WO2024170505A1

WO2024170505A1 - Methods of treatment of iron overload associated diseases

Info

Publication number: WO2024170505A1
Application number: PCT/EP2024/053500
Authority: WO
Inventors: Léon KAUTZ
Original assignee: Institut National de la Santé et de la Recherche Médicale; Université Toulouse Iii – Paul Sabatier; Institut National De Recherche Pour L’Agriculture, L’Alimentation Et L’Environnement (Inrae); Ecole Nationale Vétérinaire de Toulouse
Priority date: 2023-02-13
Filing date: 2024-02-12
Publication date: 2024-08-22

Abstract

In the present invention, inventor identified the hepatokine FGL1 as a previously undescribed suppressor of hepcidin that is highly induced in the liver in response to hypoxia during the recovery from anemia and in thalassemic mice. They demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgl1 in mice (Fgl1 -/-) results in a lower repression of hepcidin after bleeding. Finally, inventors clearly demonstrate the FGL1 is a BMP antagonist that directly binds BMP6 to impair the canonical BMP-SMAD signaling cascade that governs hepcidin regulation. So, the present invention relates to a method for preventing or treating iron overload associated diseases by targeting the new hepcidin repressor, the hepatokine FGL1.

Description

METHODS OF TREATMENT OF IRON OVERLOAD ASSOCIATED DISEASES

FIELD OF THE INVENTION:

The present invention relates to a method for preventing or treating iron overload associated diseases such as P -thalassemia, haemochromatosis, congenital dyserythropoietic anaemia and myelodysplastic syndrome by targeting the newly identified hepcidin repressor, the hepatokine FGL1 (Fibrinogen-like protein 1).

BACKGROUND OF THE INVENTION:

Anemia, defined as a decreased number of functional red blood cells, is a major cause of morbidity and mortality affecting one-third of the worldwide population¹. A large subset of conditions including iron deficiency, bleeding, infections and genetic disorders result in anemia. Iron is an essential functional component of erythrocytes hemoglobin which requires a sustained delivery of iron to the bone marrow for erythropoiesis in order to ensure proper tissue oxygenation^2,3. Iron is released from iron recycling macrophages, enterocyte and hepatocytes by the sole iron exporter ferroportin. The liver-derived hormone hepcidin regulates body iron content by binding to ferroportin leading to its occlusion and degradation^{4 5}.

Hepcidin synthesis is predominantly regulated by the canonical BMP-SMAD signaling pathway and the bone morphogenetic proteins BMP2 and BMP6⁶'⁸. Binding of BMP2/6 to a large receptor complex leads to the phosphorylation of SMAD1, 5 and 8 effectors that translocate into the nucleus to activate hepcidin transcription⁹. Hepcidin expression is rapidly suppressed by the erythroid regulator erythroferrone (ERFE) in conditions associated with expanded erythropoiesis such as anemia caused by bleeding or inflammation^{10 11}. Conversely, excessive release of ERFE in inherited conditions caused by genetic mutations (beta thalassemia, congenital dyserythropoietic anemia, myelodysplastic syndromes)¹²'¹⁴ causes iron overload and severe clinical complications threatening patients’ survival. In response to erythropoietin (EPO), ERFE is secreted by erythroid precursors in the bone marrow and the spleen and acts as a ligand trap that directly binds BMP 6 to inhibit the signaling cascade directing hepcidin expression⁷⁵.

Although ERFE is essential for the suppression of hepcidin within the first hours following an erythropoietic stress, Er/e-deficient mice recover from anemia induced by hemorrhage and chronic inflammation^{10 11}. Similarly, ablation or neutralization of ERFE in thalassemic mice^16,17 increase hepcidin levels and mitigate the systemic iron content. However, restoration of physiological levels of hepcidin is not sufficient to correct the iron overload and hepcidin synthesis remains inappropriately low in comparison to the liver iron content. Collectively, these data indicated that an ERFE-independent mechanism repressed hepcidin during anemia.

Inventors therefore examined hepcidin regulation during the recovery from hemorrhage- induced anemia in WT and Ar/c-deficient mice and confirmed the ERFE independent repression of hepcidin during anemia. Here inventors describe the identification of a new hepcidin suppressor that may contribute to hepcidin regulation during anemia: the liver produced hepatokine Fibrinogen-like protein 1 (FGL1).

SUMMARY OF THE INVENTION:

A first object of the invention relates to a Fibrinogen-like protein 1 (FGL1) inhibitor for use in the treatment of a subject affected with an iron overload associated disease.

A second object of the invention relates to a combination of a Fibrinogen-like protein 1 (FGL1) inhibitor and an Erythroferrone (ERFE) inhibitor for simultaneous or sequential use in preventing or treating iron overload associated disease.

A third object of the invention relates to a method, in particular an in vitro method, for screening a Fibrinogen-like protein 1 (FGL1) inhibitor (or antagonist) for use in the treatment or prevention of iron overload associated disease

In a particular embodiment, the iron overload associated disease is selected from the list consisting of hemochromatosis (adult and juvenile forms of hereditary hemochromatosis), iron- loading anemia and chronic liver diseases including alcoholic liver diseases and chronic hepatitis B and C.

In a particular embodiment, the iron-loading anemias is selected from the list consisting of alpha-thalassemia, P-thalassemia, congenital dyserythropoietic anemia, myelodysplastic syndrome (MDS).

DETAILED DESCRIPTION OF THE INVENTION:

In the present invention, inventor identified the hepatokine FGL1 as a previously undescribed suppressor of hepcidin that is highly induced in the liver in response to hypoxia during the recovery from anemia and in thalassemic mice. They demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgll in mice (Fgl 1 -/-) results in a blunted repression of hepcidin after bleeding. Finally, inventors clearly demonstrate that FGL1 is a BMP antagonist that directly binds BMP6 to impair the canonical BMP-SMAD signaling cascade that governs hepcidin regulation. Finally, as demonstrated in example 2, inventors developed specific antisense oligonucleotides directed against human and mouse FGL1 that were validated in hepatic cells. These antisense oligonucleotides have the potential to prevent the induction of FGL1 and the subsequent suppression of hepcidin in murine models of anemia.

Thus, the present invention provides methods and compositions (such as pharmaceutical compositions) for preventing or treating an iron overload associated disease.

In the context of the invention, the term "treatment or prevention" means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of the disorder may consist in reducing iron overload caused by low hepcidin expression in conditions such as P-thalassemia, haemochromatosis, Congenital dyserythropoietic anaemia and myelodysplastic syndromes (MDS). Most preferably, such treatment leads to the complete depletion of the pathological features as observed in iron overload associated disease.

Preferably, the individual to be treated is a human or non-human mammal (such as a rodent a feline, a canine, or a primate) affected or likely to be affected with hepcidin dysfunctions as observed in iron overload associated disease.

Preferably, the individual is a human.

Fibrinogen-like protein 1 (FGL1) inhibitors

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 affected with an iron overload associated disease.

In the present invention, inventors demonstrated that Fibrinogen-like protein 1 (FGL1) expressed in the liver is directly involved in modulating hepcidin expression, mainly via acting as a BMP6 antagonist.

In a more particular embodiment, the iron-loading anemia is selected from the list consisting of P-thalassemia, congenital dyserythropoietic anemias, myelodysplastic syndrome (MDS).

As used herein the term “Fibrinogen-like protein 1 (abbreviated as FGL1 or "fibrinogen- like protein 1" (synonyms LFIRE-1, HFREPI, Hepassocin, H P-041) refers to a protein of the fibrinogen family of proteins, which also includes fibrinogen-like protein 2 and clotting factors V, VIII, and XIII,, that, in humans, is encoded by the FGL1 gene (Gene ID: 2267). However, the term "FGL 1 expression" relates to both, protein and mRNA expression in humans unless otherwise stated. FGL1 contains in its C-terminal portion homologous to fibrinogen, which contains the four conserved cysteines that are common to all members of the fibrinogen family. However, FGL1 lacks the platelet-binding site, cross-linking region, and thrombin-sensitive site which allow the other members of the fibrinogen family to aid in fibrin clot formation. FGL1 is upregulated in regenerating liver and is abundantly associated with the fibrin matrix after clot formation. While the majority of FGL 1 is found in plasma, approximately 20% of FGL 1 remains in the serum after blood coagulation. Human FGL 1 precursor has the amino acid sequence as provided by the NCBI Reference Sequence database under ID number NP 004458 (see also Uniprot ref. Q08830), wherein amino acids 1 to 22 correspond to the signal peptide and amino acids 23 to 312 correspond to the mature protein. The N-terminal domain of fibrinogen like protein 1 (FGL1) corresponds to amino acids 23 to 78. The C-terminal globular domain of fibrinogen-like protein 1 corresponds to amino acids 79 to 312.

FGL-1 has also been observed to strongly bind to and activate LAG-3, a regulatory protein expressed on T cells. As LAG-3 has an important role in controlling activated T cells, manipulating FGL-1 binding to T cells has been proposed for both cancer immunotherapy and anti-inflammatory treatments (Wang J, et al. (2019). Cell. 176 (1-2): 334-347. el2)

A "Fibrinogen-like protein 1 (FGL1) inhibitor" or "Fibrinogen-like protein 1 (FGL1) antagonist" refers to a molecule (natural or synthetic) capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the biological activities of Fibrinogen-like protein 1 (FGL1) including, for example, reduction or blocking the interaction for instance 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, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Inhibitors or antagonists also include, antagonist variants of the protein, siRNA molecules directed to a protein, antisense molecules directed to a protein, aptamers, and ribozymes against a protein. For instance, the Fibrinogen-like protein 1 (FGL1) inhibitor or antagonist may be a molecule that binds to Fibrinogen-like protein 1 (FGL1) and neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of Fibrinogen-like protein 1 (FGL1) (such biological activity being inducing inhibition of hepcidin expression through direct inhibition of BMP6 in response to hypoxia during the recovery from anemia and in thalassemic mice).

In some embodiments, the FGL1 inhibitor is a direct Fibrinogen-like protein 1 (FGL1) inhibitor. Accordingly, a “Direct Fibrinogen-like protein 1 (FGL1) Inhibitor" is a Fibrinogen- like protein 1 (FGL1) inhibitor /antagonist which directly binds to Fibrinogen-like protein 1 (FGL1) (protein or nucleic sequence (DNA or mRNA)) and neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of Fibrinogen-like protein 1 (FGL1).

In the context of the present invention, the direct Fibrinogen-like protein 1 (FGL1) inhibitor (i) directly binds to Fibrinogen-like protein 1 (FGL1) (protein or nucleic sequence (DNA or mRNA)) and (ii) inhibits suppression of hepcidin expression (through blocking Fibrinogen-like protein 1 (FGLl)-induced BMP6 antagonism process).

