Engineered red blood cells and method of production thereof

Abstract

The invention relates to a method for the production of red blood cells, in particular including a method of enucleation of erythrocyte progenitor cells that were previously immortalized. The invention provides an approach for the ex-vivo generation of red blood cells for blood replacement therapies, and products associated with such use.

Description

[0001] ENGINEERED RED BLOOD CELLS AND METHOD OF PRODUCTION THEREOF

[0002] FIELD OF THE INVENTION

[0003] [1] The invention relates to a method for the production of red blood cells, in particular including a method of enucleation of erythrocyte progenitor cells that were previously immortalized. The invention provides an approach for the ex-vivo generation of red blood cells for blood replacement therapies, and products associated with such use. In addition to the generation of enucleated erythrocytes as disclosed herein, the invention further pertains to a use of nucleated erythrocyte precursors for diagnostic and therapeutic purposes as well as materials such as engineered erythrocytes of the invention immunological therapeutic strategies, such as vaccination against infections and cancer, or in treating autoimmunity.

[0004] DESCRIPTION

[0005] [2] Blood transfusion in anemic patients represents the most established and most common form of cell therapy. While for most patients, erythrocytes can be obtained very easily and efficiently from healthy donors, there is a small group of people displaying rare blood group constellations or multiple pre-immunizations, who cannot be supplied with compatible donor- derived blood components. Especially, when patients with very rare blood group phenotypes - that occur as founder mutation in their family - suffer from a disease with chronic transfusion requirement, such as sickle cell disease (SCD), it is often impossible to provide compatible blood components. In most cases, this is due to previous, not fully matched, transfusions of packed red blood cells (RBCs), which have resulted in multiple immunizations against common blood group antigens. Here, advances in stem cell technology have opened the door to new approaches aiming at differentiation and expansion of mature RBCs from stem cells of different tissues to provide an alternative source for packed RBCs transfusions. Recently, the term “blood pharming” has emerged to describe this innovative field of ex vivo production of blood cells. In addition to broader compatibility, cultured RBCs have several potential advantages over donor blood, such as a reduced risk of infectious disease transmission, and as the cells are all still nascent, the volume and number of transfusions could be reduced in patients requiring regular transfusions.1 , 2 In fact, the feasibility of transfusions using ex vivo generated blood stem cell derived RBCs was pioneered by the successful autologous re-infusion of 1 mL ex vivo expanded bone marrow CD34+ derived RBCs into a patient in 2011.3 In addition, the currently ongoing clinical trial “RESTORE” is studying the lifespan of the lab grown allogeneic RBCs transfused at dosages of 5-10 mL, and compares this with infusions of standard RBCs from the same donor (https: / / www.nhsbt.nhs.uk).

[0006] [3] So far, ex vivo generation of erythrocytes was described from bone marrow-derived hematopoietic stem cells (HSCs) 1 , 2, cord blood-derived stem cells 4-7, peripheral blood stem cells (steady phase or mobilized) 4, 8-10, embryonic stem cells (ESCs) 4, 6, 11 , and induced pluripotent stem cells (iPSCs). 4, 6, 12, 13 While easily accessible, all of these potential sources have significant disadvantages. Albeit HSCs have a certain proliferation and differentiation capacity, their main limitation for routine clinical adaptation is donor dependency and their limited ex vivo expansion potential.14 Cord blood (CB) progenitor cells yield RBCs, which possess fetal rather than adult hemoglobin. 2- To address these fallbacks of primary HSCs, also stem cell lines have been used as a source to generate red blood cells. Here, the use of iPSCs, despite unlimited expansion, is hampered by - an impaired enucleation capacity along with a lengthy differentiation process.12, 15 Beside iPSCs, also ESCs have been used as they provide a potentially unlimited source of pluripotent stem cells for the ex vivo generation of RBCs, which on the downside is associated with both, ethical concerns and an increased risk for genomic instability and tumor formation.16 In addition, the use of iPSCs and / or ESCs involves very lengthy and laborious in vitro protocols, since RBCs cannot be generated from iPSCs or ESCs directly and the pluripotent stem cells first need to be differentiated into HSCs. In order to shorten the process, several groups have further immortalized nucleated red blood cells obtained from iPSCs 13 and HSCs 2, 6 by introducing proto-oncogenes. In fact, Hirose et al. showed that transduction of c-myc and BCL- XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells leads to overexpression of these genes, resulting in sustained exponential self-replication of glycophorin A+ erythroblasts 12. While the growth rate of such erythroblasts seems very intriguing, these cells unfortunately largely fail to further differentiate into enucleated and fully differentiated RBCs with enucleation rates of only 0.36 %.13 It has also been shown, that RBCs derived through this complex process of iPS / ES cell differentiation into HSCs, from which nucleated RBCs will be differentiated and subsequently immortalized, yield RBCs which are very different from natural occurring RBCs with regard to fragility and deformability.14

[0007] [4] In an attempt to abbreviate the lengthy differentiation process from iPSCs and ESCs, it has recently also been suggested to immortalize primary, patient or donor derived hematopoietic CD34 cells. While CD34+ HSCs from cord blood and from bone marrow where used as a starting cell population for immortalization, immortalization of nucleated reticulocyte progenitors has only been performed on nucleated reticulocyte progenitors derived from peripheral blood mononuclear cells (PBMCs).9 However, this resulted in a heterogenic population with only around 50% of CD235a-CD71-positive cells and a limited life span of up to eight month.9

[0008] [5] Thus, it is an object of the invention to provide novel strategies to generate red blood cells in vitro that can be applied in blood replacement therapies.

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] [6] Generally, and by way of brief description, the main aspects of the present invention can be described as follows. This invention enables to generate the first human immortalized adult erythroid progenitor cell line by Dox-inducible expression of c-Myc and BCL-XL in bone marrow derived CD71-positive erythroid progenitor cells, which are termed imBMEP-A - (immortalized bone marrow erythroid progenitor adult). This is the first erythroid cell line, generated from direct immortalization of bone marrow derived reticulocyte progenitors, that produces hemoglobin in the immortalized state. The invention thus relates to the following aspects:

[0011] [7] In a first aspect, the invention pertains to a method for the in-vitro production of immortalized genetically modifiable erythrocyte progenitor cell, comprising the steps of

[0012] • Providing a nucleated erythrocyte progenitor cell,

[0013] • Immortalizing the nucleated erythrocyte progenitor cell by an inducible transgenic expression of one or more immortalization factors,

[0014] • Optionally: propagating (expanding) the immortalized nucleated erythrocyte progenitor cell to obtain a plurality of immortalized nucleated erythrocyte progenitor cells suitable for storage.

[0015] [8] In a second aspect, the invention pertains to a method for the in-vitro production of an erythrocyte, comprising the steps of

[0016] • Providing a nucleated erythrocyte progenitor cell,

[0017] • Immortalizing the nucleated erythrocyte progenitor cell by inducing an inducible transgenic expression of one or more immortalization factors,

[0018] • Optionally: propagating (expanding) the immortalized nucleated erythrocyte progenitor cell to obtain a plurality of immortalized nucleated erythrocyte progenitor cells;

[0019] • Stopping the induction of the inducible transgenic expression of the one or more immortalization factors and differentiating the nucleated erythrocyte progenitor cell to a reticulocyte or erythrocyte,

[0020] • Enucleating the differentiated reticulocyte or erythrocyte to obtain the final in vitro produced erythrocyte;

[0021] Wherein alternative to conducting steps (a) to (c), the method may comprise a step (a-c’) of providing the immortalized genetically modifiable erythrocyte progenitor cell obtained by the method of claim 1 , and then performing steps (d) and (e) with the so provided immortalized genetically modifiable erythrocyte progenitor cell.

[0022] [9] In a third aspect, the invention pertains to an immortalized genetically modifiable erythrocyte progenitor cell, or a plurality of immortalized genetically modifiable erythrocyte progenitor cells, as obtained or obtainable by, a method of the invention.

[0010] In a fourth aspect, the invention pertains to an in-vitro produced erythrocyte, or a plurality of in-vitro produced erythrocytes, obtained by or obtainable by, a method of the invention.

[0023]

[0011] In a fifth aspect, the invention pertains to a blood cell product, comprising an in-vitro produced erythrocyte, or a plurality of in-vitro produced erythrocytes according to the invention.

[0024]

[0012] In a sixth aspect, the invention pertains to a method comprising producing a red blood cell with a method of any one of the preceding claims and formulating the red blood cell with one or more additives to form a transfusion blood composition.

[0025]

[0013] In a seventh aspect, the invention pertains to a method of enucleating an erythrocyte or erythrocyte progenitor cell, the method comprising a step of d bringing into contact the erythrocyte or erythrocyte progenitor cell, such as a differentiated reticulocyte, with an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as mesalazine), and I or with one or more agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine).

[0026]

[0014] In an eight aspect, the invention pertains to a use of a substance as enucleating agent, wherein the substance is selected from an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as mesalazine), and I or an agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine).

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028]

[0015] In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0029]

[0016] In a first aspect, the invention pertains to a method for the in-vitro production of immortalized genetically modifiable erythrocyte progenitor cell, comprising the steps of

[0030] • Providing a nucleated erythrocyte progenitor cell,

[0031] Immortalizing the nucleated erythrocyte progenitor cell by an inducible transgenic expression of one or more immortalization factors, Optionally: propagating (expanding) the immortalized nucleated erythrocyte progenitor cell to obtain a plurality of immortalized nucleated erythrocyte progenitor cells suitable for storage.

