Methods for generating erythroid progenitor cells

Abstract

The present invention relates to a method for producing erythroid progenitor cells in vitro, which comprises contacting genetically modified or unmodified hematopoietic stem cells with a defined cell culture medium containing a glucocorticoid and an autophagy inducer.

Description

[0001] The present invention belongs to the field of medicine. More specifically, the present invention relates to novel methods for producing erythroid progenitor cells and erythrocytes. Background Art

[0002] Maintaining a constant supply of oxygen to tissues is essential for the survival of many organisms, especially humans. Red blood cells carry oxygen throughout the body via the bloodstream. This transport is provided by hemoglobin, a protein unique to red blood cells that is able to bind oxygen. When red blood cells reach tissues, the oxygen diffuses through the capillary walls. Therefore, the role of red blood cells is vital.

[0003] In cases of emergency (bleeding) and pathological conditions (blood disorders, cancer, etc.), red blood cell transfusions are required. In 2016, more than 100 million blood bags were collected and distributed worldwide to meet transfusion needs. 50% of these products are distributed in wealthy countries, which only cover 15% of the global population. Transfusion problems in developing countries concern transfusion supply and safety, while in wealthy countries these problems are better controlled. However, immunological complications (alloimmunization) associated with chronic transfusions can lead to transfusion impasses involving the fate of the patient.

[0004] Until now, blood transfusions have been based solely on blood from donors.

[0005] In adults, red blood cell production, or erythropoiesis, occurs in the so-called hematopoietic bone marrow, which is found in the flat bones and at the ends of long bones. In the bone marrow, multipotent stem cells called hematopoietic stem cells sequentially differentiate into different types of erythroid progenitor cells (BFU-E, CFU-E, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts). When erythroblasts leave the bone marrow, they lose their nucleus, become reticulocytes, and then become mature erythrocytes.

[0006] Differentiation culture medium for hematopoietic stem cells that allows the in vitro production of erythrocytes is known. However, current methods have serious defects for large-scale production, such as too fast orientation toward erythrocyte maturation, which significantly limits production output, and differentiation into cells that cannot correctly perform the enucleation step (Akimov S et al., 2005, "Stem Cells" (Stemcells), 23 (9): 1423-1433; Hirose SI et al., 2013, "Stem Cell Reports" (Stem Cell Reports), 1 (6): 499-508; Huang X, 2013, "Molecular Therapy" (Mol. Ther.), 22 (2): 451-463). Finally, other methods use co-cultivation with feeder cells (Kurita R et al., 2013, PloS One, 8 (3): e59890), which is both complex and expensive to implement.

[0007] It would appear that the development of novel methods to produce red blood cells in vitro would have the potential to meet supply needs, avoid the risk of infection and avoid immunological complications.

[0008] The invention described herein is particularly intended to meet these needs. Summary of the invention

[0009] The inventors have demonstrated that culturing hematopoietic stem cells in a medium containing dexamethasone, small molecule enhancer of rapamycin-28 (SMER28) and optionally dimethyloxalylglycine (DMOG) not only differentiates these stem cells into erythroid progenitor cells, but also significantly expands the progenitor cell population while retaining their ability to ultimately differentiate into erythrocytes. The erythroid progenitor cells thus obtained can be maintained in culture and expanded for more than 60 days without losing their ability to differentiate into mature enucleated cells, i.e., erythrocytes.

[0010] According to a first aspect, the present invention relates to an in vitro method for producing erythroid progenitor cells, the method comprising contacting hematopoietic stem cells with a cell culture medium, which is preferably adapted to the nutritional requirements of hematopoietic stem cells and in particular to the growth and / or differentiation of hematopoietic lineage cells and comprises a glucocorticoid and an autophagy inducer.

[0011] Preferably, the glucocorticoid is selected from cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortisol and derivatives and mixtures thereof. More particularly preferably, the glucocorticoid is selected from prednisone, prednisolone and dexamethasone. Most particularly preferably, the glucocorticoid is dexamethasone.

[0012] Preferably, the autophagy inducer is selected from the group consisting of small molecule enhancer of rapamycin-28 (SMER-28), SMER-10 and SMER-18, and combinations thereof. More particularly preferably, the autophagy inducer is small molecule enhancer of rapamycin-28 (SMER-28).

[0013] According to a specific embodiment, the culture medium further comprises a hypoxia inducible factor (HIF) pathway activator, preferably a prolyl hydroxylase inhibitor, more preferably dimethyloxalylglycine (DMOG).

[0014] Hematopoietic stem cells are preferably obtained by differentiation of pluripotent stem cells, in particular embryonic stem cells (ES) or induced pluripotent stem cells (iPS), or isolated from a patient's blood sample with or without mobilization of umbilical cord blood or placenta, or a bone marrow sample or collection. Preferably, the hematopoietic stem cells are human hematopoietic stem cells.

[0015] Hematopoietic stem cells can be genetically modified to overexpress one or more genes selected from the group consisting of human telomerase reverse transcriptase (HTERT), B lymphoma Mo-MLV insertion region 1 homolog (BMI1), c-MYC, 1-MYC and MYB. Preferably, hematopoietic stem cells can be genetically modified to overexpress the HTERT gene or overexpress the BMI1 gene. They can also be modified to overexpress the HTERT gene and the BMI1 gene.

[0016] One or more genes selected from the group consisting of human telomerase reverse transcriptase (HTERT), B lymphoma Mo-MLV insertion region 1 homolog (BMI1), c-MYC, 1-MYC and MYB are preferably placed under the control of one or more inducible promoters.

[0017] These hematopoietic stem cells can also be genetically modified to overexpress:

[0018] - one or more core erythroid network (CEN) pathway transcription factors, preferably LIM domain only 2 (LMO2); and / or

[0019] - One or more EPO-R / JAK2 / STAT5 / BCL-XL pathway genes, preferably extra large B-cell lymphoma (BCL-XL).

[0020] Preferably, the hematopoietic stem cell is genetically modified to overexpress the BCL-XL gene or overexpress the LMO2 gene.

[0021] The one or more genes selected from the group consisting of EPO-R / JAK2 / STAT5 / BCL-XL pathway genes are preferably under the control of one or more constitutive promoters.

[0022] The one or more genes selected from the core erythroid network (CEN) pathway transcription factor genes are preferably under the control of one or more inducible promoters.

[0023] Hematopoietic stem cells can also be immortalized cells.

[0024] Preferably, the cells are cultured in the culture medium of the present invention for at least 20 days, more preferably at least 40 days, particularly preferably at least 60 days.

[0025] In a second aspect, the present invention also relates to the use of a cell culture medium according to the invention for generating and / or expanding erythroid progenitor cells.

[0026] In a third aspect, the present invention relates to genetically modified hematopoietic stem cells as described above, and to the use of these hematopoietic stem cells for the in vitro production of erythroid progenitor cells and / or erythrocytes.

[0027] In a fourth aspect, the present invention relates to an in vitro method for producing red blood cells, the method comprising:

[0028] - producing erythroid progenitor cells according to the method of the present invention; and

[0029] - inducing maturation of said erythroid progenitor cells,

[0030] - and optionally, recovering the obtained red blood cells.

[0031] Preferably, maturation of the erythroid progenitor cells is induced by culturing the erythroid progenitor cells in an erythroid differentiation medium.

[0032] In another aspect, the present invention also relates to a cell culture medium, which is preferably adapted for the growth and / or differentiation of hematopoietic cells and comprises a glucocorticoid, preferably dexamethasone, and an autophagy inducer, preferably SMER-28, and optionally a HIF pathway activator, preferably DMOG.

[0033] Preferably, the culture medium comprises a glucocorticoid at a concentration of 0.01 mM to 0.1 mM, and / or an autophagy inducer at a concentration of 2 μM to 30 μM, and / or a HIF pathway activator at a concentration of 75 μM to 350 μM.

[0034] The autophagy inducer is preferably selected from small molecule enhancer of rapamycin-28 (SMER-28), SMER-10 and SMER-18 and a combination thereof, and more particularly preferably SMER-28.

[0035] The glucocorticoid is preferably selected from cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortisol and derivatives and mixtures thereof, more particularly preferably selected from prednisone, prednisolone and dexamethasone, most particularly preferably dexamethasone.

[0036] The hypoxia inducible factor (HIF) pathway activator is preferably a prolyl hydroxylase (PHIS) inhibitor, more preferably dimethyloxalylglycine (DMOG).

[0037] The culture medium may further comprise (i) transferrin, (ii) insulin, (iii) heparin and (iv) serum, plasma, serum pool or platelet lysate, preferably platelet lysate, and optionally stem cell factor (SCF), EPO and / or IL-3.

[0038] The invention further relates to the use of the cell culture medium according to the invention for generating and / or expanding erythroid progenitor cells.

[0039] In a fifth aspect, the present invention also relates to a kit for producing erythroid progenitor cells and / or erythrocytes, the kit comprising:

[0040] - a culture medium according to the invention; and / or

[0041] - genetically modified hematopoietic stem cells according to the invention; and

[0042] - Optionally, a guide comprising instructions for use of such a kit.

[0043] Finally, in a sixth aspect, the present invention relates to the use of a kit according to the present invention for producing erythroid progenitor cells and / or erythrocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Expression profiles of surface markers CD117 and CD235a on day 14 according to different culture schemes. A: Scheme 4; B: Scheme 3; C: Scheme 1; D: Scheme 2.

[0045] Figure 2 : Expression profiles of surface markers CD117 and CD235a in cells genetically modified to overexpress HTERT, BMI1, and LMO2 at day 24 according to different culture protocols. A: Protocol 4; B: Protocol 1; C: Protocol 2.

