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KR-20260062162-A - COMPOSITION FOR PREVENTING OR TREATING KIDNEY DISEASE

KR20260062162AKR 20260062162 AKR20260062162 AKR 20260062162AKR-20260062162-A

Abstract

The present invention relates to the therapeutic use of induced pluripotent stem cell-derived mesenchymal stem cells with EPO knock-in and exosomes isolated therefrom for kidney disease. According to the present invention, induced pluripotent stem cells with an EPO gene knock-in were prepared using CRISPR-Cas9 gene editing technology to maximize EPO expression, and then differentiated to construct mesenchymal stem cells with significantly increased EPO expression and secretion. It was confirmed that exosomes isolated therefrom 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, the exosomes can be utilized for the prevention or treatment of chronic kidney disease or renal anemia caused by it.

Inventors

  • 김용균
  • 김수아
  • 김진원
  • 남선아

Assignees

  • 가톨릭대학교 산학협력단
  • 주식회사알젠오가노바이오테크놀로지

Dates

Publication Date
20260507
Application Date
20241025

Claims (20)

  1. Mesenchymal stem cells (MSCs) derived from pluripotent stem cells in which a gene encoding EPO (erythropoietin, EPO) is integrated within the gene.
  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 a 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 are mesenchymal stem cells that produce exosomes in a particle count of 1 x 10⁹ to 1 x 10¹¹ particles/ml.
  7. Exosomes isolated from 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 claim 7, wherein the exosomes include 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, as an active ingredient, the mesenchymal stem cells of claim 1, a culture medium thereof, or exosomes isolated therefrom.
  14. A pharmaceutical composition for the prevention or treatment of kidney disease, wherein the kidney disease is 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 above 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), chylomicroparticle 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) A step of dissolving 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.

Description

Composition for preventing or treating kidney disease 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. 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. 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. Figure 1 is a schematic diagram showing the EPO knock-in (gene insertion) plasmid of the present invention. Figure 2 is a schematic diagram showing the break at site 2 location of AAV1 in human induced pluripotent stem cells to knock out the EPO gene using the EPO knock-in plasmid of the present invention and the plasmid that goes into it. 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. 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. Figure 5 is a figure confirming the morphological characteristics of human induced pluripotent stem cells dissolved in EPO and mesenchymal stem cells differentiated therefrom. 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: WTC11 iPSC: Control group human induced pluripotent stem cells; EPO iPSC: Human induced pluripotent stem cells lysed with EPO; and EPO iMSC: Mesenchymal stem cells differentiated from human induced pluripot