Composition for preventing or treating intraretinal angiogenic disease, which contains human retina-derived vascular endothelial cells exhibiting reduced b2m expression and increased tie2 expression

Abstract

According to an aspect, a pharmaceutical composition for the prevention or treatment of an intraretinal angiogenic disease, which contains vascular endothelial cells exhibiting reduced expression or activity of beta-2 microglobulin (B2M) and increased expression or activity of tyrosine kinase receptor 2 (Tie2) compared to parent cells, as an active ingredient, induces stabilization of vascular endothelial cells and reduces vascular permeability even in a state of inflammation or over-expression of vascular endothelial growth factors (VEGF), so as to slow down or inhibit intraretinal angiogenesis, thereby preventing or treating an intraretinal angiogenic disease. In addition, since the cells of the present invention survives for a long period due to low immunogenicity, the effect of inhibiting angiogenesis lasts for a long period after a single intraocular injection, and thus the present invention allows the treatment to proceed less invasively compared to conventional antibody therapies, enhances vascular stability, and enables early diagnosis and treatment of an intraretinal angiogenic disease.

Description

Composition for preventing or treating retinal neovascular disease comprising human retinal-derived vascular endothelial cells with decreased B2M expression and increased TIE 2 expression

[0001] The present invention relates to a composition for preventing or treating retinal neovascular disease, comprising human retinal-derived vascular endothelial cells in which the expression or activity of B2M (Beta-2 microglobulin) is reduced and the expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) is increased.

[0002] Many eye diseases, which cause blindness in millions of people worldwide each year, are primarily caused by neovascularization. Among the various eye diseases caused by abnormal blood vessels, prominent examples include age-related macular degeneration and diabetic retinopathy.

[0003] Age-related macular degeneration (AMD) is a representative neurodegenerative disease that progresses in the eye. It is a disease name referring to the collapse or destruction of the macula that appears along with the aging process, and it mainly occurs in the age group over 50 years. This disease is divided into non-exudative (dry type) in which the macula atrophies and loses visual function as small waste products called drusen accumulate, and exudative (wet type) in which exudate leaks from abnormal new blood vessels caused by the accumulation of hypoxia and oxidative stress in the retina and choroid, causing macular edema. If this process is repeated, the photoreceptor cells and retinal pigment epithelial cell layer in the macula permanently degenerate, causing vision loss.

[0004] In particular, wet macular degeneration has a high incidence of leading to blindness, and the prevalence rate in Korea is estimated to be 37 per 10,000 people. The exact cause of macular degeneration is not yet known, but age is known as a risk factor, and other notable environmental factors include smoking, high blood pressure, obesity, genetic predisposition, excessive UV exposure, and low blood antioxidant concentrations. Currently, the main treatment for wet macular degeneration is antibody injection therapy against vascular endothelial growth factor (VEGF), and other known treatments include laser photocoagulation, photodynamic therapy, and vitrectomy.

[0005] However, antibody injection therapy against vascular endothelial growth factor (VEGF) (Anti-VEGF therapy) or therapy using anti-Ang-2 antibody (Anti-Ang-2 therapy) have the inconvenience of requiring intraocular injections at intervals of 4 to 8 weeks. In addition, short intervals of intraocular injections can cause side effects such as eye pain, ocular hemorrhage or rupture of blood vessels, ocular inflammation or infection, visual changes, and changes in intraocular pressure, which are a great burden to patients. Therefore, regenerative therapy that can restore functional decline by transplanting cells with angiogenic inhibitory function is attracting attention as a final means to overcome common photoreceptor function decline caused by various etiologies.

[0006] The present inventors have developed a cell therapy agent for inhibiting retinal angiogenesis using hypoimmunogenic vascular endothelial cells with increased Tie 2 expression or Ang 1 expression, which have a long cell survival period due to low immunogenicity and thus can have a long-term angiogenesis inhibitory effect with a single intraocular injection, thereby opening a new way to treat various ocular diseases caused by abnormal blood vessels.

[0007] One aspect is to provide a pharmaceutical composition for preventing or treating neovascular disease in the retina, which comprises, as an active ingredient, vascular endothelial cells in which the expression or activity of B2M (Beta-2 microglobulin) is reduced and the expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) is increased compared to parent cells.

[0008] Another aspect is to provide a kit for preventing or treating intraretinal neovascular disease, comprising the pharmaceutical composition.

[0009] Another aspect is to provide a method for preventing or treating angiogenic disease in the retina, comprising administering to a subject in need thereof vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells.

[0010] Another aspect provides the use of vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells for use in the manufacture of a pharmaceutical preparation for preventing or treating neovascular diseases in the retina.

[0011] Another aspect provides the use of vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells for preventing or treating neovascular diseases in the retina.

[0012] One aspect provides a pharmaceutical composition for preventing or treating neovascular disease in the retina, which comprises, as an active ingredient, vascular endothelial cells in which the expression or activity of B2M (Beta-2 microglobulin) is reduced and the expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) is increased compared to parent cells.

[0013] As used herein, the term "Tie 2 (Tyrosine Kinase Receptor 2)" refers to Tyrosine Kinase Receptor 2, a receptor present on the surface of vascular endothelial cells. Tie 2 regulates signal transduction pathways related to retinal angiogenesis and maintenance of normal blood vessel function, and affects vascular stability and permeability, vascular disorders related to angiogenesis and inflammation, etc. Deficiency or dysfunction of Tie 2 can cause problems related to vascular instability, abnormal vascular proliferation, blood coagulation, etc., and overexpression of Tie 2 can improve vascular stability and suppress abnormal angiogenesis.

[0014] As used herein, the term "parent cell" means a native cell itself or a cell before its characteristics are changed by genetic mutation due to natural or artificial factors, but does not exclude a cell that contains a mutation that may occur naturally in a cell.

[0015] As used herein, the term "endothelial cells (EC)" may refer to cells that form a layer covering the interior of blood vessels, one of the components of the blood vessel wall. These vascular endothelial cells exist in various blood vessel types, such as veins, arteries, and lymphatic vessels, and are responsible for functions such as material exchange between blood and tissues, blood coagulation regulation, and vascular permeability control, and may also be involved in angiogenesis and tissue regeneration.

