Methods for treating iron deficiency-related diseases

Abstract

Anemia, defined as a reduction in the amount of functional red blood cells in circulation, is a major cause of disease affecting one-third of the world's population. Iron is essential for hemoglobin, the functional component of red blood cells, to store and transport oxygen. Hepcidin, a liver-derived peptide, is a key regulator of iron homeostasis. During anemia, the erythropoietic hormone erythroferon regulates hepcidin synthesis to ensure an adequate supply of iron to the bone marrow for red blood cell synthesis. However, accumulating evidence suggests that other factors may perform a similar function. We identified the hepatokine FGL1 as a previously undescribed hepcidin suppressor that is highly induced in the liver in response to hypoxia during recovery from anemia and in thalassemia mice. We demonstrated that FGL1 is a potent hepcidin suppressor in vitro and in vivo. Deletion of Fgl1 in mice blunts hepcidin suppression after hemorrhage.Finally, FGL1 is a BMP antagonist that directly binds to BMP6 and impairs the BMP-SMAD signaling cascade that controls hepcidin regulation.Therefore, the present invention relates to an FGL1 polypeptide for use in treating patients suffering from iron deficiency-related diseases.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention is in the field of medicine and relates to an FGL1 polypeptide for use in treating patients suffering from iron deficiency-related diseases.

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

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

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

[0005] Summary of the Invention The present invention is defined by the claims. In particular, the present invention provides an FGL1 polypeptide for use in treating a patient suffering from an iron deficiency-related disease, the polypeptide comprising: (i) the amino acid sequence shown in SEQ ID NO: 1 (FGL1); (ii) the amino acid sequence shown in SEQ ID NO: 3 (FGL1 mature protein); (iii) the amino acid sequence shown in SEQ ID NO: 4 (FGL1 C-terminal globular domain); (iv) an amino acid sequence that is substantially homologous to one of the sequences (i) to (iii), preferably an amino acid sequence that is at least 80% identical to the sequence (i) to (iii); or (v) a fragment of at least 30 consecutive amino acids selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4 The present invention relates to an FGL1 polypeptide comprising an amino acid sequence selected from the group consisting of:

[0006] Detailed Description of the Invention We investigated hepcidin regulation during recovery from hemorrhage-induced anemia in wild-type and Erfe-deficient mice and confirmed ERFE-independent repression of hepcidin during anemia. Here, we report the identification of a novel hepcidin suppressor, fibrinogen-like 1 (FGL1), a hepatokine produced in the liver, that may contribute to hepcidin regulation during anemia.

[0007] A first object of the present invention is to provide an FGL1 polypeptide for use in treating a patient suffering from an iron deficiency related disease, said polypeptide comprising: (i) the amino acid sequence shown in SEQ ID NO: 1 (FGL1); (ii) the amino acid sequence shown in SEQ ID NO: 3 (FGL1 mature protein); (iii) the amino acid sequence shown in SEQ ID NO: 4 (FGL1 C-terminal globular domain); (iv) an amino acid sequence that is substantially homologous to one of the sequences (i) to (iii), preferably an amino acid sequence that is at least 80% identical to the sequence (i) to (iii); or (v) a fragment of at least 30 consecutive amino acids selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4 The present invention relates to an FGL1 polypeptide comprising an amino acid sequence selected from the group consisting of:

[0008] In some embodiments, the present invention relates to an FGL1 polypeptide for use in treating a patient suffering from an iron deficiency-related disease, wherein the polypeptide is a fragment of at least 30 consecutive amino acids selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 14.

[0009] As used herein, the term "patient" or "subject" refers to a mammal. Typically, a subject of the present invention refers to any subject (preferably a human) suffering from or susceptible to an iron deficiency-related disorder. In a preferred embodiment, the patient is a human.

[0010] As used herein, the terms "iron deficiency related disease," "iron deficiency associated disease," or "iron deficiency related disorder" refer to a group of diseases and / or disorders associated with abnormally high levels of hepcidin, including diseases in which abnormalities in iron metabolism directly cause the disease, or diseases in which deregulation of blood iron levels causes the disease, or diseases in which deregulation of iron occurs as a result of another disease, or diseases that can be treated by modulating iron levels. In some embodiments, the iron deficiency-related disorder is anemia, anemia of chronic disease, anemia of inflammation, anemia of infection, hypochromic microcytic anemia, iron deficiency anemia, iron-resistant iron deficiency anemia, anemia of chronic kidney disease, anemia due to hepcidin-secreting tumors, cancer, erythropoietin resistance, attention deficit hyperactivity disorder (Oner et al., Pediatrics International, 2008), autism (Herguner et al. European journal of pediatrics, 2012), intellectual disability (Lozoff et al. The New England journal of medicine, 1991), anxiety (Chen et al. BMC Psychiatry, 2013), bipolar disorder (Lee et al. BMC Psychiatry, 2020), impetigo (Wright et al. Frontier in pharmacy, 2014), candidiasis (Wright et al. Frontier in pharmacy, 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), intracranial stroke (Chang et al. PLoS One, 2013), Weiers-Ectomy (Allen et al. American Journal of Hematology, 2013). 2013), chronic fatigue (Patterson et al. Quality of life research: an international journal of quality of life aspects of treatment, care and rehabilitation, 2000), fibrosis (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), heatstroke anemia (Betar et al. Burns, 2022), premenstrual syndrome (Chocano-Bedoya et al. American Journal of Epidemiology, 2013), preterm birth (Wali Lone et al.Tropical Medicine and International Health, 2004), risk of fetal death (Wali Lone et al. Tropical Medicine and International Health, 2004), and postpartum depression (Corwin et al. The Journal of Nutrition, 2003). In some embodiments, the iron deficiency-associated disease is cancer. In some embodiments, the iron deficiency-associated disease is selected from the group consisting of anemia of chronic disease, anemia of inflammation, anemia of infection, anemia of chronic kidney disease, anemia due to hepcidin-secreting tumors, and iron-resistant iron deficiency anemia.

[0011] 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" refers to both protein expression and mRNA expression, unless otherwise specified. FGL1 contains the C-terminal portion common to all members of the fibrinogen family, which contains four conserved cysteines. FGL1 lacks the platelet-binding site, bridging region, and thrombin-sensitive site required for fibrin clot formation. FGL1 is upregulated in the regenerating liver and is abundantly associated with the fibrin matrix after clotting. Most FGL1 is found in plasma, but approximately 20% FGL1 remains in serum after blood clotting. An exemplary amino acid sequence of FGL1 is set forth in SEQ ID NO: 1.

[0012] [ka]

[0013] 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, LEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVI) correspond to the mature protein. The N-terminal domain corresponds to amino acids 23-78 (SEQ ID NO: 14, LEDCAQEQMRLRAQVRLLETRVKQQQVKIKQLLQENEVQFLDKGDENTVIDLGSKR). The C-terminal globular domain of fibrinogen corresponds to amino acids 79-312 (SEQ ID NO: 4, QYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQSGWWFNRCHSANLNGVYYSGPYTAKTDNGIVWYTWHGWWYSLKSVVMKIRPNDFIPNVI).

[0014] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. These terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. When considered in the context of gene therapy, a polypeptide refers to the respective intact polypeptide or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein.

[0015] Therefore, in some embodiments, the FGL1 polypeptide is a biologically active portion of the FGL1 protein (i.e., has the desired biochemical function of the intact protein). For example, one of the desired biochemical functions of intact FGL1 is the inhibition of hepcidin expression by antagonizing BMP6 (bone morphogenetic protein 6). Thus, in some embodiments, the FGL1 polypeptide (i) directly binds to BMP6 and / or (ii) inhibits hepcidin expression.