More particularly, the direct Fibrinogen-like protein 1 (FGL1) inhibitor according to the invention is:

1) an inhibitor of Fibrinogen-like protein 1 (FGL1) activity (such as small organic molecule, antibody, aptamer, polypeptide) and/ or

2) an inhibitor of Fibrinogen-like protein 1 (FGL1) gene expression (such as antisense oligonucleotide, nuclease, siRNA, ...)

By "biological activity" of Fibrinogen-like protein 1 (FGL1) is meant in the context of the present invention, inhibition of hepcidin expression (through the BMP6 antagonism process).

Tests for determining the capacity of a compound to be a Fibrinogen-like protein 1 (FGL1) inhibitor are well known to the person skilled in the art. In a preferred embodiment, the antagonist/inhibitor specifically binds to Fibrinogen-like protein 1 (FGL1) (protein or nucleic sequence (DNA or mRNA)) in a sufficient manner 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) may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as a Fibrinogen-like protein 1 (FGL1) inhibitor to bind to Fibrinogen-like protein 1 (FGL1). The binding ability is reflected by the Kd measurement. The term "Kd", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). Kd values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, 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 IpM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of Fibrinogen-like protein 1 (FGL1) inhibition of hepcidin expression through blocking Fibrinogen-like protein 1 (FGLl)-induced BMP6 antagonism process.

By “inhibitor of the Fibrinogen-like protein 1 (FGL1) activity”, it is herein referred to a compound which is capable of reducing or suppressing the inhibition of hepcidin expression. In view of the teaching of the present disclosure, particularly of the examples, it falls within the ability of the skilled person to assess whether a compound is an inhibitor of the hepcidin- suppressive activity. For example, a suitable test consists in evaluating if said compound blocking the inhibition of hepcidin expression on hepatic cells i.e. restore the hepcidin-induced expression by BMP6/SMAD2 pathway. A suitable test for detecting hepcidin-induced expression is described in examples hereinafter.

The functional assays based on hepcidin expression may be envisioned such as evaluating the ability to block the inhibition of hepcidin expression processes (in hepatic cells or in murine beta thalassemia model) through blocking the inhibition of BMP6 by Fibrinogen- like protein 1 (FGL1). Antagonization 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 assay based experimental murine model of anemia (bleeding, chronic inflammation) or iron overload (i.e. beta thalassemia model) may also be used such as evaluating the ability to elevate hepcidin levels and decrease liver and serum iron level (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 instance serum iron level can be assessed by widely used colorimetric assays or by transferrin saturation (Biolabo, Iron direct method & TIBC), transferrin being the iron carrier in the plasma and both parameters are currently used as a diagnostic test for iron-deficiency anemia or iron-overload disease.

The skilled in the art can easily determine whether a Fibrinogen-like protein 1 (FGL1) antagonist neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of Fibrinogen-like protein 1 (FGL1). to check whether the Fibrinogen-like protein 1 (FGL1) antagonist /inhibitor binds to Fibrinogen-like protein 1 (FGL1) and/or is able to inhibit processes associated with inhibition of hepcidin expression (through the BMP6 antagonism process), , in the same way than the initially characterized inhibitor of Fibrinogen-like protein 1 (FGL1), binding assay and/or a BMP6 activity assay may be performed with each antagonist. Accordingly, the direct Fibrinogen-like protein 1 (FGL1) inhibitor may be a molecule that binds to Fibrinogen-like protein 1 (FGL1) selected from the group consisting, antibodies, aptamers.

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

The term "iron overload associated disease" refers to a group of diseases and/or disorders which are generally associated with abnormally low levels of hepcidin and includes diseases wherein aberrant iron metabolism directly causes the disease, or wherein iron blood levels are dysregulated causing disease, or wherein iron dysregulation is a consequence of another disease, or wherein diseases can be treated by modulating iron levels, and the like. More specifically, a disease of iron metabolism according to this disclosure includes iron overload diseases, disorders of iron biodistribution, other disorders of iron metabolism and other disorders potentially related to iron metabolism, etc. Diseases 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, congenital dyserythropoietic anemia.

In a particular embodiment, the iron overload associated disease is selected from the list consisting of hemochromatosis (adult and juvenile forms of hereditary hemochromatosis), iron- loading anemias and chronic liver diseases including alcoholic liver diseases and chronic hepatitis B and C.

The term “Iron loading anaemia” also called “secondary iron overload” refers to diseases characterized by anaemia, high serum iron, transferrin saturation and ferritin values, and haemosiderin deposits in parenchymal cells and reticuloendothelial tissue with or without organ dysfunction.

In a more particular embodiment, the iron-loading anemia is selected from the list consisting of P-thalassemia, congenital dyserythropoietic anemias, myelodysplastic syndrome (MDS). In some embodiments, the iron overload disease is myelodysplastic syndrome. In some cases the diseases and disorders included in the definition of "disease of iron overload" are not typically identified as being iron related. For example, hepcidin is highly expressed in the murine pancreas suggesting that diabetes (Type I or Type II), insulin resistance, glucose intolerance and other disorders may be ameliorated by treating underlying iron metabolism disorders. See Ilyin, G. et al. (2003) FEBS Lett. 542 22-26, which is herein incorporated by reference. As such, these diseases are encompassed under the broad definition. Those skilled in the art are readily able to determine whether a given disease is a "disease or iron metabolism" according to the present invention using methods known in the art, including the assays of WO 2004092405, which is herein incorporated by reference, and assays which monitor hepcidin, hemojuvelin, or iron levels and expression, which are known in the art such as those described in U.S. Patent No. 7,534,764, which is herein incorporated by reference. In some

As used herein, diseases and disorders related to hepcidin deficit include adult and juvenile forms of hereditary hemochromatosis, alpha-thalassemia, P-thalassemia and congenital dyserythropoietic anemias, and chronic liver diseases including alcoholic liver disease and chronic hepatitis B and C

Accordingly in a particular embodiment of the present invention, the diseases of iron metabolism are iron overload diseases directly related to hepcidin deficit which include hemochromatosis (adult and juvenile forms of hereditary hemochromatosis), or secondary iron overload associated diseases, also called iron-loading anemias (which include alphathalassemia, P-thalassemia, congenital dyserythropoietic anemias, myelodysplastic syndrome) and chronic liver diseases including alcoholic liver diseases, non-alcoholic steatohepatitis (NASH) and chronic hepatitis B and C.

In a more particular embodiment, the secondary iron overload associated disease (or iron-loading anemia) is selected from the list consisting of P-thalassemia, congenital dyserythropoietic anemia, myelodysplastic syndrome (MDS).

Preferably the iron overload associated disease is P -thalassemia.

Beta thalassemias (P thalassemias) are a group of inherited blood disorders. They are forms of thalassemia caused by reduced or absent synthesis of the beta chains of hemoglobin that result in variable outcomes ranging from severe anemia to clinically asymptomatic individuals. Global annual incidence is estimated at one in 100,000 (Galanello, et al (2010). Orphanet J Rare Dis. 5: 11). Beta thalassemias occur due to malfunctions in the hemoglobin subunit beta or HBB. The severity of the disease depends on the nature of the mutation. HBB blockage over time leads to decreased beta-chain synthesis and accumulation of alpha chains causing premature apoptosis of erythroid precursors. Moreover, the body's inability to construct new beta-chains leads to the underproduction of HbA (adult hemoglobin). Reductions in HbA available overall to fill the red blood cells in turn leads to microcytic anemia. Microcytic anemia ultimately develops in respect to inadequate HBB protein for sufficient red blood cell functioning. Due to this factor, the subject may require blood transfusions to make up for the blockage in the beta-chains. Repeated blood transfusions cause severe problems associated with iron overload(Galanello, et al (2010). Orphanet J Rare Dis. 5: 11).

P -thalassemia is not medically curable except for the use of blood transfusion, although frequent blood transfusions that lead to or potentiate iron overload (Greer, John P.; et al (2013). Wintrobe's Clinical Hematology. Lippincott Williams & Wilkins.. Accordingly there is a medical need to specifically treat B-thalassemia subject with new therapeutic approach.

In another embodiment the iron overload associated disease is myelodysplastic syndrome.

A myelodysplastic syndrome (MDS) is one of a group of blood cancers in which immature blood cells in the bone marrow do not mature, and as a result, do not develop into healthy blood cells. Early on, no symptoms are typically seen. Later symptoms may include fatigue, shortness of breath, bleeding disorders, anemia, or frequent infections. Some types may develop into acute myeloid leukemia. Risk factors include previous chemotherapy or radiation therapy, exposure to certain chemicals such as tobacco smoke, pesticides, and benzene, and exposure to heavy metals such as mercury or lead. Problems with blood cell formation result in some 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 or blood. While anemia is the most common cytopenia in MDS subjects, given the ready availability of blood transfusion, MDS subjects rarely experience injury from severe anemia. The two most serious complications in MDS subjects resulting from their cytopenias are bleeding (due to lack of platelets) or infection (due to lack of white blood cells). Long-term 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).

Treatments may include supportive care, drug therapy, and hematopoietic stem cell transplantation. Supportive care may include blood transfusions, medications to increase the making of red blood cells, and antibiotics. Drug therapy may include the medications lenalidomide, antithymocyte globulin, and azacitidine.

Typically, a direct Fibrinogen-like protein 1 (FGL1) inhibitors according to the invention includes but is not limited to: A) Inhibitor of Fibrinogen-like protein 1 (FGL1) activity such as, anti- FGL1 antibody and Anti- FGL1 aptamers

B) Inhibitor of Fibrinogen-like protein 1 (FGL1) gene expression selected from the list consisting of antisense oligonucleotide, nuclease, siRNA, shRNA or ribozyme nucleic acid sequence.

A) Inhibitor of Fibrinogen-like protein 1 (FGL1) activity

• Antibody

In one embodiment, the Fibrinogen-like protein 1 (FGL1) inhibitor of activity is an antibody (the term including antibody fragment or portion) that can block directly or indirectly the interaction of Fibrinogen-like protein 1 (FGL1) with BMP6 protein.

