Method for producing functionalized corneal endothelial substitute cells

Abstract

To provide corneal endothelial substitute cells derived from pluripotent stem cells, in particular iPS cells, endowed with new functions, especially multiple functions, and a method for producing the cells.SOLUTION: A method for producing corneal endothelial substitute cells endowed with two or more functions, comprising a step of introducing into cells two or more nucleic acids encoding functional molecules.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to functionalized corneal endothelial replacement cells, particularly iPS cell-derived corneal endothelial replacement cells ( C orneal E ndothelial C ell S substitute from i The present invention relates to a method for producing CEC Si cells (PS cells) and cells produced by the method. [Background technology]

[0002] iPS cell-derived corneal endothelial substitute cells (CECSi cells; C orneal E ndothelial C ell S substitute from i CECSi cells (PS cells) were previously developed for the treatment of bullous keratopathy (Patent Documents 1 to 3). On the other hand, CECSi cells have the advantage that differentiated endothelial cells can be mass-produced from iPS cells in a short period of time (approximately 2 weeks) with uniform quality and high adhesiveness to cells and extracellular matrix. If we could take advantage of this characteristic and confer additional functions to CECSi cells through gene editing or gene transfer, we could aim to expand the application beyond corneal treatment to other disease areas. Gene editing technologies that have been developed include those using endonucleases such as zinc finger nucleases, TALEN (transcription activation-like effector nucleases), and the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. Gene transfer techniques can be broadly divided into non-viral and viral transfer methods. In gene therapy, viral gene transfer techniques are used, and many viral vectors have been developed.

[0003] In addition to genes encoding proteins with desired functions, various genes useful for gene therapy can be introduced. For example, the introduction of genes encoding fluorescent proteins is widely used to track and observe cells and organisms. These proteins emit light within cells or tissues, making them useful for tracking specific cells or structures. Gene expression regulators, such as enhancers and promoters, and RNA interference technologies such as siRNA and miRNA, can also be used to suppress expression. These techniques enable the expression or suppression of specific genes. Depending on the gene being introduced, the base sequence of a specific gene can be altered to change its function. This can, for example, alter the properties of an enzyme or the structure of a protein. Drug-sensitivity genes can also be used, including genes that respond to specific drugs or compounds and promoters that induce their expression. This allows cells and tissues to be stimulated or controlled by specific drugs. [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 iPS cells, which have been endowed with new functions, particularly multiple types of functions, and to provide a method for producing such cells. [Means for solving the problem]

[0006] In view of the above problems, the present inventors have developed corneal endothelial replacement cells derived from pluripotent stem cells (CECSi cells: C orneal E ndothelial C ell S substitute from i The present inventors attempted to impart multiple functions to iPS cells (iPS cells). They prepared CEC-Si cells by inducing differentiation of iPS cells into corneal endothelial replacement cells, introduced multiple genes into the resulting CEC-Si cells, and confirmed the expression of these genes, which led to the completion of the present invention.

[0007] That is, the present invention provides the following. [1] A method for producing corneal endothelial substitute cells to which two or more types of functionality have been imparted, comprising the step of introducing nucleic acids encoding two or more types of functional molecules into cells, The method, wherein the cells are pluripotent stem cells or corneal endothelial replacement cells derived from pluripotent stem cells. [2] The method described in [1], wherein the pluripotent stem cells are iPS cells. [3] Corneal endothelial replacement cells are CEC Si cells ( C orneal E ndothelial C ell S substitute from i The method according to [1] or [2], wherein the cells are selected from the group consisting of erythrocytes, ...lysosomes, and lysosomes. [4] The method according to any one of [1] to [3], wherein the introduction of the nucleic acid into the cell is by gene transfer or genome editing. [5] (1) preparing an expression vector into which a nucleic acid encoding a functional molecule is inserted; (2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to prepare a cell containing the expression vector; and (3) culturing cells containing the expression vector; A method for producing corneal endothelial replacement cells to which two or more types of functionality have been imparted, comprising: The method, wherein the cells are pluripotent stem cells or corneal endothelial replacement cells derived from pluripotent stem cells. [6] Corneal endothelial replacement cells derived from pluripotent stem cells, characterized by expressing two or more types of exogenous functional molecules. [7] The cell according to [6], wherein the pluripotent stem cell is an iPS cell. [8] Corneal endothelial replacement cells are CEC Si cells ( C orneal E ndothelial C ell S substitute from i The cell according to [6] or [7], which is a PS cell. [9] Corneal endothelial replacement cells to which two or more types of functionality have been imparted, produced by the method described in any one of [1] to [5].

[10] A pharmaceutical composition comprising the cells according to any one of [6] to [9].

