Methods of treatment of iron overload associated diseases by administration hepcidin locally in the gut

Abstract

Hepcidin is an hyposideremic hormone made primarily by the liver. To address the role of hepcidin made specifically by the intestine in lowering serum iron, the inventors generated transgenic mice overexpressing the peptide specifically in this tissue. These mice exhibit, at one month of age, a severe hyposideremia, along with decreased haematological indices and hair loss. Mechanistically, they showed that intestinal hepcidin made by the transgenic mice had no effect on intestinal ferroportin, but, in contrast, induced a striking down-regulation of Divalent Metal Transporter 1 (DMT1) protein at the apical side of the enterocyte. Intestinal hepcidin can be produced in the apical side suggesting the direct role of apical hepcidin on DMT1. To confirm the therapeutic capacity of hepcidin on regulation of DMT1, the inventors developed probiotics (engineered recombinant lactic acid bacteria), capable of delivering hepcidin directly into the lumen of the intestine. They orally administrated daily these probiotics in hemochromatosis mice model, after 28 days of treatments they observe a decrease of iron overload. Thus, the present invention relates to a method for preventing or treating an iron overload associated disease in a subject in need thereof, comprising administering locally in the gut of the subject hepcidin.

Description

[0001] METHODS OF TREATMENT OF IRON OVERLOAD ASSOCIATED DISEASES BY ADMINISTRATION HEPCIDIN LOCALLY IN THE GUT

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a method for preventing or treating iron overload associated diseases such as P-thalassemia and haemochromatosis by administration hepcidin locally in the gut.

[0004] BACKGROUND OF THE INVENTION

[0005] Iron is an essential nutrient for vital biological process including hemoglobin synthesis, DNA replication and mitochondrial function. Despite years of research in the field, perturbations of iron homeostasis still affect more than 25% of the world’s population1,2. For instance, iron deficiency is the most common nutritional disorder in the world and is associated with anemia1. Moreover, hereditary hemochromatosis, characterized by iron overload is the genetic disorders with the higher prevalence in the world. The iron overload in hemochromatosis is due to a genetic mutation inducing a defect of systemic hepcidin and for consequences an increase of iron absorption3. Hepcidin is an hyposideremic hormone produced by the liver4. Hepcidin control iron efflux to inducing internalization of the only iron exporter: ferroportin (FPN)5. However, recent reports revealed extra-hepatic functional sources of hepcidin, acting in a paracrine / autocrine manner6 9, including the intestine10.

[0006] Intestine is the only organ enables to absorb iron, especially in the proximal part: duodenum. The only source of iron for organisms is the food (l-2mg iron absorbed each day). Non-heme iron dietary exists more largely as ferric form (Fe3+) and is insoluble and no bio- available for organisms. To be able to uptake iron from the intestinal lumen, enterocyte cells reduce ferric iron in ferrous iron (Fe2+) via the ferric reductase enzyme (DcytB) present at the brush border. Divalent Metal Transporter 1 (DMT1) is the ferrous iron importer localized at the apical side. Once in the enterocyte, iron can be stored in ferritin (FTL / FTH) or exported in the circulation via the basolateral exporter ferroportin (FPN). Systemic hepcidin regulates iron absorption by inhibiting iron export (FPN). However, numerous in vitro studies on intestinal cells line showed that hepcidin had no effect on FPN1 1 l4.

[0007] To address the role of hepcidin made specifically by the intestine in lowering serum iron, the inventors generated transgenic mice overexpressing the peptide specifically in this tissue (transgenic hepcidin). They next generate probiotics producing hepcidin to confirm the role of intestinal hepcidin on iron absorption (exogen hepcidin).

[0008] SUMMARY OF THE INVENTION

[0009] A first object of the invention relates to a method for preventing or treating an iron overload associated disease in a subject in need thereof, comprising administering locally in the gut of the subject a therapeutically effective amount of hepcidin.

[0010] A second object of the invention relates to a recombinant food-grade bacterium comprising a gene coding for hepcidin for use for preventing or treating an iron overload associated disease in a subject in need thereof, said recombinant food-grade bacterium being administered locally in the gut of the subject.

[0011] In particular, the invention is defined by the claims.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] Hepcidin is an hyposideremic hormone made primarily by the liver. However, recent reports revealed extra-hepatic functional sources of hepcidin, acting in a paracrine / autocrine manner, including the intestine the site of dietary iron absorption. To address the role of hepcidin made specifically by the intestine in lowering serum iron, the inventors generated transgenic mice overexpressing the peptide specifically in this tissue (Fig 1C). These mice exhibit, at one month of age, a severe hyposideremia (Fig 2C-D), along with decreased haematological indices (Fig 2H-K) and hair loss (Fig 2A). In response to this hyposideremia, liver hepcidin gene expression was drastically reduced (Fig 2F) but hepcidin levels were significantly increased in the plasma of HamplKI'Intmice compared to controls (Fig 3H). Mechanistically, they showed that intestinal hepcidin made by the transgenic mice had no effect on intestinal FPN (Fig 3A), but, in contrast, induced a striking down-regulation of DMT1 protein at the apical side of the enterocyte (with no change in mRNA levels) (Fig 3B-C). Intestinal hepcidin can be produced in the apical side (Fig 3G) suggesting the direct role of apical hepcidin on DMT1. To further assess the role of hepcidin, particularly in the duodenum, synthetic hepcidin 25 was injected in isolated duodenal loops. Two hours post injection, DMT1, but not FPN, was found decreased in comparison to PBS injection (Fig 3J).

[0014] To confirm the therapeutic capacity of hepcidin to regulate DMT1, the inventors developed probiotics (engineered recombinant lactic acid bacteria), capable of delivering hepcidin directly into the lumen of the intestine (Fig 4A). They orally administrated daily these probiotics (Lac-hepc at 5xlO9CFU), in hemochromatosis mice model (HamplAllver) after 28 days of treatments they observe a decrease of iron overload in HamplAllver +Lac-hepc25 (Fig 4B-J)

[0015] Collectively, their data revealed a previously unrecognized role of intestinal hepcidin as a critical regulator of systemic and local-intestinal iron homeostasis, acting on DTM1 at the apical side of the enterocyte.

[0016] Therapeutics methods and use of the invention.

[0017] Accordingly, a first aspect, the present invention relates to a method for decreasing iron overload in a subject in need thereof, comprising administering locally in the gut of the subject a therapeutically effective amount of hepcidin.

[0018] As used herein, the term "subject" or “patients” refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that has or is susceptible to have iron overload associated disease, and in particular P-thalassemia or haemochromatosis. In a particular embodiment of the present invention, the subject is a human being. The term "subject" does not denote a particular age, and thus encompasses children, teenagers, and adults.

[0019] In other words, the present invention relates to a method for preventing or treating an iron overload associated disease in a subject in need thereof, comprising administering locally in the gut of the subject a therapeutically effective amount of hepcidin.

[0020] In other words, the present invention relates to hepcidin for use for preventing or treating an iron overload associated disease in a subject in need thereof, wherein said hepcidin is administered locally in the gut of the subject.

