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WO2020009444A1 - Gène et produit de thérapie cellulaire utilisant la technologie de fusion cellulaire, et utilisation correspondante - Google Patents

Gène et produit de thérapie cellulaire utilisant la technologie de fusion cellulaire, et utilisation correspondante Download PDF

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Publication number
WO2020009444A1
WO2020009444A1 PCT/KR2019/008081 KR2019008081W WO2020009444A1 WO 2020009444 A1 WO2020009444 A1 WO 2020009444A1 KR 2019008081 W KR2019008081 W KR 2019008081W WO 2020009444 A1 WO2020009444 A1 WO 2020009444A1
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Prior art keywords
disease
cells
cell
diseases
muscle
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English (en)
Korean (ko)
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성정준
김기윤
전계선
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SNU R&DB Foundation
Seoul National University Hospital
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Seoul National University R&DB Foundation
Seoul National University Hospital
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Priority to CN202310900115.5A priority Critical patent/CN116870196A/zh
Priority to CN201980044907.9A priority patent/CN112384247B/zh
Priority to EP19831381.9A priority patent/EP3824894A4/fr
Priority to JP2021521927A priority patent/JP7300764B2/ja
Priority claimed from KR1020190079666A external-priority patent/KR102100490B1/ko
Publication of WO2020009444A1 publication Critical patent/WO2020009444A1/fr
Anticipated expiration legal-status Critical
Priority to JP2022062691A priority patent/JP2022104986A/ja
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    • 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
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to genes and cell therapies using cell fusion technology, and the use thereof. Specifically, hemagglutinin neuraminidase (HN) and F (fusion) genes are transduced into cells and overexpressed. Gene and cell therapeutics using cell fusion technology, thereby enhancing cell fusion with other cells and repairing cell damage and delivering normal genes through cell fusion with damaged or dying cells or cells with gene abnormalities. , And use in diseases associated with cell damage.
  • HN hemagglutinin neuraminidase
  • F fusion
  • Cell damage and cell death are generally progressed by cell damage and cell death.
  • Representative diseases in which cell damage and death are induced include neurodegenerative disease and muscular dystrophy.
  • neurodegenerative diseases including damage to the brain, spine and peripheral nerves, continue to increase.
  • the cause of neurodegenerative diseases is not yet clear.
  • the pathogenesis of each neurodegenerative disease is known to work slightly differently.
  • amyotrophic lateral sclerosis ALS
  • spinal muscular atrophy and Kennedy disease (spinal bulbar muscular atrophy) selectively kill only motor neurons so that only motor function is degenerated.
  • the result can be symptomatic of quadriplegia, also called motor neuropathy disease.
  • Alzheimer's disease unlike the motor neuron disease, Alzheimer's disease, Parkinson's disease, etc. are known as a typical neurodegenerative disease that the nerve cells of the brain are lost.
  • neuronal loss results in general brain atrophy.
  • These pathological findings are limited to the hippocampus and the olfactory cerebral cortex, which are the main brain areas that are primarily responsible for memory, but gradually spread throughout the brain through the parietal lobe and frontal lobe.
  • memory loss initially occurs mainly, and as the progress progresses, clinical symptoms become diverse and become more severe.
  • Parkinson's disease is caused by the loss of dopaminergic neurons in the substantia nigra (SN) located in the midbrain. Loss of dopamine nerves causes severe dopamine reduction in the striatum, resulting in impaired motor control by the extrapyramidal system, including the basal ganglia, thalamus, and motor cortex. .
  • Alzheimer's disease is a US FDA-approved drug that includes Aricept, Exelon, Namenda, and Razadyne, which activate the neurotransmitter acetylcholine, which helps the activity of brain nerve cells. In addition, they also reduce brain cell damage and alleviate the symptoms or slow the progression.
  • Levodopa is used as a dopamine agonist, but the long-term effect is limited.
  • Muscle is an essential tissue that supports our body and maintains life. Myoblasts differentiate into myoblasts to form multinucleated myotubes, form myocytes, and the death of myoblasts and muscle cells is the cause of various diseases. In addition, there are reports of diseases in which muscle cells are lost through genetic mutations and various causes, but there is no fundamental treatment to date. These muscle diseases vary in intensity depending on the type of symptom and disease, and may also show a complex form of neurological problems such as neurons and spinal cord.
  • muscle dystrophy is a disease in which muscle cells are destroyed, caused by genetic abnormalities, symptoms begin to weaken and difficult to occur, gradually worsening, dying of respiratory muscles may die.
  • the DMD and BMD are caused by abnormalities of the Dystrophin gene present on the X chromosome, and about one third of them are due to natural mutations, and the rest are due to reflection genetics.
  • DMD and BMD are both caused by the same gene abnormality, but a severe phenotype due to frame-shift mutations is called DMD.
  • DMD the disease progresses poorly, and it is impossible to walk in most patients between the ages of 9 and 13, and not only muscle weakness but also cognitive depression may occur, and cardiomyopathy and respiratory muscle dysfunction may lead to death.
  • Exon skipping targets the splicing enhancer sequence of exon 51 of the Dystrophin gene, which recovers only the reading frame and converts it into less severe genetic mutations, and thus is not a fundamental therapeutic alternative and is not a treatment for all DMDs. .
  • HN and F proteins hemagglutinin neuraminidase (HN) and F (fusion) proteins. It has been confirmed that HN and F proteins enhance cell fusion with other cells, and have a high ability to restore cell damage and deliver normal genes in damaged or dying cells or cells with gene abnormalities. Accordingly, the present invention has been completed by revealing that the cell fusion technology using HN and F proteins can be usefully used to treat neurodegenerative diseases or neurological diseases or muscle degenerative diseases or muscle diseases causing cell damage.
  • HN and F proteins hemagglutinin neuraminidase
  • the present invention is a neurodegenerative disease containing a vector containing a gene encoding hemagglutinin neuraminidase (HN) and F (fusion) protein as an active ingredient, or Provided is a pharmaceutical composition for preventing or treating neurological diseases, or muscle degenerative diseases or muscle diseases.
  • HN hemagglutinin neuraminidase
  • F fusion protein
  • the present invention is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases, containing the cells transformed with a vector containing genes encoding the HN and F proteins as an active ingredient. To provide.
  • the present invention includes administering to a subject a therapeutically effective amount of a vector comprising genes encoding HN and F proteins to a subject, preventing or treating a neurodegenerative or neurodegenerative or muscular disease or muscle disease.
  • a method for treating a neurodegenerative or neurodegenerative or muscular disease or muscle disease.
  • the present invention comprises administering to a subject a therapeutically effective amount of a cell transformed with a vector comprising genes encoding HN and F proteins to a neurodegenerative or neurological disease, or a muscle degenerative disease or Provided are methods for preventing or treating muscle diseases.
  • the present invention also provides the use of a vector comprising genes encoding HN and F proteins for use as a pharmaceutical composition for the prevention or treatment of neurodegenerative or neurological diseases, or muscle degenerative diseases or muscle diseases.
  • the present invention is the use of a cell transformed with a vector comprising a gene encoding the HN and F protein for use as a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases To provide.
  • HN and F protein overexpressing cells through the normal gene and transcription regulators in the nucleus of the damaged cells, by regulating the expression of genes involved in cellular repair of the damaged cells or by repairing normal proteins through normal gene transfer, It was confirmed to recover the cells.
  • HN and F protein overexpressing cells were fused with damaged cells or cells in which gene abnormalities were induced to recover normal cells.
  • cell fusion techniques using HN and F proteins can be used to repair cell damage and introduce normal genes, thereby allowing the vector or cells transformed with the genes encoding the HN and F proteins to be replaced with cell damage. It can be usefully used to treat related diseases, in particular neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases.
