Composition for prevention or treatment of renal disease

Abstract

The present invention relates to use of EPO knock-in pluripotent stem cell–derived mesenchymal stem cells and exosomes isolated therefrom for treating renal diseases. According to the present invention, in order to maximize the expression of EPO, induced pluripotent stem cells having the EPO gene knocked-in therein were prepared using a CRISPR-Cas9 gene editing technology, and then differentiated to establish mesenchymal stem cells in which EPO expression and secretion are significantly increased. It was confirmed that exosomes isolated therefrom contain and secrete a high concentration of EPO to treat nephrotic anemia, improve renal function, inhibit and alleviate renal fibrosis, and provide a renal protective effect in an animal model of chronic kidney disease, and thus the exosomes can be used for the prevention or treatment of chronic kidney disease or nephrotic anemia caused thereby.

Description

Composition for the prevention or treatment of kidney disease

[0001] The present invention relates to the therapeutic use of EPO-dissolved pluripotent stem cell-derived mesenchymal stem cells and exosomes isolated therefrom for kidney disease.

[0002] The kidneys are vital organs responsible for maintaining homeostasis in the body. They regulate body fluid volume, blood ion concentration, and pH; excrete metabolic waste products, toxins, and drugs; and perform blood pressure regulation as well as other metabolic and endocrine functions. Additionally, they activate Vitamin D to facilitate calcium absorption in the small intestine and participate in the synthesis of various hormones. Kidney disease refers to a condition in which the kidneys fail to perform their excretory, regulatory, metabolic, and endocrine functions normally, resulting in an overall decline in function or abnormalities. Functional decline caused by kidney damage leads to enlargement of the kidneys and related structures, renal atrophy, changes in fluid volume, electrolyte imbalance, metabolic acidosis, impaired gas exchange, reduced anti-infectious function, and the accumulation of uremic toxins. One of the kidneys' primary functions is to secrete erythropoietin (EPO) to produce red blood cells, and the majority of EPO is produced in the kidneys.

[0003] Chronic kidney disease (CKD) (chronic renal failure) is recognized as a serious disease worldwide. The main causes are diabetes and hypertension, while other causes include urinary tract obstruction, specific kidney abnormalities (such as polycystic kidney disease and glomerulonephritis), and autoimmune diseases (systemic lupus erythematosus [Lupus]) in which antibodies damage the small blood vessels (glomeruli) and small tubules (tubules) of the kidneys. Acute kidney injury becomes chronic kidney disease when irreversible kidney damage caused by these various diseases or when kidney function does not recover and persists for more than three months following treatment for an injury. Symptoms of chronic kidney disease include nocturia, fatigue, nausea, itching, muscle cramps and spasms, loss of appetite, confusion, shortness of breath, and body edema (most commonly in the legs). The condition of patients suffering from chronic kidney disease requiring renal replacement therapy, such as dialysis or transplantation, is referred to as end-stage renal disease (ESRD). Currently, there are no effective treatments available other than renin-angiotensin-aldosterone system (RAS) inhibitors, such as angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme (ACE) inhibitors used alone or in combination. However, even these treatments are only effective in delaying the onset of ESRD or inhibiting the decline in glomerular filtration rate (GFR) in some CKD patients; their effects are minimal for the majority of CKD patients. The persistence of various chronic kidney diseases ultimately leads to end-stage renal failure.Chronic renal failure is caused by chronic glomerulonephritis, diabetes, hypertension, urinary tract obstruction, renal tuberculosis, and hereditary kidney diseases. Even if the underlying cause is treated, kidney function does not recover, and while the progression to renal failure can be slowed, it cannot be prevented. In particular, in cases of chronic kidney disease, anemia occurs in 76% of patients due to insufficient EPO production, which is referred to as renal anemia. If renal anemia is left untreated, persistent anemia leads to a decrease in quality of life and an increased risk of cardiovascular complications. Consequently, the morbidity and mortality rates of patients with chronic kidney disease increase, yet there is a shortage of treatments available to address this condition.

[0004] The object of the present invention is to provide genetically modified pluripotent stem cell-derived mesenchymal stem cells.

[0005] In addition, the objective of the present invention is to provide exosomes isolated from the mesenchymal stem cells.

[0006] In addition, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of kidney disease.

[0007] In addition, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of renal anemia.

[0008] In addition, the objective of the present invention is to provide a method for producing exosomes containing or secreting EPO.

[0009] In addition, the objective of the present invention is to provide a method for preventing or treating kidney disease.

[0010] In addition, the objective of the present invention is to provide a method for preventing or treating renal anemia.

[0011] To solve the above problem, the present invention provides a pluripotent stem cell-derived mesenchymal stem cell in which a gene encoding EPO (erythropoietin, EPO) is integrated within the gene.

[0012] In addition, the present invention provides exosomes isolated from the mesenchymal stem cells.

[0013] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of kidney disease comprising the mesenchymal stem cells, the culture medium thereof, or exosomes isolated therefrom as an active ingredient.

[0014] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of renal anemia comprising the mesenchymal stem cells, the culture medium thereof, or exosomes isolated therefrom as an active ingredient.

[0015] In addition, the present invention provides a method for producing exosomes containing or secreting EPO.

[0016] In addition, the present invention provides a method for preventing or treating kidney disease, comprising the step of administering the mesenchymal stem cells, the culture medium thereof, or exosomes isolated therefrom to an individual.

[0017] In addition, the present invention provides a method for preventing or treating renal anemia, comprising the step of administering the mesenchymal stem cells, the culture medium thereof, or exosomes isolated therefrom to an individual.

[0018] According to the present invention, induced pluripotent stem cells with an EPO gene knocked in using CRISPR-Cas9 gene editing technology to maximize EPO expression were prepared and then differentiated to construct mesenchymal stem cells with significantly increased EPO expression and secretion. It was confirmed that exosomes isolated from these cells contain and secrete high concentrations of EPO, thereby treating renal anemia in an animal model of chronic kidney disease, improving kidney function, inhibiting and alleviating renal fibrosis, and having renal protective effects. Therefore, this can be utilized for the prevention or treatment of chronic kidney disease or renal anemia caused by it.