[11] The method according to any one of [1] to [5], wherein nucleic acids encoding two or more types of functional molecules are carried in a single expression vector.

[12] The method according to any one of [1] to [5], wherein nucleic acids encoding two or more types of functional molecules are carried on two or more expression vectors. [Effects of the Invention]

[0008] Creating CECSi cells that express multiple functions, i.e., designer cells that exhibit two or more distinct functions, is expected to produce synergistic effects in existing treatment methods. For example, it would be possible to introduce two genes with different mechanisms of action for a single disease, or to introduce a combination in which one gene supports the function of another. Furthermore, the high adhesiveness of CECSi cells could improve their ability to engraft at the disease site, thereby potentially increasing the therapeutic effect of the introduced genes. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows a construction diagram of a plasmid vector that simultaneously expresses sigGLP-1 and sVEGFR1, and the base sequence of a nucleic acid encoding a functional molecule introduced into the vector. [Figure 2] FIG. 2 shows the construction of two types of sVEGFR1 expression vectors and the base sequences of nucleic acids encoding functional molecules introduced into the vectors. [Figure 3] FIG. 3 shows a construction diagram of an EGFP expression vector and the base sequence of a nucleic acid encoding a functional molecule introduced into the vector. [Figure 4] FIG. 4 shows a construction diagram of a plasmid vector that simultaneously expresses sigGLP-1 and GFP, and the base sequence of a nucleic acid encoding a functional molecule introduced into the vector. [Figure 5] FIG. 5 shows a diagram of the construction of the sPD-1 expression vector and the base sequence of the nucleic acid encoding the functional molecule introduced into the vector. [Figure 6] FIG. 6 shows a construction diagram of a plasmid vector that simultaneously expresses sVEGFR1 and FGF7, and the base sequence of a nucleic acid encoding a functional molecule introduced into the vector. [Figure 7] Figure 7 shows the results of Western blotting of proteins secreted into the culture supernatant of ATCC-CECSi cells transfected with an sFLT1(AAV2) expression vector and a sigGLP-1-GFP expression vector or an EGFP expression vector, or with an sFLT1(AAV1) expression vector and a sigGLP-1-GFP expression vector. [Figure 8] Figure 8 shows the results of Western blotting of proteins secreted into the culture supernatant of ATCC-CECSi cells transfected with an sFLT1 (AAV2) expression vector and an sPD-1 expression vector or an EGFP expression vector. [Figure 9] FIG. 9 shows the results of Western blotting detection of each protein secreted into the culture supernatant of ATCC-CECSi cells transfected with an sGLP-1 expression vector and an sVEGFR1 expression vector. [Figure 10]FIG. 10 shows the results of Western blotting detection of each protein secreted into the culture supernatant of ATCC-CECSi cells transfected with a plasmid vector that simultaneously expresses sVEGFR1 and FGF7. [Figure 11] FIG. 11 is a graph showing the results of a comparison of the adhesion ability of ATCC CECSi cells and HUVEC cells to a culture substrate. n=3 DETAILED DESCRIPTION OF THE INVENTION

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

[0011] In the present invention, "corneal endothelial replacement cells" are cells obtained by inducing differentiation from pluripotent stem cells, more preferably from induced pluripotent stem cells (iPS cells), and have characteristics very similar to corneal endothelial cells derived from living organisms, but are not completely identical. Specific examples of the corneal endothelial cell-like properties and functions possessed by corneal endothelial substitute cells include the following characteristics (i) to (iv), and the corneal endothelial substitute cells used in the present invention have at least one, preferably two, more preferably three, and even more preferably all four of these characteristics. (i) Cell-cell adhesion is composed of N-cadherin. (ii) Tight junctions are formed between 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 intercellular 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 the Na,K-ATPase α1 subunit (ATP1A1) is expressed on the cell membrane can be confirmed by co-staining with ZO-1 and Na,K-ATPase α1 subunit by immunostaining. Whether or not the transcription factor PITX2 is expressed in the cell nucleus can be confirmed by immunostaining for PITX2.

[0012] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and skin stem cells.

[0013] 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), embryonic germ cells (EG 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 produced from germ cells (e.g., testes) are also included in pluripotent stem cells. When simply referring to stem cells, this is intended to encompass pluripotent stem cells.

[0014] ES cells can be produced by culturing the inner cell population on feeder cells or in medium containing leukemia inhibitory factor (LIF). They are also available from designated institutions and commercially available. Nuclear transfer ES cells (ntES cells), a type of ES cell, can be established from cloned embryos created by transplanting the nucleus of a somatic cell into an egg cell from which the nucleus has been removed.

[0015] EG cells can be produced 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).