[0016] The term “hypoimmunogenic vascular endothelial cells” as used herein refers to vascular endothelial cells that have the ability to avoid or limit attack by the immune system, and refers to hypoimmunogenic cells with minimized transplant immune responses, and may be used interchangeably with “immune-evading vascular endothelial cells” or “reduced immunogenic vascular endothelial cells.”

[0017] As used herein, the term "immunogenicity" refers to the degree and nature of the immune system's response to an antigen introduced from outside. It refers to the ability of an antigen to be recognized by the immune system and to elicit an antigen-antibody reaction or cell-based immune response. Antigens with high immunogenicity are more likely to elicit a strong immune response, while antigens with low immunogenicity are either not detected by the immune system or are weakly recognized, thus not eliciting a strong immune response.

[0018] The term “reduced immunogenicity” or “hypoimmunogenicity” in this specification refers to a case where, as a result of measuring changes in white blood cell count, changes in lymphocyte count ratio (e.g., CD4, CD8, etc.), and changes in cytokines (e.g., IL-γIL-2, TNF-α, etc.) secreted by the cell therapy agent reacting with the patient’s immune cells before and after administration of the cell therapy agent, the white blood cell count is reduced, the CD4 / CD8 ratio is reduced, and the secretion of inflammatory cytokines such as IL-γIL-2 and TNF-α is reduced.

[0019] In one specific example, the vascular endothelial cells may be genetically engineered to not express B2M (Beta-2 microglobulin). For example, the genetically engineered vascular endothelial cells may have the B2M (Beta-2 microglobulin) gene knocked out.

[0020] The term “B2M (Beta-2 microglobulin)” in this specification is a gene associated with the expression of HLA proteins on cell membranes recognized by the immune system as components of MHC class I molecules.

[0021] The terms "genetic engineering" or "genetically engineered" as used herein may refer to the act of introducing one or more genetic modifications into a cell. For example, this may refer to, but is not limited to, knocking out the B2M (Beta-2 microglobulin) gene in vascular endothelial cells that overexpress Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells. The term "knock-out" refers to a form of genetic engineering that inactivates a specific gene so that it is not expressed.

[0022] The term “reduced activity” herein may mean that the activity of a protein or enzyme of the same type has a lower activity than the activity of the endogenous protein or enzyme that a given genetically unmanipulated parent cell (e.g., wild type) does not have or does not have.

[0023] In one specific example, the vascular endothelial cells may be genetically engineered to express one or more genes selected from the group consisting of HLA-E (Human Leukocyte Antigen-E), CD47 (Cluster of Differentiation 47), and PD-L1 (Programmed Death-Ligand 1).

[0024] The term "HLA-E (Human Leukocyte Antigen-E)" used herein refers to one of the major histocompatibility complexes (HLA) of the human body, which plays a crucial role in the human immune system. HLA-E is primarily expressed on the cell surface and plays a crucial role in regulating the activities of some immune cells, particularly natural killer cells (NK cells) and T cells.

[0025] The term “CD47 (Cluster of Differentiation 47)” in this specification is one of the proteins that plays an important role in the immune system and cell-to-cell interaction, and is expressed on the cell surface to prevent attacks by immune cells, especially macrophages.

[0026] The term “PD-L1 (Programmed Death-Ligand 1)” in this specification plays a role in regulating immune responses mainly through interaction with immune cells, and is expressed on the cell surface, interacts with T cells, and suppresses the activity of T cells.

[0027] In one specific example, cells genetically engineered to not express the B2M (Beta-2 microglobulin) gene in vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to the parent cells, or genetically engineered to express one or more genes selected from the group consisting of HLA-E (Human Leukocyte Antigen-E), CD47 (Cluster of Differentiation 47), and PD-L1 (Programmed Death-Ligand 1) may exhibit reduced immunogenicity.

[0028] In one specific example, the vascular endothelial cells may be genetically engineered to have increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells.

[0029] The term “overexpression” or “increased expression” herein may mean a higher level of expression of the same type of protein compared to the endogenous expression of the same protein in a given genetically unmodified parent cell (e.g., wild type).

[0030] The term “increased activity” herein may mean that the activity of a protein or enzyme of the same type has a higher activity than the activity of the endogenous protein or enzyme that a given genetically unmanipulated parent cell (e.g., wild type) does not have or does have.

[0031] The above "genetic engineering" or "genetically engineered" may refer to the act of introducing one or more genetic modifications into a cell. For example, it may refer to, but is not limited to, transforming a genetically engineered vascular endothelial cell with a recombinant vector containing an exogenous gene encoding Tie 2.

[0032] In one specific example, the vascular endothelial cells may include a recombinant vector into which a gene encoding Tie 2 (Tyrosine Kinase Receptor 2) has been introduced. For example, a viral vector into which an exogenous gene encoding Tie 2 has been introduced can be used to produce low-immunogenic vascular endothelial cells with increased expression or activity of Tie 2 compared to parental cells.

[0033] The exogenous gene may be expressed in an amount sufficient to increase the activity of Tie 2 in the vascular endothelial cells or host cells compared to the parent cells. The exogenous gene may be introduced into the parent cells via an expression vector. Furthermore, the exogenous gene may be introduced into the parent cells in the form of a linear polynucleotide. Furthermore, the exogenous gene may be expressed within the cell from an expression vector (e.g., a plasmid). Furthermore, the exogenous gene may be expressed by being inserted into a genetic material (e.g., a chromosome) within the cell for stable expression. Furthermore, the exogenous gene may be appropriately regulated by an exogenous promoter operably linked to the gene. Enhanced expression of the polynucleotide may be due to modification by substitution or mutation of an expression control sequence, introduction of a mutation in the polynucleotide sequence itself, replacement of an initiation codon, increase in copy number by insertion into a chromosome or introduction via a vector, or a combination thereof.