[0046] Figure 3 : Expression profiles of surface markers CD117 and CD235a in cells genetically modified to overexpress HTERT, BMI1, and BCL-XL on day 24 according to different culture protocols. A: Protocol 4; B: Protocol 1; C: Protocol 2. DETAILED DESCRIPTION

[0047] The inventors have demonstrated that culturing hematopoietic stem cells in a medium containing an autophagy inducer, small molecule enhancer of rapamycin-28 (SMER28), and a glucocorticoid, dexamethasone, significantly increased the production of erythroid progenitor cells compared to a control medium supplemented with dexamethasone alone. The erythroid progenitor cells thus obtained can be maintained in culture and expanded for more than 60 days. The inventors have also demonstrated that these erythroid progenitor cells can effectively differentiate into red blood cells.

[0048] This in vitro culture method not only has the advantages of simplicity and economy, but also opens the way for large-scale industrial production of erythroid progenitor cells and then red blood cells. This can reduce the risk of blood shortages or transfusion deadlocks while providing optimal safety for transfused patients.

[0049] Therefore, according to a first aspect, the present application relates to an in vitro method for producing erythroid progenitor cells, comprising contacting hematopoietic stem cells with a culture medium comprising an autophagy inducer and a glucocorticoid. The purpose of the method is to induce hematopoietic stem cells to differentiate into erythroid progenitor cells and allow the progenitor cell population to expand, i.e., proliferate, while maintaining its ability to differentiate into red blood cells thereafter. Therefore, the method according to the present invention is a method for producing and amplifying erythroid progenitor cells.

[0050] As used herein, the term "erythroid progenitor cell" refers to a progenitor cell obtained by differentiation of hematopoietic stem cells during erythropoiesis. These progenitor cells are nucleated cells that have the ability to divide and subsequently differentiate into erythrocytes by denucleation. Erythroid progenitor cells are preferably selected from burst forming units-E (BFU-E) characterized by the expression of markers CD117, CD34, CD41, CD71 and CXCR4, colony forming units-E (CFU-E) characterized by the expression of markers CD117, CD34, CD36 and CD71, proerythroblasts characterized by the expression of markers CD117, CD71, CD36 and CD235a, basophilic erythroblasts characterized by the expression of markers CD117, CD71, CD36 and CD235a, and polychromatic erythroblasts characterized by the expression of markers CD36, CD71, CD235a, and mixtures thereof.

[0051] As used herein, the term "hematopoietic stem cell" or "HSC" refers to a multipotent stem cell that can differentiate into blood cells and immune cells such as white blood cells, red blood cells and platelets. Hematopoietic stem cells express CD45, CD133 and / or CD34 antigens. Preferably, hematopoietic stem cells express CD45 and CD34 antigens, and optionally CD133 antigen.

[0052] According to a preferred embodiment, the HSC is a human HSC.

[0053] These HSCs can be obtained from different sources and according to procedures well known to the skilled person. In particular, they can be separated from bone marrow, cytapheresis, whole blood or umbilical cord blood (or placental blood), for example using an immunomagnetic system or a screening system for the presence of specific membrane receptors (e.g., CD133, CD45 and / or CD34).

[0054] As used herein, the term "cytopheresis" refers to the removal of HSCs from the blood by apheresis. Apheresis is a technique for collecting certain blood components by extracorporeal blood circulation. The components to be collected are separated by centrifugation and extraction, and the uncollected components are reinfused into the donor or patient (therapeutic apheresis).

[0055] HSC can also be obtained by differentiation of pluripotent stem cells, particularly embryonic stem cells or induced pluripotent stem cells, preferably induced pluripotent stem cells. The technology of differentiating pluripotent stem cells into HSC is well known to technicians. Several protocols have been disclosed, particularly the protocol of Lengerke C et al. (2009, Ann NY AcadSci, 1176: 219-27), which consists of 17 days of differentiation, wherein the intermediate stage of embryoid bodies and the combination of the following cytokines: SCF, Flt-3 ligand, IL-3, IL-6, G-CSF and BMP-4.

[0056] As used herein, the term "embryonic stem cell" refers to cells derived from the internal cell mass of the blastocyst and having the ability to cause all tissues (mesoderm, endoderm, ectoderm) in the body to form, including germline cells. The pluripotency of embryonic stem cells can be assessed by the presence of markers such as transcription factors OCT4 and NANOG and surface markers such as SSEA3 / 4, Tra-1-60 and Tra-1-81. Embryonic stem cells can be obtained without destroying the embryo of its source, for example, by using the technology described in Chung et al. (" Cell Stem Cell " (Cell Stem Cell, 2008,2 (2): 113-117). In a specific embodiment, and for legal or ethical reasons, the embryonic stem cell is a non-human embryonic stem cell. In another specific embodiment, the embryonic stem cell used in the present invention is a human embryonic stem cell, preferably obtained without destroying the embryo of its source. The embryo used is preferably an excess embryo obtained during fertility treatment according to current law after obtaining supervision and ethical approval.

[0057] As used herein, the term "induced pluripotent stem cell" (iPS) refers to a pluripotent stem cell obtained by genetic reprogramming of differentiated somatic cells and having a morphology similar to that of embryonic stem cells and self-renewal and pluripotency potential. These cells are particularly positive for pluripotency markers, including alkaline phosphatase staining and expression of proteins NANOG, SOX2, OCT4 and SSEA3 / 4. The method for obtaining induced pluripotent stem cells is well known to technicians, and is particularly described in articles such as Yu et al. (Science, 2007, 318 (5858): 1917-1920), Takahashi et al. (Cell, 2007, 131 (5): 861-872) and Nakagawa et al. (Nat Biotechnol, 2008, 26 (1): 101-106).

[0058] The HSC used in the method according to the invention may also be a genetically modified HSC to increase its ability to participate in erythropoiesis and / or its ability to expand. Therefore, according to certain embodiments, the method according to the invention may include genetic modification of HSC to increase its ability to participate in erythropoiesis and / or its ability to expand. Methods for genetically modifying these cells are well known to the skilled person and include, for example, the introduction of a transgene into the cell genome via a retrovirus or a lentivirus or any other form of gene or protein transfer.

[0059] According to one embodiment, the expansion capacity of these HSCs is increased by overexpressing one or more genes selected from human telomerase reverse transcriptase (HTERT), B lymphoma Mo-MLV insertion region 1 homolog (BMI1), c-MYC, l-MYC and MYB.

[0060] According to a specific embodiment, the HSC is genetically modified to overexpress HTERT.

[0061] According to another specific embodiment, the HSCs are genetically modified to overexpress HTERT and one or more genes selected from the group consisting of B lymphoma Mo-MLV insertion region 1 homolog (BMI1), c-MYC, 1-MYC and MYB.

[0062] According to a preferred embodiment, HSCs are genetically modified to overexpress HTERT and / or BMI1, preferably HTERT and BMI1.

[0063] The ability of HSCs to participate in the erythropoietic pathway can be enhanced by overexpressing one or more genes involved in the EPO-R / JAK2 / STAT5 / BCL-XL pathway and / or the core erythroid network (CEN) pathway.

[0064] As used herein, the term "EPO-R / JAK2 / STAT5 / BCL-XL pathway" refers to a cell signaling pathway whose key proteins are erythropoietin (EPO) receptor, Janus kinase 2 (JAK2), signal transduction and transcription activator 5 (STAT5), and extra large B-cell lymphoma (BCL-XL). EPO is essential for erythropoiesis, and it can promote erythroid cell participation and cell survival. The binding of EPO to its membrane receptor causes the dimerization of EPO-R, and when activated, EPO-R induces JAK2. Then, JAK2 phosphorylates the tyrosine residues in the cytoplasmic tail of EPO-R. These phosphotyrosines allow interaction with proteins containing Src homology 2 (SH2) domains, resulting in activation of different signaling pathways, mainly the STAT5 pathway. STAT5 is first dimerized and then phosphorylated, which causes it to translocate into the nucleus, where it activates the transcription of different genes, including genes involved in cell proliferation and erythroid differentiation.

[0065] According to one embodiment, HSCs are genetically modified to overexpress one or more genes of the EPO-R / JAK2 / STAT5P / BCL-XL pathway, preferably one or more genes selected from genes encoding EPO-R, JAK2, STAT5P, BCL-XL and BCL-2, and combinations thereof.

[0066] According to a specific embodiment, the HSCs are genetically modified to overexpress the gene encoding BCL-XL.

[0067] As used herein, the term "CEN pathway" refers to a group of transcription factors that are essential for establishing or maintaining erythrocyte identity.

[0068] According to one embodiment, HSCs are genetically modified to overexpress one or more CEN pathway genes, preferably one or more genes selected from the group consisting of: genes encoding GATA1 (a transcription factor belonging to the zinc finger protein family that binds to the DNA sequence "GATA"), TAL bHLH transcription factor 1 (TAL1), Kru ppel-like factor 1 (KLF1), LIM domain binding 1 (LDB1), LIM domain only 2 (LMO2) and stem cell leukemia gene (SCL), and combinations thereof.

[0069] According to a specific embodiment, the HSCs are genetically modified to overexpress a gene encoding LMO2.

[0070] According to a particular embodiment, the HSC used in the method according to the invention is genetically modified to overexpress:

[0071] (i) one or more genes selected from the group consisting of genes encoding HTERT, BMI1, c-MYC, 1-MYC and MYB and combinations thereof, preferably HTERT and / or BMI1, and more particularly preferably HTERT and BMI1; and

[0072] (ii) one or more EPO-R / JAK2 / STAT5 / BCL-XL pathway genes, preferably selected from genes encoding EPO-R, JAK2, STAT5, BCL-XL and BCL-2 and combinations thereof, and more particularly preferably BCL-XL; and / or

[0073] (iii) one or more CEN pathway genes, preferably selected from the group consisting of genes encoding GATA1, TAL1, KLF1, LDB1, LMO2 and SCL, and combinations thereof, and more particularly preferably LMO2.