[0016] iPS cells are cells induced to pluripotency by reprogramming somatic cells using known methods. Specific examples of iPS cells include cells induced to pluripotency by reprogramming somatic cells differentiated into fibroblasts, peripheral blood mononuclear cells, etc., through the expression of multiple 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, etc. 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. Similar to embryonic stem cells, these cells possess pluripotency and self-renewal capabilities (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 direct reprogramming via gene expression, induced pluripotent stem cells can also be induced from somatic cells by the addition of chemical compounds (Science, 2013, 341, pp.651-654).

[0017] 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.), hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.

[0018] When producing induced pluripotent stem cells, if reprogramming is performed by expressing several types of genes (e.g., four factors: Oct3 / 4, Sox2, Klf4, and Myc), the means for expressing the genes is not particularly limited. Examples of means for expressing genes include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated viral vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors and episomal vectors) (e.g., calcium phosphate transfer, lipofection, retronectin transfer, and electroporation), gene transfer methods using RNA vectors (e.g., calcium phosphate transfer, lipofection, and electroporation), and direct protein injection.

[0019] It is also possible to obtain established induced pluripotent stem cells. Specifically, 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 and UTA-1-SF-2-2 strains (all from the University of Tokyo), 21526, 21528, 21530, 21531, 31536, and 31538 strains (all from the University of Tokyo). Examples of suitable strains include those from Fujifilm Cellular Dynamics, 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 nonprofit American Type Culture Collection).

[0020] "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 includes prosimians such as lemurs, lorises, and tree shrews, and anthropoids such as monkeys, apes, and humans.

[0021] The pluripotent stem cells used in the present invention 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.

[0022] Corneal endothelial substitute cells induced from pluripotent stem cells used in the present invention include, for example, corneal endothelial-like cells developed by the present inventors, which can be produced and prepared by the methods described in Patent Documents 1 to 3. Preferably, these are corneal endothelial substitute cells derived from iPS cells (Corneal Endothelial Cell Substitute from iPS cells; CECSi cells), which have corneal endothelial cell-like properties and functions and are characterized by enhanced expression of the NR3C2 (nuclear receptor subfamily 3, group C, member 2) gene (Patent Document 3). An example of a method for producing CECSi cells is as follows. iPS cells are cultured in iMatrix-511-coated culture dishes using StemFit® AK03N medium (Ajinomoto) until the cells reach a state of appropriate tight aggregation. The period required for "until the cells reach a state of appropriate tight aggregation" is not particularly limited as long as this state is reached, and varies depending on culture conditions such as the number of cells seeded and the state of the cells. However, it typically takes 5 to 20 days, preferably 6 to 18 days, more preferably 7 to 16 days, and particularly preferably 8 to 14 days. The iPS cells are then reseeded onto iMatrix-511-coated culture dishes, and differentiation-inducing culture is performed using differentiation-inducing medium to induce differentiation of the iPS cells into CEC Si cells. When using cryopreserved iPS cells, differentiation induction is performed after at least two passages and culture. The differentiation-inducing medium used is a basal medium such as DMEM / F12 supplemented with various growth factors (e.g., IGF1, LIF, IL-11, IL-6, TNF-α), supplements (e.g., ITS-supplement), etc. Whether or not differentiation of pluripotent stem cells into CECSi cells has been successfully induced can be determined by confirming that the cells after differentiation induction have corneal endothelial cell-like properties and functions, specifically, at least one, preferably two, more preferably three, and even more preferably all four of the above characteristics (i) to (iv). Furthermore, because the expression level of the NR3C2 gene is enhanced in CECSi cells (Patent Document 3), it is also preferable to confirm an increase in the expression level of the NR3C2 gene.

[0023] As used herein, "functionalized" cells refer to cells engineered to exhibit a biological activity that the cells do not inherently possess. One embodiment of the term refers to cells expressing a functional molecule associated with 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 ex vivo, or intracellularly or extracellularly. Generally, a molecule consisting of 50 or more amino acids is called a protein, and a molecule consisting of fewer than 50 amino acids is called a peptide. In this specification, "peptide" encompasses proteins. Furthermore, peptides can be classified into oligopeptides consisting of approximately 2 to 10 amino acids and polypeptides consisting of more than 50 amino acids. In this specification, "peptide" encompasses both oligopeptides and polypeptides. As used herein, a "specific biological function" is not limited to any function that can affect biomolecules, such as proteins and nucleic acids, cells, tissues, or individuals. Specific biological functions may be natural or non-natural, and examples include cell adhesion, signal transduction, binding, linking, labeling, and metabolic functions. Cell adhesion function refers to the interaction between cells and between cells and the extracellular matrix, involving cell adhesion molecules. This function plays an important role in maintaining cell morphology, tissue formation, intercellular communication, immune responses, cancer metastasis, and other areas. Signal transduction function refers to the mechanism by which external stimuli or information are received, received within the cell, and a response is triggered. Signal transduction typically occurs via cell surface receptors and through intracellular signaling pathways and molecular interactions. This function is involved in cell survival, proliferation, differentiation, response, and regulation, and plays an important role in controlling various physiological processes and cellular responses. Examples of binding function include the function of mediating antigen-antibody binding or receptor-ligand interaction, the function of binding biomolecules such as RNA and / or DNA, and the function of binding nickel ions, copper ions, etc. Linking function refers to the function of linking other biomolecules such as peptides and nucleic acids, small molecules, or metal ions that have specific biological functions. Labeling function refers to the function of labeling 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. The functional peptide can be appropriately selected depending on the purpose of use.

