, congenital dyserythroid anemia, myelodysplastic syndrome (MDS).

[0127] In some embodiments, the methods and uses of the present invention further comprise the step of applying an erythroferon (ERFE) inhibitor.

[0128] The present invention also relates to a method for preventing or treating an iron overload-related disease in a subject, comprising administering to the subject therapeutically effective amounts of a fibrinogen-like protein 1 (FGL1) inhibitor and an erythroferron (ERFE) inhibitor.

[0129] The present invention also relates to a pharmaceutical composition comprising a fibrinogen-like protein 1 (FGL1) inhibitor and an erythroferron (ERFE) inhibitor for simultaneous or sequential use in preventing or treating iron overload-related diseases.

[0130] As used herein, the term "ERFE," also known as "erythroferon," refers to a protein produced by erythroblasts that inhibits the action of hepcidin, thereby increasing the amount of iron available for hemoglobin synthesis. The sequence of the gene can be found in Ensembl Accession No. ENS00000178752.

[0131] The term "ERFE" inhibitor has its general meaning in the art and refers to a molecule (natural or synthetic) capable of neutralizing, blocking, inhibiting, suppressing, reducing, or interfering with the biological activity of erythroferon (ERFE) (the same biological activity as fibrinogen-like protein 1 (FGL1)) that inhibits hepcidin expression (through the process of BMP6 antagonism), including, for example, reducing or blocking the interaction between erythroferon (ERFE) and BMP6. Erythroferon (ERFE) inhibitors include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, drugs and their metabolites, transcriptional and translational control sequences, and the like. Inhibitors or antagonists also include antagonistic mutants of proteins, siRNA molecules against proteins, aptamers, which are antisense molecules against proteins, and ribozymes against proteins. For example, an erythroferon (ERFE) inhibitor or antagonist can be a molecule that binds to erythroferon (ERFE) and neutralizes, blocks, inhibits, suppresses, reduces or prevents the biological activity of erythroferon (ERFE) (such biological activity being the inhibition of hepcidin expression through direct inhibition of BMP6).

[0132] In some embodiments, the ERFE inhibitors of the present invention are A) Inhibitors of erythroferon (ERFE) activity, such as anti-ERFE antibodies (neutralizing antibodies) B) Inhibitors of erythroferon (ERFE) gene expression selected from the list consisting of antisense oligonucleotides, nucleases, siRNAs, shRNAs or ribozyme nucleic acid sequences. Including, but not limited to:

[0133] In a preferred embodiment, the therapeutic combination relates to inhibitors of the same type: an inhibitor of erythroferon (ERFE) activity and an inhibitor of fibrinogen-like protein 1 (FGL1) activity or an inhibitor of erythroferon (ERFE) gene expression and an inhibitor of fibrinogen-like protein 1 (FGL1) gene expression.

[0134] In a particular embodiment, the inhibitor of erythroferon (ERFE) activity is an antibody (a term including fragments or portions of antibodies, see definition above) that can directly or indirectly block the interaction of erythroferon (ERFE) with BMP6 protein.

[0135] Examples of erythroferrone (ERFE)-neutralizing monoclonal antibodies that can be used in accordance with the present invention are disclosed in Arezes, et al., "Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia," Blood. 2020 Feb 20;135(8):547-557, WO 2014071015, WO 2018027184, and WO 2019148186.

[0136] Those skilled in the art can employ routine techniques to use the antigen-binding sequences (eg, CDRs) of these antibodies to generate humanized antibodies for the treatment of iron overload-related diseases.

[0137] In yet another embodiment, the erythroferon (ERFE) antagonist is an inhibitor of erythroferon (ERFE) gene expression. An "inhibitor of expression" refers to a natural or synthetic compound that has the biological effect of inhibiting gene expression. Thus, an "inhibitor of erythroferon (ERFE) gene expression" refers to a natural or synthetic compound that has the biological effect of inhibiting the expression of the fibrinogen-like protein 1 (FGL1) gene (or gene transcript: RNA).

[0138] In a preferred embodiment of the present invention, the inhibitor of erythroferon (ERFE) gene expression is an antisense oligonucleotide, a nuclease, an siRNA, an shRNA or a ribozyme nucleic acid sequence.

[0139] Examples of siRNAs against erythroferon (ERFE) that can be used in the present invention are disclosed in Wang CY, et al. Blood. 2020 Feb 6; 135(6): 453-456.; Chen Q et al Cell Death Dis. 2021 May; 12(5): 417.

[0140] Examples of commercial siRNAs against erythroferron (ERFE) include, but are not limited to, ERFE siRNA (ID 151176) from Bioneer.

[0141] That is, the present invention relates to a fibrinogen-like protein 1 (FGL1) inhibitor and an erythroferron (ERFE) inhibitor for simultaneous or sequential use in the prevention or treatment of iron overload-related diseases in a subject in need thereof.

[0142] "Therapeutically effective amount" means an amount of a compound sufficient to prevent or treat an iron overload related disorder.

[0143] It will be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the age, weight, health, sex, and diet of the subject; the administration time, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or simultaneously with the specific inhibitor used, and similar factors well known in the medical field. For example, it is well within the skill of one in the art to start the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0144] The fibrinogen-like protein 1 (FGL1) inhibitor and erythroferon (ERFE) inhibitor of the present invention can be administered by any suitable route of administration. For example, the thrombin of the present invention can be administered orally (including buccal and sublingual), rectally, nasally, topically (intravesically), pulmonary, vaginally, or parenterally (including intramuscular, intraarterial, intrathecal, subcutaneous, and intravenous) or in a form suitable for administration by inhalation or insufflation.

[0145] Screening methods for treating iron overload-related diseases A further object of the present invention relates to a method for screening fibrinogen-like protein 1 (FGL1) inhibitors (or antagonists) for use in treating or preventing iron overload-related diseases.

[0146] For example, the screening method can measure the binding of a candidate compound to fibrinogen-like protein 1 (FGL1) or a cell tissue sample or organism expressing FGL1 or its fusion protein with a label directly or indirectly associated with the candidate compound. Furthermore, the screening method may include measuring or qualitatively or quantitatively detecting the ability of the candidate compound to inactivate the biological activity (see definition above) of FGL1.

