Transformed cell for producing red blood cells, and use thereof

Abstract

The present invention relates to a transformed cell for producing red blood cells and, more specifically: to the transformed cell for producing red blood cells; and a method for producing and differen

Description

Transformed cells for red blood cell production and uses thereof

[0001] The present invention relates to a transformed cell for producing red blood cells, and more particularly, to the transformed cell for producing red blood cells; and a method for producing and differentiating red blood cells using the same.

[0002] Red blood cells, a component of blood, play a vital role in transporting oxygen to body tissues and carbon dioxide out of the body. Shock, particularly in cases of excessive bleeding, is caused by a lack of red blood cells. However, the only way to replace red blood cells is through blood transfusions from another donor. The recent global pandemic and aging population have led to a shortage of blood donations, and the risk of infectious disease transmission through transfusions has made it impossible to maintain a stable red blood cell supply under the current system. Attempts have been made to develop hemoglobin solutions or oxygen carriers to replace red blood cells, but serious side effects have prevented their clinical application. Consequently, active research is underway to produce red blood cells ex vivo.

[0003] Stem cells are attracting attention for in vitro red blood cell production. Hematopoietic stem cells, in particular, are produced in the bone marrow and are the precursor cells of red blood cells and white blood cells. Red blood cells are produced from hematopoietic stem cells through several differentiation stages: proerythroblasts, basophils, polychromatic erythroblasts, and normochromatic erythroblasts. Their nuclei gradually condense and their cell size decreases. The condensed nuclei then enucleate, becoming reticulocytes, which then mature into red blood cells (erythrocytes). This differentiation process typically takes more than three weeks in vitro, and erythropoietin (EPO) is an essential culture additive for erythrocyte differentiation throughout the differentiation process.

[0004] Erythropoietin, a glycoprotein hormone involved in red blood cell production, is produced in the kidneys and, during fetal development, in the liver. It acts on erythroid progenitor cells to stimulate cell proliferation and differentiation, making it an essential growth factor for mature red blood cell differentiation.

[0005] Accordingly, the present inventors developed an MB-EPO cell line expressing inducible erythropoietin for in vitro red blood cell production, and confirmed that red blood cells can be produced without the addition of EPO when the MB-EPO cell line is used, thereby completing the present invention.

[0006] Accordingly, the purpose of the present invention is to provide an expression cassette for red blood cell production, which comprises an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain.

[0007] Another object of the present invention is to provide a recombinant vector for red blood cell production, comprising an expression cassette for red blood cell production, comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain.

[0008] Another object of the present invention is to provide a transformed cell for red blood cell production, into which is introduced an expression cassette for red blood cell production, including an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain; or a recombinant vector including the same.

[0009] Another object of the present invention is to provide a composition for producing red blood cells comprising the expression cassette, the recombinant vector or the transformed cell.

[0010] Another object of the present invention is to provide a red blood cell production kit comprising: a) a first compartment comprising a recombinant vector for red blood cell production; and b) a second compartment comprising an expression inducer.

[0011] Another object of the present invention is to provide a method for producing erythropoietin receptor-activated cells, comprising a step of transforming the expression cassette or the recombinant vector into a stem cell.

[0012] Another object of the present invention is to provide an in vitro erythroid differentiation inducing method, which comprises an erythroid differentiation step of culturing transformed cells for erythroid production and erythroid progenitor cells into which the expression cassette or the recombinant vector has been introduced.

[0013] To achieve the above purpose, the present invention provides an expression cassette for red blood cell production, comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain.

[0014] The present invention also provides a recombinant vector for red blood cell production, comprising an expression cassette for red blood cell production, comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain.

[0015] The present invention also provides a transformed cell for red blood cell production into which an expression cassette for red blood cell production is introduced, including an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain; or a recombinant vector including the same.

[0016] The present invention also provides a composition for producing red blood cells comprising the expression cassette, the expression cassette, the recombinant vector, or the transformed cell.

[0017] The present invention also provides a red blood cell production kit comprising: a) a first compartment comprising a recombinant vector for red blood cell production; and b) a second compartment comprising an expression inducer.

[0018] The present invention also provides a method for producing erythropoietin receptor-activated cells, comprising a step of transforming the expression cassette or the recombinant vector into a stem cell.

[0019] In addition, the present invention provides an in vitro erythroid differentiation inducing method, including an erythroid differentiation step of culturing transformed cells for erythroid production and erythroid progenitor cells into which the expression cassette or the recombinant vector has been introduced.

[0020] When the transformed cells for red blood cell production according to the present invention are applied to in vitro red blood cell differentiation and production, not only is the manufacturing cost significantly lower than the conventional method of differentiating and producing red blood cells by adding erythropoietin, but it has also been confirmed that red blood cells can be mass-produced. Therefore, the transformed cells for red blood cell production according to the present invention can be utilized in various fields such as blood-related clinical fields and the treatment of diseases related to red blood cell reduction.

[0021] Figure 1A is a diagram showing the inactive state of the EPO receptor.

[0022] Figure 1B is a diagram showing the mechanism of action of EPO inducible in MB-EPO cells according to the present invention.

[0023] Figures 2a and b are diagrams showing the cleavage map of a recombinant vector for constructing MB-EPO cells according to the present invention.

[0024] Figure 3A is a diagram showing the results of observing UT7 cells cultured in the presence and absence of EPO.

[0025] Figure 3B is a diagram showing the results of analyzing cell growth of UT cells depending on the presence or absence of EPO.

[0026] Figure 4A is a diagram showing the results of analyzing cell growth of UT7-MB-EPO cells in the presence of EPO.

