Periodic Reporting for period 1 - NITA (Novel targeted Intervention to Treat Anemia)
Reporting period: 2023-09-01 to 2025-08-31
Anemia, defined as a decreased quantity of circulating 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. In inflammatory disorders and during infections, hepcidin synthesis is stimulated by proinflammatory cytokines, which causes hypoferremia and inadequate iron supply for erythropoiesis. Conversely, hepcidin deficiency causes iron overload and severe clinical complications in hemochromatosis and β-thalassemia. Both iron overload and anemia are associated with decreased survival and impaired quality of life. During anemia, the erythroid hormone erythroferrone (ERFE) regulates hepcidin synthesis to ensure the proper supply of iron to the bone marrow for red blood cells synthesis. We had previously shown that ERFE facilitates the recovery from anemia induced by hemorrhage or chronic inflammation and that it contributes to the development of iron overload in thalassemia. However, mounting evidence indicated that another factor may exert a similar function. We found that the expression of the hepatokine Fibrinogen-like 1 (FGL1) was highly induced in the liver in response to hypoxia during the recovery from anemia and in thalassemic mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo and that it acts by antagonizing the BMP/SMAD signaling pathway directing hepcidin expression. Our aims were to confirm the role of FGL1 during anemia and to investigate the therapeutic potential of its manipulation in murine models of anemia. Successful completion of this project had the potential to lead to the development of new therapeutic strategies for the treatment of various forms of anemias for which current treatments remain largely ineffective.
We first explored the mechanism by which FGL1 trepresses hepcidin. We observed that hepcidin suppression was accompanied by a robust decrease the BMP/SMAD target gene expression. We found that FGL1 directly binds BMP6 to prevent receptor engagement and the activation of the signaling cascade regulating hepcidin. We therefore identified FGL1 as a new BMP antagonist (Sardo, Blood 2024).
Task 1: We observed that, similar to Erfe-/- mice, Fgl1-deficient mice exhibited a blunted repression of hepcidin 36 hours after hemorrhage compared to WT mice. We therefore decided to decipher the respective contribution of ERFE and FGL1 in iron metabolism by breeding Erfe-/- mice with Fgl1-/- mice. We analyzed male and female WT, Erfe-/-, Fgl1-/- and Erfe-/-; Fgl1-/- animals 48h after blood loss and at expected the time-point of recovery (6 days). In paralell, in thalassemic mice (Th3/+), hepcidin suppression is mediated in part by ERFE but our data indicated that Fgl1 expression was highly induced in the liver of Th3/+ mice suggesting that FGL1 could contribute to the negative regulation of hepcidin in thalassemia. To determine the respective contribution of each factor, we compared the iron (liver, spleen, serum iron) and hematologic (complete blood count) phenotype of Th3/+ mice deficient for Erfe or both Erfe and Fgl1. This task required the development of complex and time-consuming breeding schemes but the final sets of mice are currently being processed. We are expecting the final results and their related publication during the first trimester of 2026. To examine the contribution of FGL1 in human, we developed a specific immunoassay for FGL1 and found that plasma FGL1 levels are elevated in patients with various forms of anemia.
Task2 aimed at assessing the benefit of FGL1 manipulation in the treatment of iron disorders using antisense oligonucleotides. We have developed lipid-coupled ASOs with increased stability and efficacy (LASO) targeting mouse and human FGL1. The advantage of this strategy is that LASOs are efficiently delivered to the liver to impair both mRNA stability and translation initiation. We tested and validated in vitro a subset of ASOs in human and murine cell lines. The two most potent ASOs validated in vitro and a control scramble ASO were modified by the addition of a double-chain nucleolipid (ketal-bis-C15-Uridine) to their 5′ end PTO. These conjugates are ketal amphiphilic molecules that self-assemble to give supramolecular structures allowing a sustained release. We validated the ability of these compounds to repress FGL1 expression in vitro in human and mouse hepatic cells. However, when tested in mice, these LASOs proved largely inefficient at repressing FGL1. The two most potent 5’PTOs ASOs were therefore conjugated to N-acetygalatosamine for improved delivery to hepatocytes instead of the nucleolipids, as is customarily done by biotechs using this technology. We found that these ASOs were extremely potent at suppressing FGL1 expression in vitro in human and mouse hepatic cells and in vivo in mice.