[0016] As used herein, the term "hepcidin" refers to a protein involved in iron homeostasis and is encoded by the HAMP gene (gene ID: 57817). Hepcidin is essential for regulating iron stores in macrophages and for iron absorption in the intestinal tract. High hepcidin levels result in reduced serum iron due to iron sequestration in macrophages and hepatocytes. This typically results in anemia due to insufficient serum iron available for red blood cell production. Therefore, functional assays based on hepcidin expression can be envisioned, such as assays to evaluate the ability of FGL1 polypeptides to initiate the hepcidin expression process (e.g., in hepatocytes) through the inhibition of BMP6. The FGL1 initiation process can be monitored by RT-qPCR and / or Western blot to examine the inhibition of hepcidin and p-SMAD5 expression (see also Kautz L, et al. Nat. Genet. 2014;46(7):678-684). Other functional assays based on experimental mouse models of anemia (e.g., hemorrhage, chronic inflammation), such as assays assessing the ability to reduce hepcidin levels and increase liver and serum iron levels, can also be used. For example, serum iron levels can be assessed by a widely used chromogenic assay or by transferrin saturation. Transferrin is the iron transport protein in plasma, and both parameters are currently used as diagnostic tests for iron deficiency anemia.

[0017] In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide, wherein the at least 30 contiguous amino acids are selected from the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3.

[0018] In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide, wherein the at least 30 contiguous amino acids are selected from the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0019] In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide, wherein the at least 30 contiguous amino acids are selected from the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, a fibrinogen-like protein 1 (FGL1) polypeptide comprising at least 30 contiguous amino acids for use in treating a patient suffering from an iron deficiency-associated disease is an FGL1 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0020] In some embodiments, the FGL1 polypeptide comprises at least one of the following consecutive sequences selected from SEQ ID NO: 1: 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, 62, 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, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 14 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. In some embodiments, the FGL1 polypeptide comprises at least one of the following consecutive sequences selected from SEQ ID NO:3: 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, 62, 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, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.In some embodiments, the FGL1 polypeptide comprises at least one of the following consecutive sequences selected from SEQ ID NO:4: 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, 62, 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, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids. 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.

[0021] In some embodiments, a fragment of at least 30 consecutive amino acids selected from SEQ ID NO:4 is DLGSKRQYADCSEIFNDGYKLSGFYKIKPLQSPAEFSVYCDMSDGGGWTVIQRRSDGSENFNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKV (SEQ ID NO: 15) is.

[0022] In some embodiments, a fragment of at least 30 consecutive amino acids selected from SEQ ID NO:4 is FNRGWKDYENGFGNFVQKHGEYWLGNKNLHFLTTQEDYTLKIDLADFEKNSRYAQYKNFKVGDEKNFYELNIGEYSGTAGDSLAGNFHPEVQWWASHQRMKFSTWDRDHDNYEGNCAEEDQ (SEQ ID NO: 16) is.

[0023] In some embodiments, a fragment of at least 30 consecutive amino acids selected from SEQ ID NO:4 is FLTTQEDYTLKIDLADFEKNSRYAQYKNFKV (SEQ ID NO: 17) is.

[0024] In some embodiments, the present invention relates to a genetically engineered derivative of FGL1 polypeptide for use in treating patients suffering from iron deficiency-related diseases. As used herein, the term "derived from" refers to a process of isolating, deriving, or producing a different second component (e.g., a second polypeptide different from the first polypeptide) using a first component (e.g., a first polypeptide) or information from the first component.

[0025] In some embodiments, the FGL1 polypeptide of the present 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 at the same position. Mutations are referred to herein according to standard mutation nomenclature.

[0026] In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1.

[0027] In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0028] In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3.

[0029] In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4.

[0030] In some embodiments, the FGL1 polypeptide is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide comprises a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the FGL1 polypeptide consists of a sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:14.

[0031] As used herein, the "% identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % identity = number of identical positions / total number of positions × 100). Sequence comparison and determination of the % identity between two sequences can be accomplished using the mathematical algorithm described below. The % 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 % identity between two nucleotide or amino acid sequences can also be determined using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with the BLOSUM62 matrix, a "Gap Open Penalty" of 10, a "Gap Extend Penalty" of 0.5, an "End Gap Penalty" of false, an "End Gap Open Penalty" of 10, and an "End Gap Extend Penalty" of 0.5. Generally, "% identity" is a function of the number of matching positions divided by the number of compared positions multiplied by 100. For example, if 6 out of 10 sequence positions are identical between two compared sequences after alignment, the identity is 60%. % identity is typically determined over the entire length of the query sequence analyzed. Two molecules with the same primary amino acid or nucleic acid sequence are identical, regardless of the presence or absence of any chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 90% identity to a second amino acid sequence means that the first sequence has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the second amino acid sequence.

[0032] In a more particular embodiment, the present invention also relates to a polynucleotide encoding the FGL1 polypeptide of the present invention for use in treating a patient suffering from an iron deficiency-related disease.

[0033] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, comprising deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified, when any embodiment of the invention described herein is a polynucleotide, this embodiment encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0034] Exemplary nucleic acid sequences encoding FGL1 polypeptides are set forth at www.ncbi.nlm.nih.gov under Gene ID: 2267 or at ensembl.org under ENSG00000104760. In some embodiments, the polynucleotide comprises a DNA or RNA sequence corresponding to FGL1 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 14. In some embodiments, the polynucleotide consists of a DNA or RNA sequence corresponding to FGL1 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 14.

[0035] In some embodiments, the polynucleotide encoding the FGL1 polypeptide is contained in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. In some embodiments, the polynucleotide encoding the FGL1 polypeptide is contained in a plasmid or viral vector. Therefore, a further object of the present invention relates to a polynucleotide encoding the FGL1 polypeptide of the present invention contained in a plasmid vector or viral vector. In some embodiments, the present 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, and the viral vector is an adeno-associated virus (AAV), a retrovirus, a bovine papilloma virus, an adenovirus vector, a lentivirus vector, a vaccinia virus, a polyoma virus, or an infectious virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutants thereof. AAV vectors may have one or more AAV wild-type genes, preferably the rep and / or cap genes, deleted in whole or in part, but may retain functional flanking ITR sequences. Retroviruses can be selected as gene transfer vectors because of their ability to integrate genes into the host genome, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specific cell lines. To construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in place of a specific viral sequence to produce a replication-incompetent virus. To produce viral particles, a packaging cell line is constructed that contains the gag, pol, and / or env genes but lacks the long terminal repeats and / or packaging components.When a recombinant plasmid containing a cDNA is introduced into this cell line (e.g., by calcium phosphate precipitation) along with retroviral LTRs and packaging sequences, the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium. The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a variety of cell types. Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows the virus to modulate its life cycle during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV1, HIV2) and simian immunodeficiency virus (SIV). Lentiviral vectors were generated by multiple attenuation of HIV pathogenic genes. For example, the env, vif, vpr, vpu, and nef genes have been deleted to render 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. Typically, vectors are plasmid- or virus-based and are constructed to carry sequences essential for the integration, selection, and delivery of foreign nucleic acids into host cells. The gag, pol, and env genes of a given vector are also known in the art. Thus, the relevant genes are cloned into a selected vector and then used to transform a target cell of interest. Recombinant lentiviruses are capable of infecting non-dividing cells, where a suitable host cell is transfected with two or more vectors containing packaging functions. Namely, gag, pol, and env, as well as rev and tat, are described in U.S. Pat. No. 5,994,136. No. 6,213,199, which is incorporated herein by reference.This document describes a first vector capable of providing nucleic acid encoding the viral gag and pol genes, and another vector capable of providing nucleic acid encoding the viral env gene to produce packaging cells. By introducing a vector providing a heterologous gene into the packaging cells, producer cells are generated that release infectious viral particles carrying the foreign gene of interest. env is preferably an amphotropic envelope protein that enables transduction of human and other cell types. Typically, the polynucleotide or vector of the present invention contains a "control sequence." Control sequences collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites (IRES), enhancers, and the like. These sequences collectively provide for the replication, transcription, and translation of the coding sequence in recipient cells. Not all of these control sequences need always be present, as 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. A promoter sequence is used herein in its ordinary sense to refer to a nucleotide region containing a DNA regulatory sequence, where the regulatory sequence is derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcriptional promoters may 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."