[0027] Figure 4B is a diagram showing the results of analyzing cell growth of UT7-MB-EPO cells in the absence of EPO.

[0028] Figure 5A is a schematic diagram showing cell differentiation of UT7 cells into which the GFP gene has been introduced in the absence of EPO.

[0029] Figure 5B is a schematic diagram showing cell differentiation of UT7-MB-EPO cells into which MB-EPO was introduced in the absence of EPO.

[0030] Figure 6A is a diagram illustrating a TALEN-mediated targeting strategy of the AAVS1 locus for establishing MB-EPO-KI hiPSCs.

[0031] Figure 6B is a diagram showing the results of confirming doxycycline-dependent MB-EPO expression in MB-EPO-KI hiPSCs through flow cytometry analysis.

[0032] Figure 7 is a schematic diagram showing the in vitro red blood cell production process using hiPSCs; and the designed experimental group.

[0033] Figure 8A is a diagram showing the results of observing the cell morphology of the control group (CMC11 WT) during mesoderm induction.

[0034] Figure 8B is a diagram showing the results of observing the cell morphology of MB-EPO-KI hiPSCs during mesoderm induction.

[0035] Figure 9 is a diagram showing the results of observing the cell morphology of control (WT) and MB-EPO-hiPSCs in the presence or absence of EPO and doxycycline during endothelial-hematopoietic transition.

[0036] Figure 10A is a diagram showing the results of evaluating erythroid differentiation of the control group (CMC11 WT) according to the presence or absence of EPO and doxycycline after erythroid differentiation induction.

[0037] Figure 10B is a diagram showing the results of evaluating erythroid differentiation of MB-EPO-KI hiPSCs according to the presence or absence of EPO and doxycycline after erythroid differentiation induction.

[0038] Hereinafter, the present invention will be described in detail.

[0039] According to an aspect of the present invention, the present invention provides an expression cassette for red blood cell production, comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain.

[0040] In the present invention, an expression cassette refers to a unit cassette that includes a promoter and a gene encoding a target protein, and can be expressed to produce a target protein operably linked downstream of the promoter. Various factors that can aid in the efficient production of the target protein may be included within or outside of such an expression cassette. Specifically, the target protein expression cassette may be one in which a gene encoding the target protein is operably linked downstream of a promoter sequence.

[0041] The term "operably linked" as described above means that the gene sequence and the promoter sequence are functionally linked so that the nucleic acid sequence having the promoter activity of the present invention initiates and mediates transcription of the gene encoding the target protein. The operable linkage can be produced using genetic recombination techniques known in the art, and site-specific DNA cleavage and ligation can be produced using cleavage and ligation enzymes known in the art, but is not limited thereto.

[0042] In addition, the “gene expression cassette” can be inserted into the chromosome of a host cell and used to produce a recombinant microorganism, and it is obvious to those skilled in the art to which the present invention pertains that inserting the recombinant gene expression cassette into the genomic chromosome of a host cell will have the same effect as introducing a recombinant vector into the host cell as described above.

[0043] A commonly known genetic manipulation method can be used as a method for inserting a recombinant gene expression cassette into the chromosome of a host cell, and examples include a method using a retroviral vector, an adenoviral vector, an adeno-associated virus vector, a herpes simplex virus vector, a poxvirus vector, a lentiviral vector, or a non-viral vector.

[0044] In a specific embodiment of the present invention, the transmembrane domain is preferably at least one selected from the group consisting of a receptor, a ligand, an immunoglobulin, and a glycophorin. Specific examples of the transmembrane domain may include at least one selected from the group consisting of CD8, CD4, CD3, CD3y, CD38, CD3S, CD28, CD137, Fcely, a T cell receptor, a nicotinic acetylcholine receptor, and a gamma-aminobutyric acid receptor, and most preferably, the cell surface antigen CD8 represented by the base sequence of SEQ ID NO: 15. In the present invention, the cell surface antigen CD8 is a transmembrane glycoprotein that acts as a coreceptor of the T cell receptor. In particular, the cell surface antigen CD8 is expressed in almost all cells derived from bone marrow. Therefore, in the embodiment of the present invention, by binding self-secreted EPO to the cell surface antigen CD8, it was designed so that EPO can continuously affect cells even when the culture medium is replaced.

[0045] In a specific embodiment of the present invention, the expression cassette may further comprise a linker, and preferably, the linker is encoded by the base sequence of SEQ ID NO: 13.

[0046] In a specific embodiment of the present invention, the expression cassette may further include a reporter gene, and the reporter gene may be a gene encoding a fluorescent protein. The fluorescent protein is a protein that emits light, and the type thereof is not particularly limited as long as it is a protein known in the art, but may include mCherry, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), or enhanced cyan fluorescent protein (ECFP), and preferably, mCherry or green fluorescent protein (GFP) may be used.

[0047] In a specific embodiment of the present invention, the expression cassette and the recombinant vector for red blood cell production comprising the same may further include a gene capable of inducing expression of a target protein by an expression inducing substance. Examples of the gene include a promoter, a transcription activator, etc. expressed by the expression inducing substance. The expression inducing substance may be doxycycline or tetracycline.

[0048] In a specific embodiment of the present invention, it is preferable that the expression cassette sequentially and operably links a gene encoding an erythropoietin gene, a linker, and a transmembrane domain. In an embodiment of the present invention, an expression cassette for red blood cell production was constructed in which an erythropoietin gene (SEQ ID NO: 12), a linker (SEQ ID NO: 13), a CD8 gene (SEQ ID NO: 15), and an mCherry gene (SEQ ID NO: 19) were sequentially and operably linked, and the present inventors named it 'MB-EPO'. The constructed expression cassette MB-EPO is represented by the base sequence of SEQ ID NO: 11 and encodes a polypeptide represented by the amino acid sequence of SEQ ID NO: 1.

