Method for producing functionalized corneal endothelial substitute cells

Abstract

To provide a method for producing corneal endothelial substitute cells to which novel functionality has been imparted, particularly a method for producing corneal endothelial substitute cells derived from iPS cells.SOLUTION: A method for producing functionalized corneal endothelial substitute cells is provided, comprising the steps of: introducing a nucleic acid encoding a functional molecule into the genome of a pluripotent stem cell; and inducing the pluripotent stem cell containing the introduced nucleic acid into a corneal endothelial substitute cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a corneal endothelial replacement cell to which functionality has been imparted, in particular, an iPS cell-derived corneal endothelial replacement cell ( C orneal E Indothelial C ell S from i The present invention relates to a method for producing CECSi cells (PS cells) and cells produced by said method. [Background technology]

[0002] iPS cell-derived corneal endothelial substitute cells ( C orneal E Indothelial C ell S from i CECSi cells (iPS cells) have been developed for the treatment of bullous keratopathy (Patent Documents 1 to 3). On the other hand, CECSi cells have the advantage that they can mass-produce differentiated endothelial cells with uniform quality and high adhesiveness to cells and extracellular matrix from iPS cells in a short period of time (approximately 2 weeks). If we could give additional functions to CECSi cells while taking advantage of this characteristic, we could aim to expand the application of CECSi cells beyond corneal treatment to other disease areas. One method for giving functions is genome editing. Genome editing techniques that use endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems have been developed.

[0003] When adding new functions to CECSi cells, which are differentiated from iPS cells, by gene transfer, the greater the number of cells to be created, the greater the amount of materials required for gene transfer. In addition, the infection efficiency of the vector is not 100%, so some cells have been transferred with genes and some have not, necessitating cell removal procedures. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2013 / 051722 [Patent Document 2] WO2016 / 093359 [Patent Document 3] WO2019 / 142833 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide corneal endothelial replacement cells derived from pluripotent stem cells, particularly from iPS cells, to which a new function has been imparted, and to provide a method for producing such cells. [Means for solving the problem]

[0006] In view of the above problems, the present inventors attempted to impart functionality to corneal endothelial replacement cells derived from pluripotent stem cells that the present inventors had been developing, by introducing genes into the corneal endothelial replacement cells at the stage of pluripotent stem cells and inducing their differentiation into corneal endothelial replacement cells, rather than by introducing genes into the corneal endothelial replacement cells after differentiation induction. In particular, the present inventors used iPS cells as pluripotent stem cells, produced cells with functional factors introduced into their genomes, and succeeded in producing corneal endothelial replacement cells capable of constitutively expressing the functional factors by inducing differentiation by a direct method, thereby completing the present invention.

[0007] That is, the present invention provides the following. [1] A method for producing functionalized corneal endothelial replacement cells, comprising the following steps: Step 1: introducing a nucleic acid encoding a functional molecule into a genomic region of a pluripotent stem cell to obtain a functionalized pluripotent stem cell; and Step 2: A step of inducing the pluripotent stem cells to which the functionality has been imparted into corneal endothelial replacement cells. [2] The method described in [1], wherein the genomic region is the AAVS1 locus. [3] The method described in [1] or [2], wherein the pluripotent stem cells are iPS cells. [4] Corneal endothelial replacement cells are CECSi cells ( C orneal E Indothelial C ell S from i The method according to any one of [1] to [3], wherein the cells are PS cells. [5] The method according to any one of [1] to [4], characterized in that the step of introducing a nucleic acid encoding a functional molecule into the genome of a pluripotent stem cell is carried out by genome editing technology. [6] The method described in [5], wherein the genome editing technology is CRISPR / Cas9. [7] Corneal endothelial replacement cells, characterized by expressing exogenous functional molecules. [8] The cell described in [7], which is derived from a pluripotent stem cell. [9] Corneal endothelial replacement cells are CECSi cells ( C orneal E Indothelial C ell S from i The cell described in [7], which is a PS cell.

[10] A functionalized corneal endothelial replacement cell produced by the method described in any one of [1] to [6].

