Treatment for X-linked sideroblastic anemia

Abstract

The present invention provides: a pharmaceutical composition for treating X-linked sideroblastic anemia, the composition comprising a demethylating agent, in particular, 5−azacitidine or a derivative thereof; and normal erythroid cells that are differentiated and induced from iPS cells derived from a patient with X-linked sideroblastic anemia in the presence of a demethylating agent.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for X-linked sideroblastic anemia. This application claims priority to Japanese Patent Application No. 2020-218557, filed on December 28, 2020, and incorporates the content of the Japanese patent application into this application.

Background Art

[0002] X-linked sideroblastic anemia is a hereditary disease that causes severe erythroid differentiation disorders due to mutations in the gene for the enzyme ALAS2 (erythroid 5-aminolevulinate synthase), which catalyzes the first step of heme synthesis. Vitamin B6 administration is effective in about half of patients with X-linked sideroblastic anemia, but for about half of the patients for whom vitamin B6 administration is ineffective, there is no effective treatment other than blood transfusion and hematopoietic stem cell transplantation (Non-Patent Documents 1 and 2), and the development of alternative treatment methods is expected.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0004] There is a need to provide an effective treatment for X-linked sideroblastic anemia, particularly for patients who are unresponsive to vitamin B6 administration. [Means for Solving the Problems]

[0005] The present inventors have conducted extensive research to solve the above problems. The present inventors have found that by using δ-aminolevulinic acid, the erythroid differentiation ability of erythroblasts derived from iPS cells in which mutant ALAS2 is activated can be improved, but there is a problem that a large amount of δ-aminolevulinic acid is required. Therefore, the present inventors searched for a more effective drug in a smaller amount and found that a demethylating agent, particularly 5-azacytidine and its derivatives, can improve the above erythroid differentiation in a smaller amount, leading to the completion of the present invention. In the following part of this specification, 5-azacytidine is abbreviated as AZA.

[0006] That is, the present invention provides the following: (1) A pharmaceutical composition for treating X-linked sideroblastic anemia, comprising a demethylating agent. (2) The demethylating agent is of formula (I): [Chemical Formula] [In the formula, R 2 and R 2 are each independently hydrogen, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halogen, OH, OC 1-6 alkyl, OC 1-6 alkenyl, OC 1-6 alkynyl, OCOC 1-6 alkyl, OCOC 1-6 alkenyl, or COC 1-6 alkynyl, and R 3 is hydrogen, C 1-6 alkyl, C 1-6Alkenil, C 1-6 Alkinyl, COC 1-6 Alkyl, COC 1-6 Alkenil, COC 1-6 It is an alkynyl or phosphate group, and A is CR 4 or CR 5 R 6 And R 4 is hydrogen, C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 It is an alkynyl, OH, or halogen, R 5 and R 6 Each is independently hydrogen and C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkynyl, OH, or halogen, where Z is N, NR 7 CR 8 , or CR 9 R 10 And R 7 is hydrogen, OH, C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkynyl or halogen, R 8 is hydrogen, OH, C 1-6 Alkyl, C 1-6 Alkenil, C 1-6 Alkynyl, or halogen, R 9 and R 10 These are hydrogen, OH, and C, respectively, independently. 1-6 Alkyl, C 1-6 Alkenil, C 1-6 It is an alkynyl or halogen, where X is hydrogen or NR 11 R 12 And R 11 and R 12 Each is independently hydrogen and C 1-6 Alkyl, C 1-6 Alkenyl, or C 1-6 The alkyl, alkenyl, and alkynyl groups may be substituted with one or more hydroxyl groups or halogens. -- represents a single bond or a double bond. The pharmaceutical composition described in (1) is a compound indicated by ]. (3) The pharmaceutical composition according to (2), wherein the demethylating agent is AZA or 5-aza-2'-deoxycytidine. (4) The pharmaceutical composition according to (3), wherein the demethylating agent is AZA. (5) A pharmaceutical composition according to any one of (1) to (4) for use in patients with X-linked sideroblastic anemia in whom vitamin B6 administration is ineffective. (6) A method for producing normal red blood cells from iPS cells derived from a patient with X-linked sideroblastic anemia, comprising inducing differentiation of the iPS cells into red blood cells, wherein a demethylating agent is added to the cells during differentiation induction. (7) The demethylating agent is of formula (I): [ka] [In the formula, R 1 -R 12 , A, Z, X, and -- The same definition as in (2). The method described in (6), wherein the compound is represented by ]. (8) The method of (7), wherein the demethylating agent is AZA or 5-aza-2'-deoxycytidine. (9) The method according to (8), wherein the demethylating agent is AZA. (10) The patient is a patient with X-linked sideroblastic anemia for which vitamin B6 administration is ineffective, by any method described in (6) to (9). (11) Use of demethylating agents to treat X-linked sideroblastic anemia. (12) The demethylating agent is of formula (I): [ka] [In the formula, R 1 -R 12 , A, Z, X, and -- The definition is the same as in (2). The compound is indicated by ], as used in (11). (13) Use according to (12), wherein the demethylating agent is AZA or 5-aza-2'-deoxycytidine. (14) Use as described in (13), wherein the demethylating agent is AZA. (15) Use as described in any of (11) to (14) if the patient to be treated is a patient with X-linked sideroblastic anemia for whom vitamin B6 administration is ineffective. (16) A demethylating agent for use in the treatment of X-linked sideroblastic anemia. (17) Equation (I): [ka] [In the formula, R 1 -R 12 , A, Z, X, and -- The definition is the same as in (2). The demethylating agent described in (16) is a compound indicated by ]. (18) The demethylating agent according to (17), which is AZA or 5-aza-2'-deoxycytidine. (19) A demethylating agent as described in (18), which is AZA. (20) A demethylating agent according to any of (16) to (19), wherein the patient to be treated is a patient with X-linked sideroblastic anemia for whom vitamin B6 administration is ineffective. (21) Use of demethylating agents in the manufacture of pharmaceuticals for the treatment of X-linked sideroblastic anemia. (22) The demethylating agent is of formula (I): [ka] [In the formula, R 1 -R 12 , A, Z, X, and -- The definition is the same as in (2). The compound is indicated by ], as used in (21). (23) Use according to (22), wherein the demethylating agent is AZA or 5-aza-2'-deoxycytidine. (24) Use as described in (23), wherein the demethylating agent is AZA. (25) Use as described in any of (21) to (24) if the patient to be treated is a patient with X-linked sideroblastic anemia for whom vitamin B6 administration is ineffective. (26) A method for treating X-linked sideroblastic anemia, comprising administering a demethylating agent to a patient requiring treatment for X-linked sideroblastic anemia. (27) The demethylating agent is of formula (I): [ka] [In the formula, R 1 -R 12 , A, Z, X, and -- The method described in (26), wherein the compound is as defined in (2). (28) The method according to (27), wherein the demethylating agent is AZA or 5-aza-2'-deoxycytidine. (29) The method according to (28), wherein the demethylating agent is AZA. (30) The patient is a patient with X-linked sideroblastic anemia for which vitamin B6 administration is ineffective, by any method described in (26) to (29). A pharmaceutical composition for treating X-linked sideroblastic anemia, comprising normal red blood cells obtained by any of the methods described in (31)(6) to (10). Use of normal red blood cells obtained by any of the methods described in (32)(6) to (10) for the treatment of X-linked sideroblastic anemia. (33) Normal red blood cells obtained by any of the methods described in (6) to (10) for use in the treatment of X-linked sideroblastic anemia. Use of normal red blood cells obtained by any of the methods described in (34)(6) to (10) in the manufacture of a pharmaceutical product for the treatment of X-linked sideroblastic anemia. A method for treating X-linked sideroblastic anemia, comprising administering normal red blood cells obtained by any of the methods described in (35)(6) to (10) to a patient requiring treatment for X-linked sideroblastic anemia. [Effects of the Invention]

