Composition for preventing or treating retinal angiogenic diseases comprising vascular endothelial cells overexpressing tie2

Abstract

A pharmaceutical composition comprising vascular endothelial cells overexpressing tyrosine kinase receptor 2 (Tie2) as an active ingredient according to one aspect induces the stabilization of vascular endothelial cells, even in a state of vascular endothelial growth factor (VEGF) overexpression or inflammation, and reduces vascular permeability to slow or inhibit retinal angiogenesis, thus having the effect of preventing or treating retinal angiogenic diseases. In addition, the present invention has the effects of reducing invasiveness during treatment compared to existing antibody therapies that require intraocular injections at intervals of 4–8 weeks, enhancing vascular stability, and enabling early the diagnosis and treatment of retinal angiogenic diseases.

Description

Composition for preventing or treating retinal neovascular disease comprising vascular endothelial cells overexpressing TIE2

[0001] The present invention relates to a pharmaceutical composition for preventing or treating neovascular disease in the retina, comprising vascular endothelial cells overexpressing Tie2 as an active ingredient.

[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, age-related macular degeneration and diabetic retinopathy are prime examples.

[0003] Age-related macular degeneration (AMD) is a disease that refers to the collapse or destruction of the macula that occurs with the aging process, and it mainly occurs in people over 50 years of age. This disease is divided into non-exudative (dry type) in which the macula atrophies and loses visual function due to the accumulation of small waste products called drusen, 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. In particular, wet macular degeneration has a high incidence of leading to blindness, and the prevalence rate in Korea is estimated to be 37 cases per 10,000 people. The exact cause of macular degeneration is not yet known, but age is known to be a risk factor, and other notable environmental factors include smoking, high blood pressure, obesity, genetic predisposition, excessive UV exposure, and low blood antioxidant levels. Currently, the main treatment for wet macular degeneration is antibody injection therapy against vascular endothelial growth factor (VEGF). Other known treatments include laser photocoagulation, photodynamic therapy, and vitrectomy, but a complete cure still does not exist.

[0004] Angiopoietin-1 (Ang1) is an agonist of Tie2 and competes with an antagonistic ligand, angiopoietin-2 (Ang2), for binding to Tie2 on vascular endothelial cells. Ang1, a ligand that activates the Tie2 receptor, acts as a key regulator of vascular stability by maintaining the barrier function of vascular endothelial cells. Overexpression of vascular endothelial growth factor (VEGF) or inflammation activates vascular endothelial cells and increases vascular permeability. Ang1 promotes junctional integrity of vascular endothelial cells, thereby inducing endothelial cell stabilization and reducing vascular permeability.

[0005] Treatments for wet AMD, a type of age-related macular degeneration (AMD), include antibody injection therapy against vascular endothelial growth factor (VEGF) (Anti-VEGF therapy), therapy using anti-Ang2 antibodies that specifically bind to Ang2 and bind to Tie2 receptors together with Ang2, and therapy using recombinant proteins or antibodies that directly bind to Tie2 receptors to induce phosphorylation and activation (Anti-Ang-2 therapy).

[0006] 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-cycle 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 is a great burden to patients. Therefore, there is an urgent need for the development of a cell therapy agent for inhibiting retinal angiogenesis using vascular endothelial cells with increased expression of Tie2 and cells with enhanced Ang1 production function, which can have a long-lasting effect with a single intraocular injection.

[0007] One aspect is to provide a pharmaceutical composition for preventing or treating retinal neovascular disease, comprising vascular endothelial cells overexpressing Tie2 as an active ingredient.

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

[0009] Another aspect provides a use of the composition for preventing or treating intraretinal neovascular disease.

[0010] Another aspect provides a method for preventing or treating intraretinal neovascular disease, comprising administering an effective amount of the composition to a subject in need thereof.

[0011] Another aspect provides the use of the composition for use in the manufacture of a medicament for preventing or treating neovascular diseases in the retina.

[0012] One aspect provides a pharmaceutical composition for preventing or treating retinal neovascular disease, comprising vascular endothelial cells overexpressing Tie2 as an active ingredient.

[0013] As used herein, the term "Tie2 (Tyrosine Kinase Receptor 2)" refers to Tyrosine Kinase Receptor 2, a receptor present on the surface of vascular endothelial cells. Tie2 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 Tie2 can cause problems related to vascular instability, abnormal vascular proliferation, blood coagulation, etc., and overexpression of Tie2 can improve vascular stability and suppress abnormal angiogenesis.

[0014] As used herein, the term "endothelial cells (EC)" may refer to cells that form a layer covering the inside of a blood vessel as one of the components of the blood vessel wall. The vascular endothelial cells exist in various types of blood vessels, 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 regulation, and may also be involved in angiogenesis and tissue regeneration.

[0015] In one specific example, the vascular endothelial cells may be genetically engineered to overexpress Tie2. The genetically engineered vascular endothelial cells may comprise a recombinant vector into which a gene encoding Tie2 has been introduced.