[0032]

[0017] In a second aspect, the invention pertains to a method for the in-vitro production of an erythrocyte, comprising the steps of

[0033] • Providing a nucleated erythrocyte progenitor cell,

[0034] • Immortalizing the nucleated erythrocyte progenitor cell by inducing an inducible transgenic expression of one or more immortalization factors,

[0035] • Optionally: propagating (expanding) the immortalized nucleated erythrocyte progenitor cell to obtain a plurality of immortalized nucleated erythrocyte progenitor cells;

[0036] • Stopping the induction of the inducible transgenic expression of the one or more immortalization factors and differentiating the nucleated erythrocyte progenitor cell to a reticulocyte or erythrocyte,

[0037] • Enucleating the differentiated reticulocyte or erythrocyte to obtain the final in vitro produced erythrocyte;

[0038] Wherein alternative to conducting steps (a) to (c), the method may comprise a step (a-c’) of providing the immortalized genetically modifiable erythrocyte progenitor cell obtained by the method of claim 1 , and then performing steps (d) and (e) with the so provided immortalized genetically modifiable erythrocyte progenitor cell.

[0039]

[0018] In context of the invention an “erythrocyte progenitor cell” refers to a hematopoietic stem cell derivative within the erythroid lineage that has not yet matured into a fully differentiated erythrocyte. For example, these cells are characterized by their capacity to proliferate and differentiate into nucleated erythroid precursors, such as erythroblasts, which subsequently mature into enucleated erythrocytes (red blood cells). Erythrocyte progenitor cells are identifiable by specific surface markers, including but not limited to, CD34+, CD36+, and Glycophorin A, which differentiate them from other hematopoietic stem cells and mature erythrocytes. This definition encompasses various stages of erythroid progenitors, including erythroid burst-forming units (BFLI-E) and erythroid colony-forming units (CFLI-E), which are crucial for erythropoiesis, the process of red blood cell formation."

[0040]

[0019] In context of the invention an erythrocyte progenitor cell can be a nucleated erythrocyte progenitor cell.

[0041]

[0020] " Enucleation" refers to the process of removing the nucleus from a cell, thereby generating an enucleated cell. This procedure can be applied to various types of cells, but it is most commonly associated with the production of erythrocytes (red blood cells) in a laboratory setting. Enucleation in the prior art can be achieved through several methods, including mechanical, chemical, or genetic manipulation techniques, which facilitate the removal of the nucleus while preserving the viability and functional integrity of the remaining cell cytoplasm. The term encompasses not only the physical removal of the nucleus but also any preparatory and followup steps that ensure the stability, functionality, and purity of the enucleated cells for their intended application.

[0042]

[0021] The present invention in particular aspects and embodiments pertain to a specific method for enucleation of a nucleated erythrocyte progenitor cell.

[0043]

[0022] Preferably it is provided that the nucleated erythrocyte progenitor cell is a CD34+ hematopoietic stem cell derived ex-vivo from a donor sample of (mobilized) peripheral blood, or is a CD71+ bone marrow cell or umbilical cord blood; most preferably wherein the provided nucleated erythrocyte progenitor cell is a human CD71+ bone marrow cell.

[0044]

[0023] In another preferred embodiment the nucleated erythrocyte progenitor cell is a direct reticulocyte progenitor cell.

[0045]

[0024] In yet another embodiment, the method preferably comprises that step (b), comprises transfection or transduction of an inducible expression vector into the nucleated erythrocyte progenitor cell.

[0046]

[0025] The present invention relates to an inducible expression of a transgene in order to generate an immortalized erythrocyte progenitor cell. Inducible expression is realized by introducing a nucleic acid construct or system into cells that allow for an inducible and controlled expression of the transgene of desire (such as cMyc). Such a nucleic acid construct or system includes at least one cis-acting regulatory element for directing expression of the nucleic acid sequence. Cis-acting regulatory sequences include those that direct constitutive expression of a nucleotide sequence as well as those that direct inducible expression of the nucleotide sequence only under certain conditions. Thus, for example, a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner is included in the nucleic acid construct. In the case of mRNA, since gene expression from an RNA source does not require transcription, there is no need in a promoter sequence, or the additional sequences involved in transcription described hereinbelow. The nucleic acid construct or system (also referred to herein as an "expression vector") of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain a transcription and / or translation initiation sequence, transcription and / or translation terminator and a polyadenylation signal. By way of example, such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.

[0047]

[0026] Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.

[0048]

[0027] Enhancer elements can stimulate transcription up to 1 ,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long-term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.

[0049]

[0028] In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[0050]

[0029] Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: Gil or II rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AALIAAA, located 11- 30 nucleotides upstream. Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.

[0051]

[0030] In context of the present invention an expression vector preferably comprises at least one or more expression cassettes suitable for an inducible expression of the one or more immortalization factors; and wherein the one or more expression cassettes comprises at least in operable connection an inducible element, an erythrocyte lineage active promoter and an expressible gene sequence ending one of the one or more immortalization factors.

[0052]

[0031] In further preferred embodiments, the inducible element is a DOX binding element.

[0053]

[0032] In additional preferred embodiments, the expression vector is a lentiviral expression vector.

[0033] It is particularly preferred that in certain embodiments, the one or more immortalization factors are c-Myc and I or BCL-XL.

[0054]

[0034] In context of the invention it is preferred that the step of differentiating the nucleated erythrocyte progenitor cell to a reticulocyte or erythrocyte comprises bringing into contact the nucleated erythrocyte progenitor cell with erythropoietin (EPO). The term "erythropoietin" and its abbreviation "EPO" refer to a protein having the amino acid sequence as shown in the Uniprot database under the accession no. P01588 (https: / / www.uniprot.org / uniprotkb / P01588 / entry - Version of February 13, 2024) or a protein or polypeptide substantially homologous thereto, whose biological properties relate to the stimulation of red blood cell production and the stimulation of the division and differentiation of committed erythroid progenitors in the bone marrow. Recombinant erythropoietin may be prepared via expression in eukaryotic cells, for example in CHO cells, or BHK cells, or HeLa cells by recombinant DNA technology or by endogenous gene activation, i.e. the erythropoietin glycoprotein is expressed by endogenous gene activation. The term "erythropoietin" also denotes variants of the protein, in which one or more amino acid residues have been changed, deleted, or inserted, and which has comparable biological activity as the not modified protein. Such variants are known to the skilled artisan.

[0055]

[0035] The method of the invention in another preferred embodiment comprises a step of genetically modifying the immortalized nucleated erythrocyte progenitor cell before differentiation, for example by genetically altering the expression of a blood group antigen, for example by gene editing. In this embodiment a reduction or complete removable of the expression of the blood group antigen is preferred.

[0056]

[0036] Therefore, in specific embodiments of the invention, known blood group antigens are removed using CRISPR-Cas9 mediated gene knock-outs. Such knock-outs are preferred to increase transfusion ability of blood preparations. For example, it is a preferred embodiment to remove antigens such as Rhesus CcEe, Kell, Duffy (see Hawksworth et al. EMBO Mol Med. 2018 Apr 26;10(6):e8454. Doi, incorporated by reference in its entirety).

[0057]

[0037] The method of the invention in another preferred embodiment comprises enucleating in step (e) bringing into contact the differentiated reticulocyte or erythrocyte with an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as and preferably mesalazine), and I or with one or more agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine).

[0058]

[0038] In context of the invention any compound or procedure resulting in an inhibition of ERK1 / 2 or SMAD2 / 3 signaling activity is suitable as an agent for enucleation in accordance with the invention. Specific non limiting examples ERK1 / 2 inhibitors are molecules capable of inhibiting the activity of ERK1 / 2 as determined by Western blot protein detection of phosphorylated ERK1 / 2 proteins. Preferably according to a particular embodiment, the ERK1 / 2 inhibitor is a small molecule agent. Non-limiting examples of ERK1 / 2 inhibitors (also known as MEK1 / 2 inhibitors) include PD0325901 (AXONMEDCHEM - AXON 1408), PD98059 (AXON ME DC HEM - Axon 1223), and PD 184352 (AXONMEDCHEM - AXON 1368); and / or even inhibitors of RAF (which is upstream of MEK / ERK pathway) such as Sorafenib tosylate (also known as BAY 43-9006 AXONMEDCHEM -AXON 1397) or SB 590885 (TOCRIS #2650). Others are known to the skilled artisan.

[0059]

[0039] A SMAD2 / 3 inhibitor in context of the invention refers to a substance that blocks the SMAD2 and SMAD3 signaling pathways, which are key components of the Transforming Growth Factor-beta (TGF-P) signaling cascade. Examples of small molecular inhibitors are B-431542, LY2109761 or halofuginone.

[0060]

[0040] In a third aspect, the invention pertains to an immortalized genetically modifiable erythrocyte progenitor cell, or a plurality of immortalized genetically modifiable erythrocyte progenitor cells, as obtained or obtainable by, a method of the invention.