In preferred embodiment, the Fibrinogen-like protein 1 (FGL1) antagonist may consist in an antibody directed against the Fibrinogen-like protein 1 (FGL1), in such a way that said antibody impairs the binding of a Fibrinogen-like protein 1 (FGL1) to BMP6 and able of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the biological activities of Fibrinogen-like protein 1 (FGL1) ("neutralizing antibody"). In some embodiments, the neutralizing antibody of the invention is directed against the globular domain of FGL1 (SEQ ID NO: 11). In some embodiments, the neutralizing antibody of the invention is directed against SEQ ID NO: 12. In some embodiments, the neutralizing antibody of the invention is directed against SEQ ID NO: 13. In some embodiments, the neutralizing antibody of the invention is directed against SEQ ID NO: 14.

Then, for this invention, neutralizing antibody of Fibrinogen-like protein 1 (FGL1) are selected as above described for their capacity to (i) bind to Fibrinogen-like protein 1 (FGL1) (protein) and/or (ii), inhibiting the suppression of hepcidin expression in liver (through the BMP6 antagonism process) or restoring hepcidin expression.

In the present invention, inventors clearly demonstrate that 1) full length FGL1 (in vitro and in vivo) and FGL1 globular domain (in vitro) repress hepcidin expression (Figure 5D-E), and 2) reduce serum expression in mouse (Figure 5F-G) 3) FGL1 is a BMP antagonist that directly binds BMP6 to impair the canonical BMP-SMAD signaling cascade that governs hepcidin regulation (Figures 7). Altogether, these results indicate that also similar to erythroferrone, FGL1 may act as a ligand trap for BMP6 to repress hepcidin transcription during the recovery from hemorrhage.

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 portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody 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.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of Fibrinogen-like protein 1 (FGL1). The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

Briefly, the recombinant Fibrinogen-like protein 1 (FGL1) may be provided by expression with recombinant cell lines or bacteria. Recombinant form of Fibrinogen-like protein 1 (FGL1) may be provided using any previously described method. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as 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). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

Significantly, as it is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization 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 hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may be used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., I. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can 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, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to 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 will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. 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.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by 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 have been replaced by 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 have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or nonhuman sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.

In another embodiment, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

The skilled artisan can use routine technologies to use the antigen-binding sequences of these antibodies (e.g., the CDRs) and generate humanized antibodies for treatment of iron overload associated disease (such as B-thalassemia as disclosed herein).

Examples of neutralizing monoclonal of Fibrinogen-like protein 1 (FGL1) that can be used according to the invention are disclosed in : Qian et al. J Hematol Oncol (2021) 14: 147; WO2019241098; W02022007543). Example of such anti FGL1 are FGL1 antibody, (Proteintech) (Chiu CF, et al. Biomolecules. 2021;l 1 and Son Y,. Int J Mol Sci. 2021;22) abl97357 (Abeam) (Sun C et al. Respir Res. 2020;21 :210) and Anti- FGL1 (clone 177R4) (Wang Jet al. Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3. Cell. 2019;176:334-47) .

The skilled artisan can use routine technologies to use the antigen-binding sequences of these antibodies (e.g., the CDRs) and generate humanized antibodies for treatment of iron overload associated disease such as beta-thalassemia as disclosed herein.

• Aptamer

In another embodiment, the Fibrinogen-like protein 1 (FGL1) antagonist is an aptamer directed against Fibrinogen-like protein 1 (FGL1). aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

Then, for this invention, neutralizing aptamers of Fibrinogen-like protein 1 (FGL1) are selected as above described for their capacity to (i) bind to Fibrinogen-like protein 1 (FGL1) and/or (ii) and inhibiting suppression of hepcidin expression (through the BMP6 antagonism process) or restoring hepcidin expression.

B) Inhibitor of Fibrinogen-like protein 1 (FGL1) gene expression

In still 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 a biological effect to inhibit the expression of a gene. Therefore, an "inhibitor of Fibrinogen-like protein 1 (FGL1) gene expression" denotes a natural or synthetic compound that has a biological effect to inhibit the expression of Fibrinogen-like protein 1 (FGL1) gene (or the gene transcript : RNA).

In a preferred embodiment of the invention, said inhibitor of Fibrinogen-like protein 1 (FGL1) gene expression is antisense oligonucleotide, nuclease, siRNA, shRNA or ribozyme nucleic acid sequence.

Inhibitors of Fibrinogen-like protein 1 (FGL1) gene expression for use in the present invention may be based on antisense oligonucleotide constructs. Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, would act to directly block the translation of Fibrinogen-like protein 1 (FGL1) mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Fibrinogen-like protein 1 (FGL1), and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding Fibrinogen-like protein 1 (FGL1) can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see 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).

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 a vector or construct causing the production of a 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 sequence is known (e.g. see Tuschi, T. et al. (1999); Elbashir, S. M. 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 International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Example of siRNAs against Fibrinogen-like protein 1 (FGL1) that can be used according to the 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.|

Example of commercial siRNAs, against Fibrinogen-like protein 1 (FGL1) include, but are not limited to: FGL1 siRNA (h) sc-62453 from Santa Cruz Biotechnology

Inhibitors of Fibrinogen-like protein 1 (FGL1) gene expression for use in the present invention may be based nuclease therapy (like Talen or Crispr).

The term “nuclease” or “endonuclease” means synthetic nucleases consisting of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease which are used for gene targeting efforts. The synthetic nucleases according to the invention exhibit increased preference and specificity to bipartite or tripartite DNA target sites comprising DNA binding (i.e. TALEN or CRISPR recognition site(s)) and restriction endonuclease target site while cleaving at off-target sites comprising only the restriction endonuclease target site is prevented.

The guide RNA (gRNA) sequences direct the nuclease (i.e. Cas9 protein) to induce a site-specific double strand break (DSB) in the genomic DNA in the target sequence.

Restriction endonucleases (also called restriction enzymes) as referred to herein in accordance with the present invention are capable of recognizing and cleaving a DNA molecule at a specific DNA cleavage site between predefined nucleotides. In contrast, some endonucleases such as for example Fokl comprise a cleavage domain that cleaves the DNA unspecifically at a certain position regardless of the nucleotides present at this position. Therefore, preferably the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are identical. Moreover, also preferably the cleavage domain of the chimeric nuclease is derived from a restriction endonuclease with reduced DNA binding and/or reduced catalytic activity when compared to the wildtype restriction endonuclease. According to the knowledge that restriction endonucleases, particularly type II restriction endonucleases, bind as a homodimer to DNA regularly, the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonuclease subunits. Preferably, in accordance with the present invention the cleavage modules referred to herein have a reduced capability of forming homodimers in the absence of the DNA recognition site, thereby preventing unspecific DNA binding. Therefore, a functional homodimer is only formed upon recruitment of chimeric nucleases monomers to the specific DNA recognition sites. Preferably, the restriction endonuclease from which the cleavage module of the chimeric nuclease is derived is a type IIP restriction endonuclease. The preferably palindromic DNA recognition sites of these restriction endonucleases consist of at least four or up to eight contiguous nucleotides. Preferably, the type IIP restriction endonucleases cleave the DNA within the recognition site which occurs rather frequently in the genome, or immediately adjacent thereto, and have no or a reduced star activity. The type IIP restriction endonucleases as referred to herein are preferably selected from the group consisting of: Pvull, EcoRV, BamHl, Bcnl, BfaSORF1835P, Bfil, Bgll, Bglll, BpuJl, Bse6341, BsoBl, BspD6I, BstYl, CfirlOl, Ecll8kl, EcoO1091, EcoRl, EcoRll, EcoRV, EcoR1241, EcoR12411, HinPl l, Hindi, Hindlll, Hpy991, Hpyl881, Mspl, Muni, Mval, Nael, NgoMIV, Notl, OkrAl, Pabl, Pad, PspGl, Sau3Al, Sdal, Sfil, SgrAl, Thai, VvuYORF266P, Ddel, Eco571, Haelll, Hhall, Hindll, and Ndel.

Example of commercial gRNAs against Fibrinogen-like protein 1 (FGL1) include, but are not limited to: FGL1 sgRNA CRISPR Lentivirus set (Human : CAT.N°205331110101) from Applied Biological Materials, Human FGL1 activation kit by CRISPRa (CAT#: GA101589) from Origene, Hepassocin CRISPR/Cas9 KO Plasmid (human ref.N° sc-409612) from SANTA CRUZ BIOTECHNOLOGY, INC.

Other nuclease for use in the present invention are disclosed in WO 2010/079430, WO201 1072246, W02013045480, 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 herein incorporated by reference.

Ribozymes can 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 complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Fibrinogen-like protein 1 (FGL1) mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 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, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

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, e.g., by solid phase phosphoramadite 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 wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing 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 phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA or ribozyme nucleic acid to the cells and preferably cells expressing Fibrinogen-like protein 1 (FGL1). Preferably, the vector transports the nucleic acid within cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, gRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vectors and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

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., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adenoviruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .

In a preferred embodiment, the antisense oligonucleotide, nuclease (i.e. CrispR with gRNA), siRNA, shRNA or ribozyme nucleic acid sequences are under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for the hepatic cells.

Method of preventing or treating pathological conditions

The present invention further contemplates a method of preventing or treating iron overload associated disease in a subject comprising administering to the subject a therapeutically effective amount of a Fibrinogen-like protein 1 (FGL1) Inhibitor.

In a specific embodiment, the iron overload associated disease is selected from the list consisting of hemochromatosis (adult and juvenile forms of hereditary hemochromatosis), iron- loading anemia and chronic liver diseases including alcoholic and non-alcoholic liver diseases and chronic hepatitis B and C.

In a particular embodiment, the iron-loading anemia is selected from the list consisting of P-thalassemia, congenital dyserythropoietic anemias, myelodysplastic syndrome (MDS).

Preferably, the iron overload associated disease is beta-thalassemia.

In one aspect, the present invention provides a method of inhibiting iron overload associated disease in a subject comprising administering a therapeutically effective amount of a Direct Fibrinogen-like protein 1 (FGL1) Inhibitor.

By a "therapeutically effective amount" of a Direct Fibrinogen-like protein 1 (FGL1) Inhibitor as described above is meant a sufficient amount of the antagonist to prevent or treat an iron overload associated disease. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start with doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied 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 the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicine typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The invention also relates to a method for treating an iron overload associated disease in a subject presenting with a lower hepcidin level in a biological sample (such as in blood or urinary sample) as compared to a predetermined reference value with a Direct Fibrinogen-like protein 1 (FGL1) Inhibitor.

The invention also relates to Direct Fibrinogen-like protein 1 (FGL1) Inhibitor for use in the treatment of an iron overload associated disease in a subject presenting with a lower hepcidin level in a biological sample as compared to a predetermined reference value.

The above method and use comprise the step of measuring the level of hepcidin protein expression (protein or nucleic sequence (DNA or mRNA) in a biological sample obtained from said subject and comparing said measured level to a reference control value.