[0034] The term "vector" as used herein may refer to a polynucleotide product containing the regulatory sequence and base sequence of a gene so as to enable expression of the target gene in a suitable cell. Alternatively, it may refer to a polynucleotide product containing a base sequence capable of homologous recombination so as to change the regulatory sequence of an endogenous gene in the genome when introduced into a cell, or to insert an expressible target gene into a specific region of the genome. The vector may additionally include a selection marker to confirm whether it has been introduced into a cell or integrated into a chromosome. The selection marker may be a marker that confers a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface protein. In an environment treated with a selection agent, only cells expressing the selection marker will survive or exhibit other phenotypic traits, thereby allowing selection of transformed cells.

[0035] The above vector may include a plasmid vector, a lentiviral vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. In addition, a vector that can be used as the above recombinant vector may be produced by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λ△z1, and M13, etc.) or a virus (e.g., SV40, etc.) that is frequently used in the art.

[0036] The recombinant vector may be constructed typically as a cloning vector or an expression vector. The expression vector may be any vector commonly used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art.

[0037] In one specific example, the vascular endothelial cell (EC) may be any one selected from the group consisting of human umbilical vein endothelial cells (HUVEC), human induced pluripotent stem cell differentiated endothelial cells (iPSC differentiated endothelial cells), retinal endothelial cells, mesenchymal stem cell differentiated endothelial cells, and vascular progenitor cell differentiated endothelial cells, but is not limited thereto.

[0038] The term "active ingredient" as used herein may mean a cell population or composition that is effective in alleviating, inhibiting the progression of, or preventing a disease, disorder, or condition, or one or more symptoms thereof.

[0039] The term “treatment” as used herein refers to or includes the alleviation, inhibition of progression, or prevention of a disease, disorder, or condition, or one or more symptoms thereof.

[0040] The term “prevention” as used herein means any action that inhibits or delays the progression of an eye disease by administering a composition according to the present invention.

[0041] In one specific example, the pharmaceutical composition may further comprise vascular endothelial cells having increased expression or activity of Ang 1 (Angiopoietin 1) compared to parent cells.

[0042] The term "angiopoietin 1 (Ang 1)" used herein is a ligand of the Tie 2 receptor that exists specifically in vascular endothelial cells, and acts as a major regulator that maintains the stability of blood vessels by causing cells surrounding vascular endothelial cells to migrate toward the endothelial cells to form blood vessels and maintaining the barrier function of vascular endothelial cells. In cases of overexpression of vascular endothelial growth factor (VEGF) or inflammation, vascular endothelial cells are activated and vascular permeability increases, and Ang 1 promotes the junctional integrity of vascular endothelial cells, thereby inducing the stabilization of vascular endothelial cells and reducing vascular permeability.

[0043] In one specific example, the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) may be genetically engineered to not express B2M (Beta-2 microglobulin).

[0044] In one specific example, the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) may be genetically engineered to express one or more genes selected from the group consisting of HLA-E (Human Leukocyte Antigen-E), CD47 (Cluster of Differentiation 47), and PD-L1 (Programmed Death-Ligand 1).

[0045] In one specific example, the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) may be genetically engineered to overexpress Ang 1. More specifically, the genetically engineered vascular endothelial cells with increased expression or activity of Ang 1 may include a recombinant vector into which a gene encoding Ang 1 has been introduced.

[0046] In one specific example, the vascular endothelial cells overexpressing Ang 1 may be any one selected from the group consisting of human umbilical vein endothelial cells (HUVECs), human induced pluripotent stem cell differentiated endothelial cells (iPSC differentiated endothelial cells), retinal endothelial cells, mesenchymal stem cell differentiated endothelial cells, and vascular progenitor cell differentiated endothelial cells, but are not limited thereto.

[0047] In one specific example, the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) may be co-cultured with vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2), but is not limited thereto.

[0048] In one specific example, the ratio of vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) and vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) may be 1:0.1 to 1:10. For example, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5.0, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6.0, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, The cell number ratio can be 1:7.0, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8.0, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9.0, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10. Preferably, the ratio of vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) and vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) is 1:0.5 to 1:4.It could be 0. For example, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, It could be 1:3.9, 1:4.0.

[0049] The above “B2M (Beta-2 microglobulin)”, “HLA-E (Human Leukocyte Antigen-E)”, “CD47 (Cluster of Differentiation 47)”, “PD-L1 (Programmed Death-Ligand 1)”, “overexpression”, “genetically engineered” and “recombinant vector” are as described above.

[0050] The term "intracytoplasmic retinopathy" as used herein refers to a disease in which blood vessels in the retina grow abnormally or become deformed. This intraretinal retinopathy can cause symptoms such as decreased vision, central vision loss, and leakage of blood vessels along the retina. Neovascularization refers to the process in which new blood vessels form from existing blood vessels.

[0051] In one specific example, the intraretinal neovascular disease is Retinal Pigment Epithelial Dystrophy, Choroidal Neovascularization, Choroidal Vascular Expansion, Choroidal Vascular Occlusion, Choroidal Neovascular Membrane, Choroidal Vascular Occlusive Disease, Choroidal Hemorrhage, Choroidal Effusion, Proliferative Diabetic Retinopathy (PDR), Macular Telangiectasia, Age-related Macular Degeneration (AMD), Hypertensive Retinopathy It may be any one selected from the group consisting of, but is not limited to, hypertensive retinopathy, diabetic retinopathy, dry macular degeneration (Dry AMD), and wet macular degeneration (Wet AMD).

[0052] The term “angiogenic factor” as used herein may mean a growth factor that promotes the development of blood vessels, angiogenesis, endothelial cell growth, blood vessel stability, and / or vasculogenesis. For example, Vascular Endothelial Growth Factor (VEGF) and VEGF family members, Placental Growth Factor (PlGF), Platelet-Derived Growth Factor (PDGF) family, fibroblast growth factor family (FGF), TIE ligands (angiopoietins), ephrins, Developmental Endothelial Locus-1 (Del-1), fibroblast growth factors (acidic (aFGF) and alkaline (bFGF)), horistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF) / scatter factor (SF), interleukin-8 (IL-8), leptin, midkine, placental growth factor, platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factors, especially PDGF-BB or PDGFR-beta, proautotrophin (PTN), prograneurin, proliferin, transforming growth factor These include, but are not limited to, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), vascular endothelial growth factor (VEGF) / vascular permeability factor (VPF), etc.