[0016] The terms 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).

[0017] The term "genetic engineering" or "genetically engineered" as used herein may refer to the act of introducing one or more genetic modifications into a cell.

[0018] The genetically engineered endothelial cells may comprise a recombinant vector into which an exogenous gene encoding Tie2 has been introduced.

[0019] In one embodiment, vascular endothelial cells that overexpress Tie2 can be produced using a viral vector into which an exogenous gene encoding Tie2 has been introduced.

[0020] The exogenous gene may be expressed in an amount sufficient to increase Tie2 activity 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 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.

[0021] 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 host 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 of the host when introduced into the host 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 the host or integrated into the 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, so that transformed cells can be selected.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] In one specific example, a pharmaceutical composition for preventing or treating retinal neovascular disease, which comprises vascular endothelial cells overexpressing Tie2 as an active ingredient, may be a pharmaceutical composition further comprising cells expressing Ang1.

[0029] The term "angiopoietin-1 (Ang1)" used herein is a ligand of the Tie-2 receptor that exists specifically in vascular endothelial cells. It acts as a major regulator that maintains the stability of blood vessels by inducing the migration of cells surrounding vascular endothelial cells 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 or inflammation, vascular endothelial cells are activated and vascular permeability increases. Ang1 promotes the junctional integrity of vascular endothelial cells, thereby inducing the stabilization of vascular endothelial cells and reducing vascular permeability.

[0030] In one specific example, the Ang1-expressing cell may be genetically engineered to express or overexpress Ang1. More specifically, the genetically engineered Ang1-expressing cell may comprise a recombinant vector into which a gene encoding Ang1 has been introduced.

[0031] The above “overexpression”, “genetically engineered”, “recombinant vector”, etc. are as described above.

[0032] In one specific example, the Ang1-expressing cell may be any one selected from the group consisting of pericytes, mesenchymal stem cells (MSCs), endothelial cells (ECs), fibroblasts, retinal pigment epithelium (RPE), photoreceptors, horizontal cells, Muller cells, amacrine cells, astrocytes, etc., but is not limited thereto.

[0033] The term "mesenchymal stem cells (MSCs)" used herein may refer to stem cells that exist in cartilage, bone tissue, adipose tissue, and the stroma of bone marrow, differentiated from the mesoderm formed by the division of a fertilized egg, and may include mesenchymal stem cells of mammals, for example, animals including humans. Mesenchymal stem cells maintain stemness and self-renewal and have the ability to differentiate into various cells, including chondrocytes, osteoblasts, muscle cells, and adipocytes, and may be extracted from bone marrow, adipose tissue, umbilical cord blood, synovial membrane, trabecular bone, muscle, infrapatellar fat pad, and the like. Mesenchymal stem cells have immunomodulatory abilities that suppress the activity and proliferation of T lymphocytes and B lymphocytes, inhibit the activity of natural killer cells (NK cells), and regulate the functions of dendritic cells and macrophages, making them suitable for allotransplantation and xenotransplantation.

[0034] The term "isolated cell", e.g., "isolated mesenchymal stem cell" or "derived cell", e.g., "derived mesenchymal stem cell", as used herein, means a cell substantially isolated from the tissue from which the cell originated, e.g., umbilical cord blood.

[0035] In one specific example, the mesenchymal stem cells (MSCs) may be derived from any one selected from the group consisting of, but not limited to, umbilical cord, umbilical cord blood, placenta, bone marrow, adipose tissue, muscle, amniotic fluid, amniotic membrane, and dental pulp.

[0036] The above umbilical cord may refer to a cord that connects the mother and the abdomen so that the fetus of a mammal can grow in the placenta, and may generally refer to a tissue composed of three blood vessels, namely two umbilical arteries and one umbilical vein, surrounded by Wharton's jelly, and is also properly called that.

[0037] The above-mentioned umbilical cord blood may refer to blood collected from the umbilical vein connecting the placenta and fetus of a mammal. The umbilical cord blood can be easily collected from the donor's umbilical vein at the time of birth. Specifically, in the case of normal vaginal delivery, the blood can be collected from the umbilical vein after delivery, while the placenta is still within the uterus. In the case of a cesarean section, the blood can be collected from the umbilical vein after delivery, while the placenta has also been expelled from the uterus.

[0038] In one specific example, the cells expressing Ang1 may exhibit reduced immunogenicity.

[0039] 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.

[0040] The term "reduced immunogenicity" or "hypoimmunogenicity" as used herein 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.), changes in cytokines (e.g., IL-γ, IL-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, or the secretion of inflammatory cytokines such as IL-γ, IL-2, TNF-α is reduced.

[0041] In one specific example, the cells expressing Ang1 and the Tie2-overexpressing vascular endothelial cells may be co-cultured, but are not limited thereto.