[0024] Specific examples of "functional peptides" in the present invention include enzymes, binding proteins, marker proteins, artificial peptides, and peptide fragments thereof. "Enzymes" include, but are not limited to, DNA polymerase, RNA polymerase, and phosphorylation 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, etc. Typically, these are polypeptides whose activity can be used to determine the expression or presence of a target protein. Examples include, but are not limited to, fluorescent proteins such as GFP, luminescent proteins such as luciferin or aequorin, and enzymes such as horseradish peroxidase (HRP), and alkaline phosphatase (AP). "Artificial peptides," also known as tag peptides, are artificially synthesized oligopeptides consisting of several to a dozen amino acids. Peptides capable of exerting therapeutic effects on cells into which they are introduced are also preferred functional peptides in the present invention. Because CEC-Si cells have high adhesive properties and are expected to have localized effects, peptides that exert therapeutic effects when expressed locally are preferred. More specific examples of functional peptides include the soluble fraction of vascular endothelial growth factor (VEGF) receptor and human fibroblast growth factor 7 (FGF-7). Because the soluble fraction of VEGF receptor can locally suppress angiogenesis, it is expected to reduce side effects compared to systemic administration of sVEGF receptor antagonists. FGF7 acts specifically on epithelial cells and is expected to repair alveolar epithelium and other tissues. By inhibiting immune checkpoints, sPD-1 is expected to treat local recurrence and metastasis of cancer cells.

[0025] Biological activity can be measured and its expression evaluated by techniques well known in the art. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus), such as the affinity of a compound that directly binds to a peptide, which is a functional molecule, or, for example, the amount of an upstream or downstream protein or other similar function after some stimulus or event. The indicator can be appropriately selected depending on the desired biological activity.

[0026] 1. Method for producing functionalized corneal endothelial replacement cells The present invention provides a method for producing corneal endothelial substitute cells that are endowed with functionality, particularly two or more types of functionality (hereinafter also simply referred to as the production method of the present invention). The production method of the present invention comprises the step of introducing two or more types of nucleic acids encoding functional molecules into cells, wherein the cells into which the nucleic acids encoding the functional molecules are introduced are pluripotent stem cells or corneal endothelial substitute cells induced from pluripotent stem cells.

[0027] Two or more types of functionality may be imparted to corneal endothelial cells at any stage. For example, functionality may be imparted to pluripotent stem cells (particularly iPS cells), or to corneal endothelial replacement cells (particularly CECSi cells) obtained by inducing the differentiation of pluripotent stem cells. Specifically, nucleic acids encoding two or more types of functional molecules are inserted into an appropriate expression vector. The nucleic acids encoding two or more types of functional molecules may be incorporated into multiple expression vectors or into the same expression vector. In this case, they are incorporated into the expression vector so that they are expressed under the control of an expression control region, for example, an enhancer or promoter. Next, this (these) expression vectors are introduced into cells to exhibit the desired functionality. It is also possible to impart some of the two or more types of functionality to pluripotent stem cells (particularly iPS cells), and then impart the remaining functionality to corneal endothelial replacement cells (particularly CECSi cells) obtained by inducing the differentiation of the pluripotent stem cells. Specifically, for example, when imparting two types of functionality to corneal endothelial substitute cells (particularly CECSi cells), first a nucleic acid encoding one type of functional molecule is introduced into pluripotent stem cells (particularly iPS cells), and after inducing differentiation of the corneal endothelial substitute cells to which the one type of functionality has been imparted, a nucleic acid encoding another type of functional molecule is introduced.

