Compounds and compositions for stem cell transplantation

Abstract

The present invention relates to a Matriptase-2 (MT2) inhibitor, a TMPRSS6 inhibitor, an iron transporter blocking agent, or a hepcidin enhancer. In particular, the present invention relates to the use of compounds capable of reducing at least one of systemic iron levels, transferrin saturation (Tsat), non-transferrin binding iron (NTBI), and labile plasma iron (LPI). This can be accomplished by inhibiting the expression of a target gene, wherein the target gene can be transmembrane protease serine 6 (TMPRSS6). Further, the invention relates to compositions comprising said compounds and to methods of using such compounds and / or compositions. Uses may include therapeutic uses, such as for reducing iron overload and preventing iron-related toxicity before, during or after chemotherapy pretreatment, stem cell transplantation and colonization.

Description

Technical Field

[0001] This invention relates to the use of Matriptase-2 (MT2) inhibitors, TMPRSS6 inhibitors, iron transporter blockers, or hepcidin enhancers. Specifically, this invention relates to compounds capable of reducing at least one of systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and unstable plasma iron (LPI). This can be achieved by inhibiting the expression of a target gene, which may be a transmembrane protease serine 6 (TMPRSS6). Furthermore, this invention relates to compositions comprising said compounds and methods of using such compounds and / or compositions. Uses may include therapeutic uses, such as reducing iron overload and preventing iron-related toxicity before, during, or after explantation, implantation, or transplantation-related therapies (including chemotherapy pretreatment, stem cell transplantation, and colonization). Background Technology

[0002] Stem cell transplantation (SCT), which involves transplanting stem cells into a recipient, is the only curative treatment option for some diseases, including but not limited to hematopoietic stem cell transplantation (HSCT) for treating leukemia (such as AML) or other hematologic disorders (such as beta-thalassemia, sickle cell disease, and bone marrow failure). Certain other conditions caused by chemotherapy or radiation therapy, such as myeloablation, also benefit from SCT. Similarly, high-dose therapy and autologous HSCT are selective treatment options for patients with both hematologic and non-hematologic malignancies. Furthermore, gene therapy for correcting certain genetic defects in hematologic disorders often requires myeloablation prior to the colonization of genetically modified autologous stem cells.

[0003] Myelodysplastic syndromes (MDS) (formerly known as preleukemia because they can develop into leukemia) are a diverse collection of hematologic medical conditions involving the ineffective production (or abnormal proliferation) of myeloid-type blood cells. All MDS are disorders of hematopoietic stem cells in the bone marrow. In MDS, hematopoiesis (blood production) is disordered and ineffective. The number and quality of blood-forming cells in MDS are irreversibly reduced, further impairing blood production. Patients with MDS may develop severe anemia and require blood transfusions. In some cases, the disease worsens, and patients develop cytopenia (low blood cell count) due to progressive bone marrow failure. Because MDS is a stem cell-related condition, HSCT (hematopoietic stem cell transplantation) offers the possibility of curative treatment, especially in patients with more severe conditions.

[0004] Despite a better understanding of transplant immunology, improved graft-versus-host disease (GVHD) prevention, and enhanced supportive care, many risks remain associated with this type of transplantation, and morbidity and mortality associated with HSCT remain high (Evens et al., Bone Marrow Transplantation 2004, 34, 561-571). Toxicity primarily results from conditioning-induced organ damage, neutropenia that predisposes patients to bacterial or fungal infections, or impaired cellular immunity that makes patients susceptible to viral and other opportunistic infections.

[0005] Iron overload (primarily due to transfusion requirements and ineffective erythropoiesis) is common but often remains uncontrolled during HSCT and can affect transplant outcomes and gene therapy regimens. Iron overload can contribute to treatment-related toxicities and post-transplant mortality (Evens et al., Bone Marrow Transplantation 2004, 34, 561-571). Patients opting for HSCT often develop iron overload and elevated transferrin saturation due to repeated transfusions. Pre-existing iron overload is a poor prognostic factor for patients undergoing HSCT (Wermke et al., 2012, Clin. Cancer Res, 18 (23), Atilla et al., Turk J Hematol 2017;34:1-9). Indeed, elevated ferritin, transferrin saturation, liver iron, and non-transferrin-bound iron (NTBI) have become poor prognostic factors for post-transplant survival and complications in patients with β-thalassemia, as well as sickle cell disease, MDS, and leukemia. Furthermore, recent studies have shown that pretreatment before HSC transplantation further exacerbates the presence of circulating “free” nontransferrin-bound iron (NTBI) and unstable plasma iron (LPI) (the reactive fraction of LPI) (Naoum et al., Hematol. Oncol. Stem Cell Ther 2016 9, 165-167). Importantly, NTBI positivity predicts lower overall survival in transplant recipients of myelodysplastic syndromes (MDS) or AML (Wermke et al., Lancet Haematol. 2018 May;5(5):e201-e210).

[0006] Current treatment options for preventing iron overload to reduce iron-mediated toxicity before or during HSC transplantation and colonization:

[0007] Venous cut

[0008] Humans lack a dedicated iron excretion mechanism, and iron loss primarily occurs passively through the shedding of skin and intestinal epithelium and urine. Under normal circumstances, a loss of 1 to 2 mg of iron per day is balanced by the absorption of a similar amount ingested through diet (Isidori et al., Transplant Cell Ther. 2021 May;27(5):371-379). Therefore, iron overload can develop due to repeated transfusions or through iron homeostasis dysregulation caused by decreased hepcidin levels or activity, such as in iron-burden anemia or hereditary hemochromatosis (Camaschella et al., 2020, Vol. 105, No. 2 (2020): February 2020). Venotomy is considered the simplest method for removing excess iron from the body. In HSCT recipients, venotomy can be used to remove excess iron after successful hematopoietic stem cell colonization, but is not an option during pretreatment and colonization phases or when the patient still requires transfusions. Generally speaking, this method is not usually used because patients undergoing HSCT often have anemia.

[0009] Chelation

[0010] Iron chelation therapy is currently the pharmacological option for alleviating iron overload in patients with iron excess (review by Isidori et al., 2021 Transplant Cell Ther. 2021 May;27(5):371-379): Deferoxamine was not mentioned in this case due to its short half-life (requiring prolonged infusion) and its siderophoretic activity (allowing for the release of iron into microorganisms). The oral chelator deferiprone has a longer half-life, but it is also associated with the potential development of agranulocytosis. The latest generation chelator deferasirox has the advantage of a longer half-life and the ability to effectively clear NTBI / LPI. Some common adverse events associated with deferasirox therapy may overlap with side effects after acute HSCT and thus limit its use to better control late post-transplant iron toxicity for improved long-term patient outcomes.

[0011] References: Essmann S, Heestermans M, Dadkhah A, Janson D, Wolschke C,Ayuk F, Kröger NM, Langebrake C. Iron Chelation with Deferasirox Suppressesthe Appearance of Labile Plasma Iron During Conditioning Chemotherapy Priorto Allogeneic Stem Cell Transplantation. Transplant Cell Ther. 2023 Jan;29(1):42.e1-42.e6. doi: 10.1016 / j.jtct.2022.10.002. Epub 2022 Oct 12. PMID:36241148. Summary of the Invention

[0012] In one aspect, the present invention relates to a therapeutic agent for use in treating iron metabolism disorders or conditions, said therapeutic agent comprising an inhibitor selected from:

[0013] (a) TMPRSS6 inhibitors;

[0014] (b) MT2 inhibitors; or

[0015] (c) Iron transporter inhibitors,

[0016] The treatment includes cell explantation, implantation, or transplantation.

[0017] In some respects, the treatment of iron metabolism disorders or conditions further includes steps of explantation, implantation, or transplantation therapy. In some respects, it includes stem cell transplantation, preferably HSCT.

[0018] In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need before, during, or after pretreatment for HSCT. In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need prior to pretreatment for HSCT.

[0019] In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need before, during, or after HSCT. In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need before HSCT.

[0020] In another aspect, the present invention relates to pharmaceutical compositions for use in the treatment of iron metabolism disorders or conditions, said pharmaceutical compositions comprising an effective amount of the therapeutic agent as defined in any of the preceding claims, and further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0021] In another aspect, the present invention relates to a method for treating iron metabolism disorders or conditions in a subject in need, the method comprising administering an effective amount of the pharmaceutical composition or therapeutic agent of the present invention, wherein the treatment comprises extracellular implantation, engraftment, or transplantation.

[0022] In another aspect, the present invention relates to the use of the therapeutic agents or pharmaceutical compositions of the present invention in the preparation of medicaments for treating iron metabolism disorders or conditions, wherein said treatment includes extracellular implantation, engraftment, or transplantation. Invention Details

[0024] New treatment option: Inhibiting Matriptase-2 / TMPRSS6

[0025] The inventors have unexpectedly discovered different treatment options to reduce transferrin saturation (which further prevents NTBI, iron overload) and reduce iron-mediated toxicity before or during HSC transplantation and colonization. They have found that inhibitors of the TMPRSS6 gene, inhibitors of the Matriptase-2 (MT2) protein, iron transporter blockers, or hepcidin enhancers can be used for such treatments. MT2 is a protein product of the TMPRSS6 gene and a type II transmembrane serine protease that plays a key role in the regulation of iron homeostasis. MT2 is a negative regulator induced by the peptide hormone hepcidin synthesis. Hepcidin is a peptide hormone primarily produced by the liver and acts as a negative regulator of gastrointestinal iron absorption and release of iron from storage, thereby preventing iron from flowing out of intestinal epithelial cells and macrophages into circulation. Hepcidin regulates iron homeostasis by blocking the cellular release of iron (via the only known cellular iron export protein, iron transporters). Elevated hepcidin levels can induce iron restriction, thereby reducing iron accessibility in the body (McDonald et al., American Journal of Physiology, 2015, Vol. 8, No. 7, C539-C547). TMPRSS6 is primarily expressed in the liver, although high levels of TMPRSS6 mRNA have also been found in the kidneys, lower levels in the uterus, and much lower levels detected in many other tissues (Ramsay et al., Haematologica (2009), 94(*6), 84 849).

[0026] Inhibiting MT2 by reducing the expression of the TMPRSS6 gene appears to be a particularly promising approach. Inhibition of TMPRSS6 expression can be achieved through a variety of methods. One approach is to use repressive nucleic acids such as siRNA or antisense oligonucleotides (ASOs). These are short nucleic acids that inhibit the formation of proteins by targeting the degradation of the mRNA molecules encoding those proteins. Such gene silencing agents are becoming increasingly important for therapeutic applications in medicine. For drug development using these nucleic acids, the following are particularly necessary: ​​they are economically synthesizable, metabolically stable, specifically target tissues, capable of entering cells, and exert their effects within acceptable toxicity limits.

[0027] Double-stranded RNAs (dsRNAs) capable of binding to expressed mRNAs via base complementarity have been shown to block gene expression through an endogenous mechanism known as RNA interference (RNAi) (Fire et al., 1998, Nature. 1998 Feb19;391(6669):806-11 and Elbashir et al., 2001, Nature. 2001 May 24;411(6836):494-8). In many organisms, including vertebrates, short dsRNAs guide gene-specific posttranscriptional silencing and have become a useful tool for studying gene function. RNAi is mediated by RNA-induced silencing complexes (RISCs) (sequence-specific multicomponent nucleases) that degrade target messenger RNAs that are sufficiently complementary or homologous to the silencing trigger strand carried as siRNA duplexes within the RISC complex.