[0007] The present invention provides an effective treatment for X-linked sideroblastic anemia, particularly for patients for whom vitamin B6 administration is ineffective, and more preferably for female patients. In particular, AZA, one of the active ingredients of the therapeutic agent of the present invention, is currently used as a treatment for myelodysplastic syndrome, and its safety and pharmacokinetic studies in humans have been completed, so there are few concerns about its use. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1A shows pellets and iron-stained images of erythroblasts (day 34) derived from WT1-iPSC2 (indicated as "WT iPSC" in the figure) and MT1-iPSC3 (indicated as "MT iPSC" in the figure). Arrows indicate ring sideroblasts. The scale bar is 10 μm. Figure 1B shows May Grunwald-Giemsa-stained images of erythroblasts (day 34) derived from WT1-iPSC2 (indicated as "WT iPSC" in the figure) (top) and erythroblasts (day 34) derived from MT1-iPSC4 (indicated as "MT iPSC" in the figure) (bottom). The scale bar is 25 μm. Figure 1C is a graph showing the proportions of polychromatic megakaryocytes (Poly-M), polychromatic erythroblasts (Poly-E), and orthochromatic erythroblasts (Ortho-E) derived from three WT-iPSC strains and four MT-iPSC strains. Figure 1D shows o-dianisidine stained pellets (top) and o-dianisidine stained images (bottom) of erythroblasts (day 34) derived from WT1-iPSC3 (indicated as "WT iPSC" in the figure) and erythroblasts (day 34) derived from MT1-iPSC3 (indicated as "MT iPSC" in the figure). The scale bar is 200 μm. [Figure 2] Figure 2A is a graph showing the percentage of CD43+ cells obtained from WT-iPSCs and MT-iPSCs from different patients. Figure 2B is a graph showing the percentage of CD235a+ cells obtained from WT-iPSCs and MT-iPSCs from different patients. The above data are expressed as mean ± standard error of sample mean (SEM). P-values ​​were calculated using an unpaired two-sided Student's t-test. **** P<0.0001; NS No significant difference. Figure 2C is a graph showing the percentage of CD235a+ cells obtained from two WT-iPSC strains (WT3-iPSC1, WT3-iPSC2) and two MT-iPSC strains (MT3-iPSC1, MT3-iPSC2) from SA3. Figure 2D is a flow cytometry image showing the co-expression of CD235 and CD71 in erythroblasts differentiated using a feeder cell-free method. [Figure 3A]Figure 3A is a scheme showing the protocol for erythrocyte differentiation from iPSCs and the time points at which each sample was collected. [Figure 3B] Figure 3B shows a heatmap indicating the distance between samples and a hierarchical clustering of iPSCs. [Figure 3C] Figure 3C shows the PCA results for erythroblasts derived from three WT-iPSC strains and four MT-iPSC strains, as well as bone marrow erythroblasts from a healthy donor and SA3. [Figure 3D] Figure 3D shows the results of GO analysis, which reveals concentrated molecular functional terms for WT erythroblasts (left) and MT erythroblasts (right). [Figure 3E] Figure 3E shows the GSEA results for a heme metabolism dataset enriched in WT erythroblasts. [Figure 3F] Figure 3F is a heatmap of 13 genes that are characteristically expressed during the orthochromatic stage of erythroblasts, as explained in Figure 3C. [Figure 4] Figure 4A shows the scheme of AZA administration in erythrocyte differentiation. Figure 4B shows the percentage of CD235a+ erythroblasts treated with DMSO or AZA. Data are expressed as mean ± SEM. P values ​​were calculated using one-way ANOVA and Tukey's correction, and unpaired two-sided Student's t-test. ** P < 0.01; NS No significant difference. Figure 4C shows representative flow cytometry data of erythroblasts derived from MT1-iPSC2 differentiated without feeder cells after administration of DMSO or AZA. The upper panel of Figure 4D shows unstained (left) and stained (right) pellets of erythroblasts derived from MT1-iPSC2 treated with DMSO or AZA. The middle and lower panels of Figure 4D show o-dianisidine stained images of erythroblasts derived from WT1-iPSC1 (left) and MT1-iPSC2 (right) treated with DMSO or AZA. [Figure 5] Figure 5 shows the results of ALAS2 mutation analysis by Sanger sequencing. DMSO represents the DMSO-administered group, and AZA represents the AZA 500nM-administered group. [Figure 6] The right panel of Figure 6 shows a graph illustrating the effect of AZA on improving the erythrocyte differentiation ability of iPS cell-derived erythroblasts with activated mutant ALAS2. The left panel of Figure 6 shows the results of a similar experiment conducted on iPS cell-derived erythroblasts with activated mutant ALAS2. The vertical axis of the graph represents the percentage of CD235a-positive cells. [Figure 7] Figure 7 is a graph showing the effect of AZA on increasing the number of erythrocyte colonies from iPS cell-derived erythroblasts with activated mutant ALAS2. The upper panel shows the proportion of colonies of each cell cycle and erythrocytes relative to the total number of colonies. The proportion of erythrocyte colonies is indicated by the dark area at the top of each bar. The lower panel shows the number of erythrocyte colonies in each experimental system. [Figure 8] Figure 8A shows the results of colony formation assays at day 15 of differentiation of hematopoietic progenitor cells derived from WT1-iPSC1 and MT1-iPSC2 treated with DMSO or AZA. Figure 8B shows the number of erythrocytes and mixed colonies in Figure 8A. The above data are expressed as mean ± SEM. P values ​​were calculated using one-way ANOVA, Tukey's correction, and unpaired two-sided Student's t-test. * P < 0.05; NS no significant difference. Figure 8C is a representative image of mixed colonies derived from AZA-treated MT1-iPSC2. Scale bar is 200 μm. [Figure 9] Figure 9 shows the results of a colony formation assay at day 15 of differentiation of hematopoietic progenitor cells derived from WT1-iPSC1 and WT1-iPSC3 treated with DMSO or AZA (left), and the results of a colony formation assay at day 15 of differentiation of hematopoietic progenitor cells derived from MT1-iPSC1 and MT1-iPSC2 treated with DMSO or AZA (right). [Modes for carrying out the invention]