[0049] In addition, variants of the above base sequences are included within the scope of the present invention. Specifically, the gene refers to a sequence that exhibits substantially the same physiological activity as the base sequence of the gene encoding the erythropoietin gene, linker, and transmembrane domain, having a sequence identity of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%. The "% of sequence homology" for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences (which does not include additions or deletions).

[0050]

[0051] According to another aspect of the present invention, the present invention provides a recombinant vector for red blood cell production, comprising an expression cassette for red blood cell production, comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain.

[0052] In the present invention, a vector means a genetic construct comprising a base sequence of a gene operably linked to a suitable regulatory sequence so as to enable expression of a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, and a sequence for regulating the termination of transcription and translation.

[0053] In the present invention, a recombinant vector can be used as an expression vector of a target polypeptide that can express the target polypeptide with high efficiency in an appropriate host cell when the encoding gene of the target polypeptide to be expressed is operably linked, and the recombinant vector can be expressed in the host cell. The host cell may preferably be a eukaryotic cell, and depending on the type of host cell, expression control sequences such as a promoter, terminator, enhancer, sequences for membrane targeting or secretion, etc. can be appropriately selected and variously combined depending on the purpose.

[0054] For example, the vector of the present invention includes a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The vector that can be used as the recombinant vector can be produced by manipulating a plasmid (e.g., pGLS, pSC101, pGV1106, pACYC177, ColE1, pKT230, ME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4λB, λCharon, λΔz1, and M13, etc.) or a virus (e.g., CMV, SV40, etc.) that is frequently used in the art.

[0055] In the above recombinant vector, the polynucleotides encoding CYP3A4, CYP1A2, CYP2B6, CYP2D6, CYP2C9, CYP2C19, CYP2E1 and CYP2A6 can be operably linked to a promoter. The term "operably linked" refers to a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence can regulate transcription and / or translation of the other nucleotide sequence.

[0056] The recombinant vector may be constructed typically as a cloning vector or an expression vector. The expression vector may be any vector commonly used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art.

[0057] The above recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when a eukaryotic cell is used as a host, the replication origin that operates in the eukaryotic cell included in the vector includes, but is not limited to, the f1 replication origin, the SV40 replication origin, the pMB1 replication origin, the adeno replication origin, the AAV replication origin, the CMV replication origin, and the BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, and a tk promoter of HSV) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0058] In a specific embodiment of the present invention, the recombinant vector may include a promoter that controls gene expression, more preferably, the promoter is a tetracycline response elements (TRE) promoter, but the scope of the present invention is not limited thereto. The promoter that controls gene expression of the present invention can initiate gene expression by an expression inducing substance, and thus, expression of the target protein can be controlled according to the purpose. The expression inducing substance may be doxycycline or tetracycline.

[0059]

[0060] According to another aspect of the present invention, the present invention provides a transformed cell for red blood cell production, into which an expression cassette for red blood cell production is introduced, comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain; or a recombinant vector comprising the same.

[0061] Any host cell known in the art can be used as the host cell, and in the case of transforming a eukaryotic cell, yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, for example, SP2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines can be used as the host cell.

[0062] In a specific embodiment of the present invention, the transformed cell is preferably a stem cell, and more preferably, it may be at least one selected from the group consisting of an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), and a mesenchymal stem cell (MSC).

[0063] The pluripotent stem cells described above may include, without limitation, stem cells isolated or separated from a patient or donor, or commercially available stem cell lines. The pluripotent stem cells, also commonly known as PS cells, include any cell capable of differentiating into virtually any cell, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (inner stomach lining, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epithelial tissue and nervous system). PSCs may be derived from embryonic stem cells (including embryonic germ cells) or may be the descendants of pluripotent cells obtained by inducing non-pluripotent cells, such as adult somatic cells, by forcing the expression of specific genes.

[0064] In addition, the above-mentioned induced pluripotent stem cells, also commonly abbreviated as iPS cells, refer to a type of pluripotent stem cell that is artificially induced from a normally non-pluripotent cell, such as an adult somatic cell, by inducing the “forced” expression of a specific gene.

[0065] The origin of the above stem cells is human and non-human animals (e.g., mouse, rat, cow, horse, pig, sheep, monkey, dog, cat, etc.), and is not particularly limited, but is particularly preferably a cell of human origin.

[0066] Insertion of a recombinant vector into a host cell can be accomplished using any insertion method widely known in the art. If the host cell is a eukaryotic cell, the delivery method may include, but is not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene transfer.

[0067] The method for selecting the transformed host cells can be easily performed using methods widely known in the art, utilizing the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformants can be easily selected by culturing them in a medium containing the antibiotic.

[0068] In a specific embodiment of the present invention, the transformed cell is preferably bound to a transmembrane domain, and more preferably, the transmembrane domain may be bound to a cell membrane. That is, the transformed cell is one in which MB-EPO is bound to a cell membrane, as shown in FIG. 1B, and the MB-EPO bound to the cell membrane continuously affects the EPO receptor of the cell, thereby continuously inducing the expression of EPO-dependent genes.

[0069]

[0070] According to another aspect of the present invention, the present invention provides a composition for producing red blood cells comprising the expression cassette, the recombinant vector or the transformed cell.

[0071] The composition for red blood cell production of the present invention may further include known substances necessary for maintenance, growth, proliferation, differentiation induction, etc. of the constituent components.

[0072] The composition for red blood cell production according to the present invention can induce differentiation and production of red blood cells without the addition of erythropoietin, and thus can be usefully utilized in blood-related clinical fields and in the field of treatment of diseases related to red blood cell reduction.