[0028] Inhibition of TMPRSS6 expression in wild-type mice and animal models of hereditary hemochromatosis and β-thalassemia by GalNAc TMPRSS6 siRNA molecule increased serum hepcidin levels and decreased serum iron and transferrin saturation (Altamura et al., Hemasphere. 2019 Dec; 3(6): e301., Vadolas et al., Br. J. Hematology 2021 Jul;194(1):200-210). Specific chemical modifications of GalNAc siRNA compounds can enhance the duration of action of highly active TMPRSS6 siRNA sequences, leading to infrequent dosing (every 3-5 weeks) to induce iron restriction via hepcidin induction (Altamura et al., Hemasphere. 2019 Dec; 3(6):e301., Vadolas et al., Br. J. Hematology 2021 Jul;194(1):200-210).

[0029] Benefits of TMPRSS6 siRNA-mediated therapy

[0030] Compared to all the treatment options listed above, targeting MT2 or TMPRSS6 has advantages.

[0031] TMPRSS6 siRNA versus venous transection:

[0032] Patients undergoing chemotherapy pretreatment and HSC-T preparation may become anemic and dependent on blood transfusions. Venotomy reduces hematocrit and hemoglobin levels by removing red blood cells from circulation, which can worsen anemia. This can be avoided with new treatments that limit iron production through elevated levels of endogenous hepcidin via MT2 inhibitors (such as TMPRSS6 siRNA), leading to a redistribution of iron in the body. This allows for the restriction of serum iron, NTBI, or LPI release during or after pretreatment.

[0033] Venotomy is also associated with side effects related to fluid transfer, including dizziness, nausea, and vasovagal syncope. MT2-based therapies, such as TMPRSS6 siRNA therapy, are unlikely to affect fluid level compartments, and these complications are unlikely to occur with this treatment.

[0034] TMPRSS6 siRNA relative to chelating agents:

[0035] Inducing endogenous hepcidin by inhibiting TMPRSS6 expression via GalNAc siRNA offers a promising therapeutic strategy to reduce toxicity during and after HSCT treatment, enhance colonization, and reduce non-relapse morbidity and mortality.

[0036] Conditions with prognostic elevations in ferritin, transferrin saturation, liver iron, and non-transferrin-bound iron (NTBI), such as β-thalassemia, sickle cell disease, MDS, and leukemia, are currently treated with deficiencies and are often highly toxic to the host, contraindicated for large patient populations, and / or not provided in sufficient quantities to prevent GVHD. Therefore, there is a clear need to reduce the risks associated with HSCT and improve its effectiveness against various conditions. This invention addresses this need. A promising therapeutic strategy using MT2 inhibitors such as GalNAc-conjugated TMPRSS6 siRNA is provided for conditions with elevated ferritin, transferrin saturation, liver iron, and non-transferrin-bound iron.

[0037] The inventors have unexpectedly discovered that reducing iron overload or unstable plasma iron before, during, and after transplantation and colonization is beneficial for patients undergoing HSC transplantation.

[0038] One aspect of the present invention relates to the use of a therapeutic agent comprising or consisting of:

[0039] (a) TMPRSS6 inhibitors;

[0040] (b) MT2 inhibitors; or

[0041] (c) Iron transporter inhibitors.

[0042] In one aspect, the present invention relates to therapeutic agents for use in treating subjects before, during, or after receiving explantation, implantation, or transplantation, wherein said therapeutic agent comprises inhibitors selected from the group consisting of:

[0043] (a) TMPRSS6 inhibitors;

[0044] (b) MT2 inhibitors; or

[0045] (c) Iron transporter inhibitors,

[0046] The extracellular implantation, engraftment, or transplantation may be a stem cell transplantation, and optionally the treatment may further include the co-administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0047] In one aspect, the present invention relates to therapeutic agents used as medicines for reducing systemic iron levels, transferrin saturation, non-transferrin-bound iron (NTBI), and / or unstable plasma iron (LPI) in a subject.

[0048] The subjects described therein exhibited at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload, wherein the therapeutic agent included an inhibitor selected from:

[0049] (a) TMPRSS6 inhibitors;

[0050] (b) MT2 inhibitors; or

[0051] (c) Iron transporter inhibitors,

[0052] The treatment may further include extracellular implantation, engraftment, or transplantation.

[0053] In one aspect, the present invention relates to therapeutic agents for use in treating subjects before, during, or after extracellular implantation, engraftment, or transplantation, wherein the therapeutic agent comprises inhibitors selected from the group consisting of:

[0054] (a) TMPRSS6 inhibitors;

[0055] (b) MT2 inhibitors; or

[0056] (c) Iron transporter inhibitors.

[0057] In one aspect, the present invention relates to therapeutic agents for use in treating or preventing systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload and / or unstable plasma iron (LPI) overload in subjects of need, wherein the therapeutic agent comprises an inhibitor selected from:

[0058] (a) TMPRSS6 inhibitors;

[0059] (b) MT2 inhibitors; or

[0060] (c) Iron transporter inhibitors,

[0061] The treatment may further include steps such as cell explantation, implantation, or transplantation.

[0062] In one aspect, the present invention relates to therapeutic agents for use in treating iron metabolism disorders or conditions, wherein the therapeutic agents comprise inhibitors selected from:

[0063] TMPRSS6 inhibitors;

[0064] MT2 inhibitors; or

[0065] Iron transporter inhibitors,

[0066] The treatment may further include steps of extracellular implantation, engraftment, or transplantation, and optionally may further include co-administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0067] In one aspect, the present invention relates to a therapeutic agent for use in treating iron metabolism disorders or conditions associated with at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload, wherein said therapeutic agent comprises inhibitors selected from:

[0068] TMPRSS6 inhibitors;

[0069] MT2 inhibitors; or

[0070] Iron transporter inhibitors,

[0071] The treatment includes steps such as extracellular implantation, engraftment, or transplantation.

[0072] In one aspect, the present invention relates to therapeutic agents for use in treating diseases or conditions that benefit from reduction in at least one of: systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and unstable plasma iron (LPI), wherein the therapeutic agent comprises an inhibitor selected from the group consisting of:

[0073] (a) TMPRSS6 inhibitors;

[0074] (b) MT2 inhibitors; or

[0075] (c) Iron transporter inhibitors.

[0076] In some respects, iron metabolism disorders or conditions are those that benefit from reduced iron levels (as measured by one or more of systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), or unstable plasma iron (LPI).

[0077] In some respects, iron metabolism disorders or conditions manifest as at least one of systemic iron overload, transferrin saturation, nontransferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload.

[0078] In one aspect of the invention, the agent for reducing measurable iron is selected from the group consisting of:

[0079] (i) An antibody or an antigen-binding fragment thereof, or a variant, fusion or derivative of said antibody or antigen-binding fragment, or a fusion of said variant or derivative thereof;

[0080] (ii) Antibody mimics or analogues (e.g., based on non-antibody scaffolds);

[0081] (iii) RNA aptamers;

[0082] (iv) Small molecules;

[0083] (v) CovX-bodies;

[0084] or

[0085] (vi) Nucleic acid.

[0086] In one aspect of the invention, (i) an antibody or an antigen-binding fragment thereof (or a variant, fusion or derivative of said antibody or antigen-binding fragment), or a fusion of said variant or derivative thereof; and / or (ii) an antibody mimic selected from the group consisting of: affinity molecules, tetraconnectors, adnectin, monomers, anticalin, DARPin, ankylosing spicules, avimer, iMab, microsomes, aptamers, Kunitz domains, affilin, and any combination thereof.

[0087] Several TMPRSS6, MT2, and iron transporter inhibitors that can be used for the purposes of this invention are known in the art. The following are some examples of publications that disclose some of the mentioned inhibitors:

[0088] a) For MT2: Gütschow et al., J. Med. Chem. 2010, 53, 15, 5523–5535; Marsault et al., European Journal of Medicinal Chemistry, Vol. 129, 2017, pp. 110–123; Stirnberg et al., Pharmaceuticals 2018, 11(2), 49, all of which are incorporated herein by reference for the full content they disclose regarding MT2 inhibitors;

[0089] b) For TMPRSS6: WO2018 / 185240A1, WO2014190157A1, WO2016085852A1, WO2022 / 231999A1, WO2016161429A1; all of which are incorporated herein by reference for the full extent of their disclosure regarding TMPRSS6 inhibitors;

[0090] c) For iron transporters: WO2022157185A1, WO2017 / 068089, WO2017 / 068090, WO2018 / 192973, WO2017 / 068089, and WO2017 / 068090; all of which are incorporated herein by reference for the full extent of their disclosure regarding iron transporter inhibitors. Furthermore, hepcidin analogues, such as those acting as iron transporter blockers, are known.

[0091] Those skilled in the art are familiar with TMPRSS6, MT2, and / or iron transporter inhibitors. Any of the TMPRSS6, MT2, and / or iron transporter inhibitors known in the art may be used by those skilled in the art for the purposes of this invention, namely, for the treatment of iron metabolism disorders or conditions exhibiting at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and / or unstable plasma iron (LPI) overload.

[0092] In one aspect of the invention, the therapeutic agent is a nucleic acid, wherein the nucleic acid includes at least one double-stranded region, the double-stranded region including at least a portion of a first strand and at least a portion of a second strand, the at least a portion of the second strand being at least partially complementary to a portion of the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene and is capable of inhibiting the expression of TMPRSS6.

[0093] In some respects, one or more nucleotides on the first and / or second strand are modified to form modified nucleotides.

[0094] In some respects, the nucleic acid is conjugated to the ligand, optionally at the 5' end of the second strand.

[0095] In some respects, the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker that conjugates the GalNAc moieties to nucleic acids.

[0096] In some respects, the connectors are bivalent, trivalent, or tetravalent branched structures.

[0097] In some respects, nucleic acids are stabilized at the 5' and / or 3' ends of either or both strands.

[0098] In some respects, nucleic acids include phosphate thioester bonds or diphosphate thioester bonds between one, two, or three 3' nucleotides and / or 5' nucleotides at the ends of the first and / or second strands.

[0099] In some respects, nucleic acids contain two phosphate thioester bonds between each of the three terminal 3' nucleotides on the first strand and between each of the three terminal 5' nucleotides, and two phosphate thioester bonds between the three terminal nucleotides at the 3' end of the second strand.

[0100] In some respects, unmodified or modified nucleic acids can be selected from Table 1a:

[0101]

[0102] As disclosed in WO2018 / 185240A1. The unmodified or modified nucleic acid can bind to ligands known in the art, such as GN3-TMPRSS6-hcm9 or GN3-TMPRSS6-hc18 disclosed in the same document. Those skilled in the art will understand that any unmodified or modified sequence can bind to any linker known in the art, such as, for example, the GalNAc linker.

[0103] In some respects, nucleic acids can be selected from Table 1b:

[0104]

[0105] As disclosed in WO2018 / 185240A1. Nucleic acids bind to any ligands known in the art. Those skilled in the art will understand that any sequence named above can bind to any linker known in the art, such as, for example, the GalNAc linker, and, for example, those disclosed in the same document under SEQ ID Nos named above (i.e., for example, SEQ ID NOs 286, 288, 290, 292, etc.).

[0106] In some respects, unmodified or modified nucleic acids can be selected from Table 2:

[0107]

[0108] As disclosed in WO2022229150A1. Nucleic acids can be unmodified or modified, and the unmodified or modified nucleic acids can bind to ligands known in the art, as well as those disclosed in the same document, for example. Those skilled in the art will understand that any unmodified or modified sequence can bind to any linker known in the art, such as, for example, the GalNAc linker. Modified sequences of EU400, EU401, and EU402 are disclosed in the same document, which respectively contain the linkers under SEQ ID NO: 2, 4, and 5.

