EP4665384A1

EP4665384A1 - Methods of treating iron deficiency-related diseases

Info

Publication number: EP4665384A1
Application number: EP24704453.0A
Authority: EP
Inventors: Léon KAUTZ
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite Toulouse III Paul Sabatier; Ecole Nationale Veterinaire de Toulouse ENVT; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Ecole Nationale Veterinaire de Toulouse ENVT; Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement; Universite de Toulouse
Priority date: 2023-02-13
Filing date: 2024-02-12
Publication date: 2025-12-24
Also published as: JP2026506600A; WO2024170500A1

Abstract

Anemia, defined as a decreased quantity of circulating functional red blood cells, is a major source of morbidity and mortality affecting a-third of the worldwide population. As a functional component of erythrocytes hemoglobin, iron is essential for oxygen storage and transport. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure the proper supply of iron to the bone marrow for red blood cells synthesis. However, mounting evidence suggested that another factor may exert a similar function. Inventors 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. Inventors demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgl1 in mice results in a blunted repression of hepcidin after bleeding. Finally, FGL1 is a BMP antagonist that directly binds BMP6 to impair the canonical BMP-SMAD signaling cascade that governs hepcidin regulation. Accordingly, the present invention relates to a FGL1 polypeptide for use in the treatment of a patient affected with an iron deficiency-related disease.

Description

METHODS OF TREATING IRON DEFICIENCY-RELATED DISEASES

FIELD OF THE INVENTION:

The present invention is in the field of medicine and relates to a FGL1 polypeptide for use in the treatment of a patient affected with an iron deficiency-related disease.

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.

SUMMARY OF THE INVENTION:

The invention is defined by the claims. In particular, the present invention relates to FGL1 polypeptide for use in the treatment of a patient affected with an iron deficiency-related disease wherein the polypeptide comprises an amino acid sequence selected from the group consisting of

(i) The amino acid sequence as set forth in SEQ ID NO:1 (FGL1);

(ii) The amino acid sequence as set forth in SEQ ID NO:3 (FGL1 mature protein);

(iii) The amino acid sequence as set forth in SEQ ID NO:4 (FGL1 C-ter globular domain);

(iv) An amino acid sequence substantially homologous to one of the sequences (i) to (iii), preferentially an amino acid sequence at least 80% identical to the sequence (i) to (iii); or

(v) A fragment of at least 30 consecutive amino acids selected in the group consisting in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4

DETAILED DESCRIPTION OF THE INVENTION:

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

A first object of the present invention relates to a FGL1 polypeptide for use in the treatment of a patient affected with an iron deficiency -related disease wherein the polypeptide comprises an amino acid sequence selected from the group consisting of

(i) The amino acid sequence as set forth in SEQ ID NO:1 (FGL1);

(ii) The amino acid sequence as set forth in SEQ ID NO:3 (FGL1 mature protein);

(iii) The amino acid sequence as set forth in SEQ ID NO:4 (FGL1 C-ter globular domain); (iv) An amino acid sequence substantially homologous to one of the sequences (i) to (iv), preferentially an amino acid sequence at least 80% identical to the sequence (i) to (iv); or

(v) A fragment of at least 30 consecutive amino acids selected in the group consisting in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4;

In some embodiments, the present invention also relates to a FGL1 polypeptide for use in the treatment of a patient affected with an iron deficiency-related disease wherein the polypeptide is a fragment of at least 30 consecutive amino acids selected in the group consisting in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 14.

As used herein, the term “patient” or “subject” denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with an iron deficiency-related disease. In a preferred embodiment, the patient is a human.