[0009] In one embodiment, the present invention provides a pharmaceutical composition for treating X-linked sideroblastic anemia, comprising a demethylating agent.

[0010] In the present invention, a demethylating agent refers to a drug that inhibits DNA methylation.

[0011] In the present invention, the preferred demethylating agent is an AZA derivative. Specifically, the AZA derivative is the compound represented by formula (I). Note that the compound of formula (I) may include its structural isomers and stereoisomers.

[0012] Among the compounds represented by formula (I), R 1 , R 2 Compounds where both are OH groups, or R 1 , R 2 Compounds in which one of the atoms is hydrogen and the other is OH are preferred. Among the compounds represented by formula (I), R 3 Compounds in which hydrogen is present are preferred. Among the compounds represented by formula (I), compounds in which A is CH or CH2, Z is N, NH, CH, or C-halogen, and X is hydrogen or NH2 are preferred.

[0013] C in equation (I) 1-6 Alkyl, C 1-6 Alkenyl and C 1-6 Alkynyl refers to alkyl, alkenyl, and alkynyl molecules, respectively, that have 1 to 6 carbon atoms. 1-6 Alkyl, C 1-6 Alkenyl and C 1-6 Alkynnyls include structural isomers. C in formula (I) 1-6 Alkyl, C 1-6 Alkenyl and C 1-6 The alkynyl may be unsubstituted or substituted with one or more hydroxyl groups or halogens. The halogen is F, Cl, Br, or I, preferably F or Cl.

[0014] Examples of compounds represented by formula (I) include, but are not limited to, AZA, 5-aza-2'-deoxycytidine, fazarabine, decitabine, 5-fluoro-2'-deoxycytidine, 5,6-dihydro-5-azacitidine (DHAC), and zebralin.

[0015] In the present invention, preferred compounds include, but are not limited to, AZA and 5-aza-2'-deoxycytidine. In the present invention, a more preferred compound is AZA.

[0016] In this specification, treatment of X-linked sideroblastic anemia means alleviating, reducing, improving, or eliminating anemia-related symptoms in patients, such as palpitations, dizziness, and fatigue, and preventing heart failure, liver damage, and other conditions caused by the anemia. In this specification, treatment of X-linked sideroblastic anemia includes the prevention of X-linked sideroblastic anemia.

[0017] The therapeutic effect of the pharmaceutical composition of the present invention is exerted by the removal of methylation at the site corresponding to the mutation in the ALAS2 gene by a demethylating agent, resulting in the expression of the wild-type sequence in addition to the mutant sequence. For example, in the cDNA of ALAS2, the therapeutic effect of the pharmaceutical composition of the present invention is exerted by the removal of methylation at the site corresponding to the R227C mutation (where the arginine at position 227 from the N-terminus of the ALAS2 protein is replaced with cysteine), resulting in the expression of T (wild-type) in addition to C (mutant) at a portion of the 679th base. In particular, since efficacy has been demonstrated for the R227C mutation, which is one of the ALAS2 gene mutations in patients with X-linked sideroblastic anemia in whom vitamin B6 is ineffective, the pharmaceutical composition of the present invention is considered effective in treating patients with X-linked sideroblastic anemia, preferably female patients, in whom all vitamin B6 administrations, including the R227C mutation, are ineffective.

[0018] Ineffectiveness of vitamin B6 administration includes cases where vitamin B6 is completely ineffective, as well as cases where it is partially effective but insufficient to treat the condition.