[0109] In some respects, unmodified or modified nucleic acids can be selected from Table 3:

[0110]

[0111]

[0112] As disclosed in WO2022231999A1. Nucleic acids can be unmodified or modified, and said unmodified or modified nucleic acids can bind to ligands known in the art, as well as those disclosed, for example, in the same document. Those skilled in the art will understand that any unmodified or modified sequence can bind to any linker known in the art, such as, for example, the GalNAc linker.

[0113] In some respects, unmodified or modified nucleic acids can be selected from Table 4:

[0114]

[0115] As disclosed in WO2016161429A1. Nucleic acids can be unmodified or modified, and said unmodified or modified nucleic acids can bind to ligands known in the art, as well as those disclosed, for example, in the same document. Those skilled in the art will understand that any unmodified or modified sequence can bind to any linker known in the art, such as, for example, the GalNAc linker.

[0116] The synthesis of (vp)-mU-phos can be performed as described in Prakash, Nucleic Acids Res. 2015, 43(6), 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), 7581-7592. The synthesis of phosphoridamide derivatives of ST23 (ST23-phos) and its analogues can be performed as described in WO2017 / 174657.

[0117] In some respects, the conjugated nucleic acid conjugates with a tri-antennae ligand having one of the following structures:

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Where Z is any nucleic acid as defined in this paper.

[0129] On one hand, the conjugated nucleic acid conjugates with a tri-antennae ligand having the following structure:

[0130]

[0131] Wherein Z is any nucleic acid as defined herein, and wherein the terminal thiophosphate group of the ligand portion is bonded to the 5' position of the 5' terminal nucleotide of the second strand of the nucleic acid (represented by "Z"), or wherein the terminal thiophosphate group of the ligand portion is bonded to the 3' position of the 3' terminal nucleotide of the second strand of the nucleic acid ("Z").

[0132] In some respects, the nucleic acid (represented by "Z") is conjugated to a (triantennae) ligand via a phosphate group or thiophosphate group of the ligand portion, the ligand portion linking the ligand to the 5' position of the 5' end nucleotide of the second strand of the nucleic acid, or the ligand portion linking the ligand to the 3' position of the 3' end nucleotide of the second strand of the nucleic acid.

[0133] Ligands of formula (II), (III), or (IV), or any of the tritenoid ligands disclosed herein, may be attached to the ends of nucleic acids or to nucleotides not at the ends of nucleic acids. In the case of double-stranded nucleic acids, the ligand may be attached to either the 3' end of the first (antisense) strand or either the 3' and / or 5' ends of the second (sense) strand. Nucleic acids may include more than one ligand of formula (II), (III), or (IV), or any of the tritenoid ligands disclosed herein. However, a single ligand of formula (II), (III), or (IV), or any of the tritenoid ligands disclosed herein, is preferred because a single such ligand is sufficient for efficient targeting of nucleic acids to target cells. Preferably, in that case, at least the last two, preferably at least the last three, and more preferably at least the last four nucleotides at the ends of the nucleic acids to which the ligand is attached are linked by phosphodiester bonds.

[0134] In some respects, in the case of double-stranded nucleic acids, the 5' end of the first (antisense) strand is not attached to a ligand of formula (II), (III) or (IV) or any of the trianthal ligands disclosed herein, because a ligand at this position could potentially interfere with the biological activity of the nucleic acid.

[0135] Nucleic acids having a single ligand of formula (II), (III), or (IV) or any of the trianthal ligands disclosed herein at the 5' end of the strand are easier to synthesize and therefore less expensive than the same nucleic acid having the same ligand at the 3' end. Therefore, it is preferable to covalently attach a single ligand of formula (II), (III), or (IV) or any of the trianthal ligands disclosed herein to the 5' end of the nucleic acid strand (conjugated to the 5' end of the nucleic acid strand), and when the nucleic acid is double-stranded, it is preferable to attach it to the 5' end of the second strand.

[0136] In some respects, the nucleic acids in Table 1a or 1b are used for the purposes of this invention. In some respects, the nucleic acids in Table 1b are used for the purposes of this invention.

[0137] In some respects, the nucleic acids in Table 2 are used for the purposes of this invention. Additionally, control molecules may be used, comprising a first strand comprising the modified sequence of SEQ ID NO: 1 in WO2022229150A1 and a second strand as follows:

[0138] [ST23(ps)]3 C4XLT(ps) fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mUfC (ps) mG (ps) fA. The molecule described below will be named EU403.

[0139] In some respects, the nucleic acid is EU401 or EU402. In some respects, the nucleic acid is EU401. In some respects, the nucleic acid is EU402. In some respects, EU401 and EU402 are interchangeable and can be used in combination.

[0140] Specifically, EU400, EU401, EU402 and EU403 are as described in Table 5:

[0141]

[0142] Table 5

[0143] Those skilled in the art will recognize that any known repressive nucleic acid can be modified or unmodified, and can be used alone or in combination with any known ligands, such as those described above, for the purpose of repressing target genes.

[0144] In some respects, the nucleic acid is unmodified, meaning it does not contain any chemical modifications or conjugates as known in the art. In other respects, the nucleic acid is chemically modified to enhance stability or other beneficial properties.

[0145] Nucleic acids can be synthesized or modified using methods well established in the art. Modifications include, for example, terminal modifications such as 5' terminal modifications (phosphorylation, conjugation, reverse bonding) or 3' terminal modifications (conjugation, DNA nucleotides, reverse bonding, etc.); base modifications such as substitution with a stable base, a destabilized base, or a base paired with an expanded repertoire of partners, base removal, or conjugation; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; or backbone modifications, including modification or substitution of phosphodiester bonds. Terminal modifications may be added for a variety of reasons, including modulating activity or resistance to degradation. Terminal modifications that can be used to modulate activity include 5' terminal modification with a phosphate group or a phosphate analog. The nucleic acids of the present invention on the first or second strand may be 5' phosphorylated or contain a phosphoryl analog at the 5' end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P—O-5'); 5'-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5'); 5'-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5'-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5'); 5'-monothiophosphate (thiophosphate; (HO)2(S)P—O-5'); 5' - Mono-dithiophosphates (dithiophosphates; (HO)(HS)(S)P—O-5'), 5'-thiophosphates ((HO)2(O)P—S-5'); any other combination of oxygen / sulfur-substituted monophosphates, diphosphates, and triphosphates (such as 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-aminophosphates ((HO)2(O)P—NH-5', (HO)(NH2)(O)P—O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., such as RP(OH)(O)—O-5'-, (OH)2(O)P-5'-CH2-), 5'-vinylphosphonates, 5'-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., such as RP(OH)(O)—O-5'-). Examples of modifications are given in the aforementioned literature, and further, for example, in WO2018185241.

[0146] Further modifications to nucleic acids involve linking the nucleic acid to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake (e.g., entry into cells) of the nucleic acid. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556). In other embodiments, the ligands are bile acids (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-triphenylmethanethiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), and aliphatic chains such as dodecyl glycol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990). 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-racemic-glycerol or 1,2-di-O-hexadecyl-racemic-glycerol-3-phosphonate triethylammonium (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. AcidsRes., 1990, 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmitic moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylglucosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, like synthetic polyamino acids. Ligands may also include targeting groups that bind to specific cell types (e.g., kidney cells), such as cell or tissue-targeting agents, like lectins, glycoproteins, lipids, or proteins (e.g., antibodies). The targeting group can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide analogs. In some embodiments, the ligand is a polygalactose, such as N-acetyl-galactosamine.

[0147] In some aspects, the conjugate is a carbohydrate conjugate. In some embodiments, the carbohydrate is a monosaccharide. In some embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates are described, for example, in WO2017 / 174657, US2021 / 0017214 and WO2022 / 231999.

[0148] In some aspects, the present invention relates to any nucleic acid, conjugated nucleic acid, nucleic acid for use, method, composition or application according to any disclosure herein, wherein the nucleic acid comprises a vinyl-(E)-phosphonate modification, such as a 5' vinyl-(E)-phosphonate modification. In some aspects, the 5' vinyl-(E)-phosphonate modification is combined with a 2'-F modification at a second position on the first strand. The vinyl-(E)-phosphonate 2'OMe-uracil phosphoramidide can be synthesized according to methods disclosed in the literature (Haraszti et al., Nuc. Acids Res., 45(13), 2017, 7581–7592) and used in oligonucleotide synthesis.

[0149] In some aspects, the nucleic acids of the present invention may include one or more phosphate-thiophosphate modifications at one or more ends of the first and / or second strands. Optionally, each or any end of the first strand may include one, two, or three phosphate-thiophosphate modified nucleotides. Optionally, each or any end of the second strand may include one, two, or three phosphate-thiophosphate modified nucleotides. Optionally, both ends of the first strand and the 5' end of the second strand may include two phosphate-thiophosphate modified nucleotides. A phosphate-thiophosphate modified nucleotide is defined as a nucleotide whose bond to an adjacent nucleotide comprises a phosphate-thiophosphate group rather than a standard phosphate group.

[0150] In some aspects, the present invention relates to any nucleic acid, conjugated nucleic acid, nucleic acid for use, method, composition, or application according to any disclosure herein, wherein the terminal nucleotide at the 3' end of at least one of the first and second strands is a reverse nucleotide and is attached to an adjacent nucleotide via the 3' carbon of said terminal nucleotide, and / or the terminal nucleotide at the 5' end of at least one of the first and second strands is a reverse nucleotide and is attached to an adjacent nucleotide via the 5' carbon of said terminal nucleotide, and / or the terminal nucleotide at the 5' end of at least one of the first and second strands is a reverse nucleotide and is attached to an adjacent nucleotide via the 5' carbon of said terminal nucleotide, and the 5' carbon of said terminal nucleotide, is attached to an adjacent nucleotide.

[0151] Choose one of them

[0152] a. The 3' and / or 5' reverse nucleotides of the first and / or second strands are attached to adjacent nucleotides via phosphodiester bonds through phosphate groups; or

[0153] b. The 3' and / or 5' reverse nucleotides of the first and / or second strands are attached to adjacent nucleotides via phosphate thioses; or

[0154] c. The 3' and / or 5' reverse nucleotides of the first and / or second strands are attached to adjacent nucleotides via dithiophosphate groups.

[0155] Terminal modifications can also be used to monitor distribution, and in this case, the group to be added may include a fluorophore, such as fluorescein or Alexa dye. Terminal modifications can also be used to enhance uptake, and useful modifications include cholesterol. Terminal modifications can also be used to crosslink RNA agents with other moieties. Those skilled in the art will appreciate that the mentioned modifications (such as PS, PS2, and / or VP) can be combined such that the molecule contains at least two of the mentioned modifications, as disclosed in WO2018185241.

[0156] In one aspect of the invention, the therapeutic agent for use in treating iron metabolism disorders or conditions further comprises a pharmaceutically acceptable diluent, carrier, or excipient.

[0157] In some aspects, the therapeutic agent of the present invention is a pharmaceutical composition for use in treating iron metabolism disorders or conditions, said pharmaceutical composition comprising an effective amount of the therapeutic agent of the present invention, and further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0158] In some aspects, the pharmaceutical compositions of the present invention are suitable for delivery via routes selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal routes. In some aspects, the pharmaceutical compositions of the present invention are suitable for delivery via the subcutaneous route.

[0159] In some aspects, the route of delivery is selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal. In some embodiments, the route of delivery is subcutaneous.

[0160] In one aspect of the invention, iron metabolism disorders or conditions are those that would further benefit from cell explantation, implantation, or transplantation therapies such as hematopoietic stem cell transplantation (HSCT).

[0161] In some respects, the treatment of iron metabolism disorders or conditions further includes steps of explantation, implantation, or transplantation therapy. In some respects, it includes stem cell transplantation, preferably HSCT.