[0147] In a specific embodiment, the screening method of the present invention comprises: (i) providing a purified FGL1 protein (or the C-terminal globular domain of fibrinogen) and providing a cell, tissue sample, or organism expressing FGL1; (ii) providing a candidate compound, e.g., a small organic molecule, a nucleic acid, an antibody, a peptide, or a polypeptide; (iii) measuring the activity of FGL1; (iv) positively selecting candidate compounds that block the biological activity of FGL1 or inhibit FGL1 expression; The process includes the steps of:

[0148] In certain embodiments, the screening method of the present invention may further comprise a step of administering the candidate compound selected in step d) to an animal model of an iron overload-related disease (i.e., beta-thalassemia) to verify the protective effect of the candidate compound.

[0149] Generally, such screening methods involve providing appropriate cells that express FGL1. In particular, the cells can be transfected with a nucleic acid encoding FGL1 to express the hormone of the present invention. Such transfection can be achieved by methods well known in the art. In a specific embodiment, the cells can be selected from the group consisting of mammalian cells (e.g., hepatocytes) that have already been reported to express FGL1.

[0150] The screening method of the present invention can be used to determine FGL1 inhibitors by contacting such cells with a compound to be screened and determining whether such a compound inactivates FGL1.

[0151] According to one embodiment of the present invention, candidate compounds can be selected from a library of previously synthesized compounds, a library of compounds whose structures have been determined in a database, or a library of newly synthesized or naturally occurring compounds. Candidate compounds can be selected from the group consisting of (a) proteins or peptides, (b) nucleic acids, and (c) organic compounds or (non-natural) compounds. By way of example, a library of preselected candidate nucleic acids can be obtained by performing the SELEX method described in US Pat. Nos. 5,475,096 and 5,270,163. By way of further example, candidate compounds can be selected from the group of antibodies against FGL1.

[0152] Candidate compounds can be tested for FGL1 inhibition by various known methods. For example, hepcidin expression assay (see above) can be used to perform the screening method of the present invention or in situ zymography assay in pathological or healthy tissue (see Example 1, Figure 1 and Figure 6). [Brief explanation of the drawings]

[0153] [Figure 1] Figure 1: Recovery from hemorrhage-induced anemia in WT mice. (A) Hemoglobin levels in 7- to 9-week-old WT male mice 0, 1, 2, 3, 4, 5, and 6 days after 500 μl of bloodletting. Expression of Epo mRNA in the kidney (B), Erfe mRNA in the bone marrow and spleen (C), and Hamp, Id1, and Smad7 mRNA in the liver (E) were measured. (D) Serum Erfe mRNA concentrations over time. Data shown are means ± sem. For each time point, values ​​were compared with those of control mice at t = 0 (n = 5–7 per time point) and control mice (day 0) by one-way analysis of variance. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 2]Figure 2. Erfe-independent repression of hepcidin during recovery from anemia. Iron-related parameters in 7- to 9-week-old Erfe- / - mice 0-6 days after phlebotomy (500 μl). Parameters included hemoglobin levels (A), Epo mRNA expression in the kidney (B), Hamp, Id1, and Smad7 mRNA expression in the liver (C), serum hepcidin concentration (D), serum iron content (E), transferrin saturation (F), and liver iron content (G). (H) Western blots for P-Smad5, Smad5, and vinculin in the liver of Erfe- / - mice 0, 1, and 2 days after phlebotomy. (I) Densitometric ratios of phosphorylated Smad5 to total Smad5 or vinculin and Smad5 to vinculin. Data shown are means ± sem and were compared with the values ​​from control mice (n = 5–8) at t = 0 for each time point by one-way ANOVA (A, B, C) or Student's t test (D, E, F, G, I). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 3] Figure 3: Validation of the potential contribution of erythroid regulators. Percent reticulocytes (A), Gypa and Tfr1 mRNA expression in bone marrow (B), hemoglobin levels (C), and Hamp mRNA expression in liver (D) of 8-week-old control and irradiated (400 rad) WT and Erfe- / - mice (n = 4-9) at t = 0 (white bars) and 48 hours (blue bars) after exsanguination. (E) Gypa mRNA expression in bone marrow, spleen, and liver of Erfe- / - mice (n = 5-8) at t = 0-6 days after exsanguination. (F) Linear regression analysis of Gypa and Hamp mRNA expression in liver and spleen. (G) Hamp mRNA expression in liver of 7-9-week-old WT control and splenectomized WT and Erfe- / - mice (n = 3-6) at t = 0 (white bars) and 48 hours (blue bars) after exsanguination. Data shown are means ± sem and were compared between groups by two-way analysis of variance. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 4]Figure 4: Fgl1 mRNA expression is induced in mouse livers during anemia. Time course of Fgl1 mRNA expression in the liver (A) and bone marrow (B) of WT and Erfe- / - mice (n = 5-8) 1-6 days after exsanguination. (C) Hamp and Fgl1 mRNA expression in the liver of 7-week-old mice (n = 5) t = 0-20 h after a single intraperitoneal injection of EPO (200 μg). (D) Fgl1 mRNA expression in the liver of 8-week-old WT, Th3 / +, and Erfe- / -;Th3 / + mice (n = 7-9). Relative mRNA expression of HIF target genes Vegfa, Gapdh, and Angptl1 in mouse primary hepatocytes cultured in serum-free or serum-containing medium and incubated in the presence of the prolyl hydroxylase inhibitor DMOG or under hypoxic conditions (2%) for 15 h compared with untreated cells (E). (F) Relative Fgl1 expression in mouse primary hepatocytes incubated under hypoxic conditions or in the presence of DMOG, relative Fgl1 expression in the livers of Vhl-deficient mice (G), and relative Fgl1 expression in the livers of mice treated with the prolyl hydroxylase inhibitor vadadustat (H). Data shown are means ± sem and were compared with control WT values ​​at t = 0 for each time point by two-way ANOVA (A, B, C) or with WT mice by Student's t test. Data shown for experiments with primary hepatocytes are the means of three independent experiments and were compared with control cells by Student's t test (E, F, G). ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 5]Figure 5: FGL1 is a hepcidin suppressor in vivo and in vitro. Relative HAMP expression in Hep3B and HepG2 cells in response to BMP6 (25 ng / ml, 6 hours) (A) or BMP6 plus recombinant FGL1 (10 μg / ml) (B). Relative ID1 expression in a hepatocellular carcinoma cell line treated with BMP6 and FGL1 (C). HAMP (D) and ID1 (E) expression in Hep3B cells and primary mouse hepatocytes treated with BMP6 and either Fc, full-length FGL1, or the N-terminal or globular domain of FGL1 for 6 hours. Liver HAMP RNA expression (F), serum hepcidin concentration (G), and liver Id1 mRNA expression (H) in mice (n = 5) treated with saline, Fc, or recombinant FGL1 (10 mg / kg) for 6 hours. Data shown are means ± sem from three independent experiments (A–E) or treated mice, and for each condition, were compared with untreated cells or control mice by Student's t test. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 6] Figure 6: Fgl1- / - mice exhibit a blunted response to bloodletting. (A) Fgl1 mRNA expression in the livers of male and female WT mice compared with control mice at 36 h post-hemorrhage. Red blood cell (RBC) counts (B) and hemoglobin (Hb) (C) in WT (white bars) and Fgl1- / - (black bars) mice at t = 0 or 36 h post-hemorrhage, Erfe mRNA expression in the bone marrow (D) and spleen (E), and Hamp (F), Id1 (G), and Smad7 (H) mRNA expression in the liver. Data shown are means ± sem (n = 5–11). Comparisons between groups were performed by two-way ANOVA with correction for multiple comparisons by the Holm-Sidak test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 7]Figure 7: FGL1 is a BMP antagonist. Relative expression of Hamp (A), Id1 (B), and Smad7 (C) mRNA in mouse primary hepatocytes treated with BMP ligand (10 ng / ml) and human Fc IgG2 (10 μg / ml) or Fc-FGL1 (10 μg / ml) for 6 hours. Data shown are the mean ± sem of three independent experiments. For each BMP, comparisons were made between Fc- or FGL1-treated cells and control cells by two-way ANOVA. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. (D) Western blot analysis of P-SMAD5, SMAD5, and GAPDH in Hep3B cells treated with BMP6 (10 ng / ml), ERFE (1 μg / ml), or FGL1 (10 μg / ml) for 6 hours. (E) Western blot of BMP6 and FGL1 pull-down assay for human Fc IgG2 and BMP6. [Figure 8] Figure 8: Validation of specific ASOs against mouse and human FGL1. Hep3B cells were transfected with 100 mM of each ASO. FGL1 mRNA expression was measured after 72 hours (A). FGL1 protein expression was detected by Western blot after 48 and 72 hours (B).