[0074] According to another specific embodiment, the HSC used in the method according to the invention is genetically modified to overexpress:

[0075] (i) one or more genes selected from the group consisting of genes encoding HTERT, BMI1, c-MYC, l-MYC and MYB and combinations thereof, preferably HTERT and / or BMI1, and more particularly preferably HTERT and BMI1; and

[0076] (ii) one or more EPO-R / JAK2 / STAT5 / BCL-XL pathway genes, preferably selected from genes encoding EPO-R, JAK2, STAT5P, BCL-XL and BCL-2, and combinations thereof, and more particularly preferably BCL-XL; and

[0077] (iii) one or more CEN pathway genes, preferably selected from the group consisting of genes encoding GATA1, TAL1, KLF1, LDB1, LMO2 and SCL, and combinations thereof, and more particularly preferably LMO2.

[0078] According to a preferred embodiment, HSCs are genetically modified to overexpress

[0079] (i) a gene encoding HTERT, and optionally a gene encoding BMI1, and

[0080] (ii) a gene encoding LMO2 and / or a gene encoding BCL-XL, preferably a gene encoding BCL-XL.

[0081] In particular, HSCs may be genetically modified to overexpress a gene encoding HTERT and a gene encoding BCL-XL, and optionally a gene encoding BMI1.

[0082] According to another preferred embodiment, HSCs are genetically modified to overexpress

[0083] (i) genes encoding HTERT and BMI1, and

[0084] (ii) a gene encoding LMO2 and / or a gene encoding BCL-XL.

[0085] In particular, HSCs can be genetically modified to overexpress

[0086] (i) genes encoding HTERT and BMI1 and a gene encoding LMO2, or

[0087] (ii) Genes encoding HTERT and BMI1 and a gene encoding BCL-XL.

[0088] As used herein, the term "overexpression" refers to the expression level of a gene in a genetically modified cell being higher than the expression level of the same gene in a non-genetically modified cell. When a cell does not express the gene in question before genetic modification but expresses it after modification, the term can be replaced with "expression".

[0089] Overexpression of genes in HSCs can be achieved by any technique known to the skilled person, in particular by introducing into HSCs a nucleic acid comprising one or more genes to be overexpressed or several nucleic acids each comprising one gene to be overexpressed. Thus, one or more nucleic acids can be arranged on the same construct or in separate constructs. They can be introduced into HSCs by any method known to the skilled person, in particular by viral transduction, microinjection, transfection, electroporation and biobolus.

[0090] As used herein, the term "construct" refers to an expression cassette or an expression vector.

[0091] In the expression cassette, one or more genes to be overexpressed are operably connected to the sequences necessary for their expression. In particular, they can be under the control of a promoter that allows it to be expressed in HSC. Typically, the expression cassette includes a promoter, one or more genes, and a transcription terminator that initiates transcription, or is composed of it. The expression "operably connected" indicates that the elements are combined so that the expression of the coding sequence is under the control of the transcription promoter. Typically, the sequence of the promoter is located upstream (5') of one or more target genes. A spacer sequence may be present between the regulatory element and the gene, as long as they do not prevent the expression of the translation of the encoded protein. The expression cassette may also include at least one "enhancer" activation sequence operably connected to the promoter.

[0092] The expression vector comprises one or more of the nucleic acids or expression cassettes. The expression vector can be used to transform a host cell and allow the expression of the target nucleic acid in the cell. The vector can be constructed by conventional molecular biology techniques well known to the technician.

[0093] Advantageously, the expression vector includes regulatory elements that allow expression of the target nucleic acid. These elements may include, for example, transcription promoters, transcription activators, terminator sequences, start and stop codons. The method of selecting these elements is well known to the technician.

[0094] The vector can be circular or linear, single-stranded or double-stranded. It is advantageously selected from plasmids, phages, phagemids, viruses, cosmids and artificial chromosomes. Preferably, the vector is a viral vector.

[0095] The gene or genes to be overexpressed may be placed under the control of the same or different constitutive or inducible promoters, whether or not they are present on the same nucleic acid.

[0096] HSCs can be transiently or stably transformed / transfected, and one or more nucleic acids, cassettes or vectors can be contained in the cells as episomes or integrated into the HSC genome. They can be inserted into the same or different regions in the eukaryotic cell genome.

[0097] There are many techniques well known to technicians that allow stable or transient expression of target genes. In particular, knock-in techniques can be used, using a targeted expression system, particularly a CRISPR-Cas9 system, to integrate the target gene into the genome of the cell (see, for example, Platt et al., Cell. October 9, 2014; 159(2): 440-55 or Lo et al., Biotechniques. April 1, 2017; 62(4): 165-174). This technology allows a single copy of one or more target genes to be inserted into a predetermined locus in the cell genome, based on the transfection of one or more vectors, which allows the gene encoding the Cas9 nuclease to be co-expressed with a guide RNA (gRNA) specific to the locus into which one or more genes will be inserted. Due to the repair of the break produced by Cas9, the one or more target genes or the box comprising the one or more target genes are inserted.

[0098] As used in this application, the term "guide RNA" or "gRNA" refers to an RNA molecule capable of interacting with Cas9 to guide it to a target chromosomal region.

[0099] Each gRNA can include two regions:

[0100] - a first region at the 5' end of the gRNA (often referred to as the "SDS" region) that is complementary to the target chromosomal region and mimics the crRNA of the endogenous CRISPR system, and

[0101] - A second region at the 3' end of the gRNA (often referred to as the "handle" region) that mimics the base pairing interaction between the trans-activating crRNA (tracrRNA) and the endogenous CRISPR system crRNA and has a double-stranded stem-loop structure with a substantially single-stranded sequence at the 3' end. This second region is essential for gRNA-Cas9 binding.

[0102] The gene of interest or a cassette comprising one or more genes of interest is flanked by homologous sequences to the break site targeted by the gRNA, thereby allowing insertion of the gene or cassette by break repair using homologous recombination.

[0103] Examples of promoters that allow constitutive expression include, but are not limited to, pEF1α long, pCMV, and pCAG.

[0104] The inducible expression system that can be used in the present invention is the Tet-On system, which is based on the use of a tetracycline transactivator (tTA) protein, which is produced by fusing the tetracycline repressor (TetR) protein present in Escherichia coli with the activation domain of the VP16 protein present in herpes virus. The rtTA protein can only bind to DNA on a specific TetO operator sequence if it binds tetracycline. Several repeating TetO sequences are placed under the control of a promoter, such as the EF1α long promoter. The TetO sequence coupled to the promoter is called a tetracycline response element (TRE), and responds to the binding of the tetracycline transactivator (tTA) protein by causing increased expression of the gene under the control of the promoter.

[0105] When several genes are overexpressed, they can be placed under the control of the same promoter or of several promoters.

[0106] According to a specific embodiment, HSCs are genetically modified to overexpress one or more genes selected from the group consisting of genes encoding HTERT, BMI1, c-MYC, l-MYC and MYB and combinations thereof, preferably HTERT and / or BMI1 and more particularly preferably HTERT and BMI1, and the gene or genes are placed under the control of one or more inducible promoters.

[0107] According to a particular embodiment, HSCs are genetically modified to overexpress one or more CEN pathway genes, preferably selected from the group consisting of genes encoding GATA1, TAL1, KLF1, LDB1, LMO2 and SCL, and combinations thereof, and more particularly preferably LMO2, and the gene or genes are placed under the control of one or more inducible promoters.

[0108] According to a specific embodiment, HSCs are genetically modified to overexpress one or more EPO-R / JAK2 / STAT5 / BCL-XL pathway genes, preferably selected from genes encoding EPO-R, JAK2, STAT5P, BCL-XL and BCL-2 and combinations thereof and more particularly preferably BCL-XL, and the gene or genes are placed under the control of one or more constitutive promoters.

[0109] According to a preferred embodiment, HSCs are genetically modified to overexpress a gene encoding HTERT under the control of an inducible promoter and to overexpress a gene encoding BCL-XL under the control of a constitutive promoter.

[0110] According to another preferred embodiment, HSCs are genetically modified to overexpress genes encoding HTERT, BMI1 and LMO2 under the control of one or more inducible promoters.

[0111] According to another preferred embodiment, HSCs are genetically modified to overexpress genes encoding HTERT and BMI1 under the control of one or more inducible promoters and to overexpress a gene encoding BCL-XL under the control of a constitutive promoter.

[0112] Alternatively, or in addition to the above-mentioned genetic modifications, the HSC used in the method according to the present invention can also be genetically modified to contain a suicide gene under the control of an inducible promoter, such as the HSV-TK gene or the Casp9 gene. As used herein, the term "suicide gene" refers to any gene whose expression causes the death of cells expressing it with the ability to divide, in the presence or absence of other molecules (drugs or other), depending on the suicide gene under consideration. For example, cell death of cells expressing the HSV-TK gene or the Casp9 gene, respectively, is obtained by adding ganciclovir or AP1003.

[0113] According to certain embodiments, the HSC used in the method according to the invention is an immortalized HSC. These immortalized cells may be further genetically modified as described above.

[0114] Immortalized HSCs can be obtained from immortalized cell lines established from malignant cells.