[0019] Figure 1 is a schematic diagram showing the EPO knock-in (gene insertion) plasmid of the present invention.

[0020] Figure 2 is a schematic diagram showing the break at site 2 location of AAV1 in human induced pluripotent stem cells to knock in the EPO gene using the EPO knock-in plasmid of the present invention and the plasmid that goes into it.

[0021] Figure 3 shows the knock-in sequence inserted into the AAV1 gene location of human induced pluripotent stem cells and the confirmation of this by PCR analysis.

[0022] Figure 4 shows the results of the analysis of the characteristics of mesenchymal stem cells differentiated from human induced pluripotent stem cells that had been dissolved in EPO.

[0023] Figure 5 is a figure confirming the morphological characteristics of human induced pluripotent stem cells dissolved in EPO and mesenchymal stem cells differentiated therefrom.

[0024] Figure 6 is a figure analyzing the EPO expression levels of human induced pluripotent stem cells (iPSCs) dissolved in EPO and mesenchymal stem cells differentiated therefrom:

[0025] WTC11 iPSC: Control group human induced pluripotent stem cells;

[0026] EPO iPSC: Human induced pluripotent stem cells lysed with EPO; and

[0027] EPO iMSC: Mesenchymal stem cells differentiated from human induced pluripotent stem cells that have been dissolved in EPO.

[0028] Figure 7 shows the analysis of EPO secretion in the culture medium of human induced pluripotent stem cells (iPSCs) dissolved in EPO and mesenchymal stem cells differentiated therefrom:

[0029] WTC11 iPSC: Control group human induced pluripotent stem cells;

[0030] EPO iPSC: Human induced pluripotent stem cells lysed with EPO; and

[0031] EPO iMSC: Mesenchymal stem cells differentiated from human induced pluripotent stem cells that have been dissolved in EPO.

[0032] Figure 8 shows a TEM image of an exosome (iMSC-EPO) isolated from mesenchymal stem cells differentiated from human induced pluripotent stem cells that had been dissolved in EPO.

[0033] Figure 9 is a figure analyzing the size, distribution, and particle number characteristics of exosomes (iMSC-EPO) isolated from mesenchymal stem cells differentiated from human induced pluripotent stem cells that dissolved EPO.

[0034] Figure 10 shows the expression of extracellular vesicle markers in exosomes (iMSC-EPO) isolated from mesenchymal stem cells differentiated from human induced pluripotent stem cells that had been dissolved in EPO, as confirmed by Western blot analysis.

[0035] Figure 11 is a figure analyzing the amount of EPO secreted by exosomes isolated from human induced pluripotent stem cells (iPSCs) that had EPO dissolved and mesenchymal stem cells differentiated therefrom:

[0036] EPO iPSC exosome: Exosomes isolated from human induced pluripotent stem cells that have lysed EPO; and

[0037] EPO iMSC exosome: Exosomes isolated from mesenchymal stem cells differentiated from human induced pluripotent stem cells that have dissolved EPO.

[0038] Figure 12 is a diagram showing the analysis of microRNAs (top 50) in exosomes (iMSC-EPO) isolated from mesenchymal stem cells differentiated from human induced pluripotent stem cells that had EPO dissolved.

[0039] Figure 13 is a graph showing the percentage content of microRNA (top 23) in exosomes (iMSC-EPO) isolated from mesenchymal stem cells differentiated from human induced pluripotent stem cells that had EPO dissolved.

[0040] Figure 14 is a schematic diagram showing the process of establishing an animal model of chronic kidney disease (CKD) using an adenine diet.

[0041] Figure 15 is a figure analyzing the body weight and blood BUN, creatinine, hematocrit, and hemoglobin levels of an animal model of chronic kidney disease constructed with an adenine diet.

[0042] Figure 16 is a figure showing the CKD induction and drug administration schedule.

[0043] Figure 17 shows the results of RT-PCR analysis of EPO expression in the kidneys of mice in a CKD animal model administered iMSC-EPO exosomes and rhEPO:

[0044] Control: Control group;

[0045] Adenine: CKD-inducing animal model;

[0046] EPO iMSC exosome: group administered EPO iMSC exosomes to a CKD-induced animal model; and

[0047] rhEPO: Group administered human recombinant EPO to an animal model of CKD induction (positive control).

[0048] Figure 18 shows the results of fluorescent staining analysis of EPO expression in the kidneys of mice in a CKD animal model administered iMSC-EPO exosomes and rhEPO:

[0049] Control: Control group;

[0050] Adenine: CKD-inducing animal model; and

[0051] EPO iMSC exosome + adenine: Group administered EPO iMSC exosomes to an animal model of CKD induction.

[0052] Figure 19 is a figure analyzing the therapeutic effect of iMSC-EPO exosome administration on renal anemia in a CKD animal model:

[0053] Control: Control group;

[0054] Adenine: CKD-inducing animal model; and

[0055] EPO iMSC exosome + adenine: Group administered EPO iMSC exosomes to an animal model of CKD induction.

[0056] Figure 20 is a figure analyzing the effect of iMSC-EPO exosome administration on improving renal function in a CKD animal model:

[0057] Control: Control group;

[0058] Adenine: CKD-inducing animal model; and

[0059] EPO iMSC exosome + adenine: Group administered EPO iMSC exosomes to an animal model of CKD induction.

[0060] Figure 21 is a figure analyzing the effect of iMSC-EPO exosome administration on renal fibrosis improvement in a CKD animal model through changes in marker expression:

[0061] Control: Control group;

[0062] Adenine: CKD-inducing animal model; and

[0063] EPO iMSC exosome + adenine: Group administered EPO iMSC exosomes to an animal model of CKD induction.