[0162] In some respects, the diseases or conditions treated using the compositions and methods of the present invention may be non-malignant diseases or conditions. The non-malignant diseases or conditions are selected from the group consisting of: severe aplastic anemia, hemoglobinopathies such as thalassemia and / or sickle cell disease, aplastic anemia, Fanconi anemia, Wiskott-Aldrich syndrome, Hurlers syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Costerman syndrome, severe immunodeficiency syndrome, severe combined immunodeficiency syndrome, or autoimmune diseases such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteosclerosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic defects, and / or other immunodeficiency or autoimmune diseases.

[0163] In some respects, the diseases or conditions treated using the compositions and methods of the present invention may be malignant diseases or conditions. The malignant diseases or conditions are selected from the group consisting of: myelodysplastic syndromes (MDS), leukemias (such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and other leukemias (such as hairy cell leukemia (HCL), T-cell prolymphoblastic leukemia (T-PLL), large granular lymphoblastic leukemia and adult T-cell leukemia). (Diseases), lymphomas (e.g., precursor T-cell leukemia / lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cell carcinoma, hepatocellular carcinoma, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma (nonspecific type), Hodgkin lymphoma (nodular sclerosis), Hodgkin lymphoma (mixed cell subtype), multiple myeloma, neuroblastoma, Ewing sarcoma, and glioma.

[0164] In some aspects, the object of the present invention is to treat hematologic disorders or hematologic malignancies. In some aspects, the hematologic malignancy is leukemia, such as ALL, AML, or AMoL. In some aspects, the hematologic condition is MDS. In some aspects, the disease or condition is CML, CLL, other leukemias, and lymphoma. In some aspects, the disease or condition to be treated is a hematologic disorder requiring stem cell transplantation (such as allogeneic HSCT), such as ALL, AML, AMoL, or MDS. In some aspects, the disease or condition is AML. In some aspects, the disease or condition is MDS.

[0165] The most common types of hematopoietic stem cell transplantation are derived from bone marrow, peripheral blood, or cord blood. Hematopoietic stem cells (HSCs) are pluripotent or multipotent cells that produce multiple or all types of blood cells, namely myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). It is known that a small number of HSCs can expand to produce a large number of progeny HSCs. This phenomenon is used in hematopoietic stem cell transplantation (HSCT), where a relatively small number of HSCs are used to reconstruct the blood system. HSCTs can be allogeneic (from another individual) or autologous (from the same individual).

[0166] In some aspects of the invention, HSCT is autologous, allogeneic, syngeneic, or heterogeneous, genetically modified, or genetically edited.

[0167] In some respects, HSCT is the first HSCT. In other respects, HSCT is a re-transplantation, such as a re-transplantation after relapse.

[0168] Prior to transplantation, pretreatment for HSCT is performed, typically using methods such as radiation therapy or chemotherapy to disrupt the recipient's immune system. The aim of this step is to remove malignant cell populations from the recipient and reduce the risk of rejection of newly immune HSCs at the cost of partial or complete myeloablation (i.e., destroying the patient's bone marrow's ability to grow new blood cells). The stem cells to be transplanted are then infused into the recipient's bloodstream, where a sufficient number of stem cells find their way to the bone marrow cavity, where they replace the damaged hematopoietic system and restore the recipient's normal blood cell production.

[0169] In one aspect of the invention, the therapeutic agent or pharmaceutical composition of the invention for use in treating a disease or condition further comprises co-administration of one or more chemotherapeutic agents, radiotherapy, and / or immunotherapy.

[0170] In some respects, one or more chemotherapeutic agents may be selected from the group consisting of: busulfan, cyclophosphamide, fludarabine, treosulphane, melphalan, and thiotepa. In some respects, chemotherapy may be a low-intensity pretreatment.

[0171] In some respects, radiotherapy is selected from the following groups: whole-body irradiation, whole-lymphatic irradiation, and whole-bone marrow irradiation.

[0172] In some cases, chemotherapy and radiation therapy can be combined.

[0173] In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need before, during, or after pretreatment for HSCT. In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need prior to pretreatment for HSCT.

[0174] In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need before, during, or after HSCT. In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are administered to subjects in need before HSCT.

[0175] HSCT is a procedure with a number of potential complications, such as infection and graft-versus-host disease (GVHD). However, with improvements in treatment and survival rates, its application has expanded beyond cancer to include conditions such as congenital metabolic disorders and autoimmune diseases. If complications such as GVHD and a range of opportunistic infections can be overcome, transplantation offers the opportunity for cure or long-term remission. Therefore, there is a need for improved transplantation methods that minimize transplant-related risks and complications and provide more efficient transplantation.

[0176] In one aspect of the invention, the therapeutic agent or pharmaceutical composition of the invention improves at least one of the efficacy of HSCT, including improving colonization, improving overall survival, reducing non-relapse mortality, reducing infection and idiopathic pneumonia syndrome, and reducing the incidence of HSC-T-related conditions such as hepatic sinusoidal obstruction syndrome, chronic liver disease, GVHD, and / or GVL.

[0177] In certain aspects of the invention, the therapeutic agents or pharmaceutical compositions of the invention improve the efficacy of HSCT, including improving colonization, improving overall survival, reducing non-relapse mortality, reducing infection and idiopathic pneumonia syndrome, and reducing the incidence of HSC-T-related conditions such as hepatic sinusoidal obstruction syndrome, chronic liver disease, and GVHD.

[0178] In some aspects, the therapeutic agents or pharmaceutical compositions of the present invention are capable of preventing and / or reducing graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL). In some aspects, it is capable of preventing and / or reducing graft-versus-host disease (GVHD).

[0179] The inventors have discovered that, during the perioperative period of transplantation, treatment with TMPRSS6 siRNA to reduce TMPRSS6 gene expression in the liver increases circulating hepcidin levels, reduces circulating iron levels, and decreases the production of non-transferrin-bound iron. By reducing iron-mediated toxicity, this method surprisingly enhances HSC donor cell colonization, limits susceptibility to infection after HSCT, and reduces non-relapse mortality.

[0180] In one respect, the use of a therapeutic agent or pharmaceutical composition for use in treating the mentioned disease or condition includes or comprises the following steps:

[0181] (a) Inhibit TMPRSS6,

[0182] (b) Blocking iron transporters, or

[0183] (c) Add hepcidin,

[0184] Among them, systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels were reduced.

[0185] In one aspect, the present invention relates to a method of treating an iron metabolism disorder or condition in a subject in need, wherein said treatment comprises administering to said subject an effective amount of the therapeutic agent or pharmaceutical composition of the present invention. In some aspects, the subject in need is a subject requiring exoplantation, implantation, or transplantation therapy. In some aspects, an iron metabolism disorder is associated with at least one of systemic iron overload, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload.

[0186] In some respects, the treatment of iron metabolism disorders or conditions is associated with therapies involving cell explantation, implantation, or transplantation (such as HSCT).

[0187] In one aspect, the present invention relates to methods for treating iron metabolism disorders or conditions in subjects in need, wherein the treatment includes therapies involving explantation, implantation or transplantation steps, such as HSCT.

[0188] In one aspect, the present invention relates to the use of therapeutic agents or pharmaceutical compositions according to the present invention in the preparation of medicaments for treating iron metabolism disorders or conditions.

[0189] In some respects, the treatment of iron metabolism disorders or conditions further includes therapies involving explantation, implantation, or transplantation, such as HSCT.

[0190] In one aspect, the present invention relates to the use of therapeutic agents or pharmaceutical compositions according to the invention in the preparation of medicaments for treating iron metabolism disorders or conditions, wherein said treatment includes therapies involving extracellular implantation, engraftment, or transplantation steps, such as HSCT.

[0191] As used herein, the terms “bone marrow transplant” or “stem cell transplant” should be considered interchangeable and refer to the transplantation of stem cells into the recipient. Stem cells do not necessarily have to originate from bone marrow, but can also be derived from other sources such as umbilical cord blood.

[0192] As used herein, the term "iron metabolism disorder or condition" refers to a disorder or condition associated with at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload. Specifically, this refers to a condition in which the subject exhibits at least one of systemic iron overload, transferrin saturation, non-transferrin-bound iron (NTBI) overload, and unstable plasma iron (LPI) overload.

[0193] As used in this article, the term "HSCT" refers to the transplantation of hematopoietic stem cells into a recipient, wherein the stem cells are typically collected from bone marrow, peripheral blood, or umbilical cord blood.

[0194] As used herein, the term "pre-transplant" in the context of administering the therapeutic agents or pharmaceutical compositions of the present invention refers to a period of time several hours, days, or weeks prior to transplantation. Specifically, the therapeutic agents or pharmaceutical compositions of the present invention are administered before transplant pretreatment or several weeks to several months (e.g., 1-10 weeks or 1-4 months) prior to transplantation. The therapeutic agents or pharmaceutical compositions may be administered continuously during this period, or administered once or at a few times. In some aspects of the invention, the therapeutic agents or pharmaceutical compositions are administered in a single dose. The therapeutic agents or pharmaceutical compositions should be administered pre-transplant at an effective dose, at multiple times, and for a sufficient duration to achieve a sufficient reduction in the desired systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and / or unstable plasma iron (LPI). Such administration regimens are readily implemented by those skilled in the art who have access to the therapeutic agents or pharmaceutical compositions.

[0195] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is an animal, preferably a mammal, such as nonprimates (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys (e.g., rhesus monkeys, cynomolgus monkeys, or chimpanzees) and humans). In some aspects of the invention, the subject is a human.

[0196] As used herein, the term "recipient" in the context of transplantation refers to the subject receiving the transplant, as opposed to "donor," which is the subject from whom the transplanted material (cells) originates. In allogeneic cases, the recipient and donor are different individuals; in autologous cases, the recipient and donor are the same individual. In syngenetic cases, the donor and recipient are different individuals but share the same genes. In xenogeneic cases, the donor comes from a different species than the recipient.

[0197] As used herein, the terms “administering” or “administration” refer to the delivery of the therapeutic agent or pharmaceutical composition of the present invention by any suitable method known in the art.

[0198] As used herein, the term "effective amount" refers to an amount of therapy (e.g., a therapeutic agent) sufficient to reduce and / or alleviate the severity and / or duration of a disease or condition or its symptoms, prevent the progression of said disease or condition, induce remission, prevent recurrence, development, or onset of one or more symptoms associated with said disease or condition, or enhance or improve the preventive or therapeutic effect of other therapies (e.g., other prophylactic or therapeutic agents). Therefore, the effective amount of the therapeutic agent or pharmaceutical composition of the present invention used before, during, or after HSCT is an amount sufficient to improve HSCT efficacy, including improving colonization, improving overall survival, reducing non-relapse mortality, reducing infection and idiopathic pneumonia syndrome, and reducing HSC-T-related morbidity such as hepatic sinusoidal obstruction syndrome, chronic liver disease, GVHD, and / or GVL, in a manner sufficient to result in a reduction of at least one of systemic iron levels, transferrin saturation (Tsat), non-transferrin-bound iron (NTBI), and unstable plasma iron (LPI).

[0199] As used herein, “agent” is any purified or isolated natural or chemically synthesized entity comprising one or more molecules.

[0200] In the methods and uses for preparing the therapeutic agents or pharmaceutical compositions of the present invention, a therapeutically effective amount of the active ingredient is provided. The therapeutically effective amount can be determined by a person skilled in medicine or veterinary medicine based on patient characteristics (such as age, weight, sex, condition, complications, other diseases, etc.), as is well known in the art.