  • Figure 1 shows the effect of inhibiting apoptosis of NSC34 motor neuron cell line in death by cell fusion with hATMSCs according to an embodiment of the present invention.
  • Figure 2 shows the expression analysis of Bax and Bcl-xL in the NSC34 cell group in which cell fusion with hATMSCs according to an embodiment of the present invention.
  • Figure 3 is a schematic diagram for preparing a pcDNA3.1 expression vector, each inserted with HN and F according to an embodiment of the present invention.
  • Figure 4 is the analysis of HN and F mRNA expression in HN / F-hATMSCs according to an embodiment of the present invention.
  • FIG. 5 is an imaging analysis of HN and F protein expression in HN / F-hATMSCs according to one embodiment of the present invention.
  • Figure 6 analyzes the immunophenotyping (immunophenotyping) of HN / F-hATMSCs according to an embodiment of the present invention.
  • Figure 7 analyzes the cell fusion capacity with NSC34 motor neuron line in HN / F-hATMSCs according to an embodiment of the present invention.
  • FIG. 8 shows the expression of ChAT and CD105 in cells fused with HN / F-hATMSCs and NSC34 motor neuron cell lines according to an embodiment of the present invention.
  • Figure 9 shows the results of proteomics analysis between the NSC34 motor neuron cell line, the killing NSC34 motor neuron cell line and HN / F-hATMSCs according to an embodiment of the present invention.
  • Figure 10 shows the results of the heat map (heatmap) between the NSC34 motor neuron cell line, the killing NSC34 motor neuron cell line and HN / F-hATMSCs according to an embodiment of the present invention.
  • Figure 11 analyzes the expression of genes DDB1, HMGB1 and MSH2 mRNA associated with cell recovery in NSC34 motor neurons, dying NSC34 motor neurons and HN / F-hATMSCs according to an embodiment of the present invention.
  • FIG. 12 shows the effect of inhibiting apoptosis of C2C12 myoblast cells in death by HN / F-NSCs and cell fusion according to an embodiment of the present invention.
  • Figure 13 analyzes the HN and F mRNA expression in HN / F-HeLa according to an embodiment of the present invention.
  • Figure 14 analyzes the cell fusion ability with N2A neuroblastoma cell line in HN / F-HeLa according to an embodiment of the present invention.
  • 15 is a schematic diagram for preparing a pcDNA3.1-P2A expression vector inserted with HN and F according to an embodiment of the present invention.
  • Figure 16 analyzes the cell fusion ability with C2C12 myoblasts in HN / F-HeLa or F-P2A-HN-HeLa according to an embodiment of the present invention.
  • FIG. 17 illustrates a TDP-43 binding motif, a transcriptional factor (TF), in a promoter region of a gene DDB1 related to cell recovery selected according to an embodiment of the present invention.
  • TF transcriptional factor
  • FIG. 18 is a schematic diagram for preparing a promoter specifically binding to mouse DBB1 (mDDB1) according to an embodiment of the present invention.
  • 19 is an analysis of mDDB1 expression by TDP-43 in a cell fused with an N2A neuroblastoma cell line and a HeLa cell line transduced with GFP-TDP-43 according to an embodiment of the present invention.
  • Figure 21 illustrates the mechanism of recovery of cell damage by HN / F protein overexpressing cells in damaged cells.
  • FIG. 22 illustrates apoptosis inhibitory effect of HZ / F-hATMSCs and killed Alzheimer's disease (AD) cell model by cell fusion according to an embodiment of the present invention.
  • FIG. 23 illustrates apoptosis inhibitory effect of Huntington's disease (HD) disease cell model, which is being killed by cell fusion with HN / F-hATMSCs according to an embodiment of the present invention.
  • HD Huntington's disease
  • FIG. 24 shows apoptosis inhibitory effect of HF / F-hATMSCs and dying heart failure disease cell models by cell fusion according to an embodiment of the present invention.
  • FIG. 25 illustrates intravenous injection of HN / F-hATMSCs into a mouse model of amyotrophic lateral sclerosis (ALS) or Duchenne muscular dystrophy (DMD) disease according to one embodiment of the present invention. -Shows the cord cord injection method.
  • ALS amyotrophic lateral sclerosis
  • DMD Duchenne muscular dystrophy
  • FIG. 26 analyzes the motor performance in G93A SOD Tg mice injected with HN / F-hATMSCs according to one embodiment of the present invention.
  • Figure 27 analyzes the expression of Dystrophin and CTGF of the DMD disease cell model prepared according to the embodiment of the present invention.
  • FIG. 28 analyzes the expression of Dystrophin and CTGF in a DMD disease cell model treated with HN / F-hATMSCs according to an embodiment of the present invention.
  • FIG. 29 analyzes Dystrophin expression in skeletal muscle of mdx mouse, a DMD disease animal model.
  • FIG. 30 analyzes the engraftment ability of HN / F-hATMSCs in skeletal muscle tissue of mdx mice injected with HN / F-hATMSCs according to one embodiment of the present invention.
  • FIG. 31 analyzes dystrophin expression in skeletal muscle tissue of mdx mice injected with HN / F-hATMSCs 3 weeks after injection and 15 weeks after injection according to an embodiment of the present invention.
  • FIG. 32 shows CTGF expression in skeletal muscle tissue of mdx mice injected with HN / F-hATMSCs according to one embodiment of the present invention.
  • the present invention provides a vector comprising genes encoding hemagglutinin neuraminidase (HN) and F (fusion) proteins.
  • HN hemagglutinin neuraminidase
  • F fusion proteins
  • the hemagglutinin-neuraminidase and F protein are sentai virus (sendai virus), human immunodeficiency virus 1 (human immunodeficiency virus 1), human parainfluenza virus (Human parainfluenza virus), human respiratory cells Human respiratory syncytial virus (HRSV), influenza virus or vesicular stomatitis virus may be derived from, but are not limited to, through the same or similar mechanisms of cells Various forms of hemagglutinin neuraminidase and F proteins of various origins and / or full length and / or fragments can be used so long as they can promote fusion, inhibit death, and repair damage to damaged cells. (Eckert DM, Kim PS.
  • Hemagglutinin neuraminidase is a glycoprotein expressed in virus, a single viral protein having both the activity of hemagglutinin and neuraminidase, or This may be present as a separate protein, but is not limited thereto, and may be included in the present invention as long as the effect according to the present invention is achieved.
  • the former include mump virus, sendai virus, human parainfluenza virus, such as human parainfluenza virus type 1 (HPIV-1), type 2 (HPIV-2), or 3 Or a paramyxovirus such as avian newcastle disease virus. Examples of the latter include those derived from influenza virus.
  • hemagglutinin-neuraminidase may refer to information from Enzyme entry (http://enzyme.expasy.org) EC: 3.2.1.18, protein sequence Is derived from, for example, Sandivirus, GenBank accession no. Published as AAB06288.1.
  • a Sandyvirus derived HN monoprotein which can be prepared according to the method of Example 2 according to the present invention is used.
  • a fusion protein refers to a glycoprotein used for intercellular fusion or fusion / entry of a virus to a cell, endocytosis, and membrane trafficking.
  • a F protein from virus is used, which is divided into classes I, II and III according to structural features.
  • classes I include GP2 of Ebola virus, Mo-55 of Moloney murine leukemia virus (MoMuLv), gp41 of immunodeficiency virus HIV, Simian Virus (SIV) gp41, and the like.
  • class II include SFV E1, TBEV E, and the like.
  • class III examples include glycoprotein B (gB) of Herpes Simplex virus (HSV), gB of Epstein-Barr virus (EBV), protein G of Vesicular Stomatitis virus (VSV), and gp64, a glycoprotein of baculovirus, and the like.
  • GB Herpes Simplex virus
  • EBV Epstein-Barr virus
  • VSV Vesicular Stomatitis virus
  • gp64 a glycoprotein of baculovirus, and the like.
