WO2019119673A1 - 一种双基因修饰的干细胞及其用途 - Google Patents

一种双基因修饰的干细胞及其用途 Download PDF

Info

Publication number
WO2019119673A1
WO2019119673A1 PCT/CN2018/081616 CN2018081616W WO2019119673A1 WO 2019119673 A1 WO2019119673 A1 WO 2019119673A1 CN 2018081616 W CN2018081616 W CN 2018081616W WO 2019119673 A1 WO2019119673 A1 WO 2019119673A1
Authority
WO
WIPO (PCT)
Prior art keywords
mesenchymal stem
culture
modified mesenchymal
stem cell
protein
Prior art date
Application number
PCT/CN2018/081616
Other languages
English (en)
French (fr)
Inventor
段海峰
薛冰华
谢晶
张振利
Original Assignee
北京吉源生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京吉源生物科技有限公司 filed Critical 北京吉源生物科技有限公司
Priority to US16/954,763 priority Critical patent/US11752173B2/en
Publication of WO2019119673A1 publication Critical patent/WO2019119673A1/zh
Priority to US18/357,639 priority patent/US20240100100A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the invention relates to the field of cell therapy.
  • the present invention relates to a modified mesenchymal stem cell and a culture supernatant thereof, and a pharmaceutical composition comprising such a cell or a culture supernatant thereof.
  • the invention also relates to the use of the modified mesenchymal stem cells and their culture supernatants for the treatment of metabolic disorders in a subject (eg, a human), and in the preparation for use in a subject (eg, a human) Use in a medicament for treating a metabolic disorder.
  • the invention further relates to a method of treating a metabolic disorder comprising the step of administering to a subject in need thereof a modified mesenchymal stem cell of the invention or a culture supernatant or pharmaceutical composition thereof.
  • Metabolic disorders such as diabetes are closely related to some endogenous molecules involved in metabolic regulation.
  • endogenous molecules There are three main types of endogenous molecules that have been discovered: one is hormones, including insulin, glucagon, GLP1, glucocorticoids, etc.; the other is cell growth factors with hormone-like functions, including FGF19, FGF21, and FGF23, etc.; three are enzymes involved in metabolic regulation or cell signal transduction, including SPK1, PI3K, HSL, and the like.
  • GLP1 glucagon-like peptide 1
  • the hypoglycemic drugs developed for GLP1 are mainly DPP4 inhibitors and GLP1 analogs which inhibit GLP1 degradation, and GLP1-Fc with GLP1 and human antibody Fc have a long half-life and good tolerance.
  • Fibroblast growth factor (FGF) is an important class of tissue growth factors, including 22 family members.
  • FGF19/21/23 is a member of the FGF family, which has a hormone-like effect and plays an important role in the regulation of glycolipid metabolism.
  • FGF21 is mainly synthesized by the liver, acts on adipose tissue through the endocrine pathway, and regulates the metabolism of glycolipids.
  • FGF21 As a glycolipid metabolism regulating molecule, FGF21 has been developed as a drug for the treatment of metabolic diseases. In clinical application, GLP1-Fc and FGF21 need to face long-term medication, lifelong medication, combined medication, hypoglycemic and lipid-lowering, etc., and these drugs can only play a role in the treatment of the disease, and can not improve the cause of the disease from the root cause.
  • the inventors of the present application have unexpectedly discovered through experiments and repeated exploration that the mesenchymal stem cells modified by the FGF21 and GLP-1 genes have significantly improved biological activities such as lowering blood sugar and blood lipids. Based on this finding, the inventors have developed new modified cells for metabolic disorders as well as methods for treating metabolic disorders based on such cells.
  • the invention provides a modified mesenchymal stem cell expressing: (1) a first protein selected from: FGF21 or a variant thereof, or comprising the FGF21 or variant thereof a first fusion protein; and, (2) a second protein selected from the group consisting of: GLP-1 or a variant thereof, or a second fusion protein comprising the GLP-1 or a variant thereof.
  • the variant of FGF21 has one or more amino acid substitutions, insertions or deletions, or at least 80%, 85%, 90%, 95 compared to the sequence from which it is derived. %, 96%, 97%, 98%, or 99% sequence identity, and retains FGF21 activity.
  • the "FGF21 activity” refers to one or more physiological effects of naturally occurring FGF21, which are well known to those skilled in the art, including, but not limited to, inducing insulin-independent glucose uptake, reducing Plasma glucose, fructosamine, triglyceride, insulin, glucagon levels, reduction of LDL cholesterol and increase of HDL cholesterol, enhancement of insulin sensitivity, etc., can be found in detailed teachings, for example, Dostálová I et al. Physiol Res. 2009; 58 (1 ): 1-7; Kharitonenkov A et al. J Clin Invest. 2005 Jun; 115 (6): 1627-35.
  • the variant of GLP-1 has one or more amino acid substitutions, insertions or deletions, or at least 80%, 85%, 90% compared to the sequence from which it is derived. , 95%, 96%, 97%, 98%, or 99% sequence identity, and retains GLP-1 activity.
  • GLP-1 activity refers to one or more physiological effects of naturally occurring GLP-1, which are known to those skilled in the art, including, but not limited to, glucose secreted by insulin.
  • Dependent stimulation suppression of glucagon secretion, stimulation of insulin (original) biosynthesis, reduction of food intake, deceleration of gastric emptying, etc., for details, see, for example, Nauck MA et al. Exp Clin Endocrinol Diabetes. 1997; 105(4): 187-95.
  • the FGF21 has the amino acid sequence set forth in SEQ ID NO:1.
  • the GLP-1 has the amino acid sequence set forth in SEQ ID NO:4.
  • the modified mesenchymal stem cell expresses: (1) a first protein selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 2, or comprising SEQ ID NO: a first fusion protein of the indicated amino acid sequence; and, (2) a second protein selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 5 or the second fusion comprising the amino acid sequence set forth in SEQ ID NO: protein.
  • the modified mesenchymal stem cells are capable of secreting the first protein and the second protein.
  • the first fusion protein further comprises a first additional polypeptide.
  • the first additional polypeptide is capable of extending the half-life of the first fusion protein in vivo.
  • the first additional polypeptide is selected from the group consisting of an immunoglobulin Fc domain (eg, a human immunoglobulin Fc domain, eg, an Fc domain of a human IgG), serum albumin (eg, human Serum albumin (HSA)), an albumin binding polypeptide (eg, an HSA binding polypeptide), transferrin, and a functional fragment of any of the foregoing.
  • an immunoglobulin Fc domain eg, a human immunoglobulin Fc domain, eg, an Fc domain of a human IgG
  • serum albumin eg, human Serum albumin (HSA)
  • HSA binding polypeptide eg, an HSA binding polypeptide
  • transferrin eg, transferrin, and a functional fragment of any of the foregoing.
  • the first additional polypeptide is a human immunoglobulin Fc domain, such as the Fc domain of a human IgG, such as the Fc domain of human IgGl, IgG2, IgG3 or IgG4.
  • the first additional polypeptide has the amino acid sequence set forth in SEQ ID NO:7.
  • the first additional polypeptide is optionally fused to the FGF21 or variant thereof via a linker.
  • the first additional polypeptide is fused, optionally via a linker, to the N-terminus or C-terminus of the FGF21 or variant thereof.
  • the linker is a peptide linker rich in a combination of Gly and Ser, such as a sequence consisting of a repeating GGGGS amino acid sequence.
  • the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 9).
  • the second fusion protein further comprises a second additional polypeptide.
  • the second additional polypeptide is capable of extending the half-life of the second fusion protein in vivo.
  • the second additional polypeptide is selected from the group consisting of an immunoglobulin Fc domain (eg, a human immunoglobulin Fc domain, such as the Fc domain of a human IgG), serum albumin (eg, human Serum albumin (HSA)), an albumin binding polypeptide (eg, an HSA binding polypeptide), transferrin, and a functional fragment of any of the foregoing.
  • an immunoglobulin Fc domain eg, a human immunoglobulin Fc domain, such as the Fc domain of a human IgG
  • serum albumin eg, human Serum albumin (HSA)
  • HSA binding polypeptide eg, an HSA binding polypeptide
  • transferrin eg, transferrin, and a functional fragment of any of the foregoing.
  • the second additional polypeptide is a human immunoglobulin Fc domain, such as the Fc domain of a human IgG, such as the Fc domain of human IgGl, IgG2, IgG3 or IgG4.
  • the second additional polypeptide has the amino acid sequence set forth in SEQ ID NO:7.
  • the second additional polypeptide is optionally fused to the GLP-1 or variant thereof via a linker.
  • the second additional polypeptide is optionally fused to the N-terminus or C-terminus of the GLP-1 or variant thereof via a linker.
  • the linker is a peptide linker rich in a combination of Gly and Ser, such as a sequence consisting of a repeating GGGGS amino acid sequence.
  • the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 9).
  • the modified mesenchymal stem cell expresses: (1) a first protein selected from the group consisting of: the amino acid sequence set forth in SEQ ID NO: 2, or comprising SEQ ID NO: a first fusion protein of the indicated amino acid sequence; and, (2) a second protein selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 5 or the second fusion comprising the amino acid sequence set forth in SEQ ID NO: protein.
  • the modified mesenchymal stem cell expresses: an amino acid sequence set forth in SEQ ID NO: 2; and an amino acid sequence set forth in SEQ ID NO: 10.
  • the modified mesenchymal stem cells comprise:
  • a first exogenous nucleic acid comprising a nucleotide sequence encoding the first protein
  • a second exogenous nucleic acid comprising a nucleotide sequence encoding the second protein.
  • the first exogenous nucleic acid and/or the second exogenous nucleic acid are operably linked to a promoter (eg, a constitutive promoter, a tissue-specific promoter, or an inducible promoter) .
  • a promoter eg, a constitutive promoter, a tissue-specific promoter, or an inducible promoter
  • the first exogenous nucleic acid and/or the second exogenous nucleic acid are linked to a nucleotide sequence encoding a signal peptide.
  • the 5' end of the second exogenous nucleic acid is ligated to a nucleotide sequence encoding a signal peptide (e.g., the signal peptide set forth in SEQ ID NO: 22).
  • the first exogenous nucleic acid and the second exogenous nucleic acid can be joined by a sequence encoding a self-cleaving peptide.
  • the sequence encoding a self-cleaving peptide is linked to the 3' end of the first exogenous nucleic acid and to the 5' end of the second exogenous nucleic acid.
  • the first exogenous nucleic acid and/or the second exogenous nucleic acid are integrated into the genome of the mesenchymal stem cell.
  • the first exogenous nucleic acid and/or the second exogenous nucleic acid are independent of the genome of the mesenchymal stem cell.
  • the modified mesenchymal stem cells comprise an expression vector, and the first exogenous nucleic acid and the second exogenous nucleic acid are contained in the same or different expression vectors.
  • the expression vector comprises the first exogenous nucleic acid and the second exogenous nucleic acid.
  • the first exogenous nucleic acid and the second exogenous nucleic acid can be joined by a sequence encoding a self-cleaving peptide.
  • the sequence encoding a self-cleaving peptide is linked to the 3' end of the first exogenous nucleic acid and to the 5' end of the second exogenous nucleic acid.
  • suitable self-cleaving peptides are known to those skilled in the art, examples of which include, but are not limited to, 2A peptides derived from the genus Anopheles or the genus Cardiovirus, such as from foot-and-mouth disease virus (FMDV), equine rhinitis 2A peptide of A virus (ERAV), Thoseaasigna virus (TaV) or Porcine virus (PTV-1).
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis 2A peptide of A virus
  • TaV Thoseaasigna virus
  • PTV-1 Porcine virus
  • the first exogenous nucleic acid is linked to a second exogenous nucleic acid by a sequence encoding a 2A peptide.
  • the sequence encoding the 2A peptide is linked to the 3' end of the first exogenous nucleic acid and to the 5' end of the second exogenous nucleic acid.
  • the 2A peptide is a 2A peptide derived from the Thoseaasigna virus (TaV).
  • the 2A peptide has the amino acid sequence set forth in SEQ ID NO: 12.
  • the sequence encoding the 2A peptide has the nucleotide sequence set forth in SEQ ID NO: 13.
  • mesenchymal stem cells can be genetically modified, such as physical, chemical or biological methods, or a combination thereof, by methods and techniques well known in the art.
  • the biological methods include the use of viral vectors such as lentiviruses, retroviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.
  • the chemical means include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, etc.; lipid based systems, including oil-in-water emulsions, micelles, mixed micelles or liposomes, and the like.
  • the physical methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • the modified mesenchymal stem cells of the invention are obtained by introducing the first exogenous nucleic acid and the second exogenous nucleic acid into mesenchymal stem cells.
  • the modified mesenchymal stem cells are obtained by the following steps:
  • the expression vector in step (2), is stably transfected into the mesenchymal stem cells.
  • the expression vector in step (1), is a viral vector, such as a lentiviral vector.
  • the expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 15.
  • the mesenchymal stem cells are derived from adipose tissue, umbilical cord, bone marrow, or cord blood. In certain exemplary embodiments, the mesenchymal stem cells are derived from adipose tissue.
  • the modified mesenchymal stem cells of the present invention can be formulated and administered as a pharmaceutical composition.
  • a pharmaceutical composition may be in any form known in the medical field, preferably an injection (including an injection, a lyophilized powder).
  • the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution eg, a balanced salt solution or physiological saline
  • dispersion suspension or emulsion.
  • the modified mesenchymal stem cells of the invention are used to treat a metabolic disorder in a subject, or to prepare a medicament for treating a metabolic disorder in a subject.
  • the metabolic disorder is selected from the group consisting of obesity, type I and type II diabetes, dyslipidemia (eg, hyperlipidemia), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders, and secondary complications of these diseases (eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing).
  • dyslipidemia eg, hyperlipidemia
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • insulin resistance eg, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders
  • secondary complications of these diseases eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing.
  • the subject is a mammal, such as a human.
  • the invention provides a culture comprising the modified mesenchymal stem cells of the invention, and a culture medium.
  • a medium which can be used for stem cell culture is known to those skilled in the art, and non-limiting examples thereof include ⁇ -MEM medium, DMEM medium, IMDM medium, Ham's F12 medium, and RPMI 1640 medium. And a mixed medium formed by any combination of the above (for example, IMDM/HamF12 medium in which IMDM and HamF12 are mixed in equal amounts).
  • the above medium optionally further comprises supplemental substances such as serum (for example, fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (for example, Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA). , antibiotics, vitamins, minerals.
  • the medium is alpha-MEM medium with or without serum.
  • the culture of the present invention can be formulated and administered as a pharmaceutical composition.
  • a pharmaceutical composition may be in any form known in the medical field, preferably an injection (including an injection, a lyophilized powder).
  • the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution eg, a balanced salt solution or physiological saline
  • dispersion suspension or emulsion.
  • a culture as described herein is used to treat a metabolic disorder in a subject, or to prepare a medicament for treating a metabolic disorder in a subject.
  • the metabolic disorder is selected from the group consisting of obesity, type I and type II diabetes, dyslipidemia (eg, hyperlipidemia), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders, and secondary complications of these diseases (eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing).
  • dyslipidemia eg, hyperlipidemia
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • insulin resistance eg, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders
  • secondary complications of these diseases eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing.
  • the subject is a mammal, such as a human.
  • the invention provides a culture supernatant which is the supernatant of the culture of the invention.
  • the culture supernatant is free of the modified mesenchymal stem cells.
  • the culture supernatant is serum free.
  • the culture supernatant comprises a basal medium, or a broth in which one or more supplemental substances (eg, serum) are added to the basal medium.
  • the base medium optionally further comprises one or more supplemental substances (eg, serum).
  • the culture supernatant comprises alpha-MEM medium with or without serum.
  • the culture supernatant of the present invention can be formulated and administered as a pharmaceutical composition.
  • Such pharmaceutical compositions may be in any form known in the medical arts, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections. (including injection, lyophilized powder) and other forms.
  • the culture supernatant as described herein is used to treat a metabolic disorder in a subject, or to prepare a medicament for treating a metabolic disorder in a subject.
  • the metabolic disorder is selected from the group consisting of obesity, type I and type II diabetes, dyslipidemia (eg, hyperlipidemia), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders, and secondary complications of these diseases (eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing).
  • dyslipidemia eg, hyperlipidemia
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • insulin resistance eg, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders
  • secondary complications of these diseases eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing.
  • the subject is a mammal, such as a human.
  • the present invention is also directed to a method of making a culture supernatant as described herein, comprising the steps of:
  • the method further comprises: (3) treating the supernatant obtained in step (2), the treatment being selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, Freeze-dried, diluted, desalted, preserved, and any combination thereof.
  • the modified mesenchymal stem cells of the present invention can be cultured using any medium known in the art for use in stem cell culture and culture conditions.
  • a basal medium can be used in step (1), which optionally comprises one or more supplemental substances (eg, serum).
  • the basal medium is selected from the group consisting of alpha-MEM medium, DMEM medium, IMDM medium, Ham's F12 medium, RPMI 1640 medium, and a mixed medium formed by any combination of the above.