[0053]

[0054] Another aspect provides a kit for preventing or treating intraretinal neovascular disease, comprising the composition.

[0055] The definitions of “vascular endothelial cells overexpressing Ang 1,” “vascular endothelial cells overexpressing Tie 2,” and “intraretinal neovascular disease” are as described above.

[0056]

[0057] Another aspect provides a method for treating retinal neovascular disease, comprising administering to a subject a composition comprising, as an active ingredient, vascular endothelial cells having increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells. The vascular endothelial cells having increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) may be genetically engineered not to express B2M (Beta-2 microglobulin) or may be genetically engineered to express one or more genes selected from the group consisting of HLA-E (Human Leukocyte Antigen-E), CD47 (Cluster of Differentiation 47), and PD-L1 (Programmed Death-Ligand 1), thereby exhibiting reduced immunogenicity.

[0058] The composition comprises pharmaceutically suitable and pharmaceutically acceptable carriers, excipients, and vehicles. Typically, such carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, and specifically include saline solutions and buffered media such as phosphate-buffered saline (PBS). Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Excipients such as preservatives and other additives may also be provided, including antimicrobial agents, antioxidants, chelating agents, and inert gases. Suitable preparation aids, carriers, other excipients and methods of preparing pharmaceutical compositions are disclosed in Remington's Pharmaceutical Sciences, 14th Ed., Mack Publishing Co., 1970, especially Part VIII, "Pharmaceutical Preparation and Their Manufacture", pages 1461-1762.

[0059] The terms "administering," "introducing," and "implanting" herein are used interchangeably and may refer to placement of a composition according to one embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site. Administration may be by any suitable route that delivers at least a portion of the cells or cellular components of the composition according to one embodiment to a desired location within a viable subject. The survival period of the cells after administration to a subject may be as short as several hours, for example, 24 hours to several days, or as long as several years. The pharmaceutical composition may be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

[0060] The above "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a cell population or composition effective in preventing or treating retinal neovascular disease, which is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects. The level of the above effective amount may be determined based on factors including the patient's health condition, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0061] The above pharmaceutical composition may be administered as an individual treatment or in combination with other treatments, sequentially or simultaneously with conventional treatments, or in single or multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily available to those skilled in the art.

[0062] Specifically, the effective amount of vascular endothelial cells overexpressing Tie 2 in the pharmaceutical composition may vary depending on the patient's age, sex, and weight, and the pharmaceutical composition may be administered once or repeatedly. The range is not limited thereto, as it may increase or decrease depending on the route of administration, severity of the disease, sex, weight, age, etc.

[0063] In one specific example, the composition may further comprise vascular endothelial cells having increased expression or activity of Ang 1 (Angiopoietin 1) compared to parent cells. The vascular endothelial cells having increased expression or activity of Ang 1 (Angiopoietin 1) compared to parent cells may be genetically engineered not to express B2M (Beta-2 microglobulin) or may be genetically engineered to express one or more genes selected from the group consisting of HLA-E (Human Leukocyte Antigen-E), CD47 (Cluster of Differentiation 47), and PD-L1 (Programmed Death-Ligand 1), thereby exhibiting reduced immunogenicity.

[0064] In one specific example, the pharmaceutical composition comprising vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) as an active ingredient may be co-administered with a pharmaceutical composition comprising vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1). For example, the pharmaceutical composition comprising vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) may be administered sequentially or simultaneously with the pharmaceutical composition comprising vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) as an active ingredient, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0065]

[0066] Another aspect is that vascular endothelial cells overexpressing Tie 2 can be used for the manufacture of a therapeutic agent for preventing or treating neovascular diseases in the retina. In one specific example, vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) and vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) can be used together for the manufacture of a therapeutic agent for treating neovascular diseases in the retina.

[0067] Another aspect provides a method for preventing or treating angiogenic disease in the retina, comprising administering to a subject in need thereof vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells.

[0068] Another aspect provides the use of vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells for use in the manufacture of a pharmaceutical preparation for preventing or treating neovascular diseases in the retina.

[0069] Another aspect provides the use of vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells for preventing or treating neovascular diseases in the retina.

[0070] The terms and methods described for the above inventions apply equally to each invention.

[0071] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0072] A pharmaceutical composition for preventing or treating intraretinal neovascular disease, comprising as an active ingredient vascular endothelial cells in which the expression or activity of B2M (Beta-2 microglobulin) is reduced and the expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) is increased compared to parent cells according to the daily aspect, has the effect of preventing or treating intraretinal neovascular disease by inducing stabilization of vascular endothelial cells and reducing vascular permeability to slow or inhibit intraretinal neovascularization even in the overexpression of vascular endothelial growth factor (VEGF) or in an inflammatory state. In addition, due to low immunogenicity, the cell survival period is long, and the effect of inhibiting neovascularization for a long period of time with a single intraocular injection is maintained, thereby reducing invasiveness during treatment, enhancing vascular stability, and enabling early diagnosis and treatment of intraretinal neovascular disease.

[0073] Figure 1 is a diagram showing the results of flow cytometry analysis of retinal endothelial cells according to the daily pattern:

[0074] Figure 1a shows the results of flow cytometry analysis of purchased human retinal endothelial cells (HREC), and Figure 1b shows the results of flow cytometry analysis of universal human retinal endothelial cells (uHREC) with low immunogenicity in which some gene expressions have been regulated by gene editing.

[0075] Figure 2 is a photograph confirming whether the Ang 1 gene and Tie 2 gene were delivered to low-immunogenic vascular endothelial cells (uHREC) by GFP and RFP expression.

[0076] Figure 3 is a graph showing the results of analyzing the level of Tie 2 gene expression in a relative quantitative manner to GAPDH expression in a control group (uHREC) that was not transfected with a lentivirus vector (LV-mCherry-hTie 2) and in low-immunogenic human retinal endothelial cells (uHREC_Tie 2) overexpressing Tie 2 using RT-PCR.