[0021] In some embodiments, the hepcidin inhibits Divalent Metal Transporter 1 (DMT1).

[0022] In other words, in one embodiment, the hepcidin is an inhibitor of the Divalent Metal Transporter 1 (DMT1).

[0023] As used herein, the term “Divalent Metal Transporter 1”, also known as DMT1, has its general meaning in the art and refers to the major iron transporter and contributes non-heme iron uptake in most types of cell. DMT1 is present at the apical side of the enterocyte. Intestinal DMT1 is critical for iron absorption.

[0024] In particular embodiment, hepcidin inhibits the activity of DMT1, i.e iron absorption.

[0025] In particular embodiment, hepcidin regulates DMT1 at the post-transcriptional level. As used herein, the terms “iron overload associated disease" refers to a group of diseases and / or disorders which are generally associated with abnormally low levels of serum hepcidin and includes diseases wherein aberrant iron metabolism directly causes the disease, or wherein iron blood levels are dysregulated causing disease, or wherein iron dysregulation is a consequence of another disease, or wherein diseases can be treated by modulating iron levels, and the like. More specifically, a disease of iron metabolism according to this disclosure includes iron overload diseases, disorders of iron biodistribution, other disorders of iron metabolism and other disorders potentially related to iron metabolism, etc. Diseases of iron metabolism include hemochromatosis, HFE mutation hemochromatosis, ferroportin mutation hemochromatosis, transferrin receptor 2 mutation hemochromatosis, hemojuvelin mutation hemochromatosis, hepcidin mutation hemochromatosis, juvenile hemochromatosis, neonatal, hemochromatosis, hepcidin deficiency, thalassemia, thalassemia intermedia, alpha thalassemia, beta thalassemia, African iron overload, hyperferritinemia, ceruloplasmin deficiency, atransferrinemia, congenital dyserythropoietic anemia.

[0026] In a particular embodiment, the iron overload associated disease is selected from the list consisting of hemochromatosis (adult and juvenile forms of hereditary hemochromatosis), iron- loading anemia and chronic liver diseases including alcoholic liver diseases, non-alcoholic steatohepatitis (NASH) and chronic hepatitis B and C.

[0027] As used herein, the term “Iron loading anaemia” also called “secondary iron overload” refers to diseases characterized by anaemia, high serum iron, transferrin saturation and ferritin values, and haemosiderin deposits in parenchymal cells and reticuloendothelial tissue with or without organ dysfunction.

[0028] In a more particular embodiment, the iron-loading anemia is selected from the list consisting of alpha-thalassemia, P-thalassemia, congenital dyserythropoietic anemias, myelodysplastic syndrome (MDS).

[0029] In a particular embodiment, the iron overload associated disease is hemochromatosis or P-thalassemia.

[0030] As used herein, the term “hemochromatosis” refers to a genetic disorder characterized by excessive intestinal absorption of dietary iron, resulting in a pathological increase in total body iron stores. There are five types of hereditary hemochromatosis: type 1, 2 (2A, 2B), 3, 4 and 5, all caused by mutated genes. Hereditary hemochromatosis type 1 is the most frequent, and unique related to the HFE gene. Early diagnosis and treatment is critical to prevent complications from the disorder. Indeed, Too much iron is toxic to the body and over time the high levels of iron can damage tissues and organs and lead to cirrhosis, liver cancer, heart problems, arthritis or diabetes.

[0031] As used herein, the term “beta thalassemias (P thalassemias)” refers to a group of inherited blood disorders. They are forms of thalassemia caused by reduced or absent synthesis of the beta chains of hemoglobin that result in variable outcomes ranging from severe anemia to clinically asymptomatic individuals. Global annual incidence is estimated at one in 100,000 (Galanello, et al (2010). Orphanet J Rare Dis. 5: l l21). Beta thalassemias occur due to malfunctions in the hemoglobin subunit beta or HBB. The severity of the disease depends on the nature of the mutation. HBB blockage over time leads to decreased beta-chain synthesis and accumulation of alpha chains causing premature apoptosis of erythroid precursors. Moreover, the body's inability to construct new beta-chains leads to the underproduction of HbA (adult hemoglobin). Reductions in HbA available overall to fill the red blood cells in turn leads to microcytic anemia. Microcytic anemia ultimately develops in respect to inadequate HBB protein for sufficient red blood cell functioning. Due to this factor, the subject may require blood transfusions to make up for the blockage in the beta-chains. Repeated blood transfusions cause severe problems associated with iron overload(Galanello, et al (2010). Orphanet J Rare Dis. 5: l l21).

[0032] P -thalassemia is not medically curable except for the use of blood transfusion, although frequent blood transfusions that lead to or potentiate iron overload. Accordingly there is a medical need to specifically treat B-thalassemia subject with new therapeutic approach.

[0033] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. In particular, the treatment of the disorder may consist in reducing iron overload caused by low hepcidin expression in conditions such as P-thalassemia, haemochromatosis, congenital dyserythropoietic anaemia and myelodysplastic syndromes (MDS). Most preferably, such treatment leads to the complete depletion of the pathological features as observed in iron overload associated disease. By “therapeutic regimen” is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a “loading regimen”, which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase “maintenance regimen” or “maintenance period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0034] As used herein, a “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a patient. For example, a “therapeutically effective amount of the active agent” to a patient is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the patient. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 100 mg / kg of body weight per day.

[0035] As used herein, the term “Hepcidin” has its general meaning in the art and was first identified as a liver-derived antimicrobial peptide. The human hepcidin gene encodes an 84- residue prepropeptide that contains a 24-residue N-terminal signal peptide that is subsequently cleaved to produce pro-hepcidin. Pro-hepcidin is then processed to produce a mature 25-amino acid hepcidin (Jordan et al., The Journal of Biological chemistry, 2009,284, 36, 24155-24167).

[0036] It is now well established that hepatic hepcidin is the regulator of iron homeostasis (as disclosed by Lesbordes-Brion et al., Blood, 2006, 108, 1402-1405 and Nicolas et al., Proc. Natl. Acad. Sci. U.S. A., 2002, 99, 4596-4601). Hepcidin is a hypoferremic hormone; it binds to and degrades ferroportin (FPN, the only iron exporter known to date), thereby decreasing the iron levels in the circulation. Since its expression can be induced by inflammation (Nicolas et al., J. Clin. Invest., 2002, 110, 1037-1044), hepcidin has been proposed as an important mediator of Anemia of Chronic disease (ACD), also known as Anemia of Inflammation (Al).

[0037] The term “Hepcidin”, when used in the context of the present invention, should be understood broadly, it encompasses the mature forms of hepcidin, variants and fragments thereof having same biological activity (regulation of DMT 1 at the apical side of the enterocyte in order to limit iron overload) as well as a nucleic acid encoding said mature forms of hepcidin, variants or fragments. Biological activity of mature form of hepcidin can be measured for example by iron and iron transporters DMT1 quantification (see experimental section and figure 3). Hepcidin can be in the form of the known human 20, 22 or 25-amino acid peptide or of a functional fragment or variant thereof. As disclosed in Nemeth et al, hepcidin structure is permissive for mutations (Nemeth et al., Blood, 2006, 107, 30 328-333). Typically, the fragments of hepcidin according to the present invention comprise 10, 15, 20, 22 or 25 amino acids. Precursors of said mature forms of hepcidin, i.e. prohepcidin and preprohepcidin and nucleic acids encoding said precursors can also be used.

