WO2022080820A1 - Expression cassette and vector comprising alzheimer's disease-related mutant genes, human totipotent stem cell line transformed using same, and alzheimer's disease organoid and neuronal cell model differentiated therefrom - Google Patents

Expression cassette and vector comprising alzheimer's disease-related mutant genes, human totipotent stem cell line transformed using same, and alzheimer's disease organoid and neuronal cell model differentiated therefrom Download PDF

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WO2022080820A1
WO2022080820A1 PCT/KR2021/014022 KR2021014022W WO2022080820A1 WO 2022080820 A1 WO2022080820 A1 WO 2022080820A1 KR 2021014022 W KR2021014022 W KR 2021014022W WO 2022080820 A1 WO2022080820 A1 WO 2022080820A1
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promoter
human
alzheimer
disease
expression cassette
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Korean (ko)
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이민영
최무석
여한철
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경북대학교 산학협력단
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal 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

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  • the present invention relates to an expression cassette comprising a gene related to Alzheimer's disease, a vector, a human pluripotent stem cell line transformed using the same, and brain organoid and neuronal Alzheimer's disease modeling using the same, and more specifically, the expression cassette of the present invention contains these genes so that APP (amyloid precursor protein) and PSEN1 (preseniline 1:Preseniline-1) mutant genes associated with Alzheimer's disease can be expressed simultaneously.
  • the present invention also relates to a human totipotent stem cell line transformed using an expression cassette or vector containing the above genes, and further to a three-dimensional organoid and two-dimensional neuronal cell model derived from the cell line.
  • Alzheimer's disease is one of the degenerative brain diseases and accounts for 60% of dementias that cause cognitive loss due to the gradual degeneration of nerve cells. Depending on the cause, Alzheimer's disease is divided into familial Alzheimer's disease, which is caused by genetic factors, and sporadic Alzheimer's disease, which occurs in a large number of patients although the exact cause is unknown. When the brain tissue of a patient who died from Alzheimer's disease was examined, characteristic lesions such as neuritic plaque (or senile plaque) and neurofibrillary tangles were observed. General brain atrophy is seen.
  • neurites are formed by the accumulation of proteins and dead cells outside the cell, and their main component is a peptide called ⁇ -amyloid (A ⁇ ) consisting of 42 or 43 amino acids.
  • a ⁇ ⁇ -amyloid
  • the cytoskeleton is abnormally agglomerated around the excessively phosphorylated Tau protein and looks like a bundle of threads.
  • APP and PSEN1 which are known as the representative causative genes of Alzheimer's disease, contribute to the overexpression of ⁇ -amyloid and the aggregation of Tau protein, which are the etiological mechanisms causing Alzheimer's disease.
  • ⁇ -Amyloid is produced from amyloid precursor protein (APP) through proteolysis.
  • APP amyloid precursor protein
  • APP a proprotein
  • APP is a protein having one transmembrane domain. It is expressed in several isotypes by alternate splicing and is known to pass through two metabolic pathways in the cell. One is a pathway in which p3 and sAPP ⁇ are produced by ⁇ -secretase and ⁇ -secretase, and the other is a pathway in which ⁇ -amyloid and APP ⁇ are produced by ⁇ -secretase and ⁇ -secretase. Mutations in this APP protein are found in patients with familial Alzheimer's disease.
  • PSEN1 Another gene that exhibits a mutation that causes familial Alzheimer's disease is presenilin 1 (PSEN1).
  • PSEN1 is a protein with eight transmembrane domains that plays an important role in the development process and is known to act as a member of ⁇ -secretase itself or a complex. More than 45 mutations that cause familial Alzheimer's disease have been reported throughout PSEN1 protein, and these mutations are also known to increase the amount of ⁇ -amyloid formation.
  • Alzheimer's disease due to the produced ⁇ -amyloid is accompanied by a process of nerve cell damage caused by hyperphosphorylation of Tau protein, and it is known that several kinases act on this hyperphosphorylation of Tau protein.
  • Tau tangle formation also plays a role in damaging nerve cells, and mutations in Tau that form tangles well were found.
  • transgenic mice for the study of the pathogenesis of Alzheimer's disease.
  • the transgenes that have been the main targets of these trials are genes such as APP, PSEN1, Tau and ApoE4, which have been identified as causative genes in familial Alzheimer's disease.
  • the transgenic mouse which is mainly used in the present study, is a model that uses APP or PSEN1 gene mutations to increase the concentration of ⁇ -amyloid in the brain to form neurites.
  • APP or PSEN1 gene mutations to increase the concentration of ⁇ -amyloid in the brain to form neurites.
  • Tau mutant gene since it is difficult to know precisely the pathogenesis of Alzheimer's disease with ⁇ -amyloid alone, recent attempts have been made to simultaneously insert the Tau mutant gene. This is to create a model closer to human Alzheimer's disease by simultaneously expressing ⁇ -amyloid and Tau in the brain of transgenic mice.
  • ⁇ -amyloid can be produced at an earlier stage.
  • TG2576 the first-generation transgenic mouse, the accumulation of ⁇ -amyloid in the brain starts after 12 months of age or more, whereas in the case of 5XFAD or APP/PSEN1 mice, ⁇ -amyloid accumulation begins within 6 months.
  • the Duff group succeeded in developing a double transgenic mouse obtained by crossing APP and PSEN1 single transgenic mice in 1996, this transgenic mouse has been used in several studies to date. In fact, in the case of double transgenic mice, it can be observed that the period of spot formation is shortened from 3 months to 9 months due to the synergistic effect of the two genes.
  • the inventors of the present invention studied to produce an Alzheimer's disease model having human cell/tissue physiological characteristics using human cells, and as a result, the present invention was completed.
  • One object of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are To provide an Alzheimer's disease-related gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).
  • APP amyloid precursor protein
  • Another object of the present invention is to provide a recombinant expression vector comprising the expression cassette.
  • Another object of the present invention is to provide a human totipotent stem cell line transformed using the recombinant expression vector.
  • Another object of the present invention is to provide organoids and nerve cells derived from the cell line.
  • One aspect of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are Provided is an Alzheimer's disease-associated gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).
  • APP amyloid precursor protein
  • the APP mutant gene may have the sequence of SEQ ID NO: 1.
  • the PSEN1 mutant gene may have the sequence of SEQ ID NO: 3.
  • the expression cassette is a CMV promoter (cytomegalovirus promoter (eg, human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pL ⁇ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial Virus (Respiratory syncytial virus; RSV) promoter, metallotionin promoter, ⁇ -actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene It may contain any one promoter selected from the group consisting of a promoter, a human granulocyte-macrophage, EF1-al
  • One aspect of the present invention provides a recombinant expression vector comprising the expression cassette.
  • the recombinant expression vector may have the sequence of SEQ ID NO: 5.
  • Another aspect of the present invention provides a cell line transformed using the recombinant expression vector.
  • the cell line is human adult stem cell (hASC), human embryonic stem cell (human embryonic stem cell, hESCs) and human induced pluripotent stem cell (human induced pluripotent stem cell, hiPSC) ) may be any one of.
  • Another aspect of the present invention provides a nerve cell derived from the cell line.
  • another aspect of the present invention provides an organoid derived from the cell line.
  • the present invention can provide an Alzheimer's disease model with human cell/tissue physiological characteristics using human cells.
  • FIG. 1 is a conceptual diagram illustrating a method for producing a gene expression cassette and brain organoids of the present invention.
  • FIG. 2 is a graph showing the results of confirming the levels of A ⁇ 1-40 and 1-42 in CmCPP control, CAPPP and CACPP brain organoids.
  • FIG. 3 is a view showing the Western blot results confirming the phosphorylation of tau in CmCPP control, CAPPP and CACPP brain organoids.
  • 4 and 5 are photographs showing the results of immunochemical analysis of CmCPP control, CAPPP and CACPP brain organoids and 5xFAD model.
  • FIG. 6 is to investigate the usefulness of the FAD brain organoids (COs) model as a means for drug development, CACPP brain organoids (CO) BACE1 inhibitor IV (BSI), a ⁇ -secretase inhibitor or compound E (CE), a It is a conceptual diagram showing the process of treatment with ⁇ -secretase inhibitor.
  • COs FAD brain organoids
  • BACE1 inhibitor IV BACE1 inhibitor IV
  • CE compound E
  • FIG. 7 is a diagram showing changes in the levels of A ⁇ 1-40 and 1-42 in CACPP brain organoids (CO) treated with BSI or CE.