[0077] Figure 4 is a graph showing the results of a relative quantitative analysis of Ang 1 gene expression levels in control (uHREC) not transfected with a lentivirus vector (LV-eGFP-hANG 1) and Ang 1-overexpressing, low-immunogenic human retinal endothelial cells (uHREC_Ang 1) transfected with a lentivirus vector (LV-eGFP-hANG 1) compared to GAPDH expression.

[0078] Figure 5 is a diagram showing the results of flow cytometry analysis of uHREC, uHREC_Ang 1, and uHREC_Tie 2 according to the daily aspect.

[0079] Figure 6 is a graph showing the results of ELISA analysis performed at 24-hour intervals for 3 days after transfection for uHREC, uHREC_Ang 1, and uHREC_Tie 2.

[0080] Figure 7 shows the results of confirming the effect of reducing migration of retinal endothelial cells when co-cultured with uHREC_Ang 1 cells and uHREC_Tie 2 cells:

[0081] Figure 7a is a photograph showing the result of staining vascular cells that moved under the filter with crystal violet, and Figure 7b is a graph showing the result of measuring the absorbance at 595 nm in a spectrofluorometer by dissolving the stained cells with acetic acid.

[0082] Figure 8 is a photograph showing the results of real-time time-lapse imaging of tubular structures taken every hour for 3 hours after uHREC (uHREC_Cont. or uHREC_Tie 2) cells and uHREC (uHREC-Cont. or uHREC_Ang 1) cells were attached to Matrigel at a ratio of 2:1.

[0083] Figure 9 is a graph showing the degree of tight junction formation in hypoimmune vascular endothelial cells (uHREC_Ang 1, uHREC_Tie 2) overexpressing Ang 1 or Tie 2, measured by TEER (Trans epithelial / endothelial electrical resistance).

[0084] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0085]

[0086] Example 1. Production of low-immunogenic human retinal endothelial cells (uHREC_Ang 1, uHREC_Tie 2) overexpressing Tie 2 or Ang 1

[0087] 1.1 Preparation of low-immunogenic vascular endothelial cells (Universal Human Retinal Endothelial Cells, uHREC)

[0088] Human retinal endothelial cells (HRECs) were purchased and maintained and expanded in vascular endothelial cell culture medium. After seeding the vascular endothelial cells in cell culture dishes, when they reached 50-70% confluence, functional lentiviruses containing the CRISPR-Cas9 gene editing scissor were used to transduce B2M knockout (KO), HLA-E, and CD47.

[0089] Specifically, we induced loss of function of the hB2M gene, an accessory protein constituting MHC class I, by gene editing using genetic scissors and lentiviral vectors. Since the frameshift mutation in hB2M completely eliminates MHC class I expression, leading to NK cell-mediated recognition and elimination, we transduced the cells with other immune modulators, such as HLA-E, PD-L1, and CD47, as NK cell inhibitory molecules to limit host NK cell reactivity.

[0090] B2M knockout cells were isolated by HLA-A negative selection, and HLA-E and CD47 positive cells were isolated using antibodies. The results of flow cytometry analysis of HREC (human retinal endothelial cells) and uHREC (Universal Human Retinal Endothelial Cells) and the cell sorting process using them are shown in Figure 1.

[0091] Figure 1 is a diagram showing the results of flow cytometry analysis of retinal endothelial cells according to the daily pattern:

[0092] Figure 1a shows the results of flow cytometry analysis of purchased human retinal endothelial cells (HREC), and Figure 1b shows the results of flow cytometry analysis of universal human retinal endothelial cells (uHREC) with low immunogenicity in which some gene expressions have been regulated by gene editing.

[0093] As shown in Fig. 1, universal human retinal endothelial cells (uHREC) expressing B2M knockout (KO), HLA-E, and CD47 were established.

[0094]

[0095] 1.2 Inhibition of angiogenesis in low-immunogenic vascular endothelial cells (Universal Human Retinal Endothelial Cells, uHREC)

[0096] The low-immunogenic vascular endothelial cells (Universal Human Retinal Endothelial Cells, uHREC) prepared by the method of Example 1.1 were divided into two groups and Ang 1 and Tie 2 were transduced respectively using a gene editing method.

[0097] Specifically, cells transferred with the Tie 2 gene were separated using a positive cell sorting method using an antibody, and cells transferred with the Ang 1 gene were separated using a fluorescent flow cytometer by tagging the gene scissors with GFP.

[0098]

[0099] 1.3 Confirmation of Ang 1 or Tie 2 gene expression in low-immunogenic human retinal endothelial cells (uHREC_Ang 1, uHREC_Tie 2) overexpressing Tie 2 or Ang 1

[0100] The presence and degree of Ang 1 or Tie 2 gene expression in uHREC_Ang 1 or uHREC_Tie 2 produced by the method of Example 1.2 were confirmed.

[0101] Specifically, when Ang 1 and Tie 2 genes were successfully delivered to uHREC, the lentivirus vector was labeled with a fluorescent material (LV-eGFP-hANG 1, LV-mCherry-hTie 2) to confirm gene delivery by expression of GFP and RFP, respectively. The results of confirming whether Ang 1 and Tie 2 genes were delivered to uHREC using a fluorescent microscope by expression of GFP and RFP are shown in Fig. 2.

[0102] Figure 2 is a photograph confirming whether the Ang 1 gene and Tie 2 gene were delivered to low-immunogenic vascular endothelial cells (uHREC) by GFP and RFP expression.

[0103] As shown in Fig. 2, it was confirmed that GFP and RFP were expressed and a fluorescent signal was detected in the low-immunogenic human retinal endothelial cells (uHREC_Ang 1, uHREC_Tie 2) overexpressing Ang 1 or Tie 2 prepared by the method of Example 1.2. This means that the Ang 1 gene or Tie 2 gene is expressed in the low-immunogenic vascular endothelial cells (uHREC).

[0104]

[0105] Example 2. Confirmation of Tie 2 gene expression level in uHREC_Tie 2

[0106] To evaluate the efficiency of Tie 2 gene expression in uHREC_Tie 2 produced according to Example 1, RT-PCR was used.