  • the supplement may be selected from the group consisting of serum (eg, fetal bovine serum, human serum, sheep serum, etc.), serum replacement (eg, Knockout serum replacement (KSR), etc.), bovine serum white Protein (BSA), antibiotics, vitamins, minerals.
  • the modified mesenchymal stem cells are cultured in step (1) using alpha-MEM medium with or without serum.
  • the culture supernatant of the present invention is serum free for improved safety.
  • the modified mesenchymal stem cells can be cultured using serum-free medium (eg, basal medium or serum-free medium).
  • serum-free medium eg, basal medium or serum-free medium.
  • the serum-free medium can be cultured throughout the culture process, or in the final or last few subcultures.
  • the culture supernatant obtained in the step (2) may be subjected to dialysis or solvent replacement to remove serum, whereby a serum-free culture supernatant may also be obtained.
  • the invention provides a pharmaceutical composition comprising a modified mesenchymal stem cell, culture or culture supernatant as described herein.
  • the pharmaceutical composition comprises a therapeutically effective amount of the modified mesenchymal stem cells or culture.
  • the pharmaceutical composition comprises one or more of the modified mesenchymal stem cells as described herein.
  • the pharmaceutical composition can be in any form known in the medical arts.
  • the pharmaceutical composition may be a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, a granule, an elixir, a lozenge, a suppository, an injection (including an injection, lyophilization) Powder) and other forms.
  • the pharmaceutical composition is an injection (including injection, lyophilized powder).
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • the pharmaceutical composition can be delivered in the form of a suspension, gel, gel, serum or mixture.
  • the pharmaceutical composition comprises a therapeutically effective amount of a culture supernatant as described herein.
  • the pharmaceutical composition may be in any form known in the medical arts, for example, the pharmaceutical composition may be a tablet, a pill, a suspension, an emulsion, a solution, a gel, Capsules, powders, granules, tinctures, lozenges, suppositories, injections (including injections, lyophilized powders), and the like.
  • the pharmaceutical composition is an injection (including an injection, a lyophilized powder).
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • the pharmaceutical compositions of the present invention optionally further comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is selected from the group consisting of an anti-diabetic agent, an anti-obesity agent, an anti-hypertensive drug, an anti-atherosclerotic drug, and a lipid-lowering drug.
  • non-limiting examples of suitable antidiabetic agents include thiazolidinediones (such as rosiglitazone or pioglitazone), biguanides (such as metformin or phenformin), and sulfonylureas (such as Urea, glibenclamide, gliclazide, chlorpropamide or glipizide), glucosidase inhibitors (such as acarbose or miglitol), PPAR-alpha agonists, PPAR- Gamma agonist, PPAR- ⁇ / ⁇ dual agonist (such as motaghazole), aP2 inhibitor, DPP4 inhibitor (such as, for example, sitagliptin or vildagliptin), insulin sensitizer, insulin or phorhin Benzoic acids (such as repaglinide) and the like.
  • thiazolidinediones such as rosiglitazone or pioglitazone
  • biguanides such as
  • Non-limiting examples of suitable anti-obesity drugs include ⁇ 3 adrenergic agonists (such as AJ9677 (Taked a/Dainippon), L750355 (Merck) or CP331648 (Pfizer)), lipase inhibitors (such as orlistat) a serotonin (and dopamine) reuptake inhibitor (such as sibutramine or topiramate), a thyroid receptor beta compound (such as the compounds disclosed in WO 99/00353 and WO 00/039077), a CB-1 antagonist (such as rimonabant) or anorexia drugs (such as dextroamphetamine).
  • ⁇ 3 adrenergic agonists such as AJ9677 (Taked a/Dainippon), L750355 (Merck) or CP331648 (Pfizer)
  • lipase inhibitors such as orlistat
  • Non-limiting examples of suitable lipid lowering drugs include those selected from the group consisting of MTP inhibitors, cholesterol ester transfer protein inhibitors (such as CP-529414 (Pfizer)), and HMG CoA reductase inhibitors ( For example, pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin, cerivastatin or atorvastatin, squalene synthetase inhibitors (such as a- disclosed in US Pat. No.
  • Phosphono-sulfonate Phosphono-sulfonate
  • phenylacetic acid derivatives such as fenofibrate, gemfibrozil, clofibrate, bezafibrate, ciprofibrate, clebide, etc.
  • LDL receptor activity upregulator eg, MD-700 (Taisho Pharmaceutical Co.
  • LY295427 (Eli Lilly)
  • lipoxygenase inhibitors such as the benzimidazole derivatives disclosed in WO 97/12615, 15-LO disclosed in WO 97/12613
  • Inhibitors isothiazolones disclosed in WO 96/38144
  • ACAT inhibitors such as avasamab
  • cholesterol absorption inhibitors ileal Na ⁇ +>/bile acid cotransporter inhibitors.
  • Non-limiting examples of suitable antihypertensive drugs include beta adrenergic blockers, calcium channel blockers (such as diltiazem, verapamil, nifedipine, amlodipine), diuretics (eg, Chlorothiazine, hydrochlorothiazide, fluoromethazine, hydrofluorothiazide, benzfluorothiazide, methylchlorothiazide, trichlorothiazide, polibizine, benzylthiazine, tennic acid, chlorthalidone, fur Sami, bumetanide, amiloride or spironolactone), renin inhibitors, ACE inhibitors (eg captopril, zofenopril, fosinopril, enalapril, cilazapril) , delaipril, pentopril, quinapril, ramipril or lisinopril),
  • the pharmaceutical composition is for treating a metabolic disorder in a subject.
  • the subject is a mammal, such as a human.
  • the metabolic disorder is selected from the group consisting of obesity, type I and type II diabetes, dyslipidemia (eg, hyperlipidemia), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders, and secondary complications of these diseases (eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing).
  • dyslipidemia eg, hyperlipidemia
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • insulin resistance eg, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders
  • secondary complications of these diseases eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing.
  • the invention relates to the use of a modified mesenchymal stem cell, culture or culture supernatant as described herein for the treatment of a metabolic disorder in a subject, or for the preparation of a treatment in a subject
  • a modified mesenchymal stem cell, culture or culture supernatant as described herein for the treatment of a metabolic disorder in a subject, or for the preparation of a treatment in a subject
  • drugs for metabolic disorders are used.
  • the medicament comprises a therapeutically effective amount of the modified mesenchymal stem cells or culture.
  • the modified mesenchymal stem cells of the invention can be used in combination.
  • the medicament may comprise one or more of the modified mesenchymal stem cells of the invention.
  • the drug can be in any form known in the medical arts.
  • the drug may be a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, a granule, an elixir, a lozenge, a suppository, an injection (including an injection, a lyophilized powder) Etc.
  • the medicament is an injection (including an injection, a lyophilized powder).
  • the medicament further comprises a pharmaceutically acceptable carrier or excipient.
  • the medicament comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • the drug can be delivered in the form of a suspension, gel, gel, serum or mixture.
  • the medicament comprises a therapeutically effective amount of a culture supernatant as described herein.
  • the medicament may be in any form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, mash Agents, lozenges, suppositories, injections (including injections, lyophilized powders), and the like.
  • the medicament is an injection (including an injection, a lyophilized powder).
  • the medicament further comprises a pharmaceutically acceptable carrier or excipient.
  • the medicament comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • the medicament optionally further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is selected from the group consisting of an anti-diabetic agent, an anti-obesity agent, an anti-hypertensive drug, an anti-atherosclerotic drug, and a lipid-lowering drug.
  • the metabolic disorder is selected from the group consisting of obesity, type I and type II diabetes, dyslipidemia (eg, hyperlipidemia), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders, and secondary complications of these diseases (eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing).
  • dyslipidemia eg, hyperlipidemia
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • insulin resistance eg, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders
  • secondary complications of these diseases eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing.
  • the subject is a mammal, such as a human.
  • the invention provides a method of treating a metabolic disorder comprising administering to a subject in need thereof a modified mesenchymal stem cell, culture, culture supernatant, or the invention of the invention Pharmaceutical composition.
  • the modified mesenchymal stem cells of the invention can be used in combination.
  • one or more of the modified mesenchymal stem cells of the invention can be administered to a subject.
  • the modified mesenchymal stem cells or cultures of the invention can be formulated and administered as a pharmaceutical composition.
  • Such pharmaceutical compositions can comprise a therapeutically effective amount of the modified mesenchymal stem cells or culture.
  • the pharmaceutical composition can be in any form known in the medical arts.
  • the pharmaceutical composition may be a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, a granule, an elixir, a lozenge, a suppository, an injection (including an injection, lyophilization) Powder) and other forms.
  • the pharmaceutical composition is an injection (including an injection, a lyophilized powder).
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution eg, a balanced salt solution or physiological saline
  • dispersion suspension or emulsion.
  • a modified mesenchymal stem cell or culture as described herein, or a pharmaceutical combination comprising the modified mesenchymal stem cell or culture can be administered to a subject in various suitable manners in the present invention. Things.
  • transplantation by local injection eg, stereotactic intracerebral injection or local injection of spinal cord
  • blood circulation eg, intravenous or intraarterial injection
  • cerebrospinal fluid route A modified mesenchymal stem cell or pharmaceutical composition as described herein is administered to a subject by transplantation (eg, lumbar puncture subarachnoid injection). It is known to those skilled in the art how to select a suitable cell transplantation route depending on the location and nature of the lesion.
  • the modified mesenchymal stem cells, cultures or pharmaceutical compositions of the invention may be transplanted in the form of a suspension, gel, gel, serum or mixture.
  • the culture supernatant of the present invention can be formulated and administered as a pharmaceutical composition.
  • a pharmaceutical composition may comprise a therapeutically effective amount of the culture supernatant.
  • the pharmaceutical composition may be in any form known in the medical arts, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, Tinctures, lozenges, suppositories, injections (including injections, lyophilized powders), etc.
  • the pharmaceutical composition is an injection (including an injection, a lyophilized powder).
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension or emulsion.
  • the culture supernatant of the present invention, or a pharmaceutical composition comprising the culture supernatant can be administered to a subject in various suitable manners in the present invention.
  • the culture supernatant of the present invention, or a pharmaceutical composition comprising the culture supernatant can be administered by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, oral administration or the like.
  • the method further comprises administering an additional pharmaceutically active agent selected from the group consisting of an anti-diabetic agent, an anti-obesity agent, an anti-hypertensive drug, an anti-atherosclerotic drug And lipid-lowering drugs.
  • an additional pharmaceutically active agent selected from the group consisting of an anti-diabetic agent, an anti-obesity agent, an anti-hypertensive drug, an anti-atherosclerotic drug And lipid-lowering drugs.
  • additional pharmaceutically active agents can be administered prior to, concurrently with, or subsequent to administration of the modified mesenchymal stem cells as described herein or their culture supernatants or the pharmaceutical compositions.
  • the method further comprises administering an additional therapy.
  • additional therapies can be any therapy known to be used for metabolic disorders, such as medical therapy, surgical treatment, and the like. This additional therapy can be administered prior to, concurrently with, or after administration of the methods described above.
  • the subject is a mammal, such as a human.
  • the metabolic disorder is selected from the group consisting of obesity, type I and type II diabetes, dyslipidemia (eg, hyperlipidemia), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders, and secondary complications of these diseases (eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing).
  • dyslipidemia eg, hyperlipidemia
  • NAFLD nonalcoholic fatty liver disease
  • NASH nonalcoholic steatohepatitis
  • insulin resistance eg, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, atherosclerosis, coronary heart disease, hypertension, and other metabolic disorders
  • secondary complications of these diseases eg, diabetic complications such as retinopathy, neuropathy, kidney disease, and delayed wound healing.
  • exogenous nucleic acid refers to an artificially introduced nucleotide sequence that is foreign to a cell that has not been genetically modified. Exogenous nucleic acids include, but are not limited to, any gene or nucleotide sequence not found in the genome of the cell.
  • FGF21 refers to a member of the fibroblast growth fa ctor (FGF) protein family.
  • the amino acid sequence can be found, for example, in GenBank Accession No. NP_061986.1, and the corresponding nucleotide sequence can be found, for example, in NCBI Reference Serial No. NM_019113.2.
  • variant of FGF21 refers to a protein whose amino acid sequence is derived from the wild type sequence derived therefrom (for example, the wild type FGF21 full-length sequence, for example, as shown in SEQ ID NO: 1 Amino acid sequence) having one or more amino acid substitutions, insertions or deletions, or having at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% Sequence identity and retaining "FGF21 activity”.
  • variants of FGF21 as used herein also include truncated forms of the N-terminus and/or C-terminus of a wild-type protein.
  • FGF21 variants include Chinese patent application CN200980130476.4, CN201180065381.6, CN201280057789.3 or CN201580070276.X, international patent application WO2005/061712, WO2006/028595, WO2006/028714, WO2006/065582 or Those described in WO2008/121563.
  • the "FGF21 activity” refers to one or more physiological effects of naturally occurring FGF21, which are well known to those skilled in the art, including, but not limited to, binding to and activating the FGF21 receptor, for example, inducing insulin-independent glucose.
  • GLP-1 refers to glucagon-like peptide-1. It is known in the art that GLP-1 is obtained by post-translational modification and is derived from a glucagon preproprotein. The wild-type sequence of the proglucagon preproprotein has the NCBI accession number NP_002045 and is found in the international patent applications WO 98/19698 and WO 87/06941 A. GLP-1 is a fragment consisting of amino acid residues 92-128 of proglucagon.
  • GLP-1 (7-37) OH its wild type sequence As shown in SEQ ID NO: 4, and GLP-1(7-36)NH 2 formed after further amidation of the terminal.
  • the expression "variant of GLP-1” refers to a protein whose amino acid sequence is derived from the wild type sequence derived therefrom (for example, the wild type sequence of the active form GLP-1 (7-37) OH,
  • the amino acid sequence shown in SEQ ID NO: 4 has one or more amino acid substitutions, insertions or deletions, or has at least 60%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, or 99% sequence identity, and retains "GLP-1 activity”.
  • variants of GLP-1 as used herein also include truncated forms of the N-terminus and/or C-terminus of a wild-type protein, such as the naturally truncated form of GLP-1 as described above.
  • GLP-1 variants include those described in Chinese Patent Application No. CN200910173888.8, CN201010508567.1.
  • GLP-1 activity refers to one or more physiological effects of naturally occurring GLP-1, which are known to those skilled in the art, including, but not limited to, binding to and activating a GLP-1 receptor, for example Glucose-dependent stimulation of insulin secretion, suppression of glucagon secretion, stimulation of insulin (original) biosynthesis, reduction of food intake, deceleration of gastric emptying, etc., for details, see, for example, Nauck MA et al. Exp Clin Endocrinol Diabetes. 1997; 105(4): 187-95.
  • the expression "prolonging the half-life of a protein in vivo” means preventing or slowing the hydrolysis and enzymatic degradation of the protein in the body, increasing the half-life, and/or improving or altering other pharmacokinetics or organisms. Physical properties including, but not limited to, increased absorption rate, reduced toxicity, improved solubility, reduced protein aggregation, and the like. Substances that can be used to extend the half-life are well known to those skilled in the art, non-limiting examples of which include the Fc domain of immunoglobulins, serum albumin, transferrin, and the like.
  • the term "Fc domain” has the meaning commonly assigned to the term in the field of immunology, especially to antibody fragments that do not contain two antigen binding regions (Fab fragments) from the antibody.
  • the Fc domain consists of two heavy chain constant regions of an antibody that bind by non-covalent interactions and disulfide bonds.
  • the Fc domain may comprise a hinge region and extend to the C-terminus of the antibody via the CH2 and CH3 domains.
  • the Fc domain may also comprise one or more glycosylation sites.
  • the effector function of the Fc domain is typically mediated by interaction with the Fc receptor (Fc[gamma]R) or by binding to Clq and fixed complement.
  • Fc domain includes native Fc or variants thereof.
  • the variant has one or more mutations (eg, amino acid substitutions, insertions or deletions) compared to the wild type sequence from which it is derived, which may affect or participate, or does not affect non-participation: (1) disulfide Bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity in expression of selected host cells, (4) glycosylation, (5) interaction with complement (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
  • mutations eg, amino acid substitutions, insertions or deletions
  • linker refers to a linear polypeptide formed by the joining of multiple amino acid residues by peptide bonds.
  • the linker of the invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region.
  • linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121-1123 ).
  • culture refers to a product obtained after culturing a cell (eg, a modified mesenchymal stem cell of the invention) in a culture medium.
  • the term "culture supernatant” refers to a culture medium containing no cells itself obtained by culturing a cell (for example, a modified mesenchymal stem cell of the present invention).