[11] A pharmaceutical composition comprising the cells described in

[10] . Effect of the Invention

[0008] If a cell stock can be created through successful gene transfer at the pluripotent stem cell stage, a master cell bank can be created by expanding and culturing the cells, and corneal endothelial replacement cells (CECSi cells) with the desired functionality can be repeatedly produced from the master cell bank, with only a minimum of materials required for gene transfer. CECSi cells that constitutively express functional molecules are highly durable and can be more effective locally when used in cell therapy for various diseases for which the functional molecules are effective. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows an example of a procedure for constructing a plasmid vector for genome editing. [Diagram 2] After cloning the genome editing plasmid vector, E. coli was transformed. Inserts were confirmed in all six selected clones. Figure 2 shows the PCR results. [Diagram 3] FIG. 3 shows the results of confirming the expression of functional markers for corneal endothelial cells in CECSi cells on day 8 after differentiation induction, by fluorescent immunostaining. [Figure 4] 1 shows the results of confirming the expression of sPD-1 gene in iPS cells into which a nucleic acid encoding sPD-1 was introduced by genome editing, and in CECSi cells induced to differentiate from the iPS cells. sPD-1 gene expression was confirmed by RT-PCR in iPS cells at the first passage after genome editing, and in CECSi cells on day 9 after induction of differentiation. [Diagram 5] FIG. 13 shows the secretion of sPD-1 in the culture supernatant of CECSi cells, which were induced to differentiate from iPS cells into which a nucleic acid encoding sPD-1 had been introduced by genome editing, confirmed by Western blotting. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will now be described. Terms used in the present specification have the meanings commonly used in the art unless otherwise specified.

[0011] 1. Method for producing functionalized corneal endothelial replacement cells The present invention provides a method for producing functionalized corneal endothelial substitute cells (hereinafter also simply referred to as the production method of the present invention). The manufacturing method of the present invention includes the following steps. Step 1: A step of introducing a nucleic acid encoding a functional molecule into a genomic region of a pluripotent stem cell to obtain a functionalized pluripotent stem cell. Step 2: A step of inducing the functionalized pluripotent stem cells into corneal endothelial replacement cells.

[0012] In the present specification, a "cell to which functionality has been imparted" means a cell that has been engineered to exert a biological activity that the cell does not originally have, and in one embodiment, a cell expressing a functional molecule related to the biological activity. The functional molecule may be a functional peptide. In the present invention, a "functional peptide" refers to a peptide that has a specific biological function in vivo or in vitro, or intracellularly or extracellularly. Generally, a compound consisting of 50 or more amino acids is called a protein, and a compound consisting of less than 50 amino acids is called a peptide, but in the present specification, "peptide" is a concept that includes proteins. Peptides can be classified into oligopeptides having about 2 to 10 amino acids and polypeptides having more than 50 amino acids, but in the present specification, "peptide" includes both oligopeptides and polypeptides. In the present specification, a "specific biological function" is not limited to a function that can have any effect on a biomolecule such as a protein or a nucleic acid, a cell, a tissue, or an individual. The specific biological function may be natural or non-natural, and examples thereof include a cell adhesion function, a signal transduction function, a binding function, a linking function, a labeling function, and a metabolic function. The cell adhesion function refers to the interaction between cells or between cells and the extracellular matrix, in which cell adhesion molecules are involved, and corresponds to a function that plays an important role in maintaining cell morphology, tissue formation, intercellular communication, immune response, cancer metastasis, etc. The signal transduction function refers to a mechanism that receives stimuli or information from the outside, receives it within the cell, and causes a response, and usually, signal transduction is performed through intracellular signal transduction pathways and molecular interactions via receptors on the cell surface. This function is involved in cell survival, proliferation, differentiation, response, and regulation, and plays an important role in controlling various physiological processes and cell responses. Examples of the binding function include a function that mediates antigen-antibody binding or receptor-ligand interaction, a function that binds biomolecules such as RNA and / or DNA, and a function that binds nickel ions, copper ions, etc. The linking function corresponds to, for example, a function that links other biomolecules such as peptides and nucleic acids that have a specific biological function, low molecular weight compounds, or metal ions. The labeling function corresponds to a function that labels biomolecules such as proteins and nucleic acids, cells, tissues, and individuals. Examples include fluorescent labeling and epitope labeling.The metabolic function includes a genome editing function such as nuclease activity, etc. The functional peptide can be appropriately selected depending on the purpose of use.