[0028] There are no particular limitations on the type of expression vector that can be used, as long as it stably retains the inserted gene, and various types of vectors are available. The vector can be a viral or non-viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and vaccinia viral vectors. Among these, retroviral vectors, lentiviral vectors, and adeno-associated viral vectors integrate the target gene into the host chromosome, allowing for stable and long-term expression. Each viral vector can be prepared according to standard methods or using commercially available dedicated kits. Examples of non-viral vectors include plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, BAC vectors, and artificial chromosome vectors. These vectors can also be used in gene therapy, preferably adeno-associated virus vectors. In the case of viral vectors, expression vectors are introduced into cells by viral infection. In the case of non-viral vectors such as plasmids, conventional methods such as electroporation, lipofection, calcium phosphate injection, and nucleofection can be used for introduction into cells, and lipofection is preferred.

[0029] Functionality may be imparted to cells by genome editing. "Genome editing" is a technique for intentionally modifying a target gene or genome region by site-specific cleavage of genomic DNA strands using nucleases or chemical conversion of bases. Examples of site-specific nucleases include zinc finger nucleases (ZFN), TALEN, and CRISPR / Cas9. Genome editing technology can be used to create knockout cell lines in which specific genes are deleted, knockin cell lines in which a different sequence is artificially inserted into a specific gene locus, and the like. In the present invention, genome editing technology is used to introduce nucleic acids encoding functional molecules into pluripotent stem cells or corneal endothelial substitute cells induced from pluripotent stem cells. 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 a DNA double-strand break with a specificity of 18 to 36 bases. TALENs are artificial nucleases that contain a TALE, a DNA-binding domain found in the plant pathogenic bacterium Xanthomonas. The DNA-binding domain of TALE consists of 34 amino acids that recognize a single base, and TALENs with 15 to 20 units are engineered on the sense and antisense strands to induce double-strand breaks in DNA at the target site. The CRISPR / Cas system uses a complex of Cas nuclease, which has DNA double-strand cleavage activity by itself, and a target sequence-specific single-stranded guide RNA to introduce double-stranded DNA breaks at targeted base sequences. Due to its convenience, high efficiency, and versatility, the CRISPR / Cas system has become a standard genome editing technology used worldwide. Cas nucleases include Cas9, Cas12a (Cpf1), Cas13a (C2c2), Cas14, Cas3, and CasX, with Cas9 being 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 Cpf1 (AsCpf1) derived from Acidaminococcus sp. and Cpf1 (LbCpf1) derived from Lachnospiraceae bacterium. The CRISPR / Cas system can also use mutants (subspecies) with mutations in the amino acid sequence of the Cas nuclease. dCas9 (deadCas9) is a nuclease-deficient Cas9 that binds to DNA sequences but does not cleave them. nCas9 (nickase Cas9) introduces single-strand 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 preferred is a technology using CRISPR / Cas, particularly CRISPR / Cas9, which uses Cas9 as the Cas nuclease.

[0030] When the cells to be transfected with a nucleic acid molecule encoding a functional molecule are pluripotent stem cells (particularly iPS cells), the subsequent cell culture for expansion 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), StemFit (registered trademark) AK medium (Ajinomoto), and mixed media thereof are included. Corneal endothelial substitute cells can be obtained by inducing differentiation of the thus obtained pluripotent stem cells into which nucleic acids encoding functional molecules have been introduced, for example, according to the descriptions in Patent Documents 1 to 3. The obtained corneal endothelial substitute cells exhibit the desired functionality depending on the type of nucleic acid encoding the introduced functional molecule.

[0031] When the cells used in the step of imparting functionality to the cells are corneal endothelial substitute cells (particularly CECSi cells) that have been induced to differentiate from pluripotent stem cells, the cell culture in the subsequent expansion culture is preferably carried out in a medium for culturing corneal endothelial substitute cells. Such a medium can be any known medium, and is not particularly limited as long as it does not inhibit the proliferation of differentiated cells. Examples of such a medium 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 Examples include BMOC-3 Medium and mixed media thereof.

[0032] 2. Functionalized corneal endothelial replacement cells The present invention provides two or more types of 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 molecules" and "corneal endothelial replacement cells" are described in detail above in "1. Method for producing functionalized corneal endothelial replacement cells." In the present invention, the functionalized corneal endothelial replacement cells are preferably corneal endothelial replacement cells derived from pluripotent stem cells, particularly iPS cells, and are even more preferably CEC Si cells to which functionality has been imparted.

[0033] 3. Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising functionalized corneal endothelial substitute cells as an active ingredient. The functionalized corneal endothelial substitute cells contained as an active ingredient in the pharmaceutical composition of the present invention are the cells described above in the section "2. Functionalized corneal endothelial substitute cells" and may be cells produced by the method described above in the section "1. Method for producing functionalized corneal endothelial substitute cells."