[0073]

[0074] According to another aspect of the present invention, the present invention provides a red blood cell production kit comprising: a first compartment comprising a recombinant vector for red blood cell production; and a second compartment comprising an expression inducer.

[0075] The recombinant vector for red blood cell production comprises an expression cassette for red blood cell production, which comprises an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a CD8 gene represented by the base sequence of SEQ ID NO: 15. Preferably, the recombinant vector comprises a promoter that regulates expression of the gene; and the promoter may be a TRE (tetracycline response elements) promoter.

[0076] The first compartment of the erythrocyte production kit of the present invention may further comprise additional components for maintaining and preserving the expression cassette, recombinant vector, or transformed cells. Furthermore, the second compartment of the present invention may further comprise components for maintaining and preserving the expression inducer.

[0077] The red blood cell production kit of the present invention may additionally include reagents, materials, etc. known in the art for use in red blood cell production. Furthermore, the red blood cell production kit of the present invention may further include a user manual describing optimal performance conditions. The manual describes how to use the kit, such as the proposed reaction conditions and the order of each step. The manual includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and descriptions on the surface of the package containing the kit. Furthermore, the manual includes information disclosed or provided through electronic media, such as the Internet.

[0078] In a specific embodiment of the present invention, the expression inducer may be doxycycline or tetracycline.

[0079]

[0080] According to another aspect of the present invention, the present invention provides a method for producing erythropoietin receptor-activated cells, comprising the step of transforming a stem cell with the expression cassette; or the recombinant vector;

[0081] In a specific embodiment of the present invention, the erythropoietin receptor-activated cell can express erythropoietin bound to a transmembrane domain located in the cell membrane.

[0082] In a specific embodiment of the present invention, the expression cassette or recombinant vector may include a promoter that regulates expression of a gene.

[0083] The erythropoietin receptor-activated cells produced by the method of the present invention can continuously activate the erythropoietin receptor, as shown in Fig. 1B, since the expressed erythropoietin is bound to the cell surface antigen CD8, and expression can be induced according to the purpose by a promoter and expression-inducing substance that regulates gene expression.

[0084]

[0085] According to another aspect of the present invention, the present invention provides an in vitro erythroid differentiation inducing method, comprising an erythroid differentiation step of culturing transformed cells for erythroid production and erythroid progenitor cells into which the expression cassette or the recombinant vector has been introduced.

[0086] The above erythroid induction differentiation can be used interchangeably with 'erythroid production'.

[0087] In the present invention, cell culture means in vitro cell growth in an artificial medium for research or medical treatment.

[0088] The culture of the present invention can be carried out using any method known in the art as long as the purpose of the present invention can be achieved, and can be arbitrarily controlled by a person skilled in the art as long as no abnormality is observed in the shape and activity of the cells.

[0089] That is, the culture process of the present invention can be performed according to media and culture conditions known in the art. These culture processes can be easily adjusted and used by those skilled in the art depending on the selected cells.

[0090] The above culture can use a stationary culture or suspension culture method. Stationary culture means culturing in a state where the culture is left alone in the culture medium without agitation or shaking, and suspension culture means culturing in a state where the cells are suspended without being attached to the bottom or side of the reactor through aeration or agitation. In addition, the reactor for stationary culture and the reactor for suspension culture may be the same or different.

[0091] In a specific embodiment of the present invention, the transformed cells for red blood cell production are 0.1 X 10 5 20 X 10 5 It can be cells / ml, preferably 1 X 10 5 2 X 10 5 cells / ml, most preferably 0.75 X 10 5 It can be cells / ml.

[0092] In a specific example of the present invention, the erythrocyte differentiation is preferably induced to differentiate and enucleate erythrocyte progenitor cells into erythrocytes.

[0093] In the present invention, induction of differentiation refers to a change from a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Therefore, "induction of erythroid differentiation" refers to the induction of division of hematopoietic stem cells into progeny cells (i.e., erythrocytes) having characteristics different from those of hematopoietic stem cells, such as genotype (e.g., changes in gene expression determined by genetic analysis) and / or phenotype (e.g., changes in protein expression). In a broad sense, induction of erythroid differentiation of the present invention refers to inducing a change from hematopoietic stem cells or erythroid cells to progeny cells (i.e., erythrocytes) through endothelial-hematopoietic transition, hematopoietic induction, or enucleation induction.

[0094] In the present invention, enucleation refers to the process by which the nuclei of erythroid progenitor cells gradually condense during differentiation, and the erythroid progenitor cells lose their condensed nuclei. After enucleation, the erythroid progenitor cells become reticulocytes, and through a maturation process, ultimately become erythrocytes.

[0095] In a specific embodiment of the present invention, the recombinant vector includes a promoter that regulates expression of a gene, and further includes a step of treating an expression inducer after the erythroid differentiation step.

[0096] In a preferred embodiment of the present invention, the expression inducer may be doxycycline or tetracycline.

[0097] In a specific embodiment of the present invention, the erythroid differentiation step may include: (a) culturing erythroid progenitor cells in a medium containing IL-3, SCF (stem cell factor), and the transformed cells for erythroid production; (b) culturing erythroid progenitor cells in a medium containing SCF and the transformed cells for erythroid production; and (c) culturing erythroid progenitor cells in a medium containing the transformed cells for erythroid production.

[0098] In a specific embodiment of the present invention, the method may further include a step of 'determining the expression of an erythroid differentiation marker or an enucleation marker.' This is a process for determining whether the cells finally obtained are erythrocytes, and the expression of erythroid differentiation markers (CD71, GPA (CD235a)) and enucleation markers (DRAQ5) can be determined using an analytical method known in the art (e.g., flow cytometry, microscopic observation).