[0036] A further object of the present invention relates to a host cell transformed with a vector containing a polynucleotide encoding the FGL1 polypeptide of the present invention. The term "transformation" refers to the introduction of a "foreign" (i.e., foreign or extracellular) gene, DNA, or RNA sequence into a host cell, such that the host cell will express the introduced gene or sequence and produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that accepts and expresses the introduced DNA or RNA has been "transformed." In certain embodiments, prokaryotic cells, particularly E. coli cells, will be selected to express and produce the FGL1 polypeptide of the present invention. In some embodiments, the FGL1 polypeptide is produced in a eukaryotic environment that may facilitate post-translational modifications (e.g., glycosylation). Furthermore, prokaryotic cells have the advantage of producing large amounts of protein. When a eukaryotic environment is required, yeast (e.g., Saccharomyces strains) may be particularly suitable, as yeast allows for the production of large amounts of protein. Otherwise, typical eukaryotic cell lines, such as CHO, BHK-21, COS-7, C127, PER.C6, YB2 / 0, or HEK293, may be used due to their ability to process appropriate post-translational modifications of the FGL1 polypeptide of the present invention. Construction of expression vectors and transformation of host cells according to the present invention can be performed using conventional molecular biology techniques. The polypeptide of the present invention can be obtained, for example, by culturing cells genetically transfected according to the present invention and recovering the polypeptide expressed by the cells from the culture. The polypeptide can then be purified, if necessary, by techniques conventionally known to those skilled in the art, such as fractional precipitation, particularly ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins can be used to produce the protein of the present invention. Polynucleotides encoding FGL1 polypeptides are typically used as pharmaceuticals.In particular, a polynucleotide encoding an FGL1 polypeptide (whether inserted into a vector or not) is particularly suitable for gene therapy.

[0037] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventive treatment and curative or disease-modifying treatment, including treatment of subjects at risk of or suspected of having a disease, as well as diseased subjects or subjects diagnosed with a disease or medical condition, including suppression of clinical recurrence. Treatment can be administered to subjects with a medical disorder or subjects who are likely to eventually develop the disorder in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of, the disorder or recurrent disorder, or to prolong the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., a dosing pattern used during treatment. Therapeutic regimens can include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general purpose of an induction regimen is to provide high levels of a drug to a subject during the initial stages of the treatment regimen. An induction regimen may utilize (in part or in whole) a "loading regimen." A loading regimen may involve administering a higher dose of a drug than a physician would utilize during a maintenance regimen, administering a drug more frequently than a physician would utilize during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a subject during treatment of a disease, e.g., to maintain a subject in remission over an extended period of time (months or years). A maintenance regimen may utilize continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching certain pre-established criteria (e.g., pain, disease symptoms, etc.)).

[0038] In some embodiments, the above-mentioned method and use further comprises measuring the expression level of hepcidin (protein or nuclear DNA or mRNA) in a biological sample obtained from the subject. Here, the expression level is compared with a reference value. In some embodiments, a high level of hepcidin indicates that the subject has or is at high risk of developing iron deficiency-related diseases, and means that FGL1 polypeptide should be used. Typically, a biological sample is obtained from the subject, and the hepcidin level in this biological sample is measured. 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. In these patients who show high levels of hepcidin, increasing FGL1 levels will be particularly beneficial.

[0039] The above-described FGL1 polypeptide can be combined with a pharmaceutically acceptable excipient, and optionally with a sustained-release matrix, such as a biodegradable polymer, to form a therapeutic composition. Thus, the present invention also relates to a pharmaceutical composition comprising the FGL1 polypeptide of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention also relates to a pharmaceutical composition comprising a polynucleotide encoding the FGL1 polypeptide of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the polynucleotide encoding the FGL1 polypeptide of the present invention is present in a vector.

[0040] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation auxiliary. In therapeutic applications, the composition is administered to a patient already suffering from the described disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Appropriate doses of pharmaceutical compositions are readily determined according to any one of several well-established protocols. For example, animal studies (e.g., in mice or rats) are commonly used to determine the maximum tolerated dose per kilogram of body weight of a bioactive agent. Typically, at least one of the animal species tested is a mammal. The results of animal studies can be extrapolated to determine dosages for use in other species, such as humans. What constitutes an effective dose also depends on the nature and severity of the disease or condition and the patient's health status. In therapeutic treatments, the protein contained in the pharmaceutical composition can be administered in multiple doses or in a single dose until the desired response is achieved. Treatment is typically monitored, and repeated doses can be administered as needed. The compounds of the present invention can be administered according to established dosing regimens when activation of the FGL1 polypeptide is required. The daily dosage of the product can vary over a wide range, from 0.01 to 1,000 mg per adult per day. Preferably, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, or 500 mg of the active ingredient, with the dosage adjusted depending on the symptoms of the patient being treated. Pharmaceuticals typically contain about 0.01 mg to about 500 mg of the active ingredient, preferably 1 mg to about 100 mg. An effective amount of the drug is ordinarily supplied at a dosage level of from 0.0002 mg to about 20 mg per kg of body weight per day, especially from about 0.001 mg to 10 mg per kg of body weight per day.It will be understood, however, that the specific dose level and frequency of administration for any particular patient may vary and will depend on a variety of factors, including the activity of the specific compound employed, the metabolic stability and duration of action of that compound, the age, weight, health, sex, diet, method and time of administration, rate of excretion, drug combinations, the severity of the particular condition, and the host being treated. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active principle, alone or in combination with another active principle, can be administered to animals and humans in unit dosage forms or in admixture with a conventional pharmaceutical carrier. Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal, and intranasal dosage forms, and rectal dosage forms. Suitable unit dosage forms include oral dosage forms, such as tablets, gelatin capsules, powders, granules, and orally ingested solutions or suspensions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, intramuscular, intravenous, intranasal, or intraocular dosage forms, and rectal dosage forms. In the pharmaceutical compositions of the present invention, the active principle is generally formulated as a dosage unit containing 0.5 to 1000 mg, preferably 1 to 500 mg, and more preferably 2 to 200 mg of the active principle per dosage unit per day. When preparing solid compositions in tablet form, a wetting agent, such as sodium lauryl sulfate, can be added to the active principle, which can optionally be micronized. This is then mixed with a pharmaceutical vehicle, such as silica, gelatin, starch, lactose, magnesium stearate, talc, gum arabic, etc. Tablets can be coated with sucrose, various polymers, or other suitable materials, or they can be otherwise processed to have a sustained or delayed release, allowing a predetermined amount of the active principle to be continuously released. Preparations in the form of gelatin capsules are obtained by mixing the active principle with a diluent, for example a glycol or glycerol ester, and filling the mixture obtained into soft or hard gelatin capsules.Preparations in the form of syrups or elixirs may contain the active principle together with a sweetener (preferably calorie-free), methylparaben and propylparaben as preservatives, flavorings, and suitable colorants. Water-dispersible powders or granules may contain the active principle mixed with a dispersing or wetting agent or suspending agent, such as polyvinylpyrrolidone, and also a sweetener or taste modifier. The active principle may also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives. Among the sustained-release forms useful for chronic treatment, implants can be used. These can be prepared in the form of an oily suspension or a microsphere suspension in an isotonic medium. The FGL1 of the present invention can be administered by any suitable administration route. For example, the FGL1 of the present invention can be administered orally (including buccal and sublingual), rectally, nasally, topically (intracolonically), pulmonary, vaginally, or parenterally (including intramuscular, intraarterial, intrathecal, subcutaneous, and intravenous). In a preferred embodiment, FGL1 can be administered orally (including buccal and sublingual), rectally, or topically (intracolonic). The FGL1 polypeptide of the present invention can be formulated into a variety of oral dosage forms. The term "preparation" is intended to include formulating the active compound with an encapsulating material as a carrier, providing a capsule in which the active ingredient (with or without a carrier) is associated with the carrier and surrounded by the carrier. Similarly, cachets and lozenges are also included. Tablets, powders, capsules, pills, cachets, and lozenges may be solid preparations suitable for oral administration. Other forms suitable for oral administration include liquid preparations, including emulsions, syrups, elixirs, aqueous solutions, and aqueous suspensions, or solid preparations intended to be converted to liquid preparations immediately before use. Emulsions can be prepared in solutions, for example, in aqueous propylene glycol solutions, or can contain emulsifying agents, such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents.Aqueous suspensions can be prepared by dispersing the finely divided active ingredient in water with viscous substances, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. Solid preparations include solutions, suspensions, and emulsions, which may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, and the like.