  • FIG. 8 is a diagram showing the change in p-tau expression in CACPP brain organoids (CO) treated with BSI or CE.
  • FIG. 9 is a view showing the results of ELISA analysis showing that the levels of A ⁇ 1-40 and 1-42 significantly increased in CACPP neuronal cells compared to the CmCPP control group.
  • FIG. 10 is a view showing Western blot results showing that the level of p-tau in CACPP neuronal cells significantly changed compared to CmCPP control.
  • FIG. 11 is a view showing the results showing the change in PHF-1 expression in CACPP neurons compared to the CmCPP control.
  • FIG. 12 is a conceptual diagram illustrating an experimental procedure for confirming the effect of BSI or CE on a neuron model.
  • FIG. 13 is a diagram showing the results of changing the levels of A ⁇ 1-40 and 1-42 by treatment with BSI or CE in CACPP neurons.
  • 14 and 15 are diagrams showing the results of changing the level of p-tau by treatment with BSI or CE in CACPP neurons.
  • One aspect of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are To provide an Alzheimer's disease-associated gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).
  • APP amyloid precursor protein
  • the APP mutant gene is an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated.
  • APP amyloid precursor protein
  • the mutation of the 176 amino acid is I176V mutation (Florida, FL)
  • the 670 amino acid mutation and the 671 amino acid mutation are K670/M67L (Swedish, Swe), respectively
  • the 717 amino acid mutation is V717I (London) .Lon) mutation.
  • the PSEN1 mutant gene is a PSEN1 mutant gene encoding presenilin 1 (PSEN1) in which amino acids 146 and 286 are mutated. Specifically, the 146 amino acid mutation is M146L, and the 286 amino acid mutation is L286V.
  • the APP mutant gene may have the sequence of SEQ ID NO: 1.
  • the APP mutant gene is a mutation of amino acids 176, 670, 671 and 717 as described above compared to the normal APP gene of SEQ ID NO: 2.
  • the PSEN1 mutant gene may have the sequence of SEQ ID NO: 3.
  • the PSEN1 mutant gene is one in which amino acids 146 and 286 are mutated as compared to the normal PSEN1 gene of SEQ ID NO: 4.
  • the Alzheimer's disease-related gene expression cassette may include a promoter to further improve gene expression, and the promoter is a CMV promoter (eg, human or mouse CMV immediate -early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pL ⁇ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial virus (RSV) promoter, metallotionin promoter, ⁇ -actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene promoter, human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter
  • CMV promoter
  • the CAG promoter is a kind of modified CMV promoter, cytomegalovirus immediate-early enhancer, chicken ⁇ -actin promoter, chimeric intron (chimeric intron), exon 1 (exon) 1) and a complex promoter including exon 2 of the rabbit ⁇ -globin gene (Hitoshi Niwa et al., Gene, 108:193-199, 1991, Monahan et al., Gene Therapy, 7:24-30, 2000).
  • the CAG promoter allows expression to be maintained without disappearing for a long period of time even after differentiation induction after the expression cassette of the present invention is introduced into stem cells.
  • the gene expression cassette may have the sequence of SEQ ID NO: 5.
  • the gene expression cassette may include a sequence for its expression together with the above-described APP mutant gene and PSEN1 mutant gene.
  • the Alzheimer's disease-related gene expression cassette may further include an enhancer to further improve gene expression, and the enhancer may include a known enhancer.
  • the gene expression cassette of the present invention is contained in a recombinant expression vector, a transgenic human pluripotent stem cell line, and a three-dimensional organoid and two-dimensional neuron produced using the same, which will be described later, so as to have human cell/tissue physiological characteristics Alzheimer's disease model can be provided.
  • Another aspect of the present invention is to provide a recombinant expression vector comprising the expression cassette.
  • recombinant expression vector refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotes are known.
  • operably linked refers to a functional linkage between a gene expression control sequence and another nucleotide sequence.
  • the gene expression control sequence may be at least one selected from the group consisting of an origin of replication, a promoter, and a terminator.
  • the transcription termination sequence may be a polyadenylation sequence (pA)
  • the origin of replication may be an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, or a BBV origin of replication, but is not limited thereto. .
  • the recombinant expression vector according to an embodiment of the present invention is selected from the group consisting of viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, lentiviral vectors, retroviral vectors and adeno-associated viral vectors.
  • viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, lentiviral vectors, retroviral vectors and adeno-associated viral vectors.
  • Vectors that can be used as recombinant expression vectors include plasmids used in the art (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1).
  • phage eg, ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, M13, etc.
  • viral vectors eg, adeno-associated virus (AAV) vectors, lenti viral vectors, etc.
  • the recombinant expression vector of the present invention may further include one or more selectable markers.
  • the marker is a nucleic acid sequence having characteristics that can be selected by conventional chemical methods, and includes all genes capable of distinguishing a transfected cell from a non-transfected cell.
  • herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, Bleomycin ), hygromycin (hygromycin), may be an antibiotic resistance gene such as chloramphenicol (chloramphenicol), but is not limited thereto.
  • the production of the recombinant expression vector of the present invention can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. there is.
  • the recombinant expression vector comprises a restriction enzyme site, constructing a first vector from which the promoter and gene cluster are removed; preparing a recombinant second vector by inserting a promoter, a mutant APP gene, and a mutant PSEN 1 gene into the second vector; And inserting the second recombinant vector into the first vector: can be prepared through a recombinant expression vector manufacturing method comprising:.
  • the recombinant expression vector of the present invention can provide an Alzheimer's disease model with human cell/tissue physiological characteristics by being included in a recombinant cell line and three-dimensional organoid to be described later including the gene expression cassette described above.
  • Another aspect of the present invention is to provide a cell line transformed using the recombinant expression vector.
  • a method known in the art for introducing a nucleic acid molecule into an organism, cell, tissue or organ can be used, As is known, it can be carried out by selecting an appropriate standard technique according to the host cell. Such methods include, for example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method , and lithium acetate-DMSO method and the like may be included, but are not limited thereto.
  • CaPO4 calcium phosphate
  • CaCl2 calcium chloride
  • PEG polyethylene glycol
  • DEAE-dextran method DEAE-dextran method
  • cationic liposome method cationic liposome method
  • lithium acetate-DMSO method and the like may be included, but are not limited thereto.
  • the cell line may be a human adult stem cell (hASC), a human embryonic stem cell (hESCs), or a human pluripotent stem cell (hiPSCs). And, specifically, it may be a human embryonic stem cell.
  • hASC human adult stem cell
  • hESCs human embryonic stem cell
  • hiPSCs human pluripotent stem cell
  • the transformed cell line may be prepared by a method for producing a transformed cell line comprising the step of transforming with the recombinant expression vector.
  • Another aspect of the present invention is to provide a nerve cell derived from the cell line.
  • the nerve cell is the cell line differentiated into a nerve cell, and may be prepared by a known method including a known material necessary for differentiation.
  • Another aspect of the present invention is to provide an organoid derived from the nerve cell.
  • organoid refers to a cell mass having a structure, cell components, and functions similar to those of living (human) organs.
  • the organoid of the present invention may include a two-dimensional cell tissue cultured on a plate as well as a three-dimensional cell mass.
  • the organoid may be a tissue differentiated into a three-dimensional brain organoid or a two-dimensional nerve cell.
  • the organoid may be prepared through a method for preparing an organoid comprising the step of culturing the cell line.
  • hESC Human embryonic stem cell lines
  • SNUES31 Human embryonic stem cell lines
  • hESCs were isolated from feeder cells using 1 mg/mL dispase® (Invitrogen), and cultured in Essential-8 medium (Invitrogen) on Geltrex (Invitrogen)-coated culture plate. became hESCs were subcultured into small clusters using 0.5 mM EDTA solution every 4 days.
  • APP human amyloid precursor protein
  • K670M/N671L Swedish
  • I716V Florida
  • V717I London
  • PSEN1M146L/L286V presenilin containing M146L and L286V mutations
  • PSEN1M146L/L286V The construct encoding -1 (PSEN1) was obtained from brain tissue of an 8-month-old 5xFAD mouse (The Jackson Laboratory).
  • the primers used for replication were as follows:
  • APP SweFlLon , PSEN1 M146L/L286V , APP SweFlLon -IRES-PSEN1 M146L/L286V was inserted, which was prepared by introducing the amplified genes of APP SweFlLon or PSEN1 M146L/L286V , or the mCherry gene into MCS, and four Constructs of CAG-APP SweFlLon -IRES-PSEN1 M146L/L286V -PGK-Puro (CAPPP), CAG-APP SweFlLon -PGKPuro(CAPP), CAG-PSEN1 M146L/L286V -PGK-mCherry(CPPmC) and CAG-mCherry-PGK -Puro (CmCPP) was obtained.