[0019] Known methods can be used to confirm the efficacy of the pharmaceutical composition of the present invention. For example, efficacy may be confirmed by taking a patient interview and / or by observing the red blood cell count, MCV, MCH, MCHC values, morphological observation of red blood cells (microscopic observation), color observation of red blood cells, morphological observation of bone marrow, confirmation of bias in X chromosome inactivation, and the expression ratio of mutant and wild-type ALAS2 by red blood cell cDNA.

[0020] The pharmaceutical compositions of the present invention typically contain pharmaceutically acceptable carriers or excipients.

[0021] The pharmaceutical composition of the present invention may be administered via any route. Preferably, the pharmaceutical composition of the present invention is administered by subcutaneous injection, intravenous injection, intravenous drip infusion, or orally.

[0022] The pharmaceutical composition of the present invention can be manufactured by known methods depending on the route of administration. The pharmaceutical composition of the present invention, which is an injectable or intravenous preparation, may be manufactured by mixing and dissolving the active ingredient (e.g., AZA) in a known carrier such as water for injection, physiological saline, glucose solution, or Ringer's solution. The pharmaceutical composition of the present invention, which is an orally administered preparation, may be manufactured by mixing it with a known solid, semi-solid, or liquid carrier. Examples of dosage forms of the pharmaceutical composition of the present invention, which is an orally administered preparation, include, but are not limited to, tablets, capsules, powders, granules, tablets, gels, syrups, and drinks. The pharmaceutical composition of the present invention may appropriately contain, for example, buffering components for pH adjustment, fragrances, sweeteners, etc.

[0023] The dosage of compounds such as AZA in the pharmaceutical composition of the present invention can be determined by a physician, taking into consideration the type of compound to be administered, the severity of the patient's symptoms, age, weight, health condition, medical history, and current treatments. For example, approximately 50 to approximately 100 mg / m². 2 AZA (body surface area) may be administered once daily by subcutaneous injection or intravenous infusion.

[0024] The pharmaceutical composition of the present invention may contain anemia treatment components other than demethylating agents, such as iron preparations. Furthermore, the pharmaceutical composition of the present invention may be used in combination with other anemia treatments, such as iron supplementation therapy.

[0025] In another embodiment, the present invention provides a method for producing normal erythrocytes from iPS cells derived from patients with X-linked sideroblastic anemia, comprising inducing differentiation of the iPS cells into erythrocytes, wherein a demethylating agent is added to the cells during differentiation induction.

[0026] iPS cells can be obtained by introducing nuclear reprogramming factors into somatic cells. Methods for producing iPS cells are well-known, and any of these methods may be used. Nuclear reprogramming factors basically include Oct family genes or their products, Klf family genes or their products, Sox family genes or their products, and Myc family genes or their products. In addition to these factors, the Lin28 gene or its product, nanog gene or its product, etc., may be introduced into somatic cells to enhance the efficiency of iPS cell induction. Methods for introducing nuclear reprogramming factors into somatic cells are also well-known. Furthermore, treating cells with histone deacetylase (HDAC) inhibitors after introducing nuclear reprogramming factors may enhance the efficiency of iPS cell establishment. Methods for culturing somatic cells into which nuclear reprogramming factors have been introduced, and methods for detecting and selecting iPS cells, are also well-known.

[0027] Somatic cells may originate from any part of the body. Hematologic cells, such as peripheral blood mononuclear cells, are preferred.

[0028] Methods for differentiating stem cells into erythrocytes are also known. Generally, embryoid body (EB) formation in the presence of appropriate factors is used, but is not limited to this. For example, iPS cells may be cultured in the presence of BMP4, Rock inhibitor, bFGF, VEGF, IL6, IL3, IL11, SCF, FLT3, and even TPO and EPO to form embryoid bodies (EBs) and differentiate them into erythrocytes. The resulting EBs may be cultured in a medium containing stem cell factor (SCF) and erythropoietin (EPO) to further differentiate them into erythrocytes.

[0029] For example, cells positive for cell surface markers specific to hematopoietic stem cells and / or hematopoietic progenitor cells may be selected a certain period after the start of EB formation (e.g., 10-20 days), and the selected cells may be cultured in a medium containing SCF or EPO to obtain a cell population rich in erythrocytes. Examples of cell surface markers specific to hematopoietic stem cells and / or hematopoietic progenitor cells include, but are not limited to, CD34 and CD43. Methods for selecting cells are well known, and a cell sorter may be used, for example.

[0030] When culturing in a medium containing SCF or EPO, erythrocyte differentiation may be promoted by co-culturing with stromal cells. When co-culturing with stromal cells, the cells in the process of differentiation may be transferred back onto the stromal cells to further promote erythrocyte differentiation induction. Any type of stromal cells may be used, but bone marrow-derived stromal cells are preferred. Examples of bone marrow-derived stromal cells include, but are not limited to, OP9 cells.

[0031] Efficacy of erythrocyte differentiation can be investigated by detecting cell surface markers specific to erythrocyte progenitor cells and / or erythrocytes. Examples of such markers include, but are not limited to, CD235a, CD71, CD36, CD23, CD234, CD235b, and CD236. Preferred markers include CD235a, CD71, and CD36. Methods for detecting these markers are also known; for example, markers with visible labels may be used, or specific antibodies may be used. Methods for selecting differentiated erythrocytes are also known; for example, a cell sorter may be used.

[0032] The addition of demethylating agents to cells is carried out under conditions where the cells and the demethylating agent are in contact. Typically, the demethylating agent is added to the culture medium. The addition of demethylating agents to cells may be carried out at any stage of erythrocyte differentiation from iPS cells. Preferably, the demethylating agent is added when differentiation has progressed to a certain extent using the EB method (for example, 10 to 20 days after the start of EB formation). The demethylating agent may also be added with each change of culture medium.

[0033] The concentration of the demethylating agent added to the culture medium can be selected and changed as appropriate. For example, when AZA is added, the AZA concentration in the culture medium is usually about 100 to about 1000 nM, preferably about 300 to about 700 nM, or for example, about 500 nM.