[0013] In the present invention, specific examples of "functional peptides" include enzymes, binding proteins, marker proteins, and artificial peptides, as well as peptide fragments thereof. "Enzymes" include, but are not limited to, DNA polymerase, RNA polymerase, and phosphorylating enzymes that induce phosphorylation during intracellular signal transduction. "Binding proteins" are proteins that specifically bind to specific molecules. Examples include, but are not limited to, antibodies or antibody fragments or antigens that mediate antigen-antibody binding, (strept)avidin, maltose-binding protein (MBP), receptors or ligands that mediate receptor-ligand interactions, DNA-binding proteins, and RNA-binding proteins. "Marker proteins" are proteins that can be used as labels when detecting cells, proteins, and the like. Typically, polypeptides that can determine the expression or presence of a target protein based on their activity are relevant. Examples include, but are not limited to, fluorescent proteins such as GFP, luminescent proteins such as luciferin or aequorin, enzymes such as horseradish peroxidase (HRP), and alkaline phosphatase (AP). An "artificial peptide", also called a tag peptide, is an oligopeptide consisting of several to a dozen amino acids that are artificially synthesized. Peptides that can exert a therapeutic effect on cells into which they are introduced are also preferred as functional peptides in the present invention. In particular, since CECSi cells have high adhesiveness and can be expected to have a local action effect, peptides that can exert a therapeutic effect by being expressed locally are preferred. More specific examples of functional peptides include soluble fractions of vascular endothelial growth factor (VEGF) receptors and human fibroblast growth factor 7 (FGF-7). Since the soluble fraction of the VEGF receptor can locally suppress angiogenesis, it is expected that side effects can be suppressed compared to systemic administration of an antagonist of the sVEGF receptor. FGF7 is expected to repair alveolar epithelium and the like by acting specifically on epithelial cells. sPD-1 is expected to treat local recurrence and metastasis of cancer cells by inhibiting immune checkpoints.

[0014] Biological activity can be measured by techniques well known in the art to evaluate its expression. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect response (i.e., have a measurable effect in response to some exposure or stimulation), such as the affinity of a compound that directly binds to a peptide that is a functional molecule, or, for example, the amount of upstream or downstream protein or other similar function after some stimulation or event. It can be appropriately selected according to the desired biological activity.

[0015] Step 1: A step of introducing a nucleic acid encoding a functional molecule into a genomic region of a pluripotent stem cell to obtain a functionalized pluripotent stem cell. In the production method of the present invention, step 1 is characterized in that a nucleic acid encoding a functional molecule is introduced into the genome of a pluripotent stem cell. The nucleic acid encoding the functional molecule (described above) is not particularly limited as long as it can express the desired functional molecule in a cell, and examples thereof include DNA, RNA, or DNA / RNA chimeras, artificial nucleic acids, etc. The nucleic acid may be a wild-type nucleic acid or a mutant. Such a mutant nucleic acid preferably has 90% or more identity, more preferably 95% or more identity, even more preferably 98% or more identity, and even more preferably 99% or more identity in nucleic acid sequence to the wild-type nucleic acid.

[0016] Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), and induced pluripotent stem cells (iPS cells). Muse cells (Multi-lineage differentiating Stress Enduring cells) obtained from mesenchymal stem cells (MSCs), and GS cells prepared from germ cells (e.g., testes) are also included in pluripotent stem cells. The pluripotent stem cells are preferably derived from mammals.

[0017] ES cells can be produced by culturing the inner cell population on feeder cells or in a medium containing leukemia inhibitory factor (LIF). They are also available from designated institutions and can be purchased commercially. Nuclear transfer ES cells (ntES cells), which are one type of ES cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.

[0018] EG cells can be prepared by culturing primordial germ cells in a medium containing mouse stem cell factor (mSCF), LIF and basic fibroblast growth factor (bFGF) (Cell, 70:841-847, 1992).

[0019] An iPS cell is a cell in which pluripotency is induced by reprogramming a somatic cell by a known method. Specific examples of iPS cells include cells in which pluripotency is induced by reprogramming a somatic cell differentiated into a fibroblast, peripheral blood mononuclear cell, or the like, through expression of a plurality of genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, and the like. In 2006, Yamanaka et al. established induced pluripotent stem cells from mouse cells (Cell, 2006, 126(4) pp.663-676). In 2007, induced pluripotent stem cells were established from human fibroblasts, and they have the same pluripotency and self-renewal ability as embryonic stem cells (Cell, 2007, 131(5) pp.861-872; Science, 2007, 318(5858) pp.1917-1920; Nat. Biotechnol., 2008, 26(1) pp.101-106). In addition to the method of producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341, pp.651-654).

[0020] Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells, T cells, etc.), hepatic cells, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.