[0099] The red blood cells differentiated and produced according to the method of the present invention can be utilized in the form of artificial blood compositions, etc. The artificial blood composition of the present invention can be transfused into a subject and can contribute to resolving issues such as blood shortages and transfusion side effects. Furthermore, the produced red blood cells and artificial blood composition can be usefully utilized in blood-related clinical fields and in the treatment of diseases related to red blood cell reduction.

[0100]

[0101] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0102] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0103]

[0104] [Experimental Example]

[0105] Experimental Example 1. hPSC (human pluripotent stem cell) culture

[0106] CMC-hiPSC-011 cells, obtained from the National Stem Cell Bank, were maintained in Essential 8 (E8) medium (Gibco A1517001) in iMatrix-511 (Matrixome, 892 021) and fed daily. hPSCs were dissociated into clumps using ReLeSR (Stemcell Tech., 05873), passaged, and then reseeded in E8 medium containing Y-27632 (2 μM; Stemcell Tech, 72307), a p160-ROCK (Rho-associated coiled-coil kinase) inhibitor.

[0107]

[0108] Experimental Example 2. UT7 Cell Culture

[0109] UT7 cells were cultured at 37°C and 5% CO2 using Gibco MEM α medium (Thermo Fisher Scientific # 12571063). The Gibco MEM α medium contained 20% FBS (Gibco, Thermo Fisher Scientific # 16000044) and 100 μg / ml penicillin-streptomycin (WELGENE #LS 202-02), and was supplemented with 2 U / ml erythropoietin (EPO) (PeproTech, 100-64). The control group was cultured with Gibco MEM α medium without EPO. UT7 cells were seeded at 2.5 x 10 5 Cells were subcultured every 2-3 days at a density of 10 cells / ml.

[0110]

[0111] Experimental Example 3. Construction of MB-EPO cell lines expressing inducible EPO.

[0112] In this example, MB-EPO cells expressing inducible EPO were prepared. To prepare the MB-EPO cell line, human CMC11 iPSC cell line; or UT7 cell line; 2 x 10 6 Dogs were prepared, and the knock-in vector (pL VX CMV MB EPO mCh) (5 μg) and AAVS1-targeting TALEN (2 μg) of Figure 2 were introduced into the prepared cells by electroporation (Amaxa 4D Nucleofector system (Lonza)).

[0113] The cleavage map of the knock-in vector is as shown in Figs. 2a and b. Cells into which the knock-in vector has been introduced express the 'MB-EPO mCherry peptide' represented by the amino acid sequence of SEQ ID NO: 1, and the peptide is encoded by the base sequence of SEQ ID NO: 11. Specifically, the MB-EPO mCherry peptide is sequentially and operably linked to EPO (SEQ ID NO: 2), linker (SEQ ID NO: 3), myc (SEQ ID NO: 4), CD8 (SEQ ID NO: 5), and mCherry (SEQ ID NO: 9). The CD8 includes a hinge domain (SEQ ID NO: 6), a transmembrane domain (SEQ ID NO: 7), and a cytosolic domain (SEQ ID NO: 8). The EPO, linker, myc, CD8, and mCherry are encoded by the base sequences of SEQ ID NOs: 12, 13, 14, 15, and 19, respectively. Additionally, the above-mentioned hindge domain, transmembrane domain, and cytosolic domain are encoded by the base sequences of SEQ ID NOs. 16 to 18, respectively.

[0114]

[0115] Afterwards, E8 medium supplemented with Y-27632 (10 μM) was dispensed into 6-well plates, and serial cell dilutions were seeded into the 6-well plates. Clones were selected using antibiotics, and the selected clones were expanded and screened by Dox treatment and Cas9 staining. For genotyping, genomic DNA was extracted using the DNeasy Blood & Tissue Kit. Junction PCR using the extracted genomic DNA as a template was performed using KOD-Multi & Epi (Toyobo, KME-101).

[0116]

[0117] Experimental Example 4. Endothelial-to-hematopoietic transition (EHT) stage after mesoderm induction

[0118] Induced pluripotent stem cells (iPSCs) were maintained in Essential 8 (E8) medium (Gibco A1517001) in iMatrix-511 (Matrixome, 892 021). Cells were detached using TrypLE (Gibco 12563011) and cultured for 2 days in mTeSR™3D medium (StemCell Technologies 3950) on an orbital shaker set at 70 rpm according to the manufacturer's instructions. Pluripotent spheroids were then transferred to STEMdiff™ APEL™2 medium containing 10 μM CHIR-99021 (Selleckchem), 50 ng / ml BMP4 (Miltenyi), and 50 ng / ml VEGF-165 (Miltenyi) and cultured for 2 days. After removing CHIR, the cells were cultured for 2 days. Finally, the cells were cultured for 2 days in the presence of 50 ng / ml BMP4 (Miltenyi), 50 ng / ml VEGF-165 (Miltenyi), and 50 ng / ml SCF (Miltenyi). After 6 days of mesoderm induction, 20 embryoid bodies (EBs) were transferred to 35 mm gelatin-coated dishes (Sigma G1393) and cultured with medium for 4 days. The medium contains STEMdiff APEL2 Medium (STEMCELL Technologies) 5% PFHM-II Protein-Free Hybridoma Medium (Thermo Fisher Scientific), 10 ng / ml BMP4, 100 ng / ml SCF, 100 ng / ml FLT3-L (Miltenyi), 50 ng / ml TPO (Miltenyi), 50 ng / ml VEGF-165, 25 ng / ml IL-3 (PeproTech), 25 ng / ml IL-6 (PeproTech), 10 ng / ml IGF-II (PeproTech) and 10 ng / ml FGF-2 (R&D Systems).Afterwards, the medium was replaced with STEMdiff APEL2 medium (STEMCELL Technologies), 5% PFHM-II Protein-Free Hybridoma Medium (Thermo Fisher Scientific), 100 ng / ml SCF, 100 ng / ml FLT3-L, 50 ng / ml TPO, 25 ng / ml IL-3, and 25 ng / ml IL-6.