[0115] Preferably, the immortalized HSCs are obtained from immortalized cell lines established from non-malignant cells, such as from iPS (Kurita et al., PLoS ONE, 2013, 8, e59890), umbilical cord blood cells (Kurita et al., supra; Huang, X. et al., Mol. Ther. 2014, 22, 451-463), embryonic stem cells (Hirose, S. et al., Stem Cell Rep. 2013, 1, 499-508), or HSCs isolated from bone marrow, cell apheresis or whole peripheral blood (Trakarnsanga et al., 2017, Nature Communications, Vol. 8, 14750).

[0116] HSCs can be immortalized by any technique known to the skilled person, in particular by transduction with a lentiviral vector carrying the human papillomavirus type 16 oncogenes E6 and E7 (HPV16 E6 / E7) (Akimov et al., Stem Cells. 2005 Oct; 23(9): 1423-1433; Trakarnsanga et al., supra). Optionally, HSCs can also be transduced with a lentiviral vector carrying a gene encoding HTERT (Akimov et al., supra).

[0117] According to a preferred embodiment, the immortalized HSC used in the method according to the invention comprises a suicide gene allowing elimination of said immortalized HSC after inducing maturation of erythroid progenitor cells into mature enucleated erythrocytes.

[0118] The method according to the invention comprises contacting a HSC as described above with a glucocorticoid and an inducer of autophagy, more particularly with a culture medium adapted for growth and / or differentiation of cells of the hematopoietic lineage and comprising a glucocorticoid and an inducer of autophagy.

[0119] As used herein, the terms "autophagy," "autolysis," and "autophagy" are equivalent and can be used interchangeably. Autophagy refers to the degradation of a cell's cytoplasm by its own lysosomes.

[0120] Autophagy is a physiological process that helps eliminate certain proteins (viruses, deformities, etc.) and damaged organelles. This process can also be involved in the elimination of intracellular pathogens. Several signaling pathways detect different types of cellular stress, ranging from nutrient deprivation to microbial invasion, and converge to regulate autophagy. As used herein, the term "autophagy inducer" refers to a molecule that can induce autophagy in a cell.

[0121] In particular, the autophagy inducer can be an mTOR pathway inhibitor, such as metformin, rapamycin, perifosine, everolimus, resveratrol or tamoxifen, an autophagosome formation activator such as compound MG-132 (26S proteasome inhibitor), compound SAHA (pan-histone deacetylase inhibitor), trichostatin A or valproic acid, or a small molecule that acts independently of the mTOR pathway such as SMER-28, SMER-10 or SMER 18.

[0122] According to a specific embodiment, the autophagy inducer is an inducer that acts independently of the mTOR pathway, which is preferably selected from small molecule enhancer of rapamycin-28 (SMER-28), SMER10 and SMER 18, and combinations thereof.

[0123] According to a preferred embodiment, the autophagy inducer is SMER-28.

[0124] As used herein, the terms "glucocorticoid", "corticosteroid" or "corticoid" are equivalent and can be used interchangeably. These terms refer to natural or synthetic steroid hormones that have a pregnane nucleus and have effects on protein and carbohydrate metabolism.

[0125] According to one embodiment, the glucocorticoid is selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazole and derivatives and mixtures thereof.

[0126] According to a particular embodiment, the glucocorticoid is a synthetic hormone, preferably selected from prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortisol and derivatives and mixtures thereof.

[0127] According to a preferred embodiment, the glucocorticoid is selected from prednisolone, methylprednisolone, dexamethasone and derivatives and mixtures thereof, preferably selected from prednisolone, methylprednisolone and dexamethasone.

[0128] According to a particularly preferred embodiment, the glucocorticoid is dexamethasone.

[0129] According to a specific embodiment, the autophagy inducer is selected from SMER-28, SMER 10 and SMER 18, preferably SMER-28, and the glucocorticoid is selected from prednisolone, methylprednisolone and dexamethasone, preferably dexamethasone.

[0130] According to a very specific embodiment, the autophagy inducer is SMER-28 and the glucocorticoid is dexamethasone.

[0131] Optionally, the HSC may also be contacted with an activator of the HIF pathway. As used herein, the term "HIF pathway" refers to a signaling pathway initiated by hypoxia-inducible factor (HIF), which stimulates EPO secretion and thereby activates the EPO-R / JAK2 / STAT5 / BCL-XL pathway.

[0132] Preferably, the HIF pathway activator according to the present invention is a prolyl hydroxylase (PHIS) inhibitor.

[0133] As used herein, the terms "prolyl hydroxylase (PHIS)" and "procollagen-proline dioxygenase" are equivalent and can be used interchangeably. Prolyl hydroxylase is a hydroxylase of HIF on its prolyl residues. When hydroxylated, HIF is inhibited. Therefore, a specific inhibitor of prolyl hydroxylase is a molecule that is able to inhibit prolyl hydroxylase and thus activate the HIF pathway, which in turn activates the EPO-R / JAK2 / STAT5 / BCL-XL pathway.

[0134] Preferably, the prolyl hydroxylase inhibitor is selected from dimethyloxalylglycine (DMOG), N-oxalylglycine (NOG), deferoxamine (DFO), FG-4383, F-0041, FG-2216, FG-4592, S956711, 3,4-dihydroxy-ethyl benzoate (EDHB), TM6089, TM655, TM6008, 8-hydroxyquinoline and its derivatives.

[0135] Most particularly preferably, the HIF pathway activator is DMOG.

[0136] In the process of implementing the method according to the present invention, HSC is cultured in a culture medium adapted to the growth and / or differentiation of hematopoietic lineage cells. Many culture mediums adapted to the nutritional requirements of HSC are known to the technician and commercially available, such as StemSpan SFEM (STEMCELL Technologies) or human StemMACS HSC expansion culture medium XF (Invitrogen), which is preferably supplemented with stem cell factor (SCF), erythropoietin (EPO) and lipid.

[0137] Preferably, HSCs are cultured at a concentration of 200 to 10,000 cells / ml, preferably 500 to 2,000 cells / ml and more preferably about 1,000 cells / ml.

[0138] Preferably, the medium is changed about every 3 days so that the concentration of cells does not exceed about 4,000,000 cells / mL.

[0139] On the first day of culture or after a few days of culture, such as 10 to 15 days, HSCs can be contacted with a glucocorticoid and an autophagy inducer.

[0140] Preferably, the HSCs are contacted with a glucocorticoid and an autophagy inducer simultaneously.

[0141] Alternatively, the HSCs may first be contacted with the glucocorticoid and then with the autophagy inducer, or vice versa. The addition of the second compound may be performed, for example, several hours after contact with the first compound.

[0142] Preferably, HSC is contacted with glucocorticoid and autophagy inducer simultaneously. More particularly preferably, HSC is contacted with glucocorticoid and autophagy inducer simultaneously from the first day of culture.

[0143] Contacting can be achieved by adding glucocorticoids and / or autophagy inducers to the culture medium of the HSCs or by placing the HSCs in culture medium containing glucocorticoids and / or autophagy inducers.

[0144] The concentrations of the glucocorticoid and the autophagy inducer can be constant or varied throughout the culture or exposure period.

[0145] Preferably, when the culture medium comprises a glucocorticoid, the glucocorticoid is present in a concentration of 0.001 mM to 10 mM, preferably 0.01 mM to 1 mM, more preferably 0.01 mM to 0.5 mM and particularly preferably 0.02 mM to 0.1 mM.

[0146] According to a specific embodiment, when the culture medium comprises a glucocorticoid, the glucocorticoid is present at a concentration of about 0.1 mM.

[0147] Preferably, when the culture medium contains an autophagy inducer, the autophagy inducer is present in the culture medium at a concentration of 0.1 μM to 100 μM, preferably 0.5 μM to 50 μM, more preferably 1 μM to 30 μM and particularly preferably 2 μM to 30 μM. Alternatively, when the culture medium contains an autophagy inducer, the autophagy inducer may be present in the culture medium at a concentration of 10 μM to 30 μM.

[0148] According to a specific embodiment, when the culture medium comprises an autophagy inducer, the autophagy inducer is present in the culture medium at a concentration of about 2 μM.

[0149] As used herein, the term "about" refers to a range of values ​​of ±5%, preferably ±2%, of a specified value. For example, "about 20" includes 20±5%, or 19 to 21.

[0150] Similarly, in embodiments where the HSCs are placed in the presence of an activator of the HIF pathway, the concentration of the activator can be constant or can vary throughout the culture or contacting process.

[0151] Preferably, when the culture medium contains a HIF pathway activator, preferably DMOG, the latter is present in the culture medium at a concentration of 1 μM to 1000 μM, preferably 10 μM to 500 μM, more preferably 50 μM to 400 μM and particularly preferably 75 μM to 350 μM.

[0152] According to a specific embodiment, after 10 to 15 days of culture, HSCs are placed in the presence of a glucocorticoid, preferably dexamethasone, and an autophagy inducer, preferably SMER28. Preferably, according to this embodiment, the concentration of the glucocorticoid is 0.01 mM to 0.5 mM, more particularly preferably 0.02 mM to 0.1 mM, and the concentration of the autophagy inducer is 10 μM to 30 μM.

[0153] According to another specific embodiment, HSCs are placed in the presence of a glucocorticoid, preferably dexamethasone, and an autophagy inducer, preferably SMER28, from the first day of culture or before day 10 of culture. Preferably, according to this embodiment, the concentration of the glucocorticoid is 0.01 mM to 0.5 mM, preferably about 0.1 mM, and the concentration of the autophagy inducer is 2 μM to 30 μM, preferably about 2 μM.

[0154] Preferably, HSC is maintained in a culture medium comprising a glucocorticoid and an autophagy inducer and optionally an HIF pathway activator for at least 10 days. More particularly preferably, HSC is maintained in a culture medium containing a glucocorticoid and an autophagy inducer for at least 20, 30, 40, 50 or 60 days.

[0155] It should be understood that during this step, the cell culture includes not only HSCs but also erythroid progenitor cells.