[0038] The term Hepcidin encompasses also the analogues / variant of hepcidin which mimic the hepcidin activity of Hepcidin -25, the bioactive human 25-amino acid form. These analogues can be known in the art as “mini-hepcidins” and include PR73 that mimics the first 9 amino residue of hepcidin and analogues described in WO2015200916.

[0039] Other examples of Hepcidin or nucleic acid encoding thereof suitable for use according to the invention are vertebrate, preferably mammalian, homologous of mature forms of human hepcidin or precursors thereof. Known vertebrate homologous of human hepcidin include for instance rat hepcidin, mouse hepcidin, trout hepcidin.

[0040] Preferably, if the patient is human, the hepcidin is a mature form of human hepcidin (UniProtKB - P81172). The protein sequence of said human hepcidin, may be found in NCBI database with the following access numbers: mRNA NM_ NM_021175, and protein_id: NP_ NP 066998 (hepcidin preproprotein). Chimeric polypeptides, comprising the sequence of a mature form of hepcidin, can also be used.

[0041] In particular embodiment, the hepcidin comprises or consists of the sequence having at least 50%, 60%, 70%, 80% or more particularly 90% sequence identity with the amino acid sequence selected in the group consisting of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3.

[0042] SEQ ID NO : 1 > Hepcidin - 20 aa ICI FCCGCCHRSKCGMCCKT

[0043] SEQ ID NO : 2 > Hepcidin - 22 aa FPICI FCCGCCHRSKCGMCCKT

[0044] SEQ ID NO : 3 > Hepcidin - 25 aa

[0045] DTHFPICI FCCGCCHRSKCGMCCKT

[0046] According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8: 155- 165, 1992; and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994). Altschul et al., Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CAB IOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996).

[0047] In some embodiment, the hepcidin comprises a sequence as set forth in SEQ ID NO: 3 (Hepcidin - 25)

[0048] The invention also encompasses the use of functional equivalents of the above-defined hepcidin. Functional equivalents are herein defined as peptide variants, having the same functional biological activity as the mature forms of hepcidin.

[0049] All these polypeptides and nucleic acids can be obtained by classical methods known in themselves. For instance, the 20 amino-acids and 25 amino-acids forms of hepcidin can be obtained from plasma or from urine, as disclosed by KRAUSE et al. ( FEBS Lett., 2000, vol. 480, 147-150) ; or PARK et al. (J. Biol. Chem., 2001, vol. 276,7806-7810). Alternatively, they can be obtained by culturing cells expressing hepcidin, and recovering said polypeptide from the cell culture. According to a particular embodiment, said cells are host cells transformed by a nucleic acid encoding one of the polypeptides defined above.

[0050] Methods for producing hepcidin are well known. For example, hepcidin can be obtained by classical sequential procedures for hepcidin purification from total human plasma. Starting from human plasma, ADDO L et al (Int J Hematol. 2016 Jan;103(l):34-43) purified 3 isoform of human hepcidin by using liquid chromatography-tandem mass spectrometry (LC-tandem MS). By using liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS), a highly specific and quantitative serum hepcidin method (isoform 25) was also describe (Lefebvre T et al. Clin Chem Lab Med. 2015 Sep l;53(10): 1557-67 or Abbas et al , Anal Bioanal Chem. 2018 Apr 18. doi: 10.1007 / s00216-018-1056-0.). By using surface-enhanced laser-desorption / ionization time-of-flight mass spectrometry (SELDI-TOF MS), isoform of human hepcidin detection in serum and urine was also describe (Kemna et al., Clin Chem. 2007 Apr;53(4):620-8.).

[0051] Alternatively, hepcidin can be a recombinant hepcidin as described in Gagliardo et al., FEBS J. 2008 Aug;275(15):3793-803.

[0052] Chemical synthesis can also be used, in particular in the case of the peptide derivatives. A nucleic acid encoding hepcidin can for instance be obtained from a genomic or cDNA library of a vertebrate, using suitable primers able to hybridize selectively with said nucleic acids. It can also be obtained by the classical techniques of polynucleotide synthesis.

[0053] Typically a variant of hepcidin has at least 50%, preferably, at least 60%, preferably, at least 70%, preferably, at least 80%, preferably, at least 85% more preferably at least 90%, more preferably at least 95% and even more preferably at least 99% identity with human mature form of hepcidin.

[0054] As used herein the terms “administering” or “administration” refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., hepcidin) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0055] Accordingly, in order to be administered directly to the intestine (gut), the hepcidin is administered to the subject orally (including buccal and sublingual administration), rectally or topically (intracolic administration). When administered orally rectally or topically, the hepcidin according to the invention can be in the form of a sustained release composition, or can be produced by a recombinant bacteria so as to deliver the hepcidin directly to the intestine.

[0056] In particular embodiment, the hepcidin is administered in intestinal lumen of the subject.

[0057] In particular embodiment, the hepcidin is administered in the gut of the subject by using a gut lumen-targeted delivery system.

[0058] As used herein, the terms “gut lumen-targeted delivery system” refers to delivery system has been developed for specific delivery and controlled release of cargos to gut area, i.e the gut lumen. Gut lumen-targeted delivery system not only protects the cargos from premature degradation and controls the release of the cargos in the gut area, greatly improving its bioavailability and reducing side effects, but also has better patient compliance and less interference by a variety of immune cells and cytokine in circulatory system. Indeed direct oral administration of bioactive agents (active substances) in the intestine (gut) can be fraught with problems due to the complex and harsh gastrointestinal environment. The strong acidic environment in the stomach inactivates many bioactive substances, while digestive enzymes in the small intestine further decompose and absorb them

[0059] Gut lumen-targeted delivery system can include Gut lumen-targeted oral delivery system as described in Liu, J. et al.20and / or and / or probiotic bacterium (or food-grade bacterium) as described in Benbouziane B. et al. 2013.17and / or mucosal delivery system including lipid carrier delivery systems such as micelles, liposomes and nanoparticle-based oral delivery systems as described in Zhang, M. et al22.

[0060] In particular embodiment, the hepcidin is administered in the gut of the subject by using a food-grade bacterium or probiotic bacterium.

[0061] Use of a food-grade bacterium comprising Hepcidin polypeptide.

[0062] Use of a food-grade bacterium (or probiotic bacterium) to deliver hepcidin allow to provides a safety and a good efficiency to deliver hepcidin locally in the gut.

[0063] Accordingly further object of the invention relates to a recombinant food-grade bacterium comprising a gene coding for hepcidin for use for preventing or treating iron overload associated disease in a subject in need thereof, wherein said recombinant food-grade bacterium is being administered locally in the gut of the subject.