[0064] Figure 22 shows the analysis of the effect of iMSC-EPO exosome administration on renal fibrosis improvement in a CKD animal model using Masson's trichrome staining:

[0065] Control: Control group;

[0066] Adenine: CKD-inducing animal model; and

[0067] EPO iMSC: Group administered EPO iMSC exosomes to an animal model of CKD induction.

[0068] Figure 23 shows the renal protective effect of iMSC-EPO exosome administration in a CKD animal model, confirmed through the analysis of apoptosis levels in kidney tissue:

[0069] Control: Control group;

[0070] Adenine: CKD-inducing animal model; and

[0071] EPO iMSC / EPO iMSC exosome: Group administered EPO iMSC exosomes to an animal model of CKD induction.

[0072] Figure 24 shows the renal protective effect of iMSC-EPO exosome administration in a CKD animal model, confirmed through analysis of peritubular capillary rarefaction in kidney tissue:

[0073] Control: Control group;

[0074] Adenine: CKD-inducing animal model; and

[0075] EPO iMSC / EPO iMSC exosome: Group administered EPO iMSC exosomes to an animal model of CKD induction.

[0076] FIG. 25 is a schematic diagram showing the process of using the EPO-iMSC-derived exosomes of the present invention for the treatment of chronic kidney disease.

[0077] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0078] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0079] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0080]

[0081] In one aspect, the present invention relates to a mesenchymal stem cell (MSC) derived from a pluripotent stem cell in which a gene encoding EPO (erythropoietin, EPO) is integrated within the gene.

[0082] In one embodiment, a donor vector containing a knock-in construct containing a sequence encoding EPO can be incorporated into the AAV1 gene by knocking it in by electroporation.

[0083] In one embodiment, the knock-in construct may be DNA, may be double-stranded DNA, may be a plasmid, and may be a non-linearized plasmid.

[0084] In one embodiment, the sequence encoding EPO (erythropoietin, EPO) may include the base sequence represented by SEQ ID NO. 1.

[0085] In one embodiment, the vector may further include one or more sequences selected from the group consisting of a cell selection marker sequence, a protein purification tag, a reporter marker sequence, an IRES sequence, an exogenous gene sequence, a promoter sequence, a 2A linker sequence, a termination sequence, an mRNA stabilization sequence, or a combination thereof.

[0086] In one embodiment, the cell selection marker sequence may be a zeocin resistance marker, a neomycin resistance marker, a puromycin resistance marker, a blasticidin resistance marker, or a hygromycin resistance marker.

[0087] In one embodiment, the pluripotent stem cell may be an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC).

[0088] In one embodiment, the somatic cells prior to the dedifferentiation of the induced pluripotent stem cells may be somatic cells derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, amniotic fluid, or placenta, and the somatic cells may include fibroblasts, hepatocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, muscle cells, cardiac muscle cells, melanocytes, neural cells, glial cells, astroglial cells, monocytes, macrophages, etc.

[0089] In one embodiment, the pluripotent stem cell may be a genetically modified pluripotent stem cell in which a gene encoding EPO (erythropoietin, EPO) is integrated into the gene, and may be a pluripotent stem cell in which a gene encoding EPO is knocked in at the break at site 2 position of the AAV1 gene and a gene encoding EPO is integrated into the gene.

[0090] In one embodiment, the gRNA for knocking in the gene encoding the EPO may include the nucleotide sequence represented by SEQ ID NO. 2.

[0091] In one embodiment, the gene in which the gene encoding EPO is integrated into the AAV1 gene may include a nucleotide sequence indicated by SEQ ID NO. 3.

[0092] In the lentivirus vector of the present invention, the nucleotide sequences represented by SEQ ID NOs 1 to 3 each include modified sequences in which some nucleotides are substituted, deleted, or added.

[0093] That is, the nucleotide sequence variants represented by SEQ ID NOs 1 to 3 may be included within the scope of the present invention. The concept includes functional equivalents of the nucleotide sequences represented by SEQ ID NOs 1 to 3 and the nucleic acid molecules constituting them, for example, variants in which some nucleotide sequences of SEQ ID NOs 1 to 3 have been modified by deletion, substitution, or insertion, but which can perform the same function as the nucleotide sequences of SEQ ID NOs 1 to 3 and the nucleic acid molecules constituting them. Specifically, it may include nucleotide sequences having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with respect to the nucleotide sequences of SEQ ID NOs 1 to 3. The “% of sequence homology” for a base sequence is determined by comparing two optimally arranged sequences with a comparison region, and a portion of the base sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences.

[0094] In one embodiment, the mesenchymal stem cells of the present invention differentiated from the genetically modified pluripotent stem cells may have increased expression or secretion of EPO compared to the pluripotent stem cells.

[0095] In one embodiment, the mesenchymal stem cell of the present invention contains 1 × 10 exosomes. 9 Up to 1 × 10 11 It can be produced with a particle count of particles / ml.

[0096] As used in the present invention, the term "gene editing technology" refers to a technology capable of introducing targeted mutations into the genomic base sequences of animal and plant cells, including human cells, by knocking out or knocking in specific genes, or by introducing mutations into non-coding DNA sequences that do not produce proteins. Additionally, DNA on the genome can be deleted, duplicated, inverted, replaced, or rearranged through gene editing. Gene editing technology may include zinc finger nucleases (ZFN), transcription factor-like operator nucleases (TALEN), and clustered regularly interspaced short palindromic repeat sequences / CRISR-related systems (CRISPR / Cas9), etc.

[0097] As used in the present invention, the term "pluripotent stem cell (PSC)" refers to a stem cell capable of induced differentiation into any type of cell constituting the body, and the embryonic stem cells included therein are induced from the inner cell mass of a blastocyst at the pre-implantation stage. The induced cells are maintained in a specific environment and allow for unlimited culture and pluripotent differentiation. Furthermore, pluripotent stem cells are not limited to embryonic stem cells and induced pluripotent stem cells, but may include all cells possessing both differentiation pluripotency and self-replication ability. However, preferably, the pluripotent stem cells may be mammalian cells, and more preferably, human-derived pluripotent stem cells.