[0156] The inventors have demonstrated that the use of a culture medium containing glucocorticoids and an autophagy inducer significantly amplifies the production of erythroid progenitors that do not participate in the terminal erythroid differentiation pathway. Therefore, according to the method of the present invention, HSCs can be cultured in a culture medium containing glucocorticoids and an autophagy inducer for at least 60, 70, 80, 90 or 100 days. Preferably, HSCs are cultured in a culture medium containing glucocorticoids and an autophagy inducer for up to 70, 80, 90 or 100 days.

[0157] By assessing the proportion of erythroid progenitor cells present in the cell population, a skilled person can easily determine the duration of HSC culture and the contact time with glucocorticoids and autophagy inducers and optionally HIF pathway activators. Preferably, HSC is contacted with glucocorticoids and autophagy inducers until a population comprising at least 90% erythroid progenitor cells, preferably at least 95% erythroid progenitor cells, more preferably at least 99% erythroid progenitor cells is obtained.

[0158] The culture may be maintained until markers of maturation into mature erythrocytes appear. Preferably, the culture is stopped when the cells no longer proliferate and / or less than 10%, preferably less than 5%, of the cells express the membrane receptor CD117. Alternatively, the culture may be stopped when at least 90%, preferably at least 95%, of the cells in the culture have reached the polychromatic erythroblast stage or are at a more advanced stage of differentiation.

[0159] According to certain embodiments, the process may comprise alternating the following phases: culture phases in which the culture medium comprises a glucocorticoid and an autophagy inducer and optionally a HIF pathway activator, and culture phases in which the culture medium does not comprise a glucocorticoid, an autophagy inducer and / or a HIF pathway activator.

[0160] Glucocorticoids, autophagy inducers, and HIF pathway activators can be added to or removed from the culture medium simultaneously or sequentially.

[0161] Techniques for changing the composition of the culture medium are well known to the skilled person. In particular, the addition of molecules or the increase in their concentration can be carried out directly in the pre-existing culture medium, and the removal of molecules or the reduction in their concentration can be carried out by centrifuging the cells and resuspending them in new culture medium or by diluting the culture medium.

[0162] The method according to the present invention may further include a step of recovering the obtained erythroid progenitor cells. This step may be performed by any technique known to the technician, in particular by centrifugation and removal of culture medium. The method of the present invention may also include a cell sorting step, in particular a cell selection step based on the expression of marker CD117. CD117+ cells may be selected to extend the expansion of erythroid progenitor cells. Conversely, CD117- cells may be selected to produce erythrocytes.

[0163] The method according to the invention may also comprise a step of washing the obtained / recovered erythroid progenitor cells. This step may be performed by any technique known to the skilled person, in particular by a series of centrifugation and resuspension steps.

[0164] According to a specific aspect, the present invention also relates to a population of erythroid progenitor cells obtained by the method of the present invention.

[0165] According to another aspect, the present invention also relates to the use of erythroid progenitor cells obtained by the method according to the present invention for producing erythrocytes.

[0166] As used herein, the terms "erythrocyte", "mature erythrocyte", "erythrosome", "erythrocyte" and "mature erythrocyte" are equivalent and can be used interchangeably. The term "erythrocyte" refers to an enucleated cell with characteristic markers of erythrocyte maturation. They specifically express glycoprotein A (CD235a), but do not express the marker CD36.

[0167] Therefore, the present invention relates to an in vitro method for producing red blood cells, the method comprising:

[0168] - producing erythroid progenitor cells according to the above-mentioned method of the present invention; and

[0169] - inducing maturation of said erythroid progenitor cells.

[0170] The above-described embodiments of the method for producing erythroid progenitor cells according to the invention are also considered in this regard.

[0171] Maturation of erythroid progenitor cells may include expression of erythroid maturation markers such as CD235a and by enucleation.

[0172] The method of the present invention may further comprise a cell sorting step, in particular a CD117- cell selection step.

[0173] The maturation of progenitor cells can be induced by any method known to the technician. In particular, maturation can be induced by culturing erythroid progenitor cells in erythroid differentiation medium, for example, in a medium supplemented with erythropoietin and optionally SMER28. Preferably, maturation is induced by culturing erythroid progenitor cells in a medium not containing stem cell factor (SCF) or dexamethasone and supplemented with erythropoietin (about 2.5 IU / mL) and optionally SMER28 (about 2.5 μM). Alternatively, maturation is induced by placing cells in a serum-free medium. Preferably, at a high cell concentration, for example, higher than 5,000,000 cells / ml of culture, progenitor cell maturation is induced.

[0174] According to one embodiment, the method for producing red blood cells according to the invention further comprises a step of eliminating nucleated cells. This step results in a homogenous population containing only mature red blood cells.

[0175] According to a specific embodiment, wherein the HSC used in the method for producing erythroid progenitor cells according to the present invention comprises an inducible suicide gene, the step of eliminating nucleated cells can be performed by inducing the expression of the suicide gene.

[0176] The method for producing erythrocytes may further comprise a step of recovering the erythrocytes obtained. This step may be performed by any technique known to the skilled person, in particular by filtering, centrifuging and removing the culture medium.

[0177] The method according to the invention may also comprise a step of washing the obtained / recovered erythrocytes. This step may be performed by any technique known to the skilled person, in particular by a series of filtration, centrifugation and resuspension steps.

[0178] According to another aspect, the present invention also relates to a population of erythrocytes obtained by the method of the present invention.

[0179] According to another aspect, the present invention also relates to a pharmaceutical composition comprising erythroid progenitor cells obtained by the method of the present invention and a pharmaceutically acceptable carrier.

[0180] The invention also relates to erythroid progenitor cells according to the invention, or a pharmaceutical composition comprising erythroid progenitor cells according to the invention, for use as a hematopoietic transplant. As used herein, the term "hematopoietic transplant" refers to a group of cells intended to be administered to the bone marrow of a subject and capable of producing red blood cells.

[0181] The present invention also relates to the erythroid progenitor cells according to the present invention, or a pharmaceutical composition comprising the erythroid progenitor cells according to the present invention, for use in the treatment of anemia.

[0182] As used herein, the term "anemia" refers to an abnormal blood count characterized by abnormally low levels of healthy red blood cells and a drop in circulating hemoglobin below normal for the subject's age. By way of example, anemia is typically characterized by hemoglobin levels below 13 g / dL in males, below 12 g / dL in females, below 11 g / dL in children, and below 14 g / dL in newborns.

[0183] Anemia can have a variety of causes. Examples of anemia include, but are not limited to, anemia associated with bleeding (e.g., bleeding caused by trauma or surgery), anemia caused by drug therapy (e.g., chemotherapy) or exposure to toxic substances (e.g., lipolytic agents, oxidants, lead, venom, or poisons), hemolytic anemia caused by inherited red cell membrane disorders (e.g., hereditary spherocytosis, hereditary elliptocytosis, or hereditary thermophilic polycythemia), hemolytic anemia caused by acquired red cell membrane disorders (e.g., paroxysmal nocturnal hemoglobinuria or acanthocytosis), autoimmune The invention relates to anemia caused by an infectious agent (e.g., malaria, Babesia or Bartonella infection, trypanosomiasis, visceral leishmaniasis, sepsis or CMV infection), anemia caused by hereditary or acquired sideroblastic anemia, anemia caused by bone marrow failure (e.g., bone marrow suppression, vitamin B12 deficiency, myelodysplasia or bone marrow disease invasion by hematologic malignancies (leukemia, lymphoma, metastatic tumors)), or anemia associated with sickle cell disease or thalassemia syndrome. Preferably, the anemia is associated with sickle cell disease or thalassemia syndrome.

[0184] The present invention also relates to a method for treating a patient suffering from anemia, which method comprises administering to the patient a therapeutically effective amount of erythroid progenitor cells obtained by the method of the present invention, or a pharmaceutical composition comprising erythroid progenitor cells obtained according to the present invention.

[0185] The present invention also relates to the use of the erythroid progenitor cells obtained according to the present invention or a pharmaceutical composition comprising said progenitor cells for the preparation of a medicament, in particular a biological drug, for the treatment of anemia.

[0186] As used herein, the term "biopharmaceutical" refers to a pharmaceutical product whose active substance is produced by or extracted from a biological source.

[0187] Preferably, in this aspect, the erythroid progenitor cells are obtained from non-genetically modified HSCs.

[0188] As used herein, the terms "subject" and "patient" are equivalent and can be used interchangeably. These terms preferably refer to animals, particularly mammals, most particularly preferably humans, particularly fetuses, newborns, children, teenagers, adults or elderly people. As used herein, the term "fetus" refers to the intrauterine development stage of more than 8 weeks of gestation, the term "newborn" refers to a person under 12 months of age, the term "child" refers to a person aged 1 to 12 years, the term "adult" refers to a person aged 12 to 60 years, and the term "elderly" refers to a person aged 60 years or more.

[0189] The patient is preferably a patient who has failed a transfusion, has been transfused multiple times or has a rare blood type. According to a preferred embodiment, the patient suffers from anemia, in particular anemia associated with sickle cell disease or thalassemia syndrome.

[0190] As used herein, the term "transfusion failure" refers to a transfusion that is ineffective and / or induces pathological complications in the patient.

[0191] When the transfusion efficiency is less than 80% 24 hours after transfusion of red blood cell concentrate (RCC), the red blood cell transfusion is considered ineffective.

[0192] Red blood cell transfusion efficiency (ETE) was calculated by the following formula:

[0193]

[0194] The minimum amount of HB infused was 40 g, and the average observed was 50 g. TBV refers to total blood volume.