[0042] In one specific example, the ratio of the cells expressing Ang1 to the Tie2-overexpressing vascular endothelial cells may be 1:0.1 to 1:10. For example, the cell number ratio may be 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, 1:2.1, 1:3.1, 1:4.1, 1:5.1, 1:6.1, 1:7.1, 1:8.1, 1:9.1, 1:10. Preferably, the ratio of the cells expressing Ang1 to the Tie2-overexpressing vascular endothelial cells may be 1:0.5 to 1:3. For example, it can be 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.

[0043] The term "intracytoplasmic retinopathy" as used herein refers to a disease in which blood vessels in the retina grow or become abnormally 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.

[0044] 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).

[0045] The term "angiogenic factor" as used herein may mean a growth factor that promotes the development of blood vessels, angiogenesis, endothelial cell growth, vascular 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.

[0046]

[0047] Another aspect provides a kit for preventing or treating intraretinal neovascular disease, comprising the composition.

[0048] The above “vascular endothelial cells overexpressing Tie2”, “cells expressing Ang1”, “intraretinal neovascular disease”, etc. are as described above.

[0049]

[0050] Another aspect provides a method for treating angiogenic diseases in the retina, comprising administering to a subject in need thereof a composition comprising, as an active ingredient, vascular endothelial cells overexpressing an effective amount of Tie2 (Tyrosine Kinase Receptor 2).

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Specifically, the effective amount of the Tie2-overexpressing vascular endothelial cells 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.

[0056] In one specific example, the composition may further comprise cells expressing Ang1.

[0057] In one specific example, a pharmaceutical composition comprising vascular endothelial cells overexpressing Tie2 as an active ingredient may be co-administered with a pharmaceutical composition comprising cells expressing Ang1.

[0058]

[0059] Another aspect provides the use of a composition comprising vascular endothelial cells overexpressing Tie2 (Tyrosine Kinase Receptor 2) as an active ingredient for preventing or treating neovascular diseases in the retina.

[0060] Another aspect provides the use of Tie2-overexpressing endothelial cells for the manufacture of a medicament for the treatment of neovascular diseases in the retina.

[0061] In one specific example, vascular endothelial cells overexpressing Tie2 and cells expressing Ang1 can be used together for the manufacture of a medicament for the treatment of neovascular diseases in the retina.

[0062] The above “vascular endothelial cells overexpressing Tie2”, “cells expressing Ang1”, “intraretinal neovascular disease”, etc. are as described above.

[0063]

[0064] 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.

[0065]

[0066] A pharmaceutical composition comprising vascular endothelial cells overexpressing Tie2 as an active ingredient has the effect of preventing or treating retinal neovascular diseases by stabilizing vascular endothelial cells and reducing vascular permeability, thereby slowing or inhibiting neovascularization in the retina even in the presence of overexpression of vascular endothelial growth factor (VEGF) or inflammation. In addition, compared to existing antibody therapies that require intraocular injections at 4-8 week intervals, the composition has the effect of reducing invasiveness during treatment, enhancing vascular stability, and enabling early diagnosis and treatment of retinal neovascular diseases.

[0067] Figure 1 shows the vector structure for producing Tie2-overexpressing human umbilical vein endothelial cells (HUVEC_Tie2) and the results of confirming Tie2 gene expression in HUVEC_Tie2: Figure 1a is a schematic diagram showing the structure of the lentiviral vector pLV-mCherry-CMV encoding mCherry as a control vector, Figure 1b is a schematic diagram showing the structure of the lentiviral vector pLV-mCherry-CMV encoding human Tie2 and mCherry, Figure 1c is a photograph showing the results of detecting mCherry in human umbilical vein endothelial cells (HUVEC_Tie2) transfected with the lentiviral vector pLV-mCherry-CMV using a fluorescence microscope, and Figure 1d is a diagram showing the correlation between Tie2 gene expression levels in non-transfected HUVEC (control) and Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2) using RT-PCR with respect to GAPDH expression. This is a graph showing the results of relative quantitative analysis, and 1e is a figure showing the results of FACS analysis of HUVEC_NC, HUVEC_Cont., and HUVEC_Tie2.

[0068] Figure 2 shows the vector structure for producing Ang1 overexpressing mesenchymal stem cells (MSC_Ang1) and the results of confirming Tie2 gene expression in MSC_Ang1: Figure 2a is a schematic diagram showing the structure of the lentiviral vector pLV-EGFP-CMV encoding EGFP, Figure 2b is a schematic diagram showing the structure of the lentiviral vector pLV-EGFP-CMV encoding human Angiopoietin-1 and EGFP, Figure 2c is a photograph showing the results of detecting GFP under a fluorescence microscope in human mesenchymal stem cells (hMSCs) transfected with the lentiviral vector pLV-EGFP-CMV, Figure 2d is a graph showing the results of analyzing the Ang1 gene expression level in non-transfected human mesenchymal stem cells (control) and Ang1 overexpressing human mesenchymal stem cells (hMSC_Ang1) as a relative quantification of GAPDH expression through RT-PCR, and Figure 2e is NC, This graph shows the results of ELISA analysis performed at 2-day intervals for up to 10 days after transfection for Lv-cont. and Lv-Ang1.