  • CAPPP CAG-APP SweFlLon -IRES-PSEN1 M146L/L286V -PGK-Pur
  • each plasmid is a viral packaging plasmid [VSV-G expression envelope plasmid and a plasmid containing gag, pol and rev genes, which are prepared by Dr. HEK293T (ATCC) cells cultured in 100 mm tissue culture plates were transfected using X-tremeGene HP DNA transfection reagent (Roche) together with Yibing Qyang (provided by Yale Cardiovascular Research Center, Yale School of Medicine, CT, USA)]. was introduced, and incubated at 37° C., 5% CO 2 .
  • the culture medium was changed 24 hours after transduction, and 10 mL of the virus-containing culture medium was collected every day for 3 days. Each 30mL of the collected culture solution was concentrated to 200 ⁇ L by ultracentrifugation (Hitachi) at 55,200g and 4°C for 2 hours.
  • Hitachi ultracentrifugation
  • hESCs introduced with the CAPPP gene were selected after treatment with 5 ⁇ g/ml puromycin (Invitrogen). This cell line was designated as the CAPPP hESC line.
  • hESCs introduced with CmCPP and CPPmC viruses were separated into single cells, and then 500 cells were inoculated into a 6-well plate STO-plated well (corning). After 7 days of culture, single-cell-derived mCherry-expressing colonies were manually isolated, transferred to a Matrigel-coated 12-well tissue culture plate, and cultured in a feeder-free state. These cell lines were named CmCPP and CPPmC hESC lines, respectively.
  • CPPmC hESCs were cultured for 24 hours by replacing the culture medium with Essential-8 medium containing 20 ⁇ L of concentrated CAPP virus and 2 ⁇ g/mL polybrene, thereby induced CAPP gene introduction through virus infection. Thereafter, CPPmC hESCs into which the CAPP gene was introduced were selected using 5 ⁇ g/ml puromycin.
  • the hESC cell line double-infected with CPPmC and CAPP lentivirus was named CACPP hESC line. Each hESC was imaged using a fluorescence microscope (DM IL LED Fluo, Leica).
  • hESCs cultured without a feeder were digested into single cells by incubation in 0.5 mM EDTA for 4 min, and incubated in Accutase® for 4 min. And the cells were 96-well ultra low-attachment U- at a density of 9x10 4 cells/cm 2 using low bFGF hESC culture medium containing 4 ng/ml bFGF and 50 ⁇ M Y27632, a Rho-associated protein kinase inhibitor (Tocris). Cultured on a bottom plate (Corning). The corresponding day was designated as day 0, and on day 2, the culture medium was changed to a hESC culture medium containing 5 ng/ml bFGF and 50 ⁇ M Y27632.
  • each embryoid body was treated with 500 ⁇ l neural induction medium [1% N2 supplement (Invitrogen), 1% Glutamax (Invitrogen), MEM-NEAA, 1 ⁇ g/ml Heparin (Sigma-) aldrich) in DMEM/F-12] and transferred to a 24-well ultra low-attachment plate.
  • 500 ⁇ l of neural induction medium was added.
  • neuroepithelial tissues were transferred to 20 ⁇ l matrigel (BD Bioscience) droplets on a 3 mm grooved Parafilm, and left at 37° C. for 1 hour.
  • the matrigel droplet contains 5ml brain organoid (CO) differentiation culture medium without vitamin A [0.5% N2 supplement, B27 supplement minus vitamin A (Invitrogen), 2.5 ⁇ g/mL human insulin (Roche), 1% Glutamax, 0.5% MEMNEAA, 3.5 ⁇ l/l ⁇ -mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium (Invitrogen)] was transferred to a 60 mm tissue culture dish (corning).
  • CO brain organoid
  • the culture medium was changed every day, and on day 14, matrigel droplets containing brain organoids were placed in a 125 ml bioreactor with a brain organoid differentiation culture medium [0.5% N2 supplement, B27 supplement (Invitrogen), 2.5 ⁇ g/mL human insulin 1%, Glutamax, 0.5% MEM-NEAA, 3.5 ⁇ l/l ⁇ -mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium] was transferred together. After that, the culture medium was changed once every 7 days.
  • a brain organoid differentiation culture medium [0.5% N2 supplement, B27 supplement (Invitrogen), 2.5 ⁇ g/mL human insulin 1%, Glutamax, 0.5% MEM-NEAA, 3.5 ⁇ l/l ⁇ -mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium] was transferred together. After that, the culture medium was changed once every 7 days.
  • feeder-free hESC cell lines were separated into single cells by incubation with Accutase® for 5-8 minutes, and 10 ⁇ M Y27632, a Rhoassociated protein kinase inhibitor was added at a density of 5 x 10 4 cells/cm 2 Inoculated on Matrigel-coated 24-well plates containing mTeSR TM 1. This time was designated as day-4. The next day, the culture medium was changed to mTeSR TM 1 without Y27632, and the culture medium was changed daily for additional 2 days.
  • the mTeSR TM 1 culture medium is a neuronal cell induction medium (1:1 mixture of DMEM/F12 medium and neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 10 ⁇ g/ml human Insulin, 1% Glutamax, 1% NEAA and 3.5 ⁇ l/l ⁇ -mercaptoethanol) were replaced (day 0) and the culture medium was changed every day for 10 days.
  • Cells differentiated on day 11 were separated into single cells with Accutase®, and then inoculated again on Matrigel-coated 24-well plates at a density of 1.5x10 5 cells/cm 2 .
  • the culture medium was replaced with a neuronal cell culture medium supplemented with 100 nM LDN193189, selective BMP type I receptor inhibitor, 10 ⁇ M SB431542, a selective TGF- ⁇ receptor inhibitor and 2 ⁇ M XAV939, a tankyrase inhibitor, and the culture medium was washed every 7 days. replaced.
  • the culture medium was changed to a neuronal maturation medium (neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 1% Glutamax and 25 ng/ml BDNF), and thereafter changed daily.
  • cells were re-inoculated at a density of 5 x 10 4 cells/cm 2 after single cell isolation with Accutase®.
  • a neuronal maturation culture medium containing 1 ⁇ M ⁇ -secretase inhibitor IV (BSI) or 10 nM compound E (CE) was used ( FIG. 12 ).
  • Brain organoids were fixed with a 3.2% paraformaldehyde phosphate buffer solution at 4° C. for 2 hours, and sequentially immersed in 15% and 30% sucrose solutions until the brain organoids subsided.
  • Brain organoid samples were placed in OCT compound (Leica) and cryosectioned (cryosectioned) to a thickness of 20 ⁇ m using a Leica CM1850 cryostat (Leica).
  • tissue sections were immersed in PBST (0.1% [vol/vol] Triton X-100 in PBS) containing 10% normal goat serum at room temperature for 1 hour. Tissue sections were also incubated overnight at 4°C with primary antibody in PBST containing 2% normal goat serum.
  • the primary antibodies used were:
  • TUJ1 (mouse, R&D systems MAB1195, 1:200),
  • TBR2 (mouse, R&D systems MAB6166, 1:100),
  • PAX6 (rabbit, Biolegend 901301, 1:200),
  • N-cadherin (rabbit, Santa Cruz sc-7939, 1:100)
  • Each brain organoid or nerve cell was subjected to sonication (Vibra-Cell) with a lysis buffer (iNtRON biotechnology) on ice.
  • Cell lysates were separated by 10-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride transfer membranes (Millipore).
  • SDS sodium dodecyl sulfate
  • Millipore polyvinylidene fluoride transfer membranes
  • the blot was washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween-20), blocked with 5% skim milk (Millipore) after 1 hour, and left overnight at 4° C. together with the primary antibody.
  • the primary antibodies used were:
  • APP anti-amyloid precursor protein
  • the primary antibody was goat anti-rabbit (Santa Cruz sc-2004, 1:10000) or goat anti-mouse (Santa Cruz sc-2005, 1:10,000) conjugated to horseradish peroxidase (HRP). It was detected with IgG. The bands were visualized with an enhanced chemiluminescence solution (Thermo Scientific), and images were acquired using an ImageQuant LAS 4000 Mini (GE Healthcare).