[0041] It has been previously reported that hepcidin expression is rapidly suppressed by the erythropoiesis regulator erythroferon (ERFE) in conditions associated with increased erythropoiesis, such as anemia caused by bleeding or inflammation. 10,11 .

[0042] Therefore, another object of the present invention relates to a combination of an FGL1 polypeptide and erythroferon (ERFE) for simultaneous or sequential use in preventing or treating iron deficiency-related diseases.

[0043] As used herein, the term "erythroferon" or "ERFE" refers to a protein produced by erythroblasts that inhibits hepcidin transcription, thereby increasing the amount of iron available for hemoglobin synthesis. ERFE is encoded by the ERFE gene (Gene ID: 151176). An exemplary amino acid sequence for ERFE is set forth in SEQ ID NO: 5. [ka]

[0044] The present invention also relates to a pharmaceutical composition comprising an FGL1 polypeptide and ERFE for simultaneous or sequential use in preventing or treating an iron deficiency-associated disease. In another embodiment, the present invention also relates to a pharmaceutical composition comprising a polynucleotide encoding an FGL1 polypeptide and a polynucleotide encoding ERFE for simultaneous or sequential use in preventing or treating an iron deficiency-associated disease. In another embodiment, the present invention also relates to a pharmaceutical composition comprising an FGL1 polypeptide and a polynucleotide encoding ERFE for simultaneous or sequential use in preventing or treating an iron deficiency-associated disease. [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1. Recovery from hemorrhage-induced anemia in WT mice. (A) Hemoglobin levels in 7- to 9-week-old WT male mice on days 0, 1, 2, 3, 4, 5, and 6 after 500 μl of bloodletting. mRNA expression of Epo in the kidney (B), Erfe in the bone marrow and spleen (C), and Hamp, Id1, and Smad7 in the liver (E) of bled mice. (D) Time course of serum Erfe concentrations. Data shown are mean ± sem. For each time point, values ​​were compared with those of control mice at t = 0 (n = 5–7 per time point) and control mice (day 0) by one-way analysis of variance. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 2]Figure 2. Erfe-independent repression of hepcidin during recovery from anemia. Iron-related parameters in 7- to 9-week-old Erfe- / - mice 0-6 days after phlebotomy (500 μl). Parameters included hemoglobin levels (A), Epo mRNA expression in the kidney (B), Hamp, Id1, and Smad7 mRNA expression in the liver (C), serum hepcidin concentration (D), serum iron content (E), transferrin saturation (F), and liver iron content (G). (H) Western blots for P-Smad5, Smad5, and vinculin in the liver of Erfe- / - mice 0, 1, and 2 days after phlebotomy. (I) Densitometric ratios of phosphorylated Smad5 to total Smad5 or vinculin and Smad5 to vinculin. Data shown are means ± sem and were compared with the values ​​from control mice (n = 5–8) at t = 0 for each time point by one-way ANOVA (A, B, C) or Student's t test (D, E, F, G, I). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 3] Figure 3. Validation of the potential contribution of erythroid regulators. % reticulocytes (A), Gypa and Tfr1 mRNA expression in bone marrow (B), hemoglobin levels (C), and Hamp mRNA expression in liver (D) of 8-week-old control and irradiated (400 rad) WT and Erfe- / - mice (n=4-9) at t=0 (white bars) and 48 hours (blue bars) after exsanguination. (E) Gypa mRNA expression in bone marrow, spleen, and liver of Erfe- / - mice (n=5-8) at t=0-6 days after exsanguination. (F) Linear regression analysis of Gypa and Hamp mRNA expression in liver and spleen. (G) Hamp mRNA expression in liver of 7-9-week-old WT control and splenectomized WT and Erfe- / - mice (n=3-6) at t=0 (white bars) and 48 hours (blue bars) after exsanguination. Data shown are means ± sem and were compared between groups by two-way analysis of variance. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 4]Figure 4. Fgl1 mRNA expression is induced in mouse livers during anemia. Time course of Fgl1 mRNA expression in the liver (A) and bone marrow (B) of WT and Erfe- / - mice (n = 5-8) 1-6 days after exsanguination. (C) Hamp and Fgl1 mRNA expression in the liver of 7-week-old mice (n = 5) t = 0-20 h after a single intraperitoneal injection of EPO (200 μg). (D) Fgl1 mRNA expression in the liver of 8-week-old WT, Th3 / +, and Erfe- / -;Th3 / + mice (n = 7-9). Relative mRNA expression of HIF target genes Vegfa, Gapdh, and Angptl1 in mouse primary hepatocytes cultured in serum-free or serum-containing medium and incubated in the presence of the prolyl hydroxylase inhibitor DMOG or under hypoxic conditions (2%) for 15 h compared with untreated cells (E). (F) Relative Fgl1 expression in mouse primary hepatocytes incubated under hypoxic conditions or in the presence of DMOG, relative Fgl1 expression in the livers of Vhl-deficient mice (G), and relative Fgl1 expression in the livers of mice treated with the prolyl hydroxylase inhibitor vadadustat (H). Data shown are means ± sem and were compared with control WT values ​​at t = 0 for each time point by two-way ANOVA (A, B, C) or with WT mice by Student's t test. Data shown for experiments with primary hepatocytes are the means of three independent experiments and were compared with control cells by Student's t test (E, F, G). ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 5]Figure 5. FGL1 is a hepcidin suppressor in vivo and in vitro. Relative HAMP expression in Hep3B and HepG2 cells in response to BMP6 (25 ng / ml, 6 hours) (A) or BMP6 plus recombinant FGL1 (10 μg / ml) (B). Relative ID1 expression in a hepatocellular carcinoma cell line treated with BMP6 and FGL1 (C). HAMP (D) and ID1 (E) expression in Hep3B cells and primary mouse hepatocytes treated with BMP6 and either Fc, full-length FGL1, or the N-terminal or globular domain of FGL1 for 6 hours. Liver HAMP RNA expression (F), serum hepcidin concentration (G), and liver Id1 mRNA expression (H) in mice (n = 5) treated with saline, Fc, or recombinant FGL1 (10 mg / kg) for 6 hours. Data shown are means ± sem from three independent experiments (A–E) or treated mice, and for each condition, were compared with untreated cells or control mice by Student's t test. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. [Figure 6] Figure 6. Fgl1- / - mice exhibit a blunted response to bloodletting. (A) Fgl1 mRNA expression in the livers of male and female WT mice compared with control mice at 36 h post-hemorrhage. Red blood cell (RBC) counts (B) and hemoglobin (Hb) (C), Erfe mRNA expression in the bone marrow (D) and spleen (E), and Hamp (F), Id1 (G), and Smad7 (H) mRNA expression in the liver were measured in WT (white bars) and Fgl1- / - (black bars) mice at t = 0 or 36 h post-hemorrhage. Data shown are mean ± sem (n = 5–11). Comparisons between groups were performed by two-way ANOVA with correction for multiple comparisons by the Holm-Sidak test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. [Figure 7]Figure 7. FGL1 is a BMP antagonist. Relative expression of Hamp (A), Id1 (B), and Smad7 (C) mRNA in mouse primary hepatocytes treated with BMP ligand (10 ng / ml) and human Fc IgG2 (10 μg / ml) or Fc-FGL1 (10 μg / ml) for 6 hours. Data shown are the mean ± sem of three independent experiments. For each BMP, comparisons were made between Fc- or FGL1-treated cells and control cells by two-way ANOVA. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. (D) Western blot analysis of P-SMAD5, SMAD5, and GAPDH in Hep3B cells treated with BMP6 (10 ng / ml), ERFE (1 μg / ml), or FGL1 (10 μg / ml) for 6 hours. (E) Western blot of BMP6 and FGL1 pull-down assay for human Fc IgG2 and BMP6.