[0061]

[0041] In some additional embodiments of the invention the methods and the below described products, may pertain further to manipulation of endogenous factors of the progenitor cells (or the immortalized cells) wherein the endogenous factors are selected from micro-RNA (miR)-30a-5p or a factor associated with the restoration of the actin filament network, preferably Scinderin.

[0062]

[0042] For example in this further embodiments it might be preferred that the method comprises an additional step of reducing the expression and / or function of micro-RNA (miR)-30a-5p. MIR30A microRNA 30a is a micro RNA and is expressed by a miR gene according to the Gene ID: 407029, updated on 8-Feb-2025 of the NCBI website of February 8, 2025

[0063] (htps: / / www.ncbi.nlm.nih.gov / gene / 407029). This micro RNA is well known to the person of skill in the art.

[0064]

[0043] There are many methods known to the skilled artisan how to reduce the expression and / or function of a given miR in a cell of choice. For example, the person of skill may use miR inhibitors in order to interfere with the expression and / or function of miR-30a-5p in a cell of the invention. An miRNA inhibitior targeting miR-30a-5p may be selected from an antisense compound, or from a genetic approach targeting the miR-gene directly, for example using the CRISPR-Cas9 or similar gene editing approaches. There are many ways and possibilities to genetically edit the genome of a cell of the invention in order to interfere with the expression and / or function of the miR-30a-5p. Some preferred approaches are disclosed herein in the example section. Additionally, compounds that are used as direct inhibitors of miR-30a-5p are selected from a PNA, an LNA, a ssRNA, a dsRNA, an mRNA, an antisense RNA, a ribozyme, an antisense oligonucleotide, a bifunctional antisense oligonucleotide, a pri-miRNA, an shRNA, an antagomir, an aptamer, an siRNA, a dsDNA, a DNAzyme, a ssDNA, polypeptide or active fragment thereof, an antibody, an intrabody, a transbody, a protein, an enzyme, a peptidomimetic, a peptoid, a transcriptional factor, or a small organic molecule, and the like. Preferred are nucleic acid compounds comprising an antisense sequence to the miR mature or immature sequence, preferably antagomirs or similar molecules.

[0065]

[0044] In some embodiments, the miR inhibitor compound of the invention is a compound such as nucleic acid that hybridizes with miR-30a-5p or having sequence complementarity to that of miR-30a-5p. In some embodiments, miR inhibitor compound of the invention is a compound such as nucleic acid having at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 99 or 100% sequence complementarity to that of miR-30a-5p.

[0066]

[0045] Suitable miR inhibitor compounds include double-stranded RNA (such as short- or smallinterfering RNA or "siRNA"), antagomirs, antisense nucleic acids, and enzymatic RNA molecules such as ribozymes. Each of these compounds can be targeted to a given miRNA and destroy or induce the destruction of the target miRNA. For example, expression of a given miRNA can be inhibited by inducing RNA interference of the miRNA with an isolated double- stranded RNA ("dsRNA") molecule which has at least 90%, for example 90%; 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence homology with at least a portion of the miRNA. In some embodiments, the dsRNA molecule is a "short or small interfering RNA" or "siRNA".

[0067]

[0046] siRNA useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter "basepaired"). The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA.

[0068]

[0047] As used herein, a nucleic acid sequence in a siRNA which is "substantially identical" to a target sequence contained within the target mRNA is a nucleic acid sequence that is identical to the target sequence, or that differs from the target sequence by one or two nucleotides. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are basepaired and are covalently linked by a single-stranded "hairpin" area. The siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. One or both strands of the siRNA can also comprise a 3 overhang. As used herein, a "3' overhang" refers to at least one unpaired nucleotide extending from the 3 '-end of a duplexed RNA strand. Thus, in some embodiments, the siRNA comprises at least one 3' overhang of 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length. In some embodiments, the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length. For example, each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").

[0069]

[0048] The siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. published patent application 2002 / 0173478 to Gewirtz and in U.S. published patent application 2004 / 0018176 to Reich et al, the entire disclosures of which are herein incorporated by reference.

[0070]

[0049] Expression of a given miRNA can also be inhibited by an antisense nucleic acid. As used herein, an "antisense nucleic acid" refers to a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-peptide nucleic acid interactions, which alters the activity of the target RNA. Antisense nucleic acids suitable for use in the present methods are single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a miRNA. Preferably, the antisense nucleic acid comprises a nucleic acid sequence that is 50-100% complementary, more preferably 75-100% complementary, and most preferably 95-100% complementary to a contiguous nucleic acid sequence in a miRNA. Without wishing to be bound by any theory, it is believed that the antisense nucleic acids activate RNase H or some other cellular nuclease that digests the miRN A / anti sense nucleic acid duplex.

[0071]

[0050] In some embodiments, the miR inhibitor is an antagomir and / or an antisense oligonucleotide. The ternTantagomir” as used herein refers to a chemically engineered small RNA that is used to silence miR-30a-5p. The antagomir is complementary to the specific miRNA target with either mis-pairing or some sort of base modification. Antagomirs may also include some sort of modification to make them more resistant to degradation. In some embodiments the antagomir is a chemically engineered cholesterol- conjugated single- stranded RNA analogue. Inhibition of miR-30a-5p can also be achieved with antisense 2'-0-methyl (2 - O-Me) oligoribonucleotides, 2'- 0-methoxyethyl (2'-0-MOE), phosphorothioates, locked nucleic acid (LNA), morpholino oligomers or by use of lentivirally or adenovirally expressed antagomirs (Stenvang and Kauppinen (2008), Expert Opin. Biol. Ther. 8(1):59-81). Furthermore, MOE (2'-0-methoxy ethyl phosphorothioate) or LNA (locked nucleic acid (LNA) phosphorothioate chemistry)-modification of single-stranded RNA analogous can be used to inhibit miRNA activity.

[0051] Antisense nucleic acids can also contain modifications of the nucleic acid backbone or of the sugar and base moieties (or their equivalent) to enhance target specificity, nuclease resistance, delivery or other properties related to efficacy of the molecule. Such modifications include cholesterol moieties, duplex intercalators such as acridine or the inclusion of one or more nuclease-resistant groups.

[0072]

[0052] Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described below. Exemplary methods for producing and testing are within the skill in the art; see, e.g., Stein and Cheng (1993), Science 261 : 1004 and U.S. Pat. No. 5,849,902 to Woolf et al., the entire disclosures of which are herein incorporated by reference.

[0073]

[0053] Further the method of the invention as described herein may be preferred, wherein the additional step of reducing the expression and / or function of miR-30a-5p is performed before and / or during the step of enucleation.

[0074]

[0054] Further preferred maybe that the expression and / or function miR-30a-5p comprises a step of gene editing the genome of the nucleated erythrocyte progenitor cell to remove or mutate the miR-30a-5p gene, and / or using an antisense nucleic acid construct to reduce the expression of miR-30a-5p.

[0075]

[0055] In an additional or alternative embodiment the methods of the invention comprise a further step of restoring the actin filament network in the erythrocyte progenitor cell.

[0076]

[0056] In this embodiment it is preferred that the step of restoring the actin filament network involves reducing the expression and / or function of a Scinderin protein or mRNA in the erythrocyte progenitor cell. Human Scinderin and its sequence can be derived from the UniProt database in the Version of February 14, 2025, under the accession number Q9Y6U3 (https: / / www.uniprot.org / uniprotkb / Q9Y6U3 / entry). The gene of Scinerin is called SCIN.

[0077]

[0057] Such specific method may comprise a step of genetically modifying the erythrocyte progenitor cell, for example using gene editing, to have a reduced expression of Scinderin protein or mRNA, or comprising a step of expressing or bringing into contact the cell with an antisense nucleic acid compound, such as an siRNA or shRNA. In this context the above disclosed with regard to the inhibition equally applies to inhibiting and I or reducing Scinderin protein and / or mRNA. Here any nucleic acid based antisense inhibitor has a sequence complementary to an mRNA sequence of Scinderin.

[0078]

[0058] In another embodiment indirect methods of the inhibition of Scinderin are also preferred in context of the present invention. For example it is known that protein kinase C inhibitors (e.g., bisindolylmaleimide I) as well as the inhibition of ERK1 / 2 (e.g., through U0126) exert a positive effect on the inhibition of Scinderin Furthermore, the present invention pertains to an embodiment of indirectly inhibiting Scinderin by use of calcium-free culture medium. For example, the method of the invention may comprise a step of culturing the cells in a calcium depleted medium for a duration sufficient to mediate a reconstruction of the active filament network.

[0079]

[0059] Another option to indirectly affect Scinderin function is the use of Calcium channel inhibitors. Such compounds are well known in the art and refers to agents that block the conduction of calcium ions (Ca2+) through calcium channels. As used herein, the term “calcium channel inhibitor” is intended to be synonymous with “calcium channel blocker”. Exemplary calcium channel inhibitors include verapamil, diltiazem, nifedipine, nimodipine, nicardipine, flunarizine, and cinnarizine.

[0080]

[0060] In a fourth aspect, the invention pertains to an in-vitro produced erythrocyte, or a plurality of in-vitro produced erythrocytes, obtained by or obtainable by, a method of the invention.

[0081]

[0061] In a fifth aspect, the invention pertains to a blood cell product, comprising an in-vitro produced erythrocyte, or a plurality of in-vitro produced erythrocytes according to the invention.