[0021] When producing artificial pluripotent stem cells, in the case of initializing by the expression of several kinds of genes (for example, four factors, Oct3 / 4, Sox2, Klf4 and Myc), the means for expressing the genes is not particularly limited. Examples of the means for expressing the genes include infection methods using virus vectors (for example, retrovirus vectors, lentivirus vectors, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors), gene transfer methods using plasmid vectors (for example, plasmid vectors, episomal vectors) (for example, calcium phosphate method, lipofection method, retronectin method, electroporation method), gene transfer methods using RNA vectors (for example, calcium phosphate method, lipofection method, electroporation method), and direct protein injection method.

[0022] It is also possible to obtain strains of induced pluripotent stem cells. Specifically, the iPS cells include 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, 836B3, FF-I14s03, FF-I01s04, MH09s01, Ff-XT18s02, Ff-WIs03, Ff-WJs513, Ff-CLs14, Ff-KVs09, QHJI14s03, QHJI01s04, RWMH09s01, DRXT18s02, RJWIs03, YZWJs513, ILCLs14, GLKVs09, Ff-XT28s05-ABo_To, Ff-I01s04-ABII-KO, Ff-I14s04-ABII-KO (all from iPS Academia Japan, Inc., or Kyoto University iPS Research Foundation), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeaky (JCRB1329 strain), Toe (JCRB1338 strain), and Lollipop (JCRB1336 strain) (all from the National Center for Child Health and Development, Department of Rare and Intractable Diseases and Disease Resources, National Institute of Biomedical Innovation, JCRB Cell Bank), UTA-1 strain and UTA-1-SF-2-2 strain (all from the University of Tokyo), 21526, 21528, 21530, 21531, 31536, and 31538 strains (all from the University of Tokyo). Alternatively, any of the following strains may be used: ATCC-DYP0730, ATCC-DYP0250, ATCC-HYR0103, ATCC-DYR0100, ATCC-DYR0530, ATCC-DYS0530, ATCC-DYP0530, ATCC-DYS0100, ATCC-HYS0103, ATCC-CYS0105, KYOU-DXR0109B, ATCC-BYS0110, ATCC-BYS0111, ATCC-BYS0112, ATCC-BYS0113, ATCC-BXS0114, ATCC-BXS0115, ATCC-BXS0116, and ATCC-BXS0117 (all from the American Type Culture Collection, a non-profit organization).

[0023] "Mammals" include rodents, ungulates, felines, lagomorphs, primates, etc. Rodents include mice, rats, hamsters, guinea pigs, etc. Ungulates include pigs, cows, goats, horses, sheep, etc. Felidae include dogs, cats, etc. Lagomorphs include rabbits, etc. "Primates" refers to mammals belonging to the order Primates, and examples of primates include prosimians such as lemurs, lorises, and tree shrews, and anthropoids such as monkeys, apes, and humans.

[0024] In the present invention, pluripotent stem cells are mammalian pluripotent stem cells, preferably rodent (e.g., mouse, rat) or primate (e.g., human, monkey) pluripotent stem cells, and most preferably human pluripotent stem cells.

[0025] Introduction of a nucleic acid encoding a functional molecule into a genomic region of a pluripotent stem cell is preferably carried out by introducing an expression vector containing a nucleic acid encoding the functional molecule into a genomic region of a pluripotent stem cell. Thus, preferably, the method of the present invention comprises the step of inserting a nucleic acid encoding a functional molecule into an expression vector to prepare an expression vector containing the nucleic acid.

[0026] In the case of genome editing, a cut is made in the genome and a donor vector containing a desired sequence is introduced into a cell, so that the sequence can be inserted into the cut site of the genome. The sequence to be inserted into the genome can be a sequence for replacing the desired site (a sequence of a gene encoding a functional molecule). In addition to the desired sequence, the donor vector may contain other elements such as a regulatory sequence such as a promoter or enhancer that controls the expression of the desired sequence, or a drug resistance gene for cell selection, and may contain sequences at both ends that are homologous to the insertion site of the genome. The donor vector can be introduced into the desired site by non-homologous end joining or homologous recombination. As the donor vector, a plasmid or a viral vector such as an adeno-associated virus vector or an integrase-deficient lentivirus vector can be used.