  • a ⁇ 1-40 and A ⁇ 1-42 levels were measured with a commercially available human amyloid- ⁇ ELISA Kit (Immuno-Biological laboratories Inc.). Each brain organoid (COs) was homogenized by ultrasonication with a lysis buffer on ice, and the supernatant was collected after centrifugation. ELISA was performed according to the manufacturer's instructions. A ⁇ 1-40 and A ⁇ 1-42 ELISA signals were quantified using an ELISA plate reader (BioTek, Winooski, VT, USA).
  • a ⁇ 1-40 and 1-42 levels were measured and compared using 70-day-old controls and FAD brain organoids. As confirmed in FIG. 2 , the levels of A ⁇ 1-40 and 1-42 were significantly increased in CAPPP and CACPP brain organoids compared to CmCPP control brain organoids. In addition, a significant increase in tau phosphorylation (phosphorylated at Ser396, Ser404) was confirmed in CAPPP and CACPP brain organoids through Western blot analysis (FIG. 3). These results were also confirmed by immunochemical analysis ( FIGS. 4 and 5 ), and A ⁇ aggregation was confirmed in CACPP and CAPPP brain organoids. These brain organoids showed extensive PHF-1 expression.
  • CACPP brain organoids were treated with BACE1 inhibitor IV (BSI), a ⁇ -secretase inhibitor or compound E (CE), a ⁇ -secretase It was treated with inhibitor.
  • BSI or CE treatment significantly lowered the levels of A ⁇ 1-40 and 1-42 in CACPP brain organoids ( FIG. 7 ).
  • Western blot analysis showed that p-tau expression of CACPP brain organoids was also decreased by BSI or CE (FIG. 8).
  • FAD brain organoid model can be generated from FAD hPSCs
  • FAD neurons can also be produced from FAD hPSCs
  • FAD neurons can also be utilized as a two-dimensional FAD model.
  • CmCPP control and CACPP FAD hPSCs were differentiated into neurons, and A ⁇ and tau phosphorylation in FAD neurons, ie, pathological phenotyping of AD, was performed.
  • ELISA analysis showed that the levels of A ⁇ 1-40 and 1-42 were significantly increased in CACPP neurons compared to the CmCPP control ( FIG. 9 ).
  • FIG. 12 Western blot and immunostaining also significantly reduced p-tau levels by BSI or CE ( FIGS. 14 and 15 ).
  • the human-derived two-dimensional neuronal three-dimensional brain organoid system of the present invention can successfully reproduce the pathological characteristics of amyloid ⁇ and tau, and serves as a platform for drug screening and pathological mechanism study of Alzheimer's disease (AD).
  • the model of the present invention can provide a novel human disease model platform for the study of molecular mechanisms of Alzheimer's disease (AD).
  • the present invention which reproduces the Alzheimer's (AD) pathological phenotype in two-dimensional and three-dimensional models, may contribute to the development of precise human models of other diseases.

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Abstract

The present invention relates to an expression cassette and a vector comprising Alzheimer's disease (AD)-related genes, a cell line transformed using same, and an organoid and a neuronal cell produced using same, and, more specifically, the expression cassette of the present invention comprises mutant genes of amyloid precursor protein (APP) and preseniline-1 (PS1), which are AD-related genes so that are simultaneously expressed and maintained. Therefore, the present invention can provide an AD model having physiological characteristics of human cells/tissues by using human cells, and the disease model can be used as a platform for AD pathological mechanism research and drug screening. In addition, the present invention can provide a novel human disease model platform for AD molecular mechanism research. The present invention, reproducing pathological phenotypes of AD in two-dimensional and three-dimensional models, may contribute to the development of accurate human models for other diseases.

Description

알츠하이머병 관련 돌연변이 유전자를 포함하는 발현 카세트, 벡터, 이를 이용하여 형질 전환된 인간 전능성 줄기세포주와 이로부터 분화된 알츠하이머병 오가노이드 및 신경세포 모델Expression cassette and vector containing Alzheimer's disease-related mutant gene, human pluripotent stem cell line transformed using the same, and Alzheimer's disease organoid and neuron model differentiated therefrom
본 발명은 알츠하이머병과 관련된 유전자를 포함하는 발현 카세트, 벡터 및 이를 이용하여 형질 전환된 인간 전능성 줄기세포주 및 이를 활용한 뇌오가노이드 및 신경세포 알츠하이머병 모델링에 관한 것으로서, 보다 구체적으로 본 발명의 발현 카세트는 알츠하이머병과 관련된 APP(아밀로이드 전구 단백질) 및 PSEN1(프리세닐린 1:Preseniline-1) 돌연변이 유전자가 동시에 발현될 수 있도록 이들 유전자를 포함한다. 또한 본 발명은 상기 유전자들을 포함하는 발현 카세트 또는 벡터를 이용하여 형질 전환된 인간 전능성 줄기세포주에 관한 것이며, 나아가 상기 세포주로부터 유래된 3차원 오가노이드 및 2차원 신경세포 모델에 관한 것이다.The present invention relates to an expression cassette comprising a gene related to Alzheimer's disease, a vector, a human pluripotent stem cell line transformed using the same, and brain organoid and neuronal Alzheimer's disease modeling using the same, and more specifically, the expression cassette of the present invention contains these genes so that APP (amyloid precursor protein) and PSEN1 (preseniline 1:Preseniline-1) mutant genes associated with Alzheimer's disease can be expressed simultaneously. The present invention also relates to a human totipotent stem cell line transformed using an expression cassette or vector containing the above genes, and further to a three-dimensional organoid and two-dimensional neuronal cell model derived from the cell line.
알츠하이머병은 퇴행성 뇌질환 중 하나로서 신경세포의 점진적인 퇴화로 인해 인지능력 상실을 가져오는 치매 중 60%를 차지한다. 알츠하이머병은 원인에 따라서 유전적 요인에 의해 나타나는 가족성 알츠하이머병과 정확한 원인은 알 수 없으나 많은 수의 환자에게서 발병하는 산발성 알츠하이머병으로 나뉜다. 알츠하이머 병으로 사망한 환자의 뇌 조직을 검사하였을 때, 특징적인 병변으로 신경반 (neuritic plaque) (또는 노인반) 과 신경원섬유 다발(neurofibrillary tangles)등이 관찰되고, 육안 관찰 시에는 신경세포 소실로 인하여 전반적 뇌 위축 소견이 보인다.Alzheimer's disease is one of the degenerative brain diseases and accounts for 60% of dementias that cause cognitive loss due to the gradual degeneration of nerve cells. Depending on the cause, Alzheimer's disease is divided into familial Alzheimer's disease, which is caused by genetic factors, and sporadic Alzheimer's disease, which occurs in a large number of patients although the exact cause is unknown. When the brain tissue of a patient who died from Alzheimer's disease was examined, characteristic lesions such as neuritic plaque (or senile plaque) and neurofibrillary tangles were observed. General brain atrophy is seen.
이 중 신경반은 세포외부에 단백질과 죽은 세포 등이 축적되어 형성되는 것으로서 주 구성성분은 42개 혹은 43 개의 아미노산으로 이루어진 β-아밀로이드(Aβ)라는 펩티드이며, 신경원섬유 다발은 세포 내에서 세포골격 (cytoskeleton)이 과다하게 인산화된 Tau 단백질을 중심으로 비정상적으로 뭉쳐져 실뭉치처럼 보이는 것을 말한다.Among them, neurites are formed by the accumulation of proteins and dead cells outside the cell, and their main component is a peptide called β-amyloid (Aβ) consisting of 42 or 43 amino acids. The cytoskeleton is abnormally agglomerated around the excessively phosphorylated Tau protein and looks like a bundle of threads.
알츠하이머병의 대표적인 원인유전자로 알려진 APP 및 PSEN1이 알츠하이머병을 일으키는 병인기전인 이러한 β-아밀로이드의 과발현과 Tau 단백질의 응집에 기여 한다는 것이 알려져 있다.It is known that APP and PSEN1, which are known as the representative causative genes of Alzheimer's disease, contribute to the overexpression of β-amyloid and the aggregation of Tau protein, which are the etiological mechanisms causing Alzheimer's disease.