[0154] Example 1 method Animal models Erfe on a C57BL / 6J background - / -and Erfe+ / + mice were bred and housed in a specific pathogen-free barrier facility within the animal facility of INSERM US006. Fgl1- / - and WT controls on a C57Bl / 6N background were obtained from The European Mouse Mutant Archive (EMMA) and bred at Janvier labs (Le Genest St Isle) before being transferred to the animal facility of INSERM US006 at 4-5 weeks of age. Mice were housed under a standard 12-h light / dark cycle in accordance with European Union guidelines and provided with ad libitum access to water and standard laboratory mouse chow (Ssniff, iron 200 mg / kg). This study was approved by the Midi-Pyrenees Animal Ethics Committee. Thalassemia mice and vadadustat-treated mice were, respectively, housed in a specific pathogen-free barrier facility within the animal facility of INSERM US006. Fgl1- / - and WT controls on a C57Bl / 6N background were obtained from The European Mouse Mutant Archive (EMMA) and bred at Janvier labs (Le Genest St Isle). At 4-5 weeks of age, they were transferred to the animal facility of INSERM US006. Mice were housed under a standard 12-h light / dark cycle in accordance with European Union guidelines and provided with ad libitum access to water and standard laboratory mouse chow (Ssniff, iron 200 mg / kg). This study was approved by the Midi-Pyrenees Animal Ethics Committee. Thalassemia mice and vadadustat-treated mice were, respectively, housed in a specific pathogen-free barrier facility within the animal facility of INSERM US006. 18 and Tomas Ganz 19 Expression data for Vhl- / - mice was kindly provided by Carole Peyssonnaux 20 The samples were provided by [the author]. To study recovery from anemia, mice were exsanguinated (500 μL) by retroorbital puncture and analyzed 1–6 days later. Destruction of erythroid components was achieved by sublethal X-ray irradiation (400 rad) in mice, and the mice were exsanguinated 48 hours later. Surgical removal of spleens was performed on 7–8-week-old WT and Erfe- / - mice. The mice were allowed to recover for 7 days before exsanguination. Some WT mice received a single dose of EPO (200 U) and were analyzed 12, 15, 18, or 20 hours later. Recombinant FGL1, Fc fragment, or saline was intraperitoneally administered at a dose of 10 mg / kg to 7-week-old C57Bl / 6J mice fed an iron-replete diet (Ssniff, 50 mg / kg) for 2 weeks, and the mice were analyzed 6 hours later. For all mice, tissues were collected and divided into samples that were snap-frozen in liquid nitrogen for RNA, protein, and iron measurements, and samples in 4% formalin for paraffin embedding. Male mice were preferentially studied unless otherwise noted.