[0107] Specifically, total RNA was extracted from cultured cells using the Total RNA Isolation Kit (Macherey-Nagel) according to the manufacturer's instructions for RT-PCR. RNA concentration and purity were confirmed using Nanodrop. To detect Tie 2 mRNA expression, RT-PCR analysis was performed using the 7500 Real-Time PCR system (Applied Biosystems). Each sample was analyzed for relative quantification against GAPDH expression, and the results are shown in Figure 3.

[0108] Figure 3 is a graph showing the results of analyzing the level of Tie 2 gene expression in a relative quantitative manner to GAPDH expression in a control group (uHREC) that was not transfected with a lentivirus vector (LV-mCherry-hTie 2) and in low-immunogenic human retinal endothelial cells (uHREC_Tie 2) overexpressing Tie 2 using RT-PCR.

[0109] As shown in Fig. 3, it was confirmed that the Tie 2 gene expression level increased more in uHREC_Tie 2 compared to the control group (uHREC). In addition, when Ang 1-expressing vascular endothelial cells (uHREC_Ang 1) and Tie 2-expressing vascular endothelial cells (uHREC_Tie 2) were co-cultured (uHREC_Ang 1 / Tie 2), it was confirmed that the Tie 2 gene expression level increased the most when co-cultured for 48 hours compared to the control group (uHREC).

[0110]

[0111] Example 3. Confirmation of Ang 1 (Angiopoietin 1) gene expression level in uHREC_Ang 1

[0112] To evaluate the expression efficiency of Ang 1 gene in UHREC_Ang 1 produced according to Example 1, RT-PCR was used.

[0113] Specifically, total RNA was extracted from cultured cells using the Total RNA Isolation Kit (Macherey-Nagel) according to the manufacturer's instructions for RT-PCR. RNA concentration and purity were confirmed using Nanodrop. To detect Ang 1 mRNA expression, RT-PCR analysis was performed using the 7500 Real-Time PCR system (Applied Biosystems). Each sample was analyzed for relative quantification against GAPDH expression, and the results are shown in Figure 4.

[0114] Figure 4 is a graph showing the results of a relative quantitative analysis of Ang 1 gene expression levels in control (uHREC) not transfected with a lentivirus vector (LV-eGFP-hANG 1) and Ang 1-overexpressing, low-immunogenic human retinal endothelial cells (uHREC_Ang 1) transfected with a lentivirus vector (LV-eGFP-hANG 1) compared to GAPDH expression.

[0115] As shown in Fig. 4, it was confirmed that the Ang 1 gene expression level increased more in low-immunogenic human retinal endothelial cells (uHREC_Ang 1) overexpressing Ang 1 compared to the control group (uHREC). In addition, when Ang 1-expressing endothelial cells (uHREC_Ang 1) and Tie 2-expressing endothelial cells (uHREC_Tie 2) were co-cultured (uHREC_Ang 1 / Tie 2), the Ang 1 gene expression level increased the most when co-cultured for 24 hours compared to the control group (uHREC), and it was confirmed that the Ang 1 gene expression level was maintained for up to 72 hours.

[0116]

[0117] Example 4. FACS analysis results of uHREC, uHREC-Ang 1, and uHREC-Tie 2

[0118] To evaluate the gene expression pattern in uHREC, uHREC-Ang 1, and uHREC-Tie 2 prepared according to Example 1, FACS analysis was performed using BD Trucount™ Absolute Counting Tube. The Tie 2 antibody used for FACS analysis was BD Pharmingen™ Alexa Fluor® 647 Mouse Anti-Human Tie 2 (CD202b). The gene expression pattern was analyzed in the control group (uHREC) not transfected with lentivirus vector (LV-mCherry-hTie 2), Ang 1-overexpressing, low-immunogenic human retinal endothelial cells (uHREC_Ang 1), and Tie 2-overexpressing, low-immunogenic human retinal endothelial cells (uHREC_Tie 2).

[0119] To create low-immunogenic uHREC-Ang 1 and uHREC-Tie 2 cell lines, the composition of cells expressing negative HLA-ABC was determined. Among HLA-ABC-negative cells, HLA-E and GFP (green fluorescence) double positive cells were isolated to obtain uHREC-Ang1 cells. Among HLA-ABC-negative cells, HLA-E and RFP (red fluorescence) double positive cells were isolated to obtain uHREC-Tie2 cells.

[0120] Figure 5 is a diagram showing the results of flow cytometry analysis of uHREC, uHREC_Ang 1, and uHREC_Tie 2 according to the daily aspect.

[0121] As shown in Figure 5, HLA-ABC expression was confirmed to be relatively low in uHREC-Ang 1 and uHREC-Tie 2. HLA-ABC is a major histocompatibility complex (MHC) protein involved in the immune response, and when its expression is low, the characteristic of having low immunogenicity is strengthened. Therefore, these results imply that uHREC_Ang 1 and uHREC_Tie 2 are cell lines with low immunogenicity.

[0122]

[0123] Example 5. Measurement of Angiopoietin 1 expression in uHREC-Ang 1 and uHREC-Tie 2

[0124] Ang 1 (Angiopoietin 1) gene expression was detected through ELISA analysis for uHREC, uHREC-Ang 1, and uHREC-Tie 2 produced according to Example 1.

[0125] Ang 1-specific ELISA was performed using Quantikine Human Angiopoietin 1 Immunoassay (R&D Systems). Samples of hMSC_Ang 1 culture medium from 6-well plates were collected at 24-hour intervals for 3 days after transfection for uHREC, uHREC-Ang 1, and uHREC-Tie 2. The uHREC refers to a control group with low immunogenicity due to B2M knock out, CD47, and HLA-E gene expression. The uHREC-Ang 1 refers to cells transfected with a lentiviral vector containing Ang 1, and uHREC-Tie 2 refers to cells transfected with a lentiviral vector containing Tie 2. uHREC_Ang 1 / Tie 2 are co-cultured with uHREC_Ang 1 and uHREC_Tie 2. The results of the Ang 1-specific ELISA are shown in Figure 6.

[0126] Figure 6 is a graph showing the results of ELISA analysis performed at 24-hour intervals for 3 days after transfection for uHREC, uHREC_Ang 1, and uHREC_Tie 2.