[0098] The term "induced pluripotent stem cell (iPSC)" as used in the present invention refers to a cell that has acquired pluripotency by inducing dedifferentiation in an already differentiated cell, such as a somatic cell, to return to an initial undifferentiated state. The said pluripotency refers to the ability to differentiate into tissues or organs of the three germ layers that constitute a living organism, namely the endoderm, mesoderm, and ectoderm.

[0099] The induced pluripotent stem cells of the present invention include induced pluripotent stem cells derived from all mammals, such as humans, monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, and rabbits, but preferably are induced pluripotent stem cells derived from humans.

[0100] As used in the present invention, the term "mesenchymal stem cell" refers to a stem cell with multipotential that can differentiate into various cells, including osteoblasts, chondrocytes, muscle cells, and adipocytes. Mesenchymal stem cells are also referred to as stromal cells.

[0101] As used in the present invention, the term "genetically modified cell" refers to a eukaryotic or prokaryotic cell comprising a "construct" or an "exogenous fragment" integrated within its genome, and should be understood to refer not only to a specific target cell but also to its offspring or potential offspring. Even if said offspring are not exactly identical to the parent cell due to mutation or environmental influence, they may still be included within the scope of said term as used herein.

[0102] The above construct may be a knock-in construct or a donor vector containing it, and is "exogenous" to the cell if the nucleotide sequence is not naturally part of the cell genome or is intentionally inserted into the cell genome. The nucleotide sequence may be intentionally inserted into the cell genome by human intervention or automated means.

[0103] As used in the present invention, the term "expression" generally refers to a cellular process in which a biologically active polypeptide is generated from a DNA sequence and exhibits biological activity in a cell. In this sense, gene expression includes not only transcription and translation processes, but also post-transcriptional and post-translational processes that may affect the biological activity of the gene or gene product. These processes include, but are not limited to, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of the polypeptide.

[0104] In the present invention, the expression can be confirmed by measuring the expression level of a gene or mRNA using a polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip method using a nucleic acid sequence, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer pair and a probe that specifically recognize the nucleic acid sequence and a fragment of the sequence complementary to the nucleic acid sequence, and using an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognize the entire length of the corresponding protein or its fragment using Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Protein expression levels can be confirmed by measuring them using radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray methods.

[0105] In one aspect, the present invention relates to an exosome isolated from a mesenchymal stem cell of the present invention.

[0106] In one embodiment, the exosome may have an average size of 10 to 300 nm.

[0107] In one embodiment, the exosome may contain or secrete EPO.

[0108] In one embodiment, the exosome may have an increased amount of EPO secretion compared to an exosome isolated from a pluripotent stem cell in which a gene encoding EPO is integrated within the gene.

[0109] In one embodiment, the exosome may contain or express one or more microRNAs selected from the group consisting of hsa-miR-148a-3p, hsa-miR-143-3p, and hsa-miR-99a-5p.

[0110] In one embodiment, the exosome may exhibit expression levels of 40 to 50% of hsa-miR-148a-3p, 5 to 6% of hsa-miR-143-3p, and 4 to 5% of hsa-miR-99a-5p with respect to 100% of total microRNA expression, but is not limited thereto. Preferably, the exosome may exhibit expression levels of 43.62% of hsa-miR-148a-3p, 5.92% of hsa-miR-143-3p, and 4.93% of hsa-miR-99a-5p.

[0111] In one embodiment, the exosome is hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-let-7i-5p, hsa-miR-10b-5p, hsa-miR-21-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-miR-34c-5p, hsa-miR-100-5p, hsa-let-7a-5p, hsa-miR-27b-3p, hsa-let-7b-5p, hsa-miR-215-5p, hsa-let-7c-5p, hsa-miR-203a-3p, hsa-miR-7-5p, hsa-miR-126-3p One or more microRNAs selected from the group consisting of hsa-miR-192-5p may be additionally included or expressed.

[0112] In one embodiment, the exosomes may be separated by centrifugation, ultracentrifugation, density gradient centrifugation, chromatography, filtration, ultrafiltration, tangential flow filtration, polymer-based precipitation, a total exosome extraction kit, or an immunoaffinity separation method.

[0113] In the present invention, the exosome isolated from the mesenchymal stem cell refers to the exosome that exists within the mesenchymal stem cell described above or is secreted from the mesenchymal stem cell.

[0114] The term "exosome" as used in this invention refers to a microbody that is secreted in various forms depending on the cellular environment from the multivescular endosome (MVE), a special organelle within a cell. It contains various growth factors, cytokines, and nucleic acids (DNA and RNA) and has a structure similar to a liposome composed of a phospholipid membrane. Exosomes are a concept that encompasses microvesicles. Known marker proteins for exosomes include CD63 and CD81, and other known proteins include cell surface receptors such as EGFR, molecules involved in signal transduction, proteins involved in cell adhesion, MSC-associated antigens, heat shock proteins, and Alix, which is involved in vesicle formation.

[0115] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of kidney disease comprising, as an active ingredient, mesenchymal stem cells of the present invention, a culture medium thereof, or exosomes isolated therefrom.

[0116] In one embodiment, the kidney disease may be one or more selected from the group consisting of chronic kidney disease (CKD), polycystic kidney disease (PKD), acute kidney injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-associated nephropathy, chronic kidney disease (CKD), renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicroparticle retention disease (CRD), sitosterolemia, glomerular hyperfiltration, and pruritus of renal failure.

[0117] In one embodiment, the composition can reduce creatinine or urea nitrogen.

[0118] In one embodiment, the composition can reduce renal fibrosis, renal cell death, or peritubular capillary rarefaction.

[0119] In one embodiment, the culture medium may be a culture or the supernatant thereof obtained during or after culturing the mesenchymal stem cells of the present invention in a medium. The culture medium may be a concentrate of a culture, a filtrate thereof, a fraction thereof, or the supernatant thereof, a freeze-dried product thereof, or a supercritical dried product thereof obtained during or after culturing the mesenchymal stem cells.