β-아밀로이드는 아밀로이드 전구단백질(amyloid precursor protein; APP)로부터 프로테올리시스(proteolysis) 과정을 통해 만들어진다. 전구단백질인 APP는 하나의 트랜스맴브레인 도메인(transmembrane domain)을 가지고 있는 단백질로 교번 스플리싱(alternative splicing)에 의해 몇 가지 이소타입으로 발현되며 세포 내에서 두 가지 대사경로를 거치는 것으로 알려져 있다. 한가지는 β-시크리타아제와 γ-시크리타아제에 의해 p3와 sAPPα가 생성되는 경로이며 다른 한가지는 β-시크리타아제와 γ-시크리타아제에 의해 β-아밀로이드와 APPβ가 생성되는 경로이다. 가족성 알츠하이머병 환자들에서 이 APP 단백질에 돌연변이가 발견된다. 지금까지 발견된 돌연변이는 APP670/671(Swedish), APP672(Flemish), APP716(Florida), APP717 (London) 등이 있으며 이들 돌연변이에서 β-아밀로이드의 형성이 증가하는 것이 밝혀져 있다.β-Amyloid is produced from amyloid precursor protein (APP) through proteolysis. APP, a proprotein, is a protein having one transmembrane domain. It is expressed in several isotypes by alternate splicing and is known to pass through two metabolic pathways in the cell. One is a pathway in which p3 and sAPPα are produced by β-secretase and γ-secretase, and the other is a pathway in which β-amyloid and APPβ are produced by β-secretase and γ-secretase. Mutations in this APP protein are found in patients with familial Alzheimer's disease. Mutations discovered so far include APP670/671 (Swedish), APP672 (Flemish), APP716 (Florida), APP717 (London), and the like, and it has been found that the formation of β-amyloid is increased in these mutations.
가족성 알츠하이머병을 유발하는 돌연변이를 나타내는 또 다른 한가지의 유전자는 프리세닐린 1(PSEN1)이다. PSEN1 은 8개의 트랜스맴브레인 도메인을 가진 단백질로서 발생 과정에서 중요한 역할을 하며 γ- 시크리타아제 자체 또는 복합체의 일원으로 작용하는 것으로 알려져 있다. PSEN1은 단백질 전체에 걸쳐 가족성 알츠하이머병을 유발하는 돌연변이들이 45가지 이상이 보고되어 있으며 이들 돌연변이들 역시 β-아밀로이드의 형성량을 증가시키는 것으로 알려져 있다.Another gene that exhibits a mutation that causes familial Alzheimer's disease is presenilin 1 (PSEN1). PSEN1 is a protein with eight transmembrane domains that plays an important role in the development process and is known to act as a member of γ-secretase itself or a complex. More than 45 mutations that cause familial Alzheimer's disease have been reported throughout PSEN1 protein, and these mutations are also known to increase the amount of β-amyloid formation.
생성된 β-아밀로이드에 의한 알츠하이머병의 발병은 Tau 단백질의 과인산화에 의한 신경세포손상의 과정을 동반하며, 이러한 Tau 단백질의 과인산화에는 몇 가지 인산화효소가 작용하는 것으로 알려져 있다. Tau의 과인산 화와 더불어 Tau의 tangle 형성 또한 신경세포를 손상시키는 역할을 하며, 다발 (tangle)이 잘 형성되는 Tau의 돌연변이가 발견되었다.The onset of Alzheimer's disease due to the produced β-amyloid is accompanied by a process of nerve cell damage caused by hyperphosphorylation of Tau protein, and it is known that several kinases act on this hyperphosphorylation of Tau protein. In addition to Tau hyperphosphorylation, Tau tangle formation also plays a role in damaging nerve cells, and mutations in Tau that form tangles well were found.
인간 뇌세포의 노화 외에도, β-아밀로이드의 축적과 응집, Tau 단백질의 tangle 형성기전을 밝히는 것이 알츠하이머병의 치료방법에 있어서 중요한 역할을 할 것으로 예상되므로, 이를 연구하기 위하여 APP 유전자 돌연변이, PSEN1 유전자 돌연변이, 및 Tau 유전자 돌연변이가 동시에 발현하는 세포주 또는 동물 모델을 수립할 필요성이 대두된다.In addition to aging of human brain cells, elucidating the mechanism of β-amyloid accumulation and aggregation and Tau protein tangle formation is expected to play an important role in the treatment of Alzheimer's disease. , and the need to establish a cell line or animal model in which the Tau gene mutation is expressed simultaneously.
여러 연구에서 알츠하이머병의 병인기전의 연구를 위해서 형질전환 마우스를 수립하기 위한 시도가 있었다. 이러한 시도의 주 목표가 된 형질전환 유전자는 가족성 알츠하이머병에서 원인 유전자로 밝혀진 APP, PSEN1, Tau 및 ApoE4 같은 유전자들이다. 현재 연구에 주로 사용되고 있는 형질전환 마우스는 APP 또는 PSEN1 유전자 돌연변이를 이용하여 뇌 속에 β-아밀로이드의 농도를 올려서 신경반이 형성되게 하는 모델이다. 하지만 β-아밀로이드만으로는 알츠하이머병의 병인기전을 정확히 알기 어렵기 때문에 최근에는 Tau의 돌연변이 유전자를 동시에 넣는 시도들이 이뤄지고 있다. 이는 형질전환 마우스의 뇌 속에 β-아밀로이드 및 Tau를 동시에 발현시켜 인간의 알츠하이머병에 보다 더 근접한 모델을 만들기 위한 것이다.Several studies have attempted to establish transgenic mice for the study of the pathogenesis of Alzheimer's disease. The transgenes that have been the main targets of these trials are genes such as APP, PSEN1, Tau and ApoE4, which have been identified as causative genes in familial Alzheimer's disease. The transgenic mouse, which is mainly used in the present study, is a model that uses APP or PSEN1 gene mutations to increase the concentration of β-amyloid in the brain to form neurites. However, since it is difficult to know precisely the pathogenesis of Alzheimer's disease with β-amyloid alone, recent attempts have been made to simultaneously insert the Tau mutant gene. This is to create a model closer to human Alzheimer's disease by simultaneously expressing β-amyloid and Tau in the brain of transgenic mice.
또한 APP 및 PSEN1 이 동시에 돌연변이 형태로 존재하면 β-아밀로이드를 보다 이른 시기에 만들어 낼 수 있다. 1 세대 형질전환 마우스인 TG2576는 생후 12개월 이상 사육을 해야 뇌 속에 β-아밀로이드의 축적 현상이 시작되는데 반하여, 5XFAD 또는 APP/PSEN1 마우스의 경우에는 6개월 이내에 β-아밀로이드 축적이 시작된다. 1996년 Duff 그룹에서 APP 및 PSEN1의 단일 형질전환 마우스를 교배시켜서 얻은 이중 형질전환 마우스의 개발에 성공한 이후 현재까지 여러 연구에서 이 형질전환 마우스가 사용되고 있다. 실제로 이중 형질전환 마우스의 경우 두 가지 유전자의 상승 효과(Synergistic Effect)로 인해 반점 형성기간이 3개월에서 9개월 사이로 단축됨을 관찰할 수 있다. 하지만, 다양한 AD 동물 모델 개발에도, 동물과 인간의 유전학적, 생리/병리학적 차이로 인해 동물 모델을 활용한 연구로부터 도출된 결과를 인간에게 그대로 적용하기 어려운 경우가 많다. In addition, when APP and PSEN1 are present in mutant forms at the same time, β-amyloid can be produced at an earlier stage. In TG2576, the first-generation transgenic mouse, the accumulation of β-amyloid in the brain starts after 12 months of age or more, whereas in the case of 5XFAD or APP/PSEN1 mice, β-amyloid accumulation begins within 6 months. Since the Duff group succeeded in developing a double transgenic mouse obtained by crossing APP and PSEN1 single transgenic mice in 1996, this transgenic mouse has been used in several studies to date. In fact, in the case of double transgenic mice, it can be observed that the period of spot formation is shortened from 3 months to 9 months due to the synergistic effect of the two genes. However, even in the development of various AD animal models, it is often difficult to apply the results derived from studies using animal models to humans due to genetic, physiological/pathological differences between animals and humans.
이러한 문제점을 개선하기 위해서 본 발명의 발명자들은 인간 세포를 이용하여 인간의 세포/조직 생리학적 특성을 가진 알츠하이머 질병 모델을 제작하고자 연구하였고, 그 결과 본 발명을 완성하였다. In order to improve this problem, the inventors of the present invention studied to produce an Alzheimer's disease model having human cell/tissue physiological characteristics using human cells, and as a result, the present invention was completed.
본 발명의 일 목적은 a) 176번 아미노산, 670번 아미노산, 671번 아미노산 및 717번 아미노산이 돌연변이된 아밀로이드 전구단백질(APP)을 암호화하는 APP 돌연변이 유전자, 및 b) 146번 아미노산 및 286번 아미노산이 돌연변이된 프리세닐린 1(PSEN1)을 암호화하는 PSEN1 돌연변이 유전자를 포함하는 알츠하이머병 관련 유전자 발현 카세트를 제공하는 것이다.One object of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are To provide an Alzheimer's disease-related gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).