[0127] As shown in Figure 6, the expression level of Angiopoietin 1 was the highest in uHREC-Ang 1, and it was confirmed that the expression level of Angiopoietin 1 steadily increased over time.

[0128] These results indicate that uHREC_Ang 1 effectively induces the expression of Angiopoietin 1, and that the expression lasts for more than 72 hours. Meanwhile, when uHREC_Ang 1 and uHREC_Tie 2 were co-cultured (uHREC_Ang 1 / Tie 2), the concentration of Ang 1 in the culture medium was confirmed to be reduced compared to the culture medium consisting of only uHREC_Ang1 cells, as uHREC_Ang 1 cells expressing Ang 1 and HREC_Tie2 cells that do not express Ang 1 were mixed.

[0129]

[0130] Experimental Example 1. Confirmation of the effect of reducing retinal endothelial cell migration when co-cultivating uHREC_Ang 1 cells and uHREC_Tie 2 cells.

[0131] The effect of co-culture of uHREC_Ang 1 cells and uHREC_Tie 2 cells was evaluated through evaluation of retinal endothelial cell mobility.

[0132] Specifically, to confirm the effect of reducing migration of retinal endothelial cells when co-cultured with uHREC_Ang 1 cells and uHREC_Tie 2 cells, a Transwell chamber (CorningCostar) containing a 6.5 mm diameter polycarbonate filter with a pore size of 8 μm was used. uHREC without overexpressing Tie 2 (HREC_Cont.) or uHREC with overexpressing Tie 2 (uHREC_Tie 2) were cultured at a density of 5 x 10 4Cells were seeded at 10 cells / ml, and the culture medium of uHRECs that did not overexpress Ang 1 (uHREC_Cont.) or uHRECs that overexpressed Ang 1 (uHREC_Ang 1) was treated for 24 hours, and then 20 ng / ml of VEGF was added and cultured in an incubator for 24 hours. After culture, the cells on the filter were wiped off using a cotton swab, and the cells that migrated under the filter were washed with PBS, fixed with methanol for 20 minutes, and stained with 1% crystal violet for 20 minutes. After washing with PBS, the migrated cells were observed under a microscope. The cells stained with 1% acetic acid were dissolved and measured for absorbance at 595 nm using a spectrofluorometer, and the results are shown in Fig. 7.

[0133] Figure 7 shows the results of confirming the effect of reducing migration of retinal endothelial cells when co-cultured with uHREC_Ang 1 cells and uHREC_Tie 2 cells:

[0134] Figure 7a is a photograph showing the result of staining vascular cells that moved under the filter with crystal violet, and Figure 7b is a graph showing the result of measuring the absorbance at 595 nm in a spectrofluorometer by dissolving the stained cells with acetic acid.

[0135] As shown in Fig. 7a and Fig. 7b, uHREC_Cont. cells in the disease environment (VEGF-containing culture medium) showed very high cell motility (about twice that in the absence of VEGF), whereas when uHREC_Ang 1 cells and uHREC_Tie 2 cells were co-cultured in the disease environment (VEGF-containing culture medium), it was confirmed that the migration of retinal endothelial cells induced by VEGF was effectively reduced. In particular, when uHREC_Ang 1 cells and uHREC_Tie 2 cells were co-cultured in a ratio of 2:1 to 1:1, the migration of retinal endothelial cells induced by VEGF was most effectively reduced. This means that in the case of a cell therapy product co-cultured with uHREC_Ang 1 cells and uHREC_Tie 2 cells, the migration of retinal endothelial cells is effectively suppressed even in a disease environment.

[0136]

[0137] Experimental Example 2. Confirmation of the in vitro angiogenesis inhibition ability of uHREC_Ang 1 cells and uHREC_Tie 2 cells.

[0138] To evaluate the in vitro angiogenesis inhibition ability of uHREC_Ang 1 cells and uHREC_Tie 2 cells, uHREC_Ang 1 cells and uHREC_Tie 2 cells were cultured on gels and a capillary-like tube formation assay was performed.

[0139] Specifically, the tube formation assay of uHREC cells was performed using a tube formation assay kit (Abcam). 50 μl / well of extracellular matrix solution (ECM) was added to a 96-well plate, and the solution was allowed to solidify at 37°C for 1 hour to form a gel. 2 × 10 cells were added to each well. 4 cells / 100μl of uHREC (uHREC_Cont. or uHREC_Tie 2) cells and 1×10 4uHREC (uHREC_Cont. or uHREC_Ang 1) cells were mixed and added at a rate of 100 μl / cell, and 20 ng / ml VEGF was added. The cells were cultured at 37°C for 10 h to allow tube formation. The formation of blood vessel-like structures was observed using a Juli™Stage Real-Time History Recorder (NanoEnTek). The area covered by the tubes over time was measured using the Image J program, and the results are shown in Fig. 8.

[0140] Figure 8 is a photograph showing the results of real-time time-lapse imaging of tubular structures taken every hour for 3 hours after uHREC (uHREC_Cont. or uHREC_Tie 2) cells and uHREC (uHREC-Cont. or uHREC_Ang 1) cells were attached to Matrigel at a ratio of 2:1.

[0141] As shown in Fig. 8, the normal group (uHREC_Cont / uHREC_Cont) rapidly formed tube-forming structures in a VEGF environment (disease environment), and it was confirmed that the final tube-forming area decreased. The experimental group (uHREC_Tie 2 / uHREC_Ang 1) confirmed that tube formation progressed slowly even in a VEGF environment. This is thought to be because angiogenesis is slowed down by tight junction formation by low-immunogenic vascular endothelial cells (uHREC_Tie 2, HREC_Ang 1) that overexpress Tie 2 or Ang 1, and it means that the cell therapy agent of low-immunogenic vascular endothelial cells (uHREC_Tie 2, uHREC_Ang 1) that overexpress Tie 2 or Ang 1 slows down angiogenesis and is effective in inhibiting angiogenesis in the retina.

[0142]

[0143] Experimental Example 3. Confirmation of the degree of tight junction formation in hypoimmune vascular endothelial cells (uHREC_Ang 1, uHREC_Tie 2) overexpressing Ang 1 or Tie 2.