[0119]

[0120] Experimental Example 5. Hematopoietic induction

[0121] After 8 days of EHT, cells were cultured in hematopoietic induction medium for 7 days until erythroid progenitors were induced. The hematopoietic induction medium contained STEMdiff APEL2 Medium (STEMCELL Technologies), 5% PFHM-II Protein-Free Hybridoma Medium (Thermo Fisher Scientific), 5 ng / ml IL-3, 100 ng / ml SCF, 3 U / ml EPO (PeproTech), and 1 μM Dexamethasone (Stem Cell Technologies). For erythroid differentiation, the hematopoietic-induced cell suspension (i.e., erythroid progenitor cells) was harvested.

[0122]

[0123] Experimental Example 6. Erythrocyte Differentiation

[0124] According to the three-step erythropoiesis protocol, erythroid differentiation of erythroid progenitor cells (i.e., hematopoietic-induced cells) harvested in Example 5 was induced. To induce erythroid differentiation, erythroid progenitor cells were cultured in StemSpan™ SFEM II medium supplemented with 10% AB serum (Innovative Research ISERAB) and 330 mg / mL transferrin (R&D Systems) for 13 days. Specifically, from day 0 to day 8, 1.5 X 10 MB-EPO KI cells were cultured in 5 ng / ml IL-3, 100 ng / ml SCF, and 1.5 X 10 MB-EPO KI cells. 5 Differentiation was induced using medium containing 100 ng / ml SCF and MB-EPO KI cells (1.5 × 10 5 Differentiation was induced by replacing the medium with a medium containing 1.5 × 10 cells / 2 ml. Finally, from day 8 to day 13, MB-EPO KI cells were cultured in 1.5 × 10 5 Differentiation was induced by replacing the medium with one containing 2 cells / ml. The culture period could be extended to 18 days as needed. Hematopoietic and erythroid differentiation was confirmed by flow cytometry and microscopic evaluation at specified intervals.

[0125] Meanwhile, the control group was induced to differentiate using medium containing 5 ng / ml IL-3, 100 ng / ml SCF, and 3 U / ml EPO from days 0 to 8. In addition, differentiation was induced by changing the medium to containing 100 ng / ml SCF and 3 U / ml EPO from days 8 to 11. Finally, differentiation was induced by changing the medium to containing 3 U / ml EPO from days 8 to 13.

[0126] Recombinant human EPO, IL-3, and SCF used in this experiment were purchased from PEPROTECH.

[0127]

[0128] Experimental Example 7. Cell Counting

[0129] The number of viable cells was counted using trypan blue staining. Hematopoietic and erythrocytes were harvested, centrifuged, and the supernatant was removed. The cells were then suspended in medium and washed. The suspended cells were stained 1:1 with trypan blue solution and counted using a cell counter (LunaII cell counter, Logos Biosystems).

[0130]

[0131] Experimental Example 8. Flow Cytometry

[0132] Cells were washed with PBS, resuspended in PBS / 5% fetal bovine serum, and reacted with 10 μl of each monoclonal antibody at 4°C for 30 min. To confirm MB-EPO KI, cells were labeled with Myc-tag-Alexa 488 (Cell Signaling), and IgG2-Alexa 488 (Cell Signaling) was used for isotypic control staining. To analyze the expression of surface protein markers of in vitro differentiated erythrocytes, cells were labeled with the following anti-human antibodies: CD71-PE (BD Pharmingen), glycophorin A (GPA)-FITC (BD Pharmingen), and immunoglobulin G2 IgG2-FITC and IgG2-PE (BD Pharmingen) were used for isotypic control staining. Cells were washed twice with PBS / 5% FBS, resuspended in 300 μl of formaldehyde (2%), and analyzed using a BD FACSAria II (BD Bioscience). Flow cytometry data were analyzed using FlowJo™ Software (BD Bioscience).

[0133]

[0134] Experimental Example 9. Microscopic Evaluation

[0135] Cells were stained with trypan blue and observed using an inverted microscope (Ts2-FL, Nikon). In addition, for observation of red blood cells, cells were stained with Wright-Giemsa and observed using an optical microscope.

[0136]

[0137] [Example]

[0138] Example 1. Analysis of cell viability of UT7-MB-EPO cells in the presence or absence of EPO.

[0139] 1-1. Analysis of cell growth of UT7 cells in the presence or absence of EPO

[0140] We analyzed the cell growth of UT7 cells in the presence or absence of EPO. Specifically, UT7 cells were cultured using medium containing EPO as described in Experimental Example 2 (EPO+ group). The control group was cultured in medium without EPO (EPO- group). Cells were evaluated microscopically and counted every 24 hours after the start of culture. Cell viability was calculated after cell counting. The results of the microscopic evaluation and cell growth in the presence or absence of EPO are shown in Figures 3A and B, respectively.

[0141] As shown in Figure 3A, the EPO+ group showed normal cell morphology even on the third day (72 hours after culture). On the other hand, the EPO- group (control group) showed abnormal cell morphology and a large number of cell deaths.

[0142] As shown in Figure 3B, significant cell proliferation was observed in the EPO+ group. In contrast, no significant proliferation was observed in the EPO- group.