  • Such fusion sugar protein and nucleic acid sequences are disclosed, for example GenBank Accession no. Published as AAC82300.1.
  • genes encoding the hemagglutinin neuraminidase (HN) and F (fusion) proteins may be used in the form of full length and / or fragments.
  • HN hemagglutinin neuraminidase
  • F fusion proteins
  • the wild type gene sequence encoding the protein disclosed in the present invention and fragments thereof, as well as genes in which a part of the nucleotide sequence is artificially modified to be advantageous, such as characteristics such as expression in cells or stability of the protein, and naturally found A portion of the nucleotide sequence includes a fragment of the modified gene or both.
  • the vector means a means for expressing a gene of interest in a host cell.
  • the vector includes elements for gene expression of interest, and may include a replication origin, a promoter, an operator, a transcription terminator, and the like, into the genome of the host cell.
  • Appropriate enzyme sites eg, restriction enzyme sites
  • RBS ribosomal binding sites
  • IRES Internal Ribosome Entry Site
  • the vector may further comprise transcriptional regulatory sequences (eg, enhancers, etc.) other than the promoter.
  • the vector may be a plasmid DNA, recombinant vector or other mediator known in the art, specifically a linear DNA plasmid DNA, recombinant non-viral vector, recombinant viral vector or inducible gene expression vector system (inducible gene expression vector)
  • the recombinant viral vector may be a retrovirus, adenovirus, adeno associated virus, helper-dependent adenovirus, and herpes simplex virus. herpes simplex virus), lentivirus, or vaccinia virus vector, but is not limited thereto.
  • the vector means an expression vector for gene therapy.
  • gene therapy refers to a method of normalizing its function by inserting a normal gene or providing a new function to a cell with a genetic abnormality in order to treat various genetic diseases caused by the genetic abnormality. Therefore, the expression vector for gene therapy in the present invention transfers and expresses hemagglutinin neuraminidase (HN) and F (fusion) protein genes into normal cells in the body, thereby expressing the cells with abnormal genes and normal cells.
  • HN hemagglutinin neuraminidase
  • F fusion
  • hemagglutinin neuraminidase (HN) and F (fusion) protein genes are prepared from primers that can specifically recognize them from known sequences as described above. And, using this, the amplification of the gene through a polymerase chain amplification (Polymerase Chain Reaction) and introduced it into the expression vector as described above and can be introduced into the cell according to the invention as described below.
  • Methods of introduction include, for example, liposome mediated transduction, calcium phosphate transfection, DEAE-dextran mediated transfusion, positively charged lipid mediated transduction, electroporation, transduction using a phage system or infection with a virus. Methods, and the like, but not limited thereto.
  • the present invention also provides a cell transformed with a vector comprising a gene encoding hemagglutinin neuraminidase (HN) and F (fusion) protein.
  • HN hemagglutinin neuraminidase
  • F fusion
  • the term "transformation" is that the genetic properties of the organism is changed by the DNA given from the outside, that is, DNA, which is a kind of nucleic acid extracted from cells of certain strains of the organism, may be introduced into living cells of other strains. When DNA enters the cell, the genetic traits change.
  • the transformed cells may be stem cells, progenitor cells or animal cells, but is not limited thereto.
  • the stem cells may be specifically embryonic stem cells, adult stem cells or induced pluripotent stem cells (iPS), but is not limited thereto, stem cells differentiated from the stem cells, such as embryonic stem Mesenchymal stem cells derived from cells, mesenchymal stem cells derived from induced pluripotent stem cells, neural stem cells derived from induced pluripotent stem cells, and the like.
  • the cells may be autologous or other, or allogenic or Xenogenic cells. Most preferably, since it is autologous and derived from the recipient, there is an advantage that the administration of the pharmaceutical composition is safe and safe from the problem of the immune response.
  • the embryonic stem cells are formed from the inner cell mass of the blastocytes, which are the early stages of embryonic development, and have the advantage of being able to differentiate into any tissue cell because they have the potential to differentiate into all cells. It is used in cell therapy research.
  • the adult stem cells refer to undifferentiated stem cells found throughout the adult even after the embryonic development stage. These adult stem cells have a site-specific differentiation ability to differentiate themselves according to the characteristics of the surrounding tissues.
  • Adult stem cells may be derived from various adult cells such as bone marrow, blood, brain, skin, fat, skeletal muscle, umbilical cord, umbilical cord blood and the like.
  • mesenchymal stem cells (MSC) skeletal muscle stem cells, hematopoietic stem cells, neural stem cells (NSC), adipose derived stem cells (MSC) adipose-derived stem cells, adipose-derived progenitor cells, vascular endothelial progenitor cells, and the like, but are not limited thereto.
  • the mesenchymal stem cells are stem cells derived from adult tissues obtained from the respective parts of the body already adult, umbilical cord, cord blood, bone marrow, blood, brain, skin, fat, skeletal, muscle, nerve, periosteum, amnion Or pluripotent or multipotent cells that can be separated from the placenta and can differentiate into various cells such as adipocytes and motor neurons.
  • mesenchymal stem cells are characterized by being able to effectively engraft in allogeneic or heterologous recipients without the use of immunosuppressive agents.
  • the mesenchymal stem cells may be mesenchymal stem cells of animals, specifically mammals, and more particularly humans.
  • the mesenchymal stem cells are mesenchymal stem cells derived from adipose tissue.
  • Adipose tissue-derived mesenchymal stem cells unlike bone marrow, amniotic fluid and umbilical cord blood stem cells, have a practical advantage of being provided with a large amount, and about 1% of fat cells are estimated to be stem cells.
  • the plastic surgery being performed is liposuction, the adipose derived stem cells are particularly useful due to their infinite supply.
  • the procedure for obtaining the mesenchymal stem cells is as follows: Isolating mesenchymal stem cells from a mammal, including human or mouse, preferably a human mesenchymal stem cell source, such as adipose tissue, blood or bone marrow . The cells are then cultured in a suitable medium. Suspension cells are removed during the culturing and passaged cells attached to the culture plate are obtained to finally obtain mesenchymal stem cells.
  • any medium commonly used for culturing stem cells can be used. More specifically, a medium containing serum (eg fetal calf serum, horse serum and human serum) or serum replacement may be used.
  • Medium that can be used in the present invention is, for example, RPMI series, Eagle's MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), ⁇ -MEM (Stanner, CP et al., Nat. New Biol. 230: 52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc.
  • the medium may include other components such as antibiotics or antifungal agents (eg, penicillin, streptomycin), glutamine, and the like. General descriptions of media and cultures are described in R. Ian Freshney, Culture of Animal Cells, Alan R. Liss, Inc., New York (1984), which is incorporated herein by reference.
  • Identification of mesenchymal stem cells can be carried out, for example, via flow cytometry. Such flow cytometry is performed using specific surface markers of mesenchymal stem cells.
  • the mesenchymal stem cells according to the present invention have a phenotype of CD29, CD44 and CD90 as markers and CD34, CD45 and HLA-DR which are negative markers.
  • the induced pluripotent stem cells are introduced into four specific genes causing reverse differentiation into somatic cells, such as skin cells of adults without pluripotency, and then expressed or differentiated from cells into which four genes introducing reverse differentiation are introduced.
  • somatic cells such as skin cells of adults without pluripotency
  • stem cells having the same pluripotency as embryonic stem cells can be made. These are called induced pluripotent stem cells or dedifferentiated stem cells.
  • Stem cell therapy using induced pluripotent stem cells somatic cells obtained from patients, induced pluripotent stem cells are made and differentiated in vitro to study the progress of various diseases, and used in disease models and cell-based research in drug development.
  • the induced pluripotent stem cells are known methods, for example, Yu J et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science. 318, 1917-1920 or Takahashi K et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. It can be obtained according to the method disclosed in Cell 131, 861-872 and the like.