  • the culture supernatant usable in the present invention can be obtained by separating and removing the cell components after the culture.
  • the culture supernatant may also be subjected to other treatments such as centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze drying, dilution, desalting, storage, and the like.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • promoter has the meaning well-known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of the gene that initiates expression of the downstream gene.
  • a constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in a gene product in the cell under most or all physiological conditions of the cell. The production.
  • An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in substantially only when an inducer corresponding to the promoter is present in the cell The gene product is produced intracellularly.
  • a tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, is substantially only caused when the cell is a cell of the tissue type corresponding to the promoter Gene products are produced in the cells.
  • pharmaceutically acceptable carrier or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, ionic strength enhancers, maintenance of penetration. Pressurized reagents, delayed absorption reagents, diluents, adjuvants, preservatives, and the like.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), and the like.
  • Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvant (e.g., complete Freund's adjuvant), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • the pharmaceutically acceptable carrier or excipient is a sterile isotonic aqueous or non-aqueous solution (eg, a balanced salt solution or physiological saline), dispersion, suspension, or emulsion.
  • treating refers to treating or curing a disease (eg, a metabolic disorder), delaying the onset of symptoms of a disease (eg, a metabolic disorder), and/or delaying the progression of a disease (eg, a metabolic disorder).
  • a disease eg, a metabolic disorder
  • delaying the onset of symptoms of a disease eg, a metabolic disorder
  • delaying the progression of a disease eg, a metabolic disorder
  • the term "effective amount” refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • treating an effective amount of a disease is meant an amount sufficient to cure or at least partially arrest a disease and a complication thereof in a patient already suffering from a disease (eg, a metabolic disorder). Determination of such an effective amount is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously. and many more.
  • the term "subject” includes, but is not limited to, various animals, such as mammals, such as bovine, equine, ovine, porcine, canine, feline, A rabbit, a rodent (eg, a mouse or rat), a non-human primate (eg, a macaque or a cynomolgus monkey) or a human.
  • the subject eg, a human
  • the modified mesenchymal stem cells of the present invention have significant advantages compared to the prior art.
  • the modified mesenchymal stem cells of the present invention exhibit a remarkable synergistic effect, can significantly lower blood sugar, blood lipids, lose weight, and can be safely administered to a subject without eliciting an immunogen Sexual reaction. Therefore, the modified mesenchymal stem cells of the present invention can be used for the treatment of metabolic disorders and have great clinical value.
  • Figure 1 is an electropherogram of PCR amplification products, M represents Marker, and kFG represents a cloned FGF21/GLP1-Fc gene fragment.
  • Figure 2 is a map of the pCDH-FG plasmid.
  • Figure 3 is a flow chart showing the surface markers of MSC and MSC-FG cells.
  • Figure 4 is a graph showing the results of adipogenic and osteogenic differentiation of MSC and MSC-FG cells.
  • Figure 5 shows the results of Western blot analysis of the expression of FGF21 and GLP1-Fc in MSC and MSC-FG cells and cell supernatants.
  • Figure 6 shows the results of the effects of culture supernatants of MSC-FGF21, MSC-GLP1-Fc and MSC-FG on the secretion of Insulin in INS-1 cells after glucose stimulation.
  • Fig. 7 shows the results of examining the effect of the culture supernatant of MSC-FGF21, MSC-GLP1-Fc, and MSC-FG on the expression level of the sterol metabolic gene SREBP1C mRNA.
  • Figure 8 is a graph showing the trend of fasting body weight in diabetic model rats.
  • Liraglutide is a drug-positive control group
  • the arrow represents the time point of administration of drugs and cells
  • * represents a significant difference compared with the Con group
  • ** represents a significant difference compared with the Con group.
  • Figure 9 is a graph of fasting blood glucose in diabetic model rats.
  • Liraglutide is a drug-positive control group. Arrows represent the time points at which drugs and cells are administered. * represents a significant difference compared with Con group. ** represents a significant difference compared with Con group.
  • Figure 10 is a comparison of the body types of diabetic rats in different experimental groups after 28 days of cell therapy, and Liraglutide is a drug-positive control group.
  • Figure 11 shows HE staining of liver and abdominal subcutaneous fat in different experimental groups of diabetic rats after 28 days of cell therapy, and Liraglutide is a drug-positive control group.
  • Figure 12 is a bar graph showing the serum levels of insulin in different experimental groups of diabetic mice after 28 days of cell therapy, and Liraglutide is a drug-positive control group.
  • Figure 13 is a bar graph showing the blood lipid levels in the serum of different experimental groups of diabetic mice after 28 days of cell therapy, and Liraglutide is a drug-positive control group.
  • AD-MSCs Adipose derived mesenchymal stem cells
  • the healthy adult adipose tissue sucked by the liposuction was transferred to a 50 mL centrifuge tube, washed with PBS, and centrifuged at 1500 rpm for 5 minutes to obtain the upper adipose tissue.
  • the adipose tissue was digested in a shaker at 37 ° C for 30 minutes.
  • the digested adipose tissue was immediately added to 10% FBS ⁇ -MEM cell culture medium (purchased from Gibco) at 1500 rpm, and centrifuged for 10 minutes to precipitate cells and tissue pellets.
  • the cells were resuspended in ⁇ -MEM complete medium and undigested tissue was removed through a 100 ⁇ m pore size nylon mesh.
  • the cells were seeded in a culture flask and cultured at 37 ° C in a saturated humidity, 5% CO 2 incubator. After 2 days, the unattached cells were drained, washed gently with PBS, and the stem cell complete medium was added.
  • a DNA molecule encoding FGF21/GLP1-Fc is synthesized by Zhongmeitai Biotechnology (Beijing) Co., Ltd., wherein FGF21 (SEQ ID NO: 2, coding sequence is SEQ ID NO: 3) and GLP1-Fc fusion protein ( SEQ ID NO: 10, the coding sequence is SEQ ID NO: 11) is linked by the T2A sequence (SEQ ID NO: 12, coding sequence is SEQ ID NO: 13), and the GLP1-Fc fusion protein is from the N-terminus to the C-terminus by GLP1 ( SEQ ID NO: 5), a linker (SEQ ID NO: 9) and an IgG4-Fc (SEQ ID NO: 7), and having a signal peptide (SEQ ID NO: 22) linked at its N-terminus.
  • the DNA molecule encoding FGF21/GLP1-Fc is designated pGSI-seq, the nucleotide sequence thereof is shown in SEQ ID NO: 15, and the amino acid sequence of FGF21/GLP1-Fc is shown in SEQ ID NO: 14.
  • the target fragment kFG (1578 bp) was amplified with primer pair kspFGF21BamHIF (5' ⁇ 3':CGCGGATC CGCCACCATGGACTCGGACGAGACC)+IgG4FcSalIR (5' ⁇ 3':ACGCGTCGACTC ATTTACCCGGAGACAG) and condensed by agarose
  • the amplified products were analyzed by gel electrophoresis, and the results are shown in Fig. 1.
  • the positive and negative primers were introduced into the BamHI and SalI cleavage sites, respectively.
  • the PCR product was digested and recovered, and digested with BamHI and SalI.
  • the recombinant lentiviral vector plasmid pCDH-EF1 (purchased from Addgene) was also digested with BamHI and SalI, and the digested product gel was recovered and ligated with T4 DNA ligase. The cells were ligated overnight at 4 ° C, and DH5 ⁇ competent cells were transformed, and 100 ⁇ L of the bacterial solution was applied to an LB plate containing ampicillin resistance, and cultured at 37 ° C overnight. The monoclonal clones were picked for colony PCR, the positive clones were sampled, the clones with the correct sequencing results were saved and the plasmid was extracted and named as pCDH-FG. The schematic diagram of the plasmid is shown in Fig. 2.
  • the 293T cell culture medium was changed to 18 ml DMEM medium, 1 ml of pre-warmed DMEM medium was added to the A-sterilized centrifuge tube, and then the pCDH-FG plasmid, pHelper1 plasmid and pHelper2 prepared in the above 1.2 were added.
  • Plasmid (pCDH-FG: pHelper1: pHelper2 1:1:1, total 54 ⁇ g, pHelper1 and pHelper2 plasmids were helper plasmids for lentiviral packaging, donated by Lab217 Embryo Engineering Laboratory, Northeast Agricultural University), and mixed evenly.
  • the DNA-liposome mixture was transferred to pre-distilled 293T cells, mixed, and cultured at 37 ° C, 5% CO 2 saturated humidity. After 6-8 hours of culture, the medium containing the transfection mixture was discarded, and 20 ml of pre-warmed DMEM medium containing 5% FBS was added to each dish, and cultured at 37 ° C, 5% CO 2 saturated humidity. After changing the solution at 24 h and 48 h, the supernatant was collected and stored at 4 ° C and replaced with 20 ml of fresh medium.
  • the collected liquid was centrifuged at 3 ° C, 3500 rpm for 15 min, the precipitate was discarded, and the supernatant was concentrated with a Millipore protein ultrafiltration column (10 KD) to obtain a lentiviral vector carrying FGF21/GLP1-Fc (Lenti-FGF21/GLP1-Fc).
  • the virus titer was measured, and the virus was diluted to 1 ⁇ 10 8 TU/ml according to the measurement result, and the virus after the packaging was stored at -80 ° C.
  • the pre-frozen P3 generation adipose-derived mesenchymal stem cells were resuspended to a 150 mm culture dish, and cultured in a 20 ml serum-free medium at 37 ° C, 5% CO 2 saturated humidity. After the cells to be resuscitated were overgrown, the cells were digested with 0.05% trypsin, the digestion was stopped with serum-containing medium, the cell suspension was centrifuged at 800 rpm for 5 min, and the cells obtained by centrifugation were resuspended in MSC serum-free medium (purchased from Bioind).
  • Each 150 mm culture dish was inoculated with 2-2.5 ⁇ 10 6 cells, and the cells of the cells taken up the next day after inoculation were discarded, replaced with serum-free ⁇ -MEM medium, 20 ml medium/dish, and 16 ⁇ l of Polybrene ( From Sigma), Lenti-FGF21/GLP1-Fc lentivirus (titer 1 x 10 8 U/ml) obtained in 1.3 was added in a multiplicity of 40 MOIs, and cultured at 37 ° C, 5% CO 2 saturated humidity for 6-8 h. After 6-8 hours, the virus-containing ⁇ -MEM medium was discarded, replaced with a serum-free medium, and culture was continued for 2-3 days at 37 ° C, 5% CO 2 saturated humidity.
  • the FGF21/GLP1-Fc gene-modified mesenchymal stem cells were named MSC-FG.
  • FGF21 single gene-modified mesenchymal stem cells (MSC-FGF21) and GLP1-Fc single gene-modified mesenchymal stem cells (MSC-GLP1-Fc) were prepared according to the method described in the above 1.1-1.4.
  • P6 generation MSC and MSC-FG were selected, digested with 0.05% trypsin, washed twice with PBS and then treated with mouse anti-human CD14-PerCp-Cy5.5, CD19-PE, CD34-PE, CD45-PE Or CD45-FITC, HLA-DR-PE, CD73-PE, CD90-PE and CD105-PE (Becton, Dickinson and Company) antibody-labeled MSC, each sample to be tested about 1 ⁇ 10 6 cells, incubate at room temperature in the dark After 30 min, the PBS was washed twice, fixed with 2% paraformaldehyde, and detected by flow cytometry. The results are shown in Fig. 3.
  • Directed differentiation induction adipogenic and osteogenic induction, P6 generation MSC and MSC-FG, digested with 0.05% trypsin, plated in 12-well plates at a cell density of 2 ⁇ 10 5 /well, replaced on the 2nd day It was an adipogenic induction medium and an osteogenic induction medium (purchased from BI), and then changed every 2 days.
  • the adipogenic induction was induced by oil red O staining after 18 days, and the osteogenic induction was carried out 23 days later. S staining, the results are shown in Figure 4. The results showed that MSC cells (MSC-FG) after Lenti-FGF21-GLP1-Fc lentivirus infection still had adipogenic and osteogenic differentiation ability.
  • FGF21 and GLP1-Fc The expression of FGF21 and GLP1-Fc: MSC and MSC-FG cell lysate, and the cell culture supernatant after concentrating 10 times of MSC and MSC-FG were subjected to 15% SDS-PAGE gel electrophoresis and wet to PVDF membrane. 5% skim milk was blocked, antibody FGF21 (1:3000, Abcam, detection of FGF21 protein expression) and antibody IgG4-HRP (1:3000, Abcam, detection of GLP1-Fc protein expression) were added, and overnight at 4 ° C; TBST was washed three times.
  • the secondary antibody was incubated for 1 h; after TBST was washed three times, it was developed with a chemiluminescence kit and exposed by a computer image analysis system. The results are shown in Fig. 5. The results showed that high amounts of FGF21 and GLP1-Fc were present in the supernatant of FGF21/GLP1-Fc double gene-modified MSC cells (MSC-FG), and less was detected intracellularly.
  • MSC, MSC-FG, MSC-FGF21 and MSC-GLP1-Fc cells obtained in Example 1 were cultured in 100 mm culture respectively. When the cell confluence reached 70%-80%, the original was discarded. MSC serum-free medium, 10 ml of ⁇ -MEM medium, and incubation at 37 ° C, 5% CO 2 saturation humidity for 48 h. The culture supernatants of the four cells were collected, concentrated 10 times with an ultrafiltration column, and stored at 4 ° C until use. Long-term storage should be placed in a -80 ° C refrigerator.
  • INS-1 cell rat insulinoma cells, given by the Academy of Military Medical Sciences
  • INS-1 medium 50% FBS + 1 mM sodium pyruvate + 2 mM glutamine + 50 ⁇ M mercaptoethanol + +1640 medium
  • the supernatant was collected, and the content of Insulin in the culture solution was detected by a radioactive immunogen method.
  • the results are shown in Fig. 6.
  • the results showed that the culture supernatant of FGF21/GLP1-Fc double gene-modified MSC cells (MSC-FG) significantly stimulated the secretion of insulin by INS-1, and its value was significantly higher than that of FGF21 single gene modified MSC (MSC-FGF21).
  • the culture supernatant and the culture supernatant of GLP1-Fc single gene modified MSC (MSC-GLP1-Fc).
  • FGF21/GLP1-Fc double gene-modified MSC cells exhibited a remarkable synergistic effect in regulating insulin secretion.
  • MSC, MSC-FG, MSC-FGF21 and MSC-GLP1-Fc cells obtained in Example 1 were cultured in 100 mm culture respectively. When the cell confluence reached 70%-80%, the original was discarded. MSC serum-free medium, 10 ml of ⁇ -MEM medium, and incubation at 37 ° C, 5% CO 2 saturation humidity for 48 h. The culture supernatants of the four cells were collected and stored at 4 ° C until use. Long-term storage should be placed in a -80 ° C refrigerator.
  • HePG2 cell human hepatoma cells, donated by the Academy of Military Medical Sciences
  • a 15 ml centrifuge tube containing 5 ml of preheated medium was added dropwise.
  • the cells were centrifuged at 1200 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in HePG 2 medium (10% FBS + DMEM) and inoculated into a 100 mm culture dish, and cultured at 37 ° C, 5% CO 2 saturated humidity. Under normal circumstances, it will be transmitted once every two to three days, and the ratio of passage is 1:6.
  • the HePG2 medium in the original 6-well plate was discarded, and 2 ml of the previously collected culture supernatants of MSC, MSC-FGF21, MSC-GLP1-Fc and MSC-FG were added, and cultured at 37 ° C, 5% CO 2 saturated humidity for 24 hours. After digesting the cells, after centrifugation, the cells were collected for TRIzol RNA extraction, and after measuring the RNA concentration, 500 ng of total RNA was inverted into cDNA using a reverse transcription kit (All-in-One cDNA Synthesis SuperMix).
  • Primer sequence (5' ⁇ 3'): SEBP1C-F: CACTGTGACCTCGCAGATCC; S EBP1C-R: ATAGGCAGCTTCTCCGCATC; ⁇ -Actin-F: CCTGGCACCCAGCACAA T; ⁇ -Actin-R: GGGCCGGACTCGTCATAC
  • FGF21/GLP1-Fc double gene-modified MSC cells significantly inhibited the mRNA expression level of SREBP1c (liver tissue sterol regulatory element binding protein-1C), and its inhibition The activity was significantly higher than that of the culture supernatants of MSC-FGF21 and MSC-GLP1-Fc.
  • SREBP1c liver tissue sterol regulatory element binding protein-1C
  • mice Thirty 5-week-old male diabetic model mice, BKS.Cg-Dock7m+/+Leprdb/Nju (purchased from the Institute of Model Animals, Nanjing University), were selected. The experiment was divided into: control group (saline), liraglutide drug group (drug), MSC group (cell), MSC-FGF21 group (cell), MSC-FG group (cell) and MSC-GLP1-Fc group ( Cells), a total of 6 groups of 5 mice per group.
  • control group saline
  • liraglutide drug group drug
  • MSC group cell
  • MSC-FGF21 group cell
  • MSC-FG group Cells
  • MSC-GLP1-Fc group Cells
  • the liraglutide drug group (Liraglutide) was administered subcutaneously, and the other groups were administered intraperitoneally. All groups were required to be administered in the morning.
  • Dosing time The cell group is required to inject the cells every 7 days for a total of 3 cells.
  • the liraglutide drug group requires two doses of the drug per week.
  • Control group (saline group): Each mouse was intraperitoneally injected with 100 ⁇ l of physiological saline per injection.
  • Cell group Each mouse was injected with 1 ⁇ 10 6 cells/100 ul each time, once every 7 days, intraperitoneally.
  • Liraglutide drug group The drug concentration was 0.5 mg/Kg, once every 3-4 days, subcutaneously.
  • Serum biochemical indicators After 28 days of treatment, the mouse eyeballs were bled, and the serum was separated by centrifugation at 3000 rpm for 10 min. The samples were sent to Beijing North Biomedical Technology Co., Ltd. for triglyceride (TG), total cholesterol (TG), and high-density lipoprotein. Detection of (HDL), low density lipoprotein (LDL) and insulin (INS) indicators.
  • TG triglyceride
  • TG total cholesterol
  • INS insulin
  • Animal tissue hematoxylin-eosin (HE) staining After 28 days of treatment, after the neck was sacrificed, the midline incision was made in the abdomen, the abdomen was fully exposed, and the liver of the mouse and the subcutaneous fat of the abdomen were taken out, and 10% of the volume was mixed. Formaldehyde was fixed for 24 hours and subjected to paraffin embedding and hematoxylin-eosin (HE) staining.
  • the body size of the mice in the MSC-FG cell treatment group was also significantly smaller than that of the control mice, and the subcutaneous fat content in the abdomen was significantly reduced (Fig. 10).
  • Hepatic histopathological sections showed HE staining (Fig. 11).
  • the liver of Con mice showed severe fatty degeneration, liver tissue was filled with fat cells, and hepatocytes were necrotic.
  • liver MSC-FG treatment hepatic steatosis was significantly improved. The degree is reduced.
  • HE staining of abdominal subcutaneous fat showed (Fig. 11).
  • the fat cells of Con group were over-expanded and the nucleus was irregular. After MSC-FG treatment, the volume of adipocytes was significantly reduced.
  • MSC-FGF1 can significantly reduce the body weight and blood sugar of mice, reduce blood lipid levels, alleviate fatty liver, improve abnormal blood lipid metabolism, repair islet cell function, and its efficacy is better than MSC-FGF21 and MSC-GLP1.
  • the -Fc group with significant synergy, is also superior to the hypoglycemic drug Liraglutide in improving blood lipids.
  • the modified MSCs of the invention are particularly useful for treating metabolic disorders.