[0069] Figure 3 is a photograph showing the results of staining vascular cells that moved under the filter with crystal violet.

[0070] Figure 4 is a graph showing the results of measuring the absorbance at 595 nm using a spectrofluorometer after dissolving stained cells with acetic acid.

[0071] Figure 5 is a photograph taken with a confocal microscope showing HUVEC cells moving into the RPE layer, divided into the normal group (NC), disease group, cell therapy group #1, and cell therapy group #2.

[0072] Figure 6 is a photograph showing the results of staining the vascular cells that migrated under the filter with crystal violet after culturing HUVECs for 24 hours at 37°C, adding hMSCs on top of HUVECs, co-culturing for 6 to 8 hours at 37°C, adding 20 ng / ml of VEGF, and performing a migration assay after 18 to 20 hours in a 37°C incubator:

[0073] Figure 6a is a photograph showing the results of a cell mobility evaluation conducted after co-culture of HUVECs that did not overexpress Tie2 (HUVEC-Cont.) and hMSCs that did not overexpress Ang1 (hMSC-Cont.), Figure 6b is a photograph showing the results of a cell mobility evaluation conducted after co-culture of HUVECs that did not overexpress Tie2 (HUVEC-Cont.) and hMSCs that did not overexpress Ang1 (hMSC-Cont.), and Figure 6c is a photograph showing the results of a cell mobility evaluation conducted after co-culture of HUVECs that did not overexpress Tie2 (HUVEC_Tie2) and hMSCs that did overexpress Ang1 (hMSC_Ang1), and then adding VEGF to the culture medium.

[0074] Figure 7 is a graph showing the results of measuring the absorbance at 595 nm in a spectrophotometer after dissolving cells stained with crystal violet in acetic acid.

[0075] Figure 8 is a photograph showing the results of real-time time-lapse imaging of tubular structures after HUVEC cells and hMSC cells were attached to Matrigel at a ratio of 2:1, and images were taken every hour for 3 hours.

[0076] Figure 9 is a graph showing the area covered by the tube over time.

[0077] The following examples are provided for more detailed explanation. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0078] Example 1. Production of Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2)

[0079] 1.1 Cell culture

[0080] Human umbilical vein endothelial cells (HUVEC) were purchased from Life Technologies (Gibco; C-003-5C) and used in experiments at passages 1–8. Cells were cultured at 37°C in EBM-2 basal medium (Lonza) supplemented with the EGM-2 SingleQuot Kit.

[0081] 1.2 Production of Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2)

[0082] To produce vascular endothelial cell control (HUVEC_Cont.), human umbilical vein endothelial cells (HUVEC) were transfected with a lentiviral vector encoding mCherry using lentivirus. In addition, to produce Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2), human umbilical vein endothelial cells (HUVEC) were transfected using lentivirus, and the expression of the Tie2 gene in the transfected HUVEC was confirmed.

[0083] Specifically, the lentiviral vector pLV-mCherry-CMV was purchased prepackaged into viral particles from VectorBuilder. The structure of the viral vector is shown in Figure 1a.

[0084] Figure 1a is a schematic diagram showing the structure of the lentiviral vector pLV-mCherry-CMV encoding mCherry. In addition, Figure 1b is a schematic diagram showing the structure of the lentiviral vector pLV-mCherry-CMV encoding human Tie2 and mCherry.

[0085] HUVEC cells were seeded at 1x10 per well in a 6-well plate. 5 Cells were seeded. 1 ml of virus was infected per well at a multiplicity of infection (MOI) of 15, and the medium was replaced with 2 ml after 24 h of transfection. To evaluate the transfection efficiency, mCherry was detected under a fluorescence microscope, and the results are shown in Figure 1b.

[0086] Figure 1c is a photograph showing the results of detecting mCherry using a fluorescence microscope in human umbilical vein endothelial cells (HUVEC_Tie2) transfected with the lentiviral vector pLV-mCherry-CMV.

[0087] As shown in Fig. 1c, it was confirmed that mCherry was detected in human umbilical vein endothelial cells (HUVEC) transfected using lentivirus, which means that the Tie2 gene was expressed in HUVEC (HUVEC_Tie2) transfected with the lentiviral vector.

[0088] 1.3 Confirmation of Tie2 gene expression in Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2)

[0089] To confirm whether Tie2 gene expression was observed in Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2) transfected with lentivirus according to Example 1.2, Tie2 gene expression was detected using RT-PCR and FACS analysis.

[0090] 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 Tie2 mRNA expression, RT-PCR analysis was performed using the 7500 Real-Time PCR system (Applied Biosystems). The primers used were purchased from IDT, and the primer sequences are shown in Table 1.