A low level of hepcidin is predictive of a high risk of having or developing an iron overload associated disease (such as P-thalassemia, haemochromatosis, congenital dyserythropoietic anaemia and myelodysplastic syndrome) and means that Direct Fibrinogen- like protein 1 (FGL1) Inhibitor could be used. Typically, a biological sample is obtained from the subject and the level of hepcidin is measured in this blood sample. Indeed, decreasing Fibrinogen-like protein 1 (FGL1) levels would be particularly beneficial in those subjects displaying low levels of hepcidin.

Pharmaceutical compositions of the invention:

The Fibrinogen-like protein 1 (FGL1) Inhibitor activity / inhibitor of Fibrinogen-like protein 1 (FGL1) gene expression as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

Accordingly, the present invention relates to a pharmaceutical composition comprising a Direct Fibrinogen-like protein 1 (FGL1) Inhibitor according to the invention and a pharmaceutically acceptable carrier.

The present invention also relates to a pharmaceutical composition for use in the prevention or treatment of iron overload associated disease (such as P-thalassemia, haemochromatosis, congenital dyserythropoietic anaemia and myelodysplastic syndrome) comprising a Direct Fibrinogen-like protein 1 (FGL1) Inhibitor according to the invention and a pharmaceutically acceptable carrier.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction 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.

In therapeutic applications, compositions are administered to a subject already suffering from a disease, as described, in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. An appropriate dosage of the pharmaceutical composition is readily determined according to any one of several well-established protocols. For example, animal studies (for example on mice or rats) are commonly used to determine the maximal tolerable dose of the bioactive agent per kilogram of weight. In general, at least one of the animal species tested is mammalian. The results from the animal studies can be extrapolated to determine doses for use in other species, such as humans for example. What constitutes an effective dose also depends on the nature and severity of the disease or condition, and on the general state of the subject's health.

In therapeutic treatments, the antagonist contained in the pharmaceutical composition can be administered in several dosages or as a single dose until a desired response has been achieved. The treatment is typically monitored and repeated dosages can be administered as necessary. Compounds of the invention may be administered according to dosage regimens established whenever inactivation of Fibrinogen-like protein 1 (FGL1) is required.

The daily dosage of the products may be varied 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 the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 10 mg/kg of body weight per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability, and length of action of that compound, the age, the body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

The appropriate unit forms of administration include forms for oral administration, such as tablets, gelatine capsules, powders, granules and solutions or suspensions to be taken orally, forms for sublingual and buccal administration, aerosols, implants, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms for rectal administration.

In the pharmaceutical compositions of the present invention, the active principle is generally formulated as dosage units containing from 0.5 to 1000 mg, preferably from 1 to 500 mg, more preferably from 2 to 200 mg of said active principle per dosage unit for daily administrations. When preparing a solid composition in the form of tablets, a wetting agent such as sodium laurylsulfate can be added to the active principle optionally micronized, which is then mixed with a pharmaceutical vehicle such as silica, gelatine, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets can be coated with sucrose, with various polymers or other appropriate substances or else they can be treated so as to have a prolonged or delayed activity and so as to release a predetermined amount of active principle continuously.

A preparation in the form of gelatin capsules is obtained by mixing the active principle with a diluent such as a glycol or a glycerol ester and pouring the mixture obtained into soft or hard gelatine capsules.

A preparation in the form of a syrup or elixir can contain the active principle together with a sweetener, which is preferably calorie-free, methyl-paraben and propylparaben as an antiseptic, a flavoring and an appropriate color.

The water-dispersible powders or granules can contain the active principle mixed with dispersants or wetting agents, or suspending agents such as polyvinyl-pyrrolidone, and also with sweeteners or taste correctors.

The active principle can also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives.

Among the prolonged-release forms which are useful in the case of chronic treatments, implants can be used. These can be prepared in the form of an oily suspension or in the form of a suspension of microspheres in an isotonic medium.

The direct Fibrinogen-like protein 1 (FGL1) inhibitors according to the invention can be administered by any suitable route of administration. For example, direct Fibrinogen-like protein 1 (FGL1) inhibitor according to the invention can be administered by oral (including buccal and sublingual), rectal, nasal, topical (intracolic), pulmonary, vaginal, or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration.

For the direct Fibrinogen-like protein 1 (FGL1) inhibitors according to the invention and in the preferred embodiment the FGL1 inhibitor can be administered by oral (including buccal and sublingual), rectal or topical (intracolic) administration.

The direct Fibrinogen-like protein 1 (FGL1) inhibitors of the present invention, may be formulated in a wide variety of oral administration dosage forms.

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

Combination of FGL1 inhibitor and ERFE Inhibitor

It was already described that hepcidin expression is rapidly suppressed by the erythroid regulator erythroferrone (ERFE) in conditions associated with expanded erythropoiesis such as anemia caused by bleeding or inflammation^{10 11}. Conversely, excessive release of ERFE in inherited conditions caused by genetic mutations (beta thalassemia, congenital dyserythropoietic anemia, myelodysplastic syndromes)¹²'¹⁴ causes iron overload and severe clinical complications threatening subjects’ survival. In response to erythropoietin (EPO), ERFE is secreted by erythroid precursors in the bone marrow and the spleen and acts as a ligand trap that directly binds BMP 6 to inhibit the signaling cascade directing hepcidin expression¹⁵. The present invention relates to the identification of a new hepcidin suppressor that contributes to hepcidin regulation during anemia: the liver produced hepatokine Fibrinogen like 1 (FGL1). Altogether, similar to erythroferrone, FGL1 act as a ligand trap for BMP6 to repress hepcidin transcription during the recovery from hemorrhage.

Accordingly, in order to treat iron disorders associated with low hepcidin expression (such as P-thalassemia, haemochromatosis, congenital dyserythropoietic anaemia and myelodysplastic syndrome) means that Direct Fibrinogen-like protein 1 (FGL1) Inhibitor could be used in combination with an Erythroferrone (ERFE) inhibitor

In some embodiments, the methods and use of the present invention further comprises the step of applying an Erythroferrone (ERFE) inhibitor.

Accordingly, the present invention also relates to a method of preventing or treating iron overload associated disease in a subject comprising administering to the subject a therapeutically effective amount of a Fibrinogen-like protein 1 (FGL1) inhibitor and an an Erythroferrone (ERFE) inhibitor.

The present invention also relates to pharmaceutical composition comprising a Fibrinogen-like protein 1 (FGL1) Inhibitor and an Erythroferrone (ERFE) inhibitor for simultaneous or sequential use in preventing or treating iron overload associated disease.

As used herein the term "ERFE" also known as “Erythroferrone” refers to a protein produced by erythroblasts which inhibits the action of hepcidin, and so increases the amount of iron available for hemoglobin synthesis. The sequence of said gene can be found under the Ensembl accession number ENSG00000178752.

The term “ERFE inhibitor” has its general meaning in the art and refers to a molecule (natural or synthetic) capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the biological activities of Erythroferrone (ERFE) (the same biological activity as Fibrinogen-like protein 1 (FGL1) that is inhibition of hepcidin expression (through the BMP6 antagonism process)) including, for example, reduction or blocking the interaction for instance between Erythroferrone (ERFE) and BMP6. Erythroferrone (ERFE) inhibitors include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translation control sequences, and the like. Inhibitors or antagonists also include, antagonist variants of the protein, siRNA molecules directed to a protein, antisense molecules directed to a protein, aptamers, and ribozymes against a protein. For instance, the Erythroferrone (ERFE) inhibitor or antagonist may be a molecule that binds to Erythroferrone (ERFE) and neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of Erythroferrone (ERFE) (such biological activity being inhibiting hepcidin expression through direct inhibition ofBMP6). In some embodiments, the ERFE inhibitor of the present invention includes but is not limited to:

A) Inhibitor of Erythroferrone (ERFE) activity such as, anti- ERFE antibody (neutralizing antibody)

B) Inhibitor of Erythroferrone (ERFE) gene expression selected from the list consisting of antisense oligonucleotide, nuclease, siRNA, shRNA or ribozyme nucleic acid sequence..

In a preferred embodiment, the therapeutic combination concern the same type of inhibitors : an inhibitor of Erythroferrone (ERFE) activity with an inhibitor Fibrinogen-like protein 1 (FGL1) activity or an inhibitor of Erythroferrone (ERFE) gene expression with an inhibitor Fibrinogen-like protein 1 (FGL1) gene expression.

In a particular embodiment the of Erythroferrone (ERFE) inhibitor of activity is an antibody (the term including antibody fragment or portion see above definition) that can block directly or indirectly the interaction of Erythroferrone (ERFE) with BMP6 protein.

Examples of neutralizing monoclonal of Erythroferrone (ERFE) that can be used according to 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 W02014071015, WO2018027184 WO2019148186).

The skilled artisan can use routine technologies to use the antigen-binding sequences of these antibodies (e.g., the CDRs) and generate humanized antibodies for treatment of iron overload associated disease.

In still another embodiment, the Erythroferrone (ERFE) antagonist is an inhibitor of Erythroferrone (ERFE)gene expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. Therefore, an "inhibitor of Erythroferrone (ERFE) gene expression" denotes a natural or synthetic compound that has a biological effect to inhibit the expression of Fibrinogen-like protein 1 (FGL1) gene (or the gene transcript : RNA).

In a preferred embodiment of the invention, said inhibitor of Erythroferrone (ERFE) gene expression is antisense oligonucleotide, nuclease, siRNA, shRNA or ribozyme nucleic acid sequence.

Example of siRNAs against Erythroferrone (ERFE) that can be used according to the 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. Example of commercial siRNAs, against Erythroferrone (ERFE) include, but are not limited to: ERFE siRNA (ID 151176) from Bioneer.

In other words, the inventions relates to an inhibitor Fibrinogen-like protein 1 (FGL1) with an inhibitor of Erythroferrone (ERFE) for the simultaneous or sequential use for preventing or treating iron overload associated disease in a subject in need thereof.

By a "therapeutically effective amount" is meant a sufficient amount of compound to treat and/or to prevent iron overload associated disease.

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

The inhibitor Fibrinogen-like protein 1 (FGL1) with the inhibitor of Erythroferrone (ERFE) according to the invention can be administered by any suitable route of administration. For example, thrombin according to the invention can be administered by oral (including buccal and sublingual), rectal, nasal, topical (intravesical), pulmonary, vaginal, or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

Method of screening for treating iron overload associated disease

A further object of the invention relates a method for screening a Fibrinogen-like protein 1 (FGL1) inhibitor (or antagonist) for use in the treatment or prevention of iron overload associated disease.