[0027] The introduction of an expression vector containing a nucleic acid encoding a functional molecule into the genomic region of a pluripotent stem cell, preferably an iPS cell, is preferably performed by genome editing technology. "Genome editing (technology)" is a technology that intentionally modifies a target gene or genome region by site-specific cleavage of a genomic DNA strand using a nuclease, or by chemical conversion of bases. Specific examples include zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated proteins (Cas) CRISPR / Cas, hereinafter abbreviated as CRISPR / Cas), and the like. ZFNs are artificial nucleases that have zinc fingers as DNA-binding domains. Each zinc finger recognizes three bases, so ZFNs with three to six zinc fingers specifically bind to 9 to 18 base pairs (bp), and each pair introduces DNA double-strand breaks with specificity of 18 to 36 bases. TALEN is an artificial nuclease that has a DNA-binding domain derived from the plant pathogenic bacterium Xanthomonas. The DNA-binding domain of TALE is a unit of 34 amino acids that recognizes one base, and TALENs with 15 to 20 units are created in each of the sense and antisense strands to introduce double-stranded DNA breaks at the desired target site. The CRISPR / Cas system uses a complex of Cas nuclease, which has DNA double-strand cleavage activity alone, and a target sequence-specific single-stranded guide RNA to introduce DNA double-strand cleavage into a targeted base sequence. The CRISPR / Cas system has become a standard genome editing technology used worldwide due to its convenience, high efficiency, and versatility. Examples of Cas nuclease include Cas9, Cas12a (Cpf1), Cas13a (C2c2), Cas14, Cas3, and CasX, and Cas9 is preferred. Examples of Cas9 include Streptococcus pyogenes-derived Cas9 (SpCas9), Staphylococcus aureus-derived Cas9 (SaCas9), Francisella novicida-derived Cas9 (FnCas9), Campylobacter jejuni-derived Cas9 (CjCas9), and Streptococcus thermophilus-derived Cas9 (St1Cas9, St3Cas9). Examples of Cpf1 include Acidaminococcus sp.-derived Cpf1 (AsCpf1) and Lachnospiraceae bacterium-derived Cpf1 (LbCpf1). In the CRISPR / Cas system, mutants (subspecies) with mutations in the amino acid sequence of Cas nuclease can also be used. dCas9 (deadCas9) is a nuclease-defective Cas9 that binds to DNA sequences but does not cut them. nCas9 (nickase Cas9) introduces single-stranded nicks, and xCas9 has broad PAM compatibility and high DNA specificity. In the production method of the present invention, any genome editing technology may be used, but preferably, it is a technology using CRISPR / Cas, in particular, CRISPR / Cas9, which uses Cas9 as the Cas nuclease.

[0028] In the method of the present invention, the genomic region of a pluripotent stem cell into which a nucleic acid encoding a functional molecule is introduced is not particularly limited as long as it is a genomic region in which the gene can be stably expressed. Examples of such genomic regions include the AAVS1 locus, the hROSA26 locus, and the CCR5 locus, and the AAVS1 locus, which has been most analyzed and is used frequently, is preferred.

[0029] Although not limited thereto, the pluripotent stem cells to which functionality has been imparted are preferably expanded prior to induction of differentiation into CECSi cells. The cell culture in the expansion culture is preferably carried out in a medium for pluripotent stem cell culture. Any known medium can be used for pluripotent stem cells, and is not particularly limited as long as it does not inhibit the proliferation of pluripotent stem cells. Examples of such media include DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752 / 1, CMRL-1066, Williams' medium E, Brinster's BMOC-3 Medium, E8 medium (Nature Methods, 2011, 8, 424-429), ReproFF2 medium (ReproCell, Inc.), StemFit (registered trademark) AK medium (Ajinomoto Co., Inc.), and mixed media thereof. The thus obtained pluripotent stem cells to which functionality has been imparted can be induced to differentiate to obtain desired corneal endothelial replacement cells. For example, corneal endothelial replacement cells, which are corneal endothelial-like cells, can be obtained by inducing differentiation of the pluripotent stem cells to which functionality has been imparted, according to the descriptions in Patent Documents 1 to 3. An example of a method for producing corneal endothelial replacement cells is as follows. Functionalized iPS cells were cultured in iMatrix-511 (0.6 μg / cm 2 ) coated on a culture dish and cultured for 1 week using StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.). Then, iMatrix-511 (0.3 μg / cm 2), and the iPS cells are cultured for 7 to 14 days to induce differentiation into corneal endothelial replacement cells using the differentiation induction medium below (Table 1). When using cryopreserved iPS cells to which functionality has been imparted, differentiation is induced after two passages and culture. The composition of the differentiation induction medium in Table 1 can be changed to appropriate alternative components as long as differentiation into corneal endothelial replacement cells is possible.