[0046] Example 1 Materials and Methods Animal models Erfe on a C57BL / 6J background - / - and Erfe+ / + mice were bred and housed in a specific pathogen-free barrier facility within the animal facility of INSERM US006. Fgl1- / - and WT controls on a C57Bl / 6N background were obtained from The European Mouse Mutant Archive (EMMA) and bred at Janvier labs (Le Genest St Isle) before being transferred to the animal facility of INSERM US006 at 4-5 weeks of age. Mice were housed under a standard 12-h light / dark cycle in accordance with European Union guidelines and provided with ad libitum access to water and standard laboratory mouse chow (Ssniff, iron 200 mg / kg). This study was approved by the Midi-Pyrenees Animal Ethics Committee. Thalassemia mice and vadadustat-treated mice were, respectively, housed in a specific pathogen-free barrier facility within the animal facility of INSERM US006. Fgl1- / - and WT controls on a C57Bl / 6N background were obtained from The European Mouse Mutant Archive (EMMA) and bred at Janvier labs (Le Genest St Isle). At 4-5 weeks of age, they were transferred to the animal facility of INSERM US006. Mice were housed under a standard 12-h light / dark cycle in accordance with European Union guidelines and provided with ad libitum access to water and standard laboratory mouse chow (Ssniff, iron 200 mg / kg). This study was approved by the Midi-Pyrenees Animal Ethics Committee. Thalassemia mice and vadadustat-treated mice were, respectively, housed in a specific pathogen-free barrier facility within the animal facility of INSERM US006. 18 and Tomas Ganz 19 Expression data for Vhl- / - mice was kindly provided by Carole Peyssonnaux20 The samples were provided by [the author]. To study recovery from anemia, mice were exsanguinated (500 μL) by retroorbital puncture and analyzed 1–6 days later. Destruction of erythroid components was achieved by sublethal X-ray irradiation (400 rad) in mice, and the mice were exsanguinated 48 hours later. Surgical removal of spleens was performed on 7–8-week-old WT and Erfe- / - mice. The mice were allowed to recover for 7 days before exsanguination. Some WT mice received a single dose of EPO (200 U) and were analyzed 12, 15, 18, or 20 hours later. Recombinant FGL1, Fc fragment, or saline was intraperitoneally administered at a dose of 10 mg / kg to 7-week-old C57Bl / 6J mice fed an iron-replete diet (Ssniff, 50 mg / kg) for 2 weeks, and the mice were analyzed 6 hours later. For all mice, tissues were collected and divided into samples that were snap-frozen in liquid nitrogen for RNA, protein, and iron measurements, and samples in 4% formalin for paraffin embedding. Male mice were preferentially studied unless otherwise noted.

[0047] Production of recombinant FGL1 The mouse FGL1 cDNA sequence (full-length, N-terminal domain, and globular domain) and human FGL1 sequence were cloned into the pFUSEN-hG2Fc plasmid (Invivogen) with the following modifications: The vector signal sequence (derived from interleukin-2) was used in place of the native sequence, followed by the Fc fragment of human IgG2. Recombinant protein was produced in suspension culture in transiently transfected Freestyle 293F cells (Life Technologies) using FectroPro reagent (Polyplus). Supernatant from cells overexpressing Fc-tagged FGL1 protein was collected after 5 days and supplemented with a protease inhibitor cocktail (Sigma). Recombinant protein was purified using a Hitrap protein A HP column mounted on an AKTA pure chromatography system (GE Healthcare) and eluted with 0.1 M glycine, pH 3.5. The eluted fraction was concentrated using a Spin-X UF 20 centrifugal concentrator (Corning), and the recombinant FGL1 protein was suspended in saline (0.9% NaCl). Protein purity and concentration were measured using Coomassie Imperial Protein Stain and Pierce bicinchoninic acid protein assay (ThermoFisher Scientific).

[0048] Mouse ERFE immunoassay A human recombinant monoclonal antibody against mouse ERFE was produced by Bio-Rad using HuCAL technology. High-binding 96-well plates (Corning) were coated overnight at 4°C with 2 μg / ml capture antibody (100 μl / well) diluted in 50 mM sodium carbonate buffer, pH 9.6. The plates were washed (TBS, 0.05% Tween 20) and blocked with 300 μL / well of blocking buffer (PBS, 0.2% Na-casein, 0.05% Tween 20, 0.1 M NaCl) for 1 hour at room temperature. Recombinant mouse ERFE standards were serially diluted to 10, 5, 2.5, 1.25, and 0.625 ng / ml. Serum samples diluted in PBS and standards diluted in PBS + 5% BSA were incubated for 1 hour at room temperature. Plates were washed and incubated with 0.5 μg / ml biotinylated detection antibody (100 μL / well) in PBS + 5% BSA for 1 hour. Plates were washed and incubated with 1 / 5000 Neutravidin-HRP (Pierce) (100 μL / well) in PBS + 5% BSA for 45 minutes. Plates were developed with 100 μL / well ultrasensitive TMB substrate (Thermofisher) in the dark at room temperature. The reaction was stopped by adding 50 μL of 2N sulfuric acid, and absorbance was measured at 450 nm.

[0049] Measurement of iron and hematological parameters Serum iron concentration was measured by the direct iron method (ferene, Biolabo, 92108), and transferrin saturation was estimated by measuring unsaturated iron-binding capacity (UIBC, Biolabo, 97408). Liver iron content was measured as previously described. 21 Complete blood counts were performed using a Cell-Dyn Emerald hematology analyzer (Abbott).

[0050] Western blot analysis Liver proteins were extracted by physical dissociation using an ULTRA-TURRAX® (IKA) in PEB buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% NP-40) containing protease inhibitors (cOmplete™, Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2, Sigma). Hep3B cells were lysed in RIPA buffer (Thermofischer, 89900) containing protease and phosphatase inhibitors. Freshly extracted proteins were diluted with Laemmli buffer 2x (Sigma), incubated at 95°C for 10 min, subjected to SDS-PAGE, and electroblotted onto a nitrocellulose membrane (Biorad). The membrane was blocked for 1 hour with 5% nonfat dry milk (NFDM, Cell Signaling) diluted in TBS-T buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.15% Tween 20) and then incubated overnight at 4°C with an antibody against phospho-Smad5 (Ser463 / 465, Abcam, ab92698, 1 / 2000) diluted in TBS-T buffer (5% BSA) or for 2 hours at RT with an antibody against Smad5 (Abcam, ab40771, 1 / 5000) diluted in TBS-T buffer (5% BSA). Loading was measured using antibodies against GAPDH (Cell Signaling, D16H11, 1 / 10,000) or vinculin (Cell Signaling, 4650, 1 / 20,000) diluted in TBST-NFDM (5%) for 2 h at RT. After three washes, membranes were incubated with HRP-conjugated goat anti-human IgG (Novus Biologicals, NBP1-75006, 1 / 10,000), goat anti-rabbit IgG (Cell Signaling, 7074, 1 / 10,000), or horse anti-mouse IgG (Cell Signaling, 7076, 1 / 10,000) secondary antibodies diluted in TBST-NFDM (5%) for 2 h at RT.Enzyme activity was developed using ECL Prime reagent (GE Healthcare) on a ChemiDoc XRS+ imaging system.