Abstract

提供了一种经修饰的间充质干细胞及其培养上清，以及包含此类细胞或其培养上清的药物组合物。该间充质干细胞其能够表达：(1)第一蛋白，其选自：FGF21或其变体，或包含所述FGF21或其变体的第一融合蛋白；和，(2)第二蛋白，其选自：GLP-1或其变体，或包含所述GLP-1或其变体的第二融合蛋白。还提供了经修饰的间充质干细胞及其培养上清，以及包含此类细胞或其培养上清的药物组合物在治疗代谢病症、以及在制备用于治疗代谢病症的药物中的用途。

Description

一种双基因修饰的干细胞及其用途 技术领域
本发明涉及细胞治疗领域。具体而言，本发明涉及一种经修饰的间充质干细胞及其培养上清，以及包含此类细胞或其培养上清的药物组合物。本发明还涉及所述经修饰的间充质干细胞及其培养上清，用于在受试者(例如，人)中治疗代谢病症的用途，以及在制备用于在受试者(例如，人)中治疗代谢病症的药物中的用途。本发明还涉及一种治疗代谢病症的方法，其包括向有此需要的受试者施用本发明的经修饰的间充质干细胞或其培养上清或药物组合物的步骤。
背景技术
糖尿病等代谢病症与参与代谢调控的一些内源性分子密切相关。已发现的此类内源性分子主要有三类：一是激素类，包括胰岛素、胰高血糖素、GLP1、糖皮质激素等；二是具有激素样功能的细胞生长因子类，包括FGF19、FGF21和FGF23等；三是参与代谢调控或细胞信号转导的酶类，包括SPK1、PI3K、HSL等。
内源性的激素、细胞因子或酶类的分泌不足、活性降低或功能缺陷与代谢病的发生密切相关，如胰岛素抵抗和相对分泌不足是导致糖尿病的核心原因。因此，这些糖脂代谢平衡调控的关键分子也是治疗糖尿病等代谢综合征药物开发的重点对象，其中胰岛素和胰高血糖素样的多肽1(GLP1)已成为目前最主要的糖尿病治疗药物。
目前，针对GLP1开发的降糖药物主要是抑制GLP1降解的DPP4抑制剂和GLP1类似物，将GLP1与人抗体Fc融合后的GLP1-Fc半衰期长，耐受性良好。纤维母细胞生长因子(FGF)是一类重要的组织生长因子，包括22个家族成员。FGF19/21/23是FGF家族的一类成员，它们具有激素样的作用，在糖脂代谢调控中发挥中重要作用。其中，FGF21主要由肝脏合成，通过内分泌途径作用于脂肪组织，调控糖脂的代谢。作为一种糖脂代谢调控分子，FGF21已经被作为药物开发，用于代谢病的治疗。GLP1-Fc和FGF21在临床应用中患者均需面临长期用药、终身用药、联合用药降糖降脂等问题，而且这些药物只能起到治疗病症的效果，均无法从根源上改善疾病的诱因。
总之，鉴于目前可获得的降糖降脂药物都受限于一些缺陷，寻找一种全新的、可靠的、从根本上治疗代谢病症的方法迫在眉睫。
发明内容
本申请的发明人经过大量实验和反复摸索，出人意料地发现，经FGF21和GLP-1基因修饰的间充质干细胞具备显著提高的降低血糖、血脂等生物学活性。基于这一发现，本发明人开发了新的用于代谢病症的经修饰细胞以及基于该细胞的代谢病症治疗方法。
经修饰的间充质干细胞
因此，在一个方面，本发明提供了一种经修饰的间充质干细胞，其表达：(1)第一蛋白，其选自：FGF21或其变体，或包含所述FGF21或其变体的第一融合蛋白；和，(2)第二蛋白，其选自：GLP-1或其变体，或包含所述GLP-1或其变体的第二融合蛋白。
在某些优选的实施方案中，所述FGF21的变体与其所源自的序列相比，具有一个或多个氨基酸的替换、插入或删除，或者具有至少80％，85％，90％，95％，96％，97％，98％，或99％的序列同一性，并且保留了FGF21活性。
在本发明中，所述“FGF21活性”是指天然存在的FGF21的一种或多种生理作用，其是本领域技术人员熟知的，包括但不限于，诱导不依赖胰岛素的葡萄糖摄入，降低血浆葡萄糖、果糖胺、甘油三酯、胰岛素、高血糖素水平，减少LDL胆固醇和增加HDL胆固醇，增强胰岛素敏感性等，其详细教导可参见，例如DostálováI等人.Physiol Res.2009；58(1):1-7；Kharitonenkov A等人.J Clin Invest.2005Jun；115(6):1627-35.
在某些优选的实施方案中，所述GLP-1的变体与其所源自的序列相比，具有一个或多个氨基酸的替换、插入或删除，或者具有至少80％，85％，90％，95％，96％，97％，98％，或99％的序列同一性，并且保留了GLP-1活性。
在本发明中，所述“GLP-1活性”是指天然存在的GLP-1的一种或多种生理作用，其是本领域技术人员已知的，包括但不限于，对胰岛素分泌的葡萄糖依赖性刺激、对高血糖素分泌的阻抑、胰岛素(原)生物合成的刺激、食物摄取减少、胃排空减速等，其详细教导可参见例如Nauck MA等人.Exp Clin Endocrinol Diabetes.1997；105(4):187-95.。
在某些优选的实施方案中，所述FGF21具有如SEQ ID NO:1所示的氨基酸序列。
在某些优选的实施方案中，所述GLP-1具有如SEQ ID NO:4所示的氨基酸序列。
在某些优选的实施方案中，所述经修饰的间充质干细胞表达：(1)第一蛋白，其选自：SEQ ID NO:2所示的氨基酸序列，或包含SEQ ID NO:2所示的氨基酸序列的第一融合蛋白；和，(2)第二蛋白，其选自：SEQ ID NO:5所示的氨基酸序列，或包含SEQ ID NO:5所示的氨基酸序列的第二融合蛋白。
在某些优选的实施方案中，所述经修饰的间充质干细胞能够分泌所述第一蛋白和第 二蛋白。
在某些优选的实施方案中，所述第一融合蛋白还包含第一额外多肽。在某些优选的实施方案中，所述第一额外多肽能够延长所述第一融合蛋白在体内的半衰期。
在某些优选的实施方案中，所述第一额外多肽选自免疫球蛋白Fc结构域(例如，人免疫球蛋白Fc结构域，例如人IgG的Fc结构域)、血清白蛋白(例如，人血清白蛋白(HSA))、白蛋白结合多肽(例如，HSA结合多肽)、转铁蛋白，及前述任一项的功能性片段。
在某些优选的实施方案中，所述第一额外多肽为人免疫球蛋白Fc结构域，例如人IgG的Fc结构域，例如人IgG1、IgG2、IgG3或IgG4的Fc结构域。在某些示例性实施方案中，所述第一额外多肽具有如SEQ ID NO:7所示的氨基酸序列。
在某些优选的实施方案中，所述第一额外多肽任选地通过接头与所述FGF21或其变体融合。在某些优选的实施方案中，所述第一额外多肽任选地通过接头与所述FGF21或其变体的N端或C端融合。在某些优选的实施方案中，所述接头为富含Gly和Ser的组合的肽接头，例如由重复的GGGGS氨基酸序列组成的序列。在某些示例性实施方案中，所述接头为GGGGSGGGGSGGGGS(SEQ ID NO:9)。
在某些优选的实施方案中，所述第二融合蛋白还包含第二额外多肽。在某些优选的实施方案中，所述第二额外多肽能够延长所述第二融合蛋白在体内的半衰期。
在某些优选的实施方案中，所述第二额外多肽选自免疫球蛋白Fc结构域(例如，人免疫球蛋白Fc结构域，例如人IgG的Fc结构域)、血清白蛋白(例如，人血清白蛋白(HSA))、白蛋白结合多肽(例如，HSA结合多肽)、转铁蛋白，及前述任一项的功能性片段。
在某些优选的实施方案中，所述第二额外多肽为人免疫球蛋白Fc结构域，例如人IgG的Fc结构域，例如人IgG1、IgG2、IgG3或IgG4的Fc结构域。在某些示例性实施方案中，所述第二额外多肽具有如SEQ ID NO:7所示的氨基酸序列。
在某些优选的实施方案中，所述第二额外多肽任选地通过接头与所述GLP-1或其变体融合。在某些优选的实施方案中，所述第二额外多肽任选地通过接头与所述GLP-1或其变体的N端或C端融合。在某些优选的实施方案中，所述接头为富含Gly和Ser的组合的肽接头，例如由重复的GGGGS氨基酸序列组成的序列。在某些示例性实施方案中，所述接头为GGGGSGGGGSGGGGS(SEQ ID NO:9)。
在某些示例性实施方案中，所述经修饰的间充质干细胞表达：(1)第一蛋白，其选 自：SEQ ID NO:2所示的氨基酸序列，或包含SEQ ID NO:2所示的氨基酸序列的第一融合蛋白；和，(2)第二蛋白，其选自：SEQ ID NO:5所示的氨基酸序列，或包含SEQ ID NO:5所示的氨基酸序列的第二融合蛋白。
在某些示例性实施方案中，所述经修饰的间充质干细胞表达：如SEQ ID NO:2所示的氨基酸序列；和如SEQ ID NO:10所示的氨基酸序列。
在某些优选的实施方案中，所述经修饰的间充质干细胞包含：
(1)第一外源核酸，其包含编码所述第一蛋白的核苷酸序列；和
(2)第二外源核酸，其包含编码所述第二蛋白的核苷酸序列。
在某些优选的实施方案中，所述第一外源核酸和/或第二外源核酸可操作地连接至启动子(例如，组成型启动子、组织特异性启动子或诱导型启动子)。
在某些优选的实施方案中，所述第一外源核酸和/或第二外源核酸连接有编码信号肽的核苷酸序列。在某些示例性实施方案中，所述第二外源核酸的5’端连接有编码信号肽(例如，如SEQ ID NO:22所示的信号肽)的核苷酸序列。
在某些优选的实施方案中，所述第一外源核酸与第二外源核酸可以通过编码自我切割肽的序列连接。在某些示例性实施方案中，所述编码自我切割肽的序列连接至所述第一外源核酸的3’端，并且连接至所述第二外源核酸的5’端。
在某些优选的实施方案中，所述第一外源核酸和/或第二外源核酸整合于所述间充质干细胞的基因组中。
在某些优选的实施方案中，所述第一外源核酸和/或第二外源核酸独立于所述间充质干细胞的基因组。在此类实施方案中，所述经修饰的间充质干细胞包含表达载体，并且所述第一外源核酸和第二外源核酸包含于同一或不同的表达载体中。
在某示例性实施方案中，所述表达载体包含所述第一外源核酸和所述第二外源核酸。在此类实施方案中，所述第一外源核酸与第二外源核酸可以通过编码自我切割肽的序列连接。在某些示例性实施方案中，所述编码自我切割肽的序列连接至所述第一外源核酸的3’端，并且连接至所述第二外源核酸的5’端。
在本发明中，适宜的自我切割肽是本领域技术人员已知的，其实例包括但不限于，源自口疮病毒属或心病毒属的2A肽，例如源自口蹄疫病毒(FMDV)、马鼻炎A病毒(ERAV)、Thoseaasigna病毒(TaV)或猪捷申病毒(PTV-1)的2A肽。
在某些优选的实施方案中，所述第一外源核酸与第二外源核酸通过编码2A肽的序列连接。在某些示例性实施方案中，所述编码2A肽的序列连接至所述第一外源核酸的3’ 端，并且连接至所述第二外源核酸的5’端。在某些优选的实施方案中，所述2A肽为源自Thoseaasigna病毒(TaV)的2A肽。在某些示例性实施方案中，所述2A肽具有如SEQ ID NO:12所示的氨基酸序列。在某些示例性实施方案中，所述编码2A肽的序列具有如SEQ ID NO:13所示的核苷酸序列。
在本发明中，可以通过本领域中公知的方法和技术对间充质干细胞进行遗传修饰，例如物理、化学或生物学方法，或其组合。例如，所述生物学方法包括使用病毒载体，例如慢病毒、逆转录病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。例如，所述化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠等；基于脂质的系统，包括水包油乳剂、胶束、混合胶束或脂质体等。例如，所述物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。
在某些优选的实施方案中，本发明的经修饰的间充质干细胞通过将所述第一外源核酸和第二外源核酸导入间充质干细胞而获得。
在某些优选的实施方案中，所述经修饰的间充质干细胞通过以下步骤获得：
(1)提供包含第一外源核酸和第二外源核酸的表达载体；
(2)将步骤(1)所述的表达载体转染至间充质干细胞。
在某些优选的实施方案中，在步骤(2)中，将所述表达载体稳定转染至所述间充质干细胞。在某些优选的实施方案中，在步骤(1)中，所述表达载体为病毒载体，例如慢病毒载体。在某些优选的实施方案中，所述表达载体包含SEQ ID NO:15所示的核苷酸序列。
在某些优选的实施方案中，所述间充质干细胞来源于脂肪组织、脐带、骨髓或脐血。在某些示例性实施方案中，所述间充质干细胞来源于脂肪组织。
本发明的经修饰的间充质干细胞可以作为药物组合物被配制和施用。这样的药物组合物可以是医学领域已知的任何形式，优选为注射剂(包括注射液、冻干粉剂)。在某些优选的实施方案中，该药物组合物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。关于该药物组合物的配制的一般原则可参考由G.Morstyn和W.Sheridan编著的《细胞疗法：干细胞移植，基因疗法和细胞免疫疗法(Cell Therapy:Stem Cell Transplantation,Gene Therapy,and Cellular Immunotherapy)》，剑桥大学出版社，1996；和《造血干细胞疗法(Hematopoietic Stem  Cell Therapy)》,E.D.Ball,J.Lister&P.Law,Churchill Livingstone,2000。
在某些优选的实施方案中，本发明的经修饰的间充质干细胞用于在受试者中治疗代谢病症，或者用于制备在受试者中治疗代谢病症的药物。
在某些优选的实施方案中，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
培养物及培养上清
在另一个方面，本发明提供了一种培养物，其包含本发明的经修饰的间充质干细胞，以及培养基。
在本发明中，可用于干细胞培养的培养基是本领域技术人员已知的，其非限制性实例包括，α-MEM培养基、DMEM培养基、IMDM培养基、Ham's F12培养基、RPMI1640培养基，以及上述任一项组合形成的混合培养基(例如，IMDM与HamF12等量混合而成的IMDM/HamF12培养基)。上述培养基任选地进一步包含补充物质，例如血清(例如，胎牛血清、人血清、羊血清等)、血清替代物(例如，Knockout serum replacement(KSR)等)、牛血清白蛋白(BSA)、抗生素、维生素、矿物质。
在某些示例性实施方案中，所述培养基为含有或不含有血清的α-MEM培养基。
本发明的培养物可以作为药物组合物被配制和施用。这样的药物组合物可以是医学领域已知的任何形式，优选为注射剂(包括注射液、冻干粉剂)。在某些优选的实施方案中，该药物组合物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。关于该药物组合物的配制的一般原则可参考由G.Morstyn和W.Sheridan编著的《细胞疗法：干细胞移植，基因疗法和细胞免疫疗法(Cell Therapy:Stem Cell Transplantation,Gene Therapy,and Cellular Immunotherapy)》，剑桥大学出版社，1996；和《造血干细胞疗法(Hematopoietic Stem Cell Therapy)》,E.D.Ball,J.Lister&P.Law,Churchill Livingstone,2000。
在某些优选的实施方案中，如本文所述的培养物用于在受试者中治疗代谢病症，或者用于制备在受试者中治疗代谢病症的药物。
在某些优选的实施方案中，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在另一个方面，本发明提供了一种培养上清，其为本发明的培养物的上清液。
在某些优选的实施方案中，所述培养上清不含所述经修饰的间充质干细胞。
在某些优选的实施方案中，所述培养上清不含血清。
在某些优选的实施方案中，所述培养上清包含基础培养基、或在基础培养基中添加有一种或多种补充物质(例如血清)的培养液。所述基础培养基任选地进一步包含一种或多种补充物质(例如血清)。在某些示例性实施方案中，所述培养上清包含含有或不含有血清的α-MEM培养基。
本发明的培养上清可以作为药物组合物被配制和施用。这样的药物组合物可以是医学领域已知的任何形式，例如片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。
在某些优选的实施方案中，如本文所述的培养上清用于在受试者中治疗代谢病症，或者用于制备在受试者中治疗代谢病症的药物。
在某些优选的实施方案中，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在另一个方面，本发明还涉及制备如本文所述的培养上清的方法，其包括以下步骤：
(1)对本发明的经修饰的间充质干细胞进行培养；和
(2)回收步骤(1)获得的培养物的上清液。
在某些优选的实施方案中，所述方法还包括：(3)对步骤(2)获得的上清液进行处理， 所述处理选自离心、浓缩、溶剂的置换、透析、冷冻、干燥、冷冻干燥、稀释、脱盐、保存，及其任意组合。
在本发明中，可以使用本领域已知的任何可用于干细胞培养的培养基以及培养条件对本发明的经修饰的间充质干细胞进行培养。在某些优选的实施方案中，在步骤(1)中可以使用基础培养基，所述基础培养基任选地包含一种或多种补充物质(例如血清)。
在某些优选的实施方案中，所述基础培养基选自α-MEM培养基、DMEM培养基、IMDM培养基、Ham's F12培养基、RPMI1640培养基，以及上述任一项组合形成的混合培养基(例如，IMDM与HamF12等量混合而成的IMDM/HamF12培养基)。在某些优选的实施方案中，所述补充物质可以选自血清(例如，胎牛血清、人血清、羊血清等)、血清替代物(例如，Knockout serum replacement(KSR)等)、牛血清白蛋白(BSA)、抗生素、维生素、矿物质。
在某些示例性实施方案中，在步骤(1)中使用含有或不含有血清的α-MEM培养基对所述经修饰的间充质干细胞进行培养。
在某些优选的实施方案中，本发明的培养上清不含血清，以提高安全性。因此，在某些示例性实施方案中，在步骤(1)中，可以使用不含血清的培养基(例如，基础培养基或无血清培养基)对所述经修饰的间充质干细胞进行培养，从而获得不含血清的培养上清。在此类实施方案中，可以在整个培养过程中、或在最后或最后几次的传代培养中使用所述不含血清的培养基进行培养。在某些示例性实施方案中，可以对步骤(2)获得的培养上清进行透析或溶剂置换，从而去除血清，由此也可得到不含血清的培养上清。
药物组合物及治疗用途
在另一个方面，本发明提供了一种药物组合物，其包含如本文所述的经修饰的间充质干细胞、培养物或培养上清。
在某些优选的实施方案中，所述药物组合物包含治疗有效量的所述经修饰的间充质干细胞或培养物。
在某些优选的实施方案中，所述药物组合物包含如本文所述的经修饰的间充质干细胞中的一种或多种。
在某些优选的实施方案中，所述药物组合物可以为医学领域已知的任何形式。例如，所述药物组合物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在某些优 选的实施方案中，所述药物组合物为注射剂(包括注射液、冻干粉剂)。
在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。
在某些优选的实施方案中，所述药物组合物可以以悬浮液、凝胶、胶体、浆液或混合物的形式进行移植。
关于包含本发明的经修饰的间充质干细胞的药物组合物的配制的一般原则可参考由G.Morstyn和W.Sheridan编著的《细胞疗法：干细胞移植，基因疗法和细胞免疫疗法(Cell Therapy:Stem Cell Transplantation,Gene Therapy,and Cellular Immunotherapy)》，剑桥大学出版社，1996；和《造血干细胞疗法(Hematopoietic Stem Cell Therapy)》,E.D.Ball,J.Lister&P.Law,Churchill Livingstone,2000。
在某些优选的实施方案中，所述药物组合物包含治疗有效量的如本文所述的培养上清。