[0082]

[0062] In a sixth aspect, the invention pertains to a method comprising producing a red blood cell with a method of any one of the preceding claims and formulating the red blood cell with one or more additives to form a transfusion blood composition.

[0083]

[0063] In a seventh aspect, the invention pertains to a method of enucleating an erythrocyte or erythrocyte progenitor cell, the method comprising a step of d bringing into contact the erythrocyte or erythrocyte progenitor cell, such as a differentiated reticulocyte, with an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as mesalazine), and I or with one or more agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine).

[0084]

[0064] In preferred embodiments the enucleation agent is mesalazine.

[0085]

[0065] The method according to the invention is preferred, wherein the agent is brought into contact with the erythrocyte or erythrocyte progenitor cell for a time sufficient to enucleate the erythrocyte or erythrocyte progenitor cell.

[0086]

[0066] In an eight aspect, the invention pertains to a use of a substance as enucleating agent, wherein the substance is selected from an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as mesalazine), and I or an agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine). Definitions of such inhibitors are provided herein above.

[0087]

[0067] In addition to the generation of enucleated erythrocytes as disclosed herein, the invention further pertains to a use of nucleated erythrocyte precursors as described herein elsewhere, for diagnostic and therapeutic purposes. Furthermore, it should be understood, that the herein disclosed materials such as engineered erythrocytes are useful in immunological strategies, such as vaccination against infections and cancer, or useful in treating autoimmunity as an "adjuvant" through the expression or binding of costimulatory molecules or other binding domains.

[0088]

[0068] The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0089]

[0069] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0090]

[0070] It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

[0091]

[0071] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0092]

[0072] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety. A list of references is provided at the end of this specification / description.

[0093] BRIEF DESCRIPTION OF THE FIGURES

[0094]

[0073] The figures show:

[0074] Figure 1 : Generation of immortalized bone marrow derived erythroid progenitor cells (imBMEP-A). Human adult bone marrow CD71-positive cells were cultured for 24 h, before transduction with the Tet-inducible c-myc-p2A-BCL-XL construct. Two days after transduction, cells were transferred to expansion medium containing doxycycline and maintained in expansion medium thereafter. (A) Scheme of experimental approach. (B) Representative cytospins illustrating similar morphology of cells proliferating in continuous culture on days 0, 28, 100 and 365 compared to peripheral blood CD34-positive HSCs on days 5, 7, 11 and 15. (C) Cell pellets of imBMEP-A cells in continuous culture on days 105 and 413. (D) Growth curve of imBMEP-A cells maintained in undifferentiated, proliferative state. Cells were seeded at either 3.0 x 105 or 1.0 x 105 cells / mL on day 0 and cells were counted regularly, with total cell number extrapolated. Doxycycline and cytokines were refreshed every 3 to 4 days to maintain cells in undifferentiated state, n = 3. Data are presented as mean ± SEM. Tests were performed two-sided. **P < 0.01 ; ***P < 0.001 , ****p < 0.0001 (student t test).

[0095]

[0075] Figure 2: ImBMEP-A cells show characteristic markers for basophilic and polychromatic erythroblasts. (A) Flow cytometric analysis of imBMEP cells. Cells were positive for different erythroid surface marker (CD36, CD71 , CD44, CD105, CD235a) and negative for the stem cell markers CD34 and CD45. (B) Percentage of different erythroid cell subtypes in the heterogeneous imBMEP-A cell line. (C-D) Real-time PCR expression analysis of exogenous BCL- XL (C) and exogenous c-myc (D) in imBMEP-A cells in the presence and absence of doxycycline (n = 3 independent differentiation experiments). Relative fold changes in expression (normalized to GAPDH) were calculated by the AACT method and values are expressed as 2-AACT to the mean values of undifferentiated control (day 0). Data are presented as mean ± SEM. Tests were performed two-sided. *p < 0.05 (student t test).

[0096]

[0076] Figure 3: ImBMEP-A cells are capable for terminal differentiation. (A-B) Quantification of total BCL-XL (A) and c-myc (B) protein expression by western blot analysis during ex vivo erythropoiesis of primary HSCs and imBMEP-A cells. Expression was normalized to GAPDH protein expression as endogeneous loading control (fold change). (C) Western blot analysis from primary HSCs and imBMEP-A cells during erythropoiesis using antibodies against BCL-XL, c- myc and GAPDH as loading control. One of three representative experiments is shown. (D-E) Real-time PCR expression analysis of endogenous BCL-XL (C) and endogenous c-myc (D) in imBMEP-A cells in the presence and absence of doxycycline (n = 3 independent differentiation experiments) and primary HSCs. Relative fold changes in expression (normalized to GAPDH) were calculated by the AACT method and values are expressed as 2-AACT to the mean values of undifferentiated control (day 0). (F) Flow cytometric analysis of imBMEP-A cells during erythropoiesis in the presence and absence of SCF. Cells were double stained with FITC-labelled anti-CD36 and APC-labelled anti-CD235a antibodies. (G) Quantitative analysis of flow cytometric analysis. Distribution of erythroid differentiation stages were determined by the CD36-CD235a expression pattern of the cell population, n = 4. Data are presented as mean ± SEM. Tests were performed two-sided. *P < 0.05 (Mann-Whitney II test). (A-B, D-E) n = 3. Data are presented as mean ± SEM. Tests were performed two-sided. *P < 0.05 (student t test).

[0097]

[0077] Figure 4: ImBMEP-A cells are able to differentiate into enucleated red blood cells. (A) Evaluation of enucleated red blood cells derived from differentiated primary HSCs and imBMEP- A cells, determined via SYTO16-SYTOX flow cytometric analysis. One of four representative experiments is shown. (B) Quantitative analysis of enucleation rate, n = 4. Data are presented as mean ± SEM. Tests were performed two-sided. *P < 0.05 (Mann-Whitney II test). (C-D) Flow cytometric analysis of imBMEP-A cells and primary HSCs during erythropoiesis. Cells were dual stained by incubation with antibodies to a4 integrin and band 3 (C) as well as pi -integrin and a5- integrin (D).

[0098]

[0078] Figure 5: ImBMEP-A cells showed increased apoptosis compared to HSCs during erythroid differentiation. (A) Morphological analysis of primary HSCs and imBMEP-A cells during ex vivo erythropoiesis. Cells were stained with Giemsa solution (bar = 50pm). Arrows indicate dead cells and arrow heads indicate enucleated mature RBCs. (B) Apoptosis assay of imBMEP- A cells during erythroid differentiation. (C) Apoptosis assay of primary HSCs during erythroid differentiation. (B-C) n = 3. Data are presented as mean ± SEM. Tests were performed two-sided. *P < 0.05, **P < 0.01 , ***P < 0.001 , # significance between stem cells and imBMEP-A cells (student t test).

[0099]

[0079] Figure 6: imBMEP-A cells express globin chains from both fetal and adult hemoglobin. (A-D) Real-time PCR expression analysis of a-globin (A), p-globin (B), y-globin (C), and b-globin mRNA (D) in imBMEP-A cells in the presence and absence of doxycycline (n = 3 independent differentiation experiments) and primary HSCs. (E) Flow cytometric analysis of fetal Hb using FMH Quikquant™ kit in imBMEP-A cells and primary HSCs during erythroid differentiation. (F) Real-time PCR expression analysis of GATA-1 mRNA (D) in imBMEP-A cells in the presence and absence of doxycycline and primary HSCs. (G-H) Quantification of full length GATA-1 (G) and EDAG (H) protein expression by western blot analysis during ex vivo erythropoiesis of primary HSCs and imBMEP-A cells. Expression was normalized to GAPDH protein expression as endogenous loading control (fold change). (I) Western blot analysis from primary HSCs and imBMEP-A cells during erythropoiesis using antibodies against GATA-1 , EDAG and GAPDH as loading control. One of three representative experiments is shown. (A-D, F) Relative fold changes in expression (normalized to GAPDH) were calculated by the AACT method and values are expressed as 2-AACT to the mean values of primary HSCs (day 0). (A-D, F, G-H) n = 3 independent differentiation experiments. Data are presented as mean ± SEM. Tests were performed two-sided. *P < 0.05, n.s. not significant (student t test).

[0080] Figure 7: imBMEP-A cells are amenable to genetic manipulation. Flow cytometric confirmation of individual blood group knockouts in imBMEP-A cells. imBMEP-A blood group knockout lines were created using Cas9 mRNA and synthetic sgRNAs gene editing and single cell sorted into clonal sub-lines. Expression levels of targeted blood group antigens in knockout lines overlay with IgG controls indicating complete protein knockouts. Cells were stained with antibodies against either PE-labelled CD240DCE or APC-labelled CD238 (KEL). One of five representative single-cell clones of each individual knockout is shown.

[0100]

[0081] Figure 8: Mesalazine treatment of imBMEP-A cells leads to drastical increase of terminal erythroid differentiation. Cells were cultivated in the absence of doxycyclin and stimulated with different concentrations of mesalazine for 10 days. (A) Evaluation of enucleated red blood cells derived from imBMEP-A cells, determined via SYTO16-SYTOX flow cytometric analysis. (B) Morphological analysis of imBMEP-A cells during ex vivo erythropoiesis in the absence or presence of 20 mM mesalazine. Cells were stained with Giemsa solution (bar = 50pm).