As used herein, the term “iron deficiency-related disease”, “iron deficiency- associated disease” or “iron deficiency-related disorders” refers to a group of diseases and/or disorders which are associated with abnormally high levels of hepcidin and includes diseases wherein aberrant iron metabolism directly causes the disease, or where 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. In some embodiments, the iron deficiency-related disease is selected from the group consisting in anemia, anemia of chronic disease, anemia of inflammation, anemia of infection, hypochromic microcytic anemia, iron-deficiency anemia, iron-refractory iron deficiency anemia, anemia of chronic kidney disease, anemias due to tumors that secrete hepcidin, cancer, erythropoietin resistance, attention deficit hyperactivity disorder (Oner et al., Pediatrics International, 2008), autism (Hergiiner et al. European journal of pediatrics, 2012), mental retardation (Lozoff et al. The New Engl and journal medicine, 1991), anxiety (Chen et al. BMC Psychiatry, 2013), bipolar disorder (Lee et al. BMC Psychiatry, 2020), impetigo (Wright et al. Frontier in pharmology, 2014), candidiasis (Wright et al. Frontier in pharmology, 2014), Helicobacter pylori infection (Cardernas et al. American journal of epidemiology, 2006), Thrichuris trichiura infection (Khuroo et al. Gastrointestinal endoscopy, 2010), multiple sclerosis (Kotze et al. Blood cells, molecules and diseases, 2001), rheumatoid arthritis (Vreugdenhil et al. Annals of the rheumatic diseases, 1990), lupus (Giannouli et al. Annals of the rheumatic diseases, 2006), inflammatory bowel disease (Kaitha et al. World journal of gastrointestinal pathophysiology, 2015), Crohn’s disease, ulcerative colitis, celiac disease (Corazza et al. Scandinavian journal of gastroenterology, 1995), autoimmune gastritis (World journal of gastroenterology, 2014), obesity (Nead et al. Pediatrics, 2004), hypothyroidism (Zimmerman et al. Thyroid : official journal of the American Thyroid Association, 2002), heart failure (van Veldhuisen et al. Nature reviews, 2011), stroke (Chang et al. PLoS One, 2013), Willis-Ekbom disease (Allen et al. American Journal of Hematology, 2013), chronic fatigue (Patterson et al. Quality of life research : an international journal of quality of life aspects of treatment, care and rehabilitation, 2000), fibromyalgia (Ortancil et al. European journal of clinical nutrition, 2010), chronic obstructive pulmonary disease (Nickol et al. BMJ Open, 2015), cystic fibrosis (Reid et al. Chest,

2002), chronic kidney disease (Macdougall et al. Kidney international, 2016), bum anemia (Betar et al. Burns, 2022), premenstrual syndrome (Chocano-Bedoya et al. American Journal of Epidemiology, 2013), premature delivery (Wali Lone et al. Tropical Medicine and International Health, 2004), fetal risk of death (Wali Lone et al. Tropical Medicine and International Health, 2004), post-partum depression (Corwin et al. The journal of nutrition,

2003). In some embodiments, the iron deficiency-related disease is cancer. In some embodiments, the iron deficiency-related disease is selected from the group consisting in anemia of chronic disease, anemia of inflammation, anemia of infection, anemia of chronic kidney disease, anemias due to tumors that secrete hepcidin and iron-refractory iron-deficiency anemia.

As used herein, the term “Fibrinogen-Like Protein 1” or “FGL1” refers to a protein belonging to the fibrinogen family. FGL1 is encoded by the FGL1 gene (Gene ID: 2267). The term "FGL1 expression" relates to both protein and mRNA expression, unless otherwise stated. FGL1 contains a C-terminal portion common to all members of the fibrinogen family, which contains four conserved cysteines. FGL1 lacks the platelet-binding site, cross-linking region, and thrombin-sensitive site which are necessary for fibrin clot formation. FGL1 is upregulated in regenerating liver and is abundantly associated with the fibrin matrix after clot formation. While the majority of FGL1 is found in plasma, approximately 20% of FGL1 remains in the serum after blood coagulation. An exemplary amino acid sequence for FGL1 is depicted in SEQ ID NO:! sapiens OX=9606 GN=FGL1 PE=1 MAKVFSFILV TTALTMGREI SALEDCAQEQ MRLRAQVRLL ETRVKQQQVK IKQLLQENEV QFLDKGDENT VIDLGSKRQY ADCSEI FNDG YKLSGFYKIK PLQSPAEFSV YCDMSDGGGW TVIQRRSDGS ENFNRGWKDY ENGFGNFVQK HGEYWLGNKN LHFLTTQEDY TLKIDLADFE KNSRYAQYKN FKVGDEKNFY ELNIGEYSGT AGDSLAGNFH PEVQWWASHQ RMKFSTWDRD HDNYEGNCAE EDQSGWWFNR CHSANLNGVY YSGPYTAKTD NGIVWYTWHG WWYSLKSWM KIRPNDFI PN VI

The amino acids 1 to 22 (SEQ ID NO:2, MAKVFSFILVTTALTMGREISA) correspond to the signal peptide and amino acids 23 to 312 (SEQ ID NO:3, LEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKR QYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNR GWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKN FKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYE GNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSV VMKIRPNDFIPNVI) correspond to the mature protein. The N-terminal domain corresponds to amino acids 23 to 78 (SEQ ID NO: 14,

LEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKR) . The C-terminal globular domain of fibrinogen corresponds to amino acids 79 to 312 (SEQ ID NO:4, QYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNR GWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKN FKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYE GNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSV VMKIRPNDFIPNVI).