在某些优选的实施方案中，所述药物组合物可以为医学领域已知的任何形式，例如，所述药物组合物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在某些优选的实施方案中，所述药物组合物为注射剂(包括注射液、冻干粉剂)。
在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。
在某些优选的实施方案中，本发明的药物组合物任选地还包含另外的药学活性剂。在某些优选的实施方案中，所述另外的药学活性剂选自抗糖尿病药物、抗肥胖症药物、抗高血压药物、抗动脉粥样硬化药物和降脂药物。
在本发明中，合适的抗糖尿病药物的非限制性实例包括，噻唑烷二酮类(如罗格列酮或吡格列酮)、双胍类(如二甲双胍或苯乙双胍)、磺脲类(如格列美脲、格列本脲、格列齐特、氯磺丙脲或格列吡嗪)、葡萄糖苷酶抑制剂(如阿卡波糖或米格列醇)、PPAR-α激动剂、PPAR-γ激动剂、PPAR-α/γ双重激动剂(如莫格他唑)、aP2抑制剂、DPP4抑制剂(如如西他列汀或维格列汀)、胰岛素敏化剂、胰岛素或氯茴苯酸类(如瑞 格列奈)等。
合适的抗肥胖症药物的非限制性实例包括，β3肾上腺素能激动剂(如AJ9677(Taked a/Dainippon)、L750355(Merck)或CP331648(Pfizer))、脂酶抑制剂(如奥利司他)、5-羟色胺(和多巴胺)再摄取抑制剂(如西布曲明或托吡酯)、甲状腺受体β化合物(如WO99/00353和WO 00/039077中公开的化合物)、CB-1拮抗剂(如利莫那班)或厌食药物(如右旋苯丙胺)。
合适的降脂药物(包括抗动脉粥样硬化药物)的非限制性实例包括，选自MTP抑制剂、胆固醇酯转移蛋白抑制剂(如CP-529414(Pfizer))、HMG CoA还原酶抑制剂(如普伐他汀、洛伐他汀、辛伐他汀、阿托伐他汀、氟伐他汀、西立伐他汀或阿伐他汀)、角鲨烯合成酶抑制剂(如美国专利US 5,712,396中公开的α-膦酰基-磺酸酯)、苯乙酸衍生物(如非诺贝特、吉非贝齐、氯贝丁酯、苯扎贝特、环丙贝特、克利贝特等)、LDL受体活性上调剂(如MD-700(Taisho Pharmaceutical Co.Ltd)和LY295427(Eli Lilly))、脂氧化酶抑制剂(如WO 97/12615中公开的苯并咪唑衍生物、WO 97/12613中公开的15-LO抑制剂、WO 96/38144中公开的异噻唑酮类)、ACAT抑制剂(如阿伐麦布)、胆固醇吸收抑制剂、回肠Na<+>/胆汁酸协同转运蛋白抑制剂。
合适的抗高血压药物的非限制性实例包括，β肾上腺素能阻断剂、钙通道阻断剂(如地尔硫、维拉帕米、硝苯地平、氨氯地平)、利尿剂(如氯噻嗪、氢氯噻嗪、氟甲噻嗪、氢氟噻嗪、苄氟噻嗪、甲基氯噻嗪、三氯噻嗪、泊利噻嗪、苄噻嗪、替尼酸、氯噻酮、呋塞米、布美他尼、阿米洛利或螺内酯)、肾素抑制剂、ACE抑制剂(如卡托普利、佐芬普利、福辛普利、依那普利、西拉普利、地拉普利、喷托普利、喹那普利、雷米普利或赖诺普利)、AT-1受体拮抗剂(如氯沙坦、厄贝沙坦或缬沙坦)、ET受体拮抗剂(如西他生坦或阿曲生坦)、双重ET/AII拮抗剂(如在WO 00/01389中公开的化合物)、双重NEP-ACE抑制剂(如奥马曲拉)以及硝酸脂类。
在某些优选的实施方案中，所述药物组合物用于在受试者中治疗代谢病症。
在某些优选的实施方案中，所述受试者是哺乳动物，例如人。
在某些优选的实施方案中，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)。
在另一个方面，本发明涉及如本文所述的经修饰的间充质干细胞、培养物或培养上清用于在受试者中治疗代谢病症的用途，或者用于制备在受试者中治疗代谢病症的药物的用途。
在某些优选的实施方案中，所述药物包含治疗有效量的所述经修饰的间充质干细胞或培养物。
在某些优选的实施方案中，可以组合使用本发明的经修饰的间充质干细胞。因此，所述药物可以包括本发明的经修饰的间充质干细胞中的一种或数种。
在某些优选的实施方案中，所述药物可以为医学领域已知的任何形式。例如，所述药物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在某些优选的实施方案中，所述药物为注射剂(包括注射液、冻干粉剂)。
在某些优选的实施方案中，所述药物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。
在某些优选的实施方案中，所述药物可以以悬浮液、凝胶、胶体、浆液或混合物的形式进行移植。
在某些优选的实施方案中，所述药物包含治疗有效量的如本文所述的培养上清。
在某些优选的实施方案中，所述药物可以为医学领域已知的任何形式，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在某些优选的实施方案中，所述药物为注射剂(包括注射液、冻干粉剂)。
在某些优选的实施方案中，所述药物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。
在某些优选的实施方案中，所述药物任选地还包含另外的药学活性剂。在某些优选的实施方案中，所述另外的药学活性剂选自抗糖尿病药物、抗肥胖症药物、抗高血压药物、抗动脉粥样硬化药物和降脂药物。
在某些优选的实施方案中，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)。
在某些优选的实施方案中，所述受试者是哺乳动物，例如人。
在另一个方面，本发明提供了一种治疗代谢病症的方法，其包括向有此需要的受试者施用本发明的经修饰的间充质干细胞、培养物、培养上清、或本发明的药物组合物。
在某些优选的实施方案中，可以组合使用本发明的经修饰的间充质干细胞。因此，可以向受试者施用本发明的经修饰的间充质干细胞中的一种或多种。
在某些优选的实施方案中，可以将本发明的经修饰的间充质干细胞或培养物，作为药物组合物进行配制和施用。这样的药物组合物可包含治疗有效量的所述经修饰的间充质干细胞或培养物。在某些优选的实施方案中，所述药物组合物可以是医学领域已知的任何形式。例如，所述药物组合物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在某些优选的实施方案中，所述药物组合物为注射剂(包括注射液、冻干粉剂)。在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。关于该药物组合物的配制的一般原则可参考由G.Morstyn和W.Sheridan编著的《细胞疗法：干细胞移植，基因疗法和细胞免疫疗法(Cell Therapy:Stem Cell Transplantation,Gene Therapy,and Cellular Immunotherapy)》，剑桥大学出版社，1996；和《造血干细胞疗法(Hematopoietic Stem Cell Therapy)》,E.D.Ball,J.Lister&P.Law,Churchill Livingstone,2000。
在本发明中可以通过各种合适的方式，向受试者施用如本文所述的经修饰的间充质干细胞或培养物，或包含所述经修饰的间充质干细胞或培养物的药物组合物。在某些优选的实施方案中，通过局部注射移植(例如，立体定向脑内注射移植或脊髓局部注射移植)、血液循环途径移植(例如，静脉内注射移植或动脉内注射移植)或经脑脊液途径移植(例如，腰椎穿刺蛛网膜下腔注射移植)等途径向受试者施用如本文所述的经修饰的间充质干细胞或药物组合物。本领域技术人员已知如何根据病灶的位置和性质等选择合 适的细胞移植途径。
在某些优选的实施方案中，本发明的经修饰的间充质干细胞、培养物或药物组合物可以以悬浮液、凝胶、胶体、浆液或混合物的形式进行移植。
在某些优选的实施方案中，可以将本发明的培养上清，作为药物组合物进行配制和施用。这样的药物组合物可包含治疗有效量的所述培养上清。在某些优选的实施方案中，所述药物组合物可以是医学领域已知的任何形式，例如片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在某些优选的实施方案中，所述药物组合物为注射剂(包括注射液、冻干粉剂)。在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。
在本发明中可以通过各种合适的方式，向受试者施用本发明的培养上清，或包含所述培养上清的药物组合物。在某些优选的实施方案中，可以通过皮内注射、皮下注射、肌肉注射、静脉注射、口服给予等途径施用本发明的培养上清，或包含所述培养上清的药物组合物。
在某些优选的实施方案中，所述方法还包括施用另外的药学活性剂，所述另外的药学活性剂选自抗糖尿病药物、抗肥胖症药物、抗高血压药物、抗动脉粥样硬化药物和降脂药物。这种另外的药学活性剂可以在施用如本文所述的经修饰的间充质干细胞或其培养上清或所述药物组合物之前、同时或之后施用。
在某些优选的实施方案中，所述方法还包括施用额外的疗法。这种额外的疗法可以是已知用于代谢病症的任何疗法，例如药物治疗、手术治疗等。这种额外的疗法可以在施用如上所述的方法之前、同时或之后施用。
在某些优选的实施方案中，所述受试者是哺乳动物，例如人。
在某些优选的实施方案中，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、细胞培养、生物化学、细胞生物学等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“外源核酸”是指，人工引入的核苷酸序列，其相对于未经遗传修饰的细胞而言是外来的。外源核酸包括但不限于，未在所述细胞基因组中发现的任何基因或核苷酸序列。
如本文中所使用的，术语“FGF21”是指，成纤维细胞生长因子(fibroblast growth fa ctor，FGF)蛋白质家族的成员。其氨基酸序列可参见例如GenBank登录号NP_061986.1，相应的核苷酸序列可参见例如NCBI参考序列号NM_019113.2。
在本发明中，表述“FGF21的变体”是指这样的蛋白，其氨基酸序列与其所源自的野生型序列(例如，野生型FGF21全长序列，例如，如SEQ ID NO:1所示的氨基酸序列)相比，具有一个或多个氨基酸的替换、插入或删除，或者具有至少60％，80％，85％，90％，95％，96％，97％，98％，或99％的序列同一性，并且保留了“FGF21活性”。因此，本发明所述的“FGF21的变体”也包括野生型蛋白的N端和/或C端的截短形式。“FGF21变体”的非限制性实例包括，中国专利申请CN200980130476.4、CN201180065381.6、CN201280057789.3或CN201580070276.X、国际专利申请WO2005/061712、WO2006/028595、WO2006/028714、WO2006/065582或WO2008/121563中所描述的那些。所述“FGF21活性”是指天然存在的FGF21的一种或多种生理作用，其是本领域技术人员熟知的，包括但不限于，结合并激活FGF21受体，例如诱导不依赖胰岛素的葡萄糖摄入，降低血浆葡萄糖、果糖胺、甘油三酯、胰岛素、高血糖素水平，减少LDL胆固醇和增加HDL胆固醇，增强胰岛素敏感性等，其详细教导可参见，例如DostálováI等人.Ph ysiol Res.2009；58(1):1-7；Kharitonenkov A等人.J Clin Invest.2005Jun；115(6):1627-35.
如本文中所使用的，术语“GLP-1”是指，胰高血糖素样肽-1(glucagon-like peptide-1)。本领域已知，GLP-1是经翻译后修饰获得的，并且源自前胰高血糖素原(glucagon preproprotein)。前胰高血糖素原(glucagon preproprotein)的野生型序列具有NCBI登录号NP_002045，并且可见于国际专利申请WO98/19698和WO87/06941A。GLP-1是由前胰高血糖素原的第92-128位氨基酸残基构成的片段。GLP-1野生型序列的第6位和第 7位残基之间的内源性裂解产生了两种活性形式(天然截短形式)：GLP-1(7-37)OH(其野生型序列如SEQ ID NO:4所示)，以及末端进一步酰胺化后形成的GLP-1(7-36)NH 2。
在本发明中，表述“GLP-1的变体”是指这样的蛋白，其氨基酸序列与其所源自的野生型序列(例如，活性形式GLP-1(7-37)OH的野生型序列，例如SEQ ID NO:4所示的氨基酸序列)相比，具有一个或多个氨基酸的替换、插入或删除，或者具有至少60％，80％，85％，90％，95％，96％，97％，98％，或99％的序列同一性，并且保留了“GLP-1活性”。因此，本发明所述的“GLP-1的变体”也包括野生型蛋白的N端和/或C端的截短形式，例如如上所述的GLP-1天然截短形式。“GLP-1变体”的非限制性实例包括，中国专利申请CN200910173888.8、CN201010508567.1中所描述的那些。所述“GLP-1活性”是指天然存在的GLP-1的一种或多种生理作用，其是本领域技术人员已知的，包括但不限于，结合并激活GLP-1受体，例如对胰岛素分泌的葡萄糖依赖性刺激、对高血糖素分泌的阻抑、胰岛素(原)生物合成的刺激、食物摄取减少、胃排空减速等，其详细教导可参见例如Nauck MA等人.Exp Clin Endocrinol Diabetes.1997；105(4):187-95.。
如本文中所使用的，表述“延长蛋白在体内的半衰期”是指，能够阻止或减缓该蛋白在体内的水解和酶促降解，增加半衰期，和/或改进或改变其他药代动力学或生物物理学特性，包括但不限于增加吸收速率、降低毒性、改进溶解度、减少蛋白质聚集等。能够用于延长半衰期的物质是本领域技术人员熟知的，其非限制性实例包括，免疫球蛋白的Fc结构域、血清白蛋白、转铁蛋白等。
如本文中所使用的，术语“Fc结构域”具有免疫学领域通常赋予该术语的涵义，尤其指不含有来自该抗体两个抗原结合区(Fab片段)的抗体片段。Fc结构域由通过非共价相互作用和二硫键结合的抗体的两个重链恒定区组成。Fc结构域可以包含铰链区，并经CH2和CH3结构域延伸到抗体C末端。Fc结构域还可以包含一个或多个糖基化位点。Fc结构域的效应子功能通常由与Fc受体(FcγR)的相互作用或通过结合Clq和固定补体介导。与FcγR的结合可以导致抗体依赖性细胞介导的细胞毒性(ADCC)，而与补体因子的结合可以导致补体依赖的细胞毒性(CDC)。因此，当仅利用Fc结构域的延长半衰期活性时，尽量减小效应子功能是重要的，例如可以使用结合FcγR和补体因子的能力相对较低的免疫球蛋白亚型的Fc结构域(例如IgG4的Fc)，或者在天然Fc结构域进入突变(例如，一个或多个氨基酸的替换)来减小效应子功能。因此，在本发明中，术语“Fc结构域”包括天然Fc或其变体。所述变体与其所源自的野生型序列相比，具有一个或多个突变(如，氨基酸替换、插入或删除)，该突变可能影响或参与，或者不影响不参与：(1)二 硫键形成，(2)与选择的宿主细胞的不相容性，(3)在选择的宿主细胞表达时N-末端异质性，(4)糖基化作用，(5)与补体的相互作用，(6)与补救受体以外的Fc受体结合，或(7)抗体依赖性的细胞毒性(ADCC)。
如本文中所使用的，术语“接头”是指，由多个氨基酸残基通过肽键连接形成的线性多肽。本发明的接头可以为人工合成的氨基酸序列，或天然存在的多肽序列，例如具有铰链区功能的多肽。此类接头多肽是本领域众所周知的(参见例如，Holliger,P.等人(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448；Poljak,R.J.等人(1994)Structure2:1121-1123)。
如本文中所使用的，术语“培养物”是指，将细胞(例如，本发明的经修饰的间充质干细胞)在培养基中培养后获得的产物。
如本文中所使用的，术语“培养上清”是指，对细胞(例如，本发明的经修饰的间充质干细胞)进行培养而得到的不含细胞本身的培养液。因而，例如可以通过在培养后分离去除细胞成分来得到可用于本发明的培养上清。该培养上清也可以经过其他处理，例如离心、浓缩、溶剂的置换、透析、冷冻、干燥、冷冻干燥、稀释、脱盐、保存等。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“启动子”具有本领域技术人员公知的含义，其是指一段位于基因的上游能启动下游基因表达的非编码核苷酸序列。组成型(constitutive)启动子是这样的核苷酸序列：当其与编码或者限定基因产物的多核苷酸可操作地相连时，在细胞的大多数或者所有生理条件下，其导致细胞中基因产物的产生。诱导型启动子是这样的核苷酸序列，当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当对应于所述启动子的诱导物在细胞中存在时，其导致所述基因产物在细胞内产生。组织特异 性启动子是这样的核苷酸序列：当可操作地与编码或者限定基因产物的多核苷酸相连时，基本上只有当细胞是该启动子对应的组织类型的细胞时，其才导致在细胞中产生基因产物。
如本文中所使用的，术语“药学上可接受的载体或赋形剂”是指，在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，离子强度增强剂，维持渗透压的试剂，延迟吸收的试剂，稀释剂，佐剂，防腐剂等。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。在某些实施方案中，所述药学上可接受的载体或赋形剂是无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液。
如本文中所使用的，术语“治疗”是指，治疗或治愈疾病(例如代谢病症)，延缓疾病(例如代谢病症)的症状的发作，和/或延缓疾病(例如代谢病症)的发展。
如本文中所使用的，术语“有效量”是指，足以获得或至少部分获得期望的效果的量。“治疗疾病有效量”是指，足以治愈或至少部分阻止已患有疾病(例如，代谢病症)的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄、体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“受试者”包括但不限于各种动物，例如哺乳动物，例如牛科动物、马科动物、羊科动物、猪科动物、犬科动物、猫科动物、兔科动物、啮齿类动物(例如，小鼠或大鼠)、非人灵长类动物(例如，猕猴或食蟹猴)或人。在某些实施方式中，所述受试者(例如人)患有代谢病症，或者，具有患代谢病症的风险。
发明的有益效果
与现有技术相比，本发明的经修饰的间充质干细胞具有显著的有利方面。特别地，本发明的经修饰的间充质干细胞展现出显著的协同效应，能够显著降低受试动物的血糖、血脂，减轻体重，且可安全地施用给人受试者，而不引发免疫原性反应。因此，本发明的经修饰的间充质干细胞能够用于治疗代谢病症，具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1为PCR扩增产物的电泳图谱，M代表Marker，kFG代表克隆的FGF21/GLP1-Fc基因片段。
图2为pCDH-FG质粒图谱。
图3为MSC和MSC-FG细胞表面标志流式检测图。
图4为MSC和MSC-FG细胞成脂和成骨分化染色结果图。
图5为Western blot检测MSC和MSC-FG细胞及细胞上清中FGF21和GLP1-Fc表达量的检测结果。
图6为MSC-FGF21、MSC-GLP1-Fc、MSC-FG的培养上清对葡萄糖刺激后INS-1细胞Insulin分泌量影响的检测结果。
图7为MSC-FGF21、MSC-GLP1-Fc、MSC-FG的培养上清对固醇代谢基因SREBP1C mRNA表达水平的影响的检测结果。
图8为糖尿病模型鼠空腹体重趋势图，Liraglutide为药物阳性对照组，箭头代表给予药物和细胞的时间点，*代表和Con组相比差异显著，**代表和Con组相比差异极显著。