[0101]

[0082] Figure 9 Generation of miR-30a-5p knockout in imBMEP-A cell line. Human adult bone marrow CD71 -positive cells were cultured for 24 h, before transduction with the Tet-inducible c-myc-p2A-BCL-XL construct. Two days after transduction, cells were transferred to expansion medium containing doxycycline and maintained in expansion medium thereafter. Two years after transduction imBMEP-A cells were knocked out for miR-30a-5p using CRISPR / Cas9 technology and single cell clones were generated. (A) Scheme of experimental approach. (B) K.O. of miRNA- 30a-5p in imBMEP-A cells was performed using two sgRNAs for dual-targeting miRNA-30a-5p in opposing direction. (C) Real-time PCR expression analysis of miR-30a-5p (n = 3 independent differentiation experiments). Relative fold changes in expression (normalized to U6) were calculated by the AACT method and values are expressed as 2'AACTto the mean values of HSCs (day 0). Data are presented as mean ± SEM. Tests were performed two-sided. (D) Sequence of the human miR-30a stem loop. Mature miRNAs were marked in red (miR-30a-5p) and blue (miR- 30a-3p), respectively. The sequence which was cut out is highlighted in grey. (E-F) Real-time PCR expression analysis of exogenous BCL-XL (E) and exogenous c-myc (F) in imBMEP-A cells in the presence (day 0) and absence of doxycycline (n = 3 independent differentiation experiments). Relative fold changes in expression (normalized to GAPDH) were calculated by the AACT method and values are expressed as 2'AACTto the mean values of imBMEP-A cells (day 0). Data are presented as mean ± SEM. Tests were performed two-sided. *p < 0.05 (student t test). (G-H) Quantification of total BLC-XL (G) and c-myc (H) protein expression by western blot analysis during in vitro erythropoiesis of primary HSCs and imBMEP-A cell lines. Expression was normalized to GAPDH protein expression as endogeneous loading control (fold change). (I) Western blot analysis from primary HSCs and imBMEP-A cell lines during erythropoiesis using antibodies against BCL-XL, c-myc and GAPDH as loading control. One of three representative experiments is shown. (J-K) Cell growth and viability in the proliferation and differentiation phase. Development of total cell number (J) and viability (K) during the transition from proliferation to differentiation. (L) Proliferative growth of both imBMEP-A cell lines during cell culture routine, adjusting cell density to 3x105cells / ml every 3-4 days, n = 4 biological replicates. Data are presented as mean ± SEM. Tests were performed two-sided. **p < 0.01 ; ***p < 0.001 (student t test).

[0102]

[0083] Figure 10: miR-30a-5p knockout facilitates terminal erythroid differentiation. (A) Morphologic analysis of both imBMEP-A cell lines during ex vivo erythropoiesis. Cells were stained with Giemsa solution (bar = 50pm). (B) Quantitative analysis of flow cytometric analysis. Distribution of erythroid differentiation stages were determined by the CD36-CD235a expression pattern of the cell population. (C) Quantitative analysis of enucleation rate, n = 4. Data are presented as mean ± standard deviation. Tests were performed two-sided. *p <0.05, **p <0.01 , ***p <0.001 (Mann-Whitney II test). (D) Evaluation of enucleated RBCs derived from differentiated primary HSCs and the imBMEP-A cell lines, determined via SYTO16-SYTOX flow cytometric analysis. One of four representative experiments is shown.

[0103]

[0084] Figure 11 : miR-30a-5p knockout promotes the temporary switch from fetal to adult hemoglobin production. (A-D) Real-time PCR expression analysis of a-globin (A), p-globin (B), y-globin (C) and b-globin mRNA (D) in imBMEP-A cell lines in the presence (day 0) and absence of doxycycline (n = 3 independent differentiation experiments) and primary HSCs. Relative fold changes in expression (normalized to GAPDH) were calculated by the AACT method and values are expressed as 2-AACTto the mean values of HSCs (day 0). Data are presented as mean ± SEM. Tests were performed two-sided. *p < 0.05 (student t test). (E) Flow cytometric analysis of fetal Hb using FMH Quikquant kit in imBMEP-A cell lines and primary HSCs during erythroid differentiation.

[0104]

[0085] Figure 12: Transcriptomic and proteomic analysis during erythroid differentiation. (A-B) Transcriptomic analysis. (C, D) Proteomic analysis. Comparative analysis of the signalling pathways between imBMEP-A cells at day 0 and HSCs at day 7 of erythroid differentiation (A, C). Comparative analysis of the signalling pathways between imBMEP-A cells at day 7 and HSCs at day 11 of erythroid differentiation (B, D). The plots show 20 altered pathways with the highest significance between the individual comparisons. Pathways involved in the regulation of apoptosis are highlighted in light blue, those involved in the regulation of the cytoskeleton in red. (E) Western blot analysis from primary HSCs and imBMEP-A cell lines during erythropoiesis using antibodies against actin, a-tubulin, vimentin, scinderin, gelsolin and GAPDH as loading control. One of four representative experiments is shown. (F-J) Quantification of total scinderin (F) and gelsolin (G), -tubulin (H), actin (I) and vimentin (J) protein expression by western blot analysis during in vitro erythropoiesis of primary HSCs and imBMEP-A cell lines. Expression was normalized to GAPDH protein expression as endogenous loading control (fold change).

[0105]

[0086] Figure 13: Scinderin knockout promotes terminal erythroid differentiation by restoring the actin filament network (A) Morphological analysis of imBMEP-miR30a-K.O. cells and imBMEP-SCIN-K.O. cells. Cells were stained with Giemsa solution (bar = 50pm). The red arrow heads indicate enucleating cells, the turquoise arrow heads indicate reticulocytes. (B) Quantitative analysis of flow cytometric analysis. Percentage of different erythroid cell subtypes in the imBMEP-miR30a-K.O and imBMEP-SCIN-K.O. cell lines. (C) Immunofluorescence analysis. Undifferentiated and differentiated cells were stained with Alexa Fluor 488-labelled phalloidin and DAPI. (D) Quantitative analysis of flow cytometric analysis. Distribution of erythroid differentiation stages were determined by the CD36-CD235a expression pattern of the cell population. (E) Quantitative analysis of enucleation rate, n = 4. Data are presented as mean ± standard deviation. Tests were performed two-sided. *p <0.05, **p <0.01 , ***p <0.001 (Mann- Whitney Utest).

[0106]

[0087] Figure 14: imBMEP-A cells show a high rate of apoptosis, which cannot be rescued by SCIN knockout. (A) Live cell imaging of imBMEP-miR30a-K.Q. cells. Nuclei were labelled with Incucyte Nuclight Rapid Red Dye. The yellow circles mark an enucleating cell and an apoptotic cell. Bar = 50pm. (B) Flow cytometric analysis of ROS production during erythroid differentiation of HSCs and immortalized cell lines. (C-E) Flow cytometric analysis of viable cells (C), early apoptotic cells (D) and late apoptotic cells using the FITC Annexin V Apoptosis Detection Kit I. (F) Development of viability during erythroid differentiation, n = 3 independent differentiation experiments. Data are presented as mean ± SEM. Tests were performed two sided. *p <0.5, **p <0.01 (student t test).

[0107]

[0088] Figure 15: Constitutive upregulation of endogenous BCL-XL in imBMEP-A cells delays apoptosis. (A) Real-time PCR expression analysis of BCL-XL mRNA in imBMEP-A cell lines in the presence (day 0) and absence of doxycycline (n = 3 independent differentiation experiments) and primary HSCs. Relative fold changes in expression (normalized to GAPDH) were calculated by the AACT method and values are expressed as 2'AACTto the mean values of HSCs (day 0). NTC = non-target control, ABCB10 = ATP binding cassette subfamily B member 10, BCL-XL = B-cell lymphoma extra-large. (B) Quantitative analysis of flow cytometric analysis. Distribution of erythroid differentiation stages were determined by the CD36-CD235a expression pattern of the cell population. (C-E) Flow cytometric analysis of viable cells (C), early apoptotic cells (D) and late apoptotic cells using the FITC Annexin V Apoptosis Detection Kit I. (F) Development of viability during erythroid differentiation. Data are presented as mean ± SEM. Tests were performed two-sided. *p <0.5, **p <0.01 , ***p <0.001 (student t test). EXAMPLES

[0108]

[0089] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0109]

[0090] The examples show:

[0110]

[0091] Example 1 : Generation of stable erythroid progenitor cell lines from human CD71- positive cells with inducible c-myc-p2A-BCL-XL expression

[0111]

[0092] Isolated CD71-positive cells from human bone marrow (BM) were transduced with c-myc- p2A-BCL-XL using an antibiotic-inducible lentiviral vector system. 48 h after transduction, the induction phase was initiated by adding doxycycline and puromycin to select transduced cells. At day 100, CD235a-positive cells were isolated from the heterogenous immortalized cell population and single-cell clones were generated with proven monoclonality by using the single-cell-printing technology (SOP).17Figure 1A visualizes the outline of the protocol. Based on the extent of proliferation rate and expression of erythroid markers, as well as hemoglobin formation, we selected clone 34 (hereinafter referred to as imBMEP-A) for all further analyses. The imBMEP-A cells displayed continuous proliferation and were frozen after 170 days - well beyond the Hayflick limit18. Additionally, samples were frozen at regular intervals throughout this period and reestablished efficiently in culture following freezing and thawing. The mean doubling time of the cells after one year in continuous culture was 49.1 h ± 6.5 h. Morphological analysis of the immortalized cells showed that they are nucleated cells with largely basophilic erythroblast character as compared to peripheral blood CD34+ hematopoietic stem cells, which were mostly enucleated after 15 days (Fig. 1B). There were no changes in morphology over time. Furthermore, pelleted erythroid cells from imBMEP-A line showed the typical red color, which indicates hemoglobin production (Fig. 1C). The expansion profile of imBMEP-A after 200 days in culture shows an exponential growth over 7 days until a plateau is finally reached (Fig. 1D). Transduction with other oncogenes, such as SV40 LargeT antigen, LhX-2, Bmi-1 or c-myc and BCL-XL alone did not result in continuous proliferation and cells died after a maximum of 40 days (data not shown).