[0064] In other words, another aspect of the invention relates to a method for preventing or treating iron overload associated disease in a subject in need thereof, comprising administering to said subject a food-grade bacterium comprising a gene coding for hepcidin, said food-grade bacterium is used to deliver directly the hepcidin in the gut of said subject.

[0065] In other words, another aspect of the invention relates to hepcidin for use preventing or treating iron overload associated disease in a subject in need thereof, wherein a food-grade bacterium comprising a gene coding for said hepcidin is used to deliver directly the hepcidin in the gut.

[0066] In particular embodiment, the recombinant food-grade bacterium comprising a gene coding for hepcidin having sequence set forth as SEQ ID NO:3.

[0067] As used herein, the term “food-grade bacterium” denotes a bacterium that is widely used in fermented foods and possesses a perfect safety profile recognized by the GRAS (Generally Recognized As Safe) and QPS (Qualified Presumption of Safety) status in USA and European Community, respectively. Such bacterium can be safely in functional foods or food additives with allegations concerning maintain in good health and well-being or prevention of disease. The term food-grade bacterium includes also probiotic bacterium.

[0068] As used herein, the term “probiotic bacterium” denotes a bacterium which ingested live in adequate quantities can exert beneficial effects on the human health. They are now widely used as a food additive for their health-promoting effects. Most of the probiotic bacteria are Lactic Acid Bacterium (LAB) and among them strains of the genera Lactobacillus spp. and Bifidobacterium spp. are the most widely used probiotic bacteria.

[0069] In a preferred embodiment, the food-grade bacterium strain according to the invention is a Lactococcus lactis strain or a Lactobacillus casei strain or a Lactococcus lactis htrA strain [Poquet et al., 2000] or a Lactobacillus plantarum strain or a Bifidobacterium longum strain.

[0070] In a preferred embodiment, the food-grade bacterium strain according to the invention is a Lactobacillus lactis strain.

[0071] In a preferred embodiment, the food-grade bacterium strain according to the invention is a lactic acid bacteria.

[0072] Indeed, lacid acid bacteria (LAB) are well known as mucosal delivery vehicles, as described in Benbouziane B. et al. 2013.17

[0073] In a preferred embodiment, the food-grade bacterium strain according to the invention is an engineered Lactobacillus lactic strain.

[0074] In a preferred embodiment, the food-grade bacterium strain according to the invention is an engineered Lactobacillus lactic strain comprising a controlled expression system.

[0075] As used herein, the term “controlled expression system” refers to a system comprising inducible promoter enabling to regulates the expression of the gene of interest and to ensure that the protein of interest are coded when and where they are needed.

[0076] In a preferred embodiment, the food-grade bacterium strain according to the invention is an engineered Lactobacillus lactic strain comprising a Stress-Inducible Controlled Expression (SICE) system.

[0077] In a preferred embodiment, the food-grade bacterium strain according to the invention is an engineered Lactobacillus lactic strain comprises the groESL operon promoter.

[0078] In a preferred embodiment, the food-grade bacterium strain according to the invention is an engineered Lactobacillus lactic strain comprising a Stress-Inducible Controlled Expression (SICE) system as described in Benbouziane B. et al. 2013.17 In other word, in a preferred embodiment, the food-grade bacterium strain according to the invention is an engineered Lactobacillus lactic strain comprising the groESL operon promoter and a gene coding for hepcidin having sequence set forth as SEQ ID NO:3.

[0079] Accordingly, in order to be administered directly to the intestine (gut), the recombinant food-grade bacterium according to the invention is administered to the gut’s subject preferably orally (including buccal and sublingual administration), rectally or topically (intracolic administration).

[0080] Pharmaceutical compositions of the invention:

[0081] The hepcidin and recombinant food-grade bacterium as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0082] Accordingly, the present invention relates to a pharmaceutical composition for use in the prevention or treatment of iron overload associated disease in a subject in need thereof, said pharmaceutical composition comprises hepcidin or a recombinant food-grade bacterium as defined above.

[0083] The present invention also relates to a pharmaceutical composition for use in the prevention or treatment of iron overload associated disease in a subject in need thereof, said pharmaceutical composition comprises i) hepcidin or a recombinant food-grade bacterium as defined above, and ii) a pharmaceutically acceptable carrier.

[0084] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0085] In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described, in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. An appropriate dosage of the pharmaceutical composition is readily determined according to any one of several well-established protocols. For example, animal studies (for example on mice or rats) are commonly used to determine the maximal tolerable dose of the bioactive agent per kilogram of weight. In general, at least one of the animal species tested is mammalian. The results from the animal studies can be extrapolated to determine doses for use in other species, such as humans for example. What constitutes an effective dose also depends on the nature and severity of the disease or condition, and on the general state of the patient's health.

[0086] In therapeutic treatments, the antagonist contained in the pharmaceutical composition can be administered in several dosages or as a single dose until a desired response has been achieved. The treatment is typically monitored and repeated dosages can be administered as necessary.

[0087] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0088] The appropriate unit forms of administration include forms for oral administration, such as tablets, gelatine capsules, powders, granules and solutions or suspensions to be taken orally, forms for sublingual and buccal administration, aerosols, implants, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms for rectal administration.

[0089] In the pharmaceutical compositions of the present invention, the active principle is generally formulated as dosage units containing from 0.5 to 1000 mg, preferably from 1 to 500 mg, more preferably from 2 to 200 mg of said active principle per dosage unit for daily administrations.

[0090] When preparing a solid composition in the form of tablets, a wetting agent such as sodium laurylsulfate can be added to the active principle optionally micronized, which is then mixed with a pharmaceutical vehicle such as silica, gelatine, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets can be coated with sucrose, with various polymers or other appropriate substances or else they can be treated so as to have a prolonged or delayed activity and so as to release a predetermined amount of active principle continuously.

[0091] A preparation in the form of gelatin capsules is obtained by mixing the active principle with a diluent such as a glycol or a glycerol ester and pouring the mixture obtained into soft or hard gelatine capsules. A preparation in the form of a syrup or elixir can contain the active principle together with a sweetener, which is preferably calorie-free, methyl-paraben and propylparaben as an antiseptic, a flavoring and an appropriate color.

[0092] The water-dispersible powders or granules can contain the active principle mixed with dispersants or wetting agents, or suspending agents such as polyvinyl-pyrrolidone, and also with sweeteners or taste correctors.

[0093] The active principle can also be formulated as microcapsules or microspheres, optionally with one or more carriers or additives.

[0094] Among the prolonged-release forms which are useful in the case of chronic treatments, implants can be used. These can be prepared in the form of an oily suspension or in the form of a suspension of microspheres in an isotonic medium.

[0095] Hepcidin or food-grade bacterium according to the invention can be administered by any suitable route of administration. For example, hepcidin or food-grade bacterium according to the invention can be administered by oral (including buccal and sublingual), rectal, nasal, topical (intracolic), pulmonary, vaginal, or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration.

[0096] The hepcidin or food-grade bacterium of the present invention, may be formulated in a wide variety of oral administration dosage forms.