[0155] Production of recombinant FGL1 The mouse FGL1 cDNA sequence (full-length, N-terminal domain, and globular domain) and human FGL1 sequence were cloned into the pFUSEN-hG2Fc plasmid (Invivogen) with the following modifications: The vector signal sequence (interleukin-2) was used instead of the native sequence, followed by the Fc fragment of human IgG2. Recombinant protein was produced in suspension culture in transiently transfected Freestyle 293F cells (Life Technologies) using FectroPro reagent (Polyplus). Supernatant from cells overexpressing Fc-tagged FGL1 protein was collected after 5 days and supplemented with a protease inhibitor cocktail (Sigma). Recombinant protein was purified using a Hitrap protein A HP column mounted on an AKTA pure chromatography system (GE Healthcare) and eluted with 0.1 M glycine, pH 3.5. The eluted fraction was concentrated using a Spin-X UF 20 centrifugal concentrator (Corning), and the recombinant FGL1 protein was suspended in saline (0.9% NaCl). Protein purity and concentration were measured using Coomassie Imperial Protein Stain and Pierce bicinchoninic acid protein assay (ThermoFisher Scientific).

[0156] Mouse ERFE immunoassay A human recombinant monoclonal antibody against mouse ERFE was produced by Biorad using HuCAL technology. High-binding 96-well plates (Corning) were coated overnight at 4°C with 2 μg / ml capture antibody (100 μl / well) diluted in 50 mM sodium carbonate buffer, pH 9.6. The plates were washed (TBS, 0.5% Tween 20) and blocked with 300 μL / well of blocking buffer (PBS, 0.2% Na-casein, 0.05% Tween 20, 0.1 M NaCl) for 1 hour at room temperature. Recombinant mouse ERFE standards were serially diluted to 10, 5, 2.5, 1.25, and 0.625 ng / ml. Serum samples diluted in PBS and standards diluted in PBS + 5% BSA were incubated for 1 hour at room temperature. Plates were washed and incubated with 0.5 μg / ml biotinylated detection antibody (100 μL / well) in PBS + 5% BSA for 1 hour. Plates were washed and incubated with 1 / 5000 Neutravidin-HRP (Pierce) (100 μL / well) in PBS + 5% BSA for 45 minutes. Plates were developed with 100 mL / well Ultrasensitive TMB Substrate (Thermofisher) in the dark at room temperature. The reaction was stopped by adding 50 μL of 0.2 N sulfuric acid, and absorbance was measured at 450 nm.

[0157] Measurement of iron and hematological parameters Serum iron concentration was measured by the iron direct method (ferene dBiolabo, 92108), and transferrin saturation was estimated by measuring unsaturated iron binding capacity (UIBC, Biolabo, 97408). Liver iron content was measured as previously described. 21 Complete blood counts were performed using a Cell-Dyn Emerald hematology analyzer (Abbott).

[0158] Western blot analysis Liver proteins were extracted by physical dissociation using an ULTRA-TURRAX® (IKA) in PEB buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% NP-40) containing protease inhibitors (cOmplete™, Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2, Sigma). Hep3B cells were lysed in RIPA buffer (Thermofischer, 89900) containing protease and phosphatase inhibitors. Freshly extracted proteins were diluted with Laemmli buffer 2x (Sigma), incubated at 95°C for 10 min, subjected to SDS-PAGE, and electroblotted onto a nitrocellulose membrane (Biorad). The membrane was blocked for 1 hour with 5% nonfat dry milk (NFDM, Cell Signaling) diluted in TBS-T buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.15% Tween 20) and then incubated overnight at 4°C with an antibody against phospho-Smad5 (Ser463 / 465, Abcam, ab92698, 1 / 2000) diluted in TBS-T buffer (5% BSA) or for 2 hours at RT with an antibody against Smad5 (Abcam, ab40771, 1 / 5000) diluted in TBS-T buffer (5% BSA). Loading was measured using antibodies against GAPDH (Cell Signaling, D16H11, 1 / 10,000) or vinculin (Cell Signaling, 4650, 1 / 20,000) diluted in TBST-NFDM (5%) for 2 h at RT. After three washes, membranes were incubated with HRP-conjugated goat anti-human IgG (Novus Biologicals, NBP1-75006, 1 / 10,000), goat anti-rabbit IgG (Cell Signaling, 7074, 1 / 10,000), or horse anti-mouse IgG (Cell Signaling, 7076, 1 / 10,000) secondary antibodies diluted in TBST-NFDM (5%) for 2 h at RT.Enzyme activity was developed using ECL Prime reagent (GE Healthcare) on a ChemiDoc XRS+ imaging system.

[0159] Pull-down assay One microgram of Fc-tagged recombinant proteins (Fc alone, full-length FGL1, the globular portion of FGL1, and the N-terminal portion of FGL1) was incubated with protein A magnetic beads (Dynabeads, Pierce) in NETN buffer (20 mM Tris-HCl pH 8.0, 0.5% NP-40, 100 mM NaCl, 1 mM EDTA pH 8.0, protease inhibitor cocktail (Sigma P8340)) overnight at 4°C with or without hBMP6 (Biotech). Proteins were eluted using Laemmli buffer and analyzed by Western blot using goat anti-human IgG Fc fragment secondary antibody [HRP] (Novus biological NBP1-75006) or anti-BMP6 antibody (R&D systems, AF6325).

[0160] Cell treatment Hep3B and HepG2 cells were cultured in Dulbecco's modified Eagle's medium-high glucose GlutaMAX, 10% fetal bovine serum, and 1% penicillin-streptomycin unless otherwise noted. Cells were seeded 24 hours prior to treatment and treated with serum-free medium for 6 hours. Hepatocytes were perfused with collagenase via the portal vein as previously described. 22Cells were isolated from wild-type C57BL / 6 mice by ELISA. Cells were cultured overnight (15 h) in fresh Williams E medium (Gibco) supplemented with 200 μM L-glutamine and 10% FBS. Hep3B and HepG2 cells, as well as primary hepatocytes, were treated with 25 ng / ml BMP6 (Peprotech) or BMPs 2, 4, and 7 (R&D Systems) and Fc (hIgG2), full-length Fc-FGL1 (FL), its N-terminal domain (Nter), or globular domain for 6 h. For hypoxia experiments, cells were maintained in serum-free medium for 15 h in a hypoxia chamber (Whitley, H35 Hypoxystation) at 2% oxygen or in the presence of 1 mM DMOG (dimethyloxalylglycine, N-(methoxyoxoacetyl)-glycine methylmester, Sigma) in a conventional CO2 incubator. To examine FGL1 regulation by inflammatory cytokines, HepB cells were treated with 20 ng / ml IL-6 or 50 ng / ml TNFα (R&D Systems) for 6 hours.