  • stem cells derived from induced pluripotent stem cells such as induced Pluripotent Stem Cells-derived mesemchymal stem cells (iPSCs-MSCs) or induced pluripotent stem cells-derived neural stem cells (induced Pluripotent Stem) Cells-derived neural stem cells, iPSCs-NSCs).
  • iPSCs-MSCs induced Pluripotent Stem Cells-derived mesemchymal stem cells
  • induced Pluripotent Stem induced Pluripotent Stem Cells-derived neural stem cells
  • Mesenchymal stem cells derived from the induced pluripotent stem cells or neural stem cells derived from induced pluripotent stem cells are known methods, for example, L.G. VILLA-DIAZ et al. (2012) Derivation of Mesenchymal Stem Cells from Human Induced Pluripotent Stem Cells Cultured on Synthetic Substrates. STEM CELLS. 30, 1174-1181 or Hyun Soo Choi et al. (2014) Neural Stem Cells Differentiated From iPS Cells Spontaneously Regain Pluripotency. STEMCELLS. 32, 2596-2604, etc. can be obtained according to the method disclosed.
  • progenitor cells are cells prior to the morphology and function of specific cells, and may be differentiated into cells of specific cell lines or formed into specific types of tissue, and have self-renewal. It means cells with extremely limited differentiation capacity. Endoderm progenitor cells, mesoderm progenitor cells, and ectoderm progenitor cells are all included here.
  • the animal cell is a functional and structural basic unit derived from an animal including a human, and if the cell is derived from an animal including a human (for example, a mammal such as a monkey, dog, goat, pig or state) is within the scope of the present invention. May be included.
  • the animal cells of the present invention include, but are not limited to, epithelial cells, endothelial cells, muscle cells, germ cells, skin cells (eg, fibroblasts, keratinocytes), immune cells, cancer cells and the like.
  • CHO Chinese hamster ovary
  • mouse myeloma (NS0) cells mouse myeloma (NS0) cells
  • baby hamster kidney (BHK) cells mouse myeloma (Sp2 / 0) cells
  • human retinal cells HUVEC cells, HMVEC cells
  • COS-1 cells COS-7 cells
  • HeLa cells HeLa cells
  • HEK-293 cells HepG-2 cells
  • HL-60 cells IM-9 cells
  • Jurkat cells MCF-7 cells or T98G cells, etc.
  • T98G cells human retinal cells
  • HUVEC cells human retinal cells
  • HMVEC cells human retinal cells
  • COS-1 cells COS-7 cells
  • HeLa cells HeLa cells
  • HEK-293 cells HepG-2 cells
  • HL-60 cells HepG-2 cells
  • IM-9 cells Jurkat cells
  • MCF-7 cells T98G cells
  • a neurodegenerative disease or neurological disease, or muscle containing a vector containing a gene encoding a hemagglutinin neuraminidase (HN) and the gene encoding the F (fusion) protein as an active ingredient It provides a pharmaceutical composition for preventing or treating degenerative diseases or muscle diseases.
  • HN hemagglutinin neuraminidase
  • the vector is the same as the description of the vector comprising the gene encoding the hemagglutinin neuraminidase (HN) and F (fusion) protein, the specific description is the above In the following, only the specific configuration of the pharmaceutical composition for preventing or treating neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases will be described.
  • HN hemagglutinin neuraminidase
  • F fusion protein
  • the present invention is a cell overexpressing the HN and F protein is enhanced cell fusion capacity with other cells by the HN and F protein, the normal gene is delivered and expressed through the fusion with damaged cells, the cell damage is repaired It was confirmed that the expression of the gene is controlled. In addition, in various disease models related to cell damage, it was confirmed that HN and F protein overexpressing cells were fused with damaged cells or cells in which gene abnormalities were induced to recover normal cells.
  • the vector containing the gene encoding the HN and F protein can be usefully used as an active ingredient for the prevention or treatment of neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases.
  • the neurodegenerative disease or neurological disease, or muscle degenerative disease or muscle disease is a disease associated with cell damage.
  • the neurodegenerative disease or neurological disease may cause nerve cell damage, spinal nerve damage, peripheral nerve damage or neuronal cell death.
  • the neurodegenerative disease or neurological disease is a neurodegenerative disease or neurological disease causing motor neuron damage or death and a neurodegenerative disease or neurological disease causing neuronal cell damage or death, spinal nerve injury or peripheral nerve damage. Can be divided into
  • neurodegenerative diseases or neurological diseases causing motor neuron damage or death In order to improve or treat neurodegenerative diseases or neurological diseases causing motor neuron damage or death, preservation and recovery of motor neuron cells must be made.
  • neurodegenerative or neurological diseases include spinal muscular atrophy, kennedy (spinal bulbar muscular atrophy), amyotrophic lateral sclerosis (ALS), multiple sclerosis, primary sclerosis ( primary lateral sclerosis or progressive bulbar palsy. It is not limited to this.
  • Neurodegenerative diseases or neurological diseases causing nerve cell damage or death of the brain, spinal nerve damage or peripheral nerve damage may be prevented or treated through the recovery of nerve cells, peripheral nerves of the damaged brain or spinal cord.
  • Examples of such neurodegenerative or neurological diseases include Alzheimer's disease (AD), dementia, multi-infact dementia (mid), frontotemporal dementia, and Lewy body dementia (AD).
  • dementia with Lewy bodies mild cognitive impairment, corticobasal degeneration, Parkinson's disease (PD), depression, metabolic brain disease, multiple system atrophy Multiple system atrophy (MSA), Huntington's disease, progressive supranuclear palsy; PSP, epilepsy, dentatorubropallidoluysian atrophy (DRPLA), spinocerebellar ataxia, glaucoma, stroke, brain ischemia, encephalitis Post-encephalitic parkinsonism, Tourette's syndrome, restless legs syndrome, or attention deficit disorders with hyperactivity.
  • the muscle degenerative disease or muscle disease may cause muscle cell damage or muscle cell death.
  • muscle degenerative diseases or muscle diseases include myopathy, congenital myopathy, congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy muscular dystrophy, Limb Girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, oculopharyngeal muscular atrophy, distal muscular dystrophy, adriatic muscular dystrophy (Emery-Dreifuss muscular dystrophy), Myotonic dystrophy, Barth syndrome, heart failure, sarcopenia or X-linked dilated cardiomyopathy It may be, but is not limited thereto.
  • compositions according to the invention may be formulated in a suitable form with the pharmaceutically acceptable carriers generally used.
  • Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, carriers for parenteral administration such as aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • composition for cell therapy according to the present invention if necessary according to the administration method or dosage form, suspensions, dissolution aids, stabilizers, tonicity agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH adjusters, analgesics , Buffers, antioxidants, and the like may be appropriately included.
  • Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the pharmaceutical composition may be administered by any device that can move the active ingredient to the target cell.
  • the pharmaceutical composition of the present invention may comprise a therapeutically effective amount for the treatment of a disease.
  • treatment means to reverse, alleviate, inhibit, or prevent the progression of one or more symptoms of a disease or condition to which the term applies.
  • therapeutically effective amount also refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human being contemplated by a researcher, veterinarian, physician or other clinical person, Amounts that induce alleviation of the symptoms of the disease or disorder being treated. It is apparent to those skilled in the art that the active ingredient included in the pharmaceutical composition of the present invention will vary depending on the effect.
  • the optimal pharmaceutical composition content can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex and It can be adjusted according to various factors including the diet, the time of administration, the route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used concurrently.
  • the pharmaceutical composition is administered intravenously or intraspinally.