[0034] Confirmation of whether red blood cells are normal can be performed by examining whether the ALAS2 gene mutation has been normalized. This confirmation can also be performed by comparing the nucleotide sequence of the ALAS2 gene of red blood cells or erythroblasts obtained by differentiation with the nucleotide sequence of a non-mutated ALAS2 gene (which is publicly known). Methods for analyzing the nucleotide sequence of the gene are publicly known, and an automated sequencer may be used, for example.

[0035] Furthermore, whether or not the red blood cells are normal can be confirmed by known methods. For example, confirmation may be made by microscopic observation, confirmation of gene expression such as Hmox-1 and globin, o-dianisidine staining, confirmation of the base sequence of the ALAS2 mutation site, or confirmation of X chromosome inactivation. Alternatively, confirmation may be made by comparing the color of the red blood cells obtained from the patient's iPS cells with the color of red blood cells from a healthy person (a person without impairment in red blood cell differentiation) (for example, with the naked eye). If the color of the red blood cells obtained from the patient's iPS cells is the same as or close to the color of red blood cells from a healthy person, the obtained red blood cells may be judged as normal.

[0036] In a further embodiment, the present invention provides the following: • Use of demethylating agents to treat X-linked sideroblastic anemia; • Demethylating agents for use in the treatment of X-linked sideroblastic anemia; • Use of demethylating agents in the manufacture of pharmaceuticals for the treatment of X-linked sideroblastic anemia; and A method for treating X-linked sideroblastic anemia, comprising administering a demethylating agent to patients requiring treatment for X-linked sideroblastic anemia. The demethylating agents and patients in these embodiments are as described above.

[0037] In further embodiments, the present invention provides the following: A pharmaceutical composition for treating X-linked sideroblastic anemia comprising normal red blood cells obtained by a method for producing normal red blood cells from iPS cells derived from a patient with X-linked sideroblastic anemia, the method comprising differentiating the iPS cells into red blood cells, and adding a demethylating agent to the cells during differentiation induction; • Use of normal red blood cells to treat X-linked sideroblastic anemia; • Normal red blood cells for use in the treatment of X-linked sideroblastic anemia; • Use of the normal red blood cells in the manufacture of pharmaceuticals for the treatment of X-linked sideroblastic anemia; and A method for treating X-linked sideroblastic anemia, comprising administering the aforementioned normal red blood cells to a patient requiring treatment for X-linked sideroblastic anemia. The demethylating agents and patients in these embodiments are as described above.

[0038] The present invention will be described in more detail and specifically below with reference to examples, but the description of the examples is not intended to limit the scope of the present invention. [Examples]

[0039] Obtaining iPS cells from patients with X-linked sideroblastic anemia iPS cells were generated from the blood cells (peripheral blood mononuclear cells) of a patient with X-linked sideroblastic anemia (42 years old (at the time of iPS cell establishment), female, hereinafter also referred to as "SA1"), her sister (40 years old (at the time of iPS cell establishment), female, hereinafter also referred to as "SA2"), and their mother (70 years old (at the time of iPS cell establishment), female, hereinafter also referred to as "SA3"). Similar to SA1, SA2 and SA3 were also patients with X-linked sideroblastic anemia, with the ALAS2 gene on the X chromosome being heterozygous and carrying the R227C mutation. Vitamin B6 administration was ineffective for SA1. The procedure for generating iPS cells was as follows.

[0040] Peripheral blood was collected from patients, and peripheral blood mononuclear cells were isolated using Ficoll-Paque Plus (GE Healthcare). CD3-positive T cells and CD3-negative non-T cells were separated using MACS CD3 MicroBeads (Mlltenyi Biotec, 130-050-101). Gene transfection was performed using the Amaxa kit (Lonza) with pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1 (these vectors were obtained from Addgene, USA) (Amaxa Human T Cell Nucleofector Kit (Lonza) was used for T cells, and Amaxa Human Monocyte Nucleofector Kit (Lonza) was used for non-T cells). For T cells, 50 μLM of Dynabeads CD3 / CD28 was dissolved in 1 cc of hematopoietic medium X VIVO-10 (Lonza) in a 1.5 ml tube, the tube was placed in DynaMag-2, and the supernatant was removed. Subsequently, the cells were cultured on an MEF feeder in a medium of X VIVO-10 supplemented with 3 ng / ml of IL-2. Non-T cells were cultured in a medium of αMEM supplemented with 10% FBS, and 10 ng / ml each of IL-3, IL-6, G-CSF, and GM-CSF.

[0041] Because the ALAS2 gene is located on the X chromosome, one of the two ALAS2 genes is randomly inactivated by X chromosome inactivation in female patients. Therefore, the iPS cells obtained from each patient were of the following two types: iPS cells in which the X chromosome containing the ALAS2 mutation is inactivated, and only wild-type ALAS2 is expressed (referred to as "wild-type ALAS2 active iPS cells," "active wild-type iPSCs," or "WT-iPSCs"). iPS cells in which the X chromosome without the ALAS2 mutation is inactivated, and only the mutant ALAS2 is expressed (referred to as "active mutant iPSCs" or "MT-iPSCs"). In this specification, iPS cells may be referred to as "iPSCs". 58 iPSC strains were obtained from SA1. 94 iPSC strains were obtained from SA2. 47 iPSC strains were obtained from SA3. In this specification, WT-iPSC strains obtained from SA1 are referred to as "WT1-iPSC1," "WT1-iPSC2," and "WT1-iPSC3," and MT-iPSC strains obtained from SA1 are referred to as "MT1-iPSC1," "MT1-iPSC2," "MT1-iPSC3," and "MT1-iPSC4." Similarly, WT-iPSC strains obtained from SA3 are referred to as "WT3-iPSC1" and "WT3-iPSC2," and MT-iPSC strains obtained from SA3 are referred to as "MT3-iPSC1" and "MT3-iPSC2." One iPSC strain (692D2) obtained from a healthy donor was used as a control. In this specification, a cell being positive for a certain marker is indicated by "positive" or "+", and a cell being negative is indicated by "negative" or "-". For example, a CD235a-positive cell is referred to as a "CD235a-positive cell" or a "CD235a+ cell".