[0161] Quantification of mRNA levels Total RNA was extracted from mouse tissues using the Trizol (MRC) / chloroform (Sigma) method. Complementary cDNA was synthesized using M-MLV reverse transcriptase (Promega). Messenger RNA (mRNA) expression levels were assessed by quantitative polymerase chain reaction (RT-qPCR) (primers are listed in Table 1) using Takyon SYBR Green (Eurogentec). RT-qPCR was performed in duplicate on a LightCycler480 (Roche) instrument. Transcription levels were normalized to the reference gene Hprt and expressed as the difference between the reference and target genes within each group of mice (-ΔCt) ± standard error of the mean (SEM). Data from albumin-Cre / VHLflox / flox mice were normalized to the reference gene 36B4. Results from in vitro treatment were expressed as fold changes, i.e., log-transformed data (2 ΔΔCt) which indicates expression relative to the control condition. Expression data for the control condition were normalized to the control mean to obtain distribution within the control group (Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-408). Statistical significance was determined using Student's t-test or analysis of variance (ANOVA).

[0162] Microarray Total RNA was extracted from mouse liver and bone marrow of Erfe- / - mice at t = 0, 24, and 48 h post-exsanguination using Trizol (MRC) / chloroform (Sigma). RNA quality was assessed with an RNA 6000 Nano chip using a Bioanalyzer 2100 (Agilent Technologies). Gene-level expression profiling of liver and bone marrow from exsanguinated mice was performed at the GeT-TriX facility (GenoToul, Genopole Toulouse Midi-Pyrenees) using Agilent SurePrint G3 Mouse GE v2 microarrays (8x60K, design 074809) according to the manufacturer's instructions. For each sample, cyanine-3 (Cy3)-labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling Kit (Agilent Technologies) followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) according to the manufacturer's instructions. Dye incorporation and cRNA yield were confirmed using a Dropsense 96 UV / VIS drop reader (Trinean, Belgium). 600 ng of Cy3-labeled cRNA was hybridized onto the microarray slides according to the manufacturer's instructions. Immediately after washing, the slides were scanned on an Agilent G2505C microarray scanner using Agilent Scan Control A.8.5.1 software, and the fluorescent signal was extracted using Agilent Future Extraction v10.10.1.1 (using default parameters). Microarray data and experimental details are available in NCBI's Gene Expression Omnibus7 and can be accessed through GEO series accession number GSE229041 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE229041).Expression data were analyzed using R (Rv3.1.2)-bioconductor and iDEP (Integrated Differential Expression and Pathway analysis). 23 Control mice (n=3) were analyzed by comparing them with mice at 24 hours (n=3) and 48 hours (n=3) using the ELISA.

[0163] Statistical analysis of microarray data Microarray data were analyzed using R (R Core Team, 2018) and Bioconductor packages8, as described in GEO accession number GSE229041. Raw data (median signal intensities) were filtered, log2-transformed, and normalized using the quantile method (Bolstad BM, Irizarry RA, Astrand M, Speed ​​TP. A comparison of normalization methods for high-density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19(2):185-193). Initial exploratory and statistical analyses indicated the possibility of correlation structure between gene expression, which could negatively affect the multi-test approach. To mitigate the dependency structure, the FAMT method10 was applied using a model including one additional factor. Models were fitted using the limma lmFit function (Ritchie ME, Phipson B, Wu D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47). Pairwise comparisons between biological conditions were applied using specific contrasts. Corrections for multiple testing were applied using the Benjamini-Hochberg method to control the false positive rate (FDR). Probes with an FDR ≤ 0.01 were considered differential between conditions. Hierarchical clustering was applied to samples and differential probes using the 1-Pearson correlation coefficient as the distance and Ward's criterion as the agglomeration criterion. Expression data were further analyzed using R iDEP (Integrated Differential Expression and Pathway analysis) by performing three comparisons: control mice (n=4) vs. mice 24 hours post-phlebotomy (n=4), control mice vs. mice 48 hours post-phlebotomy, and mice 48 hours post-phlebotomy vs. mice 24 hours post-phlebotomy.We focused on transcripts encoding secreted proteins that were significantly upregulated 24 hours after exsanguination compared with control mice and whose expression remained stable or increased between 24 and 48 hours. Liver and bone marrow were analyzed separately.

[0164] statistical analysis Statistical significance was assessed by Student's t-test, one-way analysis of variance, or two-way analysis of variance (ANOVA) using Prism 9 (GraphPad). Statistics shown for two-way ANOVA are the results of the Holm-Sidak multiple comparison test.