[0091] Sequence SEQ ID NO: Primer1(Tie2)5'-GAGGCAATCAGGATACGAACC-3'1Primer2(Tie2)5'-TCTCCCTTGTCCACAGTCAT-3'2

[0092] Each sample was analyzed for relative quantification of GAPDH expression, and the results are shown in Fig. 1c.

[0093] Figure 1d is a graph showing the results of analyzing the level of Tie2 gene expression in non-transfected HUVEC (control) and Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2) using RT-PCR as a relative quantitative measure of GAPDH expression.

[0094] As shown in Fig. 1d, it was confirmed that the amount of Tie2 gene expression was higher in Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2) compared to non-transfected HUVEC (control).

[0095] Meanwhile, to evaluate the efficiency of Tie2 gene expression in Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2) transfected with lentivirus according to Example 1.2, FACS analysis was performed using BD Trucount® Absolute Counting Tube. The Tie2 antibody used for FACS analysis was BD Pharmingen® Alexa Fluor® 647 Mouse Anti-Human Tie2 (CD202b). The efficiency of Tie2 expression in transfected HUVEC is shown in Fig. 1d.

[0096] Figure 1e shows the results of FACS analysis of HUVEC_NC, HUVEC_Cont., and HUVEC_Tie2. HUVEC_NC represents a negative control without antibody, HUVEC_Cont. represents a control in which antibody was added to cells that did not overexpress Tie2, and HUVEC_Tie2 represents HUVEC in which Tie2 was overexpressed.

[0097] As shown in Fig. 1e, it was confirmed that Tie2 was overexpressed in HUVEC_Tie2 transfected using the lentiviral vector pLV-mCherry-CMV.

[0098]

[0099] Example 2. Production of Ang1-expressing mesenchymal stem cells (MSC_Ang1)

[0100] 2.1 Cell culture

[0101] Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSCs) were obtained from the Stem Cell Center at Asan Medical Center, Seoul. hMSCs used in this experiment were passage 5–12. Cells were cultured at 37°C in DMEM medium containing 10% FBS and 1% penicillin-streptomycin solution.

[0102] 2.2 Production of Ang1-overexpressing mesenchymal stem cells (MSC_Ang1)

[0103] To produce mesenchymal stem cell control (hMSC_Cont.), human mesenchymal stem cells (hMSCs) were transfected with a lentiviral vector encoding EGFP using lentivirus. In addition, to produce Ang1 (Angiopoietin-1)-overexpressing mesenchymal stem cells (MSC_Ang1), human mesenchymal stem cells (hMSCs) were transfected using lentivirus, and the expression of the Ang1 gene in the transfected hMSCs was confirmed.

[0104] Specifically, the lentiviral vector pLV-EGFP-CMV encoding EGFP was purchased pre-packaged into viral particles from VectorBuilder. The structure of the viral vector is shown in Fig. 2a. In addition, Fig. 2b is a schematic diagram showing the structure of the lentiviral vector pLV-EGFP-CMV encoding human Angiopoietin-1 and EGFP.

[0105] 1x10 hMSCs per well in a 6-well plate 5Cells were seeded. 1 ml of virus was infected per well at a multiplicity of infection (MOI) of 10, and the medium was replaced with 2 ml after 24 h of transfection. To evaluate the transfection efficiency, GFP was detected under a fluorescence microscope, and the results are shown in Figure 2b.

[0106] Figure 2c is a photograph showing the results of detecting GFP using a fluorescence microscope in human mesenchymal stem cells (hMSCs) transfected with the lentiviral vector pLV-EGFP-CMV.

[0107] As shown in Fig. 2c, it was confirmed that GFP was detected in human mesenchymal stem cells (hMSCs) transfected using lentivirus, which confirmed that the Ang1 gene was expressed in human mesenchymal stem cells (hMSCs-Ang1) transfected with the lentiviral vector.

[0108] 2.3 Confirmation of Ang1 gene expression in Ang1-overexpressing mesenchymal stem cells (MSCs-Ang1)

[0109] To confirm whether Ang1 gene expression was observed in Ang1-overexpressing human mesenchymal stem cells (hMSC_Ang1) transfected with lentivirus according to Example 2.2, RT-PCR and ELISA analyses were used to detect Ang1 gene expression.

[0110] 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 Ang1 mRNA expression, RT-PCR analysis was performed using the 7500 Real-Time PCR system (Applied Biosystems). The primers used were purchased from IDT, and the primer sequences are shown in Table 2.

[0111] Sequence number Primer 1 (Ang1) 5'-AACCAGCCTCCTCTCTCAG-3'3 Primer 2 (Ang1) 5'-GCAGCTGTATCTCAAGTCGAG-3'4

[0112] Each sample was analyzed for relative quantification of GAPDH expression, and the results are shown in Fig. 2c.

[0113] Figure 2d is a graph showing the results of analyzing the level of Ang1 gene expression in non-transfected human mesenchymal stem cells (control) and Ang1-overexpressing human mesenchymal stem cells (hMSC_Ang1) using RT-PCR as a relative quantitative analysis of GAPDH expression.