[0030] [Table 1]

[0031] The obtained corneal endothelial substitute cells exhibit biological activity that depends on the functional molecule encoded by the introduced nucleic acid.

[0032] Here, the corneal endothelium alternative The term "cells" refers to cells obtained by inducing differentiation from stem cells, preferably pluripotent stem cells, and more preferably induced pluripotent stem cells (iPS cells), and has characteristics very similar to corneal endothelial cells derived from a living body, but is not completely identical. Specific examples of the corneal endothelial cell-like properties and functions possessed by the corneal endothelial substitute cells include the following characteristics (i) to (iv), and among these characteristics, the cells have at least one, preferably two, more preferably three, and even more preferably all four. (i) Cell-cell adhesion is composed of N-cadherin. (ii) Tight junctions are formed between the cells. (iii) Express the Na,K-ATPase α1 subunit on the cell membrane. (iv) Expression of the transcription factor PITX2 is observed in the cell nucleus. Whether or not the cell-cell adhesion is composed of N-Cadherin can be confirmed by immunostaining for N-Cadherin. Whether or not tight junctions are formed between cells can be confirmed by observing the presence of ZO-1, a protein that constitutes tight junctions, using immunostaining for ZO-1, or by directly observing the structure using an electron microscope. Whether or not Na,K-ATPase α1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by co-staining of ZO-1 and Na,K-ATPase α1 subunit by immunostaining for both. Whether or not the transcription factor PITX2 is expressed in the cell nucleus can be confirmed by immunostaining for PITX2.

[0033] 2. Functionalized corneal endothelial replacement cells The present invention provides corneal endothelial replacement cells to which functionality has been imparted. "Functionalized" means that the cells express an exogenous functional molecule (i.e., a functional molecule other than a functional molecule that is inherently expressed by the cells). "Functional molecule" and "corneal endothelial replacement cells" are described in detail in "1. Method for producing functionalized corneal endothelial replacement cells" above. In the present invention, the functionalized corneal endothelial replacement cells are preferably corneal endothelial replacement cells derived from pluripotent stem cells, particularly from iPS cells, and are more preferably CECSi cells to which functionality has been imparted.

[0034] 3. Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising functionalized corneal endothelial replacement cells as an active ingredient. The functionalized corneal endothelial replacement cells contained as an active ingredient in the pharmaceutical composition of the present invention are the cells described in the above section "2. Functionalized corneal endothelial replacement cells" and may be cells produced by the method described in the above section "1. Method for producing functionalized corneal endothelial replacement cells".

[0035] The pharmaceutical composition of the present invention can be prepared by mixing the functionalized corneal endothelial replacement cells of the present invention, which are the active ingredient, with a pharma- ceutical acceptable carrier. The term "pharma-ceutical acceptable carrier" as used herein includes diluents, adjuvants, excipients, stabilizers, vehicles, or supports (such as cell fiber (alginate hydrogel)) that are non-toxic to cells exposed thereto at the doses and concentrations used. In many cases, the carrier is an aqueous pH buffer solution, an antioxidant, a low molecular weight (less than about 10 residues) polypeptide, a hydrophilic polymer, an amino acid, a monosaccharide, a disaccharide, a chelating agent such as EDTA, a salt-forming counterion such as sodium; and a non-ionic surfactant such as TWEEN (registered trademark), polyethylene glycol (PEG), and PLURONICS (registered trademark), and a preferred carrier is a physiological saline solution, particularly for compositions administered intravenously.

[0036] The pharmaceutical composition of the present invention contains the functionalized corneal endothelial substitute cells of the present invention as an active ingredient, and the cells can express (and in some cases secrete) a therapeutically effective amount of a functional molecule. The therapeutically effective amount is an amount that, when the pharmaceutical composition of the present invention is administered to a subject, can provide a therapeutic effect against diseases such as those described below, compared to a subject not administered the pharmaceutical composition. The specific therapeutically effective amount is appropriately determined depending on the administration method, the purpose of use, and the age, weight, symptoms, etc. of the subject. EXAMPLES

[0037] The present invention will be described in detail below using examples, but the present invention is not limited thereto. In addition, the reagents and materials used are commercially available unless otherwise specified. Abbreviations used in this specification are the same as those commonly used in the art unless otherwise specified.