[0042] Maintaining iPSCs All iPSC lines were maintained on STO feeder cells in primate ES cell medium (ReproCell) supplemented with 4 ng / mL recombinant bFGF. After one passage on a plate coated with Matrigel to remove the feeder cells, the iPSC lines were differentiated. [Examples]

[0043] Induction of erythrocyte differentiation of iPS cells I. Experimental Methods iPS cells were differentiated in Stem Pro-34 medium (gibco) using the EB method (Grigoriadis AE et.al Blood. Apr. 8; 115(14): 2769-76 (2010)). On day 0, cells were cultured in medium containing BMP4 and Rock inhibitor; from day 1 to 4, in medium containing BMP4 and bFGF; from day 4 to 8, in medium containing VEGF, IL6, IL3, IL11, SCF, bFGF, and optionally Flt3a; and from day 8 to 15, in medium containing VEGF, IL6, IL3, IL11, SCF, TPO, EPO, and optionally Flt3a.

[0044] Flow cytometry analysis and cell sorting To prepare iPSC-derived hematopoietic cells for flow cytometry analysis and sorting, germ layers were dissociated using 0.25% trypsin, followed by 10-20 pipettes. The cells were then subjected to flow cytometry analysis and sorting using FACS ARIA II (Becton-Dickinson). Dead cells were removed using 7-AAD (BD Pharmingen). The antibodies used for sorting hematopoietic progenitor cells (HPCs) were CD34-PE (BD Biosciences, 348057) and CD43-FITC (BD Biosciences, 555475).

[0045] Erythrocyte differentiation was performed on OP9 stromal feeder cells. Hematopoietic progenitor cells sorted by FACS on day 15 were plated onto OP9 cells and cultured and maintained in Alpha Minimal Essential Medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 mM glutamine (Invitrogen), 100 μM monothioglycerol (Sigma Aldrich), 5 μg / mL transferrin (Roche), and 50 μg / mL ascorbic acid (Sigma Aldrich). The cytokines and compounds used for differentiation were as follows: Days 15-20, hSCF (100 ng / mL), human Fms-like tyrosine kinase 3 ligand (hFlt-3; 100 ng / mL, R&D Systems), hEPO (4 U / mL), and human thrombopoietin (hTPO; 50 ng / mL, R&D Systems); Days 20-27, hSCF (100 ng / mL) and hEPO (4 U / mL); Days 27-34, hEPO (4 U / mL) and ferrous citrate (SFC; 250 μM, Nihon Generic Co., Ltd).

[0046] May Grunwald-Giemsa staining May Grunwald-Giemsa staining was performed using Giemsa staining solution (Wako) and Giemsa solution (Wako) according to the manufacturer's instructions.

[0047] Berlin Blue Dye A fe stain kit (Muto Pure Chemicals Co., Ltd.) was used to perform berlin blue staining according to the manufacturer's instructions.

[0048] o-dianisidine staining O-dianisidine staining was performed for 15 minutes in 0.01 M sodium acetate, 0.65% water, 40% ethanol, and 0.6 mg / mL o-dianisidine (Sigma).

[0049] II. Experimental Results Pellets of erythroblasts derived from active wild-type iPSCs (WT erythroblasts) were red, while pellets of erythroblasts derived from active mutant iPSCs (MT erythroblasts) were white (Figure 1A). Iron staining showed that ring sideroblasts were present in MT erythroblasts but not in WT erythroblasts (Figure 1A). May Grunwald-Giemsa staining showed that most WT erythroblasts were mature, while most MT erythroblasts exhibited an immature morphological phenotype. Furthermore, dysplastic changes such as irregular nuclear margins and nuclear disintegration were observed in MT erythroblasts, similar to those in patient erythroblasts (Figure 1B). Most WT erythroblasts were orthochromatic erythroblasts, while most MT erythroblasts were polychromatic erythroblasts and megaloblasts (Figure 1C). Furthermore, while o-dianisidine staining of WT erythroblasts was normal, MT erythroblasts were only slightly stained (Figure 4D), suggesting that heme synthesis was significantly impaired in MT erythroblasts.

[0050] Furthermore, the erythrocyte differentiation ability of MT-iPSCs was investigated under erythrocyte culture conditions without feeder cells. To induce erythrocyte differentiation under conditions without feeder cells, hematopoietic progenitor cells derived from iPSCs, sorted by fluorescence-activated cell sorting (FACS), were differentiated in StemPro-34 medium (Invitrogen). The cytokines used for differentiation were as follows: Days 15-23, human stem cell factor (hSCF; 100 ng / mL, R&D Systems), human erythropoietin (hEPO; 4 U / mL, Kyowa Kirin Co., Ltd.), and human IL-3 (hIL-3; 5 ng / mL, R&D Systems); Days 23-26, hSCF (100 ng / mL) and hEPO (4 U / mL); Days 26-29, hEPO (4 U / mL).