[0112]

[0093] Example 2: Characterization of the imBMEP-A line

[0113]

[0094] To confirm the basophilic erythroblast character of the imBMEP-A line we performed flow cytometric analysis for lineage-specific surface markers. ImBMEP-A cells highly expressed erythroid markers such as CD71 , CD36, CD44, and CD235a, and were negative for the hematopoietic stem cell markers CD34 and CD45 (Fig. 2A). Furthermore, we subdivided the cells into different subpopulations according to the expression pattern of specific surface markers such as CD36 and CD235a.19The preferred cell type of the imBMEP-A line corresponded to basophilic erythroblasts. A small fraction showed characteristic expression patterns for polychromatic erythroblasts (Fig 2B).

[0114]

[0095] Example 3: ImBMEP-A cells are capable of terminal differentiation

[0115]

[0096] Removal of doxycycline from the culture medium led to silencing of exogenous BCL-XL and c-myc, measured at both the mRNA (Fig. 2C, D) and protein levels (Fig. 3A-C). Interestingly, during normal erythropoiesis of HSCs, endogenous BCL-XL was upregulated more than 600-fold until day 15 of erythroid differentiation (Fig. 3D). In contrast, endogenous BCL-XL expression was upregulated by only one-tenth of the physiological level observed in HSCs, once the exogenous BCL-XL gene was silenced in imBMEP-A cells. The highest levels of endogenous BCL-XL were observed at day 5 of erythroid differentiation and afterwards it was downregulated. An opposite effect was observed for the expression of endogenous c-myc mRNA (Fig. 3E). During physiological erythropoiesis of HSCs c-myc is downregulated, whereas imBMEP-A cells showed a slightly but significant upregulation of c-myc mRNA.

[0116]

[0097] Furthermore, the number of orthochromatic erythroblasts increased dramatically over differentiation time. The absence of SCF during differentiation yielded significantly more orthochromatic erythroblasts from day 5 onward compared to cell cultures with SCF supplementation (Fig. 3F, G). Orthochromatic erythroblasts were highly positive for CD235a, but negative for CD36.19

[0117]

[0098] The enucleation process was monitored using two different DNA-staining dyes, SYTO16 and SYTOX blue, which are cell-permeable and cell-impermeable fluorescent dyes, respectively20(Fig. 4A, B). Whereas HSCs yielded ~ 40 % enucleated cells after 15 days of erythroid differentiation culture, the same conditions resulted in only a small amount of enucleated reticulocytes in the imBMEP-A line (~ 5 % at day 10 of erythroid differentiation).

[0118]

[0099] To assess differentiation in more detail, the expression of Band 3 (CD233) vs a4-integrin and a5-integrin vs pi-integrin was analyzed using dual staining. During erythroid differentiation, the expression of Band 3 protein increased, whereas that of a4-integrin expression strongly decreased. In contrast, the expression of a4-integrin in HSCs was only slightly decreased at the end of differentiation (day 10) (Fig. 4C). A similar pattern was seen when cells were stained with anti-a5-integrin (CD49e) (Fig. 4D), whereas pi-integrin (CD29) was not detected in both HSCs and imBMEP-A cells during erythroid differentiation. However, undifferentiated imBMEP-A cells still expressed p 1 -integrin in ~50 % of cells.

[0119]

[0100] Example 4: ImBMEP-A cells showed increased apoptosis compared to HSCs during erythroid differentiation

[0101] Morphologically, imBMEP-A cells showed decreased cell size, more condensed nuclei, and a lighter cytoplasm during differentiation, which is comparable to the differentiation process of HSCs (Fig. 5A). At day 7 of differentiation, imBMEP-A cells corresponded to orthochromatic erythroblasts, resembling day 11 of HSC differentiation. In HSC-derived differentiation cultures, we observed many enucleated reticulocytes at day 15 and day 18, whereas differentiation of imBMEP-A led to only very low numbers of enucleated cells (arrowheads in Fig. 5A). However, increased cell death was detectable (arrows). Thus, apoptosis was subsequently evaluated by Annexin V and PI staining in both differentiated imBMEP-A cells (Fig. 5B) and HSCs (Fig. 5C). There was a strong increase of early apoptotic cells from day 3 and late apoptotic / necrotic cells from day 5, respectively. Due to the thawing process, HSC cultures contained a large population of early apoptotic cells, which decreased during the first three days until it finally normalized. However, the population of late apoptotic cells was significantly lower compared to imBMEP-A cells and did not increase significantly throughout the course of differentiation.

[0120]

[0102] Example s: imBMEP-A cells express globin chains of both fetal and adult hemoglobin

[0121]

[0103] We next examined differential globin chain expression in imBMEP-A cells and HSCs at the mRNA level (Fig. 6A-D). In contrast to HSCs, inBMEP-A cells already expressed a-, p-, y- and b-globin at day 0 (undifferentiated state). The respective levels of globin chains expression was similar to that of HSCs of erythroid differentiation at day 5, except for the 6-globin , which was similar to the expression in HSCs at day 0. Flow cytometry revealed further differences in hemoglobin type composition between HSCs and imBMEP-A cells. Whereas the majority of HSCs produce adult hemoglobin (FITC-negative section) after induction of hemoglobinization at day 7 - 15, imBMEP-A cells show a predominant proportion of fetal hemoglobin (FITC-positive section) throughout the entire differentiation period. Instead of a clear switch from fetal to adult hemoglobin type, only a slight shift is detectable after 10 days of differentiation (Fig. 6E).

[0122]

[0104] Regarding TF GA TA 1, which plays a central role in erythropoiesis21, there was a slight increase in mRNA expression until day 5 which dropped to the original level on day 10 (Fig. 6F). In HSCs, mRNA expression of GATA 1 was continuously upregulated during erythropoiesis. Similar, protein levels of GATA1 increased steadily during erythropoiesis in HSCs, unlike in imBMEP-A cells, where GATA1 expression increased until day 5 and then decreased again (Fig. 6G). Another important factor of erythroid differentiation, the erythroid differentiation-associated gene (EDAG)22was upregulated during normal erythropoiesis (Fig. 6H, I), but markedly downregulated during differentiation of imBMEP-A cells.

[0123]

[0105] Example 7: imBMEP-A cells are amenable to genetic manipulation

[0106] As it is our aim to produce universal compatible RBCs, we next investigated, whether the cells are amenable to genetic manipulation i.e. to the knock out of different blood group antigens. Since the imBMEP-A cell line was derived from a donor of Rh type D+C-c+E+e-, both RhD and RhCE required removal. Thus, single sgRNAs targeting the RH-associated glycoprotein (RHAG) were designed. Mutations in RHAG result in Rhnuii erythrocytes, demonstrating that RHAG is essential for stable expression of Rh.23’24The Rhnuii phenotype is associated with mild compensated anemia.25Therefore, in parallel, we designed single sgRNAs targeting only RHD / CE rather than completely removing RhAG. Furthermore, we knocked out KEL2 expression, which corresponds to the KEL negative phenotype. Afterwards, single-cell clones were generated and analyzed for absence of CD240DCE and CD238 expression. We provided two targeted sgRNAs and generated single-cell clones from those cells in which the knock out with the corresponding sgRNA was most successful. Fig. 7 shows the complete absence of expression of proteins targeted by CRISPR gRNAs in knockout imBMEP-A cells compared with unedited imBMEP-A cells, as measured by flow cytometry. In parallel, 3x knockout was performed using single gRNAs to target RhAG along with single gRNAs to target KEL2.

[0124]

[0107] Example s: Enucleation method

[0125]

[0108] Currently, the highest enucleation rate achieved during differentiation of immortalized erythroid cells is approximately 30% (doi: 10.1016 / j.omtm.2021.06.002). However, a much higher rate of at least 70-80% is required for successful practicable clinical application. Therefore, we performed optimization of culture conditions and tested different substances that influence different pathways involved in enucleation process. We treated imBMEP-A cells with different concentrations of mesalazine in the absence of doxycycline and measured the enucleation rate at day 10 of differentiation. The enucleation process was monitored using two different nucleic acid dyes. While the green fluorescent SYTO16 is membrane-permeable, SYTOX Blue only penetrates dead cells with compromised plasma membranes (doi:10.1038 / nature03964). Mesalazine treatment of imBMEP-A cells leads to drastical increase of terminal erythroid differentiation (Fig. 8A). Giemsa staining of cells treated with 20mM mesalazine showed more than 80% enucleated reticulocytes (Fig. 8B).