[0143]

[0144] 1-2. Construction of UT7-MB-EPO cells

[0145] UT7 cells were cultured using the method of Experimental Example 2, and lentiviral particles containing a gene encoding the MB-EPO mCherry peptide were introduced into the cells as in Experimental Example 3. The UT7 cells introduced with the MB-EPO mCherry peptide were named 'UT7-MB-EPO cells'. In the UT7-MB-EPO cells, EPO is linked to the cell surface antigen CD8, and the CD8 is bound to the cell membrane. Therefore, EPO bound to the UT7-MB-EPO cell membrane continuously affects the EPO receptor of the cell, resulting in the induction of EPO-dependent gene expression (Fig. 1).

[0146] The control group was formed by introducing a gene encoding GFP instead of the MB-EPO mCherry peptide into the cells and was named 'UT-GFP cells'.

[0147]

[0148] 1-3. Cell growth analysis of UT7-MB-EPO cells in the presence or absence of EPO

[0149] The cell growth of UT7-MB-EPO cells (MB-EPO) constructed in Examples 1-2 was analyzed in the presence or absence of EPO. Cell numbers were counted every three days of culture and analyzed for 12 days. Controls included wild-type UT7 cells (WT) and UT-GFP cells (GFP) into which the GFP gene had been introduced. The results of the cell growth analysis in the presence and absence of EPO are shown in Figures 4A and B, respectively.

[0150] As shown in Figure 4A, when cells were cultured in the presence of EPO, the cell growth of the experimental group (MB-EPO) and the control group (WT, GFP) showed similar trends.

[0151] As shown in Figure 4B, the control group (WT, GFP) died when cultured in the absence of EPO. In contrast, MB-EPO showed cell growth similar to that in the presence of EPO (Figure 4A).

[0152] Based on the above results, cell growth according to the introduction of a gene encoding the MB-EPO mCherry peptide was schematically depicted in Figure 5. Specifically, UT7 cells transduced with GFP undergo cell death in the absence of EPO (Figure 5A). In contrast, MB-EPO was confirmed to promote cell growth even in the absence of EPO supplied to the medium. This is thought to be because EPO bound to the UT7-MB-EPO cell membrane continuously affects the cell receptor, resulting in EPO-dependent gene expression induction without the need for additional EPO during the culture process.

[0153]

[0154] Example 2. Construction of MB-EPO-KI hiPSCs for in vitro erythropoiesis and erythropoiesis.

[0155] 2-1. Construction of MB-EPO-KI hiPSCs

[0156] MB-EPO-KI hiPSCs were constructed in which the expression of intracellular MB-EPO was induced by doxycycline (dox). The TALEN-mediated targeting strategy of the AAVS1 locus for constructing CMC11-MB-EPO cells is shown in Figure 6A. The AAVS1 donor vector contains an MB-EPO gene cassette that can be controlled by the dox-inducible TRE3G promoter. The vector in Figure 6A was intracellularly introduced into CMC11 cells, and these were designated 'MB-EPO-KI hiPSCs.' The constructed MB-EPO-KI hiPSCs express MB-EPO in the presence of dox.

[0157]

[0158] We verified MB-EPO expression in the constructed MB-EPO-KI hiPSCs. Specifically, the constructed MB-EPO-KI hiPSCs were cultured in doxycycline-containing culture medium to induce MB-EPO expression. MB-EPO-expressing cells were then identified using flow cytometry. The flow cytometry results are shown in Figure 6B.

[0159] As shown in Figure 6B, it was confirmed that 97.8% of the cell population expressed MB-EPO.

[0160]

[0161] 2-2. In vitro erythropoiesis of MB-EPO-KI hiPSCs

[0162] hiPSCs are cultured in 3D to form hiPSC spheroids, and then erythrocytes are produced through mesoderm induction and endothelial-hematopoietic transition (Figure 7).

[0163] In this example, in vitro erythrocyte production was performed using MB-EPO-KI hiPSCs constructed in Example 2-1, as shown in Fig. 7. The control group is wild-type hiPSCs (WT).

[0164] In the experiments described below, MB-EPO-KI hiPSCs and the control group (WT) were divided into five groups according to the presence of EPO and doxycycline, and the morphology of cells at each differentiation stage was analyzed under a microscope.

[0165]

[0166] - Morphological analysis of MB-EPO-KI hiPSCs during mesoderm induction

[0167] Morphological analyses of control (CMC11 WT) and MB-EPO-KI hiPSCs were performed during mesoderm induction. The results of morphological analyses of control (CMC11 WT) and MB-EPO-KI hiPSCs are shown in Figures 8A and B, respectively.

[0168] As shown in Figures 8A and B, both the control group (CMC11 WT) and MB-EPO-KI hiPSCs formed embryoid bodies (EBs) normally, and it was confirmed through a microscope that the shape was maintained even after EB plating.

[0169]

[0170] - Morphological analysis of MB-EPO-KI hiPSCs undergoing endothelial-to-hematopoietic transition (EHT)

[0171] Morphological analyses of control (WT) and MB-EPO-KI hiPSCs were performed during the endothelial-hematopoietic transition after mesoderm induction. The results of the morphological analyses of control (WT) and MB-EPO-KI hiPSCs in the presence or absence of EPO and doxycycline during the endothelial-hematopoietic transition are shown in Figure 9.

[0172] As shown in Figure 9, it was confirmed through microscopy that hematopoietic single suspension cells were formed during endothelial-hematopoietic transition in all experimental groups.