[0034] The pharmaceutical compositions of the present invention can generally be prepared by mixing the functionalized corneal endothelial substitute cells of the present invention, which are the active ingredient, with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" encompasses diluents, adjuvants, excipients, stabilizers, vehicles, or supports (such as cell fiber (alginate hydrogel)) that are non-toxic to cells exposed to them at the dosages and concentrations used. In many cases, the carrier is an aqueous pH buffer solution, antioxidants, low-molecular-weight (less than about 10 residues) polypeptides, hydrophilic polymers, amino acids, monosaccharides, disaccharides, chelating agents such as EDTA, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. For compositions administered intravenously, a preferred carrier is saline solution.

[0035] The pharmaceutical composition of the present invention contains, as an active ingredient, the functionalized corneal endothelial substitute cells of the present invention, and the cells can express (and, in some cases, secrete) a therapeutically effective amount of a functional molecule. A therapeutically effective amount is an amount that, when 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 determined appropriately depending on the method of administration, the purpose of use, and the age, weight, symptoms, etc. of the subject. [Example]

[0036] The present invention will be described in detail below using examples, but the present invention is not limited thereto. Furthermore, 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.

[0037] Materials and Methods

[0038] 1. Preparation of CEC Si Cells CECSi cells were prepared by inducing differentiation from iPS cells purchased from ATCC (ATCC-BYS0112 Human [Non-Hispanic Caucasian Male] Induced Pluripotent Stem (IPS) Cells (ATCC ACS-1026)) (hereinafter referred to as ATCC CECSi cells in this example). Differentiation into CECSi cells was performed according to a conventional method, for example, the following method described in Patent Document 3. iPS cells were cultured in iMatrix-511 (0.6 μg / cm 2 The cells were cultured for 1 week on a culture dish coated with iMatrix-511 (0.3 μg / cm ) in StemFit® AK03N medium (Ajinomoto). 2 ) and cultured for 8 to 14 days in the differentiation-inducing medium shown in Table 1 below.

[0039] [Table 1]

[0040] 2. Vector Construction (1) sigGLP-1-(Flag)-sVEGFR1 A plasmid vector (sigGLP-1-(Flag)-sVEGFR1) that simultaneously expresses Flag-tagged sigGLP-1 and sVEGFR1 was constructed using serotype 2 (AAV2). The structure of the vector is shown in Figure 1. The nucleotide sequence encoding Flag-tagged sigGLP-1 is shown in SEQ ID NO: 1, and the nucleotide sequence encoding sVEGFR1 is shown in SEQ ID NO: 2. SEQ ID NO: 2 corresponds to the sequence of a portion of the extracellular domain of VEGFR-1. (2) sVEGFR1 (AAV1 and AAV2) Two types of viral vector plasmids were used. The serotype 1 (AAV1) vector was constructed by adding a partial sequence of the extracellular domain of VEGFR-1 behind the CAG promoter to the pAAV expression vector (Figure 2(A), sFLT-1(AAV1)). The serotype 2 (AAV2) vector was constructed by adding a partial sequence of the extracellular domain of VEGFR-1 behind the EF1α promoter to the pAAV expression vector (Figure 2(B), sFLT-1(AAV2)). The nucleotide sequence of the partial extracellular domain of VEGFR-1 used is shown in SEQ ID NO: 3. (3) EGFP An EGFP expression vector was constructed using serotype 2 (AAV2). The EGFP sequence was added to the end of the EF1α promoter in the pAAV expression vector (Figure 3). The nucleotide sequence of the EGFP used is shown in SEQ ID NO: 4. (4) sigGLP-1-GFP A plasmid vector (sigGLP-1-GFP) that simultaneously expresses sigGLP-1 and GFP was constructed using serotype 2 (AAV2). The structure of the vector is shown in Figure 4. The nucleotide sequences encoding sigGLP-1 and GFP to be inserted into the vector are shown in SEQ ID NO:5. (5)sPD-1 An sPD-1 expression vector was constructed using serotype 1 (AAV1). The sequence of exon 1 and part of exon 2 of PD-1 was added downstream of the CAG promoter to form sPD-1 (Figure 5). The sequence of sPD-1 consisted of exon 1 and part of exon 2 of PD-1 [NM_005018.3]. The nucleotide sequence with the termination codon TGA at the end is shown in SEQ ID NO: 6. (6)sVEGFR1-FGF7 A plasmid vector (sVEGFR1-FGF7) that simultaneously expresses sVEGFR1 and FGF7 was constructed using serotype 1 (AAV1). The structure of the vector is shown in Figure 6. The nucleotide sequence encoding VEGFR1 was that shown in SEQ ID NO: 3. The nucleotide sequence encoding T2A is shown in SEQ ID NO: 7, and the nucleotide sequence encoding FGF7 is shown in SEQ ID NO: 8.