[0051] Pull-down assay One microgram of Fc-tagged recombinant proteins (Fc alone, full-length FGL1, the globular portion of FGL1, and the N-terminal portion of FGL1) was incubated with protein A magnetic beads (Dynabeads, Pierce) in NETN buffer (20 mM Tris-HCl pH 8.0, 0.5% NP-40, 100 mM NaCl, 1 mM EDTA pH 8.0, protease inhibitor cocktail) overnight at 4°C with or without hBMP6 (Biotech). Proteins were eluted using Laemmli buffer and analyzed by Western blot using goat anti-human IgG Fc fragment secondary antibody [HRP] (Novus biological NBP1-75006) or anti-BMP6 antibody (R&D systems, AF6325).

[0052] Cell treatment Hep3B and HepG2 cells were cultured in Dulbecco's modified Eagle's medium-high glucose GlutaMAX, 10% fetal bovine serum, and 1% penicillin-streptomycin unless otherwise noted. Cells were seeded 24 hours prior to treatment and treated with serum-free medium for 6 hours. Hepatocytes were perfused with collagenase via the portal vein as previously described. 22Cells were isolated from wild-type C57BL / 6 mice by ELISA. Cells were cultured overnight (15 h) in fresh Williams E medium (Gibco) supplemented with 200 μM L-glutamine and 10% FBS. Hep3B and HepG2 cells, as well as primary hepatocytes, were treated with 25 ng / ml BMP6 (Peprotech) or BMPs 2, 4, and 7 (R&D Systems) and Fc (hIgG2), full-length Fc-FGL1 (FL), its N-terminal domain (Nter), or globular domain for 6 h. For hypoxia experiments, cells were maintained in serum-free medium for 15 h in a hypoxia chamber (Whitley, H35 Hypoxystation) at 2% oxygen or in the presence of 1 mM DMOG (dimethyloxalylglycine, N-(methoxyoxoacetyl)-glycine methylmester, Sigma) in a conventional CO2 incubator. To examine FGL1 regulation by inflammatory cytokines, HepB cells were treated with 20 ng / ml IL-6 or 50 ng / ml TNFα (R&D Systems) for 6 hours.

[0053] Quantification of mRNA levels Total RNA was extracted from mouse tissues using the Trizol (MRC) / chloroform (Sigma) method. Complementary cDNA was synthesized using M-MLV reverse transcriptase (Promega). Messenger RNA (mRNA) expression levels were assessed by quantitative polymerase chain reaction (RT-qPCR) (primers are listed in Table 1) using Takyon SYBR Green (Eurogentec). RT-qPCR was performed in duplicate on a LightCycler480 (Roche) instrument. Transcription levels were normalized to the reference gene Hprt and expressed in vivo as the difference between the reference and target genes (-ΔCt) ± standard error of the mean (SEM) within each group of mice. Data from albumin-Cre / VHLflox / flox mice were normalized to the reference gene 36B4. Results from in vitro treatment were expressed as fold changes, i.e., log-transformed data (2 ΔΔCt) which indicates expression relative to the control condition. Expression data for the control condition were normalized to the control mean to obtain distribution within the control group (Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-408). Statistical significance was determined using Student's t-test or analysis of variance (ANOVA).

[0054] Microarray Total RNA was extracted from mouse liver and bone marrow of Erfe- / - mice at t = 0, 24, and 48 h post-exsanguination using Trizol (MRC) / chloroform (Sigma). RNA quality was assessed with an RNA 6000 Nano chip using a Bioanalyzer 2100 (Agilent Technologies). Gene-level expression profiling of liver and bone marrow from exsanguinated mice was performed at the GeT-TriX facility (GenoToul, Genopole Toulouse Midi-Pyrenees) using Agilent SurePrint G3 Mouse GE v2 microarrays (8x60K, design 074809) according to the manufacturer's instructions. For each sample, cyanine-3 (Cy3)-labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling Kit (Agilent Technologies) followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) according to the manufacturer's instructions. Dye incorporation and cRNA yield were confirmed using a Dropsense 96 UV / VIS drop reader (Trinean, Belgium). 600 ng of Cy3-labeled cRNA was hybridized onto the microarray slides according to the manufacturer's instructions. Immediately after washing, the slides were scanned on an Agilent G2505C microarray scanner using Agilent Scan Control A.8.5.1 software, and the fluorescent signal was extracted using Agilent Future Extraction v10.10.1.1 (using default parameters). Microarray data and experimental details are available in NCBI's Gene Expression Omnibus7 and can be accessed through GEO series accession number GSE229041 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE229041).Expression data were analyzed using R (Rv3.1.2)-bioconductor and iDEP (Integrated Differential Expression and Pathway analysis). 23 Control mice (n=3) were analyzed by comparing them with mice at 24 hours (n=3) and 48 hours (n=3) using the ELISA.

[0055] Statistical analysis of microarray data Microarray data were analyzed using R (R Core Team, 2018) and Bioconductor packages8, as described in GEO accession number GSE229041. Raw data (median signal intensities) were filtered, log2-transformed, and normalized using the quantile method (Bolstad BM, Irizarry RA, Astrand M, Speed ​​TP. A comparison of normalization methods for high-density oligonucleotide array data based on variance and bias. Bioinformatics. 2003;19(2):185-193). Initial exploratory and statistical analyses indicated the possibility of correlation structure between gene expression, which could negatively affect the multi-test approach. To mitigate the dependency structure, the FAMT method10 was applied using a model including one additional factor. Models were fitted using the limma lmFit function (Ritchie ME, Phipson B, Wu D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47). Pairwise comparisons between biological conditions were applied using specific contrasts. Corrections for multiple testing were applied using the Benjamini-Hochberg method to control the false positive rate (FDR). Probes with an FDR ≤ 0.01 were considered differential between conditions. Hierarchical clustering was applied to samples and differential probes using the 1-Pearson correlation coefficient as the distance and Ward's criterion as the agglomeration criterion. Expression data were further analyzed using R iDEP (Integrated Differential Expression and Pathway analysis) by performing three comparisons: control mice (n=4) vs. mice 24 hours post-phlebotomy (n=4), control mice vs. mice 48 hours post-phlebotomy, and mice 48 hours post-phlebotomy vs. mice 24 hours post-phlebotomy.We focused on transcripts encoding secreted proteins that were significantly upregulated 24 hours after exsanguination compared with control mice and whose expression remained stable or increased between 24 and 48 hours. Liver and bone marrow were analyzed separately.

[0056] statistical analysis Statistical significance was assessed by Student's t-test, one-way analysis of variance, or two-way analysis of variance (ANOVA) using Prism 9 (GraphPad). Statistics shown for two-way ANOVA are the results of the Holm-Sidak multiple comparison test.