[0120] In one embodiment, the culture medium may include substances such as cell-derived proteins, cytokines, growth factors, exosomes, and nucleic acids (DNA and RNA) secreted by the mesenchymal stem cells of the present invention.

[0121] In one embodiment, the exosome may be extracted from the culture medium of the mesenchymal stem cells of the present invention.

[0122] In one embodiment, the composition of the present invention may additionally include a known kidney disease treatment in addition to the active ingredient and may be used in combination with other known treatments for the treatment of these diseases.

[0123] The term "containing as an active ingredient" as used in the present invention means that exosomes isolated from mesenchymal stem cells contain an amount sufficient to achieve preventive or therapeutic activity for kidney disease.

[0124] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of renal anemia comprising, as an active ingredient, mesenchymal stem cells of the present invention, a culture medium thereof, or exosomes isolated therefrom.

[0125] In one embodiment, renal anemia may be caused by chronic kidney disease, end-stage renal disease (ESKD), renal failure, polycystic kidney disease (PKD), acute kidney injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-related nephropathy, renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicroparticle retention disease (CRD), sitosterolemia, or glomerular hyperfiltration.

[0126] In one embodiment, the composition of the present invention can increase blood hemoglobin or hematocrit.

[0127] As used in the present invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, spread, and recurrence of the said disease by administering the pharmaceutical composition according to the present invention, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of the said disease by administering the composition of the present invention. A person skilled in the art to which the present invention pertains would be able to determine the precise criteria for diseases to which the composition of the present invention is effective, and to judge the degree of improvement, enhancement, and treatment, by referring to materials provided by organizations such as the Korean Medical Association.

[0128] In this invention, the term "therapeutically effective amount" used in combination with the active ingredient refers to an amount effective for preventing or treating the said disease, and the therapeutically effective amount of the composition of this invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in the human body, the dosage should be determined as an appropriate amount by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments.

[0129] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health status, type of disease, cause of onset, severity, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0130] The pharmaceutical composition of the present invention may include a carrier, a diluent, an excipient, or a combination of two or more of these commonly used in biological preparations. The term "pharmaceuticalally acceptable" as used in the present invention means exhibiting properties that are non-toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition and may be used as a compound, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, or into pills, capsules, granules, or tablets. Furthermore, the composition may be preferably formulated according to each disease or component by appropriate methods in the art.

[0131] In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, and an oral or injectable formulation is more preferred.

[0132] As used in the present invention, the term "administration" means providing a specific substance to an individual or patient by any appropriate method. Depending on the intended method, it may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally. The dosage varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid formulations, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc. The pharmaceutical composition of the present invention may also be administered by any device capable of delivering the active substance to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip infusions, etc. Injectables can be prepared using aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleate), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), non-aqueous solvents, and may include pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0133] As used in the present invention, the term "individual" refers to any animal, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, that has developed or may develop the said disease, and the said diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the individual. The pharmaceutical composition of the present invention may be administered in conjunction with existing therapeutic agents.

[0134] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0135] In one aspect, the present invention relates to a food composition for the prevention or improvement of kidney disease comprising the mesenchymal stem cells of the present invention, or a culture medium thereof, or exosomes isolated therefrom.

[0136] In one embodiment, the food-grade acceptable salt may include a salt derived from a food-grade acceptable organic acid, inorganic acid, or base.

[0137] When the composition of the present invention is used as a food composition, the compound may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The composition may include a food-grade additive that is food-gradely acceptable in addition to the active ingredient, and the amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0138] As used in the present invention, the term "food" refers to a natural product or processed product containing one or more nutrients, preferably one that has undergone a certain degree of processing to become edible, and in a conventional sense includes health functional foods, beverages, food additives, beverage additives, etc.

[0139] The term "food auxiliary additive" as used in the present invention refers to a component that can be added to food as an auxiliary component, and is added to manufacture health functional foods of each formulation, and can be appropriately selected and used by a person skilled in the art. Examples of food auxiliary additives include various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food auxiliary additives of the present invention are not limited by the above examples.

[0140] The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having functional properties useful to the human body. Here, "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the ordinary technical field, and during such manufacturing, raw materials and ingredients commonly added in the ordinary technical field may be added. Furthermore, the formulation of the health functional food may also be manufactured without restriction as long as it is a formulation recognized as a health functional food. The health functional food of the present invention may be consumed as an adjuvant to enhance the effects of a kidney disease treatment.

[0141] In addition, there are no restrictions on the types of health foods to which the composition of the present invention can be used. Furthermore, the composition of the present invention may be prepared by mixing other appropriate auxiliary ingredients and known additives that may be contained in health functional foods, depending on the choice of a person skilled in the art. Examples of foods to which it can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and it may be prepared by adding it to juices, teas, jellies, and juices prepared using the composition of the present invention as a main ingredient.

[0142] As used in the present invention, the term "improvement" refers to any act of reducing parameters related to a target disease, such as the degree of symptoms, by administering a composition according to the present invention.

[0143] In one aspect, the present invention relates to a method for producing exosomes containing or secreting EPO, comprising the steps of: dissolving a gene encoding EPO into pluripotent stem cells; differentiating into mesenchymal stem cells; and isolating exosomes.

[0144] In one aspect, the present invention provides a method for preventing or treating kidney disease, comprising the step of administering mesenchymal stem cells according to the present invention, a culture medium thereof, or exosomes isolated therefrom thereof to an individual.

[0145] In one embodiment, the kidney disease may be one or more selected from the group consisting of chronic kidney disease (CKD), polycystic kidney disease (PKD), acute kidney injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-associated nephropathy, chronic kidney disease (CKD), renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chyloblastic retention disease (CRD), sitosterolemia, glomerular hyperfiltration, and pruritus of renal failure, but is not limited thereto.