[0051] Hematopoietic differentiation potential was comparable between the two iPSC strains (Figure 2A). However, compared to WT-iPSC strains obtained from both SA1 and SA3, MT-iPSC strains showed significantly impaired further differentiation into CD235+ erythrocytes (Figures 2B, 2C, and 2D). [Examples]

[0052] Expression of erythrocyte maturation-related genes in erythroblasts I. Experimental Methods RNA-seq RNA was extracted from TRA1-60+ iPSCs obtained from three WT-iPSC strains and four MT-iPSC strains that had been pre-sorted using FACS, as well as from CD34+ cells at day 8, CD43+CD34+CD38-Lin- cells at day 15, and CD235a+ erythroblasts at day 34. RNA was also extracted from CD235a+ erythroblasts sorted by MACS in patient bone marrow mononuclear cells isolated using Ficoll-Paque (GE Healthcare), and from bone marrow mononuclear cells (Lonza) from healthy donors. Libraries were constructed using the TruSeq Stranded Total RNA with Ribo-Zero Gold LT Sample Prep Kit, Sets A and B (Illumina) according to the manufacturer's instructions. Sequencing was performed using the NextSeq 500 / 550 High Output Kit v2 (75 cycles) (Illumina). Adapter sequences in the reads were trimmed using cutadapt-1.15. Bowtie2 and Samtools were used to remove reads mapped to ribosomal RNA. Reads were mapped to the human genome (GRCh38 from the UCSC Genome Browser) using STAR (version 2.5.4a), and RSeQC (version 2.6.4) was used for quality checks. Reads were counted using HTSeq (version 0.9.1) according to the GENCODE annotation file (version 27). The counts were then normalized using DESeq2 (version 1.24.0), a package in R (version 3.6.1). GO analysis, GSEA, PCA, and Wald tests were performed using the DESeq2 package, clusterProfiler (version 3.12.0), and enrichplot (version 1.6.1) packages.

[0053] II. Experimental Results To compare transcriptional expression patterns during erythrocyte differentiation in WT and MT cells, RNA-seq was performed on iPSCs, CD34+ cells at day 8, CD43+CD34+CD38-Lin- cells at day 15, and CD235a+ erythroblasts at day 34 in three WT-iPSC strains and four MT-iPSC strains (Figure 3A). No differences were observed between WT and MT at the iPSC, CD34+ cell, and CD43+CD34+CD38-Lin- cell stages. However, hierarchical clustering revealed that erythroblasts were divided into two clusters (Figure 3B). Principal component analysis (PCA) of erythroblasts differentiated from five iPSC strains, as well as bone marrow erythroblasts obtained from healthy donors and SA3, showed that iPSC-derived erythroblasts had a similar clustering pattern to bone marrow erythroblasts from the corresponding patients. MT erythroblasts and SA3-derived bone marrow erythroblasts were located on the positive side of PC2, while WT erythroblasts and healthy donor bone marrow erythroblasts were located on the negative side (Figure 3C). To confirm this observation, cells were evaluated using Gene Ontology (GO) analysis of RNA-seq data. Upregulated genes in WT cells showed enrichment of GO terms associated with mature erythroblasts, including Ras GTPase binding, Rho GTPase binding, and actin binding, as well as GO terms regulated by iron transporters, such as transition metal ion transmembrane transporter activity (Figure 3D). This analysis showed that WT erythroblasts differentiated to a more mature stage than MT erythroblasts, despite the same differentiation method. Gene set enrichment analysis (GSEA) was also used to investigate enrichment of gene expression in WT and MT erythroblasts regarding sets of genes expressed differently during erythrocyte differentiation. The heme metabolism dataset was significantly enriched in WT erythroblasts (Figure 3E and Table 1). Hierarchical cluster analysis was performed on iPSC-derived and bone marrow-derived erythroblasts for genes specifically expressed in early orthochromatic erythroblasts (Figure 3F). Expression of these genes was lower in MT erythroblasts and SA3 bone marrow erythroblasts than in WT erythroblasts and healthy donor bone marrow erythroblasts.These gene expression data support findings from phenotypic experiments, demonstrating that erythroblasts derived from MT-iPSCs cease differentiation at the immature erythrocyte stage. [Table 1] [Examples]

[0054] We investigated the changes in heme synthesis and erythrocyte differentiation caused by AZA. I. Experimental Methods Processing in AZA From the start of erythrocyte differentiation, 100 nM or 500 nM AZA (Nacalai Tesque) was added to the culture medium. AZA was added to the culture medium during medium changes (on days 19, 23, and 26 using the EB method without feeder cells).

[0055] II. Experimental Results When AZA was added during the erythrocyte differentiation stage (Figure 4A), the production of CD235+ erythroblasts in MT hematopoietic progenitor cells was significantly improved (Figures 4B and 4C). Furthermore, in o-dianisidine staining, a considerable number of erythroblasts differentiated from MT hematopoietic progenitor cells were not positive in the absence of AZA, but a considerable number of erythroblasts differentiated from MT hematopoietic progenitor cells were positive in the presence of AZA (Figure 4D). [Examples]

[0056] We investigated the nucleotide sequence changes in the ALAS2 gene caused by AZA. I. Experimental Methods Induction of erythrocyte differentiation of iPS cells WT-iPSCs and MT-iPSCs derived from SA1 obtained in Example 1 were used. iPS cells were differentiated in Stem Pro-34 medium (gibco) using the EB method (Grigoriadis AE et.al Blood. Apr. 8; 115(14): 2769-76 (2010)). On day 0, cells were cultured in medium containing BMP4 and Rock inhibitor; from day 1 to 4, in medium containing BMP4 and bFGF; from day 4 to 8, in medium containing VEGF, IL6, IL3, IL11, SCF, bFGF, and optionally Flt3a; and from day 8 to 15, in medium containing VEGF, IL6, IL3, IL11, SCF, TPO, EPO, and optionally Flt3a.

[0057] Cells differentiated for 15 days were reacted with FITC CD43 (BD ​​Biosciences, 555475) / PE CD34 (BD Biosciences, 348057), and CD43+CD34+ cells were FACS sorted. The sorted cells were differentiated into erythrocytes for 14 days. From day 15 to 23 after the start of EB formation, cells were cultured in Stem Pro-34 medium (gibco) containing SCF, EPO, and IL3; from day 23 to 26, in medium containing SCF and EPO; and from day 26 to 29, in medium containing EPO. Culture was performed under feeder-free conditions. With each medium change, the AZA-administered group used medium containing 500 nM AZA (Nacalai Tesque Co., Ltd.), while the control group used medium containing DMSO instead of AZA.