As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labelling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.

Thus, in some embodiments, the FGL1 polypeptide is a biologically active part of the FGL1 protein (i.e. having a desired biochemical function of the intact protein). As an example, one of the desired biochemical functions of the intact FGL1 is the inhibition of hepcidin expression through the antagonization of BMP6 (Bone Morphogenic Protein 6). Accordingly, in some embodiments, the FGL1 polypeptide (i) directly binds BMP6 and/or (ii) inhibits hepcidin expression. As used herein, the term “hepcidin” refers to a protein involved in the maintenance of iron homeostasis and encoded by the HAMP gene (Gene ID: 57817). Hepcidin is necessary for the regulation of iron storage in macrophages and for intestinal iron absorption. When hepcidin level is high, serum iron falls due to iron trapping within the macrophages and hepatocytes. This typically leads to anemia due to an inadequate amount of serum iron being available for developing red blood cells. Thus, the functional assays based on hepcidin expression may be envisioned such as evaluating the ability to initiate hepcidin expression processes (e.g. in hepatic cells) through the inhibition of BMP6 by FGL1 polypeptide. Initiation process of FGL1 can be monitored by examining the inhibition 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 on experimental murine models of anemia (e.g. bleeding, chronic inflammation) may also be used such as evaluating the ability to lower hepcidin levels and increase liver and serum iron level. For instance, serum iron level can be assessed by widely used colorimetric assays or by transferrin saturation, transferrin being the iron carrier in the plasma and both parameters are currently used as a diagnostic test for iron-deficiency anemia.

In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino for use in the treatment of a patient affected with an iron deficiency- related disease is a FGL1 polypeptide wherein the at least 30 consecutive amino acids are selected in the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency-related disease is a FGL1 polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency- related disease is a FGL1 polypeptide consisting in the amino acid sequence as set forth in SEQ ID NO:3

In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency-related disease is a FGL1 polypeptide wherein the at least 30 consecutive amino acids are selected in the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency- related disease is a FGL1 polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency-related disease is a FGL1 polypeptide consisting in the amino acid sequence as set forth in SEQ ID NO:4.

In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency-related disease is a FGL1 polypeptide wherein the at least 30 consecutive amino acids are selected in the amino acid sequence as set forth in SEQ ID NO:14. In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency- related disease is a FGL1 polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 consecutive amino acids for use in the treatment of a patient affected with an iron deficiency-related disease is a FGL1 polypeptide consisting in the amino acid sequence as set forth in SEQ ID NO: 14.

In some embodiments, the FGL1 polypeptide comprises at least 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

61 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,

108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 consecutive amino acids selected in SEQ ID NO: 1 In some embodiments, the FGL1 polypeptide comprises at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,

44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 61, 63, 64, 65, 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,

114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 consecutive amino acids selected in SEQ ID NO:3 In some embodiments, the FGL1 polypeptide comprises at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,

47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 61, 63, 64, 65, 66, 67, 68, 69, 70, 71,

72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 consecutive amino acids selected in SEQ ID NO:4 In some embodiments, the FGL1 polypeptide comprises at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 consecutive amino acids selected in SEQ ID NO: 14

In some embodiments, the fragment of at least 30 consecutive amino acids selected in SEQ ID NO: 4 is DLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDG SENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRY AQYKNFKV (SEQ ID NO: 15).

In some embodiments, the fragment of at least 30 consecutive amino acids selected in SEQ ID NO: 4 is FNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQ YKNFKVGDEKNFYELNIGEYSGTAGD SL AGNFHPEVQWWASHQRMKF STWDRDHD NYEGNCAEEDQ (SEQ ID NO: 16).

In some embodiments, the fragment of at least 30 consecutive amino acids selected in SEQ ID NO: 4 is

FLTTQEDYTLKIDLADFEKNSRY AQYKNFKV (SEQ ID NO: 17).

In some embodiments, the present invention relates to a FGL1 polypeptide genetically engineered derivative for use in the treatment of a patient affected with an iron deficiency- related disease. As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).