[0114] As shown in Fig. 2d, it was confirmed that the amount of Ang1 gene expression was higher in human mesenchymal stem cells (hMSC_Ang1) overexpressing Ang1 compared to human mesenchymal stem cells (hMSC_control) that did not overexpress Ang1.

[0115] Meanwhile, Ang1 gene expression was detected through ELISA analysis for Ang1-overexpressing human mesenchymal stem cells (hMSC_Ang1) transfected with lentivirus according to Example 2.1.

[0116]

[0117] Ang1-specific ELISA was performed using the Quantikine Human Angiopoietin-1 Immunoassay (R&D Systems). Samples of hMSC_Ang1 cultures from 6-well plates were collected and analyzed at 2-day intervals for up to 10 days after transfection.

[0118] The results of the Ang1-specific ELISA are shown in Fig. 2d.

[0119] Figure 2e is a graph showing the results of ELISA analysis performed at 2-day intervals for up to 10 days after transfection for NC, Lv-cont., and Lv-Ang1. NC refers to a negative control of untransfected hMSCs, Lv-cont. refers to a control transfected with a lentiviral vector not containing Ang1, and Lv-Ang1 refers to a control transfected with a lentiviral vector containing Ang1.

[0120] As shown in Fig. 2e, Ang1 was confirmed to be overexpressed in human mesenchymal stem cells (hMSCs-Ang1) transfected with pLV[Exp]-EGFP:T2A:PuroCMV>hANGPT1.

[0121]

[0122] Example 3. Manufacturing of cell therapy using co-culture method of HUVEC and hMSC

[0123] A cell therapy product was manufactured by co-culturing HUVEC cells and hMSC cells. Co-culturing was performed considering the size difference between HUVEC cells and hMSC cells and the difference in the expression of Tie2 and Ang1 in each cell. Specifically, 3x10 HUVECs were cultured in a Transwell. 4 cells / ml were seeded and cultured in a 37°C incubator for 24 hours. hMSCs were seeded at 1.5x10 4The cells were co-cultured on HUVEC cells at 10 cells / ml (0.5 times the number of HUVECs), and after 6–8 hours of incubation at 37°C, 20 ng / ml of VEGF was added. Migration assays were performed after 18–20 hours in a 37°C incubator.

[0124]

[0125] Experimental Example 1. Confirmation of the migration-reducing effect of Tie2-overexpressing vascular endothelial cells (HUVECs).

[0126] 1.1 Migration assay of Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2)

[0127] Transwell chambers (CorningCostar) containing 6.5 mm diameter polycarbonate filters with 8 μm pore size were used. 5 × 10 HUVECs without Tie2 overexpression (HUVEC-Cont.) or HUVECs with Tie2 overexpression (HUVEC_Tie2) were seeded. 4 Cells were seeded at a density of cells / ml, and cultured with a culture medium of hMSCs that did not overexpress Ang1 (hMSC-Cont.) or hMSCs that overexpressed Ang1 (hMSC_Ang1) for 24 hours. 20 ng / ml of VEGF was added and cultured in an incubator for 24 hours. After culture, cells on the filter were wiped off with a cotton swab, and 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, migrated cells were observed under a microscope. Cells stained with 1% acetic acid were dissolved and measured for absorbance at 595 nm using a spectrofluorometer, and the results are shown in Figs. 3 and 4.

[0128] Figure 3 is a photograph showing the results of staining vascular cells that moved under the filter with crystal violet. As shown in Figure 3, the amount of vascular cell migration varied depending on whether VEGF was added and the amount added, but it was confirmed that there was no significant difference in the amount of vascular cell migration when VEGF 20 ng / ml and VEGF 40 ng / ml were added.

[0129] Figure 4 is a graph showing the results of measuring the absorbance at 595 nm using a spectrofluorometer after dissolving stained cells with acetic acid.

[0130] As shown in Figures 3 and 4, it was confirmed that HUVEC migration significantly increased compared to the normal group (A) when VEGF was added (B: disease group), and it was confirmed that the migration of Tie2-expressing vascular endothelial cells (HUVEC_Tie2) was decreased compared to the disease group (B) in the culture medium with VEGF and without Ang-1 (C: Cell therapy agent #1). In addition, it was confirmed that the migration of Tie2-expressing vascular endothelial cells was significantly reduced compared to the disease group (B) in the culture medium with VEGF and Ang-1 and was at a similar level to the normal group (A) (D: Cell therapy agent #1+Ang_1).

[0131] 1.2 Establishment of a retinal tissue model through co-culture of retinal pigment epithelial cells (RPE) and human umbilical vein endothelial cells (HUVECs).

[0132] Using the Transwell system, a retinal tissue model was constructed by culturing retinal pigment epithelial cells (RPE) and vascular endothelial cells (HUVEC) to confirm the migration-reducing effect of Tie2-overexpressing vascular endothelial cells (HUVEC).