[0097] The term “preparation” is intended to include the formulation of the active compound with an encapsulating material as carrier, providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pulls, cachets, and lozenges may be as solid forms suitable for oral administration. Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents, for example, such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizers, and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0098] The hepcidin or food-grade bacterium of the present invention of the present invention may be formulated for administration as suppositories. Typically, a low melting wax, such as a mixture of fatty-acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously.

[0099] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0100] FIGURES:

[0101] Figure 1 : Hepcidin is expressed all along the intestine.

[0102] A. Hampl expression mRNA (qPCR) in the gut of 8-10 weeks old C57B16j mice (n=4). B. Hampl expression mRNA (qPCR) along the duodenum villi in 8-10 weeks old C57B16j mice (n=4). C. Genetic construction of mouse model of Hampl overexpression specific in the gut (Hampl KI- Vil). D-G. Hampl expression mRNA (qPCR) in epithelial enrichment cells from duodenum, jejunum, ileum and colon. All analyses were performed on males and females. 26 days old Hampl CMV-1 ox-STOP -lox (round) and Hamp 1KI- Vil (square). Unpaired Mann- Whitney test used : **P < 0.01 and ***P < 0.001.

[0103] Figure 2_: Hampl Kl-Vil mice developped en iron deficiency anemia

[0104] A. Hamp 1KI'V11phenotype, percentage of mice who lost hair in female and male HamplKI’V11B. HamplCMV-lox-STOp-loxanc[ HamplKI'V1124-26 days old weight (g). C-E. Plasma iron parameters : Iron (pmol / L), Ferritin (pg / L) and Fixation Capacity of Transferrin (FCT %) . F. Hampl expression mRNA (qPCR) in liver. G-K. Blood count parameters : Red blood cells (1012 / L), Hemoglobine (g / dL), Hematocrit (%), Mean Corpusula Volume (MCV fL) and Mean Corpuscular Hemoglobin (MCH pg). All of analyses was done on male and female HamplCMV' 1OX-STOP-1OX (rounc[)anc[ Hamp 1KI'V11(square) aged of 26 days old. Mean + / - SD. Unpaired Mann- Whitney test used : *P<0.05, **P < 0.01 and ***P < 0.001.

[0105] Figure 3 : Apical hepcidin decreased iron absorption via downregulation of DMT1 A. FPN western blots on epithelial cell enrichment from duodenum of 24-26 days old HamplCMV'lox'STOP'loxand Hamp 1KI'V11female mice . B. DMT1 western blot on epithelial cell enrichment from duodenum of 24-26 days old female HamplCMV-lox-STOp-lox(round)and HamplKI'V11(square) mice. C-F. mRNA expression (qPCR) of Dmtl+IRE, Cybdr, Ftl and SlclOal (FPN) on epithelial cells enrichment from duodenum of 24-26 days old male and female HamplCMV-lox-STOp-loxand Hamp 1KI'V11mice. G. Luminal duodenum hepcidin content in 15 days old male and female HamplCMV-lox-STOp-loxand HamplKI'V11H. Serum hepcidin in 24- 26 days old male and female HamplCMV-lox-STOp-loxand HamplKI'V11. I. Concentration of 57Fe absorbed by the duodenum (normalized by dry weight and endogenous 54Fe) of male and female HamplCMV-lox-STOp-loxmice anc[ HamplKI'V11aged of 17 days old measured by ICP-MS J. DMT1 and FPN western blot on duodenum of C57B16 mice (n=3) after injection of hepcidin (1 pM) in the duodenum loops lumen during 2 hours.

[0106] Mean + / - SD. Unpaired Mann-Whitney test used : **P <0,01.

[0107] Figure 4 : Lactoccocus lactis probiotics producing hepcidin decreased iron overload in hemochromatosis mouse models

[0108] Lac-pGroESL and Lac-hepc constructs and quantification of hepcidin production (n=3) by ELISA after stimulation of bacteria at 37°C for 2 hours. B. DMT1 western blot on epithelial cell enrichment from duodenum of HamplA11Vmice after 3 hours of Lac-pGroESL or Lac-hepc treatment C. Scheme of protocol treatment. D. Evolution of weight in mice during treatment. E-G. Plasma iron parameters: Iron (pmol / L), Ferritin (pg / L) and Fixation Capacity of Transferrin (FCT %). H-K. Tissus iron content (pg / g) in liver, pancreas, spleen and heart respectively. L. Peris' blue staining on liver section after 4 weeks of treatment in HamplAllvand control Hamplfl / fltreated with Lac-pGroESL or Lac-hepc. M. Serum hepcidin concentration in HamplAllvand control Hamp lfl / fltreated with Lac-pGroESL or Lac-hepc after 4 weeks of treatment N. DMT1 western blot on duodenum in HamplAllvand control Hamplfl / flmice after 4 weeks of treatment.

[0109] EXAMPLE

[0110] Material & Methods

[0111] Mice

[0112] To generate Hampl KLvil mice, we first generated a Rosa26-pCAG-LSL-Hamp mouse line by homologous recombination Knock-in technology into Rosa26 locus. Hampl cDNA was inserted downstream the cytomegalovirus (CMV), immediate enhancer / p-actin (CAG) promoter and a loxP-STOP-loxP (LSL) cassette. The vector was transfected into embryonic stem cell (ESC) line and the selection was made in G418. ES cell clones were screened for proper homologous recombination by DNA-PCR before injection into BALB / c blastocysts. Chimeric male mice were crossed with C57BL / 6N female mice to obtain the ROSAHampl Fl mice. The identification of Fl mice was performed by PCR of tail DNA using primers that amplify a 310 bp band in case of the mutant allele, and primers that amplify a 192 bp band in the case of the WT Rosa Allele. ROSAHampl Fl mice don’t express the Hampl cDNA but after breeding with B6.Cg-Tg(Vill-cre)1000Gum / J mice (Jackson Laboratory)15, the LSL cassette were deleted and Hampl mRNA was expressed in the gut epithelial cells.

[0113] Hemochromatosis mouse model (HamplAllver) was generated by our laboratory as described before16

[0114] 57Fe isotope experiment

[0115] The stable iron isotope57Fe (57Fe at 96% enrichment, MFE57M, Eurisotop) was used as tracer. The procedure was adapted from Fiorito et al. 17 days oldHamP1CMV-lox-STOp-loxanc[ Hamp lKlv11mice were force-fed with a solution of 20mM57FeSO4, 5.38mM ascorbic acid and 10%sucrose. 90min after oral administration, mice were anesthetized, perfused and their duodenum collected. Iron isotope measurement was performed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) on the Nexion 5000® spectrometer (Perkin Elmer, Les Ulis, France).

[0116] Mice Surgery and hepcidin treatment

[0117] 7 weeks old male C57B16 / J mice were anesthetized with pentobarbital. Next, mice were open and the duodenum was ligatured twice, first at the pylore and 3cm after pylore. Before to finish the second ligature, 400pL PBS or 400pL hepcidin-25 (IpM) were injected in the duodenum lumen. Mice have been closed during 2 hours. Mice were maintained under isoflurane during the surgery. 2 hours later, sleeping mice were open and the duodenum were collected.