[0144] To evaluate the degree of tight junction formation between uHREC_Ang 1 cells and uHREC_Tie 2 cells, TEER (Trans epithelial / endothelial electrical resistance) was measured.

[0145] Specifically, uHREC, uHREC_Ang 1, and uHREC_Tie 2 cells were seeded onto Transwell inserts equipped with a permeable membrane and cultured for 3 days until the cells reached full confluency. Cell morphology was observed under a microscope to confirm that the cells had proliferated densely. Then, the electrodes were thoroughly washed and sterilized in preparation for TEER measurement. The electrodes were washed with sterile PBS or cell culture medium and then soaked in cell culture medium before use. The culture medium in the Transwell was completely replaced with fresh medium to ensure smooth electrical contact, and an appropriate amount of cell culture medium was added to the upper and lower chambers to complete the preparation. The electrodes were placed in the upper chamber of the Transwell insert, and the opposite electrode was placed in the lower chamber. Care was taken to ensure that the electrodes did not disturb or damage the cell layer. Once the electrodes were positioned, the TEER measurement device was operated to measure the resistance value. The resistance value was measured several times and the average value was obtained. The TEER value was calculated by converting it and multiplying the measured resistance value by the surface area of ​​the Transwell insert, and the results are shown in Figure 9. If the TEER value remains at a constant level or increases, it can be interpreted that the single layer is healthy and properly formed. Conversely, if the TEER value decreases, it can mean that there is damage to the cell layer or that permeability has increased.

[0146] Figure 9 is a graph showing the degree of tight junction formation in hypoimmune vascular endothelial cells (uHREC_Ang 1, uHREC_Tie 2) overexpressing Ang 1 or Tie 2, measured by TEER (Trans epithelial / endothelial electrical resistance).

[0147] As shown in Fig. 9, in the case of uHREC+VEGF, the TEER value decreased over time, indicating that the tight junctions between cells were weakened, and in the case of uHREC_Ang 1 / Tie 2, the TEER value did not decrease even when treated with VEGF. In addition, the TEER values ​​of uHREC_Ang 1 / Tie 2 and uHREC_Ang 1 / Tie 2+VEGF did not show a significant difference.

[0148] These results indicate that tight junctions are not weakened even when VEGF (Vascular Endothelial Growth Factor), which increases proliferation and vascular permeability of vascular endothelial cells, is treated in uHREC_Ang 1 / Tie 2, indicating that uHREC_Ang 1 / Tie 2 is effective in inhibiting neovascularization in the retina.

Claims

1. A pharmaceutical composition for preventing or treating neovascular disease in the retina, comprising, as an active ingredient, vascular endothelial cells in which the expression or activity of B2M (Beta-2 microglobulin) is reduced and the expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) is increased compared to parent cells.

2. A pharmaceutical composition according to claim 1, wherein the vascular endothelial cells are genetically engineered to express at least one gene selected from the group consisting of HLA-E (Human Leukocyte Antigen-E), CD47 (Cluster of Differentiation 47), and PD-L1 (Programmed Death-Ligand 1).

3. A pharmaceutical composition according to claim 1, wherein the vascular endothelial cells have a B2M (Beta-2 microglobulin) gene knocked out.

4. A pharmaceutical composition according to claim 1, wherein the vascular endothelial cells include a recombinant vector into which a gene encoding Tie 2 (Tyrosine Kinase Receptor 2) is introduced.

5. A pharmaceutical composition according to claim 1, wherein the vascular endothelial cell is any one selected from the group consisting of human umbilical vein endothelial cells (HUVEC), iPSC differentiated endothelial cells, retinal endothelial cells, mesenchymal stem cell differentiated endothelial cells, and vascular progenitor cell differentiated endothelial cells.

6. In claim 1, the pharmaceutical composition further comprises vascular endothelial cells having increased expression or activity of Ang 1 (Angiopoietin 1) compared to parent cells, or is administered in combination therewith.

7. A pharmaceutical composition according to claim 6, wherein the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) are genetically engineered to express at least one gene selected from the group consisting of HLA-E (Human Leukocyte Antigen-E), CD47 (Cluster of Differentiation 47), and PD-L1 (Programmed Death-Ligand 1).

8. A pharmaceutical composition according to claim 6, wherein the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) include a recombinant vector into which a gene encoding Ang 1 (Angiopoietin 1) is introduced.

9. A pharmaceutical composition according to claim 6, wherein the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) are any one selected from the group consisting of human umbilical vein endothelial cells (HUVEC), iPSC differentiated endothelial cells, retinal endothelial cells, mesenchymal stem cell differentiated endothelial cells, and vascular progenitor cell differentiated endothelial cells.

10. A pharmaceutical composition according to claim 8, wherein the vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) are co-cultured with the vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) of claim 1.

11. A pharmaceutical composition according to claim 10, wherein the ratio of vascular endothelial cells with increased expression or activity of Ang 1 (Angiopoietin 1) and vascular endothelial cells with increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) is 1:0.1 to 1:

10.

12. In claim 1, the intraretinal neovascular disease is retinal pigment epithelial dystrophy, choroidal neovascularization, choroidal vascular expansion, choroidal vascular occlusion, choroidal neovascular membrane, choroidal vascular occlusive disease, choroidal hemorrhage, choroidal effusion, proliferative diabetic retinopathy (PDR), macular telangiectasia, age-related macular degeneration (AMD), hypertensive A pharmaceutical composition, wherein the pharmaceutical composition is any one selected from the group consisting of hypertensive retinopathy, diabetic retinopathy, dry macular degeneration (Dry AMD), and wet macular degeneration (Wet AMD).

13. A kit for preventing or treating intraretinal neovascular disease, comprising a composition according to any one of claims 1 to 16.

14. A method for preventing or treating angiogenic disease in the retina, comprising administering to a subject in need thereof vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells.

15. Use of vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells for use in the manufacture of pharmaceutical preparations for preventing or treating intraretinal angiogenic diseases.

16. Use of vascular endothelial cells having reduced expression or activity of B2M (Beta-2 microglobulin) and increased expression or activity of Tie 2 (Tyrosine Kinase Receptor 2) compared to parent cells for preventing or treating neovascular diseases in the retina.

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