For example, the screening method may measure the binding of a candidate compound to Fibrinogen-like protein 1 (FGL1), or to cells tissue sample or organism expressing FGL1, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Furthermore, the screening method may involve measuring or, qualitatively or quantitatively, detecting ability of said candidate compound to inactivate FGL1 biological activity (see above definition).

In a particular embodiment, the screening method of the invention comprises the step consisting of:

(i) providing purified FGL1 protein (or C-terminal globular domain of fibrinogen), providing a cell, tissue sample or organism expressing the FGL1,

(ii) providing a candidate compound such as small organic molecule, nucleic acids, antibodies, peptide or polypeptide,

(iii) measuring the activity of the FGL1,

(iv) and selecting positively candidate compounds that, blocks the biological activity ofFGLl or inhibits FGL1 expression. .

In a particular embodiment, the screening method of the invention may further comprise a step consisting in administering the candidate compound selected at step d) to an animal model of iron overload associated disease (i.e. beta thalassemia) to validate the protective effects of said candidate compound.

In general, such screening methods involve providing appropriate cells which express FGL1. In particular, a nucleic acid encoding FGL1 may be employed to transfect cells to thereby express the hormone of the invention. Such a transfection may be accomplished by methods well known in the art. In a particular embodiment, said cells may be selected from the group consisting of the mammal cells reported yet to express FGL1 (e.g. hepatic cells).

The screening method of the invention may be employed for determining a FGL1 inhibitor by contacting such cells with compounds to be screened and determining whether such compound inactivates FGL1.

According to a one embodiment of the invention, the candidate compounds may be selected from a library of compounds previously synthesized, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesized de novo or natural compounds. The candidate compound may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) organic or chemical compounds (natural or not). Illustratively, libraries of pre-selected candidate nucleic acids may be obtained by performing the SELEX method as described in documents US 5,475,096 and US 5,270,163. Further illustratively, the candidate compound may be selected from the group of antibodies directed against FGL1.

FGL1 inhibition with the candidate compound can be tested by various known methods. For example hepcidin expression assay (see above) may be used for performing the screening method of the invention, or in situ zymography assays in pathological or healthy tissues (see example 1, figure 1 and figure 6).

FIGURES:

Figure 1: Recovery from hemorrhage-induced anemia in WT mice. (A) Hemoglobin levels of 7-9 week-old WT male mice 0, 1, 2, 3, 4, 5 and 6 days after phlebotomy (500pl). mRNA expression of Epo in the kidney (B), Erfe in the bone marrow and spleen (C) and Hamp, Idl and Smad7 (E) in the liver of phlebotomized mice. (D) Time course of serum ERFE concentration. Data shown are means ± s.e.m and were compared for each time point to values for control mice at t = 0 (n = 5 to 7 per time-point) to control mice (day 0) by One-way ANOVA. **** < 0.0001, *** < 0.001, **P < 0.01, *P < 0.05.

Figure 2: ERFE-independent repression of hepcidin during the recovery from anemia. Iron-related parameters in 7-9 week-old Erfe-/- mice 0-6 days after phlebotomy (500pl). Parameters included hemoglobin levels (A), kidney Epo mRNA expression (B), liver Hamp, Idl and Smad7 mRNA expression (C), serum hepcidin concentration (D), serum iron content (E), transferrin saturation (F) and liver iron content (G). (H) Western blotting for P- Smad5, Smad5 and vinculin in the liver of Erfe-/- mice 0, 1 and 2 days after phlebotomy. (I) Densitometric ratio of phosphorylated Smad5 to total Smad5 or Vinculin and Smad5 to Vinculin. Data shown are means ± s.e.m and were compared for each time point to values for control mice at t = 0 (n = 5-8) by One-way ANOVA (A, B, C) or Student t-test (D, E, F, G, I). **** < 0.0001, *** < 0.001, **P < 0.01, *P < 0.05.

Figure 3: Testing the potential contribution of an erythroid regulator. Percentage of reticulocytes (A), Gypa and Tfrl mRNA expression in the bone marrow (B), hemoglobin levels (C) and liver Hamp mRNA expression (D) in 8 week-old control and irradiated (400 radian) WT and Erfe-/- at t=0 (white bar) and 48 (blue bars) hours after phlebotomy (n=4-9). (E) Gypa mRNA expression in the bone marrow, spleen and liver of Erfe-/- at t=0 to 6 days after phlebotomy (n=5-8). (F) Linear regression analysis of Gypa and Hamp mRNA expression in the liver and the spleen. (G) Liver Hamp mRNA expression in 7-9 week-old WT control and splenectomized WT and Erfe-/- at t=0 (white bar) and 48 (blue bars) hours after phlebotomy (n=3-6). Data shown are means ± s.e.m and were compared between each group by Two-way ANOVA. **** < 0.0001, *** < 0.001, **P < 0.01, *P < 0.05.

Figure 4: Fgll mRNA expression is induced in mouse liver during anemia. Time course of Fgll mRNA expression in the liver (A) and the bone marrow (B) 1 to 6 days after phlebotomy in WT and Erfe-/- mice (n=5-8). (C) Hamp and Fgll mRNA expression in the liver of 7 w.o. mice at t=0 to 20h after a single intra-peritoneal injection of EPO (200u) (n=5). (D) Fgll mRNA expression in liver of 8 w.o WT, Th3/+ and Erfe-/-; Th3/+ mice (n=7-9). Relative mRNA expression of HIF target genes Vegfa, Gapdh an AngptH in mouse primary hepatocytes cultured in serum free or serum-containing media and incubated for 15 hours in presence of prolyl hydroxylases inhibitor DMOG or in low oxygen condition (2%) compared to untreated cells (E). (F) Relative Fgll expression in mouse primary hepatocytes incubated in hypoxic conditions or with DMOG, in the liver of fTzZ-deficient mice (G), and in the liver of mice treated with prolyl hydroxylase inhibitor vadadustat (H). Data shown are means ± s.e.m and were compared for each time point to values for control WT at t = 0 by Two-way ANOVA (A, B, C) or to WT mice by Student t-test. Data shown for experiment in primary hepatocytes are means of three independent experiments and were compared to control cells by Student t-test (E, F, _G) ****p < o.OOOl, *** < 0.001, **P < 0.01, *P < 0.05.

Figure 5: FGL1 is a suppressor of hepcidin in vivo and in vitro. Relative HAMP expression in Hep3B and HepG2 cells in response to BMP6 (25 ng/ml, 6h) (A) or BMP6 + recombinant FGL1 (lOpg/ml) (B). Relative ID1 expression in hepatoma cell lines treated with BMP6 and FGL1 (C). HAMP (D) and ID1 (E) expression in Hep3B cells and mouse primary hepatocytes treated for 6h with BMP6 and either Fc, full length FGL1 and the N-terminal or globular domain of FGL1. Hepatic Hamp RNA expression (F), serum hepcidin concentration (G) and liver Idl mRNA expression (H) in mice treated for 6 hours with saline, Fc or recombinant FGL1 (10 mg/kg) (n=5). Data shown are means ± s.e.m of three independent experiments (A-E) or treated mice and were compared for each condition to untreated cells or control mice by Student t-test. **** < 0.0001, *** < 0.001, **P < 0.01, * < 0.05.

Figure 6: Fgll-/- mice exhibit a blunted response to phlebotomy. (A) Fgll mRNA expression in the liver of male and female WT mice 36 hours after bleeding compared to control mice. Red blood cell count (RBC) (B) and hemoglobin (Hb) (C), marrow (D) and spleen (E) Erfe mRNA expression, liver Hamp (F), Idl (G) and Smad7 (H) mRNA expression in WT (white bar) and Fgll-/- (black bar) mice at t=0 or 36 hours after phlebotomy. Data shown are means ± s.e.m (n= 5-11) and were compared between each group by Two-way ANOVA and corrected for multiple comparisons by Holm-Sidak method. ****P < 0.0001, ***p < 0.001, **P < 0.01, *P < 0.05.

Figure 7: FGL1 is a BMP antagonist. Relative expression of Hamp (A), Idl (B), Smad7 (C) mRNA expression in mouse primary hepatocytes treated with BMP ligand (10 ng/ml) and human Fc IgG2 (10 pg/ml) or Fc-FGLl (10 pg/ml) for 6 hours. Data shown are means ± s.e.m of three independent experiments and were compared for each BMP 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 blotting of Hep3B cells treated for 6 hours with BMP6 (10 ng/ml), ERFE (1 pg/ml), or FGL1 (10 pg/ml) for P-SMAD5, SMAD5 and GAPDH. (E) Western blotting of pull-down assay of BMP6 and FGL1 for human Fc IgG2 and BMP6.

Figure 8: Validation of specific ASOs to mouse and human FGL1. Hep3B cells were transfected with lOOmM of each ASO. FGL1 mRNA expression was measured after 72h (A). FGL1 protein expression was detected by western blot after 48h and 72h (B).

EXAMPLE 1:

Method

Animal models

Erfe'¹' and Erfe+/+ on a C57BL/6J background were bred and housed in a specific- pathogen-free barrier facility in the animal facilities of INSERM US006. Fgll-/- and WT controls on a C57B1/6N background were obtained from The European Mouse Mutant Archive (EMMA), bred by Janvier labs (Le Genest St Isle) and transferred in the animal facilities of INSERM US006 at the age of 4-5 weeks. Mice were housed under a standard 12-hour light/dark cycle with water and standard laboratory mouse chow diet (Ssniff, 200 mg iron/kg) ad libitum, in accordance with the European Union guidelines. The study was approved by the MidiPyrenees Animal Ethics Committee. Thalassemic mice and mice treated with Vadadustat were provided by Yelena Ginzburg¹⁸ and Tomas Ganz¹⁹ respectively. Expression data from Vhl-/- mice were provided by Carole Peyssonnaux²⁰. To study the recovery from anemia, mice were phlebotomized by a retro-orbital puncture (500pL) and analyzed after 1 to 6 days. Disruption of the erythroid compartment was achieved by exposing mice to a sublethal dose of X-ray (400 rads) and mice were phlebotomized 48 hours later. Surgical ablation of the spleen was performed on 7-8 week-old WT and Erfe-/- mice. Mice were allowed to recover for 7 days before phlebotomy. A subset of WT mice was given a single dose of EPO (200U) and were analyzed 12, 15, 18 or 20 hours later. Recombinant FGL1, Fc fragment or saline were administered intra-peritoneally to 7-week-old C57B1/6J mice fed for two weeks with an iron adequate diet (Ssniff, 50 mg/kg) at a dose of lOmg/kg and the mice were analyzed after 6 hours For all mice, tissue were harvested and divided into flash frozen sample in liquid nitrogen for RNA, protein and iron measurements and in 4% formalin for paraffin embedding. Male mice were preferentially studied unless otherwise specified.