[0165] result ERFE-independent repression of hepcidin during anemia. First, we characterized the timeline for recovery from phlebotomy-induced anemia in WT and Erfe-deficient mice. In both genotypes, hemoglobin and hematocrit levels decreased for 3 days after phlebotomy and significantly improved by day 6 (Figure 1A, data not shown). Consequently, Epo mRNA expression in the kidney was rapidly induced within 2 days and gradually returned to normal by day 6 (Figure 1B). Erfe mRNA expression in the bone marrow and spleen increased to a maximum after 24 hours and then gradually returned to baseline, whereas Erfe mRNA levels remained slightly elevated even after 6 days (Figure 1C). In contrast, serum ERFE levels were highest 24 hours after phlebotomy and declined below the detection limit within 3–4 days (Figure 1D). On the other hand, a significant decrease in hepcidin mRNA expression in the liver was maintained for 5 days (Figure 1E), accompanied by a mild decrease in the BMP target genes Id1 and Smad7 mRNA 3 days after phlebotomy. Therefore, we investigated whether hepcidin might be regulated independently of ERFE during recovery from anemia in Erfe- / - mice. Similar to WT mice, hemoglobin levels in Erfe- / - mice reached a nadir 3 days after phlebotomy (Figure 2A) and nearly recovered by day 6. Erfe- / - mice showed a slight increase in MCV, MCH, and RDW 3 days after phlebotomy compared with WT mice (data not shown). Epo mRNA expression was rapidly induced in the kidney 24 hours after phlebotomy and gradually returned to normal after 6 days (Figure 2B). Consistent with the stress hormone properties of ERFE, Hamp mRNA expression remained unchanged after 24 hours but declined to levels comparable to those in WT mice 2–3 days after phlebotomy and then returned to normal after 6 days. Meanwhile, Id1 and Smad7 mRNA expression remained unchanged (Figure 2C). Similar results were observed in phlebotomized female mice (data not shown). Therefore, we decided to focus on mechanisms induced within 24–48 hours after phlebotomy, but not beyond 48 hours.Serum hepcidin concentrations and hepcidin / liver iron content ratios were consistent with liver Hamp mRNA expression in WT and Erfe- / - mice at 1 and 2 days after phlebotomy, whereas liver iron content and Bmp6 mRNA expression remained unchanged (Figure 2D and data not shown). Therefore, we focused on mechanisms induced within 24–48 h after phlebotomy that may contribute to hepcidin suppression in Erfe- / - mice. The changes in hepcidin synthesis in Erfe- / - mice occurred without any changes in serum iron concentration, transferrin saturation, or liver iron content compared with control mice at 1 and 2 days after phlebotomy (Figure 2E, 2F, 2G). Similarly, SMAD5 phosphorylation was not reduced in the liver of Erfe- / - mice at 2 days after phlebotomy (Figure 2H–I). No statistically significant increases in Gdf15 and Twsg1 mRNA expression were detected in the bone marrow and spleen of exsanguinated WT and Erfe- / - mice (data not shown). These data indicate that hepcidin expression is negatively regulated by an ERFE-independent mechanism. ERFE was initially identified by searching for transcripts induced in the bone marrow 9–15 h after exsanguination. We hypothesized that another bone marrow-derived erythroid regulator or a factor derived directly from the liver might repress hepcidin 24–48 h after exsanguination. To this end, we analyzed the transcriptome profiles of exsanguinated Erfe- / - mice compared with control mice at 1 and 2 days after exsanguination using microarrays.

[0166] Examine the potential contribution of erythroid regulators. Hepcidin suppression may be related to the role of another erythroid regulator, 24,25To determine whether hepcidin is mediated by hemorrhage, we abolished erythroid components by irradiation and examined the hepcidin response to hemorrhage in WT and Erfe- / - mice. 48 h after hemorrhage, WT and Erfe- / - mice showed an increase in reticulocytes, whereas irradiated mice showed a significant decrease in circulating reticulocytes (Figure 3A). The reduction in reticulocyte production was accompanied by a decrease in the expression of erythroid markers Gypa and Tfr1 in the bone marrow of irradiated WT and Erfe- / - mice compared with their controls. Thus, successful erythroid depletion was confirmed (Figure 3B). Hemoglobin levels were reduced in all hemorrhagic groups, more significantly in irradiated mice, but no difference was observed between WT and Erfe- / - mice (Figure 3C). Hepcidin mRNA expression in the liver was repressed in control WT and Erfe- / - mice at 48 h after phlebotomy, but not in irradiated mice (Figure 3D). Interestingly, recovery from anemia was paralleled by increased Gypa mRNA expression in the spleen and liver compared with control mice 1–6 days after phlebotomy (Figure 3E). Hamp mRNA expression was inversely correlated with Gypa mRNA expression in the liver and spleen (Figure 3F). This suggests that hepcidin suppressors may be released from the liver and spleen. Therefore, we performed surgical removal of the spleens of WT and Erfe- / - mice to assess their response to hemorrhage. However, splenectomized WT and Erfe- / - mice showed decreased Hamp mRNA expression in the liver at 48 h after phlebotomy, indicating that the hepcidin suppressor is not derived from the spleen (Figure 3G). Therefore, to explore the liver and bone marrow responses to anemia, we analyzed the transcriptome profiles of exsanguinated Erfe − / − mice at 1 and 2 days post-exsanguination compared with control mice.