[0195] Pathological complications associated with transfusion failure may be the result of transfusional immunization (transfusional alloimmunization) that may occur years later and impair the patient's future transfusions. Indeed, prior immunization may cause an immediate risk of hemolysis (if antibodies are present at sufficient titers) or, more often, delayed hemolysis (if antibodies are present at low titers or even undetectable by serology) during a new transfusion.

[0196] As used herein, the term "multiple transfusions" refers to a patient who has undergone several transfusions and / or a patient who has received a transfusion and is about to receive another transfusion.

[0197] As used herein, the term "rare blood type" refers to a blood type with a frequency of less than 1 / 250 in the French and / or European and / or global population and / or a blood type for which patients cannot be transfused with type O blood. Rare blood is difficult to supply.

[0198] In the field of veterinary applications, the subject of the invention may be non-human animals, preferably pets or farm animals, for example selected from dogs, cats, cows, sheep, rabbits, pigs, goats, horses, rodents, non-human primates and poultry.

[0199] As used herein, the term "treatment" refers to any action intended to improve or eliminate symptoms, slow the progression of a disease, stop the progression of a disease, or eliminate a disease. This term more specifically refers to an increase in the level of healthy red blood cells and circulating hemoglobin, preferably to normal values ​​for the age of the subject. The term includes both preventive and therapeutic treatments. As used herein, the term "therapeutically effective amount" refers to an amount sufficient to produce an effect on at least one symptom of a disorder, and more specifically, sufficient to increase the level of healthy red blood cells and circulating hemoglobin in the subject being treated.

[0200] As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable carrier" are equivalent and refer to any substance present in a pharmaceutical composition other than the active ingredient. Its addition is particularly intended to promote the preservation and administration of the cells without changing their properties. The pharmaceutically acceptable carrier for preparing a composition containing red blood cells or red blood cell progenitor cells according to the present invention can be, for example, selected from saline, PBS solutions with added human serum albumin, and mixtures thereof, or any other saline solution having an osmotic pressure concentration suitable for preserving red blood cells and / or progenitor cells and preferably directly applicable to a subject. In order to prepare a composition containing red blood cells, saline-adenine-glucose-mannitol (SAGM) culture medium can also be used as a pharmaceutically acceptable carrier alone or in combination with other pharmaceutically acceptable carriers mentioned above.

[0201] Preferably, administration of the progenitor cells or transplant is performed in the patient's bone marrow or by intravenous injection.

[0202] According to a preferred embodiment, the hematopoietic stem cells for producing erythroid progenitor cells are from donor samples or from cells obtained from donors, and the erythroid progenitor cells are intended to be transplanted into the recipient patient. The donor and recipient can be the same individual (autologous transplantation) or different individuals (allogeneic transplantation). Preferably, the donor and recipient are the same individual.

[0203] According to another aspect, the present invention relates to a pharmaceutical composition or a medicament, in particular a biopharmaceutical, comprising red blood cells obtained by the method of the present invention and a pharmaceutically acceptable carrier.

[0204] The present invention also relates to erythrocytes obtained by the method of the present invention or a pharmaceutical composition comprising erythrocytes obtained according to the present invention for transfusion into a patient suffering from anemia, ie in need of a supply of erythrocytes.

[0205] The present invention also relates to a method for treating a patient suffering from anemia, ie requiring a supply of red blood cells, comprising administering a therapeutically effective amount of red blood cells obtained by the method of the present invention, or a pharmaceutical composition comprising red blood cells according to the present invention.

[0206] The present invention also relates to erythrocytes obtained by the method of the present invention or a pharmaceutical composition comprising erythrocytes obtained according to the present invention, for use in treating anemia.

[0207] Preferably, the anemia is anemia associated with sickle cell disease or thalassemia syndrome.

[0208] Preferably, the patient has failed a transfusion, has been transfused multiple times, or has a rare blood type.

[0209] In the context of personalized medicine, red blood cells to be administered or administered to a patient are obtained according to an in vitro method for producing red blood cells, the method comprising:

[0210] - obtaining HSCs from a sample of said patient;

[0211] - obtaining erythroid progenitor cells from said HSCs according to the method of the present invention; and

[0212] - Obtaining erythrocytes from said erythroid progenitor cells according to the method of the invention.

[0213] HSCs can be obtained from a patient's blood or bone marrow sample. Alternatively, HSCs can be obtained from iPSCs obtained by genetically reprogramming differentiated somatic cells obtained from a patient.

[0214] HSCs may optionally be genetically modified as described above.

[0215] According to another aspect, the present invention also relates to HSCs that are genetically modified to overexpress:

[0216] (i) one or more genes selected from the group consisting of genes encoding HTERT, BMI1, c-MYC, 1-MYC and MYB and combinations thereof, preferably HTERT and / or BMI1 and more particularly preferably HTERT and BMI1; and

[0217] (ii) one or more EPO-R / JAK2 / STAT5 / BCL-XL pathway genes, which are preferably selected from genes encoding EPO-R, JAK2, STAT5P, BCL-XL and BCL-2 and combinations thereof, and more particularly preferably BCL-XL; and / or

[0218] (iii) one or more CEN pathway genes, which are preferably selected from the group consisting of genes encoding GATA1, TAL1, KLF1, LDB1, LMO2 and SCL, and combinations thereof, and more particularly preferably LMO2.

[0219] In this regard, embodiments involving HSCs for use in methods for generating erythroid progenitor cells are also contemplated.

[0220] According to a preferred embodiment, HSCs are genetically modified to overexpress a gene encoding HTERT under the control of an inducible promoter and a gene encoding BCL-XL under the control of a constitutive promoter.

[0221] According to another preferred embodiment, HSCs are genetically modified to overexpress genes encoding HTERT, BMI1 and LMO2 under the control of one or more inducible promoters.

[0222] In another preferred embodiment, HSCs are genetically modified to overexpress genes encoding HTERT and BMI1 under the control of one or more inducible promoters and to overexpress a gene encoding BCL-XL under the control of a constitutive promoter.

[0223] Genetically modified HSCs may also contain suicide genes as described above or be immortalized.

[0224] The present invention also relates to the use of the genetically modified HSCs according to the present invention for generating erythroid progenitor cells and / or erythrocytes, preferably in vitro, in particular according to the above-described method of the present invention.

[0225] According to another aspect, the present invention relates to a cell culture medium adapted to the nutritional requirements of HSCs, and in particular to the growth and / or differentiation of cells of the hematopoietic lineage, and comprising a glucocorticoid and an autophagy inducer, and optionally an activator of the HIF pathway.

[0226] Also contemplated in this regard are embodiments relating to a culture medium comprising a glucocorticoid and an inducer of autophagy and optionally an activator of the HIF pathway for use in a method of generating erythroid progenitor cells.

[0227] The glucocorticoid and the autophagy inducer are as defined above for the method according to the invention. Preferably, the glucocorticoid is dexamethasone and / or the autophagy inducer is SMER28 and / or the HIF pathway activator is DMOG.

[0228] The matrix of the cell culture medium adapted for the growth and / or differentiation of hematopoietic lineage cells may be any matrix known to the skilled person to meet the requirements of HSCs and / or erythroid progenitor cells.

[0229] As used herein, the term "growth" refers to the proliferation of cells. The term "differentiation" refers to the acquisition of characteristics that are not present in the cells that were initially used to seed the culture medium by the cultured cells. In the present case, the term refers to the acquisition of characteristics of erythroid progenitor cells. Therefore, a culture medium adapted to the growth and / or differentiation of hematopoietic cells is a culture medium that allows HSC to differentiate into erythroid progenitor cells and the proliferation of HSC and erythroid progenitor cells. The term should not be confused with "maturation", which defines the process by which erythroid progenitor cells become red blood cells and which particularly involves the enucleation of cells. Therefore, a culture medium adapted to the growth and / or differentiation of hematopoietic cells preferably does not contain any compound that induces this maturation.

[0230] The matrix of the cell culture medium may include Iscove's modified Dulbecco's medium (IMDM) or an equivalent medium adapted to the nutritional requirements of HSCs (e.g., StemSpan SFEM (STEMCELL Technologies) or human StemMACS HSC Expansion Medium XF (Invitrogen)), supplemented with insulin, transferrin, stem cell factor (SCF), heparin, IL-3, EPO, growth factors and / or serum, plasma, platelet lysate and / or serum pools. The compounds added to the culture medium are preferably human compounds obtained by recombinant or purification techniques. The concentrations of these different compounds can be easily determined by a technician based on the recommendations of the supplier or common knowledge in the art.

[0231] As used herein, the term "serum pool" refers to a mixture of attenuated human AB plasma (most often a mixture of more than 100 different plasmas). For this purpose, AB plasma from transfusion centers is mixed, attenuated and finally aliquoted. The serum pool can then be used fresh or stored frozen.

[0232] As used herein, the term "platelet lysate" refers to a product rich in growth factors obtained due to the lysis of platelet concentrates. For this reason, platelet concentrates from transfusion centers can be mixed before lysis. There are different lysis methods well known to technicians, in particular ultrasonic lysis, the use of solvents and / or detergents, or cryolysis. Platelet concentrates are preferably lysed by cryolysis. Cryolysis consists of a freeze / thaw cycle, usually two cycles, which causes platelets to rupture and releases the growth factors contained therein into the plasma. Optionally, centrifugation and / or filtration can be performed after lysis. The platelet lysate can then be used fresh or stored frozen.