[0133] Specifically, RPE cells were seeded on the bottom of the Transwell by fixing the Transwell upside down on a 6-well plate coated with Vitronectin (VTN). RPE cells were seeded at 1x10 in 100ul of medium. 5The cells / ml were seeded in the form of droplets on the bottom of the transwell and cultured for 2 hours in a 37°C incubator. After confirming that the RPE cells were well attached, the transwell was turned upside down and placed in a 24-well plate containing medium. HUVECs were seeded 1x10 on the inner surface of the transwell. 5 cells / ml and cultured for 24 hours. After adding hMSC (hMSC-Cont. or hMSC_Ang1) cell culture medium, the cells were cultured for another 24 hours, and VEGF 20 ng / ml was added and cultured for another 24 hours. Images of HUVEC cells migrating to the RPE layer were obtained using a confocal microscope.

[0134] 1.3 Evaluation of vascular endothelial cell motility in retinal tissue models

[0135] The effect of the Ang1-Tie2 system on HUVEC cell migration was confirmed using the Transwell system constructed using the method of Experimental Example 1.2 above. The image of HUVEC cells migrating into the RPE layer using a confocal microscope is shown in Figure 5.

[0136] Figure 5 is a confocal microscope photograph of HUVEC cells migrating toward the RPE layer, divided into the normal group (NC), disease group, cell therapy group #1, and cell therapy group #2. The normal group (NC) is a group in which HUVECs that did not overexpress Tie2 were not treated with VEGF, the disease group is a group treated with 20 ng / ml of VEGF, cell therapy group #1 is a group in which HUVECs that overexpressed Tie2 (HUVEC_Tie2) were treated with 20 ng / ml of VEGF, and cell therapy group #2 is a group in which MSC Lv-Ang-1 culture medium was added to HUVECs that overexpressed SMS Tie2 (HUVEC_Tie2) and VEGF was treated with 20 ng / ml. Vascular endothelial cells express red fluorescence, and the more red fluorescence is distributed, the more the migration of vascular endothelial cells increases.

[0137] As shown in Fig. 5, it can be seen that the red distribution is high in the disease group, and the cell therapy group using HUVEC_Tie2 that overexpressed Tie2 did not have much red distribution. This means that the migration of vascular endothelial cells increased in the disease group, and the number of migrated vascular endothelial cells in the cell therapy group (HUVEC_Tie2) was significantly lower than that in the disease group, and it means that the angiogenesis inhibitory effect occurred even in the VEGF environment.

[0138] 1.4. Evaluation of cell migration and motility during co-culture of HUVEC and hMSC cells

[0139] Considering the size difference between the two cells and the difference in the expression of Ang1 and Tie2 expressed in each cell, as in Example 3, HUVEC cells were first cultured on one side of a porous transwell that allows cell migration, and then hMSC cells were cultured at 0.5 times the number. After culturing HUVEC cells first to stabilize the tight junctions between cells, hMSC cells were added at 0.5 times the number of cells and co-cultured on top of the HUVEC cells, and VEGF (vascular endothelial growth factor) was added to compare and evaluate the degree of cell migration in a VEGF environment, and the results are shown in Figs. 6 and 7.

[0140] Figure 6 is a photograph showing the results of staining the blood vessel cells that moved under the filter with crystal violet after culturing HUVECs at 37°C for 24 hours, adding hMSCs on top of HUVECs, co-culturing for 6 to 8 hours at 37°C, and adding 20 ng / ml of VEGF, and performing a migration assay after 18 to 20 hours in a 37°C incubator: Figure 6a is a photograph showing the results of evaluating cell mobility after co-culturing HUVECs that did not overexpress Tie2 (HUVEC-Cont.) and hMSCs that did not overexpress Ang1 (hMSC-Cont.), and Figure 6b is a photograph showing the results of evaluating cell mobility after co-culturing HUVECs that did not overexpress Tie2 (HUVEC-Cont.) and hMSCs that did not overexpress Ang1 (hMSC-Cont.), and adding VEGF. 6c is a photograph showing the results of co-culture of HUVECs overexpressing Tie2 (HUVEC_Tie2) and hMSCs overexpressing Ang1 (hMSC_Ang1), and then adding VEGF to evaluate cell mobility.

[0141] Figure 7 is a graph showing the results of measuring the absorbance at 595 nm in a spectrofluorometer after dissolving cells stained with crystal violet in acetic acid.

[0142] As shown in Figures 6 and 7, it was confirmed that normal HUVECs showed very high cell mobility in a disease environment (VEGF-containing culture medium), and when HUVEC_Tie2 and MSC_Ang1 were co-cultured in a disease environment (VEGF-containing culture medium), cell migration was confirmed to show results similar to those of the normal group. This means that in the case of the cell therapy product co-cultured with HUVEC_Tie2 and MSC_Ang1, angiogenesis was suppressed to a level similar to that in normal people even in a disease environment.