[0118] Recombinant Lactoccocus Lactis

[0119] Development of a Stress-Inducible Controlled Expression (SICE) system in Lactococcus lactis for the production and delivery of therapeutic molecules at mucosal surfaces as described in Benbouziane, B. et al11. The expression of hepcidin via the Lactococcus lactis was induced at 37°C for 2h.

[0120] Lactoccocus Lactis culture and aliquot preparation

[0121] Lac-pGroESL and Lac-hepc was grown in agar-M17 medium (Difco Laboratories) supplemented with 0.5% glucose and antibiotic Chloramphenicol (Cm, lOpg / mL). The next day, one colony was resuspended in 5mL of M17 + 0.5% glucose + Cm and growing at 30°C all day. Ten hours later, 5mL of preculture was added to IL of M17 + 0.5% glucose + Cm at 30°C overnight. DO (600nm) was measured after overnight incubation to determine L. lactis concentration. Bacteria were centrifugated at 6,000 rpm, 4°C, 15min. Supernatent was removed and bacteria was washed with PBS IX and centrifugated again (6,000 rpm, 4°C, 15min). Bacteria pellet was resuspended in lOOmL of sterilizes PBSlX-20% glycerol, then aliquoted and freeze directly in liquid nitrogen and conseved at -80°C.

[0122] To determine the aliquot’s concentration of Lac-pGroESL and Lac-hepc preparation’s, a serial dilution was performed on agar-M17 + 0.5% glucose + Cm.

[0123] Lactoccocus Lactis treatment

[0124] Hamplfl / fland HamplAllverwere weaned at 28 days old and directly treated with frozen aliquot of Lac-pGroESL or Lac-hepc. Male and female are included. Mice were treated every day (at 6 pm) by oral gavage with 5xlO9CFU of Lac-pGroESL or Lac-hepc. Mice were separated in cage by group sex and treatment. They received food and water ad libitum.

[0125] RNA extraction and quantitative PCR

[0126] RNA extraction, reverse transcription and quantitative PCR have been performed as previously described16. Mouse samples were normalized to the threshold cycle value for 36B

[0127] Western-blot

[0128] Epithelial cells from duodenum were harvested and resuspended in a solution of 0.25 M sucrose / 0.03 M histidine (pH 7.2) supplemented with l x complete inhibitor cocktail EDTA- free (Roche) (Volume is 4 times the weight of tissue). After a 30min incubation on ice, cells were subjected to brief sonication and centrifugation at 6,000 g for 10 min. To obtain crude membrane fraction the supernatant was subsequently isolated by ultracentrifugation at 41 000 rpm (TLA-45 rotor; Beckman-Coulter) for 1 hour. Protein concentration was determined using the Pierce™ BCA Protein Assay Kits (23225, Thermo Scientific).

[0129] Lysates containing 15pg of proteins were subjected to standard methos of Western blot. DMT1 primary antibody (gift of Francois Cannone-Hergaux, CNRS [ICSN], Gif-sur-Yvette, France) was diluted at 1 :500 and FPN primary antibody (NBP1-21502, Novus) at 1 : 1000. Loading control P-actin (A5316, Sigma Aldrich at 1 :5000) antibodies were also used. Immunoblots were quantified using ImageJ software.

[0130] Iron, Transferrin, ferritin measurement and Blood Count parameters

[0131] Plasma and iron tissues (heart, liver, pancreas and spleen) levels were quantified colorimetrically by a previously described method18. Hematological parameters were determined using a OX-560 RET automatic analyzer (Baliodiasgnotics). Perl’s staining

[0132] Liver tissues were fixed in 4% formaldehyde and embedded in paraffin. Sections were cut at a thickness of 4 pm. Following dewaxing in xylene baths and rehydratation, sections were either stained with Peris’s Prussian blue and nuclear fast red counterstain.

[0133] ELISA, enzyme-linked immunosorbent assay

[0134] The serum from mice were directly used in Hepcidin Murine-Compete™ ELISA (HMC-001, Intrinsic Lifesciences) and in mouse TNFa Quantikine ELISA (MTAOOB, R&Dsy stems). Luminal duodenum content was collected in ImL PBS IX supplemented with inhibitor protease (A32965, Thermo Scientific). Tissues was homogenized by vortex and directly used in Hepcidin Murine-Compete™ ELISA (HMC-001, Intrinsic Lifesciences). After induction of hepcidin production by Lac-hepc and Lac-pGroESL, supernatant was used in mouse hepcidin ELISA (S-1465, BMA Biomedicals).

[0135] Statistical analysis

[0136] Values in the figures are expressed as mean + / - SEM. Analyses were performed using GraphPad Prism 9.0. The significance of experimental differences was evaluated by an unpaired (2 -tailed) Mann-Whitney test, when comparing two groups. A one-way or a two-way ANOVA followed by a Tukey analysis was used when more than two groups were compared. Statistical significance is indicated by * symbols (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). ns indicates not significant.

[0137] Study approval

[0138] The animal studies described here were reviewed and approved (Agreement n°APAFIS#39956- 2022090310098896) by the Ministry of Higher Education, Research and Innovation and are in accordance with the principles and guidelines established by the European Convention for the Protection of Laboratory Animals (Council of Europe, ETS 123, 1991).

[0139] Results

[0140] Hepcidin is expressed by the gut

[0141] Although the main site of hepcidin production is the liver, it has been now clearly established several peripheral organs can locally produce that hepcidin, where the hepcidin- ferroportin axis may be crucial, in pathophysiological conditions, for maintaining iron homeostasis in these organs. Here, we investigated Hampl mRNA expression all along the gut tractus in isolated epithelial cells from WT mice by qPCR analysis, and observed similar expression in the duodenumjejunum, ileum and colon (Fig 1A). Using a sequential isolation of intestinal epithelial cells along the crypt villus, we further determined that hepcidin expression was restricted to mature differentiated intestinal cells in the duodenum, its expression being undetectable in the proliferative crypt compartment (Fig IB).

[0142] To probe the function of gut-derived hepcidin, we generated a mouse model overexpressing hepcidin in intestinal epithelium. For that, the mouse Hampl gene sequence was inserted into the ROSA26 locus downstream of a strong CMV promoter and a lox-STOP- lox (LSL) sequence (referenced as HamplCMV'lox'STOP'lox). After breeding with a transgenic strain expressing the Cre recombinase under the control of the murine villin promoter (Villinl - CRE), the LSL cassette is deleted and Hampl is overexpressed. This newly generated model is referenced as Hamp 1KI'V11(See Methods, Fig 1C).

[0143] Comparing to control HamplCMV'lox'STOP'loxanimals, we demonstrated that hepcidin mRNA was dramatically overexpressed in the gut of the HamplKI'V11mice, as assessed by qPCR in isolated epithelial cells, from duodenumjejunum, ileum and colon (Fig 1D-G). We verified that Hampl overexpression was limited to the intestinal epithelium and not found in other organs (such as skin, spleen, lung, stomach, data not shown).