[0167] Fgl1 mRNA expression is induced in the liver of mice during anemia. To identify potential regulators of hepcidin, we searched for transcripts encoding secreted proteins whose expression was induced 24 and 48 h after phlebotomy compared with control mice. 63 and 38 transcripts were found to be induced (fold change >2; p value <0.05) in the liver and bone marrow, respectively, 24 h after phlebotomy compared with control mice (data not shown). Six transcripts in the liver and 23 transcripts in the bone marrow remained induced 48 h after phlebotomy compared with control mice (data not shown). In the liver, only Fgl1, Gdf15, and Cxcl1 encoded secreted proteins. Interestingly, fibrinogen-like 1 (Fgl1) mRNA expression was increased in both the liver and bone marrow. qRT-PCR revealed that Fgl1 mRNA expression was significantly induced in the liver (Figure 4A) and bone marrow (Figure 4B) of WT and Erfe- / - mice 1 to 3 days after phlebotomy, although the expression level was several orders of magnitude higher in the liver. In contrast, Gdf15 mRNA expression was mildly induced 1 to 2 days after phlebotomy, although it has been reported that Gdf15 does not contribute to hepcidin regulation during hemorrhage-induced anemia. Cxcl1 mRNA expression was only induced in Erfe- / - mice after 24 hours (data not shown). This did not correlate with the time course of hepcidin repression. Furthermore, unlike Fgl1, stimulation of Gdf15 and Cxcl1 was restricted to the liver. Therefore, we focused on FGL1 as a remaining potential candidate. Fibrinogen-like 1 (FGL1), also known as hepacosin 26 or HFREP-127, is a member of the fibrinogen protein family produced by hepatocytes and is an angiopoietin-like protein (ANGPTL). 28 It shares structural similarities with fibrinogen beta and gamma subunits, including a C-terminal globular domain that is homologous to the fibrinogen beta and gamma subunits. In contrast to other fibrinogen-related factors, FGL1 lacks the platelet-binding and thrombin-sensitive sites involved in blood clot formation. 27,29 Instead, FGL1 was induced during liver regeneration and showed growth-promoting activity on hepatocytes. 30FGL1 is also involved in evading tumor invasion in certain cancers through its interaction with the LAG-3 receptor. 31 Intraperitoneal injection of EPO (200 μg) into WT mice significantly reduced Hamp mRNA expression and increased Erfe mRNA expression in the bone marrow, but did not stimulate Fgl1 expression (Figure 4C). In contrast, Fgl1 mRNA expression was upregulated in the livers of Erfe-deficient thalassemic Th3 / + and Th3 / + mice (Figure 4D). Analysis of the mouse Fgl1 promoter revealed two HIF-binding sites (data not shown). To examine whether Fgl1 expression was stimulated in the livers of anemic mice by oxygen desaturation, we compared Fgl1 expression in mouse primary hepatocytes incubated under hypoxic conditions (2% O2) or in the presence of the prolyl hydroxylase inhibitor DMOG with control conditions. Increased expression of Vegfa, Gapdh, and Angptl1, target genes of hypoxia-inducible factors, was observed in cells cultured in serum-free or serum-containing medium and incubated in the presence of DMOG or hypoxic conditions (2%) for 15 hours compared to untreated cells (Figure 4E). Similarly, Fgl1 mRNA expression was induced in primary mouse hepatocytes incubated under hypoxic conditions or in the presence of DMOG (Figure 4F). A tendency for increase was observed in albumin-Cre / Vhl-deficient mice. 20 On the other hand, a significant increase in Fgl1 mRNA expression was observed in the liver of Hif2α-overexpressing mice (Fig. 4G). 32 and mice chronically treated with the prolyl hydroxylase inhibitor vadadustat. 19Fgl1 mRNA expression was detected in the liver of WT mice fed diets containing 10, 50, 200, or 8000 mg / kg iron for 2 weeks (Figure 4H). These results suggest that Fgl1 expression may be regulated by hypoxia-inducible factors. Consistent with previous studies, WT mice fed diets containing 10, 50, 200, or 8000 mg / kg iron for 2 weeks showed no change in Fgl1 mRNA expression in the liver, whereas Hamp and Id1 mRNA expression varied according to the iron content of the diet (data not shown). This indicates that Fgl1 is not regulated by iron. FGL1 has been reported as an acute-phase protein, and like hepcidin, its expression has been reported to be mildly induced by IL-6 and repressed by TNFα (data not shown).

[0168] FGL1 is a hepcidin suppressor in vivo and in vitro. Next, we evaluated the contribution of FGL1 to hepcidin regulation. HAMP mRNA expression was induced 600-fold and 200-fold in response to BMP6 (25 ng / ml, 6 h) in Hep3B and HepG2 cells, respectively (Fig. 5A). Treatment with recombinant Fc-tagged FGL1 for 6 h under serum-free conditions significantly reduced HAMP and ID1 expression in both hepatoma cell lines (Fig. 5B, 5C). In serum-containing medium, higher doses of FGL1 were required to repress HAMP and ID1 expression (data not shown). Mouse and human FGL1 share 82% identity, and human FGL1 also suppressed HAMP and ID1 mRNA expression in Hep3B cells, but at higher concentrations (data not shown). Therefore, we decided to use mouse FGL1 in this study. Injection of recombinant FGL1 (10 mg / kg) into WT mice significantly reduced Hamp RNA expression in the liver and serum hepcidin levels compared with Fc-treated mice (n = 5) at 6 h postinjection (Figures 5F–G), whereas no change in Id1 mRNA expression in the liver was observed (Figure 5H). Consistent with hepcidin repression, serum iron levels increased and liver iron levels decreased after FGL1 treatment (Figure 4H). In female WT mice, FGL1 treatment did not repress hepcidin expression. The significant inflammatory response to FGL1 administration (indicated by increased Saa1 levels), which was not observed in male mice, may have prevented the effect of FGL1. Finally, FGL1 was able to repress hepcidin expression in Hep3B cells, even in the presence of IL-6 (data not shown). Taken together, these results indicate that ERFE is a potent suppressor of hepcidin.

[0169] Fgl1- / - mice exhibit a blunted response to phlebotomy. To determine whether FGL1 contributes to hepcidin regulation during recovery from anemia, we compared WT and Fgl1- / - mice at 36 h after phlebotomy. First, we confirmed that Fgl1 mRNA expression was significantly increased in the livers of male and female WT mice compared with control mice at 36 h after phlebotomy (Figure 6A). Red blood cell counts and hemoglobin levels (Figure 6B-C) were decreased in WT and Fgl1- / - mice at 36 h after phlebotomy. Similar increases in Erfe mRNA expression in the bone marrow and spleen were detected in both genotypes at 36 h after phlebotomy (Figure 6D-E). Interestingly, Hamp mRNA expression in the liver was found to be reduced in both male and female exsanguinated WT and Fgl1- / - mice compared with control mice, but to a lesser extent in Fgl1- / - mice (Figure 6F). This suggests that FGL1 contributes to hepcidin suppression. No changes in Id1 or Smad7 mRNA expression were observed (Figures 6G-H). Consistent with the blunted hepcidin repression, serum iron concentrations were lower in exsanguinated Fgl1- / - mice compared with WT mice (Figure 6G). These results indicate that FGL1 increases during recovery from anemia and contributes to hepcidin suppression.

[0170] The globular domain of FGL1 is responsible for hepcidin suppression. Mouse FGL1 consists of a signal peptide for secretion, a short coil-coil N-terminal domain, and a C-terminal globular domain homologous to the fibrinogen β and γ chains (data not shown). To confirm these results and identify the active domain of FGL1, mouse primary hepatocytes were treated with Fc, full-length FGL1, or its N-terminal and globular domains. Full-length FGL1 and the globular domain repressed HAMP and ID1 expression (Figures 5D and 5E), whereas the N-terminal domain was inactive.