图9为糖尿病模型鼠空腹血糖趋势图，Liraglutide为药物阳性对照组，箭头代表给予药物和细胞的时间点，*代表和Con组相比差异显著，**代表和Con组相比差异极显著。
图10为细胞治疗28天后不同实验组糖尿病模型鼠体型对比图，Liraglutide为药物 阳性对照组。
图11为细胞治疗28天后不同实验组糖尿病模型鼠肝脏和腹部皮下脂肪HE染色图，Liraglutide为药物阳性对照组。
图12为细胞治疗28天后不同实验组糖尿病模型鼠血清中胰岛素水平检测柱状图，Liraglutide为药物阳性对照组。
图13为细胞治疗28天后不同实验组糖尿病模型鼠血清中血脂水平检测柱状图，Liraglutide为药物阳性对照组。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2018081616-appb-000001
Figure PCTCN2018081616-appb-000002
Figure PCTCN2018081616-appb-000003
Figure PCTCN2018081616-appb-000004
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.FGF21/GLP1-Fc修饰的间充质干细胞的制备
1.1自体脂肪干细胞分离培养
采用混合胶原酶消化法分离培养脂肪间充质干细胞(Adipose derived mesenchymal stem cells,AD-MSCs)，具体方法如下：
将吸脂手术吸取的健康成人脂肪组织，转移至50mL离心管中，加入PBS充分洗涤，1500rpm，离心5分钟，获取上层脂肪组织。按照1:1:1比例将I、II及IV型胶原酶混合，配制为0.2％混合胶原酶，并按脂肪组织:胶原酶＝1:1的比例将脂肪组织加入混合胶原酶消化液中，置于37℃摇床中消化脂肪组织30分钟。将消化好的脂肪组织立刻加入10％FBS的α-MEM细胞培养基(购自Gibco)，1500rpm，离心10分钟，沉降细胞及组织团块。用α-MEM完全培养基重悬细胞，通过100μm孔径的尼龙网去除未消化的组织。将细胞接种于培养瓶，置于37℃，饱和湿度，5％CO 2培养箱中静止培养。2天后，将未贴壁的细胞倒掉，PBS轻轻洗一遍，加入干细胞完全培养基，待细胞克隆长至80％融合时，0.05％胰酶消化传代至新培养瓶中。选择P3代细胞，用0.05％胰酶消化，PBS洗两遍后分别用小鼠抗人CD11b-PE、CD45-PE、HLA-DR-PE、CD73-PE、CD90-PE、CD105-PE、CD34-FITC及CD19-FITC抗体标记5×10 5个MSCs，室温避光放置30min，再用PBS洗两遍后，4％多聚甲醛固定，FACS检测。鉴定合格的细胞冻存于液氮罐中，用时复苏并做后期处理。
1.2FGF21/GLP1-Fc基因的克隆与载体构建
由中美泰和生物技术(北京)有限公司全基因合成编码FGF21/GLP1-Fc的DNA分子，其中，FGF21(SEQ ID NO:2，编码序列为SEQ ID NO:3)与GLP1-Fc融合蛋白 (SEQ ID NO:10，编码序列为SEQ ID NO:11)通过T2A序列(SEQ ID NO:12，编码序列为SEQ ID NO:13)连接，GLP1-Fc融合蛋白从N端至C端由GLP1(SEQ ID NO:5)、接头(SEQ ID NO:9)及IgG4-Fc(SEQ ID NO:7)组成，并且其N端连接有信号肽(SEQ ID NO:22)。该编码FGF21/GLP1-Fc的DNA分子，命名为pGSI-seq，其核苷酸序列如SEQ ID NO:15所示，FGF21/GLP1-Fc的氨基酸序列如SEQ ID NO:14所示。
以100ng/μl pGSI-seq为模板，用引物对kspFGF21BamHIF(5’→3’:CGCGGATC CGCCACCATGGACTCGGACGAGACC)+IgG4FcSalIR(5’→3’:ACGCGTCGACTC ATTTACCCGGAGACAG)扩增目的片段kFG(1578bp)，并通过琼脂糖凝胶电泳分析扩增产物，其结果如图1所示，正反向引物分别引入BamHI和SalI酶切位点。将PCR产物切胶回收后，用BamHI和SalI双酶切，将重组慢病毒载体质粒pCDH-EF1(购自Addgene)也用BamHI和SalI双酶切，酶切产物胶回收后用T4DNA连接酶连接，4℃连接过夜，转化DH5α感受态细胞，取100μL菌液涂布至含有氨苄抗性的LB板上，37℃过夜培养。挑取单克隆进行菌落PCR，将阳性克隆送样测序，保存测序结果正确的克隆并提取质粒，命名为pCDH-FG，该质粒示意图如图2所示。
1.3携带FGF21/GLP1-Fc的慢病毒载体的制备
从液氮中取出1支冻存的293T细胞(东北农业大学Lab217胚胎工程实验室赠予)迅速放到37℃水浴中直至冰块消失，逐滴加入含有5ml预热培养基的15ml离心管中，1200rpm离心3min，弃上清，用293T培养基(10％FBS+DMEM)重悬细胞接种至150mm培养皿中，37℃、5％CO 2饱和湿度培养。待细胞汇合度达90％以上时，弃去旧培养基，加入5ml灭菌PBS溶液，轻轻晃动，洗涤细胞后弃去PBS溶液，加入2ml0.25％Trypsin-EDTA消化液，消化1-2min直到细胞完全消化下来。加入含血清的培养基终止消化，细胞悬液1200rpm离心3min，离心所得细胞用培养基重悬，每个150mm培养皿细胞接种1.2×10 7细胞用于包装慢病毒，37℃、5％CO 2饱和湿度培养，20ml培养基/皿。
转染前2h，将293T细胞培养基更换为18ml DMEM培养基，向A灭菌离心管中加入1ml预热的DMEM培养基，然后加入上述1.2中所制备的pCDH-FG质粒、pHelper1质粒和pHelper2质粒(pCDH-FG:pHelper1:pHelper2＝1:1:1，共54μg，pHelper1和pHelper2质粒为慢病毒包装的辅助质粒，由东北农业大学Lab217胚胎工程实验室赠予)，混合均匀。向B灭菌离心管中加入1ml预热的DMEM培养基，然后加入108μl Lipofectamin 2000(购自Invitrogen)溶液，混合均匀。A管和B管在室温下温育5分 钟。将B管中的液体成滴的加入到A管中，混合均匀，室温孵育20min，以便形成DNA-脂质体转染复合物。
将DNA-脂质体混合液转移至预先换液的293T细胞中，混匀，37℃、5％CO 2饱和湿度培养。培养6-8h后吸弃含有转染混和物的培养基，每皿细胞加入20ml预热的含5％FBS的DMEM培养基，37℃、5％CO 2饱和湿度培养。换液后分别在24h和48h，收集上清液暂存储于4℃，并换20ml新鲜培养基。将收集到的液体4℃、3500rpm离心15min，弃沉淀，将上清用Millipore蛋白超滤柱(10KD)进行浓缩，从而获得携带FGF21/GLP1-Fc的慢病毒载体(Lenti-FGF21/GLP1-Fc)；同时进行病毒滴度测定，根据测定结果将病毒稀释为1×10 8TU/ml，分装后的病毒置于-80℃保存。
1.4间充质干细胞的基因修饰
复苏预先冻存的P3代脂肪间充质干细胞至一个150mm培养皿，20ml无血清培养基37℃、5％CO 2饱和湿度培养。待复苏细胞长满后，0.05％胰蛋白酶消化细胞，用含血清的培养基终止消化，细胞悬液800rpm离心5min，离心所得细胞用MSC无血清培养基(购自Bioind)重悬。
每个150mm培养皿细胞接种2-2.5×10 6细胞，接种后的第二天吸取细胞的培养基弃掉，更换为无血清的α-MEM培养基，20ml培养基/皿，加入16μl Polybrene(购自Sigma)，按照40MOIs感染复数加入1.3中获得的Lenti-FGF21/GLP1-Fc慢病毒(滴度为1×10 8U/ml)，37℃、5％CO 2饱和湿度培养6-8h。6-8小时后弃掉含有病毒的α-MEM培养基，更换为无血清培养基，37℃、5％CO 2饱和湿度继续培养2-3天。待基因编辑后的细胞长满，0.05％胰蛋白酶消化细胞，用含血清的培养基终止消化，细胞悬液800rpm离心5min，离心所得细胞用无血清培养基重悬，按照1:6的传代比例进行传代，无血清培养基37℃、5％CO 2培养3天。FGF21/GLP1-Fc基因修饰的间充质干细胞命名为MSC-FG。
另外，按照上述1.1-1.4中描述的方法制备获得FGF21单基因修饰的间充质干细胞(MSC-FGF21)以及GLP1-Fc单基因修饰的间充质干细胞(MSC-GLP1-Fc)。
1.5MSC-FG生物学功能的验证
细胞表型鉴定：选择P6代MSC和MSC-FG，用0.05％胰酶消化，PBS洗两遍后用小鼠抗人CD14-PerCp-Cy5.5、CD19-PE、CD34-PE、CD45-PE或CD45-FITC、HLA-DR-PE、CD73-PE、CD90-PE及CD105-PE(Becton，Dickinson and Company)抗体标记MSC，每个待测样品约1×10 6个细胞，室温避光孵育30min，PBS洗两遍后，用2％ 多聚甲醛固定，用流式细胞仪检测，结果如图3所示。结果显示Lenti-FGF21/GLP1-Fc慢病毒感染后的MSC细胞(MSC-FG)其表面marker仍然保持MSC细胞的特性，即CD73、CD90和CD105呈阳性(>90％)，CD14、CD19、CD34、CD45和HLA-DR呈阴性(<1％)。
定向分化诱导：即成脂和成骨诱导，取P6代MSC和MSC-FG，用0.05％胰酶消化，按照2×10 5个/孔的细胞密度铺于12孔板中，第2天更换为成脂诱导培养基和成骨诱导培养基(购买自BI公司)，之后每隔2天换液，成脂诱导于18天后进行油红O染色，成骨诱导于23天后进行茜素红-S染色，结果如图4所示。结果显示，Lenti-FGF21-GLP1-Fc慢病毒感染后的MSC细胞(MSC-FG)仍然具有成脂和成骨分化能力。
FGF21及GLP1-Fc表达情况：取MSC和MSC-FG细胞裂解液，以及MSC和MSC-FG浓缩10倍后的细胞培养上清进行15％SDS-PAGE凝胶电泳，湿转至PVDF膜。5％脱脂牛奶封闭，加入抗体FGF21(1:3000，Abcam，检测FGF21蛋白表达)和抗体IgG4-HRP(1:3000，Abcam，检测GLP1-Fc蛋白表达)，4℃过夜；TBST洗膜三次，二抗孵育1h；TBST洗膜三次后，用化学发光试剂盒显色，用计算机图像分析系统曝光，结果如图5所示。结果显示，FGF21/GLP1-Fc双基因修饰的MSC细胞(MSC-FG)上清中有高量的FGF21和GLP1-Fc存在，细胞内检测到的较少。
实施例2.FGF21/GLP1-Fc修饰的间充质干细胞的体外生物学活性评价
2.1MSC-FG对葡萄糖刺激的胰岛素分泌的影响
预先准备：在100mm培养中，分别培养实施例1中所获得的MSC、MSC-FG、MSC-FGF21及MSC-GLP1-Fc细胞，当细胞汇合度达70％-80％时，吸弃原有MSC无血清培养基，10mlα-MEM培养基，37℃、5％CO 2饱和湿度继续培养48h。收集四种细胞的培养上清，用超滤柱浓缩10倍，4℃保存备用。长期保存需放置于-80℃冰箱。
从液氮中取出1支冻存的INS-1细胞(大鼠胰岛素瘤细胞，由军事医学科学院赠予)迅速放入37℃水浴中直至冰块消失，逐滴加入含有5ml预热培养基的15ml离心管中，1200rpm离心3min，弃上清，用INS-1培养基(10％FBS+1mM丙酮酸钠+2mM谷氨酰胺+50μM巯基乙醇+1640培养基)重悬细胞接种至100mm培养皿中，37℃、5％CO 2饱和湿度培养。一般情况下两到三天传一次，传代比例1:3。细胞长满后，弃去旧培养基，加入2ml灭菌PBS溶液，轻轻晃动，洗涤细胞后弃去PBS溶液，加入2ml0.25％Trypsin-EDTA消化液，消化2-3min直到细胞完全消化下来。加入含血清的培养 基终止消化，细胞悬液1200rpm离心3min，离心所得细胞用培养基重悬，在6孔板每孔中接种为1×10 6个细胞，加入2ml INS-1培养基，37℃、5％CO 2饱和湿度培养，待细胞密度达70％-80％时即可用于功能验证。
吸弃原有6孔板中INS-1培养基，加入2ml预热的低糖KRBH缓冲液(129mM NaCl+4.8mM KCl+1.2mM KH2PO4+1.2mM MgSO4+2mM CaCl2+20mM HEPES+24mM NaHCO3+0.2％BSA+0.4mg/ml glucose)饥饿处理INS-1细胞两小时。吸弃低糖KRBH缓冲液，加入2ml预热的高糖KRBH缓冲液(129mM NaCl+4.8mM KCl+1.2mM KH2PO4+1.2mM MgSO4+2mM CaCl2+20mM HEPES+24mM NaHCO3+0.2％BSA+3mg/ml glucose)，分别加入500μl预先收集的浓缩的MSC、MSC-FGF21、MSC-GLP1-Fc和MSC-FG的培养上清，37℃、5％CO 2饱和湿度培养2小时。
收集上清，利用放射性免疫原法检测检测培养液中Insulin含量，结果如图6所示。结果显示，FGF21/GLP1-Fc双基因修饰的MSC细胞(MSC-FG)的培养上清能够显著刺激INS-1分泌表达胰岛素，且其值显著高于FGF21单基因修饰的MSC(MSC-FGF21)的培养上清以及GLP1-Fc单基因修饰的MSC(MSC-GLP1-Fc)的培养上清。由此可见，FGF21/GLP1-Fc双基因修饰的MSC细胞在调节胰岛素分泌方面展现出显著的协同作用。
2.2MSC-FG对固醇代基因SREBP1c表达水平的影响
预先准备：在100mm培养中，分别培养实施例1中所获得的MSC、MSC-FG、MSC-FGF21及MSC-GLP1-Fc细胞，当细胞汇合度达70％-80％时，吸弃原有MSC无血清培养基，10mlα-MEM培养基，37℃、5％CO 2饱和湿度继续培养48h。收集四种细胞的培养上清，4℃保存备用。长期保存需放置于-80℃冰箱。
从液氮中取出的1支冻存的HePG2细胞(人肝癌细胞，由军事医学科学院赠予)迅速的放37℃水浴中直到冰块消失，逐滴加入含有5ml预热培养基的15ml离心管中，1200rpm离心3min，弃上清，用HePG2培养基(10％FBS+DMEM)重悬细胞接种至100mm培养皿中，37℃、5％CO 2饱和湿度培养。一般情况下两到三天传一次，传代比例1:6。细胞长满后，弃去旧培养基，加入2ml灭菌PBS溶液，轻轻晃动，洗涤细胞后弃去PBS溶液，加入2ml 0.25％Trypsin-EDTA消化液，消化2-3min直到细胞完全消化下来。加入含血清的培养基终止消化，细胞悬液1200rpm离心3min，离心所得细胞用培养基重悬，在6孔板每孔中接种为1×10 6-2×10 6个细胞，加入2ml培养基，37℃、 5％CO 2饱和湿度培养，待细胞密度达70％-80％时即可用于功能验证。
吸弃原有6孔板中HePG2培养基，加入2ml预先收集的MSC、MSC-FGF21、MSC-GLP1-Fc和MSC-FG的培养上清，37℃、5％CO 2饱和湿度培养24小时。消化细胞，终止离心后，收集细胞用于TRIzol法RNA提取，测量RNA浓度后，利用反转录试剂盒(All-in-One cDNA Synthesis SuperMix)将500ng总RNA反转为cDNA。
进行RT-qPCR检测，根据Takara的荧光实时定量试剂盒(
Figure PCTCN2018081616-appb-000005
Premix Ex Taq TM)说明书进行。引物序列(5’→3’)：SEBP1C-F：CACTGTGACCTCGCAGATCC；S EBP1C-R：ATAGGCAGCTTCTCCGCATC；β-Actin-F：CCTGGCACCCAGCACAA T；β-Actin-R：GGGCCGGACTCGTCATAC
结果如图7所示，FGF21/GLP1-Fc双基因修饰的MSC细胞(MSC-FG)的培养上清能够显著抑制SREBP1c(肝组织固醇调节元件结合蛋白-1C)的mRNA表达水平，其抑制活性显著高于MSC-FGF21及MSC-GLP1-Fc的培养上清。由此可见，FGF21/GLP1-Fc双基因修饰的MSC细胞在调节脂代谢方面展现出显著的协同作用。
实施例3.FGF21/GLP1-Fc修饰的间充质干细胞的体内生物学活性评价
3.1实验分组
选取30只5周龄的雄性糖尿病模型鼠BKS.Cg-Dock7m+/+Leprdb/Nju(购自南京大学模式动物研究所)。实验分为：对照组(生理盐水)、利拉鲁肽药物组(药物)、MSC组(细胞)、MSC-FGF21组(细胞)、MSC-FG组(细胞)和MSC-GLP1-Fc组(细胞)，共6个组，每组5只小鼠。
3.2治疗方案
利拉鲁肽药物组(Liraglutide)给药方式为皮下注射，其余各组给药方式均为腹腔注射，所有组均要求上午给药。
给药时间：细胞组要求每7天注射一次细胞，一共给予3次细胞。利拉鲁肽药物组要求每周给予两次药物。
各组给药剂量：
对照组(生理盐水组)：每只小鼠每次注射100μl生理盐水，腹腔注射。
细胞组：每只小鼠每次注射量为1×10 6细胞/100ul，每7天一次，腹腔注射。
利拉鲁肽药物组：药物作用浓度为0.5mg/Kg，每3-4天一次，皮下注射。
3.3检测指标
空腹血糖和空腹体重的测量：注射细胞当天晚上小鼠进行饥饿处理(断食、水正常供应)，小鼠饥饿处理12小时，次日早上进行血糖测量和体重称重。根据小鼠平均血糖和体重绘制体重变化曲线。
血清生化指标检测：治疗28天后，小鼠眼球取血，3000rpm离心10min分离血清，样品送至北京北方生科医学技术有限公司进行甘油三脂(TG)、总胆固醇(TG)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)和胰岛素(INS)指标检测。
动物组织苏木素-伊红(HE)染色：治疗28天后，小鼠断颈处死后，做腹部正中切口，充分暴露腹部，取出小鼠肝脏、和腹部皮下脂肪，以10倍体积的4％多聚甲醛固定24小时，并进行石蜡包埋、苏木素-伊红(HE)染色。
3.4实验结果
对BKS糖尿病小鼠进行3次细胞治疗，并从第一次治疗开始每周测量一次小鼠空腹血糖和体重，停止治疗后继续观测两周数据。小鼠平均体重和血糖变化曲线分别如图8及图9所示。结果显示，治疗28天后，MSC-FG细胞治疗组小鼠与对照组小鼠相比体重明显减轻(*P<0.05)，其数值与阳性对照药物Liraglutide相近，且明显优于MSC-FGF21和MSC-GLP1-Fc组(图8)；同时，MSC-FG细胞治疗组小鼠与对照组小鼠相比空腹血糖差异极显著(*P<0.01)，且在第三次细胞治疗就呈现明显的治疗效果，其趋势与阳性对照药物Liraglutide相同，且明显优于MSC-FGF21和MSC-GLP1-Fc组(图9)。
治疗28天后，MSC-FG细胞治疗组小鼠的体型也明显小于对照小鼠体型，且腹部皮下脂肪含量明显减少(图10)。肝组织病理切片HE染色显示(图11)，Con小鼠肝脏呈现严重的脂肪变性，肝组织为脂肪细胞填充，肝细胞大片坏死，而经MSC-FG细胞治疗后肝脏脂肪变性明显改善，纤维化程度下降。腹部皮下脂肪HE染色显示(图11)，Con组小鼠脂肪细胞过度膨大，且细胞核较不规则；经MSC-FG治疗后，脂肪细胞体积显著减小。
治疗28天后，经小鼠血清生化检测发现，经MSC-FG治疗后小鼠在进食后，血清中胰岛素含量明显上升，甚至明显优于阳性对照药物Liraglutide(图12)；并且，经MSC-FG治疗后，小鼠血清中TC(总胆固醇)，LDL(低密度脂蛋白)和HDL(高密度脂蛋白)等物质均显著下降，且要优于MSC-FGF21和MSC-GLP1-Fc组(图13)。
由此可见，MSC-FGF1可明显降低小鼠体重和血糖，同时能够降低血脂含量，缓解脂肪肝发生，改善血脂代谢异常，修复胰岛细胞功能，且其疗效要优于MSC-FGF21和 MSC-GLP1-Fc组，具备显著的协同作用，在改善血脂方面也明显优于降糖药物Liraglutide。因此，本发明的经修饰的MSC特别适用于治疗代谢病症。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (11)