[0233] Preferably, the base of the culture medium according to the invention is IMDM or an equivalent medium as defined above, supplemented with:

[0234] - transferrin, preferably human transferrin, at a concentration of about 200 μg / mL to about 400 μg / mL, preferably about 300 μg / mL to about 350 μg / mL, more preferably at a concentration of about 330 μg / mL; and / or

[0235] - Insulin, preferably human insulin, at a concentration of about 1 μg / mL to about 50 μg / mL, preferably about 5 μg / mL to about 20 μg / mL, more preferably at a concentration of about 10 μg / mL; and / or

[0236] - serum, plasma or a pool of serum, preferably human serum, plasma or a pool of serum, in a concentration of about 1% to about 10%, preferably about 3% to about 7%, more preferably about 5%, and / or platelet lysate, preferably platelet lysate of human origin, in a concentration of about 0.05% to about 0.5%, preferably about 0.1% to about 0.5%, more preferably about 0.3%; and / or

[0237] - Heparin, preferably human heparin, at a concentration of about 0.5 U / mL to about 10 U / mL, preferably about 1 U / mL to about 5 U / mL, more preferably at a concentration of about 3 U / mL.

[0238] Optionally, particularly for medium adapted for the growth and / or differentiation of hematopoietic cells, the medium may also be supplemented with:

[0239] - IL-3, preferably human IL-3, at a concentration of about 1 ng / mL to about 20 ng / mL, preferably about 3 ng / mL to about 7 ng / mL, more preferably at a concentration of about 5 ng / mL;

[0240] - SCF, preferably human SCF, at a concentration of about 10 ng / mL to about 200 ng / mL, preferably about 50 ng / mL to about 150 ng / mL, more preferably at a concentration of about 100 ng / mL; and / or

[0241] - EPO, preferably human EPO, at a concentration of about 0.5 IU / mL to about 10 IU / mL, preferably about 1 IU / mL to about 5 IU / mL, more preferably at a concentration of about 2 IU / mL.

[0242] In a specific embodiment, the matrix of the culture medium according to the present invention is preferably IMDM or equivalent culture medium as defined above, and comprises transferrins, insulin, heparin and serum, blood plasma, serum pool or platelet lysate, preferably transferrins, insulin, heparin and serum pool or platelet lysate, more particularly preferably transferrins, insulin, heparin and platelet lysate. These compounds are preferably used with concentration as described above.

[0243] In a preferred embodiment, the culture medium further comprises IL-3, SCF and EPO, preferably at the concentrations described above.

[0244] Alternatively, the culture medium may contain SCF and EPO, preferably at the concentrations described above.

[0245] According to a particular embodiment, the matrix of the culture medium according to the invention comprises:

[0246] - transferrin, preferably human transferrin, at a concentration of 300 μg / mL to 350 μg / mL;

[0247] - Insulin, preferably human insulin, at a concentration of 5 μg / mL to 20 μg / mL;

[0248] - serum, plasma or a pool of sera, preferably human serum, plasma or a pool of sera, in a concentration of 3% to 7%, and / or platelet lysate, preferably platelet lysate of human origin, in a concentration of 0.1% to 0.5%; and

[0249] - heparin, preferably human heparin, in a concentration of 1 U / mL to 5 U / mL,

[0250] and optionally,

[0251] - IL-3, preferably human IL-3, at a concentration of 3 ng / mL to 7 ng / mL;

[0252] - SCF, preferably human SCF, at a concentration of 50 ng / mL to 150 ng / mL; and / or

[0253] - EPO, preferably human EPO, at a concentration of 1 IU / mL to 5 IU / mL.

[0254] The culture medium according to the present invention comprises added to the matrix (i) an autophagy inducer, preferably SMER-28, (ii) a glucocorticoid, preferably dexamethasone, and optionally (iii) a HIF pathway activator, preferably DMOG.

[0255] According to a particular embodiment, the culture medium according to the invention comprises, added to the matrix:

[0256] - a glucocorticoid, preferably dexamethasone, in a concentration of 0.001 mM to 10 mM, preferably 0.002 mM to 1 mM, more preferably 0.005 mM to 0.5 mM, and particularly preferably 0.01 mM to 0.1 mM; and

[0257] - an autophagy inducer, preferably SMER-28, at a concentration of 0.1 μM to 100 μM, preferably 0.5 μM to 50 μM, more preferably 1 μM to 30 μM, and particularly preferably 2 μM to 30 μM; and

[0258] - Optionally, a HIF pathway activator, preferably DMOG, at a concentration of 1 μM to 1000 μM, preferably 10 μM to 500 μM, more preferably 50 μM to 400 μM, and particularly preferably 75 μM to 350 μM.

[0259] In a specific embodiment, the culture medium according to the invention comprises 0.005 mM to 0.5 mM of a glucocorticoid, preferably dexamethasone, 0.5 μM to 50 μM of an autophagy inducer, preferably SMER-28, and optionally 50 μM to 400 μM of a HIF pathway activator, preferably DMOG.

[0260] In another specific embodiment, the culture medium according to the invention comprises 0.01 mM to 0.1 mM of a glucocorticoid, preferably dexamethasone, 2 μM to 30 μM of an autophagy inducer, preferably SMER-28, and optionally 75 μM to 350 μM of a HIF pathway activator, preferably DMOG.

[0261] Preferably, the culture medium according to the invention does not contain serum of non-human origin (eg fetal calf serum), thrombopoietin, vascular endothelial growth factor (VEGF), IL-6, bone morphogenetic proteins (BMP), FLT3-ligand and / or hydrocortisone.

[0262] The present invention also relates to the use of a cell culture medium according to the invention as described above for producing and / or expanding erythroid progenitor cells and / or producing erythrocytes, in particular according to the method of the invention described above, and more particularly for stimulating the differentiation of HSCs into erythroid progenitor cells and / or expanding erythroid progenitor cells.

[0263] In another aspect, the present invention further relates to a kit comprising:

[0264] - a cell culture medium according to the invention; and / or

[0265] - a genetically modified HSC or a gene construct according to the invention, to obtain a genetically modified HSC according to the invention; and

[0266] - Optionally, a guide comprising instructions for use of such a kit.

[0267] The present invention also relates to the use of a kit according to the present invention for producing erythroid progenitor cells and / or erythrocytes, in particular according to the method of the present invention described above.

[0268] All references cited in this application, including journal articles or abstracts, published patent applications, issued patents, or any other references, are hereby fully incorporated by reference, including all results, tables, figures, and text presented in these references.

[0269] Although having different meanings, throughout the specification of the present invention, the terms "comprising", "having", "containing" and "consisting of" may be replaced with each other.

[0270] Other characteristics and advantages of the invention will emerge more clearly on reading the following illustrative and non-limiting examples.

[0271] Example

[0272] Example 1: Expansion of erythroid progenitor cells from HSC derived from cord blood and cytopheresis

[0273] Materials and methods

[0274] Medium used: IMDM (Biochrom), supplemented with transferrin (330 μg / mL), insulin (10 μg / mL), 5% AB serum pool (EFS) and heparin (3 U / mL). From day 0 to day 7, the medium was supplemented with IL-3 (5 ng / mL), SCF (100 ng / mL) and EPO (2 IU / mL). From day 7 to the end of erythroblast progenitor expansion, the medium was supplemented with SCF (100 ng / mL) and EPO (2 IU / mL).

[0275] HSC:

[0276] HSCs were obtained after magnetic separation using CD34+ beads according to the protocol provided by Milenyi (see Human CD34 MicroBead Kit from Miltenyi Biotec).

[0277] Cell maintenance:

[0278] Cells were seeded at 10,000 cells / ml on day 0, diluted 1 / 5 in new medium on day 4, washed and cultured at 100,000 cells / ml in new medium on day 7. Cells were diluted to 100,000 cells / ml and seeded in new medium on day 11. Cells were reseeded in new medium at 300,000 cells / ml on day 14. Cells were diluted to 500,000 / ml on day 18. Cells were systematically restored to 1 million / ml on each maintenance day (i.e., twice a week) from day 21 onwards.

[0279] Training program:

[0280] Protocol 1: From day 0 to day 11, cells were cultured in stromal medium. On day 11, the medium was supplemented with 253.9 μM DMOG. On day 14, 333 μM DMOG, 0.02 mM dexamethasone (DEX), and 30 μM SMER28 were added to the medium. On day 18, the medium was supplemented with 0.09 mM DEX and 20.1 μM SMER28. On day 21, 0.10 mM DEX and 24.5 μM SMER28 were added to the medium, while on day 25, 0.10 mM DEX and 17.4 μM SMER28 were added thereto. Finally, from day 28 onwards, the medium was systematically supplemented with 0.10 mM DEX and 13.8 μM SMER28.

[0281] Protocol 2: From day 0 to the end of culture, the culture medium was supplemented with 0.1 mM DEX and 2.27 μM SMER28.

[0282] Protocol 3: From day 0 to the end of culture, the culture medium was supplemented with 0.1 mM DEX.

[0283] Protocol 4: This protocol was a control protocol; it was performed using stromal medium without the addition of any factors.

[0284] Flow cytometry:

[0285] According to the supplier's instructions, 100,000 cell samples were collected, washed, and then exposed to CD235a and CD117 antibodies. After 30 minutes at room temperature and in the dark, the cells were washed twice with PBS. The cells were then ready for cell counting analysis.

[0286] The CD235a antibody specifically recognizes glycoprotein A, which indicates the involvement of mature erythroid cells.

[0287] The CD117 antibody specifically recognizes the receptor c-kit (i.e. stem cell factor receptor), which indicates the immaturity, lineage and self-renewal capacity of cells.

[0288] Cell counting:

[0289] Dilute cells to 1 / 10 in trypan blue solution and assess cell mortality if necessary.

[0290] result

[0291]

[0292] Table 1: Cell counts after culture according to different protocols

[0293] Protocols 1 and 2 allow exponential expansion of erythroblasts. The expansion obtained with protocols 1 and 2 is also substantially higher than that obtained with dexamethasone alone (see Table 1). It is also interesting to note that protocols 1 and 2 allow expansion of erythroid progenitors from CD34+ cells derived from cell apheresis or cord blood.