[0173]

[0174] - Evaluation of erythroid differentiation

[0175] Erythroid differentiation is an EPO-dependent process. Erythroid differentiation was induced by culturing cells in EPO-containing medium, as described in Experimental Example 6. After induction of erythroid differentiation, erythroid differentiation was assessed through cell counting, visual observation, and microscopic observation. The results of the erythroid differentiation evaluation of control (WT) and MB-EPO-KI hiPSCs are shown in Figures 10A and B, respectively.

[0176] As shown in Figure 10A, the control group (WT) was confirmed to produce red blood cells only in the presence of EPO.

[0177] As shown in Figure 10B, MB-EPO-KI hiPSCs were confirmed to produce erythrocytes under EPO+ / DOX- and EPO- / DOX+ conditions. On the other hand, MB-EPO-KI hiPSCs did not produce erythrocytes under EPO- / DOX- conditions. These results demonstrate that MB-EPO-KI hiPSCs induce MB-EPO expression in the presence of doxycycline, thereby producing erythrocytes even without EPO.

[0178]

[0179] In summary, the present inventors have constructed an MB-EPO cell line expressing inducible EPO. The MB-EPO cell line according to the present invention has EPO linked to the cell surface antigen CD8, which is bound to the cell membrane. Therefore, EPO bound to the cell membrane continuously influences the cell receptor, inducing the expression of EPO-dependent genes (i.e., the expression of genes related to erythroid differentiation). This suggests that the MB-EPO cell line expressing the inducible EPO of the present invention can induce erythroid differentiation without the addition of EPO, and thus, the cell line can be utilized in various fields related to artificial blood.

[0180]

[0181] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The erythropoietin (EPO) gene represented by the base sequence of sequence number 12; and An expression cassette for erythrocyte production comprising a gene encoding a transmembrane domain.

2. An expression cassette for red blood cell production, wherein the transmembrane domain is at least one selected from the group consisting of a receptor, a ligand, an immunoglobulin, and a glycophorin.

3. An expression cassette for red blood cell production in the second paragraph, wherein the transmembrane domain is at least one selected from the group consisting of CD8, CD4, CD3, CD3y, CD38, CD3S, CD28, CD137, Fcely, T cell receptor, nicotinic acetylcholine receptor, and gamma-AminoButyric Acid receptor.

4. An expression cassette for red blood cell production, wherein the expression cassette further comprises a linker.

5. An expression cassette for red blood cell production, wherein the expression cassette comprises a gene encoding an erythropoietin gene, a linker, and a transmembrane domain, which are sequentially and operably linked.

6. A recombinant vector for red blood cell production comprising an expression cassette for red blood cell production, comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain.

7. A recombinant vector according to claim 6, wherein the recombinant vector comprises a promoter that controls the expression of a gene.

8. A recombinant vector according to claim 7, wherein the promoter is a TRE (tetracycline response elements) promoter.

9. An expression cassette for red blood cell production comprising an erythropoietin (EPO) gene represented by the base sequence of SEQ ID NO: 12; and a gene encoding a transmembrane domain; or A transformed cell for producing red blood cells, into which a recombinant vector comprising the same has been introduced.

10. A transformed cell for producing red blood cells, wherein the transformed cell is a stem cell in the 9th paragraph.

11. A transformed cell for red blood cell production in claim 10, wherein the stem cell is at least one selected from the group consisting of induced pluripotent stem cells (iPSC), hematopoietic stem cells (HSC), and mesenchymal stem cells (MSC).

12. In claim 11, the transformed cell is a transformed cell for producing red blood cells, wherein erythropoietin is bound to a transmembrane domain.

13. A transformed cell for producing red blood cells, wherein the transmembrane domain is bound to a cell membrane in the 12th paragraph.

14. A composition for producing red blood cells, comprising an expression cassette according to any one of claims 1 to 5; a recombinant vector according to any one of claims 6 to 8; or a transformed cell according to any one of claims 9 to 13. 15.a) A first compartment comprising the recombinant vector of claim 7 or 8; and b) A red blood cell production kit comprising a second compartment containing an expression inducer; 16. A method for producing erythropoietin receptor-activated cells, comprising the step of transforming a stem cell with an expression cassette according to any one of claims 1 to 5; or a recombinant vector according to any one of claims 6 to 8.

17. A method for producing erythropoietin receptor-activated cells in claim 16, wherein the erythropoietin receptor-activated cells express erythropoietin bound to a transmembrane domain located in the cell membrane.

18. A method for producing erythropoietin receptor-activated cells, wherein the expression cassette or recombinant vector comprises a promoter that regulates expression of a gene.

19. A method for inducing erythroid differentiation in vitro, comprising a step of culturing transformed cells for erythroid production into which the expression cassette of any one of claims 1 to 5 or the recombinant vector of any one of claims 6 to 8 has been introduced; and erythroid progenitor cells.

20. In paragraph 19, the transformed cells for red blood cell production are 0.1 X 10 5 20 X 10 5 cells / ml, method.

21. A method according to claim 19, wherein the erythrocyte differentiation is induced to differentiate and enucleate erythrocyte progenitor cells into erythrocytes.

22. In paragraph 19, the recombinant vector includes a promoter that controls the expression of a gene, A method further comprising a step of treating an expression inducer after the erythroid differentiation step.

23. A method according to claim 22, wherein the expression inducer is doxycycline or tetracycline.

24. In paragraph 19, the erythrocyte differentiation step is (a) a step of culturing erythroid progenitor cells in a medium containing IL-3, SCF (stem cell factor) and the transformed cells for erythrocyte production; (b) culturing erythroid progenitor cells in a medium containing SCF and the transformed cells for erythrocyte production; and (c) a method comprising a step of culturing erythroid progenitor cells in a medium containing the transformed cells for erythrocyte production.

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