[0057] result ERFE-independent repression of hepcidin during anemia. First, we characterized the timeline for recovery from phlebotomy-induced anemia in WT and Erfe-deficient mice. In both genotypes, hemoglobin and hematocrit levels decreased for 3 days after phlebotomy and significantly improved by day 6 (Figure 1A, data not shown). Consequently, Epo mRNA expression in the kidney was rapidly induced within 2 days and gradually returned to normal by day 6 (Figure 1B). Erfe mRNA expression in the bone marrow and spleen increased to a maximum after 24 hours and then gradually returned to baseline, whereas Erfe mRNA levels remained slightly elevated even after 6 days (Figure 1C). In contrast, serum ERFE levels were highest 24 hours after phlebotomy and declined below the detection limit within 3–4 days (Figure 1D). On the other hand, the significant decrease in hepcidin mRNA expression in the liver was maintained for 5 days (Figure 1E), accompanied by a mild decrease in the BMP target genes Id1 and Smad7 mRNA 3 days after phlebotomy. Therefore, we investigated whether hepcidin might be regulated independently of ERFE during recovery from anemia in Erfe- / - mice. Similar to WT mice, hemoglobin levels in Erfe- / - mice reached a nadir 3 days after phlebotomy (Figure 2A) and nearly recovered by day 6. Erfe- / - mice showed a slight increase in MCV, MCH, and RDW 3 days after phlebotomy compared with WT mice (data not shown). Epo mRNA expression was rapidly induced in the kidney 24 hours after phlebotomy and gradually returned to normal after 6 days (Figure 2B). Consistent with the stress hormone properties of ERFE, Hamp mRNA expression remained unchanged after 24 hours but declined to levels comparable to those in WT mice 2–3 days after phlebotomy and then returned to normal after 6 days. Meanwhile, Id1 and Smad7 mRNA expression remained unchanged (Figure 2C). Similar results were observed in phlebotomized female mice (data not shown). Therefore, we decided to focus on mechanisms induced within 24–48 hours after phlebotomy, but not beyond 48 hours.Serum hepcidin concentrations and hepcidin / liver iron content ratios were consistent with liver Hamp mRNA expression in WT and Erfe- / - mice at 1 and 2 days after phlebotomy, whereas liver iron content and Bmp6 mRNA expression remained unchanged (Figure 2D and data not shown). Therefore, we focused on mechanisms induced within 24–48 h after phlebotomy that may contribute to hepcidin suppression in Erfe- / - mice. The changes in hepcidin synthesis in Erfe- / - mice occurred without any changes in serum iron concentration, transferrin saturation, or liver iron content compared with control mice at 1 and 2 days after phlebotomy (Figure 2E, 2F, 2G). Similarly, SMAD5 phosphorylation was not reduced in the livers of Erfe- / - mice at 2 days after phlebotomy (Figure 2H, 2I). No statistically significant increases in Gdf15 and Twsg1 mRNA expression were detected in the bone marrow and spleen of exsanguinated WT and Erfe- / - mice (data not shown). These data indicate that hepcidin expression is negatively regulated by an ERFE-independent mechanism. ERFE was initially identified by searching for transcripts induced in the bone marrow 9–15 h after exsanguination. We hypothesized that another bone marrow-derived erythroid regulator or a factor derived directly from the liver might repress hepcidin 24–48 h after exsanguination. To this end, we analyzed the transcriptome profiles of exsanguinated Erfe- / - mice compared with control mice at 1 and 2 days after exsanguination using microarrays.

[0058] Examine the potential contribution of erythroid regulators. Hepcidin suppression may be related to the role of another erythroid regulator, 24,25To determine whether hepcidin is mediated by hemorrhage, we abolished erythroid components by irradiation and examined the hepcidin response to hemorrhage in WT and Erfe- / - mice. 48 h after hemorrhage, WT and Erfe- / - mice showed an increase in reticulocytes, whereas irradiated mice showed a significant decrease in circulating reticulocytes (Figure 3A). The reduction in reticulocyte production was accompanied by a decrease in the expression of erythroid markers Gypa and Tfr1 in the bone marrow of irradiated WT and Erfe- / - mice compared with their controls. Thus, successful erythroid depletion was confirmed (Figure 3B). Hemoglobin levels were reduced in all hemorrhagic groups, more significantly in irradiated mice, but no difference was observed between WT and Erfe- / - mice (Figure 3C). Hepcidin mRNA expression in the liver was repressed in control WT and Erfe- / - mice at 48 h after phlebotomy, but not in irradiated mice (Figure 3D). Interestingly, recovery from anemia was paralleled by increased Gypa mRNA expression in the spleen and liver compared with control mice 1–6 days after phlebotomy (Figure 3E). Hamp mRNA expression was inversely correlated with Gypa mRNA expression in the liver and spleen (Figure 3F). This suggests that hepcidin suppressors may be released from the liver and spleen. Therefore, we performed surgical removal of the spleens of WT and Erfe- / - mice to assess their response to hemorrhage. However, splenectomized WT and Erfe- / - mice showed decreased Hamp mRNA expression in the liver at 48 h after phlebotomy, indicating that the hepcidin suppressor is not derived from the spleen (Figure 3G). Therefore, to explore the liver and bone marrow responses to anemia, we analyzed the transcriptome profiles of exsanguinated Erfe − / − mice at 1 and 2 days post-exsanguination compared with control mice.

[0059] Fgl1 mRNA expression is induced in the liver of mice during anemia. To identify potential regulators of hepcidin, we searched for transcripts encoding secreted proteins whose expression was induced 24 and 48 h after phlebotomy compared with control mice. 63 and 38 transcripts were found to be induced (fold change >2; p value <0.05) in the liver and bone marrow, respectively, 24 h after phlebotomy compared with control mice (data not shown). Six transcripts in the liver and 23 transcripts in the bone marrow remained induced 48 h after phlebotomy compared with control mice (data not shown). In the liver, only Fgl1, Gdf15, and Cxcl1 encoded secreted proteins. Interestingly, fibrinogen-like 1 (Fgl1) mRNA expression was increased in both the liver and bone marrow. qRT-PCR revealed that Fgl1 mRNA expression was significantly induced in the liver (Figure 4A) and bone marrow (Figure 4B) of WT and Erfe- / - mice 1 to 3 days after phlebotomy, although the expression level was several orders of magnitude higher in the liver. In contrast, Gdf15 mRNA expression was mildly induced 1 to 2 days after phlebotomy. However, it has been reported that Gdf15 does not contribute to hepcidin regulation during hemorrhage-induced anemia. Cxcl1 mRNA expression was only induced in Erfe- / - mice after 24 hours (data not shown). This did not correlate with the time course of hepcidin repression. Furthermore, unlike Fgl1, stimulation of Gdf15 and Cxcl1 was restricted to the liver. Therefore, we focused on FGL1 as a remaining potential candidate. Fibrinogen-like 1 (FGL1) regulates hepatocellular carcinoma (HC). 26 or HFREP-1 27 Also known as angiopoietin-like protein (ANGPTL), it is a member of the fibrinogen protein family produced by hepatocytes. 28 It shares structural similarities with fibrinogen beta and gamma subunits, including a C-terminal globular domain that is homologous to the fibrinogen beta and gamma subunits. In contrast to other fibrinogen-related factors, FGL1 lacks the platelet-binding and thrombin-sensitive sites involved in blood clot formation. 27,29Instead, FGL1 was induced during liver regeneration and showed growth-promoting activity on hepatocytes. 30 FGL1 is also involved in evading tumor invasion in certain cancers through its interaction with the LAG-3 receptor. 31 Intraperitoneal injection of EPO (200 μg) into WT mice significantly reduced Hamp mRNA expression and increased Erfe mRNA expression in the bone marrow, but did not stimulate Fgl1 expression (Figure 4C). In contrast, Fgl1 mRNA expression was upregulated in the livers of Erfe-deficient thalassemic Th3 / + and Th3 / + mice (Figure 4D). Analysis of the mouse Fgl1 promoter revealed two HIF-binding sites (data not shown). To examine whether Fgl1 expression was stimulated in the livers of anemic mice by oxygen desaturation, we compared Fgl1 expression in mouse primary hepatocytes incubated under hypoxic conditions (2% O2) or in the presence of the prolyl hydroxylase inhibitor DMOG with control conditions. Increased expression of Vegfa, Gapdh, and Angptl1, target genes of hypoxia-inducible factors, was observed in cells cultured in serum-free or serum-containing medium and incubated in the presence of DMOG or hypoxic conditions (2%) for 15 hours compared to untreated cells (Figure 4E). Similarly, Fgl1 mRNA expression was induced in primary mouse hepatocytes incubated under hypoxic conditions or in the presence of DMOG (Figure 4F). A tendency for increase was observed in albumin-Cre / Vhl-deficient mice. 20 On the other hand, a significant increase in Fgl1 mRNA expression was observed in the liver of Hif2α-overexpressing mice (Fig. 4G). 32 and mice chronically treated with the prolyl hydroxylase inhibitor vadadustat. 19Fgl1 mRNA expression was detected in the liver of WT mice fed diets containing 10, 50, 200, or 8000 mg / kg iron for 2 weeks (Figure 4H). These results suggest that Fgl1 expression may be regulated by hypoxia-inducible factors. Consistent with previous studies, WT mice fed diets containing 10, 50, 200, or 8000 mg / kg iron for 2 weeks showed no change in Fgl1 mRNA expression in the liver, whereas Hamp and Id1 mRNA expression varied according to the iron content of the diet (data not shown). This indicates that Fgl1 is not regulated by iron. FGL1 has been reported as an acute-phase protein, and like hepcidin, its expression has been reported to be mildly induced by IL-6 and repressed by TNFα (data not shown).