[0038] Example 1: Preparation of a plasmid vector for genome editing (Figure 1) Genome editing was performed using the AAVS1 Safe Harbor Targeting System (Systems Biosciences (SBI) #GE622-KIT). To insert the soluble programmed death protein 1 (sPD-1) sequence into the multiple cloning site of AAVS1 Safe Harbor Targeting Vector 2.0, GOI Knock-in Donor (AAVS1-SA-puro-EF1-MCS) (SBI#GE622A-1), a plasmid vector carrying a sequence with a BsrGI (TGTACA) restriction enzyme site at the 5' end and an AgeI (ACCGGT) restriction enzyme site at the 3' end of sPD-1 was cloned. The insert was prepared by amplifying the target sequence by conventional PCR using the following primers with the sPD1 expression vector (pAAV plasmid vector) as bait. Forward primer: 5'-aaaaTGTACAGCCACCATGCAGATCCCAC (29 bp) (SEQ ID NO: 1) Reverse primer: 5'-aaaaACCGGTTTGGAACTGGCCGGCTGG (28 bp) (SEQ ID NO: 2) The PCR product was separated on a 1% agarose gel, and a band around 500 bp was excised and DNA was extracted from the gel using a QIAquick PCR Purification Kit (QIAGEN 28104). This was reacted at 37°C for 2 hours in the presence of AgeI-HF restriction enzyme (New England Biolabs R3552) and BrGSI-HF restriction enzyme (New England Biolabs R3535S), separated on a 1% agarose gel, and extracted from the gel to produce an insert that can be inserted into a vector. The vector was prepared by treating the HR vector provided in the kit with restriction enzyme AgeI, separating it on a 1% agarose gel, extracting it with the kit, reacting it with BrsHI at 37°C for 2 hours for restriction enzyme treatment, separating it on a 1% agarose gel, and extracting it with the kit. The insert and vector were mixed at a ratio of 3:1 and ligated (Takara Mighty Max) for 2 hours at 16°C. Transformation was performed using competent cells, which were then plated on an ampicillin-containing agar plate and incubated overnight at 37°C. The next day, colonies were picked and grown on a small scale in the presence of ampicillin, and E. coli pellets were prepared. DNA was prepared from the E. coli pellet using a miniprep kit (Wizard, Promega), and then subjected to restriction enzyme treatment with AgeI and BrsHI to confirm the presence of the insert. Inserts were confirmed in all six clones (Figure 2).

[0039] Example 2: Fluorescent immunostaining of CECSi cells expressing sPD-1 The clones containing the insert were used and transfected into iPS cells together with the Cas9 SmartNuclease AAVS1-gRNA Targeting Vector (#CAS601A-1) provided in the kit using a transfection reagent (Lipfectamin 3000 Thermo Fisher L3000001). The medium was changed 36 hours after transfection, and the cells were allowed to grow until colonies were formed. At the next passage, puromycine treatment was performed at a concentration of 0.5μg-1μg / ml to select only the cells into which the gene had been introduced. After expansion culture in the absence of puromycine, 1x10 5 ~2x10 5 The number of cells was seeded onto a 6-well plate, and differentiation induction was performed. Differentiation induction into CECSi cells was performed according to a conventional method, for example, the method described in Patent Document 3 below. After expansion, the cells (iPS cells with inserts) were cultured in iMatrix-511 (0.6 μg / cm 2 ) coated on a culture dish and cultured for 1 week using StemFit (registered trademark) AK03N medium (Ajinomoto Co., Inc.). Then, iMatrix-511 (0.3 μg / cm 2 ) and cultured for 8 days in the differentiation-inducing medium shown in Table 1 above. After differentiation induction, the cells formed an endothelial cell structure. Differentiation into endothelial cells was confirmed by the following fluorescent immunostaining. The results are shown in Figure 3. (primary antibody) Anti-PITX2 Rabbit-Polyclonal antibody (ARP32431), N-cadherin Monoclonal antibody (Thermo Fisher 333900), ZO-1 Polyclonal antibody (Thermo Fisher 402200), Na,K-ATPase Mouse antibody (Novas Biologicals, NB300-146), (Secondary antibody) Cy3-conjugated Donkey Anti-Rabbit IgG (Jackson Immuno Research 711-165-152), Donkey anti-Mouse IgG (H+L) Alexa Fluor 488 (Thermo Fisher A21202), DAPI (DOJINDO 340-07971) Differentiation into CECSi cells was confirmed by staining for ZO-1, N-cadherin, and PITX2.