본 발명의 다른 일 목적은 상기 발현 카세트를 포함하는 재조합 발현 벡터를 제공하는 것이다. Another object of the present invention is to provide a recombinant expression vector comprising the expression cassette.
본 발명의 다른 일 목적은 상기 재조합 발현 벡터를 이용하여 형질 전환된 인간 전능성 줄기세포주를 제공하는 것이다.Another object of the present invention is to provide a human totipotent stem cell line transformed using the recombinant expression vector.
본 발명의 다른 일 목적은 상기 세포주로부터 유래된 오가노이드 및 신경세포를 제공하는 것이다. Another object of the present invention is to provide organoids and nerve cells derived from the cell line.
본 발명의 일 양상은 a) 176번 아미노산, 670번 아미노산, 671번 아미노산 및 717번 아미노산이 돌연변이된 아밀로이드 전구단백질(APP)을 암호화하는 APP 돌연변이 유전자, 및 b) 146번 아미노산 및 286번 아미노산이 돌연변이된 프리세닐린 1(PSEN1)을 암호화하는 PSEN1 돌연변이 유전자를 포함하는, 알츠하이머병 관련 유전자 발현 카세트를 제공한다.One aspect of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are Provided is an Alzheimer's disease-associated gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).
본 발명의 일 구체예로 상기 APP 돌연변이 유전자는, 서열번호 1의 서열을 갖는 것일 수 있다.In one embodiment of the present invention, the APP mutant gene may have the sequence of SEQ ID NO: 1.
본 발명의 다른 일 구체예로 상기 PSEN1 돌연변이 유전자는, 서열번호 3의 서열을 갖는 것일 수 있다. In another embodiment of the present invention, the PSEN1 mutant gene may have the sequence of SEQ ID NO: 3.
본 발명의 일 구체예로 상기 발현 카세트는 CMV 프로모터(cytomegalovirus promoter(예를 들어, 인간 또는 마우스 CMV immediate-early 프로모터), U6 프로모터, EF1-alpha(elongation factor 1-a) 프로모터, EF1-alpha short(EFS) 프로모터, SV40 프로모터, 아데노바이러스 프로모터(major late promoter), pLλ 프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터, 백시니아 바이러스 7.5K 프로모터, HSV의 tk 프로모터, SV40E1 프로모터, 호흡기 세포융합 바이러스(Respiratory syncytial virus; RSV) 프로모터, 메탈로 티오닌 프로모터(metallothionin promoter), β-액틴 프로모터, 유비퀴틴 C 프로모터, 인간 IL-2(human interleukin-2) 유전자 프로모터, 인간 림포톡신(human lymphotoxin) 유전자 프로모터, 인간 GM-CSF(human granulocyte-macrophage colony stimulating factor) 유전자 프로모터 및 CAG 프로모터로 이루어진 군으로부터 선택되는 어느 하나의 프로모터를 포함하는 것 일 수 있다. In one embodiment of the present invention, the expression cassette is a CMV promoter (cytomegalovirus promoter (eg, human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial Virus (Respiratory syncytial virus; RSV) promoter, metallotionin promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene It may contain any one promoter selected from the group consisting of a promoter, a human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter, and a CAG promoter.
본 발명의 일 양상은 상기 발현 카세트를 포함하는, 재조합 발현 벡터를 제공한다.One aspect of the present invention provides a recombinant expression vector comprising the expression cassette.
본 발명의 일 구체예로, 상기 재조합 발현 벡터는, 서열번호 5의 서열을 갖는 것일 수 있다. In one embodiment of the present invention, the recombinant expression vector may have the sequence of SEQ ID NO: 5.
본 발명의 다른 일 양상은 상기 재조합 발현 벡터를 이용하여 형질전환된 세포주를 제공한다. Another aspect of the present invention provides a cell line transformed using the recombinant expression vector.
본 발명의 일 구체예로, 상기 세포주는 인간 성체줄기세포(human adult stem cell, hASC), 인간 배아줄기세포 (human embryonic stem cell, hESCs) 및 인간 유도만능줄기세포 (human induced pluripotent stem cell, hiPSC) 중 어느 하나일 수 있다. In one embodiment of the present invention, the cell line is human adult stem cell (hASC), human embryonic stem cell (human embryonic stem cell, hESCs) and human induced pluripotent stem cell (human induced pluripotent stem cell, hiPSC) ) may be any one of.
본 발명의 다른 일 양상은 상기 세포주로부터 유래된 신경세포를 제공한다.Another aspect of the present invention provides a nerve cell derived from the cell line.
또한, 본 발명의 다른 일 양상은 상기 세포주로부터 유래된 오가노이드를 제공한다.In addition, another aspect of the present invention provides an organoid derived from the cell line.
본 발명은 인간 세포를 이용하여 인간의 세포/조직 생리학적 특성을 가진 알츠하이머 질병 모델을 제공할 수 있다.The present invention can provide an Alzheimer's disease model with human cell/tissue physiological characteristics using human cells.
도 1은 본 발명의 유전자 발현 카세트 및 뇌오가노이드의 제조방법을 설명하는 개념도이다.1 is a conceptual diagram illustrating a method for producing a gene expression cassette and brain organoids of the present invention.
도 2는 CmCPP 대조군, CAPPP 및 CACPP 뇌오가노이드에서의 Aβ 1-40 및 1-42의 수준을 확인한 결과를 나타내는 그래프이다.2 is a graph showing the results of confirming the levels of Aβ 1-40 and 1-42 in CmCPP control, CAPPP and CACPP brain organoids.
도 3은 CmCPP 대조군, CAPPP 및 CACPP 뇌오가노이드에서의 tau phosphorylation를 확인한 웨스턴 블롯 결과를 나타내는 도면이다.3 is a view showing the Western blot results confirming the phosphorylation of tau in CmCPP control, CAPPP and CACPP brain organoids.
도 4 및 5는 CmCPP 대조군, CAPPP 및 CACPP 뇌오가노이드 및 5xFAD 모델의 면역화학분석 결과를 나타내는 사진이다.4 and 5 are photographs showing the results of immunochemical analysis of CmCPP control, CAPPP and CACPP brain organoids and 5xFAD model.
도 6은 약물 개발을 위한 수단으로서 FAD 뇌오가노이드 (COs) 모델의 유용성을 조사하기 위해, CACPP 뇌오가노이드 (CO)를 BACE1 inhibitor IV (BSI), aβ -secretase inhibitor 또는 compound E (CE), a γ-secretase inhibitor 로 처리하는 과정을 나타내는 개념도이다.Figure 6 is to investigate the usefulness of the FAD brain organoids (COs) model as a means for drug development, CACPP brain organoids (CO) BACE1 inhibitor IV (BSI), aβ -secretase inhibitor or compound E (CE), a It is a conceptual diagram showing the process of treatment with γ-secretase inhibitor.
도 7은 BSI 또는 CE 처리는 CACPP 뇌오가노이드 (CO)에서 Aβ 1-40 및 1-42의 수준 변화를 나타내는 도면이다.7 is a diagram showing changes in the levels of Aβ 1-40 and 1-42 in CACPP brain organoids (CO) treated with BSI or CE.
도 8은 BSI 또는 CE 처리는 CACPP 뇌오가노이드 (CO)에서 p-tau 발현의 변화를 나타내는 도면이다.8 is a diagram showing the change in p-tau expression in CACPP brain organoids (CO) treated with BSI or CE.
도 9는 CmCPP 대조군에 비교하여 CACPP 뉴런 세포에서 Aβ 1-40 및 1-42의 수준이 유의적으로 증가한 것을 보여주는 ELISA분석 결과를 나타내는 도면이다.9 is a view showing the results of ELISA analysis showing that the levels of Aβ 1-40 and 1-42 significantly increased in CACPP neuronal cells compared to the CmCPP control group.
도 10은 CmCPP 대조군에 비교하여 CACPP 뉴런 세포에서 p-tau의 수준이 유의적으로 변화하는 것을 보여주는 웨스턴 블롯 결과를 나타내는 도면이다.FIG. 10 is a view showing Western blot results showing that the level of p-tau in CACPP neuronal cells significantly changed compared to CmCPP control.
도 11는 CmCPP 대조군에 비교하여 CACPP 신경세포에서 PHF-1 발현 변화를 보여주는 결과를 나타내는 도면이다.11 is a view showing the results showing the change in PHF-1 expression in CACPP neurons compared to the CmCPP control.