[0126]

[0109] Example 9: miR-30a-5p knockout facilitates terminal erythroid differentiation

[0127]

[0110] Since previous studies have shown that miR-30a-5p negatively affects terminal erythroid differentiation, we used our imBMEP-A cell line [2] to knock out miR-30a-5p using CRISPR / Cas technology. K.O. of miRNA-30a-5p in imBMEP-A cells was performed using two sgRNAs for dualtargeting miRNA-30a-5p in opposing direction (Fig 9A) and led to downregulation of miR-30a-5p by about 60%, measured by qPCR (Fig. 9B). However, DNA sequencing of the miRNA stem loop demonstrated a complete absence of the miR-30a-5p, whereas the DNA sequence of miR-30a- 3p still existed (Fig. 9C). The resulting cell line imBMEP-miR30a-K.O. closely resembled the original cell line in terms of morphology, expression of specific surface markers, hemoglobin production and doubling time (Fig. 9D). The phenotype of both cell lines corresponded to mature erythroblasts, with the predominant cell type being basophilic erythroblasts (-60%). A small fraction showed expression patterns characteristic of polychromatic erythroblasts (~ 25%). The number of orthochromatic erythroblasts was approximately 10-fold higher after miR-30a-5p K.O. compared to the original cell line imBMEP-A. Both exogenous BCL-XL and c-myc were similarly silenced compared to imBMEP-A cells after removal of doxycycline from the culture medium, as measured at both the RNA (Fig. 9E, F) and protein levels (Fig. 9G-I).

[0128]

[0111] To investigate the proliferation potential of the imBMEP-A and imBMEP-miR30a-K.O. cell lines, the total cell number was determined over the course of 21 days of cultivation. During the first 7 days, the cells were cultured under proliferative conditions, i.e. the expression of exogenic c-Myc and BCL-XL was induced by the addition of doxycycline. This led to a continuous increase in total cells, suggesting a proliferation-triggering effect initiated by the proto-oncogene expression in both cell lines. After 7 days, the cells were transferred to differentiation medium without the addition of doxycycline, resulting in the termination of exogenous proto-oncogene expression. This resulted in the cessation of the proliferation trigger, leading to a stable cell number over the next 14 days of cultivation. In contrast, the human erythroleukaemia cell lines K562 and HEL showed continuous growth over the first 14 days of cultivation, followed by a decline in cell number induced by decreasing cell viability (Fig. 9J-L). During the proliferation phase, the cells grew stably during cell culture maintenance when the cell density was adjusted to 3x105cells / mL every 3-4 days (Fig. 9L).

[0129]

[0112] In addition, the absence of miR-30a-5p resulted in significantly more orthochromatic erythroblasts from day 5 compared to the wild type (wt) imBMEP-A cell line (Figure 10A,B), as measured by the expression of CD36 and CD235a.

[0130]

[0113] The enucleation process was monitored and quantified using the green fluorescent SYTO16 and SYTOX Blue. Although, K.O. of miR-30a-5p led to a 3-fold increase of enucleation rate up to 8.9 ± 1.7%, the efficiency of enucleation remained very low compared to RBCs derived from HSCs ex vivo (Fig. 10C,D).

[0131]

[0114] Example 10: miR-30a knockout promotes the temporary switch from fetal to adult hemoglobin production

[0132]

[0115] We next examined the mRNA expression levels of the adult and fetal globin chains in imBMEP-miR30a-K.O. cells compared to the wt cell line and HSCs (Fig. 11A-D). We found no changes in the mRNA expression of a-, -, y- and b-globin in the absence of miR-30a-5p compared to the wt cell line. In contrast, expression levels of a-, p- and y-globin in HSCs were significantly higher compared to the immortalized cells, while y-globin expression was significantly increased in the immortalized cell lines (Fig. 11C). However, flow cytometric analysis of fetal hemoglobin (HbF) showed a temporary switch from fetal to adult hemoglobin in the imBMEP- miR30a-K.O. cells at day 7 of erythroid differentiation, which was not seen in the original cell line and was reversible by day 10 (Fig. 11E). Interestingly, during in vitro erythropoiesis, HSCs also expressed ~25 % HbF.

[0133]

[0116] Example 11 : Transcriptomic and proteomic analysis during erythroid differentiation

[0134]

[0117] To identify the molecular mechanisms that may be altered in our immortalized cell lines and negatively affect terminal erythropoiesis, we compared the gene and protein expression profiles of our immortalized cell lines with differentiated HSCs (Fig. 12 A-D). Therefore, on day 7 of erythroid differentiation, HSCs were chosen as a comparison to imBMEPs on day 0, as they show a comparable phenotype at these time points. In addition, at day 11 of differentiation HSCs were compared to imBMEPs at day 7, as most cells are in the process of enucleation at this time. Principal component analysis (PCA) revealed clear differences in gene and protein expression between HSCs and imBMEPs during erythroid differentiation. Although there are changes in the expression profile between the proliferation and differentiation stages of the immortalized cells, there are clear differences between imBMEPs and HSCs during differentiation. More than 6000 genes were up- or downregulated in the immortalized cell line in both the undifferentiated and differentiated stages compared to HSCs. Furthermore, proteomic analysis revealed more than 1300 proteins that were dysregulated in the imBMEP-miR30a-K.O. cell line compared to HSCs. In addition, more than 100 proteins are up- or down- regulated in the imBMEP-miR30a-K.O. cell line compared to the original cell line, which is reduced to about 20 proteins that are differentially expressed in the course of differentiation.

[0135]

[0118] Furthermore, using Ingenuity Pathway Analysis (IPA) on the DEG data to map the gene expression differences between the immortalized cells and HSCs to specific functions and regulatory networks, these differences were mainly associated with metabolic changes, apoptosis-promoting pathways and negative regulation of cytoskeletal dynamics (Fig. 12A-D). With regard to the cytoskeleton, it was predicted that actin polymerisation in particular is inhibited at several levels within the regulatory networks. The actin capping and severing proteins scinderin (SCIN) and gelsolin were highly expressed in the imBMEP-A cells and completely absent or downregulated in the HSCs (Fig. 12E-G). While, a-tubulin is significantly increased in the immortalized cell lines compared to HSCs (Fig. 12H), the expression of both actin and vimentin does not seem to be affected by the immortalisation (Fig. 121, J).

[0119] Example 12: Scinderin knockout promotes terminal erythroid differentiation by restoring the actin filament network

[0136]

[0120] Since SCIN is highly expressed in the imBMEP-miR30a-K.O. cell line and absent in HSCs, we next performed SCIN K.O. using CRISPR / Cas9 technology. After generation of single cell clones, knockout was confirmed by Sanger sequencing. Morphological analysis revealed an increased number of spontaneously enucleating cells in the SCIN K.O. cell line compared to imBMEP-miR30a-K.O. wt cells in the presence of DOX (Fig. 13A). Furthermore, the number of polychromatic erythroblasts in SCIN knockout cells was approximately 10% higher than in wt cells (Fig. 13B). Actin filament network formation was observed during erythroid differentiation by immunofluorescence staining of F-actin using fluorescence-labelled phalloidin. A uniform distribution of actin filaments appeared in HSCs at day 7 of differentiation, whereas the formation of a contractile actomyosin ring (CAR) was observed in cells undergoing enucleation (Fig. 13C). Further differentiated HSCs at day 11 showed thicker F-actin bundles, while in some cells ongoing enucleation was detectable by the presence of CARs. Compared to HSCs, imBMEP-A cells at the proliferation stage (day 0) showed small accumulations of F-actin, which increased during erythroid differentiation, while the formation of a uniform actin network decreased. The same was observed in imBMEP-miR30a-K.O. cells, with the difference that some cells at day 0 showed CAR formation. Cells with additional K.O. of SCIN showed a higher intensity of the F-actin network and CAR formation. As differentiation progressed, no accumulated F-actin spots were observed, while an evenly distributed actin filament network was restored.

[0137]

[0121] Although, SCIN knockout resulted in an increase in terminal erythropoiesis, with a mean value of 22.1 ± 1.7% more orthochromatic erythroblasts, enucleation rates remained unaffected (Fig. 13D, E).

[0138]

[0122] Example 13: imBMEP-A cells show a high rate of apoptosis, which cannot be rescued by SCIN knockout

[0139]

[0123] Live cell imaging using the IncuCyte live-cell imaging system confirmed the low enucleation efficiency of the immortalized cells, while cell death was detectable in parallel (Fig. 14A). Since reactive oxygen species (ROS) are indicators of oxidative stress and apoptosis, we next measured the ROS production in our immortalized cell lines compared to HSCs during erythroid differentiation (Fig. 14B). Determination of the total amount of ROS in HSCs showed a low level of ROS at day 0 of erythroid differentiation, which increased sharply until day 5 and then decreased completely with further progression of erythroid differentiation. Compared to HSCs, imBMEP-A cells showed a high level of ROS at day 0, which decreased to a constant but not completely absent level throughout the differentiation period. The detection of ROS in imBMEP- miR30a-K.O. cells showed a pattern similar to that of HSCs during erythroid differentiation, with a low baseline level followed by a strong increase until day 5 and day 7 of erythroid differentiation and a subsequent decrease back to baseline. In contrast to HSCs, the amount of ROS did not decrease completely, but remained at a low concentration. Evaluation of apoptosis by annexin V and propidium iodide staining in both differentiated imBMEP-A and imBMEP-miR30a-K.O. cells showed a reduced number of late apoptotic cells after miR-30a-5p knockout, whereas the number of total live and early apoptotic cells was unaffected (Fig. 14C-F). However, SCIN knockout led to an increase in apoptosis at the end of erythroid differentiation.