  • the dosage of the composition of the present invention is 0.1 ⁇ 10 5 to 1.0 ⁇ 10 8 cells / cells transformed with a vector comprising the gene encoding the hemagglutinin-neuramidase and F protein as active ingredients. kg (body weight), more preferably 0.5 ⁇ 10 6 to 1.0 ⁇ 10 7 cells / kg body weight.
  • the dosage may be variously prescribed by such factors as the formulation method, the mode of administration, the age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient. These factors can be taken into account to properly adjust the dosage.
  • the number of administrations may be one or two or more times within the range of clinically acceptable side effects, and may be administered to one site or two or more sites for the administration site.
  • the same dosage as that of humans per kg or the volume ratio (for example, average value) of the ischemic organ (heart, etc.) between the animal of interest and humans can be adjusted.
  • Equivalent amounts may be administered.
  • the animal to be treated according to the present invention include humans and mammals for other purposes, and specifically, humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, goats, horses, pigs, and the like. This includes.
  • the present invention is a neurodegenerative disease containing a cell transformed with a vector comprising a gene encoding a hemagglutinin neuraminidase (HN) and a gene encoding F (fusion), or neurodegenerative disease
  • HN hemagglutinin neuraminidase
  • F fusion
  • the cell is the same as the description of the cell transformed with a vector comprising a gene encoding the hemagglutinin neuraminidase (HN) and F (fusion) protein,
  • HN hemagglutinin neuraminidase
  • F fusion protein
  • the present invention is a cell overexpressing the HN and F protein is enhanced cell fusion capacity with other cells by the HN and F protein, the normal gene is delivered and expressed through the fusion with damaged cells, the cell damage is repaired It was confirmed that the expression of the gene is controlled. In addition, in various disease models related to cell damage, it was confirmed that HN and F protein overexpressing cells were fused with damaged cells or cells in which gene abnormalities were induced to recover normal cells.
  • the cells transformed with the vector containing the genes encoding the HN and F proteins can be usefully used as an active ingredient of a composition for preventing or treating neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases. .
  • the present invention also includes administering to a subject a therapeutically effective amount of a vector comprising a gene encoding hemagglutinin neuraminidase (HN) and a gene encoding F (fusion) protein,
  • HN hemagglutinin neuraminidase
  • F F
  • the present invention provides a method for administering to a subject a therapeutically effective amount of cells transformed with a vector comprising a gene encoding hemagglutinin neuraminidase (HN) and a gene encoding F (fusion) protein.
  • HN hemagglutinin neuraminidase
  • F F
  • a gene encoding the hemagglutinin neuraminidase (HN) and the F (fusion) protein may be used for the vector or cells transformed with the vector and a method of administration, dosage, etc. thereof.
  • HN hemagglutinin neuraminidase
  • F fusion protein
  • the subject is a subject suffering from a neurodegenerative disease, a neurological disease, a muscle degenerative disease or a muscle disease, and more specifically, includes the gene encoding the above-described hemagglutinin-neuramidase and F protein.
  • Mammals such as humans, monkeys, mice, rats, which can be alleviated and / or repaired by administering a vector or a cell transformed with said vector, such as neuronal damage or myocyte damage caused by said disease, Rabbits, sheep, cattle, dogs, goats, horses, pigs, and the like.
  • the neurodegenerative disease or neurological disease may cause nerve cell damage, spinal cord injury, peripheral nerve damage or neuronal cell death.
  • neurodegenerative diseases or neurological diseases are neurodegenerative diseases or neurological diseases causing motor neuron damage or death and neurodegenerative diseases or neurological diseases causing neuronal cell damage or death, spinal cord injury or peripheral nerve damage. Can be divided.
  • neurodegenerative diseases or neurological diseases causing motor neuron damage or death In order to improve or treat neurodegenerative diseases or neurological diseases causing motor neuron damage or death, preservation and recovery of motor neuron cells must be made.
  • neurodegenerative or neurological diseases include spinal muscular atrophy, kennedy (spinal bulbar muscular atrophy), amyotrophic lateral sclerosis (ALS), multiple sclerosis, primary sclerosis ( primary lateral sclerosis or progressive bulbar palsy. It is not limited to this.
  • Neurodegenerative diseases or neurological diseases that cause nerve cell damage or death of the brain, spinal cord injury, or peripheral nerve damage may be prevented or treated through recovery of nerve cells or peripheral nerves of the damaged brain or spinal cord.
  • Examples of such neurodegenerative or neurological diseases include Alzheimer's disease (AD), dementia, multi-infact dementia (mid), frontotemporal dementia, and Lewy body dementia (AD).
  • dementia with Lewy bodies mild cognitive impairment, corticobasal degeneration, Parkinson's disease (PD), depression, metabolic brain disease, multiple system atrophy Multiple system atrophy (MSA), Huntington's disease, progressive supranuclear palsy (PSP), epilepsy, dentatorubropallidoluysian atrophy (DRPLA), spinal cord ventricle (spinocerebellar ataxia), glaucoma, stroke, brain ischemia, post-encephalitic parkinsonism, Tourette's syndrome, restless legs syndrome, or Uiryeok be deficient hyperactivity disorder (attention deficit disorders with hyperactivity), but is not limited thereto.
  • the muscle degenerative disease or muscle disease may cause muscle cell damage or muscle cell death.
  • muscle degenerative diseases or muscle diseases include myopathy, congenital myopathy, congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy muscular dystrophy, Limb Girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, oculopharyngeal muscular atrophy, distal muscular dystrophy, adriatic muscular dystrophy (Emery-Dreifuss muscular dystrophy), Myotonic dystrophy, Barth syndrome, heart failure, sarcopenia or X-linked dilated cardiomyopathy It may be, but is not limited thereto.
  • a cell overexpressing HN and F proteins enhances cell fusion ability with other cells by HN and F proteins, expresses normal genes through fusion with damaged cells, and restores cell damage. It was confirmed that the expression of. In addition, in various disease models related to cell damage, it was confirmed that HN and F protein overexpressing cells were fused with damaged cells or cells in which gene abnormalities were induced to recover normal cells.
  • the vector containing the gene encoding the HN and F protein or cells transformed therewith can be usefully used for preventing or treating neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases.
  • the present invention also provides hemagglutinin neuraminidase (HN) and F (fusion) proteins for use as a composition for preventing or treating neurodegenerative or neurological or muscular degenerative or muscular diseases. It provides a use of a vector comprising a gene encoding the.
  • HN hemagglutinin neuraminidase
  • F fusion proteins
  • hemagglutinin neuraminidase (HN) and F (fusion) protein for use as a composition for the prevention or treatment of neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases
  • HN hemagglutinin neuraminidase
  • F fusion protein
  • a gene encoding the hemagglutinin neuraminidase (HN) and the F (fusion) protein may be used for the vector or cells transformed with the vector and a method of administration, dosage, etc. thereof.
  • HN hemagglutinin neuraminidase
  • the specific description is used above.
  • the disease is the same as the description of the pharmaceutical composition for preventing or treating the neurodegenerative or neurological disease, or the muscle degenerative disease or the muscle disease, the specific description is used for the above description.
  • a cell overexpressing HN and F proteins enhances cell fusion ability with other cells by HN and F proteins, expresses normal genes through fusion with damaged cells, and restores cell damage. It was confirmed that the expression of. In addition, in various disease models related to cell damage, it was confirmed that HN and F protein overexpressing cells were fused with damaged cells or cells in which gene abnormalities were induced to recover normal cells.
  • the vector containing the gene encoding the HN and F protein or cells transformed therewith can be usefully used for preventing or treating neurodegenerative diseases or neurological diseases, or muscle degenerative diseases or muscle diseases.
  • hATMSCs Human adipose tissue-derived mesenchymal stem cells obtained with the consent and understanding of K-STMECELL Institutional Review Board (IRB) were treated with RKCM culture medium (10% FBS, K- Incubated in STEMCELL).