도 12는 신경세포 모델에 대한 BSI 또는 CE의 영향을 확인하기 위한 실험과정을 나타내는 개념도이다.12 is a conceptual diagram illustrating an experimental procedure for confirming the effect of BSI or CE on a neuron model.
도 13은 CACPP 신경세포에서 BSI 또는 CE의 처리에 의해 Aβ 1-40 및 1-42의 수준이 변화되는 결과를 나타내는 도면이다.13 is a diagram showing the results of changing the levels of Aβ 1-40 and 1-42 by treatment with BSI or CE in CACPP neurons.
도 14 및 15는 CACPP 신경세포에서 BSI 또는 CE의 처리에 의해 p-tau의 수준이 변화되는 결과를 나타내는 도면이다.14 and 15 are diagrams showing the results of changing the level of p-tau by treatment with BSI or CE in CACPP neurons.
본 발명의 일 양상은 a) 176번 아미노산, 670번 아미노산, 671번 아미노산 및 717번 아미노산이 돌연변이된 아밀로이드 전구단백질(APP)을 암호화하는 APP 돌연변이 유전자, 및 b) 146번 아미노산 및 286번 아미노산이 돌연변이된 프리세닐린 1(PSEN1)을 암호화하는 PSEN1 돌연변이 유전자를 포함하는, 알츠하이머병 관련 유전자 발현 카세트를 제공하는 것이다.One aspect of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are To provide an Alzheimer's disease-associated gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).
상기 APP 돌연변이 유전자는 176번 아미노산, 670번 아미노산, 671번 아미노산 및 717번 아미노산이 돌연변이된 아밀로이드 전구단백질(APP)을 암호화하는 APP 돌연변이 유전자이다. 구체적으로, 상기 176번 아미노산의 돌연변이는 I176V 돌연변이 (Florida, FL)이고, 상기 670번 아미노산 돌연변이 및 671번 아미노산 돌연변이는 각각 K670/M67L (Swedish, Swe)이며, 상기 717번 아미노산 돌연변이는 V717I (London. Lon) 돌연변이이다. The APP mutant gene is an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated. Specifically, the mutation of the 176 amino acid is I176V mutation (Florida, FL), the 670 amino acid mutation and the 671 amino acid mutation are K670/M67L (Swedish, Swe), respectively, and the 717 amino acid mutation is V717I (London) .Lon) mutation.
상기 PSEN1 돌연변이 유전자는 146번 아미노산 및 286번 아미노산이 돌연변이된 프리세닐린 1(PSEN1)을 암호화하는 PSEN1 돌연변이 유전자이다. 구체적으로, 상기 146번 아미노산 돌연변이는 M146L이고, 286번 아미노산 돌연변이는 L286V이다. The PSEN1 mutant gene is a PSEN1 mutant gene encoding presenilin 1 (PSEN1) in which amino acids 146 and 286 are mutated. Specifically, the 146 amino acid mutation is M146L, and the 286 amino acid mutation is L286V.
본 발명의 일 구체예로서 상기 APP 돌연변이 유전자는 서열번호 1의 서열을 갖는 것일 수 있다. 구체적으로 상기 APP 돌연변이 유전자는 서열번호 2의 정상 APP 유전자에 비해 전술한 176번 아미노산, 670번 아미노산, 671번 아미노산 및 717번 아미노산이 돌연변이 된 것이다. As an embodiment of the present invention, the APP mutant gene may have the sequence of SEQ ID NO: 1. Specifically, the APP mutant gene is a mutation of amino acids 176, 670, 671 and 717 as described above compared to the normal APP gene of SEQ ID NO: 2.
본 발명의 다른 일 구체예로서 상기 PSEN1 돌연변이 유전자는 서열번호 3의 서열을 갖는 것일 수 있다. 구체적으로 상기 PSEN1 돌연변이 유전자는 서열번호 4의 정상 PSEN1 유전자에 비해 전술한 146번 아미노산 및 286번 아미노산이 돌연변이 된 것이다. As another embodiment of the present invention, the PSEN1 mutant gene may have the sequence of SEQ ID NO: 3. Specifically, the PSEN1 mutant gene is one in which amino acids 146 and 286 are mutated as compared to the normal PSEN1 gene of SEQ ID NO: 4.
본 발명의 일 구체예로서 상기 알츠하이머병 관련 유전자 발현 카세트는, 유전자의 발현을 보다 향상시키기 위하여, 프로모터를 포함할 수 있고, 상기 프로모터는 CMV 프로모터(cytomegalovirus promoter(예를 들어, 인간 또는 마우스 CMV immediate-early 프로모터), U6 프로모터, EF1-alpha(elongation factor 1-a) 프로모터, EF1-alpha short(EFS) 프로모터, SV40 프로모터, 아데노바이러스 프로모터(major late promoter), pLλ 프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터, 백시니아 바이러스 7.5K 프로모터, HSV의 tk 프로모터, SV40E1 프로모터, 호흡기 세포융합 바이러스(Respiratory syncytial virus; RSV) 프로모터, 메탈로 티오닌 프로모터(metallothionin promoter), β-액틴 프로모터, 유비퀴틴 C 프로모터, 인간 IL-2(human interleukin-2) 유전자 프로모터, 인간 림포톡신(human lymphotoxin) 유전자 프로모터, 인간 GM-CSF(human granulocyte-macrophage colony stimulating factor) 유전자 프로모터 및 CAG 프로모터로 이루어진 군으로부터 선택되는 것일 수 있으나, 구체적으로는 CAG 프로모터일 수 있다. As an embodiment of the present invention, the Alzheimer's disease-related gene expression cassette may include a promoter to further improve gene expression, and the promoter is a CMV promoter (eg, human or mouse CMV immediate -early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial virus (RSV) promoter, metallotionin promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene promoter, human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter and CAG promoter selected from the group consisting of However, specifically, it may be a CAG promoter.
상기 CAG프로모터는 변형된 CMV 프로모터의 일종으로 시토메갈로바이러스 즉시초기 인핸서(cytomegalovirus immediate-early enhancer), 닭 β-액틴 프로모터(chicken β-actin promoter), 키메릭 인트론(chimeric intron), 엑손 1(exon 1) 과 토끼 β-글로빈 유전자(rabbit β-globin gene)의 엑손 2(exon 2)를 포함하는 복합 프로모터(Hitoshi Niwa et al., Gene, 108:193-199, 1991, Monahan et al., Gene Therapy, 7:24-30, 2000)이다. 본 발명에서 CAG 프로모터는 본 발명의 발현 카세트가 줄기세포에 도입된 후 분화 유도후에도 장기간 사라지지 않고 발현이 유지될 수 있도록 한다. The CAG promoter is a kind of modified CMV promoter, cytomegalovirus immediate-early enhancer, chicken β-actin promoter, chimeric intron (chimeric intron), exon 1 (exon) 1) and a complex promoter including exon 2 of the rabbit β-globin gene (Hitoshi Niwa et al., Gene, 108:193-199, 1991, Monahan et al., Gene Therapy, 7:24-30, 2000). In the present invention, the CAG promoter allows expression to be maintained without disappearing for a long period of time even after differentiation induction after the expression cassette of the present invention is introduced into stem cells.
본 발명의 다른 일 구체예로서, 상기 유전자 발현 카세트는 서열번호 5의 서열을 갖는 것일 수 있다. 구체적으로 상기 유전자 발현 카세트는 전술한 APP 돌연변이 유전자와 PSEN1 돌연변이 유전자와 함께 이의 발현을 위한 서열을 포함하는 것일 수 있다.In another embodiment of the present invention, the gene expression cassette may have the sequence of SEQ ID NO: 5. Specifically, the gene expression cassette may include a sequence for its expression together with the above-described APP mutant gene and PSEN1 mutant gene.
상기 알츠하이머병 관련 유전자 발현 카세트는, 유전자의 발현을 보다 향상시키기 위하여 인핸서를 더 포함할 수 있으며, 상기 인핸서는 공지의 인핸서를 포함할 수 있다The Alzheimer's disease-related gene expression cassette may further include an enhancer to further improve gene expression, and the enhancer may include a known enhancer.