[0146] In one aspect, the present invention provides a method for preventing or treating renal anemia, comprising the step of administering to an individual mesenchymal stem cells according to the present invention, a culture medium thereof, or exosomes isolated therefrom thereof.

[0147] In one embodiment, the renal anemia may be caused by chronic kidney disease, end-stage renal disease (ESKD), renal failure, polycystic kidney disease (PKD), acute kidney injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-related nephropathy, renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicroparticle retention disease (CRD), sitosterolemia, or glomerular hyperfiltration, but is not limited thereto.

[0148] The above-mentioned individuals are preferably mammals, including humans, and include all patients requiring treatment for kidney disease or renal anemia, including patients currently undergoing treatment, patients who have received treatment, and patients who require treatment; patients who have undergone surgical procedures for the treatment of kidney disease or renal anemia may also be included.

[0149]

[0150] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0151]

[0152] Example 1. Preparation of EPO-Effective Stem Cells

[0153] 1-1. Preparation of EPO-Dissolving Plasmid

[0154] To edit the gene by knocking in the gene encoding erythropoietin (EPO) (SEQ No. 1) into human induced pluripotent stem cells, an EPO sgRNA plasmid was constructed using gRNA (SEQ No. 2) and the Plasmid MidiPrep kit (Fig. 1), and its identity and integrity were confirmed by gel electrophoresis.

[0155]

[0156] 1-2. Preparation of EPO-Melted iPSCs

[0157] Human induced pluripotent stem cells (hiPSC) WTC11 were cultured in mTeSR1 medium on 0.1% Geltrex and isolated as single cells using Accutase. To knock-in the EPO gene into the AAV1 break-at site 2, known as the safe harbor site of the four cultured human pluripotent stem cell types (four types within the same WTC11), hiPSC 1 × 10 6 The EPO sgRNA plasmid prepared in Example 1-1 was electroporated into dogs using a NEPA21 instrument under conditions of ~50% transfection efficiency and >70% cell viability (Fig. 2). After 48 hours of electroporation, genetically edited hiPSCs were selected using puromycin (0.5–1 μg / mL) and cultured in a culture medium containing puromycin for 7 to 10 days. PCR analysis was performed to confirm whether the knock-in sequence was accurately inserted into the AAVS1 gene locus of the thus prepared EPO-iPSCs, and the results showed that the EPO gene was accurately inserted into four types of human induced pluripotent stem cells (Fig. 3).

[0158]

[0159] 1-3. Preparation of EPO-Melted Mesenchymal Stem Cells

[0160] 1 × 10⁶ EPO-lyzed iPSCs (EPO-iPSCs) of Examples 1-2 prepared using CRISPR / Cas9 technology were placed in low attachment 96-wells. 4 cells / cm 2 Embryoid bodies (EBs) were formed by segmentation at a density and suspension culture in EB medium (DMEM / F12, 1% Glutamax, 1% Non-Essential Amino Acid, 4 ng / ml bFGF, and 20 μM Rock inhibitor) supplemented with 20% knockout serum replacement. After 1 week of culture, the embryos were transferred to a T25 flask coated with 0.1% gelatin and cultured for 1 week in MSC (mesenchymal stem cell) growth medium (α-MEM, 10% FBS, 2 mM L-Glutamine, 10 mM Non-Essential Amino Acid, and 8 ng / ml FGF2). Subsequently, cells were detached and subcultured at a ratio of 1:3 in MSC induction medium (α-MEM, 10% FBS, 2 mM L-Glutamine, 100 μM L-ascorbic acid, 1 mM sodium Pyruvate, and 10 mM Non-Essential Amino Acid) in 100 mm cell culture dishes coated with 0.1% gelatin. Subsequently, subculture was continued until a uniform fibroblastic morphology appeared. The characteristics of the EPO-mocked mesenchymal stem cells (EPO-MSCs) prepared in this way were confirmed through FACS analysis (Positive maker: CD105, CD90, CD73; and Negative maker: CD45, CD34) and microscopic observation.

[0161] As a result, it was analyzed that more than 90% of the EPO-knock-in mesenchymal stem cells (MSCs) expressed three positive markers and less than 5% expressed negative markers (Fig. 4), confirming that they exhibit the morphological characteristics of mesenchymal stem cells (Fig. 5).

[0162]

[0163] Example 2. Verification of EPO expression

[0164] The gene expression levels of the EPO-iPSC and EPO-iMSC prepared in Example 1 above were confirmed by RT-PCR analysis, and the EPO secretion levels in the EPO-iPSC and EPO-iMSC differentiation media were analyzed by ELISA.

[0165] RT-PCR analysis confirmed that EPO gene expression was increased in EPO-iPSC and EPO-iMSC compared to the control WTC11 iPSC (CTR) that did not knock out EPO (Fig. 6), and that the secretion of EPO in EPO-iPSC and EPO-iMSC was increased by approximately 200 times compared to the control group (Fig. 7). In particular, it was confirmed that EPO gene expression was significantly increased in EPO-iMSC compared to EPO-iPSC, and the secretion of EPO was increased by approximately 1.5 times.

[0166]

[0167] Example 3. Preparation of EPO-Melted Mesenchymal Stem Cell-Derived Exosomes

[0168] 3-1. Exosome Separation

[0169] The culture supernatant of the EPO-iMSC prepared in Example 1 above was collected and centrifuged sequentially at 300 g for 10 minutes and at 2000 g for 10 minutes. The supernatant thus obtained was further centrifuged at 10,000 g for 30 minutes to obtain a pelletized large EV fraction. The supernatant was further ultracentrifuged at 100,000 g for 70 minutes at 4°C to separate the small EV fraction. All EV fractions were washed with PBS before use and centrifuged or ultracentrifuged one or more times.