[0201] Abbreviation Summary Table - Table 6

[0202]

[0203]

[0204]

[0205]

[0206] Preferred, non-limiting embodiments embodying certain aspects of the present invention are described with reference to the following drawings: Attached Figure Description

[0207] Figure 1 illustrates the effects of different chemotherapy pretreatment regimens, combined with siRNA (e.g., EU401), on TMPRSS6 target gene expression, HAMP mRNA induction, and systemic iron levels on day +2. The total doses of busulfan and fludarabine administered at low, intermediate, or high doses of chemotherapy are depicted. The low-dose regimen consisted of 0.8 mg / kg fludarabine administered intraperitoneally (ip) daily from day -6 to day -2, and 3.2 mg / kg busulfan administered ip daily on days -4 and -3. The intermediate-dose regimen consisted of 10 mg / kg fludarabine administered ip daily from day -6 to day -2, and 10 mg / kg busulfan administered ip daily on days -4 and -3. The high-dose regimen consisted of 10 mg / kg fludarabine administered ip daily from day -6 to day -2, and 16.25 mg / kg busulfan administered ip twice daily on days -4 and -3.

[0208] Figure 2. EU401 treatment reduced TMPRSS6 mRNA and increased HAMP mRNA in the livers of mice exposed to different doses of chemotherapy pretreatment (low and high doses, see Figure 1). Mean + / - SD.

[0209] Figure 3. EU401 treatment eliminated and attenuated the induction of serum iron, transferrin saturation, and NTBI in mice pretreated with medium and high doses of chemotherapy, respectively. Mean + / - SEM.

[0210] Figure 4. The effect of TMPRSS6 siRNA treatment on bone marrow transplantation was evaluated under two different experimental conditions: A) Mice were pretreated with a medium dose of chemotherapy after receiving EU403 or EU402 siRNA at 5 mg / kg on days -28 and -7. B) Mice were pretreated with a high dose of chemotherapy after receiving EU403 or EU402 siRNA at 5 mg / kg on days -42, -21, and -1. BM donor cells were transplanted on day 0, and chimerism was assessed as a measure of colonization in peripheral blood at 4, 6, and 8 weeks post-transplantation, and in tissues at 8 weeks post-transplantation.

[0211] Figure 5 illustrates the effect of TMPRSS6 siRNA treatment on post-transplant blood chimerism under two different experimental conditions, as shown in Figure 4. CD45.1 levels at each blood collection time point are displayed. +Percentage of donor-derived blood cells. Mice receiving medium-dose pretreatment and EU402 treatment showed better blood chimerism at 4 and 8 weeks post-transplantation (A), while mice receiving high-dose pretreatment and EU402 treatment showed better blood chimerism at 4, 6, and 8 weeks post-transplantation (B). Data are presented as mean + / - SEM.

[0212] Figure 6 illustrates the effect of TMPRSS6 siRNA treatment on post-transplantation blood myeloid and lymphoid colonization under two different experimental conditions. CD45.1 levels at each blood collection time point are shown. + Donor source CD11b + CD3 - CD19 - Myeloid and CD11b - CD3 + CD19 + Percentage of lymphoid cells. Mice receiving medium-dose pretreatment and EU402 treatment showed better blood myeloid and lymphoid chimerism at 4 and 8 weeks post-transplantation (A), while mice receiving high-dose pretreatment and EU402 treatment showed better blood myeloid and lymphoid chimerism at 4, 6, and 8 weeks post-transplantation (B). Data are presented as mean + / - SEM.

[0213] Figure 7 illustrates the effect of TMPRSS6 siRNA treatment on post-transplantation myeloid colonization under two different experimental conditions. It shows the CD45.1 concentration at each blood collection time point. + Donor source CD11b + Ly6G - L6C + / - Monocytes and CD11b + Ly6G + Percentage of neutrophils. Mice receiving medium-dose pretreatment and EU402 treatment showed better blood myeloid chimerism at 4 and 8 weeks post-transplantation (A), while mice receiving high-dose pretreatment and EU402 treatment showed better blood myeloid chimerism at 4, 6, and 8 weeks post-transplantation (B). Data are presented as mean + / - SEM.

[0214] Figure 8 illustrates the effect of TMPRSS6 siRNA treatment on post-transplantation blood lymphoid colonization under two different experimental conditions. It shows the CD45.1 concentration at each blood collection time point. + Donor source CD11b - CD19 + B cells and CD11b - CD3 +Percentage of T cells. Mice receiving medium-dose pretreatment and EU402 treatment showed better hematolymphoid chimerism at 4 and 8 weeks post-transplantation (A), while mice receiving high-dose pretreatment and EU402 treatment showed better hematolymphoid chimerism at 4, 6, and 8 weeks post-transplantation (B). Data are presented as mean + / - SEM.

[0215] Figure 9 illustrates the effect of TMPRSS6 siRNA treatment on bone marrow and spleen chimerism under two different experimental conditions. CD45.1 levels in the bone marrow and spleen are shown at 8 weeks post-transplantation. + Percentage of donor-derived cells. Mice receiving medium-dose (A) and high-dose (B) pretreatment and EU402 treatment showed better bone marrow and spleen chimerism at 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0216] Figure 10 illustrates the effect of TMPRSS6 siRNA treatment on post-transplantation bone marrow myeloid and lymphoid colonization under two different experimental conditions: CD45.1 in the bone marrow is shown at 8 weeks post-transplantation. + Donor source CD11b + Myeloid and CD11b - CD19 + CD3 + Percentage of lymphoid cells. Mice pretreated with medium (A) and high (B) doses and treated with EU402 showed better myeloid and lymphoid chimerism in the bone marrow at 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0217] Figure 11 illustrates the effect of TMPRSS6 siRNA treatment on multi-lineage colonization of bone marrow after transplantation under two different experimental conditions. CD45.1 levels in the bone marrow are shown at 8 weeks post-transplantation. + Donor source CD11b + Ly6G - Ly6C + / - Monocytes, CD11b + Ly6G + Neutrophils, CD11b - CD19 + B cells and CD11b - CD3 + Percentage of T cells. Mice pretreated with medium (A) and high (B) doses and treated with EU402 showed better bone marrow multilineage chimerism at 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0218] Figures 12 and 4 illustrate the effect of TMPRSS6 siRNA treatment on multilineage colonization of the spleen after transplantation under two different experimental conditions. CD45.1 levels in the spleen are shown at 8 weeks post-transplantation. + Donor source CD11b + Ly6G - Ly6C + / - Monocytes, CD11b + Ly6G + Neutrophils, CD11b - CD19 + B cells and CD11b - CD3 + Percentage of T cells. Mice receiving medium-dose (A) and high-dose (B) pretreatment and EU402 treatment showed better multilineage chimerism in the spleen at 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0219] Figures 13 and 4 illustrate the effect of TMPRSS6 siRNA treatment on the number of hematopoietic stem cells and progenitor cells (HSPCs) under two different experimental conditions. Bone marrow Lin was shown at 8 weeks post-transplantation. - Sca + cKit + (LSK) HSPC accounts for a total of Lin - Percentage of cells. The percentage of HSPCs remained unchanged 8 weeks post-transplantation in mice pretreated with medium (A) and high (B) doses, and in mice treated with EU402 relative to EU403. Data are presented as mean + / - SEM.

[0220] Figure 14 illustrates the effect of TMPRSS6 siRNA treatment on bone marrow hematopoietic stem cell and progenitor cell (HSPC) chimerism under two different experimental conditions. CD45.1 levels in the bone marrow are shown at 8 weeks post-transplantation. + donor source Lin - Sca + cKit + (LSK) HSPC and Lin - Sca1 - cKit + Percentage of myeloid progenitor cells (MPs). Mice pretreated with medium (A) and high (B) doses and treated with EU402 showed better chimerism in LSK HSPCs and MPs at 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0221] Figure 15. Experiments were established to test the effect of TMPRSS6 siRNA treatment on bone marrow transplantation outcomes in an MDS mouse model. NUP98-HOXD13 mice were treated with 5 mg / kg EU400 or EU401 and pretreated with a moderate dose of chemotherapy on days -35, -14, and 7. BM donor cells were transplanted on day 0, and chimerism was assessed in peripheral blood at 3, 8, and 12 weeks post-transplantation, and in tissues at 12 weeks post-transplantation as a measure of colonization.

[0222] Figure 16. Effect of TMPRSS6 siRNA treatment on blood colonization after transplantation in MDS mice pretreated with a medium dose. The CD45.1 concentration at each blood collection time point is shown. + Donor-derived blood cells (A) and CD45.1 + Donor source CD11b + Ly6G - Ly6C + / - Monocytes and CD11b + Ly6G + Percentage of neutrophils. MDS mice receiving moderate pretreatment and EU401 treatment showed better chimerism in blood monocytes and neutrophils at 3 and 8 weeks post-transplantation (B). Data are presented as + / - SEM.

[0223] Figure 17. Effects of TMPRSS6 siRNA treatment on bone marrow and spleen colonization in MDS mice after transplantation, pretreated with a moderate dose. CD45.1 levels are shown at 12 weeks post-transplantation. + Donor-derived bone marrow and spleen cells (A) and CD45.1 + Donor source CD11b + Myeloid cells and CD11b - CD19 + CD3 + Percentage of lymphocytes. Bone marrow and spleen donor chimerism in MDS mice treated with a moderate dose of pretreatment and EU401 showed an increasing trend. Data are presented as mean + / - SEM.

[0224] Figure 18. EU401 treatment reduced the expression of inflammatory cytokines in bone marrow macrophages and neutrophils of transplanted MDS mice. Bone marrow samples were collected 12 weeks post-transplantation. A) CD11b levels in bone marrow of transplanted MDS mice treated with EU400 or EU401 at 12 weeks post-transplantation. low F4 / 80 + Macrophages (A) and CD11b + Ly6G +Expression of inflammatory cytokines IL1β, TNF-α, and IFN-γ in neutrophils (B). Cytokines expressed by each cell population are expressed as fold changes relative to EU400-treated transplanted MDS mice. Mean + / - SEM.

[0225] Figure 19. Treatment of mouse hepatocytes with EU401 reduced TMPRSS6 mRNA levels to a level similar to that of EU402. Values ​​obtained for TMPRSS6 mRNA were normalized to values ​​generated for the housekeeping gene ACTIN and correlated with the mean of untreated samples (ut) set to 1-fold target gene expression. Each bar represents the mean + / - SD of three biological replicates. Mean + / - SD.

[0226] Figure 20. Design of the experiment to test the effect of TMPRSS6 siRNA treatment on bone marrow transplantation outcomes in an MDS mouse model. NUP98-HOXD13 mice were subcutaneously treated with 5 mg / kg EU403 or EU402 on days -60, -30, and -1, and they received either low-dose or high-dose chemotherapy pretreatment. The total doses of busulfan and fludarabine administered in either A) low-dose or B) high-dose chemotherapy are depicted. The low-dose consisted of 10 mg / kg fludarabine administered intraperitoneally (ip) daily from day -6 to day -2, and 2.5 mg / kg busulfan administered ip twice daily on days -4 and -3. The high-dose consisted of 10 mg / kg fludarabine administered ip daily from day -6 to day -2, and 16.25 mg / kg busulfan administered ip twice daily on days -4 and -3. BM donor cells were transplanted on day 0, and chimerism was assessed in peripheral blood at 4, 6, and 8 weeks post-transplantation, and in tissues at 8 weeks post-transplantation as a measure of colonization.