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

Mouse ERFE Immuno-Assay

Human recombinant monoclonal antibodies to mouse ERFE were produced by Biorad using the HuCAL technology. High binding 96 well plate (Corning) was coated overnight at 4°C with 100 pL/well of 2 pg/ml capture antibody diluted in 50 mM sodium carbonate buffer pH 9.6. Plate was washed (TBS, 0.5% Tween 20) and blocked for an hour with 300 pL/well blocking buffer (PBS, 0.2% Na casein, 0.05% Tween 20, 0.1 M NaCl) at room temperature. Recombinant mouse ERFE standard was 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 incubation at room temperature. Plate was washed and incubated for 1 hour with lOOpL/well of biotinylated detection antibody at 0.5pg/ml in PBS + 5% BSA. Plate was washed, and incubated for 45 minutes with lOOpl/well of 1/5000 Neutravidin-HRP (Pierce) in PBS + 5% BSA. Plate was developed with 100 mL/well Supersensitive TMB substrate (Thermofisher) in the dark at room temperature, the reaction was stopped by adding 50 pL of 0.2N sulfuric acid, and the absorbance was measured at 450 nm.

Measurement of iron and hematological parameters

Serum iron concentration was determined by iron direct method (ferene dBiolabo, 92108) and transferrin saturation was deduced by measuring the unsaturated iron binding capacity (UIBC, Biolabo, 97408). Liver iron content was determined as previously described²¹. Complete blood count was performed with a Cell-Dyn Emerald hematology analyzer (Abbott).

Western Blot Analysis Liver proteins were extracted by physical dissociation using ULTRA-TURRAX® (IKA) in PEB Buffer (150 mM de NaCl, 50 mM Tris-HCl, 5mM EDTA, 1% NP-40) containing proteases (cOmplete™, Roche) and phosphatase (Phosphatase Inhibitor Cocktail 2, Sigma) inhibitors. Hep3B cells were lyzed in RIPA Buffer (Thermofi scher, 89900) containing protases and phosphatases inhibitors. Freshly extracted proteins were diluted in Laemmli buffer 2x (Sigma), incubated 10 min at 95°c, subjected to SDS-PAGE and electroblotted to nitrocellulose membrane (Biorad). Membranes were blocked 1H with 5% of non-Fat dry milk (NFDM, Cell signaling) diluted in TBS-T buffer (lOmM Tris-HCl, pH 7.5, 150mM NaCl, 0.15% Tween 20) and incubated overnight at 4°C with phospho-Smad 5 (Ser463/465, Abeam, ab92698, 1/2000) or 2 hours at RT with antibody to Smad5 (Abeam, ab40771, 1/5000) diluted in TBS-T buffer 5% BSA. Loading was determined using antibodies to GAPDH (Cell signaling, D16H11, 1/10 000) or Vinculin (Cell signaling, 4650, 1/20 000) diluted in TBST- NFDM (5%) (2h, RT). Incubation with primary antibody was followed by 3 washes and membranes were incubated 2 hours with goat anti-human IgG (Novus biological, NBP1-75006, 1/10000), goat anti-rabbit IgG (Cell signaling, 7074, 1/10000) or horse anti-mouse IgG (Cell signaling, 7076, 1/10000) secondary antibodies conjugated with HRP and diluted in TBST- NFDM (5%). Enzyme activity was developed using ECL prime reagent (GE Healthcare) on ChemiDoc XRS+ imaging system.

Pull down assay

One microgram of Fc tagged recombinant proteins (Fc alone, FGL1 full length, FGL1 globular and FGL1 Nter) was incubated overnight at 4°C 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)) with or without 500 ng of hBMP6 (Biotechne). Protein 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).

Cell treatment

Hep3B and HepG2 cells were culture in Dulbecco’s modified Eagle medium-high glucose GlutaMAX, 10% fetal bovine serum, 1% penicillin-streptomycin unless otherwise indicated. Cells were plated 24 hours before treatments and treated for 6 hours in serum free medium. Hepatocytes were isolated from wild-type C57BL/6 mice by a portal vein collagenase perfusion method as previously described²². Cells were incubated overnight (15 hours) in fresh Williams E Medium (Gibco) supplemented with 200 pM L-glutamine, 10% FBS. Hep3B and HepG2 cells and primary hepartocytes were treated with or 25 ng/ml of BMP6 (Peprotech) or BMPs 2, 4, 7, (R&D Systems) and with Fc (hIgG2), Fc-FGLl full length (FL), its N-terminal (Nter) or globular (glob) domains for 6 hours. For hypoxia experiments, cells were maintained in serum free medium in a hypoxia chamber (Whitley, H35 Hypoxy station) with 2% of oxygen for 15 hours or in a conventional CO2 incubator in presence of ImM DMOG (Dimethyloxalylglycine, N-(Methoxyoxoacetyl)-glycine methyl ester, Sigma). To test the regulation of FGL1 by inflammatory cytokines, HepB cells were treated with 20 ng/ml IL-6 or 50 ng/ml TNFa (R&D Systems) for 6 hours.

Quantification of mRNA levels

Total RNA from mouse tissues was extracted by Trizol (MRC) / Chloroform (Sigma) method. Complementary cDNA was synthetized using M-MLV Reverse transcriptase (Promega). Messenger RNA (mRNA) expression levels were assessed by quantitative polymerase chains reactions (RT-qPCR) by using Takyon SYBR green (Eurogentec) (primers indicated in Table 1) and run in duplicate on a LightCycler480 (Roche) apparatus. Transcript abundance was normalized to the reference gene Hprt and represented as a difference between reference and target genes within each group of mice (-ACt) ± standard error of the mean (SEM). Data from Albumin-Cre/VHLflox/flox mice were normalized to the reference gene 36B4. Results from in vitro treatments are represented as a fold change i.e. Log transformed data (2^AACt) showing expression relative to control conditions. Expression data for control conditions were normalized on control average to obtain a distribution also in 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 a student t test or the analysis of variance (ANOVA).

Microarray

Total RNA from mouse liver and bone marrow from Erfe-/- mice at t= 0, 24 and 48h hours after phlebotomy were extracted using Trizol (MRC) / Chloroform (Sigma). RNA quality was assessed on RNA 6000 Nano chips using a Bioanalyzer 2100 (Agilent Technologies). Gene-level expression profiling of livers and bone marrow from phlebotomized mice were performed at the GeT-TriX facility (GenoToul, Genopole Toulouse Midi-Pyrenees) using Agilent SurePrint G3 Mouse GE v2 microarrays 8x60K, design 074809) following 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) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts). Dye incorporation and cRNA yield were checked using Dropsense 96 UV/VIS droplet reader (Trinean, Belgium). 600 ng of Cy3- labelled cRNA were hybridized on the microarray slides following the manufacturer’s instructions. Immediately after washing, the slides were scanned on Aglient G2505C Microarray Scanner using Agilent Scan Control A.8.5.1 software and fluorescence signal extracted using Agilent Feature Extraction v 10.10.1.1 with default parameters. Microarray data and experimental details are available in NCBI's Gene Expression Omnibus? and are accessible 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 by comparing control mice (n=3) to mice at the time points 24h (n=3) and 48h (n=3).

Microarray data statistical analysis

Microarray data were analyzed using R (R Core Team, 2018) and Bioconductor packages8 as described in GEO accession GSE229041. Raw data (median signal intensity) were filtered, log2 transformed and normalized using 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). A first exploratory and statistical analysis showed a possible correlation structure among gene expression which could negatively impact the multiple testing procedures. We applied the FAMT methodlO to reduce the dependence structure using a model with one extra factor. A model was fitted using the limma ImFit 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). Pair-wise comparisons between biological conditions were applied using specific contrasts. A correction for multiple testing was applied using Benjamini-Hochberg procedure to control the False Discovery Rate (FDR). Probes with FDR < 0.01 were considered to be differentially expressed between conditions. Hierarchical clustering was applied to the samples and the differentially expressed probes using 1 -Pearson correlation coefficient as distance and Ward’s criterion for agglomeration. Expression data were further analyzed using R iDEP (Integrated Differential Expression and Pathway analysis)12 by performing three comparisons: control mice (n=4) to mice 24h (n=4) after phlebotomy, control mice to mice 48h after phlebotomy and mice 48h after phlebotomy to mice 24h after phlebotomy. We focused on transcripts encoding secreted proteins that were significantly upregulated 24h after phlebotomy compared to control mice and whose expression remained stable or increased between 24 and 48 hours. Liver and bone marrow were analyzed separately.

Statistical analysis The statistical significances were assessed by Student t test, one or two-way analysis of variance (ANOVA) using Prism 9 (GraphPad). Statistics shown for Two-way ANOVAs are results of Holm-Sidak's multiple comparisons test.