  • the mouse motor neuron line (NSC34 Motor Neuron-Like Hybrid Cell line, CEDARLANE, USA) was cultured in DMEM (Dulbecco's Modified Eagle's Medium) culture medium with 10% FBS.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Mouse neuroblastoma cell line N2A cell line, ATCC was cultured in an antibiotic-added Eagle's Minimum Essential Medium (EMEM) culture medium (10% FBS addition).
  • HeLa cell line Human cervical cancer cell lines (HeLa cell line, ATCC) were cultured in an antibiotic-added Eagle's Minimum Essential Medium (EMEM) culture medium (with 10% FBS).
  • EMEM Eagle's Minimum Essential Medium
  • Mouse C2C12 muscle myoblast cell line was incubated in DMEM culture medium (10% FBS) added with antibiotics.
  • Rat heart myoblast cell line (ATC) was incubated in DMEM culture medium (10% FBS) with antibiotics.
  • iPSCs human induced pluripotent stem cells prepared by receiving blood with consent from a normal person were cultured in a floating plate containing hES cell culture medium (ThermoFisher) containing no bFGF for 4 days. It was. Next, the cells were cultured in hES cell culture medium containing Retinoic acid (RA, 5 x 10 -7 M) for 4 days, and then neurobasal medium [EGF (20 ng / ml), bFGF (10 ng / ml), B27 (2) for 7 days. %), Serum-free DMEM / F12 (1: 1) culture medium containing LIF (10 ng / ml) and heparin sodium (2 ⁇ g / ml).
  • RA Retinoic acid
  • bFGF 10 ng / ml
  • B27 (2) for 7 days.
  • Serum-free DMEM / F12 (1: 1) culture medium containing LIF (10 ng / ml) and he
  • iPSCs-NSCs induced Pluripotent Stem Cells-derived neural stem cells
  • Example 1 Motor neuron and Local origin Mesenchymal stem cells Inhibitory Effect of Apoptosis on Motor Neuronal Cell Line by Cell Fusion
  • the cell tracker (CM-DiI (Thermofisher) -labeled motor neuron cell line NSC34 cells were incubated for 24 hours after treatment with 2.5 ⁇ M of thapsigargin, apoptosis inducing factor. After Annexin V staining, Annexin V-positive NSC34 cells were isolated by flow cytometry using Annexin V antibody.
  • CM-DiI Thermofisher
  • HATMSCs and Annexin V-positive NSC34 cells labeled with green dyes were transferred to Sandai virus (HVJ) Envelope Cell fusion Kit (GenomONETM-CF EX Sendai virus (HVJ) Envelope Cell Fusion Kit, COSMO BIO, Japan) was used as the manufacturer's method for cell fusion and then V-positve cells were analyzed by flow cytometry.
  • HVJ Sandai virus
  • hSCMS and NSC34 cells with cell fusion were isolated by flow cytometry, followed by Reverse Transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR.
  • RT-PCR Reverse Transcription polymerase chain reaction
  • Bcl-xL an anti-apoptotic gene.
  • FIG. 2 the expression of Bax mRNA was significantly decreased in the NSC34 cell group in which hATMSCs and cell fusion occurred, and the expression of Bcl-xL mRNA was significantly increased in comparison with the NSC34 cell group in which hATMSCs and cell fusion did not occur.
  • Hemagglutinin Neuraminidase hemagglutinin neuraminidase , HN
  • F fusion protein overexpressing human fat Mesenchymal stem cells
  • HN hemagglutinin neuraminidase
  • F F protein gene
  • clones obtained after cloning were amplified and cultured in LB liquid medium for 24 hours, plasmid was extracted using a plasmid extraction kit (Midiprep kit (Invitrogen, USA), and then liposomes (lipofectamine 3000, Invitrogen) was transduced into hATMSCs using the manufacturer's method to obtain hATMSCs (HN / F-hATMSCs) into which the HN / F gene was introduced.
  • Total RNA was extracted from the transduced cell lines and analyzed for expression of HN and F genes via RT-PCR and real-time quantitative RT-PCR. HATMSCs were used as a control. Partial HN and F primers were used to confirm expression for HN or F mRNA using RT-PCR and real-time quantitative PCR.
  • the base sequence encoding myc protein was attached to the forward primer for amplification of the HN gene, and the base sequence encoding histidine was attached to the forward primer for amplification of the F gene.
  • Santcruz Biotechnology, USA was used to analyze the expression of HN and F proteins in HN / F-hATMSCs through confocal laser microscopy (Nicon, Japan).
  • HN and F proteins are expressed on the cell surface of HN / F-hATMSCs as shown in FIG. 5. These results indicate that human adipose-derived mesenchymal stem cells overexpressing HN and F proteins were successfully produced.
  • Fusion assay with NSC34 motor neuron cell line labeled with green dye-labeled HN / F-hATMSCs and cell tracker CM-DiI to assess cell fusion capacity of HN / F-hATMSCs prepared as in Example 2 ) was performed. Two kinds of cells were mixed in a 1.5 ml test tube in a ratio of cell number 1: 1, and reacted at 4 ° C. for 5 minutes, followed by reaction at 37 ° C. for 15 minutes. The plate was transferred to a 6-well plate containing antibiotic-containing DMEM medium (10% FBS) and incubated for 16 hours at 37 ° C. under 5% CO 2 .
  • HN / F-hATMSCs were harvested and analyzed for cell fusion rate of HN / F-hATMSCs and NSC34 motor neuron cells by flow cytometry.
  • HATMSCs were used as a control.
  • FIG. 7 the cell fusion rate of HN / F-hATMSCs and NSC34 motor neuron cell lines was increased by more than 3.5 times compared to the control group.
  • an image analysis using confocal laser microscopy confirmed an image of cells fused with HN / F-hATMSCs showing green fluorescence and NSC34 motor neuron cell lines with red fluorescence.
  • Example 5 HN / F- hATMSCs NSC34 Motor neuron Expression analysis of markers of hATMSCs and motor neuron markers in fused cells
  • Example 4 Confocal using the motor neuron marker marker choline acetyltransferase (ChAT) and the antibody against adipose derived mesenchymal stem cell marker marker (Santcruz Biotechnology, USA) for the fusion cells of Example 4
  • ChAT motor neuron marker marker choline acetyltransferase
  • CD105 adipose derived mesenchymal stem cell marker
  • FIG. 8 the expression of CD105, a marker of ChAT adipose-derived mesenchymal stem cell marker, which was a motor neuron marker, was confirmed in cells fused with HN / F-hATMSCs and NSC34 motor neuron.
  • the annexin V-positive NSC34 motor neuron cell line of Example 2 and the fusion cells of Example 4 were analyzed for expression of DDB1, HMGB1 and MSH2 via RT-PCR and quantitative RT-PCR.
  • NSC34 motor neuron line was used as a control.
  • the expression of DDB1, HMGB1, and MSH2 mRNAs was decreased in dying NSC34 motor neurons, but significantly increased in cells fused with HN / F-hATMSCs and NSC34 motor neurons.
  • Example 7 Killing C2C12 Myoblasts Hemagglutinin Neuraminidase (hemagglutinin) neuraminidase , HN ) / Overexpressing F (fusion) protein Induced pluripotent stem cells origin Of neural stem cells (HN / F-NSCs) By cell fusion C2C12 Myoblasts Apoptosis inhibitory effect analysis
  • HN / F-NSCs induced pluripotent stem cell-derived neural stem cells
  • iPSCs-NSCs pluripotent stem cell-derived neural stem cells
  • liposomes lipofectamine 3000, Invitrogen
  • Transduction was performed to obtain iPSCs-NSCs (HN / F-NSCs) into which the HN / F gene was introduced.
  • Total RNA was extracted from the transduced cell lines and analyzed for expression of HN and F genes via RT-PCR and real-time quantitative RT-PCR (results not shown).