[0227] Figure 21 illustrates the effect of TMPRSS6 siRNA treatment on blood chimerism in MDS mice after transplantation under two different experimental conditions. The CD45.1 concentration at each blood collection time point is shown. + Percentage of donor-derived blood cells. Mice receiving low-dose pretreatment and EU402 treatment (A) and mice receiving high-dose pretreatment and EU402 treatment (B) showed better blood chimerism at 4, 6, and 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0228] Figure 22 and Figure 4 illustrate the effects of TMPRSS6 siRNA treatment on post-transplantation blood myeloid and lymphoid colonization under two different experimental conditions. CD45.1 levels at each blood collection time point are shown. + Donor source CD11b +Myeloid cells and CD11b - Percentage of lymphoid cells. Mice receiving medium-dose pretreatment and EU402 treatment showed better blood myeloid and lymphoid chimerism at 4 and 8 weeks post-transplantation (A), and mice receiving high-dose pretreatment and EU402 treatment showed better myeloid and lymphoid chimerism at 4, 6, and 8 weeks post-transplantation (B). Data are presented as mean + / - SEM.

[0229] Figure 23 illustrates the effect of TMPRSS6 siRNA treatment on blood myeloid colonization in MDS mice under two different experimental conditions. It shows the CD45.1 concentration at each blood collection time point. + Donor source CD11b + Ly6G - L6C + / - Monocytes and CD11b + Ly6G + Percentage of neutrophils. Mice receiving low-dose and EU402 treatment (A) and high-dose pretreatment and EU402 treatment (B) showed better blood myeloid chimerism at 6 and 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0230] Figure 24 illustrates the effect of TMPRSS6 siRNA treatment on blood lymphoid colonization in MDS mice under two different experimental conditions. The CD45.1 concentration at each blood collection time point is shown. + Donor source CD11b - CD19 + B cells and CD11b - CD3 + Percentage of T cells. Mice receiving low-dose pretreatment and EU402 treatment (A) and high-dose pretreatment and EU402 treatment (B) showed better blood T-lymphoid chimerism at 4, 6, and 8 weeks post-transplantation. Data are presented as mean + / - SEM.

[0231] Figure 25 illustrates the effect of TMPRSS6 siRNA treatment on bone marrow chimerism after transplantation under two different experimental conditions. At 8 weeks post-transplantation, MDS mice treated with EU402 showed better bone marrow chimerism under either or both of low-dose (A, C, E) or high-dose (B, D, F) pretreatment. Data are presented as mean + / - SEM. Total CD45.1 in bone marrow is shown in A) and B). + Percentage of donor-derived cells and CD45.1 in bone marrow. + Donor source CD11b + Myeloid and CD11b - CD19 + CD3 +Percentage of lymphoid cells. C) and D) show donor-derived CD45.1 in bone marrow at 8 weeks post-transplantation. + CD11b + Ly6G - Ly6C + / - Monocytes, CD11b + Ly6G + Neutrophils, CD11b - CD19 + B cells and CD11b - CD3 + The percentage of T cells. E) and F) show CD45.1 levels in the bone marrow at 8 weeks post-transplantation. + donor source Lin - Sca + cKit + (LSK)HSPC and Lin - Sca1 - cKit + Percentage of myeloid progenitor cells (MP). Example

[0232] Example 1 - In a rodent model of chemotherapy pretreatment and stem cell transplantation, using TMPRSS6 siRNA treatment inhibits TMPRSS6 expression and increases hepcidin levels.

[0233] This example demonstrates the inhibition of TMPRSS6 expression and the induction of hepcidin expression by EU401 in mice pretreated with chemotherapy.

[0234] As described above and illustrated in Figure 1, wild-type mice were treated with either low-dose or high-dose chemotherapy pretreatment (days -6 to -2). Prior to pretreatment, mice received a single dose of 5 mg / kg EU400 or EU401 subcutaneously on day -14, or two doses of 5 mg / kg EU400 or 5 mg / kg EU401 via the same route on days -28 and -7. Liver tissue samples were collected on day +2 for total RNA extraction and target gene expression assessment by qRT-PCR. Treatment with a single dose of EU401 (5 mg / kg) on ​​day -14 reduced TMPRSS6 mRNA expression in the livers of mice pretreated with high-dose chemotherapy (Figure 2A). Similarly, two doses of EU401 reduced TMPRSS6 expression in mice pretreated with either low-dose or high-dose chemotherapy (Figures 2B and 2C). Two doses of EU401 also effectively induced Hamp expression in the livers of mice pretreated with either low-dose or high-dose chemotherapy (Figures 1D and 1E). Compared with untreated mice, high-dose pretreatment led to a significant increase in hepatic HAMP expression, and treatment with EU401 further increased this expression. The siRNA molecules used in this study are described in Table 5. The experimental setup is shown in Figure 1, and the results are shown in Figure 2.

[0235] Example 2 - In a rodent model of chemotherapy pretreatment and stem cell transplantation, TMPRSS6 siRNA was detected at... The treatment reduced serum iron, transferrin saturation and NTBI.

[0236] This example demonstrates that EU401 treatment reduced the increases in serum iron levels, transferrin saturation (Tsat), and nontransferrin-bound iron (NTBI) induced by medium- or high-dose chemotherapy pretreatment.

[0237] Wild-type mice were either untreated (UT) or administered 5 mg / kg EU401 subcutaneously on days -28 and -7, or subsequently pretreated with medium or high doses of chemotherapy (days -6 to -2), as depicted in Figure 1. Blood samples were collected on day +2 for assessment of serum iron, transferrin saturation (Tsat), and non-transferrin-bound iron (NTBI). Treatment with EU401 alone reduced serum iron levels and transferrin saturation in untreated wild-type mice. In mice pretreated with medium or high doses of chemotherapy, treatment with siRNA EU401 reduced serum iron levels, Tsat, and NTBI compared to mice pretreated with the non-targeted siRNA control EU400 (Figure 3). NTBI was completely suppressed in mice pretreated with medium doses of chemotherapy and partially suppressed in mice pretreated with high doses of chemotherapy. The siRNA molecules used in this study are depicted in Table 5. The experimental setup is shown in Figure 1, and the results are shown in Figure 3.

[0238] Example 3 - Effect of TMPRSS6 siRNA treatment on bone marrow transplantation outcomes in bone marrow-suppressed wild-type mice

[0239] This example demonstrates that treatment of recipient mice with TMPRSS6 siRNA EU401 enhances bone marrow transplantation colonization and donor chimerism.

[0240] To investigate the effect of TMPRSS6 siRNA treatment on bone marrow transplantation outcomes, we used a previously described chemotherapy-pretreated mouse model (see Figure 1) and performed bone marrow cell transplantation (treatment protocols are shown in Figures 4A and B). CD45.2 + Wild-type recipient mice were first treated with medium- or high-dose chemotherapy, and then received CD45.1 via intravenous injection into the tail vein on day 0. + Wild-type donors One donor cell.

[0241] The effect of TMPRSS6 inhibition on colonization was assessed by treating mice with TMPRSS6 siRNA EU402 and comparing it with treatment with the non-targeted control siRNA EU403. Mice pretreated with intermediate-dose chemotherapy were treated with 5 mg / kg siRNA on days -28 and -7, while mice pretreated with high-dose chemotherapy received 5 mg / kg siRNA on days -42, -21, and -1. CD45.1 levels in blood samples collected at 4, 6, and 8 weeks post-transplantation were assessed using flow cytometry with appropriate markers. + Chimerism, as a measure of colonization. Colonization was higher in mice that had received high-dose chemotherapy compared to those pretreated with a medium dose. Treatment with TMPRSS6 siRNA EU402 enhanced donor cell colonization after both medium and high-dose pretreatment. The siRNA molecules used in this study are depicted in Table 5. The experimental setup is depicted in Figure 4, and the results are shown in Figures 5 through 14.

[0242] Example 4 - Results of bone marrow transplantation in a mouse model of myelodysplastic syndrome after treatment with TMPRSS6 siRNA Impact

[0243] This example demonstrates that treatment with TMPRSS6 siRNA enhances colonization and donor chimerism in a mouse model of myelodysplastic syndrome. The effect of TMPRSS6 siRNA treatment on post-transplant colonization was also tested in NUP98-HOXD13 transgenic mice (a mouse model of myelodysplastic syndrome (MDS)) (Lin et al., Blood 2005 Jul 1; 106(1):287-295). MDS mice were treated as depicted in Figure 15: they were treated subcutaneously with 5 mg / kg EU401 or EU400 on days -35, -14, and 7, and pretreated with a moderate dose of chemotherapy. On day 0, cells derived from CD45.1...+ donor mice One bone marrow cell was transplanted into CD45.2. + Blood samples were collected from MDS mice at weeks 3, 8, and 12.

[0244] This example demonstrates that EU401 treatment improves bone marrow transplantation outcomes in MDS mice pretreated with a moderate dose. This effect is reflected in CD45.1 levels at 3 and 8 weeks post-transplantation. + The study observed an increasing trend in chimerism in both the donor-derived blood population and the myeloid population. Similarly, at the end of the study (week 12 post-transplantation), the proportion of donor-derived cells in the bone marrow and spleen of MDS mice treated with EU401 showed an increasing trend compared to those treated with EU400.

[0245] The siRNA molecules used in this study are depicted in Table 5. The experimental setup is shown in Figure 15, and the results are shown in Figures 16 and 17.

[0246] Example 5 – Reducing Inflammatory Cytokines in Bone Marrow via TMPRSS6

[0247] This embodiment shows that in an animal model of myelodysplastic syndrome, treatment with TMPRSS6 siRNA reduced the expression of inflammatory cytokines after transplantation.

[0248] The effects of TMPRSS6siRNA treatment on the production of inflammatory cytokines after transplantation were evaluated in NUP98-HOXD13 transgenic mice (a mouse model of myelodysplastic syndrome) (Blood 2005 Jul1; 106(1): 287-295).

[0249] MDS mice were treated as depicted in Figure 15 below: they were treated subcutaneously with 5 mg / kg EU401 or EU400 on days -35, -14, and -7, and pretreated with a moderate dose of chemotherapy. On day 0, [the treatment was initiated from CD45.1]. + donor mice One bone marrow cell was transplanted into CD45.2. + Blood samples were collected from MDS mice at weeks 3, 8, and 12 post-transplantation.

[0250] Bone marrow CD11b was assessed by flow cytometry. low F4 / 80 + Macrophages and CD11b + Ly6G + Cytokine expression in neutrophils. The siRNA molecules used in this study are depicted in Table 5. The experimental setup is shown in Figure 15, and the results are shown in Figure 18.

[0251] Example 6 - Reduction of TMPRSS6 mRNA levels in primary hepatocytes

[0252] This example demonstrates that, following receptor-mediated uptake, EU401 and EU402 dose-dependently reduce TMPRSS6 mRNA levels in primary hepatocytes.

[0253] Primary mouse hepatocytes were seeded at a density of 25,000 cells per well in 96-well plates. Cells were then incubated with the TMPRSS6 siRNA conjugate in cell culture medium at the indicated concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.41 nM, and 0.14 nM. The following day, cells were lysed for RNA extraction, and TMPRSS6 and Actin mRNA levels were determined by TaqMan qRT-PCR. Values ​​obtained for TMPRSS6 mRNA were normalized to values ​​generated for the housekeeping gene Actin and correlated with the mean of untreated samples (ut) set at 1-fold target gene expression. Each bar represents the mean + / - SD of three biological replicates. The siRNA conjugates used in this study are listed in Table 5. The experimental setup is shown in Figure 15, and the results are shown in Figure 19.