  1. 一种经修饰的间充质干细胞，其能够表达：(1)第一蛋白，其选自：FGF21或其变体，或包含所述FGF21或其变体的第一融合蛋白；和，(2)第二蛋白，其选自：GLP-1或其变体，或包含所述GLP-1或其变体的第二融合蛋白；
    优选地，所述FGF21的变体与其所源自的序列相比，具有一个或多个氨基酸的替换、插入或删除，或者具有至少80％，85％，90％，95％，96％，97％，98％，或99％的序列同一性，并且保留了FGF21活性；
    优选地，所述GLP-1的变体与其所源自的序列相比，具有一个或多个氨基酸的替换、插入或删除，或者具有至少80％，85％，90％，95％，96％，97％，98％，或99％的序列同一性，并且保留了GLP-1活性；
    优选地，所述FGF21具有如SEQ ID NO:1所示的氨基酸序列；
    优选地，所述GLP-1具有如SEQ ID NO:4所示的氨基酸序列；
    优选地，所述经修饰的间充质干细胞表达：(1)第一蛋白，其选自：SEQ ID NO:2所示的氨基酸序列，或包含SEQ ID NO:2所示的氨基酸序列的第一融合蛋白；和，(2)第二蛋白，其选自：SEQ ID NO:5所示的氨基酸序列，或包含SEQ ID NO:5所示的氨基酸序列的第二融合蛋白；
    优选地，所述经修饰的间充质干细胞表达：第一蛋白，其具有如SEQ ID NO:2所示的氨基酸序列；和，第二蛋白，其具有如SEQ ID NO:10所示的氨基酸序列；
    优选地，所述经修饰的间充质干细胞能够分泌所述第一蛋白和第二蛋白。
  2. 权利要求1所述的经修饰的间充质干细胞，其中，所述第一融合蛋白还包含第一额外多肽；
    优选地，所述第一额外多肽能够延长所述第一融合蛋白在体内的半衰期；
    优选地，所述第一额外多肽选自免疫球蛋白Fc结构域(例如，人免疫球蛋白Fc结构域，例如人IgG的Fc结构域)、血清白蛋白(例如，人血清白蛋白(HSA))、白蛋白结合多肽(例如，HSA结合多肽)、转铁蛋白，及前述任一项的功能性片段；
    优选地，所述第一额外多肽为人免疫球蛋白Fc结构域，例如人IgG的Fc结构域，例如人IgG1、IgG2、IgG3或IgG4的Fc结构域；优选地，所述第一额外多肽具有如 SEQ ID NO:7所示的氨基酸序列；
    优选地，所述第一额外多肽任选地通过接头与所述FGF21或其变体融合。
  3. 权利要求1或2所述的经修饰的间充质干细胞，其中，所述第二融合蛋白还包含第二额外多肽；
    优选地，所述第二额外多肽能够延长所述第二融合蛋白在体内的半衰期；
    优选地，所述第二额外多肽选自免疫球蛋白Fc结构域(例如，人免疫球蛋白Fc结构域，例如人IgG的Fc结构域)、血清白蛋白(例如，人血清白蛋白(HSA))、白蛋白结合多肽(例如，HSA结合多肽)、转铁蛋白，及前述任一项的功能性片段；
    优选地，所述第二额外多肽为人免疫球蛋白Fc结构域，例如人IgG的Fc结构域，例如人IgG1、IgG2、IgG3或IgG4的Fc结构域；优选地，所述第二额外多肽具有如SEQ ID NO:7所示的氨基酸序列；
    优选地，所述第二额外多肽任选地通过接头与所述GLP-1或其变体融合。
  4. 权利要求1-3任一项所述的经修饰的间充质干细胞，其中，所述经修饰的间充质干细胞包含：
    (1)第一外源核酸，其包含编码所述第一蛋白的核苷酸序列；和
    (2)第二外源核酸，其包含编码所述第二蛋白的核苷酸序列；
    优选地，所述第一外源核酸和/或第二外源核酸可操作地连接至启动子(例如，组成型启动子、组织特异性启动子或诱导型启动子)；
    优选地，所述第一外源核酸和/或第二外源核酸整合于所述间充质干细胞的基因组中；或者，所述第一外源核酸和/或第二外源核酸独立于所述间充质干细胞的基因组。
  5. 权利要求1-4任一项所述的经修饰的间充质干细胞，其中，所述间充质干细胞来源于脂肪组织、脐带、骨髓或脐血；
    优选地，所述间充质干细胞来源于脂肪组织。
  6. 一种培养物，其包含权利要求1-5任一项所述的经修饰的间充质干细胞，以及培养基；
    优选地，所述培养基为含有或不含有血清的α-MEM培养基。
  7. 一种培养上清，其为权利要求6所述的培养物的上清液；
    优选地，所述培养上清不含所述经修饰的间充质干细胞；
    优选地，所述培养上清不含血清。
  8. 制备权利要求7所述的培养上清的方法，其包括以下步骤：
    (1)对权利要求1-5任一项所述的经修饰的间充质干细胞进行培养；和
    (2)回收步骤(1)获得的培养物的上清液；
    优选地，所述方法还包括：(3)对步骤(2)获得的上清液进行处理，所述处理选自离心、浓缩、溶剂的置换、透析、冷冻、干燥、冷冻干燥、稀释、脱盐、保存，及其任意组合；
    优选地，在步骤(1)中使用含有或不含有血清的α-MEM培养基对所述经修饰的间充质干细胞进行培养。
  9. 一种药物组合物，其包含权利要求1-5任一项所述的经修饰的间充质干细胞、权利要求6所述的培养物或权利要求7所述的培养上清；
    优选地，所述药物组合物包含治疗有效量的所述经修饰的间充质干细胞、培养物或培养上清；
    优选地，所述药物组合物为注射剂(包括注射液、冻干粉剂)；
    优选地，所述药物组合物还包含药学上可接受的载体或赋形剂；优选地，所述药物组合物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液；
    优选地，所述药物组合物任选地还包含另外的药学活性剂；优选地，所述另外的药学活性剂选自抗糖尿病药物、抗肥胖症药物、抗高血压药物、抗动脉粥样硬化药物和降脂药物。
  10. 权利要求1-5任一项所述的经修饰的间充质干细胞、权利要求6所述的培养物、权利要求7所述的培养上清或权利要求9所述的药物组合物用于在受试者中治疗代谢病症 的用途，或者用于制备在受试者中治疗代谢病症的药物的用途；
    优选地，所述药物包含治疗有效量的所述经修饰的间充质干细胞培养物、培养上清或药物组合物；
    优选地，所述药物为注射剂(包括注射液、冻干粉剂)；
    优选地，所述药物还包含药学上可接受的载体或赋形剂；优选地，所述药物包含药学上可接受的无菌等渗水性或非水性溶液(例如，平衡盐溶液或生理盐水)、分散液、悬浮液或乳液；
    优选地，所述药物任选地还包含另外的药学活性剂；优选地，所述另外的药学活性剂选自抗糖尿病药物、抗肥胖症药物、抗高血压药物、抗动脉粥样硬化药物和降脂药物；
    优选地，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)；
    优选地，所述受试者是哺乳动物，例如人。
  11. 一种治疗代谢病症的方法，其包括向有此需要的受试者施用权利要求1-5任一项所述的经修饰的间充质干细胞、权利要求6所述的培养物、权利要求7所述的培养上清或权利要求9所述的药物组合物；
    优选地，所述方法还包括施用另外的药学活性剂，所述另外的药学活性剂选自抗糖尿病药物、抗肥胖症药物、抗高血压药物、抗动脉粥样硬化药物和降脂药物；优选地，所述另外的药学活性剂在施用所述经修饰的间充质干细胞、培养上清或药物组合物之前、同时或之后施用；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述代谢病症选自肥胖、I型和II型糖尿病、血脂异常(例如，高脂血症)、非酒精性脂肪肝病(NAFLD)、非酒精性脂肪性肝炎(NASH)、胰岛素耐受、高胰岛素血症、葡萄糖不耐受、高血糖、代谢综合征、动脉粥样硬化、冠心病、高血压和其他的代谢病症，及这些疾病的继发性并发症(例如，糖尿病并发症，如视网膜病、神经病、肾病以及延缓的创伤愈合)。
PCT/CN2018/081616 2017-12-19 2018-04-02 一种双基因修饰的干细胞及其用途 WO2019119673A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/954,763 US11752173B2 (en) 2017-12-19 2018-04-02 FGF21 and GLP1 double gene-modified mesenchymal stem cell and use in treating a metabolic disease
US18/357,639 US20240100100A1 (en) 2017-12-19 2023-07-24 FGF21 and GLP1 DOUBLE GENE-MODIFIED MESENCHYMAL STEM CELL AND USE IN TREATING A METABOLIC DISEASE