[0040] Example 3: Functionalized CECSi cells obtained by inducing differentiation from functionalized pluripotent stem cells (1) 5x10 iPS cells and CECSi cells transfected with nucleic acids 5 The mRNA was extracted using RNeasy mini kit (QIAGEN 74004) and reverse transcribed using ReveTra Ace (Toyobo TRT-101). The reverse transcribed cDNA was subjected to RT-PCR using the following primers, and the target band was confirmed at around 500 bp when separated and confirmed on an agarose gel (Figure 4). The annealing temperature was 64°C. The PCR reaction program is as follows: After 25 cycles of denaturation at 94°C for 30 seconds, annealing at 64°C for 30 seconds, and extension at 72°C for 1 minute, the mixture was reacted at 72°C for 5 minutes and stored at 4°C. The primers used are as follows: Forward Primer: 5'-CTTCACCTGCAGCTTCTCCA (SEQ ID NO: 3) Reverse Primer: 5'-CCGCAGGCTCTCTTTGATCT (SEQ ID NO: 4)

[0041] Example 4: Functionalized CECSi cells obtained by inducing differentiation from functionalized pluripotent stem cells (2) Using iPS cells transfected with a nucleic acid encoding sPD-1, differentiation induction into CECSi cells was carried out twice (lot 1, lot 2) in the same manner as in Example 2, and the sPD-1 protein secreted into each culture supernatant was confirmed by Western blotting. For the positive control (Posi), a culture supernatant sample of CECSi cells transfected with an sPD-1 plasmid was used. 6x Sample buffer (Nacalai Tesque 09499-14) containing 10% β-mercaptoethanol was added to the culture supernatant on the 8th day to prepare an electrophoresis sample. After separating proteins using e-PAGEL mini-sized pre-made gel (10-20%) on ATTO Pagelan Ace, the proteins were transferred to PVDF Membrane (ATTO WSE4057 QBlot kit M) using ATTO Powered Blot 2M (ATTO WSE-4125) at 24V for 20 minutes, and blocked with Blocking One (Nacalai Tesque 03953-95) for 2 hours. The primary antibody was α-Human PD-1 (R&D Systems #MAB10864-100) diluted 1000 times and incubated overnight at 4°C. After washing the membrane with 1x TBS / 0.05% Tween buffer, the secondary antibody was anti-mouse HRP-linked IgG (CST #7076P2) diluted 5000 times and incubated at room temperature for 1 hour. The ECL reaction was performed using Nacalai Chemi-lumi One Ultra (Nacalai Tesque 11644), and detection was performed using iBright FL1000 Imaging Systems (Thermofisher) to confirm the presence or absence of a band around 32 kDa. The results are shown in Figure 5. sPD-1 protein was detected in the culture supernatant of CECSi cells produced by inducing differentiation using iPS cells transfected with nucleic acid encoding sPD-1. sPD-1 protein was not detected in the culture supernatant of CECSi cells produced by inducing differentiation from iPS cells not transfected with nucleic acid encoding sPD-1. [Industrial Applicability]

[0042] According to the present invention, it is possible to impart various biological functions to corneal endothelial substitute cells.

Claims

1. A method for producing functionalized corneal endothelial replacement cells, comprising the steps of: Step 1: introducing a nucleic acid encoding a functional molecule into a genomic region of a pluripotent stem cell to obtain a functionalized pluripotent stem cell; and Step 2: A step of inducing the pluripotent stem cells to which the functionality has been imparted into corneal endothelial replacement cells.

2. The method of claim 1 , wherein the genomic region is the AAVS1 locus.

3. The method of claim 1 , wherein the pluripotent stem cells are iPS cells.

4. The method according to claim 1, wherein the corneal endothelial substitute cells are CECSi cells (Corneal Endothelial Cell Substitute from iPS cells).

5. The method according to claim 1, characterized in that the step of introducing a nucleic acid encoding a functional molecule into the genome of a pluripotent stem cell is carried out by genome editing technology.

6. The method according to claim 5, wherein the genome editing technology is CRISPR / Cas9.

7. A corneal endothelial replacement cell characterized by expressing an exogenous functional molecule.

8. The cell of claim 7 , which is derived from a pluripotent stem cell.

9. The cells according to claim 7, wherein the corneal endothelial substitute cells are CECSi cells (Corneal Endothelial Cell Substitute from iPS cells).

10. A functionalized corneal endothelial replacement cell produced by the method according to any one of claims 1 to 5.

11. A pharmaceutical composition comprising the cells described in claim 10.

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