In some embodiments, the FGL1 polypeptide according to the invention comprises at least one mutation. As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. Within the specification, the mutation are references according to the standard mutation nomenclature. In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:1 In some embodiments, the FGL1 polypeptide comprises a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide comprises a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 1 In some embodiments, the FGL1 polypeptide comprises a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide consists in a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide consists in a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:1 In some embodiments, the FGL1 polypeptide consists in a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:1.

In some embodiments, the FGL1 polypeptide comprises a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:2. In some embodiments, the FGL1 polypeptide comprises a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:2 In some embodiments, the FGL1 polypeptide comprises a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:2.

In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:3 In some embodiments, the FGL1 polypeptide comprises a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide comprises a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:3 In some embodiments, the FGL1 polypeptide comprises a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide consists in a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide consists in a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide consists in a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:3.

In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:4 In some embodiments, the FGL1 polypeptide comprises a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide comprises a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:4 In some embodiments, the FGL1 polypeptide comprises a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide consists in a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide consists in a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:4 In some embodiments, the FGL1 polypeptide consists in a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:4.

In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:14. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:14 In some embodiments, the FGL1 polypeptide comprises a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide comprises a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:14. In some embodiments, the FGL1 polypeptide comprises a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:14 In some embodiments, the FGL1 polypeptide consists in a sequence at least 80% identical to the amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide consists in a sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 14 In some embodiments, the FGL1 polypeptide consists in a sequence at least 95% identical to the amino acid sequence as set forth in SEQ ID NO: 14. As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotides or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

In a more particular aspect, the present invention also relates to a polynucleotide that encodes a FGL1 polypeptide according to the invention for use in the treatment of a patient affected with an iron deficiency-related disease.

As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogues thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single- stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the doublestranded form.

Exemplary nucleic acid sequences encoding FGL1 polypeptides are depicted in www.ncbi.nlm.nih.gov, Gene ID: 2267, or in ensembl.org, ENSG00000104760. In some embodiments, the polynucleotide comprises FGL1, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 14 corresponding DNA or RNA sequence. In some embodiments, the polynucleotide consists in FGL1, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO: 14 corresponding DNA or RNA sequence.

In some embodiments, the polynucleotide encoding the FGL1 polypeptide is comprised in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. In some embodiments, the polynucleotide encoding the FGL1 polypeptide is comprised in a plasmid or a viral vector. So, a further object of the invention relates to a polynucleotide encoding for the FGL1 polypeptide according to the invention comprised in a plasmid vector or a viral vector. In some embodiments, the invention also relates to a vector comprising the FGL1 polypeptide. In some embodiments, the vector is a pFUSEN-hG2Fc plasmid. Typically, the vector is a viral vector which is an adeno-associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special celllines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994, 136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the polynucleotide or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”.

A further object of the present invention relates to a host cell transformed with the vector comprising the polynucleotide encoding FGL1 polypeptide according to the invention. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed". In a particular embodiment, for expressing and producing the FGL1 polypeptide according to the present invention, prokaryotic cells, in particular E. coli cells, will be chosen. In some embodiments, FGL1 polypeptide is produced in an eukaryotic context that will favour post-translational modifications (e.g. glycosylation). Furthermore, prokaryotic cells have the advantages to produce protein in large amounts. If a eukaryotic context is needed, yeasts (e.g. saccharomyces strains) may be particularly suitable since they allow production of large amounts of proteins. Otherwise, typical eukaryotic cell lines such as CHO, BHK-21, COS-7, C127, PER.C6, YB2/0 or HEK293 could be used, for their ability to process to the right post- translational modifications of FGL1 polypeptide according to the present invention. The construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques. The polypeptides of the invention, can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering said polypeptides expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention. The polynucleotides encoding FGL1 polypeptides are typically used as medicament. In particular, the polynucleotides encoding FGL1 polypeptides (inserted or not into a vector) are particularly suitable for gene therapy. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In some embodiments, the above method and use further comprise the step of measuring the expression level of hepcidin (protein or nucleic DNA or mRNA) in a biological sample obtained from said subject wherein said expression level is compared to a reference value. In some embodiments, a high level of hepcidin is predictive of a high risk of having or developing an iron deficiency-related disease and means thatFGLl polypeptides should be used. Typically, a biological sample is obtained from the subject and the level of hepcidin is measured in this biological sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a serum sample. Increasing FGL1 levels would be particularly beneficial in these patients displaying high levels of hepcidin.