[0058] Analysis of ALAS2 mutation sites using Sanger sequencing Cells differentiated for 29 days were lysed in QIAZOL, and RNA was extracted using the miRNeasy Micro kit (QIAGEN NV). Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.), and the mutation site in exon 6 of ALAS2 was sequenced using Sanger sequencing with the Big Dye reaction.

[0059] II. Experimental Results The results are shown in Figure 5. In iPS cell-derived erythroblasts with active wild-type ALAS2, the DMSO-administered group showed only the normal CGC sequence. In the AZA 500nM-administered group, the mutant TGC sequence was expressed in addition to the normal CGC sequence. On the other hand, in iPS cell-derived erythroblasts with active mutant ALAS2, the DMSO-administered group showed only the mutant TGC sequence. In the AZA 500nM-administered group, the normal CGC sequence was expressed in addition to the mutant TGC sequence. From these findings, it can be inferred that AZA administration leads to the expression of both the mutant and wild-type ALAS2 in addition to the original wild-type and mutant ALAS2 in both iPS cell-derived erythroblasts with active wild-type ALAS2 and iPS cell-derived erythroblasts with active mutant ALAS2, suggesting that the methylation of the inactivated ALAS2 is removed. [Examples]

[0060] We investigated the improvement of erythrocyte differentiation ability by AZA.

[0061] I. Experimental Methods Using the SA1-derived iPS cells obtained in Example 1, differentiation was induced for 29 days using the EB method, similar to Example 5. CD43+CD34+ cell sorting was performed in the same manner as in Example 1. AZA administration was also performed in the same manner as in Example 5. The obtained cells were analyzed by FACS using APC CD235a as an indicator.

[0062] II. Experimental Results In erythroblasts derived from iPS cells with active wild-type ALAS2, both the DMSO-administered group (far left in Figure 6) and the AZA 500nM-administered group (second from the left in Figure 6) showed good erythroid differentiation potential. On the other hand, in erythroblasts derived from iPS cells with active mutant ALAS2, the DMSO-administered group (second from the right in Figure 6) showed a significant decrease in erythroid differentiation potential, while the AZA 500nM-administered group (far right in Figure 6) showed a remarkable improvement in erythroid differentiation potential. [Examples]

[0063] We investigated the increase in red blood cell colony count caused by AZA. I. Experimental Methods Using SA1-derived iPS cells obtained in Example 1, differentiation was induced for 29 days using the EB method, similar to Example 5. CD43+CD34+ cell sorting was also performed in the same manner as in Example 5. After sorting, the cells were lysed in MethoCult (H4435) and cultured for 14 days. The AZA-administered group used a medium containing 100 nM or 500 nM AZA, while the control group used a medium containing DMSO instead of AZA. Colony counts were measured using the following CFU assay.

[0064] CFU assay CFU assays were performed based on the method described by Nishizawa M et al. in Cell Stem Cell. 2016;19(3):341-354. Lineage assignments were determined by morphological analysis, and several typical colonies (especially erythrocytes and mixed colonies) were selected and evaluated using May Grunwald-Giemsa staining.

[0065] II. Experimental Results In iPS cells with active wild-type ALAS2, the number of red blood cell colonies was within the normal range in both the DMSO-administered group (top and bottom left of Figure 7) and the AZA 500nM-administered group (second from the left in the top and bottom of Figure 7). On the other hand, in erythroblasts derived from iPS cells with active mutant ALAS2, no red blood cell colonies were observed in the DMSO-administered group (second from the right in the top and bottom of Figure 7), but red blood cell colonies appeared in the AZA-administered group (top and bottom right of Figure 7), and the number of colonies improved to about half that of the AZA-administered group of iPS cells with active wild-type ALAS2.

[0066] We investigated the changes in erythroid colony formation ability of MT-iPSC-derived HPCs after AZA administration using multiple cell lines. The CFU assay was performed according to the procedure described above. There was no difference in the total number of colonies between the AZA-administered group and the control group (Figure 8A). In MT hematopoietic progenitor cells of the control group, no erythrocyte colonies were observed, and only a few mixed colonies were seen. On the other hand, in MT hematopoietic progenitor cells of the AZA-administered group, the formation of erythrocyte colonies and mixed colonies was significantly improved (Figures 8B, 8C). The same procedure was used for WT-iPSC3 and MT-iPSC1, and similar results were obtained (Figure 9). These results indicate that AZA administration improves defective heme synthesis and ineffective hematopoiesis in MT hematopoietic progenitor cells. [Industrial applicability]

[0067] This invention is useful in the field of pharmaceuticals for the treatment of hereditary diseases, particularly hematopoietic disorders, and in the research field of such diseases.

Claims

1. A pharmaceutical composition for treating X-linked sideroblastic anemia, comprising 5-azacitidine (AZA) or 5-aza-2'-deoxycytidine, for use in female patients with X-linked sideroblastic anemia who are heterozygous for a mutation in the ALAS2 gene.

2. The pharmaceutical composition according to claim 1, wherein the mutation in the ALAS2 gene is the R227C mutation.

3. The pharmaceutical composition according to claim 1 or 2, wherein the demethylating agent is AZA.

4. A pharmaceutical composition according to any one of claims 1 to 3, for use in patients with X-linked sideroblastic anemia in whom vitamin B6 administration is ineffective.

5. A method for producing normal red blood cells from iPS cells derived from a patient with X-linked sideroblastic anemia, comprising differentiating the iPS cells into red blood cells, wherein AZA or 5-aza-2'-deoxycytidine is added to the cells during differentiation induction, wherein the patient is a female patient with X-linked sideroblastic anemia who is heterozygous for a mutation in the ALAS2 gene.

6. The method according to claim 5, wherein the mutation in the ALAS2 gene is the R227C mutation.

7. The method according to claim 5 or 6, wherein the demethylating agent is AZA.

8. The method according to any one of claims 5 to 7, wherein the patient is a patient with X-linked sideroblastic anemia for whom vitamin B6 administration is ineffective.

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