[0170]

[0171] 3-2. Analysis of Exosome Characteristics

[0172] Exosomes isolated from EPO-iMSC culture medium were imaged by TEM (Fig. 8), and their size, distribution, and particle count were analyzed by Nanoparticle Tracking Analysis (NTA). In addition, the expression of extracellular vesicle markers CD81, Alix, and TSG101 in the isolated exosomes was confirmed by Western blot analysis.

[0173] As a result of nanoparticle tracking analysis, the size of the exosomes was found to be 30 to 200 nm, and the particle number was 1.0 5 × 10 10 It was confirmed to be particles / ml (Fig. 9). In addition, Western blot analysis confirmed that the marker was expressed only in exosomes isolated from EPO-iMSC, not in EPO-iMSC (Fig. 10).

[0174]

[0175] 3-3. Analysis of EPO Expression in Exosomes

[0176] The amount of EPO secreted by exosomes isolated from EPO-iMSC culture medium and exosomes isolated from EPO-iPSC was confirmed by ELISA analysis.

[0177] As a result, it was found that the amount of EPO secreted by EPO-iMSC exosomes increased by about 3.5 times compared to EPO-iPSC exosomes (Fig. 11).

[0178]

[0179] 3-4. Analysis of microRNA expression in exosomes

[0180] The top 50 microRNAs in exosomes isolated from EPO-iMSC culture medium were analyzed, and the top 23 microRNAs among them were calculated as a percentage. As a result, hsa-miR-148a-3p was found to be the most expressed (Figs. 12 and 13).

[0181]

[0182] Example 4. Analysis of the therapeutic effect of EPO-knock-in mesenchymal stem cell-derived exosomes on chronic kidney disease

[0183] 4-1. Preparation of Adenine Diet Animal Model and Exosome Administration

[0184] Six-week-old male C57BL / 6J mice were acclimatized for one week and then divided into four groups: a control group with a normal diet and vehicle administration; a control group with a diet containing 0.2 to 0.3% adenine and vehicle administration; a group with a diet containing 0.2 to 0.3% adenine and iMSC-EPO exosomes; and a group with a diet containing 0.2 to 0.3% adenine and human recombinant erythropoietin (rhEPO). An adenine diet-induced chronic kidney disease (CKD) animal model was prepared by providing the adenine diet groups with a 0.3% adenine diet for 10 days, followed by a maintenance diet supplemented with a 0.2% adenine diet for 4 weeks (Fig. 14), and then resting for 12 weeks. Analysis of body weight, blood BUN, creatinine, hemoglobin, and hematocrit levels in an animal model of chronic renal failure anemia with an adenine diet revealed that the adenine diet induced renal anemia caused by elevated blood BUN and creatinine levels and decreased hemoglobin and hematocrit levels (Fig. 15). In the drug administration group, after a 12-week rest period, 100 μg of iMSC-EPO exosomes and 28 ng of rhEPO were administered via tail vein every 3 days for 4 weeks, and the therapeutic effect was observed for 3 weeks thereafter (Fig. 16).

[0185]

[0186] 4-2. Effect of increasing EPO expression

[0187] EPO expression in the kidneys of mice in the above-mentioned CKD animal model, administered iMSC-EPO exosomes and rhEPO, was analyzed by RT-PCR and fluorescence staining.

[0188] RT-PCR analysis showed that EPO expression was increased in the kidneys of the group administered iMSC-EPO exosomes compared to the group administered rhEPO (Fig. 17), and fluorescence staining analysis also showed that EPO expression was increased in the kidneys of the group administered iMSC-EPO exosomes compared to the group administered rhEPO (Fig. 18), confirming that iMSC-EPO exosomes produce EPO in the kidneys.

[0189]

[0190] 4-3. Analysis of Treatment Efficacy for Renal Anemia

[0191] To confirm the effect of iMSC-EPO exosomes on renal anemia, blood hemoglobin and hematocrit levels were analyzed in the iMSC-EPO exosome administration group of Example 4-1, in which iMSC-EPO exosomes were administered to a CKD-induced animal model for 4 weeks (weeks 17–21). As a result, it was found that blood hemoglobin and hematocrit levels were significantly increased by the administration of MSC-EPO exosomes (Fig. 19), confirming that the MSC-EPO exosomes of the present invention have a therapeutic effect on renal anemia induced by chronic kidney disease.

[0192]

[0193] 4-4. Analysis of the Effect on Kidney Function Improvement

[0194] To confirm the effect of iMSC-EPO exosomes on kidney function, creatinine and urea nitrogen levels were analyzed in the iMSC-EPO exosome administration group of Example 4-1, in which iMSC-EPO exosomes were administered to an animal model of CKD for 4 weeks (weeks 17–21).

[0195] As a result, creatinine and urea nitrogen levels increased by CKD induction were found to be significantly reduced by the administration of iMSC-EPO exosomes (Fig. 20).

[0196]

[0197] 4-5. Analysis of the Effect on Improving Renal Fibrosis

[0198] To confirm the effect of iMSC-EPO exosomes on improving renal fibrosis caused by CKD, renal fibrosis in the iMSC-EPO exosome administration group of Example 4-1, in which iMSC-EPO exosomes were administered to an animal model of CKD-induced renal disease for 4 weeks (weeks 17-21), was confirmed by analyzing changes in the expression of related markers via RT-PCR analysis and fibrosis scores via histological staining.

[0199] RT-PCR analysis showed that the expression of α-SMA, which was increased by CKD induction, was significantly reduced by iMSC-EPO exosome administration (Fig. 21), and the fibrosis score confirmed by Masson's trichrome staining was also significantly reduced by iMSC-EPO exosome administration (Fig. 22).

[0200]

[0201] 4-6. Analysis of Renal Protective Effects

[0202] To confirm the renal protective effect of iMSC-EPO exosomes, the kidney tissue of the iMSC-EPO exosome administration group of Example 4-1, in which iMSC-EPO exosomes were administered to an animal model of CKD-induced disease for 4 weeks (weeks 17-21), was stained with TUNEL to analyze the degree of apoptosis, and the degree of peritubular capillary rarefaction was confirmed by fluorescence staining analysis of the expression of CD31, a vascular cell marker, and LTL, a proximal tubule marker.