[0144] Mice overexpressing gut hepcidin developed an iron deficiency anemia

[0145] From the age of 24-26 days, the HamplKI'V11mice presented with a striking phenotype. First, we observed a hair loss, beginning by patches localized on the lower back. This hair loss was present, both in female and male HamplKI’V11, respectively, in 35.3% and 53.3% cases (Fig 2A). No difference in the weight of mice was measured between HamplKI'V11and HamplCMV' 1OX-STOP-1OX The iron status of the mice was next carefully examined. We demonstrated that the HamplKI'V11mice presented an iron deficiency anemia, with a significant decrease of plasma iron, ferritin and fixation capacity of transferrin (Fig 2C-E), as well as a significant decrease of hemoglobin, HCT, MCV and MCH (respectively by: -14,85%, -12,18%, -11,2% and -14,17%) (Fig 2G-K) in male and female HamplKI'V11mice as compared to HamplCMV'lox' STOP-lox

[0146] In response to this systemic iron deficiency, Hampl expression was found drastically repressed in the liver of the HamplKI'V11compared to HamplCMV'lox'STOP'lox(Fig 2F).

[0147] Altogether, these results clearly established that gut hepcidin overexpression is sufficient to induce a microcytic iron deficiency anemia in young mice.

[0148] Duodenal lumen hepcidin decreased iron absorption via DMT1 inhibition

[0149] To explore the mechanisms underlying this phenotype of systemic iron deficiency leading to microcytic anemia in 24-26 days old HamplKI'V11mice, we looked at the iron transporters in the duodenum; DMT1, Divalent Metal Transporter 1, present at the apical side of the enterocyte; and ferroportin, FPN, present at the basolateral side, responsible for iron export into the circulation.. Intriguingly, FPN, the only known target of hepcidin, was found unchanged with regards to the huge hepcidin overexpression in the duodenum of the HamplKI'V11mice compared to HamplCMV'lox'STOP'lox(Fig 3A). In contrast, DMT1 levels were found significantly decreased. (Fig 3B). At the mRNA level, no change was observed for DMT1 (nor for the other iron-related proteins), suggesting a post transcriptional regulation of DMT 1, by the transgenic hepcidin (Fig 3C-F).

[0150] The effect of transgenic hepcidin on DMT1 at the apical side of the enterocyte prompted us to ask whether the transgenic hepcidin peptide could be secreted in the intestinal lumen. To test this hypothesis, we collected the luminal content of the duodenum of 15 days old mice (male and female) and measured hepcidin concentration using an ELISA. As shown in Fig 3G, we did observe the presence of hepcidin in the intestinal lumen, with a twofold increase of hepcidin in the HamplKI'V11mice as compared to HamplCMV'lox'STOP'loxmice. Moreover, systemic hepcidin was slighty increased in HamplKI'V11compared to Hamp lCMV'lox'STOP'loxmice (Fig 3H). To examine whether gut hepcidin prevented iron absorption by inhibiting apical iron uptake, a FeSCU solution, specifically enriched in iron isotope57Fe (Fig 31), was administered by oral gavage to HamplKI'V11mice and control littermates.57Fe was measured, 90 min postgavage, in the duodenum by Inductively Coupled Plasma Mass Spectrometry method (ICP- MS). We observed that HamplKI'V11mice exhibited lower levels of57Fe in the duodenum compared to HamplCMV'lox'STOP'loxmice (Fig 31) confirming the inhibition of iron uptake at the apical side of enterocytes.

[0151] To confirm, more directly, a potential effect of hepcidin present in the lumen on DMT1 protein levels, we injected synthetic hepcidin in duodenal loops of WT mice. Two hours after hepcidin injection, duodenum wascollected and western-blot performed on isolated enterocytes. As show in Fig 3J, when the duodenum is exposed to hepcidin in the lumen, we observed a significant decrease of DMT1, with no change in FPN. As in the transgenic enterocyte, the effect was at the protein level, with no change in the mRNA in the duodenal loop after hepcidin injection (data no shown).

[0152] Taken together, our data strongly suggest that duodenal hepcidin may regulate iron absorption by controlling the levels of apical DMT1 and may induce, when overexpressed as in the HamplKI'V11mice, iron deficiency anemia Probiotic L. lactis producing hepcidin decrease iron overload in hemochromatosis mouse model

[0153] The HamplKI'V11mouse model showed that hepcidin can be secreted into the lumen to diminish DMT1 and iron absorption. In consideration of this, hepcidin could be a new therapeutic target to inhibit iron absorption in case of pathologies linked to iron hyperabsorption. Hereditary hemochromatosis is a genetic disorder characterized by an iron overload. In these patients, hepatic hepcidin is abnormally low. This defect in liver hepcidin expression induces an iron hyperabsorption. To counteract this hyperabsorption of iron we propose to generate probiotics enable to produce hepcidin as therapeutic tools. The most common probiotic strain belongs to Lactococcus, Lactobacillus and Bifidobacterium. Lactococcus lactis (L. lactis), considered a "neutral strain", is a microbial model widely used as an expression system for molecules of interest19. It has the advantage of being a GRAS (Generally Recognized As Safe) bacterium. In this line, we generated L. lactis recombinant for hepcidin (Lac-hepc). The murine hepcidin gene was cloned into the Histidine-tagged pSICE plasmid under the control of the L. lactis GroESL promoter (Fig 4A). This promoter is inducible by stress, notably heat and pH, and will thus enable the induction of hepcidin in vivo. The plasmid was sequenced and transformed into the L. lactis strain. We verified hepcidin secretion in vitro in the culture supernatant after heat induction (Fig 4A).

[0154] After validation of production of hepcidin by Lac-hepc, we administered by oral gavage of L-hepc to hemochromatosis mice (HamplAllv) and we showed a decrease in membrane duodenal DMT 1 expression as early as 3 hours (Fig 4B). We then administered daily (5X109CFU) by oral gavage L. lactis strains expressing hepcidin (Lac-hepc) or the empty vector (Lac-pGroESL) to 28-day-old (weaning) hemochromatosis mice (HamplAllver) and their control littermates (Hamplfl / fl) (Fig4C). After 4 weeks of treatment, mice were sacrified. Indeed, Iron overload appears as early as 8 weeks of age in HamplAllver 16. Lac-pGroESL and Lac-hepc strains had no effect on the growth of Hamplflfand HamplAllverfemale and male mice (Fig 4D). While hemochromatosis mice inoculated with Lac-pGroESL showed iron accumulation at 2 months, characterized by elevated plasma ferritin, treatment with Lac-hepc decreased by twice ferritin plasma levels (Fig 4F). However, no difference in other iron blood parameters as iron and FCT was detected (Fig 4E and Fig 4G). In hemochromatosis, the liver is the first organ to become iron overloaded. In Fig 4H, we observed that hepatic iron is significant decreased in HamplAllver-Lac-hepc-treated mice compared to HamplAllver-Lac-pGroESL-treated mice (1,5- fold). We observed no effect of Lac-hepc on other extrahepatic organs as pancreas, spleen and heart (Fig 4I-K). However, in the pancreas, Hamp lAllvermice tended to become iron overloaded. This trend is not observed in HamplAllver-Lac-hepc-treated mice compared to HamplAllver-Lac- pGroESL-treated mice (Fig 4H). To confirm the effect on the liver, Perl’s blue staining was performed. Blue staining was drastically decreased in HamplAllver-Lac-hepc-treated mice compared to HamplAllver-Lac-pGroESL-treated mice (Fig 4L). We also confirmed by ELISA, that hepcidin produced by the L.Lactis did not pass through the epithelial barrier to act at the basolateral side (Fig 4M)

[0155] To verify that the rescue of iron overload in HamplAllver-Lac-hepc-treated mice was the consequence of the action of luminal hepcidin on DMT1, a western blot on duodenum was performed. We observed a decrease in DMT1 expression in mice treated with Lac-hepc (Hamplflfand HamplAllver) compared to their control groups treated with Lac-pGroESL (Fig 4N).