[0294] The persistence of C-KIT markers under these conditions was much longer than that under control conditions (see Figure 1 ); This membrane marker indicates the youthful state of cells and their proliferation capacity.

[0295] This study shows that culturing human hematopoietic stem cells in a culture medium according to the present invention can obtain:

[0296] - Cells with total erythroid involvement;

[0297] -Exponential enrichment and expansion of erythroid progenitor cells.

[0298] In particular, expansion may continue for up to 78 days.

[0299] Example 2: Expansion of erythroid progenitors from engineered HSCs derived from cytopheresis that inducibly overexpress HTERT, BMI1 and constitutively overexpress BCL-XL or inducibly overexpress HTERT, BMI1 and LMO2

[0300] Materials and methods

[0301] Medium used: IMDM (Biochrom), supplemented with transferrin (330 μg / mL), insulin (10 μg / mL), 5% AB serum pool (EFS) and heparin (3 U / mL). From day 0 to day 7, the medium was supplemented with IL-3 (5 ng / mL), SCF (100 ng / mL) and EPO (2 IU / mL). From day 7 to the end of erythroblast progenitor expansion, the medium was supplemented with SCF (100 ng / mL) and EPO (2 IU / mL).

[0302] HSC:

[0303] HSCs were obtained from cytopheresis following magnetic separation using Miltenyi CD34+ beads.

[0304] Cell maintenance:

[0305] Cells were seeded at 100,000 cells / ml on day 0 for two consecutive infections with HTERT BMI1 lentiviral supernatant and BCL-XL retroviral supernatant or HTERT BMI1 lentiviral supernatant and LMO2 lentiviral supernatant; cells were washed 3 times and re-seeded at 10,000 cells / ml on day 3, and cells were diluted to 1 / 5 in new medium on day 4. Cells were washed on day 7 and cultured at 100,000 cells / ml in new medium. Cells were diluted to 100,000 cells / ml on day 11 and seeded in new medium. Cells were re-seeded to 300,000 cells / ml in new medium on day 14. Cells were diluted to 500,000 / ml on day 18. From day 21 onwards, cells were systematically reset to 1 million / ml on each maintenance day (i.e., twice a week).

[0306] Training program:

[0307] Protocol 1: From day 0 to day 11, cells were cultured in stromal medium. On day 11, the medium was supplemented with 253.9 μM DMOG. On day 14, 333 μM DMOG, 0.02 mM dexamethasone (DEX), and 30 μM SMER28 were added to the medium. On day 18, the medium was supplemented with 0.09 mM DEX and 20.1 μM SMER28. On day 21, 0.10 mM DEX and 24.5 μM SMER28 were added to the medium, while on day 25, 0.10 mM DEX and 17.4 μM SMER28 were added. Finally, from day 28 onwards, the medium was systematically supplemented with 0.10 mM DEX and 13.8 μM SMER28.

[0308] Protocol 2: From day 0 to the end of culture, the culture medium was supplemented with 0.1 mM DEX and 2.27 μM SMER28.

[0309] Protocol 3: From day 0 to the end of culture, the culture medium was supplemented with 0.1 mM DEX.

[0310] Protocol 4: This protocol was a control protocol; it was performed using stromal medium without the addition of any factors.

[0311] result

[0312]

[0313] Table 2: Cell counts on day 65 after culture according to different protocols

[0314] Protocols 1 and 2 allowed exponential expansion of erythroblasts using both engineered HSC models (see Table 2). The expansion obtained with protocols 1 and 2 was also significantly greater than that obtained with dexamethasone alone.

[0315] The C-KIT marker persists much longer under these conditions than in controls; this membrane marker indicates the youthful state of the cells and their ability to proliferate (see Figure 2 and Figure 3 ). Protocol 2 allows for superior expansion using two types of engineered HSCs.

[0316] Since engineered cells are obtained from CD34 + , which is known for its low expansion rate (compared to CD34+ from umbilical cord blood), so these expansions are very significant.

[0317] Using these engineered cell protocols, CD34 + Compared with CD34 + The cells gain greater proliferation capacity.

Claims

1. An in vitro method for producing erythroid progenitor cells, the method comprising contacting hematopoietic stem cells with a cell culture medium comprising an autophagy inducer and a glucocorticoid, wherein the autophagy inducer is selected from the group consisting of SMER-28, SMER-10 and SMER-18, and combinations thereof.

2. The method of claim 1, wherein the autophagy inducer is SMER-28.

3. according to the method for claim 1 or 2, wherein said glucocorticoid is selected from cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazole and derivatives and mixtures thereof.

4. The method according to claim 1 or 2, wherein the glucocorticoid is selected from prednisone, prednisolone and dexamethasone.

5. The method of claim 1 or 2, wherein the glucocorticoid is dexamethasone. The method according to claim 1 , wherein the culture medium further comprises a HIF pathway activator.

7. The method according to claim 1, wherein the hematopoietic stem cells are obtained by differentiation of pluripotent stem cells, or are isolated from a patient's blood sample, from umbilical cord blood or placental blood, or from a bone marrow sample. The method according to claim 1 , wherein the hematopoietic stem cells are human hematopoietic stem cells.

9. The method of claim 1, wherein the hematopoietic stem cell is genetically modified to overexpress one or more genes selected from the group consisting of HTERT, BMI1, c-MYC, 1-MYC and MYB.

10. The method of claim 1, wherein the hematopoietic stem cell is genetically modified to overexpress the HTERT gene. The method according to claim 1 , wherein the hematopoietic stem cell is genetically modified to overexpress the BMI1 gene. 12 . The method according to claim 1 , wherein the hematopoietic stem cell is genetically modified to overexpress the HTERT gene and the BMI1 gene.

13. The method of claim 1, wherein the one or more genes selected from the group consisting of HTERT, BMI1, c-MYC, 1-MYC and MYB are placed under the control of one or more inducible promoters.

14. The method according to any one of claims 9 to 13, wherein the hematopoietic stem cell is further genetically modified to overexpress: - one or more core erythroid network pathway transcription factors; and / or - One or more EPO-R / JAK2 / STAT5 / BCL-XL pathway genes.

15. The method according to any one of claims 9 to 13, wherein the hematopoietic stem cells are further genetically modified to overexpress the BCL-XL gene.

16. The method according to any one of claims 9 to 13, wherein the hematopoietic stem cells are further genetically modified to overexpress the LMO2 gene.

17. The method of claim 14, wherein the one or more genes selected from the group consisting of EPO-R / JAK2 / STAT5 / BCL-XL pathway genes are placed under the control of one or more constitutive promoters.

18. The method of claim 14, wherein the one or more genes selected from the group consisting of core erythroid network pathway transcription factor genes are placed under the control of one or more inducible promoters.

19. The method of claim 1, wherein the hematopoietic stem cells are immortalized and / or comprise a suicide gene.

20. The method of claim 1, wherein the culture medium is a culture medium adapted to the nutritional requirements of hematopoietic stem cells.

21. The method of claim 1, wherein the cells are cultured in the culture medium for at least 20 days.

22. Use of a cell culture medium as defined in any one of claims 1 to 6 and 20 for producing and / or expanding erythroid progenitor cells.

23. An in vitro method for producing red blood cells, the method comprising: - Producing erythroid progenitor cells according to the method according to any one of claims 1 to 21; and - inducing maturation of said erythroid progenitor cells, - and optionally recovering the obtained red blood cells.

24. The method of claim 23, wherein maturation of the erythroid progenitor cells is induced by culturing the erythroid progenitor cells in an erythroid differentiation medium.

25. A cell culture medium adapted for the growth and / or differentiation of hematopoietic cells and comprising a glucocorticoid, and an autophagy inducer selected from the group consisting of SMER-28, SMER-10 and SMER-18, and combinations thereof, and optionally a HIF pathway activator.

26. The cell culture medium according to claim 25, comprising - a glucocorticoid at a concentration of 0.01 mM to 0.1 mM, and / or - an autophagy inducer at a concentration of 2 μM to 30 μM, and / or - HIF pathway activator at a concentration of 75 μM to 350 μM.

27. The cell culture medium of claim 25, wherein the autophagy inducer is SMER-28.

28. The cell culture medium according to any one of claims 25 to 27, wherein the glucocorticoid is selected from the group consisting of cortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone, triamcinolone, paramethasone, betamethasone, dexamethasone, cortivazole and derivatives and mixtures thereof.

29. The cell culture medium according to any one of claims 25 to 27, wherein the glucocorticoid is selected from prednisone, prednisolone and dexamethasone.

30. The cell culture medium according to any one of claims 25 to 27, wherein the glucocorticoid is dexamethasone.

31. The cell culture medium of claim 25, wherein the culture medium further comprises a HIF pathway activator.

32. The cell culture medium of claim 25, further comprising (i) transferrin, (ii) insulin, (iii) heparin, and (iv) serum, plasma, serum pool, or platelet lysate.

33. The cell culture medium of claim 25, further comprising SCF and EPO, optionally IL-3.

34. Use of the cell culture medium according to any one of claims 25 to 33 for producing and / or expanding erythroid progenitor cells.

35. A kit for producing erythroid progenitor cells and / or erythrocytes, the kit comprising: - A cell culture medium as defined in any one of claims 1 to 6, 20 and 25 to 33; and / or - A genetically modified hematopoietic stem cell as defined in any one of claims 9 to 18; and - Optionally, a guide comprising instructions for use of such a kit.

36. Use of the kit according to claim 35 for producing erythroid progenitor cells and / or erythrocytes.

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