[0140]

[0124] Example 14: Constitutive upregulation of endogenous BCL-XL in imBMEP-A cells delays apoptosis

[0141]

[0125] To overcome the hurdle of increased apoptosis during erythroid differentiation, we activated endogenous BCL-XL by CRISPR / dCas9 technology using lentiviral vector gene transfer (Fig. 15), since it is downregulated in the imBMEP-A cell lines compared to HSCs (Fig. 9G,I). As controls, a non-target control (NTC) and sgRNAs specific for ABCB10, which is not related to apoptosis or erythropoiesis, was used. Constitutive upregulation of genes was confirmed by qPCR (Fig. 15A). Although there is a significant increase in expression of endogenous BCL-XL compared to the NTC and ABCB10 overexpressing cells, the expression remains significantly lower compared to the expression in HSCs. Furthermore, upregulation of BCL-XL expression in the imBMEP-miR30a-K.O. cells had no effect on erythroid differentiation compared to NTC and ABCB10 overexpressing cells (Fig. 15B). However, cells overexpressing BCL-XL showed a delay in apoptosis, resulting in higher levels of early apoptotic cells but lower levels of late apoptotic cells at the end of erythroid differentiation (Fig. 15C-F).

[0142] REFERENCES

[0143]

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[0166] 25. Sturgeon P. Hematological observations on the anemia associated with blood type Rhnull. Blood 1970 Sep; 36(3): 310-320. Mei Y, Liu Y, Ji P. Understanding terminal erythropoiesis: An update on chromatin condensation, enucleation, and reticulocyte maturation. Blood reviews 2020 Mar; 46: 100740. Menon V, Ghaffari S. Erythroid enucleation: a gateway into a "bloody" world. Experimental hematology 2021 Mar; 95: 13-22. Kurita R, Funato K, Abe T, Watanabe Y, Shiba M, Tadokoro K, et al. Establishment and characterization of immortalized erythroid progenitor cell lines derived from a common cell source. Experimental hematology 2019 Jan; 69: 11-16. Soboleva S, Kurita R, Kajitani N, Akerstrand H, Miharada K. Establishment of an immortalized human erythroid cell line sustaining differentiation potential without inducible gene expression system. Hum Cell 2022 Jan; 35(1): 408-417. Hafid-Medheb K, Poindessous-Jazat V, Augery-Bourget Y, Hanania N, Robert- Lezenes J. Bcl-XL induction during terminal differentiation of friend erythroleukaemia cells correlates with delay of apoptosis and loss of proliferative capacity but not with haemoglobinization. Cell Death Differ 1999 Feb; 6(2): 166-174. You X, Liu F, Zhang T, Lv N, Liu Q, Shan C, et al. Hepatitis B virus X protein upregulates Lin28A / Lin28B through Sp-1 / c-Myc to enhance the proliferation of hepatoma cells. Oncogene 2014 Jan 23; 33(4): 449-460. Lee YT, de Vasconcellos JF, Yuan J, Byrnes C, Noh SJ, Meier ER, et al. LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo. Blood 2013 Aug 8; 122(6): 1034-1041.

Claims

CLAIMS1. A method for the in-vitro production of immortalized genetically modifiable erythrocyte progenitor cell, comprising the steps of(a) Providing a nucleated erythrocyte progenitor cell,(b) Immortalizing the nucleated erythrocyte progenitor cell by an inducible transgenic expression of one or more immortalization factors,(c) Optionally: propagating (expanding) the immortalized nucleated erythrocyte progenitor cell to obtain a plurality of immortalized nucleated erythrocyte progenitor cells suitable for storage.

2. A method for the in-vitro production of an erythrocyte, comprising the steps of(a) Providing a nucleated erythrocyte progenitor cell,(b) Immortalizing the nucleated erythrocyte progenitor cell by inducing an inducible transgenic expression of one or more immortalization factors,(c) Optionally: propagating (expanding) the immortalized nucleated erythrocyte progenitor cell to obtain a plurality of immortalized nucleated erythrocyte progenitor cells;(d) Stopping the induction of the inducible transgenic expression of the one or more immortalization factors and differentiating the nucleated erythrocyte progenitor cell to a reticulocyte or erythrocyte,(e) Enucleating the differentiated reticulocyte or erythrocyte to obtain the final in vitro produced erythrocyte;Wherein alternative to conducting steps (a) to (c), the method may comprise a step (a- c’) of providing the immortalized genetically modifiable erythrocyte progenitor cell obtained by the method of claim 1 , and then performing steps (d) and (e) with the so provided immortalized genetically modifiable erythrocyte progenitor cell.

3. The method of claim 1 or 2, wherein the provided nucleated erythrocyte progenitor cell is a CD34+ hematopoietic stem cell derived ex-vivo from a donor sample of (mobilized)peripheral blood, or is a CD71+ bone marrow cell or umbilical cord blood; most preferably wherein the provided nucleated erythrocyte progenitor cell is a human CD71+ bone marrow cell.

4. The method of any one of claims 1 to 3, wherein the nucleated erythrocyte progenitor cell is a direct reticulocyte progenitor cell.

5. The method of claim 4, wherein the expression vector comprises at least one or more expression cassettes suitable for an inducible expression of the one or more immortalization factors; and wherein the one or more expression cassettes comprises at least in operable connection an inducible element, an erythrocyte lineage active promoter and an expressible gene sequence ending one of the one or more immortalization factors.

6. The method of any one of claims 1 to 5, wherein the one or more immortalization factors are c-Myc and I or BLC-XL.

7. The method of any one of claims 2, and 3 to 6 when referring to claim 2, wherein the step of differentiating the nucleated erythrocyte progenitor cell to a reticulocyte or erythrocyte comprises bringing into contact the nucleated erythrocyte progenitor cell with erythropoietin (EPO).

8. The method of any one of the preceding claims, comprising a step of genetically modifying the immortalized nucleated erythrocyte progenitor cell before differentiation, for example by genetically altering the expression of a blood group antigen, for example by gene editing.

9. The method of any one of claims 2, and 3 to 8 when referring to claim 2, wherein enucleating in step (e) comprises bringing into contact the differentiated reticulocyte or erythrocyte with an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as mesalazine), and I or with one or more agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine).

10. The method of any one of the preceding claims, wherein the method comprises an additional step of reducing the expression and / or function of micro-RNA (miR)-30a-5p.

11. The method of claim 10, wherein the additional step of reducing the expression and / or function of miR-30a-5p is performed before and / or during the step of enucleation.

12. The method of claim 10 or 11 , wherein reducing the expression and / or function miR-30a- 5p comprises a step of gene editing the genome of the nucleated erythrocyte progenitor cell to remove or mutate the miR-30a-5p gene, and / or using an antisense nucleic acid construct to reduce the expression of miR-30a-5p.

13. The method of any one of the preceding claims, comprising a further step of restoring the actin filament network in the erythrocyte progenitor cell.

14. The method of claim 13, wherein the step of restoring the actin filament network involves the reducing the expression and / or function of a Scinderin protein or mRNA in the erythrocyte progenitor cell.

15. The method of claim 14, comprising a step of genetically modifying the erythrocyte progenitor cell, for example using gene editing, to have a reduced expression of Scinderin protein or mRNA, or comprising a step of expressing or bringing into contact the cell with an antisense nucleic acid compound, such as an siRNA or shRNA.

16. An immortalized genetically modifiable erythrocyte progenitor cell, or a plurality of immortalized genetically modifiable erythrocyte progenitor cells, as obtained or obtainable by, a method of any one of claims 1 to 15.

17. An in-vitro produced erythrocyte, or a plurality of in-vitro produced erythrocytes, obtained by or obtainable by, a method of any one of claims 2 or 3 to 15 when referring to claim 2.

18. A blood cell product, comprising an in-vitro produced erythrocyte, or a plurality of in- vitro produced erythrocytes according to claim 17.

19. A method of producing transfusion blood compositions, the method comprising producing a red blood cell with a method of any one of the preceding claims and formulating the red blood cell with one or more additives to form a transfusion blood composition.

20. A method of enucleating an erythrocyte or an (nucleated) erythrocyte progenitor cell, the method comprising a step of d bringing into contact the erythrocyte or erythrocyte progenitor cell, such as a differentiated reticulocyte, with an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as mesalazine), and I or with one or more agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine).

21. A use of a substance as enucleating agent, wherein the substance is selected from an antioxidant, an agent inhibiting the ERK1 / 2-SMAD2 / 3 signaling (such as mesalazine), and I or an agent modulating hypoxy-sensitive signaling pathways such as HIF-PH pathway (such as dimethyloxalyl glycine).

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