  • the C2C12 myoblast cell line labeled with the cell tracker CM-DiI was incubated for 24 hours after treatment with apoptosis inducing factor, tapxigin 2.5 ⁇ g / ml.
  • Annexin V staining Annexin V-C2C12 myoblast line was isolated by flow cytometry using Annexin V antibody.
  • the cells were recovered and the V-positive cells were collected by flow cytometry. Analyzed. As a result, as shown in FIG.
  • Annexin V-positive cells were identified in the C2C12 cell group without HN / F-NSCs and cell fusion, and in the C2C12 cell group with HN / F-NSCs and cell fusion. 21.6% of Annexin V-positive cells were identified.
  • Example 8 N2A Neuroblastoma cell lines and Hemagglutinin Neuraminidase (hemagglutinin) neuraminidase , HN ) / Overexpressing F (fusion) protein HeLa Evaluation of cell fusion ability of cell line (HN / F-HeLa)
  • HeLa cell line (HN / F-HeLa) overexpressing HN and F proteins was constructed to investigate whether cell fusion ability was observed in normal cell lines overexpressing HN and F proteins.
  • the GD-HN-inserted pcDNA3.1 expression vector was cloned using the GFP-pcDNA3.1 expression vector and the HN protein gene clone obtained in Example 2.
  • the pcDNA3.1 expression vector into which the RFP-F protein was inserted was cloned using the RFP-pcDNA3.1 expression vector and the F protein gene clone obtained in Example 2.
  • liposomes lipofectamine 3000, Invitrogen
  • heLa HeLa
  • HN / F-HeLa HeLa
  • Total RNA was extracted from the transduced cell lines and analyzed for expression of HN and F genes via RT-PCR and real-time quantitative RT-PCR.
  • HeLa cell line was used as a control.
  • overexpression of the HN and F genes was confirmed as shown in FIG. 13.
  • the cells were recovered after fusion of the N2A neuroblastoma cell line labeled with HN / F-HeLa and DAPI by the same method as described in Example 4.
  • Cell fusion rates of HN / F-HeLa and N2A neuroblastoma cell lines were analyzed by flow cytometry and confocal laser microscopy.
  • GFP-transduced N2A neuroblastoma cell line and mCherry-transduced HeLa cell line were fused in the same manner as described in Example 4, and then cells were recovered and analyzed for cell fusion rate by confocal laser microscopy. It was. As a result, as shown in FIG.
  • (fusion) protein gene was introduced into the HeLa cell line.
  • the pcDNA3.1-P2A expression vector into which HN and F proteins were inserted was cloned using the pcDNA3.1-P2A expression vector and the HN protein gene and the F protein gene clone obtained in Example 2.
  • liposomes liposomes (lipofectamine 3000, Invitrogen) were transduced into HeLa cell lines using the manufacturer's method to obtain HeLa cell lines (F-P2A-HN-HeLa) into which the HN / F gene was introduced.
  • the pcDNA3.1 expression vector cloned with the HN and F proteins cloned in Example 2 was transduced into the HeLa cell line as described above to obtain a HeLa cell line (HN / F-HeLa) into which the HN / F gene was introduced.
  • Total RNA was extracted from the transduced cell lines and analyzed for expression of HN and F genes via RT-PCR and real-time quantitative RT-PCR (results not shown).
  • each of the HeLa cell lines obtained above was labeled with GFP, and after cell fusion with a C2C12 myoblast cell line labeled with RFP in the same manner as described in Example 4, cells were recovered.
  • Cell flow rate of HeLa cell line and C2C12 myoblast cell line were analyzed by flow cytometry.
  • GFP-labeled HeLa cell line and RFP-labeled C2C12 myoblast cell line were fused in the same manner as described in Example 4, and then cells were recovered and analyzed for cell fusion rate by flow cytometry.
  • the cell fusion rate of F-P2A-HN-HeLa was 26.9%, and the cell fusion rate of HN / F-HeLa was 50.1%, as shown in FIG. It was.
  • HN and F proteins are overexpressed in cells through various vectors, and cells expressing HN and F proteins are enhanced in cell fusion with other cells.
  • the promoter region of the gene DDB1 related to cell repair selected in Example 6 was screened and a transcriptional factor as shown in FIG. 17. It was confirmed that a binding motif of TDP-43 (TAR DNA-binding protein 43), RF), was present at the promoter region of DDB1.
  • a primer specifically binding to mouse DDB1 (mDDB1) is prepared as shown in FIG.
  • Total DNA was extracted from blastoma cell line and HeLa cell line to identify mouse specific DDB1 gene by PCR.
  • TDP-43 was overexpressed in N2A neuroblastoma cell line and RNA was extracted and analyzed using quantitative RT-PCR.
  • the N2A neuroblastoma cell line, the HeLa cell line transduced with GFP, and the HeLa cell line transduced with the N2A neuroblastoma cell line and GFP-TDP-43 were respectively introduced into the Sandai virus (HVJ) Envelope Cell fusion Kit (GenomONETM-CF EX Sendai).
  • HVJ Sandai virus
  • Cell fusion was performed using virus (HVJ) Envelope Cell Fusion Kit, COSMO BIO, Japan) according to the manufacturer's method, followed by ChIP (Chromatin immunoprecipitation) assay (Millipore) using a fusion GFP antibody (Rockland).
  • mDDB1 increased with fusion time in the N2A neuroblastoma cell line and HeLa cell line transduced with GFP-TDP-43, and the occupancy of TDP-43 at the mDDB1 promoter region was four times or more. Increasing TDP-43 was found to regulate the expression of DDB1.
  • the cell lines were subjected to cell fusion in the same manner as described above, and analyzed for translocation of TDP-43 through confocal laser microscopy after DAPI staining, and stained with NcA neuroblastoma cell line using NucBlue (ThermoFisher) and GFP-TDP.
  • Example 11 Alzheimer's disease Alzheimer's disease; In AD) cell models HN Analysis of Therapeutic Effects Using Cell Fusion with / F-hATMSCs
  • AD Alzheimer's disease
  • TG2576 mice 80 days old
  • stem cells were isolated and filtered using trypsin from the isolated tissues.
  • EGF and FGF were added to DMEM culture medium containing B27 supplement, respectively, and cultured in sphere form for 4 days. After 4 days, the cells were transferred to well-plates and cultured for 7 days in DMEM medium containing 5% FBS and B27 supplement to induce differentiation of AD mouse-derived neural stem cells.
  • Example 2 HN / F-hATMSCs obtained in Example 2 and the AD mouse-derived neural stem cells obtained above were fused in the same manner as in Example 4.
  • 5 ⁇ l of Annexin V antibody was added and reacted at room temperature for 15 minutes, and then V-positive cells were analyzed by flow cytometry.
  • FIG. 22 50.7% of Annexin V-positive cells were identified in AD mouse-derived neural stem cells, and 15.9% of Annexin V- was found in AD mouse-derived neural stem cells in which cell fusion with HN / F-hATMSCs occurred. Positive cells were identified.
  • Huntington's disease cell model was constructed to determine the therapeutic effect using cell fusion by cells overexpressing HN / F protein in Huntington's disease.
  • a Huntington's disease animal model R6 / 2 Tg mice (80 days old), was provided from the Jackson laboratory. Then, subventricular zones of R6 / 2 mice were separated, and stem cells were isolated and filtered using trypsin in the separated tissues. Next, EGF and FGF were added to DMEM culture medium containing 20 ng / ml, respectively, incubated for 4 days in the form of a sphere, and then transferred to a well-plate and DMEM culture medium containing 5% FBS and B27 supplement. Incubation for 7 days at induced the differentiation of Huntington's disease-derived neural stem cells.