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711374615.0 2017-12-19
CN201711374615 2017-12-19

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/954,763 A-371-Of-International US11752173B2 (en) 2017-12-19 2018-04-02 FGF21 and GLP1 double gene-modified mesenchymal stem cell and use in treating a metabolic disease
US18/357,639 Continuation US20240100100A1 (en) 2017-12-19 2023-07-24 FGF21 and GLP1 DOUBLE GENE-MODIFIED MESENCHYMAL STEM CELL AND USE IN TREATING A METABOLIC DISEASE

Publications (1)

Publication Number Publication Date
WO2019119673A1 true WO2019119673A1 (zh) 2019-06-27

Family

ID=66984409

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/081616 WO2019119673A1 (zh) 2017-12-19 2018-04-02 一种双基因修饰的干细胞及其用途

Country Status (3)

Country Link
US (2) US11752173B2 (zh)
CN (2) CN111518770B (zh)
WO (1) WO2019119673A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11752173B2 (en) 2017-12-19 2023-09-12 Beijing Jiyuan Biological Technology Co., Ltd. FGF21 and GLP1 double gene-modified mesenchymal stem cell and use in treating a metabolic disease
EP4006058A4 (en) * 2019-07-25 2023-11-01 Ampsource Biopharma Shanghai Inc. FC FUSION PROTEIN FGF21, FC FUSION PROTEIN GLP-1, COMBINATION THERAPEUTIC AGENT COMPRISING THE SAME AND USE THEREOF

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110423728A (zh) * 2019-08-27 2019-11-08 吉林大学 一种间充干细胞的制备方法
CN110749579A (zh) * 2019-08-29 2020-02-04 西安医学院 一种通过免疫荧光检测药物对胰岛细胞摄脂影响的方法
CN111849911A (zh) * 2020-06-28 2020-10-30 北京纽莱福生物科技有限公司 一种经修饰的干细胞及其制备方法和用途
CN112175978A (zh) * 2020-09-22 2021-01-05 北京贝来生物科技有限公司 用于治疗高级别脑胶质瘤的基因修饰间充质干细胞及其制备方法
CN112143705B (zh) * 2020-09-23 2022-09-06 北京华奥玄德生物医药科技有限公司 一种双基因修饰的干细胞及其用途
CN112138025A (zh) * 2020-09-27 2020-12-29 东北师范大学 一种长效glp-1基因修饰的干细胞制剂、制备方法及其应用
CN114525254A (zh) * 2020-10-30 2022-05-24 未来智人再生医学研究院(广州)有限公司 一种表达fgf-21的多能干细胞或其衍生物及应用
CN112516168B (zh) * 2020-12-11 2022-07-26 中国人民解放军军事科学院军事医学研究院 用于干预应激性认知障碍的间充质干细胞
CN112553165A (zh) * 2020-12-11 2021-03-26 北京双因生物科技有限公司 以修饰的msc培养nk细胞的方法
CN112941032A (zh) * 2021-02-23 2021-06-11 北京双因生物科技有限公司 以修饰的msc培养记忆型t细胞的方法
CN117586423A (zh) * 2021-07-14 2024-02-23 北京质肽生物医药科技有限公司 用于代谢病症的融合多肽
CN113730439A (zh) * 2021-09-09 2021-12-03 陕西中鸿瑞康健康管理有限公司 能降低甘油三酯的干细胞因子冻干粉及其制备方法和应用
CN113801853B (zh) * 2021-11-19 2022-03-15 山东兴瑞生物科技有限公司 Exendin-4融合基因修饰的MSC及其应用
CN114381425A (zh) * 2021-12-10 2022-04-22 深圳市弘际生物科技有限责任公司 经修饰的间充质干细胞及其制备方法和药用组合物

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102802657A (zh) * 2009-06-11 2012-11-28 诺沃-诺迪斯克有限公司 用于治疗2型糖尿病的glp-1和fgf21组合
CN104024273A (zh) * 2011-09-26 2014-09-03 诺华股份有限公司 用于治疗代谢疾病的双功能蛋白
CN104805058A (zh) * 2014-01-23 2015-07-29 上海欧易生物医学科技有限公司 一种稳定表达外源性glp-1基因的人脐带源间充质干细胞株

Family Cites Families (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1341320C (en) 1986-05-05 2001-11-20 Joel Habener Insulinotropic hormone
US5470845A (en) 1992-10-28 1995-11-28 Bristol-Myers Squibb Company Methods of using α-phosphonosulfonate squalene synthetase inhibitors including the treatment of atherosclerosis and hypercholesterolemia
US5620997A (en) 1995-05-31 1997-04-15 Warner-Lambert Company Isothiazolones
AU6966696A (en) 1995-10-05 1997-04-28 Warner-Lambert Company Method for treating and preventing inflammation and atherosclerosis
UA65549C2 (uk) 1996-11-05 2004-04-15 Елі Ліллі Енд Компані Спосіб регулювання ожиріння шляхом периферійного введення аналогів та похідних glp-1 (варіанти) та фармацевтична композиція
GB9713739D0 (en) 1997-06-27 1997-09-03 Karobio Ab Thyroid receptor ligands
AU767456B2 (en) 1998-07-06 2003-11-13 Bristol-Myers Squibb Company Biphenyl sulfonamides as dual angiotensin endothelin receptor antagonists
GB9828442D0 (en) 1998-12-24 1999-02-17 Karobio Ab Novel thyroid receptor ligands and method II
CA2434237C (en) 2000-12-07 2012-05-15 Eli Lilly And Company Glp-1 fusion proteins
AU2004251145C1 (en) 2003-06-12 2011-04-14 Eli Lilly And Company GLP-1 analog fusion proteins
EP1702067A1 (en) 2003-12-10 2006-09-20 Eli Lilly And Company Muteins of fibroblast growth factor 21
WO2006028595A2 (en) 2004-09-02 2006-03-16 Eli Lilly And Company Muteins of fibroblast growth factor 21
WO2006028714A1 (en) 2004-09-02 2006-03-16 Eli Lilly And Company Muteins of fibroblast growth factor 21
JP2008522617A (ja) 2004-12-14 2008-07-03 イーライ リリー アンド カンパニー 線維芽細胞成長因子２１の突然変異タンパク質
DE602006009631D1 (de) * 2006-05-10 2009-11-19 Biocompatibles Uk Ltd GLP-1 Peptide enthaltende kugelförmige Mikrokapseln, deren Produktion und deren Verwendung
KR101476472B1 (ko) 2007-03-30 2015-01-05 암브룩스, 인코포레이티드 변형된 ｆｇｆ-21 폴리펩티드 및 그 용도
EP3260129A1 (en) * 2007-08-03 2017-12-27 Eli Lilly and Company An fgf-21 compound and a glp-1 compound for use in the treatment of obesity
JOP20190083A1 (ar) 2008-06-04 2017-06-16 Amgen Inc بولي ببتيدات اندماجية طافرة لـfgf21 واستخداماتها
US20120294855A1 (en) 2009-11-03 2012-11-22 Eli Lilly & Company Glp-1 receptor agonist compounds for obstructive sleep apnea
US9023791B2 (en) 2010-11-19 2015-05-05 Novartis Ag Fibroblast growth factor 21 mutations
TWI593708B (zh) 2011-09-26 2017-08-01 諾華公司 治療代謝病症之融合蛋白質
MX2015002985A (es) * 2012-09-07 2015-06-22 Sanofi Sa Proteinas de fusion para el tratamiento de un sindrome metabolico.
AU2014236559B2 (en) * 2013-03-14 2020-03-12 Julie HUGHES Cholestosome vesicles for incorporation of molecules into chylomicrons
CN104250655A (zh) * 2014-08-29 2014-12-31 杭州市萧山区中医院 Bmp-2/vegf165双基因修饰的骨髓间充质干细胞及其制备方法
KR20240024362A (ko) 2014-10-24 2024-02-23 브리스톨-마이어스 스큅 컴퍼니 변형된 fgf-21 폴리펩티드 및 그의 용도
CN111518770B (zh) 2017-12-19 2023-01-06 北京吉源生物科技有限公司 一种表达glp1和fgf21的干细胞及其用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102802657A (zh) * 2009-06-11 2012-11-28 诺沃-诺迪斯克有限公司 用于治疗2型糖尿病的glp-1和fgf21组合
CN104024273A (zh) * 2011-09-26 2014-09-03 诺华股份有限公司 用于治疗代谢疾病的双功能蛋白
CN104805058A (zh) * 2014-01-23 2015-07-29 上海欧易生物医学科技有限公司 一种稳定表达外源性glp-1基因的人脐带源间充质干细胞株

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11752173B2 (en) 2017-12-19 2023-09-12 Beijing Jiyuan Biological Technology Co., Ltd. FGF21 and GLP1 double gene-modified mesenchymal stem cell and use in treating a metabolic disease
EP4006058A4 (en) * 2019-07-25 2023-11-01 Ampsource Biopharma Shanghai Inc. FC FUSION PROTEIN FGF21, FC FUSION PROTEIN GLP-1, COMBINATION THERAPEUTIC AGENT COMPRISING THE SAME AND USE THEREOF

Also Published As

Publication number Publication date
CN111518770A (zh) 2020-08-11
CN111518770B (zh) 2023-01-06
CN109929806B (zh) 2020-05-08
US20240100100A1 (en) 2024-03-28
CN109929806A (zh) 2019-06-25
US11752173B2 (en) 2023-09-12
US20210085722A1 (en) 2021-03-25

Similar Documents

Publication Publication Date Title
WO2019119673A1 (zh) 一种双基因修饰的干细胞及其用途
RU2681857C2 (ru) Антитело, специфически связывающееся с glp-1r, и его слитый с glp-1 белок
TWI708613B (zh) 使用glp-1受體促效劑與抑胃肽受體(gipr)結合蛋白之偶聯物治療或改善代謝病症之方法
KR20140039322A (ko) 대사성 장애 및 질환의 치료를 위한 조성물, 용도 및 방법
JP2022078246A (ja) 組換え免疫細胞、作製方法、および使用方法
US20210388326A1 (en) Sphingosine kinase 1 and fusion protein comprising the same and use thereof
JPH01502669A (ja) 精製された血小板由来の成長因子及びその精製方法
TW201522379A (zh) Ｃｈｒｄｌ－１抗原結合蛋白及治療方法
US20230248780A1 (en) Cell-based gene therapy for neurodegenerative diseases
US20210196795A1 (en) Therapeutic use of vegf-c and ccbe1
KR20130094337A (ko) 개량형 이듀로네이트-2-설파타제 및 이의 용도
BG107214A (bg) Пептид, модулиращ тромбопоетиновия рецептор
CN115916831A (zh) 包含抗lag-3抗体和il-2的融合蛋白及其用途
KR20150140686A (ko) 신증후군 및 관련 병태의 치료 방법
US20040086507A1 (en) Antibody inhibiting vplf activity
WO2021228052A1 (zh) 生物大分子靶向特异性补体抑制剂及其制备方法与应用
WO2021128919A1 (zh) Cst1在预防和/或治疗肝脏免疫失调疾病中的应用
KR20210149593A (ko) 엔지니어링된 안지오텐신 변환 효소 ii 및 이의 용도
US20210403564A1 (en) Oncolytic virus expressing pd-1 binding protein and application of oncolytic virus
CN114686429B (zh) 一种激活nk细胞的肿瘤免疫治疗方法
KR101699567B1 (ko) ＪｕｎＢ의 발현 또는 활성 억제제를 유효성분으로 포함하는 면역질환의 예방 또는 치료용 조성물
CA3225321A1 (en) Ptprs in autoimmunity
JP2023509189A (ja) Ｒｓｐｏ１タンパク質およびその使用
KR20240040068A (ko) 메조텔린을 특이적으로 표적화하는 조작된 면역 세포 및 이의 용도
WO2023094507A1 (en) Improved ace2 fusion proteins

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18893054

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18893054

Country of ref document: EP

Kind code of ref document: A1