[0143]

[0144] Experimental Example 2. Confirmation of the in vitro angiogenesis inhibition ability of Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2)

[0145] To evaluate the in vitro angiogenesis inhibition ability of Tie2-overexpressing vascular endothelial cells (HUVEC_Tie2), vascular endothelial cells (HUVEC) were cultured on gels and a capillary-like tube formation assay was performed. Specifically, a tube formation assay kit (Abcam) was used for the capillary-like tube formation assay of HUVEC cells. 50 µl / well of extracellular matrix solution (ECM) was added to a 96-well plate, and the solution was solidified at 37°C for 1 hour to form a gel. 2 Х10 µl was added to each well. 4 cells / 100μl of HUVEC (HUVEC_Cont. or HUVEC_Tie2) cells and 1Х10 4 hMSC (hMSC-Cont. or hMSC_Ang1) 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 hours 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 Figs. 8 and 9.

[0146] Figure 8 is a photograph showing the results of real-time time-lapse imaging of tubular structures after HUVEC cells and hMSC cells were attached to Matrigel at a ratio of 2:1, and images were taken every hour for 3 hours.

[0147] Figure 9 is a graph showing the area covered by the tube over time.

[0148] As shown in Figures 8 and 9, the normal group (Control) rapidly formed a tube-forming structure in a VEGF environment (disease environment), and it was confirmed that the final tube-forming area decreased. The experimental group (cell therapy group) confirmed that tube formation progressed slowly even in a VEGF environment, and this is thought to be due to the slowing down of angiogenesis by the formation of tight junctions by Tie2 of vascular endothelial cells and Ang1 of mesenchymal stem cells. This means that the cell therapy agent of vascular endothelial cells overexpressing Tie2 and mesenchymal stem cells overexpressing Ang1 is effective in inhibiting angiogenesis in the retina by slowing down angiogenesis.

Claims

1. A pharmaceutical composition for preventing or treating neovascular disease in the retina, comprising vascular endothelial cells overexpressing Tie2 (Tyrosine Kinase Receptor 2) as an active ingredient.

2. A pharmaceutical composition according to claim 1, wherein the vascular endothelial cells are genetically engineered to overexpress Tie2 (Tyrosine Kinase Receptor 2).

3. A pharmaceutical composition according to claim 2, wherein the genetically engineered vascular endothelial cells include a recombinant vector into which a gene encoding Tie2 (Tyrosine Kinase Receptor 2) is introduced.

4. 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.

5. A pharmaceutical composition according to claim 1, further comprising or co-administering cells expressing Ang1 (Angiopoietin-1).

6. A pharmaceutical composition according to claim 5, wherein the cells expressing Ang1 (Angiopoietin-1) are genetically engineered to express or overexpress Ang1.

7. A pharmaceutical composition according to claim 6, wherein the genetically engineered Ang1 (Angiopoietin-1) expressing cell comprises a recombinant vector into which a gene encoding Ang1 (Angiopoietin-1) has been introduced.

8. A pharmaceutical composition according to claim 5, wherein the cell expressing Ang1 (Angiopoietin-1) is any one selected from the group consisting of pericytes, mesenchymal stem cells (MSCs), endothelial cells (ECs), fibroblasts, retinal pigment epithelium (RPE), photoreceptors, horizontal cells, Muller cells, amacrine cells, astrocytes, etc.

9. A pharmaceutical composition according to claim 8, wherein the mesenchymal stem cells (MSCs) are derived from any one selected from the group consisting of umbilical cord, umbilical cord blood, placenta, bone marrow, adipose tissue, muscle, amniotic fluid, amniotic membrane, and dental pulp.

10. A pharmaceutical composition according to claim 5, wherein the cells expressing Ang1 (Angiopoietin-1) exhibit reduced immunogenicity.

11. A pharmaceutical composition according to claim 5, wherein the cells expressing Ang1 (Angiopoietin-1) and the vascular endothelial cells overexpressing Tie2 are co-cultured.

12. A pharmaceutical composition according to claim 5, wherein the ratio of cells expressing Ang1 (Angiopoietin-1) and vascular endothelial cells overexpressing Tie2 (Tyrosine Kinase Receptor 2) is 1:0.1 to 1:

10.

13. 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).

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

15. Use of a composition comprising vascular endothelial cells overexpressing Tie2 (Tyrosine Kinase Receptor 2) as an active ingredient for preventing or treating intraretinal angiogenic disease.

16. A method for preventing or treating neovascular disease in the retina, comprising administering to a subject in need thereof a composition containing, as an active ingredient, vascular endothelial cells overexpressing an effective amount of Tie2 (Tyrosine Kinase Receptor 2).

17. Use of a composition comprising vascular endothelial cells overexpressing Tie2 (Tyrosine Kinase Receptor 2) as an active ingredient for use in the manufacture of a medicine for preventing or treating intraretinal angiogenic diseases.

Images