The FGL1 polypeptide 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 also relates to a pharmaceutical composition comprising a FGL1 polypeptide according to the invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention also relates to a pharmaceutical composition comprising a polynucleotide encoding a FGL1 polypeptide according to the invention and a pharmaceutically acceptable carrier. In some embodiments, the polynucleotide encoding a FGL1 polypeptide according to the invention is in a vector.

"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 patient 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 patient's health. In therapeutic treatments, the protein 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 activation of FGL1 polypeptide 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 patient 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 patient 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 lauryl sulfate 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 FGL1 according to the invention can be administered by any suitable route of administration. For example, FGL1 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. In a preferred embodiment, the FGL1 can be administered by oral (including buccal and sublingual), rectal or topical (intracolic) administration. The FGL1 polypeptide 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. 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}.

Accordingly, another object of the present invention relates to a combination of a FGL1 polypeptide and Erythroferrone (ERFE) for simultaneous or sequential use in preventing or treating iron deficiency-related disease.

As used herein, the term “Erythroferrone” or “ERFE” refers to a protein produced by erythroblasts which inhibits the transcription of hepcidin, and so increases the amount of iron available for hemoglobin synthesis. ERFE is encoded by ERFE gene (Gene ID: 151176). An exemplary amino acid sequence for ERFE is depicted is SEQ ID NO:5. sapiens OX=9606 GN=ERFE PE=2

MAPARRPAGA RLLLVYAGLL AAAAAGLGSP EPGAPSRSRA RREPPPGNEL PRGPGESRAG PAARPPEPTA ERAHSVDPRD AWMLFVRQSD KGVNGKKRSR GKAKKLKFGL PGPPGPPGPQ GPPGPI I PPE ALLKEFQLLL KGAVRQRERA EPEPCTCGPA GPVAASLAPV SATAGEDDDD WGDVLALLA APLAPGPRAP RVEAAFLCRL RRDALVERRA LHELGVYYLP DAEGAFRRGP GLNLTSGQYR APVAGFYALA ATLHVALGEP PRRGPPRPRD HLRLLICIQS RCQRNASLEA IMGLESSSEL FTI SVNGVLY LQMGQWTSVF LDNASGCSLT VRSGSHFSAV LLGV

The present invention also relates to a pharmaceutical composition comprising a FGL1 polypeptide and ERFE for simultaneous or sequential use in preventing or treating iron deficiency-related disease. In another embodiment, the present invention also relates to a pharmaceutical composition comprising a polynucleotide encoding a FGL1 polypeptide and a polynucleotide encoding ERFE for simultaneous or sequential use in preventing or treating iron deficiency-related disease. In another embodiment, the present invention also relates to a pharmaceutical composition comprising a polynucleotide encoding a FGL1 polypeptide and ERFE for simultaneous or sequential use in preventing or treating iron deficiency-related disease.

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, ***P < 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, ***P < 0.001, ** < 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 m Arig tll 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 EAZ-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 < 0.0001, ***P < 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. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 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, ** < 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.

EXAMPLE 1:

Material and Methods

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 (from 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 Glycine 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 ERI E Immunoassay

Human recombinant monoclonal antibodies to mouse ERFE were produced by Bio-rad using the HuCAL technology. High binding 96 well plate (Coming) 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, 0.05% 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 pL/well Supersensitive TMB substrate (Thermofisher) in the dark at room temperature, the reaction was stopped by adding 50 pL of 2Nsulfuric 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, Biolabo, 92108) and transferrin saturation was deduced by measuring the unsaturated iron binding capacity (UTBC, 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 (Therm ofi 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) with or without 500 ng of hBMP6 (Biotechne). Proteins were eluted using Laemmli buffer and analyzed by western blot using Goat antiHuman 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 hepatocytes 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 for in vivo as a difference between reference and target genes within each group of mice (-ACt) ± standard error of the mean (SEM). Data from Atbumin-Ere VHI.flox 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 marrows from phlebotomized mice were performed at the GeT-TriX facility (GenoToul, Genopole Toulouse Mi di -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 Erfe -defi ci ent 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 1A 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 iron 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-I- mice. The changes in hepcidin synthesis mErfe-/- 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, Figure 2F, Figure 2G). Similarly, SMAD 5 phosphorylation was not decreased in the liver of Erfe-/- mice 2 days after phlebotomy (Figure 2H, Figure 21). 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-/- 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-/- mice. Phlebotomized WT and Erfe-/- 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 E/'/c- - 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-/- mice (Figure 3C). Hepatic hepcidin mRNA expression was repressed in control WT and Erfe -/- 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 E/;/c- /- mice and evaluated the response to bleeding. However, splenectomized WT and Erfe-/- 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-/- 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-/- 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 formation^27,29. 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 m 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, Gapdip 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 of Albumin- C/v^? ( '///-deficient mice²⁰ (Figure 4G). However, a significant increase in Fgll mRNA expression was detected in livers of ////2/z-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 thatFg/7 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 5A). Treatment with recombinant Fc-tagged FGL1 for 6 hours under serum free conditions led to a significant reduction in HAMP and ID1 (Figure 5B, Figure 5C) expression in both hepatoma cell lines. A higher dose of FGL1 was required to repress HAMP and ID1 expression in serum-containing 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, Figure 5G) 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, Figure 6C) 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, Figure 6E). 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 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, Figure 7B) 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.