[0041] 3.Adhesion ability measurement A 96-well plate was used, coated with iMatrix511 at 37°C. After removing the supernatant from the cell-containing plate, the cells were washed with 1x PBS. 1 ml of Accutase was added to a 6 cm dish, and 2 ml to a 9 cm dish, and the plate was incubated at 37°C. ATCC-CECSi cells were detached for 10 minutes, and HUVEC cells for 5 minutes. The cells were suspended in 1x PBS and collected in a 15ml stem-fill centrifuge tube (low-adhesion tube). After centrifugation at 200G for 5 minutes at room temperature, the supernatant was removed and the cells were resuspended in 1ml of 1x PBS and transferred to an Eppendorf tube. Low-adhesion tubes are preferred. After thorough suspension, the number of cells was counted using 100 μl of the cell suspension. 3 x 10 per well 4 The cells were counted to ensure a uniform staining, and the cell suspension was transferred to a new Eppendorf tube. After centrifugation at 200G for 5 minutes at room temperature, the PBS was removed and 500 μl of 2 μM Calcein M / PBS was added. The cells were thoroughly suspended and incubated at 37°C for 30 minutes to stain live cells. The tube was inverted every 10 minutes to mix evenly. After the reaction was completed, the mixture was centrifuged at 200 G for 5 minutes at room temperature, the supernatant was removed, and the cells were suspended in culture medium. 5 The cells were seeded into 96 wells so that the cells were 100% pure. After 16 hours of incubation in a 37°C, 5% CO2 incubator, fluorescence was measured using a plate reader (530 nm / 475 nm). This was used to measure the total number of cells. After removing the supernatant, 150 μl of 1×PBS was added and the solution was pipetted up and down five times. The same procedure was repeated three times. 150 μl of 1×PBS was added, and the cells were counted using a plate reader to determine the number of cells after washing. The survival rate was calculated from the number of cells after washing and the total number of cells.

[0042] Example 1 Two or more different genes were introduced into cells by transfection, and the respective proteins were expressed. (1) Co-expression of sVEGFR1 and GLP1 (or EGFP) ATCC-CECSi cells prepared in 24-well plates were transfected with 1 μg each of sFLT1 (AAV2) and sigGLP-1-GFP or EGFP, or sFLT1 (AAV1) and sigGLP-1-GFP using Lipofectamin 3000. Secretion of sVEGFR1 protein and GLP-1-EGFP in the culture supernatant over time (3, 5, and 7 days; D3, D5, and D7, respectively) was confirmed by Western blotting. Sample preparation was performed by adding 5 μl or 25 μl of culture supernatant to 6x concentrated SDS sample buffer (Nacalai) supplemented with reducing agent (b-me) to a 1x sample buffer. The entire sample was loaded onto an e-PAGEL mini-size pre-cast gel (5-20%) prepared in an ATTO Pagelan Ace, and electrophoresis was performed at 21 mA for 60 minutes. After electrophoresis, proteins were transferred to a membrane using an ATTO Powered Blot 2M (WSE-4125) at 25 mV for 20 minutes. After transfer, the membrane was blocked with Blocking One (Nacalai) at room temperature for at least 30 minutes. Then, primary antibodies sVEGFR1 (Abcam #32152) and GFP (Anti-GFP (Green Fluorescent Protein) pAb (MBL, 598)) were diluted 1:1000 and incubated overnight at 4°C. After washing the membrane with 1x TBS / 0.05% Tween buffer, the membrane was incubated with a 2000-fold diluted secondary antibody, rabbit IgG (anti-rabbit IgG, HRP-linked Antidody, Cell Signaling #7074), for 1 to 2 hours. After the incubation, the membrane was washed with 1x TBS / 0.05% Tween buffer and subjected to electroluminescence (ELC) reaction. ChemiLumi Ultra (Nacalai Tesque) was used for ELC, and iBright FL1000 Imaging Systems and Chemidoc touch were used for chemiluminescence detection. The results are shown in Figure 7. Expression of two types of genes (sVEGFR1 and sigGLP-1-GFP, or sVEGFR1 and EGFP) was confirmed in the culture supernatant.

[0043] (2) Co-expression of sPD-1 and sVEGFR1 ATCC-CECSi cells prepared in 24-well plates were transfected with 1 μg each of sFLT1 (AAV2) and sPD-1 or EGFP using Lipofectamin 3000, and the secretion of sVEGFR1 protein and sPD-1 in the culture supernatant over time (3 days, 5 days, and 7 days; D3, D5, and D7, respectively) was confirmed by Western blotting. Western blotting was performed in the same manner as in Example 1(1), except that anti-sPD-1 (Human PD-1 Antibody (R&D Systems, MAB10864-100)) was used as the primary antibody against sPD-1. The results are shown in Figure 8. Expression of two types of genes (sVEGFR1 and sPD-1) was confirmed in the culture supernatant.