[0171] FGL1 is a BMP antagonist. Next, we investigated the mechanism by which FGL1 represses hepcidin. Treatment of hepatocytes with FGL1 downregulated ID1 mRNA expression, so we tested whether FGL1 could function as a BMP antagonist. We observed that FGL1 repressed the induction of Hamp and Id1 mRNA expression by BMP6 and BMP7, but not by BMP2 and BMP4, in mouse primary hepatocytes compared with control and Fc-treated cells (Figures 7A-7B). On the other hand, treatment of cells with FGL1 had no effect on Smad7 mRNA expression (Figure 7C). Pretreatment of cells with BMP6 before addition of FGL1 confirmed its ability to repress hepcidin and BMP target genes (data not shown). In addition, FGL1 significantly reduced SMAD5 phosphorylation in BMP6-treated Hep3B cells (Figure 7D). Furthermore, Fc or Fc-FGL1 (FL, glob, and Nter) were incubated with BMP6 and subjected to pull-down assays using protein A magnetic beads. FGL1 FL and glob interacted with BMP6, but the N-terminal domain interacted to a lesser extent, and the Fc fragment did not (Figure 7E). These results suggest that, similar to erythropoietin, FGL1 functions as a ligand trap for BMP6 to repress hepcidin transcription during hemorrhage recovery.

[0172] [Table 1]

[0173] Example 2 Methods: Antisense oligonucleotides specific for mouse and human FGL1 were synthesized. Hep3B cells were transfected with 100 mM of each ASO using INTERFERin (Polyplus). FGL1 mRNA expression was measured by qRT-PCR 72 hours later. FGL1 protein expression was detected by Western blot using an anti-FGL1 antibody (Santa Cruz Biotechnologies sc-514057) 48 and 72 hours later.

[0174] Results: FGL1 mRNA expression was repressed by 50-60% in Hep3B cells 72 hours after transfection with ASOs 1, 4, 5, 6, and 7 compared with cells transfected with a scrambled ASO (Figure 8A). Compared with the scrambled ASO, FGL1 protein levels were reduced in Hep3B cells transfected with ASOs 1 and 6 after 48 and 72 hours (Figure 8B).

[0175] Example 3 The effect of three fragments of the globular domain (SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14) was tested and demonstrated the ability to reduce hepcidin expression in a cell model (data not shown).

[0176] Table Section [Table 2] TIFF2026506602000003.tif246165 TIFF2026506602000004.tif30165

[0177] References Throughout this application, various references are made to describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure. [Table 3] TIFF2026506602000006.tif233165 TIFF2026506602000007.tif74165

Claims

1. A fibrinogen-like protein 1 (FGL1) inhibitor for use in treating a subject suffering from an iron overload-related disease.

2. Fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 1, wherein the inhibitor directly binds to the fibrinogen-like protein 1 (FGL1) protein or the nucleic acid sequence (DNA or mRNA) of FGL1 and (ii) inhibits the suppression of hepcidin expression.

3. 3. The fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 1 or 2, wherein the iron overload-related disease is selected from the list consisting of hemochromatosis (adult and juvenile hereditary hemochromatosis), iron-loading anemia and chronic liver diseases including alcoholic liver disease and chronic hepatitis B and C.

4. 4. The fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 3, wherein the iron-loaded anemia is selected from the list consisting of alpha-thalassemia, beta-thalassemia, congenital dyserythroid anemia, and myelodysplastic syndrome (MDS).

5. The fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 4, wherein the iron-loading anemia is beta-thalassemia.

6. The inhibitor is 1) an inhibitor of fibrinogen-like protein 1 (FGL1) activity and / or 2) Inhibitors of fibrinogen-like protein 1 (FGL1) gene expression 6. The fibrinogen-like protein 1 (FGL1) inhibitor for use according to any one of claims 1 to 5, wherein

7. Fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 6, wherein the inhibitor of fibrinogen-like protein 1 (FGL1) activity is selected from the list consisting of fibrinogen-like protein 1 (FGL1) neutralizing antibodies, fibrinogen-like protein 1 (FGL1) aptamers.

8. Fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 6, wherein the inhibitor of fibrinogen-like protein 1 (FGL1) gene expression is selected from the list consisting of antisense oligonucleotides, nucleases, siRNA, shRNA or ribozyme nucleic acid sequences.

9. The fibrinogen-like protein 1 (FGL1) inhibitor for use according to any one of claims 1 to 8 in a subject exhibiting a low hepcidin level in a biological sample compared to a predetermined reference value.

10. 1. A method of preventing or treating an iron overload-related disorder in a subject, comprising: administering to the subject therapeutically effective amounts of a fibrinogen-like protein 1 (FGL1) inhibitor and an erythroferon (ERFE) inhibitor; method.

11. The method for prevention or treatment according to claim 10, wherein the inhibitors directly bind to fibrinogen-like protein 1 (FGL1) (protein or nucleic acid sequence (DNA or mRNA)) and erythroferon (ERFE) (protein or nucleic acid sequence (DNA or mRNA)), respectively, and (ii) inhibit the suppression of hepcidin expression.

12. 12. The method for prevention or treatment according to claim 10 or 11, wherein the iron overload-related disease is selected from the list consisting of hemochromatosis (adult-onset and juvenile-onset hereditary hemochromatosis), iron-loading anemia, and chronic liver diseases including alcoholic liver disease and chronic hepatitis B and C.

13. 13. The method for prevention or treatment according to claim 12, wherein the iron-loaded anemia is selected from the list consisting of alpha-thalassemia, beta-thalassemia, congenital dyserythropoietic anemia, and myelodysplastic syndrome (MDS).

14. The method for prevention or treatment according to claim 13, wherein the iron-loading anemia is beta-thalassemia.

15. below: (i) providing a purified FGL1 protein (or the C-terminal globular domain of fibrinogen) and providing a cell, tissue sample or organism (the organism is neither human nor animal) expressing FGL1; (ii) providing a candidate compound, e.g., a small organic molecule, a nucleic acid, an antibody, a peptide, or a polypeptide; (iii) measuring the activity of FGL1; (iv) positively selecting candidate compounds that block the biological activity of FGL1 or inhibit FGL1 expression; The process comprises the steps of: An in vitro method for screening fibrinogen-like protein 1 (FGL1) inhibitors for use in the treatment or prevention of iron overload-related diseases.

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