CONCLUSION

Here, we describe the identification of a new hepcidin suppressor that may contribute to hepcidin regulation during anemia: the liver produced hepatokine Fibrinogen like 1 (FGL1).

EXAMPLE 2

The effect of three fragments of the globular domain (SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17) was tested and demonstrated an ability to reduce the expression of hepcidin in a cellular model (data not shown).

TABLES

Table 1: Primers used for quantification of mRNA levels 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.

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Claims

CLAIMS:

1. A FGL1 polypeptide for use in the treatment of a patient affected with an iron deficiency-related disease wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:

(i) The amino acid sequence as set forth in SEQ ID NO:1 (FGL1);

(ii) The amino acid sequence as set forth in SEQ ID NO:3 (FGL1 mature protein);

2. The FGL1 polypeptide for use according to claim 1, wherein the FGL1 polypeptide consists in the amino acid sequence as set forth in SEQ ID NO:1.

3. The FGL1 polypeptide for use according to claim 1, wherein the FGL1 polypeptide consists in the amino acid sequence as set forth in SEQ ID NO:4.

4. The FGL1 polypeptide for use according to claim 1 to 3, wherein the FGL1 polypeptide (i) directly binds BMP6 and/or (ii) inhibits hepcidin expression.

5. A polynucleotide encoding the FGL1 polypeptide according to anyone of claim 1 to 4 for use in the treatment of a patient affected with an iron deficiency -related disease.

6. The polynucleotide for use according to claim 5, wherein the polynucleotide is comprised in a plasmid vector or a viral vector.

7. A host cell transformed with the polynucleotide encoding the FGL1 polypeptide according to claim 5 or 6 for use in the treatment of a patient affected with an iron deficiency-related disease.

8. A pharmaceutical composition comprising a FGL1 polypeptide for use according to claim 1 to 7 and a pharmaceutically acceptable carrier.

9. A combination of a FGL1 polypeptide and Erythroferrone (ERFE) for simultaneous or sequential use in preventing or treating iron deficiency-related disease.

10. The FGL1 polypeptide for use according to anyone of claim 1 to 9, wherein the iron deficiency-related disease is selected from the group consisting in anemia of chronic disease; anemia of inflammation; anemia of infection; hypochromic microcytic anemia; iron-deficiency anemia; iron-refractory iron deficiency anemia; anemia of chronic kidney disease; anemias due to tumors that secrete hepcidin; attention deficit hyperactivity disorder; erythropoietin resistance; autism; mental retardation; anxiety; bipolar disorder; impetigo; candidiasis; Helicobacter pylori infection; Thrichuris trichiura infection; multiple sclerosis; rheumatoid arthritis; lupus; inflammatory bowel disease; Crohn’s disease; ulcerative colitis; celiac disease; autoimmune gastritis; obesity; hypothyroidis; heart failure; stroke; Willis-Ekbom disease; chronic fatigue; fibromyalgia; chronic obstructive pulmonary disease; cystic fibrosis; premenstrual syndrome; premature delivery; fetal risk of death or post-partum depression.

11. The FGL1 polypeptide for use according to claim 10, wherein the iron deficiency- related disease is selected from the group consisting in anemia of chronic disease, anemia of inflammation, anemia of infection, anemia of chronic kidney disease, anemias due to tumors that secrete hepcidin and iron-refractory iron-deficiency anemia.