  • Example 2 HN / F-hATMSCs obtained in Example 2 and the Huntington disease mouse-derived neural stem cells obtained above were fused in the same manner as in Example 4.
  • 5 ⁇ l of Annexin V antibody was added and reacted at room temperature for 15 minutes, and then V-positive cells were analyzed by flow cytometry.
  • 37.5% of Annexin V-positive cells were identified in Huntington's disease-derived neural stem cells as shown in FIG. Positive cells were identified.
  • Example 13 Heart failure disease cell model HN / F- hATMSCs Treatment effect analysis using cell fusion
  • a cell model of heart failure disease was prepared.
  • a heart failure disease cell model was obtained by inducing oxidative stress by incubating for 200 hours with hydrogen peroxide (H 2 O 2 ) in a H9c2 cardiomyocyte line labeled with a cell tracker CMDFA (Invitrogen).
  • Example 2 the HN / F-hATMSCs obtained in Example 2 were labeled with CM-DiI, and the cells were recovered after cell fusion in the same manner as described in Example 4 for the heart failure disease cell model obtained above. It was.
  • the recovered cells were stained with Annexin V and isolated from Annexin V-positive cells by flow cytometry, and analyzed for green fluorescent positive cells.
  • FIG. 24 29% of annexin V-positive cells were identified in a heart failure disease cell model, and 13.4% of annexin V-positive cells in a heart failure disease cell model in which cell fusion with HN / F-hATMSCs occurred. It was confirmed.
  • Example 14 Amyotrophic Lateral Sclerosis (amyotrophic lateral sclerosis, ALS ) Analysis of therapeutic effect using cell fusion of HN / F-hATMSCs in disease animal models
  • G93A SOD1 Tg mouse (ALS disease animal model) 80 days of age) were provided by the Jackson laboratory. Then, G93A SOD1 Tg mice were divided into a control group (Tg-saline), Tg-MSC and Tg-fusogenic MSC group, and using the intra-spinal cord injection method as shown in FIG. HATMSCs were injected into the Tg-MSC group and HN / F-hATMSCs prepared in Example 2 were injected into the Tg-fusogenic MSC group in 0.5-2 ⁇ 10 6 cells and rota rod test was performed. As a result, in the Tg-fusogenic MSC group injected with HN / F-hATMSCs as shown in FIG. It was.
  • Example 15 Duchesne Muscle retraction axis ( Duchenne muscular dystrophy, DMD Analysis of therapeutic effect using cell fusion of HN / F-hATMSCs in disease cell models
  • DMD disease is a muscle disease caused by Dystrophin deficiency of muscle fibers, so that siRNA (siDystrophin, GenePharma) specific to Dystrophin is obtained and introduced into C2C12 myoblasts using the manufacturer's method to suppress Dystophin expression. C2C12 myoblasts were prepared.
  • HN / F-hATMSCs prepared as in Example 2 and DMD disease prepared above
  • cells were recovered and analyzed for expression of Dystrophin and CTGF using quantitative RT-PCR and Western blot.
  • Dystophin expression was decreased and CTGF expression was increased in DMD disease cell model, while Dystrophin expression was significantly increased and CTGF expression was significantly increased in cells fused with HN / F-hATMSCs and DMD disease cell model. It was confirmed to decrease.
  • Example 16 DMD Disease in animal models HN / F- hATMSCs Treatment effect analysis using cell fusion
  • mdx mouse (2-4 weeks after birth), a DMD disease animal model, was provided from the Jackson laboratory. Skeletal muscle was isolated from mdx mice, and immunohistochemistry was performed using Dystrophin antibody (abcam) to confirm that Dystrophin was decreased as shown in FIG. 29. Then, mdx mice were divided into saline, MSC and fMSC groups, and hATMSCs labeled with saline in the control group and green dye in the MSC group using the intra-spinal cord injection method as shown in FIG. 25.
  • HN / F-hATMSCs of Example 2 labeled with green dye, were injected with 0.5-2 ⁇ 10 6 cell numbers, and skeletal muscle was isolated after 1 week. Then, the cells were stained with hNuclei antibody (abcam) and analyzed for engraftment ability in the extracted skeletal muscle through confocal laser microscopy. In addition, after 3 weeks or 15 weeks after injection, skeletal muscle was isolated, and immunohistochemistry was performed using Dystrophin antibody (abcam). One week after injection, skeletal muscle was isolated, total RNA was extracted from the isolated skeletal muscle, and CTGF gene expression was analyzed using quantitative RT-PCR.
  • HN and F protein overexpressing cells are fused with cells damaged by HN and F proteins, and through fusion with the damaged cells, transfer of transcriptional regulators in HN and F protein overexpressing cells into the nucleus of the damaged cells, By repairing damaged cells by regulating the expression of genes involved in intracellular repair, cell fusion techniques using HN and F proteins can be used to treat neurodegenerative or neurological, muscular degenerative or muscular diseases associated with cell damage. it means.
  • cell fusion technology using HN and F proteins is used for gene therapy. It can be used as a delivery tool for gene therapy.
  • the present invention relates to genes and cell therapies using cell fusion technology and the use thereof, and specifically, to repair cell damage and introduce normal genes by using cell fusion technology by HN and F proteins, whereby HN and F A vector comprising a gene encoding a protein or a cell transformed therefrom can be usefully used for treating diseases related to cell damage, more specifically neurodegenerative or neurological diseases, or muscle degenerative diseases or muscle diseases.

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Abstract

La présente invention concerne un gène et un produit de thérapie cellulaire utilisant une technologie de fusion cellulaire, et une utilisation de ces derniers. Plus particulièrement, il a été montré que des cellules qui surexpriment l'hémagglutinine-neuraminidase (HN) et la protéine F (fusion) avaient un meilleur potentiel de fusion cellulaire avec d'autres cellules grâce aux protéines HN et F et pouvaient restaurer des cellules qui avaient été endommagées ou étaient engagées dans le processus de mort, ou restaurer des cellules génétiquement aberrantes en les transformant en des cellules normales par fusion cellulaire avec ces dernières. Ainsi, un vecteur portant un gène codant pour la protéine HN/F ou des cellules transformées par ce dernier peuvent être avantageusement utilisés en tant que principe actif d'une composition destinée à la prévention ou au traitement de maladies associées à des lésions cellulaires, telles que des maladies neurodégénératives ou des neuropathies, ou des maladies dégénératives musculaires ou des myopathies.
PCT/KR2019/008081 2018-07-02 2019-07-02 Gène et produit de thérapie cellulaire utilisant la technologie de fusion cellulaire, et utilisation correspondante Ceased WO2020009444A1 (fr)

Priority Applications (5)

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CN202310900115.5A CN116870196A (zh) 2018-07-02 2019-07-02 利用细胞融合技术的基因及细胞治疗剂以及其的用途
CN201980044907.9A CN112384247B (zh) 2018-07-02 2019-07-02 利用细胞融合技术的基因及细胞治疗剂以及其的用途
EP19831381.9A EP3824894A4 (fr) 2018-07-02 2019-07-02 Gène et produit de thérapie cellulaire utilisant la technologie de fusion cellulaire, et utilisation correspondante
JP2021521927A JP7300764B2 (ja) 2018-07-02 2019-07-02 細胞融合技術を用いた遺伝子及び細胞治療剤、並びにその用途
JP2022062691A JP2022104986A (ja) 2018-07-02 2022-04-04 細胞融合技術を用いた遺伝子及び細胞治療剤、並びにその用途

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KR10-2018-0076719 2018-07-02
KR10-2018-0089538 2018-07-31
KR20180089538 2018-07-31
KR1020190079666A KR102100490B1 (ko) 2018-07-02 2019-07-02 세포융합 기술을 이용한 유전자 및 세포 치료제 및 이의 용도
KR10-2019-0079666 2019-07-02

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