[0254] Example 7 - Results of bone marrow transplantation in a mouse model of myelodysplastic syndrome treated with TMPRSS6 siRNA Impact

[0255] This example demonstrates that treatment with TMPRSS6 siRNA enhances colonization and donor chimerism in a mouse model of myelodysplastic syndrome (MDS) pretreated with low- and high-dose chemotherapy. The effect of TMPRSS6 siRNA treatment on post-transplantation colonization was tested in NUP98-HOXD13 transgenic mice (a mouse model of MDS) (Lin et al., Blood 2005 Jul1; 106(1): 287-295). MDS mice were treated as depicted in Figure 20 below. They were treated with 5 mg / kg EU403 or EU402 subcutaneously on days -60, -30, and -1, and pretreated with low- or high-dose chemotherapy, as depicted in Figure 20 below. On day 0, the siRNA from CD45.1 was used to treat the mice. + donor mice One bone marrow cell was transplanted into CD45.2. + In MDS mice, blood samples were collected at weeks 4, 6, and 8 post-transplantation. CD45.1 was assessed in blood samples collected at 4, 6, and 8 weeks post-transplantation using appropriate markers by flow cytometry. +Chimerism was used as a measure of colonization. As expected, mice that had received high-dose chemotherapy had higher colonization rates compared to those pretreated with low doses. Treatment with TMPRSS6siRNA EU402 enhanced donor cell colonization following both low- and high-dose pretreatment. The siRNA molecules used in this study are depicted in Table 1. The experimental setup is depicted in Figure 20. The results are shown in Figures 21–25.

[0256] statement

[0257] 1. A therapeutic agent for use in the treatment of iron metabolism disorders or conditions, wherein the therapeutic agent comprises inhibitors selected from:

[0258] (g) TMPRSS6 inhibitors;

[0259] (h) MT2 inhibitors; or

[0260] (i) Iron transporter inhibitors,

[0261] The treatments described therein include cell implantation, explantation, or transplantation.

[0262] 2. The therapeutic agent for use according to Statement 1, wherein the therapeutic agent is selected from:

[0263] (i) An antibody or an antigen-binding fragment thereof, or a variant, fusion or derivative of said antibody or antigen-binding fragment, or a fusion of said variant or derivative thereof;

[0264] (ii) Antibody mimics (e.g., based on non-antibody scaffolds); (hepcidin analogs) selected from the group consisting of: affinis, tetraconnector, adnectin (monomer), anticalin, DARPin (anchorin), avimer, iMab, microsomes, peptide aptamers, Kunitz domains and affilin.

[0265] (iii) RNA aptamers;

[0266] (iv) Small molecules;

[0267] (v) CovX-body; or

[0268] (vi) Nucleic acid;

[0269] (i) or (ii) can be selected from the group consisting of: affinity molecules, tetraconnectors, adnectin (monomers), anticalin, DARPin (anchorin), avimer, iMab, microsomes, peptide aptamers, Kunitz domains, and affilin, and any combination thereof.

[0270] 3. The therapeutic agent for use according to Statement 2, wherein the nucleic acid comprises at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand, wherein at least a portion of the second strand is at least partially complementary to a portion of the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene and is capable of inhibiting the expression of TMPRSS6.

[0271] 4. A therapeutic agent for use according to any one of claims 2 or 3, wherein one or more nucleotides on the first and / or second strand are modified to form modified nucleotides.

[0272] 5. A therapeutic agent for use according to any one of statements 2 to 4, wherein the nucleic acid is conjugated to a ligand, optionally at the 5' end of the second strand.

[0273] 6. The therapeutic agent for use according to Statement 5, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker wherein the linker conjugates the GalNAc moieties to the nucleic acid.

[0274] 7. A therapeutic agent for use according to any one of claims 5 or 6, wherein the linker is a divalent, trivalent, or tetravalent branched structure.

[0275] 8. A therapeutic agent for use according to any one of statements 2 to 7, wherein the nucleic acid is any one of those mentioned in Tables 1, 2, 3, 4 or 5.

[0276] 9. A therapeutic agent for use according to any one of statements 1 to 8, wherein the nucleic acid is EU401 or EU402.

[0277] 10. A pharmaceutical composition for use in the treatment of iron metabolism disorders or conditions, said pharmaceutical composition comprising an effective amount of a therapeutic agent as defined in any of the foregoing statements, further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0278] 11. The pharmaceutical composition for use according to Statement 10 is suitable for delivery via a route selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal routes.

[0279] 12. The pharmaceutical composition for use according to any one of statements 10 to 11, wherein the route of delivery is selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal.

[0280] 13. The therapeutic agent or pharmaceutical composition for use according to any one of the foregoing statements, wherein the disease or condition is:

[0281] a) Non-malignant, wherein the non-malignant disease or condition is selected from the group consisting of: severe aplastic anemia, hemoglobinopathies such as thalassemia and / or sickle cell disease, aplastic anemia, Fanconi anemia, Wiskott-Aldrich syndrome, Hurlers syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Costman syndrome, severe immunodeficiency syndrome, severe combined immunodeficiency syndrome, or autoimmune diseases such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteosclerosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic defects, and / or other immunodeficiency or autoimmune diseases;

[0282] or

[0283] b) The disease is malignant, and the malignant disease or condition is selected from the following groups: myelodysplastic syndromes (MDS), leukemias (such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and other leukemias (such as hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell prolymphocytic leukemia). Cellular leukemia), lymphoma (e.g., precursor T-cell leukemia / lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cell carcinoma, hepatocellular carcinoma, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma (nonspecific type), Hodgkin lymphoma (nodular sclerosis), Hodgkin lymphoma (mixed cell subtype), multiple myeloma, neuroblastoma, Ewing sarcoma, and glioma.

[0284] 14. A therapeutic agent or pharmaceutical composition for use according to any one of the foregoing statements, wherein the treatment comprises or consists of the following steps:

[0285] (a) Inhibit TMPRSS6,

[0286] (b) Inhibit MT2,

[0287] (c) Blocking iron transporters, or

[0288] (d) Increase hepcidin,

[0289] Among them, systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels were reduced.

[0290] 15. The therapeutic agent or pharmaceutical composition for use according to any one of the foregoing statements, wherein the treatment further comprises co-administration of one or more chemotherapeutic agents, radiotherapy and / or immunotherapy.

[0291] 16. A therapeutic agent or pharmaceutical composition for use according to any one of the foregoing statements, wherein the extracellular implantation, engraftment, or transplantation therapy includes hematopoietic stem cell transplantation (HSCT), optionally wherein the therapeutic agent or pharmaceutical composition is administered to the subject in need before, during, or after pretreatment for HSCT.

[0292] or

[0293] The agent is administered to the subject in need before, during, or after HSCT.

[0294] 17. The therapeutic agent or pharmaceutical composition for use as described in Statement 16, wherein the agent is capable of preventing and / or reducing graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL), and optionally further improving HSCT efficacy, including improving colonization, improving overall survival, reducing non-relapse mortality, reducing infection and idiopathic pneumonia syndrome, and / or reducing HSC-T-related morbidity, such as hepatic sinusoidal obstruction syndrome and chronic liver disease.

[0295] 18. A method of treating an iron metabolism disorder or condition in a subject of need, the method comprising administering an effective amount of a pharmaceutical composition or therapeutic agent as defined in any of the preceding statements, wherein the treatment includes cell implantation, explantation, or transplantation.

[0296] 19. The method according to statement 18, wherein the agent is selected from:

[0297] (i) An antibody or an antigen-binding fragment thereof, or a variant, fusion or derivative of said antibody or antigen-binding fragment, or a fusion of said variant or derivative thereof;

[0298] (ii) Antibody mimics (e.g., based on non-antibody scaffolds); (hepcidin analogs) selected from the group consisting of: affinities, tetraconnectors, adnectin (monomers), anticalin, DARPin (anchorin), avimer, iMab, microbodies, peptide aptamers, Kunitz domains, and affilin.

[0299] (iii) RNA aptamers;

[0300] (iv) Small molecules;

[0301] (v) CovX-body; or

[0302] (vi) Nucleic acid;

[0303] (i) or (ii) can be selected from the group consisting of: affinity molecules, tetraconnectors, adnectin (monomers), anticalin, DARPin (anchorin), avimer, iMab, microsomes, peptide aptamers, Kunitz domains, and affilin, and any combination thereof.

[0304] 20. The method according to Statement 19, wherein the nucleic acid comprises at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand, wherein at least a portion of the second strand is at least partially complementary to a portion of the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene and is capable of inhibiting the expression of TMPRSS6.

[0305] 21. The method according to statement 20, wherein one or more nucleotides on the first and / or second strand are modified to form modified nucleotides.

[0306] 22. The method according to statement 20, wherein the nucleic acid is conjugated to the ligand, optionally at the 5' end of the second strand.

[0307] 23. The method according to statement 22, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker wherein the linker conjugates the GalNAc moieties to nucleic acids.

[0308] 24. The method according to statement 23, wherein the connector is a divalent, trivalent, or tetravalent branch structure.

[0309] 25. The method according to statement 20, wherein the nucleic acid is any one of those mentioned in Tables 1, 2, 3, 4 or 5, preferably wherein the nucleic acid is EU401 or EU402.

[0310] 26. The method according to Statement 19, wherein the pharmaceutical composition comprises an effective amount of a therapeutic agent as defined in any of the preceding statements, further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0311] 27. The method according to statement 26, wherein the pharmaceutical composition is suitable for delivery via a route selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal routes.

[0312] 28. The method according to statement 26, wherein the delivery route of the pharmaceutical composition is selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal.

[0313] 29. The method described in Statement 18, wherein the disease or condition is:

[0314] a) Non-malignant, wherein the non-malignant disease or condition is selected from the group consisting of: severe aplastic anemia, hemoglobinopathies such as thalassemia and / or sickle cell disease, aplastic anemia, Fanconi anemia, Wiskott-Aldrich syndrome, Hurlers syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Costman syndrome, severe immunodeficiency syndrome, severe combined immunodeficiency syndrome, or autoimmune diseases such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteosclerosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic defects, and / or other immunodeficiency or autoimmune diseases;

[0315] or

[0316] b) The disease is malignant, and the malignant disease or condition is selected from the following groups: myelodysplastic syndromes (MDS), leukemias (such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and other leukemias (such as hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell prolymphocytic leukemia). Cellular leukemia), lymphoma (e.g., precursor T-cell leukemia / lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cell carcinoma, hepatocellular carcinoma, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma (nonspecific type), Hodgkin lymphoma (nodular sclerosis), Hodgkin lymphoma (mixed cell subtype), multiple myeloma, neuroblastoma, Ewing sarcoma, and glioma.

[0317] 30. The method according to Statement 18, wherein the treatment comprises or consists of the following steps:

[0318] (a) Inhibit TMPRSS6,

[0319] (b) Inhibit MT2,

[0320] (c) Blocking iron transporters, or

[0321] (d) Increase hepcidin,

[0322] Among them, systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels were reduced.

[0323] 31. The method according to Statement 18, wherein the treatment further comprises co-administration of one or more chemotherapeutic agents, radiotherapy and / or immunotherapy.

[0324] 32. The method according to Statement 18, wherein the explantation or transplantation therapy includes hematopoietic stem cell transplantation (HSCT), optionally wherein the therapeutic agent or pharmaceutical composition is administered to the subject in need before, during, or after pretreatment for HSCT.

[0325] or

[0326] The agent is administered to the subject in need before, during, or after HSCT.

[0327] 33. The method according to statement 32, wherein the agent is capable of preventing and / or reducing graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL), and optionally further improving HSCT efficacy, including improving colonization, improving overall survival, reducing non-relapse mortality, reducing infection and idiopathic pneumonia syndrome, and / or reducing HSC-T-related morbidity, such as hepatic sinusoidal obstruction syndrome and chronic liver disease.

[0328] 34. Use of the therapeutic agent or pharmaceutical composition according to any one of the foregoing statements in the preparation of a medicament for treating iron metabolism disorders or conditions, wherein said treatment includes explantation or transplantation.