[0156] Altogether, the use of probiotics producing hepcidin in the intestinal lumen to limit iron overload in hemochromatosis patients seems to be relevant.

[0157] REFERENCES:

[0158] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0159] 1. Ganz, T. Systemic iron homeostasis. Physiol Rev 93, 1721-1741 (2013).

[0160] 2. Steinbicker, A. U. & Muckenthaler, M. U. Out of balance— systemic iron homeostasis in iron-related disorders. Nutrients 5, 3034-3061 (2013).

[0161] 3. Pietrangelo, A. Hereditary Hemochromatosis: Pathogenesis, Diagnosis, and Treatment. Gastroenterology 139, 393-408. e2 (2010).

[0162] 4. Nicolas, G. et al Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci U S H 98, 8780-8785 (2001).

[0163] 5. Nemeth, E. et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 306, 2090-2093 (2004).

[0164] 6. Malerba, M. et al. Epidermal hepcidin is required for neutrophil response to bacterial infection. J Clin Invest 130, 329-334 (2020).

[0165] 7. Peyssonnaux, C. et al. TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens. Blood 107, 3727-3732 (2006).

[0166] 8. Deschemin, J.-C., Mathieu, J. R. R., Zumerle, S., Peyssonnaux, C. & Vaulont, S. Pulmonary Iron Homeostasis in Hepcidin Knockout Mice. Front Physiol 8, 804 (2017). 9. Lakhal -Littleton, S. et al. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis. Elife 5, el9804 (2016).

[0167] 10. Schwartz, A. J. et al Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells. Nat Metab 3, 969-982 (2021).

[0168] 11. Yamaji, S., Sharp, P., Ramesh, B. & Srai, S. K. Inhibition of iron transport across human intestinal epithelial cells by hepcidin. Blood 104, 2178-2180 (2004).

[0169] 12. Brasse-Lagnel, C. et al Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation. Gastroenterology 140, 1261-1271. el (2011).

[0170] 13. Mena, N. P., Esparza, A., Tapia, V., Valdes, P. & Nunez, M. T. Hepcidin inhibits apical iron uptake in intestinal cells. Am J Physiol Gastrointest Liver Physiol 294, G192-198 (2008).

[0171] 14. Chaston, T. et al. Evidence for differential effects of hepcidin in macrophages and intestinal epithelial cells. Gut 57, 374-382 (2008).

[0172] 15. Madison, B. B. et al. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J Biol Chem 277, 33275-33283 (2002).

[0173] 16. Zumerle, S. et al. Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype. Blood 123, 3646-3650 (2014).

[0174] 17. Benbouziane, B. etal. Development of a Stress-Inducible Controlled Expression (SICE) system in Lactococcus lactis for the production and delivery of therapeutic molecules at mucosal surfaces. J Biotechnol 168, 120-129 (2013).

[0175] 18. Mastrogiannaki, M. et al. HIF-2a, but not HIF-la, promotes iron absorption in mice. J Clin Invest 119, 1159-1166 (2009).

[0176] 19. Bermudez-Humaran, L. G. & Langella, P. Live bacterial biotherapeutics in the clinic. Nat Biotechnol 36, 816-818 (2018).

[0177] 20. Liu, J. et al. Gut lumen-targeted oral delivery system for bioactive agents to regulate gut microbiome. Journal of Future Foods. 202218

[0178] 21. Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis. 2010 May 21;5:1 I. doi: 10.1186 / 1750-1172-5-11. PMID: 20492708; PMCID: PMC2893117.

Claims

CLAIMS:

1. A method for decreasing iron overload in a subject in need thereof, comprising administering locally in the gut of the subject a therapeutically effective amount of hepcidin.

2. A method for preventing or treating an iron overload associated disease in a subject in need thereof, comprising administering locally in the gut of the subject a therapeutically effective amount of hepcidin.

3. The method according to claim 2, wherein the iron overload associated disease is selected from the list consisting of hemochromatosis (adult and juvenile forms of hereditary hemochromatosis), iron-loading anemia and chronic liver diseases including alcoholic liver diseases, non-alcoholic steatohepatitis (NASH) and chronic hepatitis B and C.

4. The method according to claim 2 or 3, wherein the iron overload associated disease is hemochromatosis or P-thalassemia.

5. The method according to claim 1 to 4, wherein the hepcidin comprises or consists of the sequence having at least 50%, 60%, 70%, 80% or more particularly 90% sequence identity with the amino acid sequence selected in the group consisting of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

6. The method according to claim 5, wherein the hepcidin comprises a sequence as set forth in SEQ ID NO: 3 (“Hepcidin-25aa”)7. The method according to claim 1 to 6, wherein the hepcidin is administered in the gut of the subject by using a gut lumen-targeted delivery system.

8. The method according to claim 1 to 7, wherein the hepcidin is administered in the gut of the subject by using a food-grade bacterium or probiotic bacterium.

9. A recombinant food-grade bacterium comprising a gene coding for hepcidin for use for preventing or treating iron overload associated disease in a subject in need thereof, wherein said recombinant food-grade bacterium is being administered locally in the gut of the subject.

10. The recombinant food-grade bacterium for use according to claim 9, comprising a gene coding for hepcidin having sequence set forth as SEQ ID NO:3.

11. The recombinant food-grade bacterium for use according to claim 9 or 10, wherein the food-grade bacterium is a Lactobacillus lactis strain.

12. The recombinant food-grade bacterium for use according to claim 11, wherein the foodgrade bacterium is an engineered Lactobacillus lactic strain comprising a Stress- Inducible Controlled Expression (SICE) system.

13. The recombinant food-grade bacterium for use according to claim 9 to 11 , or the method according to claim 8, wherein the recombinant food-grade bacterium is administered to the gut’s subject preferably orally (including buccal and sublingual administration), rectally or topically (intracolic administration).

14. A pharmaceutical composition for use in the prevention or treatment of iron overload associated disease in a subject in need thereof, said pharmaceutical composition comprises a hepcidin or a recombinant food-grade bacterium according to claim 9 to 11.

15. The method according to claims 1 to 8 or the recombinant food-grade bacterium according to claims 9 to 13 or the pharmaceutical composition according to the claim 14 wherein the hepcidin is an inhibitor of the Divalent Metal Transporter 1 (DMT1).

Images