Task3: Inflammatory cytokines stimulate the production of hepcidin leading to functional iron deficiency and iron-restricted erythropoiesis. The therapeutic approach of choice is to treat the underlying disease but this is often unfeasible or unsuccessful. Management of anemia of inflammation currently includes a combination of erythropoiesis stimulating agents and intravenous iron supplementation but these options remain moderately effective or responsible for dangerous side effects. Preclinical studies have demonstrated the therapeutic benefit of inhibiting the BMP signaling to reduce hepcidin levels during AI. As potent BMP antagonist, FGL1 is a promising therapeutic candidate to alleviate iron restriction in inflammatory settings. We produced and purified short versions of recombinant FGL1 (60 amino acids) that retained a potency comparable to that of full-length proteins in vitro. Preliminary data also suggest that FGL1 can overcome the induction of hepcidin by inflammatory cytokines in vitro. The most potent minimal versions of FGL1 (miniFGL1) will now require further validation in vivo.
In summary, this project unraveled the mechanism of a previously unknown regulator of iron metabolism and led to the design and in vitro validation of new therapeutic tools for the treatment of anemia.
Task 1: We observed that, similar to Erfe-/- mice, Fgl1-deficient mice exhibited a blunted repression of hepcidin 36 hours after hemorrhage compared to WT mice. We therefore decided to decipher the respective contribution of ERFE and FGL1 in iron metabolism by breeding Erfe-/- mice with Fgl1-/- mice. We analyzed male and female WT, Erfe-/-, Fgl1-/- and Erfe-/-; Fgl1-/- animals 48h after blood loss and at expected the time-point of recovery (6 days). In paralell, in thalassemic mice (Th3/+), hepcidin suppression is mediated in part by ERFE but our data indicated that Fgl1 expression was highly induced in the liver of Th3/+ mice suggesting that FGL1 could contribute to the negative regulation of hepcidin in thalassemia. To determine the respective contribution of each factor, we compared the iron (liver, spleen, serum iron) and hematologic (complete blood count) phenotype of Th3/+ mice deficient for Erfe or both Erfe and Fgl1. This task required the development of complex and time-consuming breeding schemes but the final sets of mice are currently being processed. We are expecting the final results and their related publication during the first trimester of 2026. To examine the contribution of FGL1 in human, we developed a specific immunoassay for FGL1 and found that plasma FGL1 levels are elevated in patients with various forms of anemia.
Task2 aimed at assessing the benefit of FGL1 manipulation in the treatment of iron disorders using antisense oligonucleotides. We have developed lipid-coupled ASOs with increased stability and efficacy (LASO) targeting mouse and human FGL1. The advantage of this strategy is that LASOs are efficiently delivered to the liver to impair both mRNA stability and translation initiation. We tested and validated in vitro a subset of ASOs in human and murine cell lines. The two most potent ASOs validated in vitro and a control scramble ASO were modified by the addition of a double-chain nucleolipid (ketal-bis-C15-Uridine) to their 5′ end PTO. These conjugates are ketal amphiphilic molecules that self-assemble to give supramolecular structures allowing a sustained release. We validated the ability of these compounds to repress FGL1 expression in vitro in human and mouse hepatic cells. However, when tested in mice, these LASOs proved largely inefficient at repressing FGL1. The two most potent 5’PTOs ASOs were therefore conjugated to N-acetygalatosamine for improved delivery to hepatocytes instead of the nucleolipids, as is customarily done by biotechs using this technology. We found that these ASOs were extremely potent at suppressing FGL1 expression in vitro in human and mouse hepatic cells and in vivo in mice.
Task3: Inflammatory cytokines stimulate the production of hepcidin leading to functional iron deficiency and iron-restricted erythropoiesis. The therapeutic approach of choice is to treat the underlying disease but this is often unfeasible or unsuccessful. Management of anemia of inflammation currently includes a combination of erythropoiesis stimulating agents and intravenous iron supplementation but these options remain moderately effective or responsible for dangerous side effects. Preclinical studies have demonstrated the therapeutic benefit of inhibiting the BMP signaling to reduce hepcidin levels during AI. As potent BMP antagonist, FGL1 is a promising therapeutic candidate to alleviate iron restriction in inflammatory settings. We produced and purified short versions of recombinant FGL1 (60 amino acids) that retained a potency comparable to that of full-length proteins in vitro. Preliminary data also suggest that FGL1 can overcome the induction of hepcidin by inflammatory cytokines in vitro. The most potent minimal versions of FGL1 (miniFGL1) will now require further validation in vivo.
In summary, this project unraveled the mechanism of a previously unknown regulator of iron metabolism and led to the design and in vitro validation of new therapeutic tools for the treatment of anemia.
The discovery of FGL1 has high translational potential for the treatment of anemia of various origins. These applications were protected by two international patents (PCT/EP2024/053492 and PCT/EP2024/053500). Further research will be necessary to confirm in vivo the therapeutic potential of the compounds generated during this project before FGL1-based therapies can be envisioned.