[0329] 35. The use as described in statement 34, wherein the agent is selected from:

[0330] (i) An antibody or an antigen-binding fragment thereof, or a variant, fusion or derivative of said antibody or antigen-binding fragment, or a fusion of said variant or derivative thereof;

[0331] (ii) Antibody mimics (e.g., based on non-antibody scaffolds); (hepcidin analogs) selected from the group consisting of: affinity molecules, tetraconnectors, adnectin (monomers), anticalin, DARPin (anchorin), avimer, iMab, microsomes, peptide aptamers, Kunitz domains, and affilin.

[0332] (iii) RNA aptamers;

[0333] (iv) Small molecules;

[0334] (v) CovX-body; or

[0335] (vi) Nucleic acid;

[0336] (i) or (ii) can be selected from the group consisting of: affinity molecules, tetraconnectors, adnectin (monomers), anticalin, DARPin (anchorin), avimer, iMab, microsomes, aptamers, Kunitz domains, and affilin, and any combination thereof.

[0337] 36. The use as described in statement 35, wherein the nucleic acid comprises at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand, wherein at least a portion of the second strand is at least partially complementary to a portion of the first strand, wherein the first strand is at least partially complementary to at least a portion of RNA transcribed from the TMPRSS6 gene and is capable of inhibiting the expression of TMPRSS6.

[0338] 37. In accordance with the use described in statement 36, one or more nucleotides on the first and / or second strand are modified to form modified nucleotides.

[0339] 38. The use as described in statement 36, wherein the nucleic acid is conjugated to the ligand, optionally at the 5' end of the second strand.

[0340] 39. The use as described in statement 38, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker wherein the linker conjugates the GalNAc moieties to nucleic acids.

[0341] 40. The use as described in statement 39, wherein the connector is a divalent, trivalent, or tetravalent branch structure.

[0342] 41. The use as described in Statement 36, wherein the nucleic acid is any one of those mentioned in Tables 1, 2, 3, 4 or 5, preferably wherein the nucleic acid is EU401 or EU402.

[0343] 42. The use as described in Statement 36, wherein the pharmaceutical composition comprises an effective amount of the therapeutic agent as defined in any of the preceding statements, further comprising a pharmaceutically acceptable diluent, carrier, or excipient.

[0344] 43. The use as described in Statement 36, wherein the pharmaceutical composition is suitable for delivery via a route selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal routes.

[0345] 44. The use according to statement 36, wherein the delivery route of the pharmaceutical composition is selected from the group consisting of intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular, and intrathecal.

[0346] 45. According to the use described in Statement 36, the disease or condition described is:

[0347] a) Non-malignant, wherein the non-malignant disease or condition is selected from the group consisting of: severe aplastic anemia, hemoglobinopathies such as thalassemia and / or sickle cell disease, aplastic anemia, Fanconi anemia, Wiskott-Aldrich syndrome, Hurlers syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Costman syndrome, severe immunodeficiency syndrome, severe combined immunodeficiency syndrome, or autoimmune diseases such as SLE, multiple sclerosis, IBD, Crohn's disease, Sjögren's syndrome, vasculitis, lupus, myasthenia gravis, Wegener's disease, malignant infantile osteosclerosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic defects, and / or other immunodeficiency or autoimmune diseases;

[0348] or

[0349] b) The disease is malignant, and the malignant disease or condition is selected from the following groups: myelodysplastic syndromes (MDS), leukemias (such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and other leukemias (such as hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell prolymphocytic leukemia). Cellular leukemia), lymphoma (e.g., precursor T-cell leukemia / lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., renal cell carcinoma, hepatocellular carcinoma, and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma (nonspecific type), Hodgkin lymphoma (nodular sclerosis), Hodgkin lymphoma (mixed cell subtype), multiple myeloma, neuroblastoma, Ewing sarcoma, and glioma.

[0350] 46. ​​The use as described in statement 36, wherein the treatment comprises or consists of the following steps:

[0351] (a) Inhibit TMPRSS6,

[0352] (b) Inhibit MT2,

[0353] (c) Blocking iron transporters, or

[0354] (d) Increase hepcidin,

[0355] Among them, systemic iron levels, NTBI, transferrin saturation, unstable plasma iron, and / or eLPI levels were reduced.

[0356] 47. The use as described in Statement 36, wherein the treatment further comprises co-administration of one or more chemotherapeutic agents, radiotherapy and / or immunotherapy.

[0357] 48. The use as described in Statement 36, wherein the explantation or transplantation therapy includes hematopoietic stem cell transplantation (HSCT), optionally wherein the therapeutic agent or pharmaceutical composition is administered to the subject in need before, during, or after pretreatment for HSCT.

[0358] or

[0359] The agent is administered to the subject in need before, during, or after HSCT.

[0360] 49. The use as described in Statement 36, wherein the agent is capable of preventing and / or reducing graft-versus-host disease (GVHD) and / or graft-versus-leukemia (GVL), and optionally further improving HSCT efficacy, including improving colonization, improving overall survival, reducing non-relapse mortality, reducing infection and idiopathic pneumonia syndrome, and / or reducing HSC-T-related morbidity, such as hepatic sinusoidal obstruction syndrome and chronic liver disease.

Claims

1. A therapeutic agent for use in treating an iron metabolism disease or condition, wherein the therapeutic agent comprises an inhibitor selected from the group consisting of: (g) a TMPRSS6 inhibitor; (h) a MT2 inhibitor; or (i) an ferroportin inhibitor, and wherein the treatment comprises cell implantation, explantation or transplantation.

2. The therapeutic agent for use according to claim 1, wherein the agent is selected from the group consisting of: (i) an antibody or antigen binding fragment thereof, or a variant, fusion or derivative of the antibody or antigen binding fragment, or a fusion of the variant or derivative thereof; (ii) an antibody mimetic (e.g. based on a non-antibody scaffold); (a hepcidin analogue) selected from the group consisting of an affibody, a tetranectin, an adnectin (monobody), an anticalin, a DARPin (ankyrin), an avimer, an iMab, a microbody, a peptide aptamer, a Kunitz domain and an affilin; (iii) an RNA aptamer; (iv) a small molecule; (v) a CovX-Body; or (vi) a nucleic acid; wherein (i) or (ii) can be selected from the group consisting of an affibody, a tetranectin, an adnectin, a monobody, an anticalin, a DARPin, an ankyrin, an avimer, an iMab, a microbody, a peptide aptamer, a Kunitz domain, an affilin and any combination thereof.

3. The therapeutic agent for use according to claim 2, wherein the nucleic acid comprises at least one double-stranded region comprising at least a portion of a first strand and at least a portion of a second strand, the at least a portion of the second strand being at least partially complementary to the portion of the first strand, wherein the first strand is at least partially complementary to at least a portion of an RNA transcribed from a TMPRSS6 gene and is capable of inhibiting expression of TMPRSS6.

4. The therapeutic agent for use according to any one of claims 2 or 3, wherein one or more nucleotides on the first strand and / or the second strand are modified to form a modified nucleotide.

5. The therapeutic agent for use according to any one of claims 2 to 4, wherein the nucleic acid is conjugated to a ligand, optionally at the 5' end of the second strand.

6. The therapeutic agent for use according to claim 5, wherein the ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moiety to the nucleic acid.

7. The therapeutic agent for use according to any one of claims 5 or 6, wherein the linker is a bivalent, trivalent or tetravalent branched structure.

8. The therapeutic agent for use according to any one of claims 2 to 7, wherein the nucleic acid is any one mentioned in Tables 1, 2, 3, 4 or 5.

9. The therapeutic agent for use according to any one of claims 1 to 8, wherein the nucleic acid is EU401 or EU402.

10. A pharmaceutical composition for use in treating an iron metabolism disease or condition, comprising an effective amount of a therapeutic agent as defined in any one of the preceding claims, further comprising a pharmaceutically acceptable diluent, carrier or excipient.

11. The pharmaceutical composition for use of claim 10, which is suitable for delivery by a route selected from the group comprising: intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular and intrathecal.

12. The pharmaceutical composition for use of any one of claims 10 to 11, wherein the route of delivery is selected from the group comprising: intravenous, intramuscular, subcutaneous, intra-articular, pulmonary, intranasal, intraocular and intrathecal.

13. The therapeutic agent for use or pharmaceutical composition for use of any one of the preceding claims, wherein the disease or condition is: a) non-malignant, wherein the non-malignant disease or condition is selected from the group consisting of: severe aplastic anemia, hemoglobinopathies such as thalassemia and / or sickle cell disease, aplastic anemia, Fanconi anemia, Wiskott Aldrich syndrome, Hurlers syndrome, familial hemophagocytic lymphohistiocytosis (FHL), chronic granulomatous disease (CGD), Kostmann syndrome, severe combined immunodeficiency syndrome, or an autoimmune disorder such as SLE, multiple sclerosis, IBD, Crohn’s disease, Sjogren’s syndrome, vasculitis, lupus, myasthenia gravis, Wegener’s disease, malignant infantile osteopetrosis, mucopolysaccharidosis, paroxysmal nocturnal hemoglobinuria, pyruvate kinase deficiency, congenital metabolic defects, and / or other immunodeficiency or autoimmune disease; or b) wherein the disease is malignant, wherein the malignant disease or condition is selected from the group consisting of: myelodysplastic syndrome (MDS), leukemia (such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute monocytic leukemia (AMoL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and other leukemias such as hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia and adult T-cell leukemia), lymphoma (e.g., precursor T-cell leukemia / lymphoma, Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, MALT lymphoma), solid tumors (e.g., kidney cancer, liver cancer and pancreatic cancer), mycosis fungoides, peripheral T-cell lymphoma not otherwise specified, Hodgkin’s lymphoma nodular sclerosis type, Hodgkin’s lymphoma mixed cellularity subtype, multiple myeloma, neuroblastoma, Ewing’s sarcoma and glioma.

14. The therapeutic agent for use or pharmaceutical composition for use of any one of the preceding claims, wherein the treatment comprises or consists of: (a) inhibiting TMPRSS6, (b) inhibiting MT2, (c) blocking a ferroportin, or (d) increasing hepcidin, wherein systemic iron levels, NTBI, transferrin saturation, labile plasma iron and / or eLPI levels are reduced.

15. The therapeutic agent for use or the pharmaceutical composition for use according to any one of the preceding claims, wherein the treatment further comprises co-administration of one or more chemotherapeutic agents, radiotherapy and / or immunotherapy.

16. The therapeutic agent for use or the pharmaceutical composition for use according to any one of the preceding claims, wherein the extracorporeal, implant or transplant treatment comprises hematopoietic stem cell transplantation (HSCT), optionally wherein the therapeutic agent or pharmaceutical composition is administered to a subject in need thereof prior to, during or after conditioning for HSCT, or wherein the agent is administered to a subject in need thereof prior to, during or after receiving HSCT.

17. The therapeutic agent for use or the pharmaceutical composition for use according to claim 16, wherein the agent is capable of preventing and / or reducing graft versus host disease (GVHD) and / or graft versus leukemia (GVL), and optionally further improving HSCT efficacy, including improving engraftment, improving overall survival, reducing non-relapse mortality, reducing infections and idiopathic pneumonia syndrome, and / or reducing HSC-T associated morbidity, such as sinusoidal obstruction syndrome and chronic liver disease.

18. A method of treating an iron metabolism disease or condition in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition or therapeutic agent as defined in any one of the preceding claims, wherein the treatment comprises extracorporeal, implant or transplant.

19. Use of a therapeutic agent or pharmaceutical composition according to any one of the preceding claims for the manufacture of a medicament for the treatment of an iron metabolism disease or condition, wherein the treatment comprises extracorporeal, implant or transplant.

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