[0203] TUNEL staining results confirmed that the increased renal cell death in the CKD-induced group was significantly reduced by the administration of iMSC-EPO exosomes (Fig. 23). In addition, peritubular capillary thinning was alleviated in the iMSC-EPO exosome administration group compared to the CKD group (Fig. 24).

Claims

1. A mesenchymal stem cell (MSC) derived from a pluripotent stem cell in which a gene encoding EPO (erythropoietin, EPO) is integrated.

2. In claim 1, the pluripotent stem cells are mesenchymal stem cells that are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

3. In claim 1, the mesenchymal stem cell is a mesenchymal stem cell in which a gene encoding EPO is knocked in at the break at site 2 position of the AAV1 gene and the gene encoding EPO is integrated into the gene.

4. In claim 1, the gene in which the gene encoding EPO is integrated into the AAV1 gene comprises a nucleotide sequence represented by SEQ ID NO. 3, a mesenchymal stem cell.

5. In claim 1, the mesenchymal stem cells are mesenchymal stem cells in which the expression or secretion of EPO is increased compared to pluripotent stem cells in which a gene encoding EPO is integrated within the gene.

6. In claim 1, the mesenchymal stem cells contain 1 × 10 exosomes 9 Up to 1 × 10 11 Mesenchymal stem cells produced at a particle / ml particle count.

7. Exosomes isolated from the mesenchymal stem cells of claim 1.

8. In claim 7, the exosome is an exosome having an average size of 10 to 300 nm.

9. In claim 7, the exosome is an exosome containing or secreting EPO.

10. In claim 7, the exosome expresses one or more microRNAs selected from the group consisting of hsa-miR-148a-3p, hsa-miR-143-3p, and hsa-miR-99a-5p.

11. In claim 7, the exosome is an exosome showing expression levels of 40 to 50% of hsa-miR-148a-3p, 5 to 6% of hsa-miR-143-3p, and 4 to 5% of hsa-miR-99a-5p at 100% of total microRNA expression.

12. The method of item 7, wherein the exosome is hsa-miR-26a-5p, hsa-miR-151a-3p, hsa-let-7i-5p, hsa-miR-10b-5p, hsa-miR-21-5p, hsa-miR-199a-3p, hsa-miR-199b-3p, hsa-let-7f-5p, hsa-let-7g-5p, hsa-miR-34c-5p, hsa-miR-100-5p, hsa-let-7a-5p, hsa-miR-27b-3p, hsa-let-7b-5p, hsa-miR-215-5p, hsa-let-7c-5p, hsa-miR-203a-3p, hsa-miR-7-5p, Exosomes additionally expressing one or more microRNAs selected from the group consisting of hsa-miR-126-3p and hsa-miR-192-5p.

13. A pharmaceutical composition for the prevention or treatment of kidney disease comprising the mesenchymal stem cells of claim 1, a culture medium thereof, or exosomes isolated therefrom thereof as an active ingredient.

14. A pharmaceutical composition for the prevention or treatment of kidney disease according to claim 13, wherein the kidney disease is one or more selected from the group consisting of chronic kidney disease (CKD), polycystic kidney disease (PKD), acute renal injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-associated nephropathy, chronic kidney disease (CKD), renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicroparticle retention disease (CRD), sitosterolemia, glomerular hyperfiltration, and pruritus of renal failure.

15. In claim 13, the above composition is a pharmaceutical composition for the prevention or treatment of kidney disease that reduces creatinine or urea nitrogen.

16. In claim 13, the composition is a pharmaceutical composition for the prevention or treatment of kidney disease that reduces renal fibrosis, renal cell death, or peritubular capillary rarefaction.

17. A pharmaceutical composition for the prevention or treatment of renal anemia comprising the mesenchymal stem cells of claim 1, a culture medium thereof, or exosomes isolated therefrom thereof as an active ingredient.

18. A pharmaceutical composition for the prevention or treatment of renal anemia according to claim 17, wherein the renal anemia is caused by chronic kidney disease, end-stage renal disease (ESKD), renal failure, polycystic kidney disease (PKD), acute renal injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-associated nephropathy, renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chyloblastic retention disease (CRD), sitosterolemia, or glomerular hyperfiltration.

19. In claim 17, the above composition is a pharmaceutical composition for the prevention or treatment of renal anemia that increases blood hemoglobin or hematocrit. 20.1) Step of melting in the gene encoding EPO into pluripotent stem cells; 2) a step of differentiating into mesenchymal stem cells; and 3) A method for producing exosomes containing or secreting EPO, comprising the step of isolating exosomes.

21. A method for preventing or treating kidney disease, comprising the step of administering the mesenchymal stem cells of claim 1, a culture medium thereof, or exosomes isolated therefrom thereof to an individual.

22. A method for preventing or treating kidney disease according to claim 21, wherein the kidney disease is one or more selected from the group consisting of chronic kidney disease (CKD), polycystic kidney disease (PKD), acute renal injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-associated nephropathy, chronic kidney disease (CKD), renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicroparticle retention disease (CRD), sitosterolemia, glomerular hyperfiltration, and pruritus of renal failure.

23. A method for preventing or treating renal anemia, comprising the step of administering the mesenchymal stem cells of claim 1, a culture medium thereof, or exosomes isolated therefrom thereof to an individual.

24. A method for preventing or treating renal anemia according to claim 23, wherein the renal anemia is caused by chronic kidney disease, end-stage renal disease (ESKD), renal failure, polycystic kidney disease (PKD), acute kidney injury, end-stage renal disease (ESKD), renal failure, systemic lupus erythematosus, diabetic nephropathy (DN), IgA nephritis (IgAN), HIV-associated nephropathy, renal sclerosis, focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCD), xanthine oxidase deficiency, abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicroparticle retention disease (CRD), sitosterolemia, or glomerular hyperfiltration.

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