Results

ERFE-independent repression of hepcidin during anemia. We first delineated the timeline of the recovery from anemia induced by phlebotomy in WT and /T'/c-deficient mice. In both genotypes, we observed that hemoglobin and hematocrit levels decreased for three days after phlebotomy and were significantly improved by day 6 (Figure 1 A and data not shown). As a result, Epo mRNA expression in the kidney was rapidly induced during 2 days and progressively returned to normal after 6 days (Figure IB). Erfe mRNA expression was maximally increased in the bone marrow and spleen after 24 hours before progressively returning toward baseline but Erfe mRNA levels were still somewhat elevated after 6 days (Figure 1C). In contrast, serum ERFE levels were highest 24h after phlebotomy and decreased below detection levels within 3-4 days (Figure ID). However, a significant reduction in liver hepcidin mRNA expression was maintained for 5 days (Figure IE) and accompanied by a mild decrease in BMP-target genes Idl and Smad7 mRNA 3 days after phlebotomy. We therefore investigated whether hepcidin could be regulated independently of ERFE during the recovery from anemia in Erfe-deficient mice. Similar to WT mice, in Erfe-/- mice, hemoglobin levels reached a nadir three days after phlebotomy (Figure 2A) and were almost recovered by day 6. Erfe deficient exhibited slightly elevated MCV, MCH and RDW compared to WT mice 3 days after phlebotomy (data not shown). Epo mRNA expression was rapidly induced in the kidney 24 hours after bleeding and progressively returned to normal after 6 days (Figure 2B). Consistent with the stress hormone property of ERFE, Hamp mRNA expression was unchanged after 24 hours but dropped to levels comparable to those of WT mice 2-3 days after phlebotomy before returning to normal after 6 days while Idl and Smad7 mRNA expression was unchanged (Figure 2C). Comparable results were observed in phlebotomized female mice (data not shown). We therefore decided to focus on the mechanisms triggered within 24-48h after phlebotomy and not after 48h. Serum hepcidin concentration and hepcidin/liver iron content ratio mirrored liver Hamp mRNA expression 1 and 2 days after phlebotomy in WT and Erfe-/- mice but the liver content and Bmp6 mRNA expression were unchanged (Figure 2D and data not shown). We therefore decided to focus on the mechanisms triggered within 24-48 hours after phlebotomy that could contribute to hepcidin suppression in Erfe-/- mice. The changes in hepcidin synthesis in Erfe-/- mice occurred without any variation in serum iron concentration, transferrin saturation and liver iron content 1 and 2 days after bleeding compared to control mice (Figure 2E, F, G). Similarly, SMAD 5 phosphorylation was not decreased in the liver of Erfe-I- mice 2 days after phlebotomy (Figure 2H-I). We did not detect any statistically significant increase in Gdfl5 and Twsgl mRNA expression in the bone marrow and the spleen of phlebotomized WT and Erfe-I- mice (Data not shown). These data indicate that hepcidin expression is negatively regulated by an ERFE-independent mechanism. ERFE was originally identified by searching for transcripts that were induced in the bone marrow 9-15 hours after bleeding. We hypothesized that another erythroid regulator derived from the bone marrow or a factor directly derived from the liver could repress hepcidin 24-48 hours after phlebotomy. We thus analyzed by microarray the transcriptomic profiles of phlebotomized Erfe-/- mice 1 and 2 days after phlebotomy compared to control mice.

Testing the potential contribution of an erythroid regulator. To determine whether the suppression of hepcidin is mediated by another erythroid regulator^24,25, we disrupted the erythroid compartment by irradiation and tested the response of hepcidin to bleeding in WT and Erfe-I- mice. Phlebotomized WT and Erfe-I- showed increased reticulocytosis 48h after phlebotomy whereas irradiated mice exhibit a massive decrease in circulating reticulocytes (Figure 3A). Decreased reticulocytes production was accompanied a reduced expression of erythroid markers Gypa and Tfrl in the bone marrow of irradiated WT and Erfe-I- mice compared to their respective controls thus confirming the successful depletion of the erythroid compartment (Figure 3B). Hemoglobin levels were deceased in all phlebotomized groups and to a greater extent in irradiated mice but no difference between was observed WT and Erfe-I- mice (Figure 3C). Hepatic hepcidin mRNA expression was repressed in control WT and Erfe - I- mice but not in irradiated mice 48 hours after phlebotomy (Figure 3D). Interestingly, the recovery from anemia was paralleled by an increase in Gypa mRNA expression in the spleen and the liver 1-6 days after phlebotomy compared to control mice (Figure 3E). Hamp mRNA expression inversely correlated with Gypa mRNA expression in the liver and the spleen (Figure 3F) suggesting that an hepcidin suppressor could be released from the liver and the spleen. We therefore performed surgical ablation of the spleen of WT and Erfe-I- mice and evaluated the response to bleeding. However, splenectomized WT and Erfe-I- mice exhibited reduced liver Hamp mRNA expression 48 hours after phlebotomy indicating that the hepcidin suppressive factor does not originate from the spleen (Figure 3G). To explore the liver and bone marrow response to anemia, we therefore analyzed the transcriptomic profiles of phlebotomized Erfe-I - mice 1 and 2 days after phlebotomy compared to control mice. Fgll mRNA expression is induced in mouse liver during anemia. To identify potential regulators of hepcidin, we searched for transcripts encoding secreted proteins and whose expression was induced 24 and 48 hours after phlebotomy compared to control mice. We found that 63 and 38 transcripts were induced (fold change > 2; p-value < 0.05) 24 hours after phlebotomy compared to control mice in the liver and the bone marrow respectively (data not shown). Six transcripts in the liver and 23 in the bone marrow were still induced 48 hours after phlebotomy compared to control mice (data not shown). In the liver, only Fgll, Gdfl5 and Cxcll encoded secreted proteins. Interestingly, Fibrinogen-like 1 (Fgll mRNA expression was increased in both the liver and the bone marrow. We confirmed by qRT-PCR that Fgll mRNA expression was significantly induced in the liver (Figure 4A) and the bone marrow (Figure 4B) 1-3 days after phlebotomy in WT and Erfe-I- mice but was several order of magnitude higher in the liver. In contrast, Gdfl5 mRNA expression was mildly induced 1-2 days after phlebotomy but Gdfl5 was reported not to contribute to hepcidin regulation during hemorrhage-induced anemia. Cxcll mRNA expression was only induced in Erfe-/- mice after 24h (data not shown) which did not correlate with the time course of hepcidin repression. Moreover, unlike Fgll, the stimulation of Gdfl5 and Cxcll was restricted to the liver. We therefore focused on FGL1 as the remaining potential candidate. Fibrinogen-like 1 (FGL1), also known as hepassocin26 or HFREP-127, is a member of the fibrinogen family of proteins produced by hepatocytes that share structural homologies to angiopoietin-like proteins (ANGPTL)²⁸, including a C-terminal globular domain homologous to fibrinogen beta and gamma subunits. In contrast with other fibrinogen-related factors, FGL1 lacks the plateletbinding and thrombin-sensitive sites involved in clot formation²⁷’²⁹. Instead, FGL1 was induced during liver regeneration and showed mitogenic activity on hepatocytes³⁰. It is also involved in tumor evasion of certain cancers through its interaction with LAG-3 receptor³¹. Intra-peritoneal injection of EPO (200u) in WT mice led to a significant reduction in Hamp mRNA expression and increase in bone marrow Erfe mRNA expression but did not stimulate Fgll expression (Figure 4C). However, Fgll mRNA expression was upregulated in the liver of thalassemic Th3/+ and Th3/+ mice deficient for Erfe (Figure 4D). Mouse Fgll promoter analysis revealed two HIF binding sites (data not shown). To examine whether Fgll expression was stimulated by the decreased oxygen saturation in the liver of anemic mice, we compared Fgll expression in mouse primary hepatocytes incubated in low oxygen conditions (2% 02) or in presence prolyl-hydroxylases inhibitor DMOG to control conditions. We observed that expression of hypoxia inducible factor target genes Vegfa, Gapdh, Angptll was increased in cells cultured in serum free or serum-containing media and incubated for 15 hours in presence of DMOG or in low oxygen condition (2%) (Figure 4E) compared to untreated cells. Similarly, Fgll mRNA expression was induced in mouse primary hepatocytes incubated in hypoxic conditions or with DMOG (Figure 4F). A trend toward an increase was observed and in livers o Albumin-Cre Vhl- deficient mice²⁰ (Figure 4G). However, a significant increase in Fgll mRNA expression was detected in livers of F/z 2a-overexpressing mice³² and of mice treated chronically with proly hydroxylase inhibitor vadadustat¹⁹ (Figure 4H). These results suggest that Fgll expression may be regulated by hypoxia inducible factors. . In accordance with a previous study, WT mice fed a 10-50-200 or 8000 mg/kg iron diet for two weeks did not exhibit any change in liver Fgll mRNA expression while Hamp and Idl mRNA expression followed the dietary iron content (data not shown) indicating that Fgll is not regulated by iron. FGL1 was described as an acute phase protein and, similar to hepcidin, its expression was mildly induced and repressed by IL- 6 and TNFa, respectively (data not shown).

FGL1 is a suppressor of hepcidin in vivo and in vitro. We next evaluated the contribution of FGL1 in hepcidin regulation. HAMP mRNA expression was induced 600 and 200-fold in response to BMP6 (25 ng/ml, 6h) in Hep3B and HepG2 cells respectively (Figure 5 A). Treatment with recombinant Fc-tagged FGL1 for 6 hours under serum free conditions led to a significant reduction in HAMP and ID1 (Figure 5B-C) expression in both hepatoma cell lines. A higher dose of FGL1 was required to repress HAMP and ID1 expression in serumcontaining media (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). We therefore decided to use mouse FGL1 in this study. Injection of recombinant FGL1 (10 mg/kg) into WT mice led to a significant reduction in hepatic Hamp RNA expression and serum hepcidin concentration (Figure 5F-G) compared to Fc-treated mice (n=5) 6 hours after injection but no change in liver Idl mRNA expression was observed (Figure 5H). Consistent with hepcidin repression, serum and liver iron concentration were respectively increased and decreased after FGL1 treatment (Figure 4H). In female WT mice, treatment with FGL1 did not repress hepcidin expression. A marked inflammatory reaction to FGL1 administration that was not seen in male mice, as shown by increased Saal levels, may hinder the effect of FGL1. Finally, FGL1 was also able to repress hepcidin expression in presence of IL-6 in Hep3B cells (Data not shown). Collectively, these results indicate that ERFE is a potent suppressor of hepcidin.

Fgll-/- mice exhibit a blunted response to phlebotomy. To determine whether FGL1 contributes to hepcidin regulation during the recovery from anemia, we compared WT and Fgll-/- mice 36 hours after phlebotomy. We first confirmed that Fgll mRNA expression was significantly increased in livers of male and female WT mice 36 hours after bleeding compared to control mice (Figure 6A). Red blood cell count and hemoglobin (Figure 6B-C) levels were reduced 36 hours after phlebotomy in WT and Fgll-/- mice. A comparable increase in Erfe mRNA expression in the bone marrow and the spleen was detected 36 hours after bleeding in both genotypes (Figure 6D-E). Interestingly, we found that liver Hamp mRNA expression was reduced in male and female phlebotomized WT and Fgll-/- compared to control mice but to a lower extent in Fgll-/- mice (Figure 6F) suggesting that FGL1 contributes to hepcidin suppression. No change in Idl or Smad7 mRNA expression was observed (Figure 6G-H) but, in accordance with blunted hepcidin repression, serum iron concentration was lower in bled Fgll-/- mice compared to WT mice (Figure 6G). These results indicate that FGL1 is increased during recovery from anemia and contributes to hepcidin suppression.

The globular domain of FGL1 is responsible for hepcidin suppression. Mouse FGL1 composed of a signal peptide for secretion, a short coil-coil N-terminal domain and a C-terminal globular domain homologous to fibrinogen P and y chains (data not shown). To confirm these results and identify the active domain of FGL1, we treated mouse primary hepatocytes with Fc, full length FGL1 or its N-terminal and globular domain. We found that full length FGL1 and the globular domain repress HAMP and ID1 expression (Figure 5D, Figure 5E) whereas the N- terminal domain is inactive.