[0044] Example 2 Two or more types of genes were carried in one vector, and the vector was introduced into cells by transfection to express each protein. (1) sGLP-1 and sVEGFR1 ATCC-CECSi cells prepared in 24-well plates were transfected with 1 μg of a plasmid vector co-expressing Flag-tagged sigGLP-1 and sVEGFR1 (sigGLP-1-(Flag)-sVEGFR1) using Lipofectamin 3000. Secretion of sVEGFR1 protein in the culture supernatant over time (4 and 5 days; D4 and D5, respectively) was confirmed by Western blotting. Western blotting was carried out in the same manner as in Example 1(1). The results are shown in Figure 9. Expression of sVEGFR1 was confirmed in the culture supernatant four days after transfection, suggesting that expression of sigGLP-1-Flag was also possible (because both genes were carried on the same vector).

[0045] (2) sVEGFR and FGF7 ATCC-CECSi cells prepared in a 24-well plate were transfected with 1 μg of a plasmid vector (sVEGFR1-FGF7) that simultaneously expresses sVEGFR1 and FGF7 using Lipofectamin 3000. Four days later, secretion of sVEGFR1 protein and FGF7 protein in the culture supernatant was confirmed by Western blotting. Western blotting was carried out in the same manner as in Example 1(1), except that anti-FGF7 (Santa Cruz, sc-365440, Lot No. 02318) was used as the primary antibody for FGF7. The results are shown in Figure 10. Expression of sVEGFR1 and FGF7 was confirmed in the culture supernatant 4 days after transfection.

[0046] Example 3: Examination of adhesive ability The adhesive ability of corneal endothelial substitute cells (CECSi) and vascular endothelial cells (HUVEC) was compared. Calcein-stained ATCC CECSi and HUVEC (human umbilical vein endothelial cells, PromoCell C-12200) were seeded onto iMatrix-coated dishes and incubated in a 37°C, 5% CO2 incubator for 4 and 16 hours. The number of viable cells remaining in the dish was then determined after thorough rinsing with PBS. After 4 and 16 hours of cell culture, the cells were thoroughly washed with PBS by pipetting. The number of adherent cells was measured, confirming that more CECSi cells remained than HUVEC cells. The results are shown in Figure 11. These results indicate that CECSi cells have high adhesive ability and are effective as cells for local cell therapy. [Industrial Applicability]

[0047] According to the present invention, it has become possible to endow corneal endothelial substitute cells with multiple functions. CECSi cells, which combine multiple functions, are able to exhibit multiple biological activities in addition to the excellent adhesive ability that the cells themselves possess. For example, it is possible to treat a single disease by introducing two genes with different mechanisms of action, or by using a combination in which one gene complements the function of another gene.

Claims

1. A method for producing corneal endothelial substitute cells to which two or more types of functionality have been imparted, the method comprising the step of introducing nucleic acids encoding two or more types of functional molecules into cells, The method, wherein the cells are pluripotent stem cells or corneal endothelial replacement cells derived from pluripotent stem cells.

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

3. The method according to claim 2, wherein the corneal endothelial substitute cells are CECSi cells (Corneal Endothelial Cell Substitute from iPS cells).

4. The method of claim 1, wherein the introduction of the nucleic acid into the cell is by gene transfer or genome editing.

5. (1) preparing an expression vector into which a nucleic acid encoding a functional molecule is inserted; (2) introducing the nucleic acid into a cell using an expression vector containing the nucleic acid to prepare a cell containing the expression vector; and (3) culturing cells containing the expression vector; A method for producing corneal endothelial replacement cells to which two or more types of functionality have been imparted, comprising: The method, wherein the cells are pluripotent stem cells or corneal endothelial replacement cells derived from pluripotent stem cells.

6. Corneal endothelial replacement cells derived from pluripotent stem cells, characterized by expressing two or more types of exogenous functional molecules.

7. The cell according to claim 6, wherein the pluripotent stem cell is an iPS cell.

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

9. Corneal endothelial substitute cells having two or more types of functionality imparted thereto, produced by the method according to any one of claims 1 to 5.

10. A pharmaceutical composition comprising the cells of claim 9.

11. The method according to any one of claims 1 to 5, wherein two or more types of nucleic acids encoding functional molecules are carried in a single expression vector.

12. The method according to any one of claims 1 to 5, wherein two or more types of nucleic acids encoding functional molecules are carried on two or more expression vectors.

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