[0060] FGL1 is a hepcidin suppressor in vivo and in vitro. Next, we evaluated the contribution of FGL1 to hepcidin regulation. HAMP mRNA expression was induced 600-fold and 200-fold in response to BMP6 (25 ng / ml, 6 h) in Hep3B and HepG2 cells, respectively (Fig. 5A). Treatment with recombinant Fc-tagged FGL1 for 6 h under serum-free conditions significantly reduced HAMP and ID1 expression in both hepatoma cell lines (Fig. 5B, 5C). In serum-containing medium, higher doses of FGL1 were required to repress HAMP and ID1 expression (data not shown). Mouse and human FGL1 share 82% identity, and human FGL1 also suppressed HAMP and ID1 mRNA expression in Hep3B cells, but at higher concentrations (data not shown). Therefore, we decided to use mouse FGL1 in this study. Injection of recombinant FGL1 (10 mg / kg) into WT mice significantly reduced Hamp RNA expression in the liver and serum hepcidin levels compared with Fc-treated mice (n = 5) at 6 h postinjection (Figures 5F and 5G), whereas no change in Id1 mRNA expression in the liver was observed (Figure 5H). Consistent with hepcidin repression, serum iron levels increased and liver iron levels decreased after FGL1 treatment (Figure 4H). In female WT mice, FGL1 treatment did not repress hepcidin expression. The significant inflammatory response to FGL1 administration (indicated by increased Saa1 levels), which was not observed in male mice, may have prevented the effect of FGL1. Finally, FGL1 was able to repress hepcidin expression in Hep3B cells, even in the presence of IL-6 (data not shown). Taken together, these results indicate that ERFE is a potent suppressor of hepcidin.

[0061] Fgl1- / - mice exhibit a blunted response to phlebotomy. To determine whether FGL1 contributes to hepcidin regulation during recovery from anemia, we compared WT and Fgl1- / - mice at 36 h after phlebotomy. First, we confirmed that Fgl1 mRNA expression was significantly increased in the livers of male and female WT mice compared with control mice at 36 h after phlebotomy (Figure 6A). Red blood cell counts and hemoglobin levels (Figure 6B, 6C) were decreased in WT and Fgl1- / - mice at 36 h after phlebotomy. Similar increases in Erfe mRNA expression in the bone marrow and spleen were detected in both genotypes at 36 h after phlebotomy (Figure 6D, 6E). Interestingly, Hamp mRNA expression in the liver was found to be reduced in both male and female exsanguinated WT and Fgl1- / - mice compared with control mice, but to a lesser extent in Fgl1- / - mice (Figure 6F). This suggests that FGL1 contributes to hepcidin suppression. No changes in Id1 or Smad7 mRNA expression were observed (Figures 6G-H). Consistent with the blunted hepcidin repression, serum iron concentrations were lower in exsanguinated Fgl1- / - mice compared with WT mice (Figure 6G). These results indicate that FGL1 increases during recovery from anemia and contributes to hepcidin suppression.

[0062] The globular domain of FGL1 is responsible for hepcidin suppression. Mouse FGL1 consists of a signal peptide for secretion, a short coil-coil N-terminal domain, and a C-terminal globular domain homologous to the fibrinogen β and γ chains (data not shown). To identify the active domain of FGL1, mouse primary hepatocytes were treated with Fc, full-length FGL1, or its N-terminal and globular domains. Full-length FGL1 and the globular domain repressed HAMP and ID1 expression (Figures 5D and 5E), whereas the N-terminal domain was inactive.

[0063] FGL1 is a BMP antagonist. Next, we investigated the mechanism by which FGL1 represses hepcidin. Treatment of hepatocytes with FGL1 downregulated ID1 mRNA expression, so we examined whether FGL1 could function as a BMP antagonist. We observed that FGL1 repressed the induction of Hamp and Id1 mRNA expression by BMP6 and BMP7, but not by BMP2 and BMP4, in mouse primary hepatocytes compared with control and Fc-treated cells (Figures 7A and 7B). On the other hand, treatment of cells with FGL1 had no effect on Smad7 mRNA expression (Figure 7C). Pretreatment of cells with BMP6 before addition of FGL1 confirmed its ability to repress hepcidin and BMP target genes (data not shown). In addition, FGL1 significantly reduced SMAD5 phosphorylation in BMP6-treated Hep3B cells (Figure 7D). Furthermore, Fc or Fc-FGL1 (FL, glob, and Nter) were incubated with BMP6 and subjected to pull-down assays using protein A magnetic beads. FGL1 FL and glob interacted with BMP6, but the N-terminal domain interacted to a lesser extent, and the Fc fragment did not (Figure 7E). These results suggest that, similar to erythropoietin, FGL1 functions as a ligand trap for BMP6 to repress hepcidin transcription during hemorrhage recovery.

[0064] conclusion Here, we identified a novel hepcidin suppressor: fibrinogen-like 1 (FGL1), a hepatokine produced in the liver, that may contribute to hepcidin regulation during anemia.

[0065] 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 the ability to reduce hepcidin expression in a cell model (data not shown).

[0066] table [Table 1]

[0067] References Throughout this application, various references are made to describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure. [Table 2] TIFF2026506600000005.tif234165 TIFF2026506600000006.tif66165

Claims

1. 1. An FGL1 polypeptide for use in treating a patient suffering from an iron deficiency-related disease, comprising: The polypeptide may be: (i) the amino acid sequence shown in SEQ ID NO: 1 (FGL1); (ii) the amino acid sequence shown in SEQ ID NO: 3 (FGL1 mature protein); (iii) the amino acid sequence shown in SEQ ID NO: 4 (FGL1 C-terminal globular domain); (iv) an amino acid sequence that is substantially homologous to one of sequences (i) to (iii), preferably an amino acid sequence that is at least 80% identical to sequences (i) to (iii); or (v) a fragment of at least 30 consecutive amino acids selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:

4. comprising an amino acid sequence selected from the group consisting of: FGL1 polypeptide.

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

1.

3. 2. The FGL1 polypeptide for use according to claim 1, wherein the FGL1 polypeptide consists of the amino acid sequence shown in SEQ ID NO:

4.

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

5. A polynucleotide encoding the FGL1 polypeptide according to any one of claims 1 to 4 for use in treating patients suffering from iron deficiency-related diseases.

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 a polynucleotide encoding the FGL1 polypeptide according to claim 5 or 6 for use in treating patients suffering from iron deficiency-related diseases.

8. An FGL1 polypeptide for use according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

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

10. Iron deficiency-related diseases include anemia of chronic disease; Inflammatory anemia; Infectious anemia; hypochromic microcytic anemia; iron deficiency anemia; iron-resistant iron deficiency anemia; anemia of chronic kidney disease; anemia due to hepcidin-secreting tumors; attention-deficit hyperactivity disorder; erythropoietin resistance; autism; intellectual disability; anxiety; bipolar disorder; impetigo; candidiasis; Helicobacter pylori infection; Thrichuris trichiura infection; multiple sclerosis; rheumatoid arthritis; lupus; 10. The FGL1 polypeptide for use according to any one of claims 1 to 9, selected from the group consisting of inflammatory bowel disease; Crohn's disease; ulcerative colitis; celiac disease; autoimmune gastritis; obesity; hypothyroidism; heart failure; stroke; Viral-Ekbom disease; chronic fatigue; fibromyalgia; chronic obstructive pulmonary disease; cystic fibrosis; premenstrual syndrome; preterm birth; risk of fetal death or postpartum depression.

11. The FGL1 polypeptide for use according to claim 10, wherein the iron deficiency-related disease is selected from the group consisting of anemia of chronic disease, anemia of inflammation, anemia of infection, anemia of chronic kidney disease, anemia caused by tumors secreting hepcidin, and iron-resistant iron deficiency anemia.

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