FGL1 is a BMP antagonist. We next examined the mechanism by which FGL1 represses hepcidin. Since treatment of hepatic cells with FGL1 leads to a downregulation if ID1 mRNA expression, we tested whether FGL1 could act as a BMP antagonist. We observed that FGL1 can repress the induction of Hamp and Idl mRNA expression by BMP 6 and 7 but not by BMP2 and 4 (Figure 7A-B) in mouse primary hepatocytes compared to control cells and Fc- treated cells. However, we did not observe any effect on Smad7 mRNA expression When cells are treated with FGL1 (Figure 7C). Pre-treatment of cells with BMP6 prior to the addition of FGL1 confirmed its ability to repress hepcidin and BMP target genes (data not shown). In addition, we found that FGL1 led to a significant reduction in SMAD5 phosphorylation in BMP6-treated Hep3B cells (Figure 7D). Moreover, we incubated Fc or Fc-FGLl (FL, glob, Nter) with BMP6 and performed pull-down assay using protein A magnetic beads. We found that FGL1 FL and glob and to a lower extent the N-terminal domain but not the Fc fragment interacted with BMP6 (Figure 7E). Altogether, these results indicate that, similar to erythroferrone, FGL1 acts as a ligand trap for BMP6 to repress hepcidin transcription during the recovery from hemorrhage. Table 1

EXAMPLE 2

Method: We synthesized antisense oligonucleotides specific for mouse and human FGL1. Hep3B cells were transfected with lOOmM of each ASO using INTERFERin (Polyplus). FGL1 mRNA expression was measured after 72h by qRT-PCR. FGL1 protein expression was detected by western blot after 48h and 72h using anti-FGLl antibody (Santa Cruz Biotechnologies sc-514057).

Results: FGL1 mRNA expression was repressed 50-60% in Hep3B cells 72 hours after transfection with ASOs 1, 4, 5, 6 and 7 compared to cells transfected with scramble ASO (Figure 8A). In comparison with scramble ASO, FGL1 protein abundance was decreased after 48h and 72h in Hep3b cells transfected with ASO 1 and 6 (Figure 8B).

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 an ability to reduce the expression of hepcidin in a cellular model (data not shown). Table section

Table 2: Useful nucleotide or amino acid sequences for practicing the invention

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Sankaran VG, Weiss MJ. Anemia: progress in molecular mechanisms and therapies. Nat Med. 2015;21(3):221-230.

2. Camaschella C. Iron deficiency. Blood. 2019; 133(l):30-39.

3. Finch C. Regulators of iron balance in humans. Blood. 1994;84(6): 1697-1702.

4. Nemeth E, Tuttle MS, Powelson J, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306(5704):2090-2093.

5. Aschemeyer S, Qiao B, Stefanova D, et al. Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin. Blood.

2018;131(8):899-910.

6. Koch PS, Olsavszky V, Ulbrich F, et al. Angiocrine Bmp2 signaling in murine liver controls normal iron homeostasis. Blood. 2017;129(4):415-419.

7. Meynard D, Kautz L, Damaud V, Canonne-Hergaux F, Coppin H, Roth MP. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet.

2009;41(4):478-481.

8. Andriopoulos B, Jr., Corradini E, Xia Y, et al. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet. 2009;41(4):482-487.

9. Wang CY, Babitt JL. Liver iron sensing and body iron homeostasis. Blood. 2019;133(l):18-29.

10. Kautz L, Jung G, Nemeth E, Ganz T. Erythroferrone contributes to recovery from anemia of inflammation. Blood. 2014;124(16):2569-2574.

11. Kautz L, Jung G, Valore EV, Rivella S, Nemeth E, Ganz T. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014;46(7):678-684.

12. Ganz T, Jung G, Naeim A, et al. Immunoassay for human serum erythroferrone. Blood. 2017; 130(10): 1243-1246.

13. Bondu S, Alary AS, Lefevre C, et al. A variant erythroferrone disrupts iron homeostasis in SF3Bl-mutated myelodysplastic syndrome. Sci Transl Med. 2019; 11(500).

14. Russo R, Andolfo I, Manna F, et al. Increased levels of ERFE-encoding FAM132B in patients with congenital dyserythropoietic anemia type II. Blood. 2016;128(14):1899-1902.

15. Arezes J, Foy N, McHugh K, et al. Erythroferrone inhibits the induction of hepcidin by BMP6. Blood. 2018;132(14): 1473-1477. 16. Kautz L, Jung G, Du X, et al. Erythroferrone contributes to hepcidin suppression and iron overload in a mouse model of beta-thalassemia. Blood. 2015;126(17):2031-2037.

17. Arezes J, Foy N, McHugh K, et al. Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia. Blood.

2020;135(8):547-557.

18. Castro-Mollo M, Gera S, Ruiz-Martinez M, et al. The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry. Elife. 2021; 10.

19. Hanudel MR, Wong S, Jung G, et al. Amelioration of chronic kidney disease- associated anemia by vadadustat in mice is not dependent on erythroferrone. Kidney Int.

2021;100(l):79-89.

20. Mastrogiannaki M, Matak P, Mathieu JR, et al. Hepatic hypoxia-inducible factor-2 down-regulates hepcidin expression in mice through an erythropoietin-mediated increase in erythropoiesis. Haematologica. 2012;97(6):827-834.

21. Kautz L, Meynard D, Monnier A, et al. Iron regulates phosphorylation of Smadl/5/8 and gene expression of Bmp6, Smad7, Idl, and Atoh8 in the mouse liver. Blood.

2008;112(4): 1503-1509.

22. Goodnough JB, Ramos E, Nemeth E, Ganz T. Inhibition of hepcidin transcription by growth factors. Hepatology. 2012;56(l):291-299.

23. Ge SX, Son EW, Yao R. iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC Bioinformatics . 2018; 19(1): 534.

24. Vokurka M, Krijt J, Sulc K, Necas E. Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis. Physiol Res. 2006;55(6):667-674.

25. Pak M, Lopez MA, Gabayan V, Ganz T, Rivera S. Suppression of hepcidin during anemia requires erythropoietic activity. Blood. 2006;108(12):3730-3735.

26. Hara H, Yoshimura H, Uchida S, et al. Molecular cloning and functional expression analysis of a cDNA for human hepassocin, a liver-specific protein with hepatocyte mitogenic activity. Biochim Biophys Acta. 2001;1520(l):45-53.

27. Yamamoto T, Gotoh M, Sasaki H, Terada M, Kitajima M, Hirohashi S. Molecular cloning and initial characterization of a novel fibrinogen-related gene, HFREP-1. Biochem Biophys Res Commun. 1993;193(2):681-687.

28. Demchev V, Malana G, Vangala D, et al. Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization. PLoS One. 2013;8(3):e58084. 29. Rijken DC, Dirkx SP, Luider TM, Leebeek FW. Hepatocyte-derived fibrinogen- related protein-1 is associated with the fibrin matrix of a plasma clot. Biochem Biophys Res Commun. 2006;350(l): 191-194.

30. Yan J, Ying H, Gu F, et al. Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration. Cell Res. 2002;12(5-6):353-361.

31. Wang J, Sanmamed MF, Datar I, et al. Fibrinogen-like Protein 1 Is a Major Immune Inhibitory Ligand of LAG-3. Cell. 2019;176(l-2):334-347 e312.

32. Kim WY, Safran M, Buckley MR, et al. Failure to prolyl hydroxylate hypoxiainducible factor alpha phenocopies VHL inactivation in vivo. EMBO J. 2006;25(19):4650- 4662.

Claims

CLAIMS:

1. A Fibrinogen-like protein 1 (FGL1) inhibitor for use in the treatment of a subject affected with an iron overload associated disease.

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

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

4. The Fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 3 wherein iron-loading anemia is selected from the list consisting of alphathalassemia, P-thalassemia, congenital dyserythropoietic anemia, myelodistrophic syndrome (MDS).

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

6. The Fibrinogen-like protein 1 (FGL1) inhibitor for use according to any one of claim 1 to 5 wherein said inhibitor is

1) an inhibitor of Fibrinogen-like protein 1 (FGL1) activity and/or

2) an inhibitor of Fibrinogen-like protein 1 (FGL1) gene expression.

7. The Fibrinogen-like protein 1 (FGL1) inhibitor for use according to claim 6 wherein said inhibitor of Fibrinogen-like protein 1 (FGL1) activity is selected from the list consisting of an anti-Fibrinogen-like protein 1 (FGL1) neutralizing antibody, an anti-Fibrinogen-like protein 1 (FGL1) aptamer.

8. The 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 oligonucleotide, nuclease, siRNA, shRNA or ribozyme nucleic acid sequence.

9. The Fibrinogen-like protein 1 (FGL1) inhibitor for use according to any one of claim 1 to 8, in a subject presenting with a lower hepcidin level in a biological sample as compared to a predetermined reference value.

10. A method of preventing or treating iron overload associated disease in a subject comprising administering to the subject a therapeutically effective amount of a Fibrinogen-like protein 1 (FGL1) inhibitor and an Erythroferrone (ERFE) inhibitor.

11. The method of preventing or treating according to claim 10 wherein said inhibitors directly binds respectively to Fibrinogen-like protein 1 (FGL1) (protein or nucleic acid sequence (DNA or mRNA) an Erythroferrone (ERFE) (protein or nucleic acid sequence (DNA or mRNA)) and (ii) inhibits suppression of hepcidin expression.

12. The method of preventing or treating according to claim 10 and l lwherein iron overload associated disease is selected from the list consisting of hemochromatosis (adult and juvenile forms of hereditary hemochromatosis), iron-loading anemia, and chronic liver diseases including alcoholic liver diseases and chronic hepatitis B and C.

13. The method of preventing or treating according to claim 12 wherein iron-loading anemia is selected from the list consisting of alpha-thalassemia, P-thalassemia, congenital dyserythropoietic anemias, myelodistrophic syndrome (MDS).

14. The method of preventing or treating according to claim 13 wherein iron-loading anemia is beta thalassemia.

15. An in vitro method for screening a Fibrinogen-like protein 1 (FGL1) inhibitor for use in the treatment or prevention of iron overload associated disease which comprises the step consisting of: (i) providing purified FGL1 protein (or C-terminal globular domain of fibrinogen), providing a cell, tissue sample or organism expressing the FGL1, said organism being neither human nor animal

(iii) measuring the activity of the FGL1,

(iv) and selecting positively candidate compounds that, blocks the biological activity ofFGLl or inhibits FGL1 expression.