본 발명의 상기 유전자 발현 카세트는 후술되는 재조합 발현 벡터, 형질전환 인간 전능성 줄기세포주 및 이를 활용하여 제작된 3차원 오가노이드 및 2차원 신경세포에 포함됨으로써 인간의 세포/조직 생리학적 특성을 가진 알츠하이머 질병 모델을 제공할 수 있다. The gene expression cassette of the present invention is contained in a recombinant expression vector, a transgenic human pluripotent stem cell line, and a three-dimensional organoid and two-dimensional neuron produced using the same, which will be described later, so as to have human cell/tissue physiological characteristics Alzheimer's disease model can be provided.
본 발명의 다른 일 양상은 상기 발현 카세트를 포함하는 재조합 발현 벡터를 제공하는 것이다. Another aspect of the present invention is to provide a recombinant expression vector comprising the expression cassette.
본 발명에서 사용되는 용어, "재조합 발현벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 진핵세포에서 이용 가능한 프로모터, 인핸서, 종결신호 및 폴리아데닐레이션 신호는 공지되어 있다.As used herein, the term "recombinant expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotes are known.
본 발명에서 사용되는 용어, "작동 가능하게 연결된"은 유전자 발현 조절 서열과 다른 뉴클레오티드 서열사이의 기능적인 결합을 의미한다. 상기 유전자 발현 조절 서열은 복제원점(replication origin), 프로모터 및 전사 종결 서열(terminator) 등으로 이루어진 군으로부터 선택되는 1종 이상일 수 있다. 전사 종결 서열은 폴리아데닐화 서열(pA)일 수 있으며, 복제 원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점 또는 BBV 복제원점 등일 수 있으나, 이에 한정되는 것은 아니다.As used herein, the term "operably linked" refers to a functional linkage between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be at least one selected from the group consisting of an origin of replication, a promoter, and a terminator. The transcription termination sequence may be a polyadenylation sequence (pA), and the origin of replication may be an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, or a BBV origin of replication, but is not limited thereto. .
본 발명의 일 구체예에 따른 재조합 발현벡터는 플라스미드 벡터, 코즈미드 벡터 및 박테리오파아지 벡터, 아데노바이러스 벡터, 렌티바이러스 벡터, 레트로바이러스 벡터 및 아데노-연관 바이러스 벡터와 같은 바이러스 벡터로 이루어진 군으로부터 선택되는 것일 수 있다. 재조합 발현벡터로 사용될 수 있는 벡터는 당업계에서 사용되는 플라스미드(예를 들어, pcDNA 시리즈, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX 시리즈, pET 시리즈, pUC19 등), 파지(예를 들어, λgt4λB, λ-Charon, λ△z1, M13 등) 또는 바이러스 벡터(예를 들어, 아데노-연관 바이러스(AAV) 벡터, 렌티바이러스 벡터 등) 등을 기본으로 하여 제작될 수 있으나, 이에 한정되는 것은 아니다.The recombinant expression vector according to an embodiment of the present invention is selected from the group consisting of viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, lentiviral vectors, retroviral vectors and adeno-associated viral vectors. it could be Vectors that can be used as recombinant expression vectors include plasmids used in the art (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1). , pHV14, pGEX series, pET series, pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or viral vectors (eg, adeno-associated virus (AAV) vectors, lenti viral vectors, etc.), but is not limited thereto.
본 발명의 재조합 발현벡터는 하나 이상의 선택성 마커를 더 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질주입된 세포를 비형질주입 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 예를 들어, 글리포세이트(glyphosate), 글루포시네이트암모늄(glufosinate ammonium) 또는 포스피노트리신(phosphinothricin)과 같은 제초제 저항성 유전자, 암피실린(ampicillin), 카나 마이신(kanamycin), G418, 블레오마이신(Bleomycin), 하이그로마이신(hygromycin), 클로람페니콜 (chloramphenicol)과 같은 항생제 내성 유전자일 수 있으나, 이에 한정되는 것은 아니다.The recombinant expression vector of the present invention may further include one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can be selected by conventional chemical methods, and includes all genes capable of distinguishing a transfected cell from a non-transfected cell. For example, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, Bleomycin ), hygromycin (hygromycin), may be an antibiotic resistance gene such as chloramphenicol (chloramphenicol), but is not limited thereto.
본 발명의 재조합 발현벡터의 제작은 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용하여 수행될 수 있다.The production of the recombinant expression vector of the present invention can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. there is.
상기 재조합 발현 벡터는 제한효소부위를 포함하고 프로모터 및 유전자 클러스터가 제거된 제1 벡터를 구축하는 단계; 제2 벡터 내에 프로모터, 돌연변이 APP 유전자 및 돌연변이 PSEN 1 유전자를 삽입하여 재조합 제2 벡터를 제조하는 단계; 및 상기 제1 벡터 내에 상기 재조합 제2 벡터를 삽입하는 단계:를 포함하는, 재조합 발현 벡터 제조방법을 통해 제조될 수 있다. The recombinant expression vector comprises a restriction enzyme site, constructing a first vector from which the promoter and gene cluster are removed; preparing a recombinant second vector by inserting a promoter, a mutant APP gene, and a mutant PSEN 1 gene into the second vector; And inserting the second recombinant vector into the first vector: can be prepared through a recombinant expression vector manufacturing method comprising:.
본 발명의 재조합 발현 벡터는 전술한 유전자 발현 카세트를 포함하여 후술되는 재조합 세포주 및 3차원 오가노이드에 포함됨으로써 인간의 세포/조직 생리학적 특성을 가진 알츠하이머 질병 모델을 제공할 수 있다. The recombinant expression vector of the present invention can provide an Alzheimer's disease model with human cell/tissue physiological characteristics by being included in a recombinant cell line and three-dimensional organoid to be described later including the gene expression cassette described above.
본 발명의 다른 일 양상은 상기 재조합 발현 벡터를 이용하여 형질전환된 세포주를 제공하는 것이다. Another aspect of the present invention is to provide a cell line transformed using the recombinant expression vector.
본 발명의 일 구체예에 따른 재조합 발현 벡터가 도입된 형질전환 된 세포주를 제조하기 위하여, 핵산 분자를 유기체, 세포, 조직 또는 기관에 도입하는 당 분야에서 공지된 방법을 사용할 수 있으며, 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 예를 들어, 전기천공법(electroporation), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌글리콜(PEG)법, DEAE-덱스트란법, 양이온성 리포좀법, 및 초산 리튬-DMSO법 등이 포함될 수 있으나, 이에 한정되는 것은 아니다.In order to prepare a transformed cell line into which the recombinant expression vector according to an embodiment of the present invention is introduced, a method known in the art for introducing a nucleic acid molecule into an organism, cell, tissue or organ can be used, As is known, it can be carried out by selecting an appropriate standard technique according to the host cell. Such methods include, for example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method , and lithium acetate-DMSO method and the like may be included, but are not limited thereto.
본 발명의 일 구체예에서 상기 세포주는 인간 성체줄기세포(human adult stem cell, hASC), 인간 배아줄기세포 (human embryonic stem cell, hESCs) 또는 인간 전능성 줄기세포 (human pluripotent stem cell, hiPSCs)일 수 있고, 구체적으로는 인간 배아줄기세포일 수 있다. In one embodiment of the present invention, the cell line may be a human adult stem cell (hASC), a human embryonic stem cell (hESCs), or a human pluripotent stem cell (hiPSCs). And, specifically, it may be a human embryonic stem cell.
상기 형질전환 세포주는 상기 재조합 발현 벡터로 형질전환하는 단계를 포함하는 형질전환 세포주 제조방법에 의해 제조될 수 있다.The transformed cell line may be prepared by a method for producing a transformed cell line comprising the step of transforming with the recombinant expression vector.
본 발명의 다른 일 양상은 상기 세포주로부터 유래된 신경세포를 제공하는 것이다. Another aspect of the present invention is to provide a nerve cell derived from the cell line.
상기 신경세포는 상기 세포주가 신경세포로 분화된 것으로, 분화에 필요한 공지의 물질을 포함하여 공지의 방법으로 제조된 것일 수 있다. The nerve cell is the cell line differentiated into a nerve cell, and may be prepared by a known method including a known material necessary for differentiation.
본 발명의 다른 일 양상은 상기 신경세포로부터 유래된 오가노이드를 제공하는 것이다. Another aspect of the present invention is to provide an organoid derived from the nerve cell.
본 발명에서 사용된 "오가노이드 (organoid)"는 생체(인체) 장기와 유사한 구조 (Structure), 세포의 구성 (Cellular components), 기능 (function)을 보유한 세포 덩어리를 말한다. 본 발명의 오가노이드는 3차원 세포 덩어리뿐만 아니라 평판에 배양된 2차원 세포 조직을 포함할 수 있다. 구체적으로, 상기 오가노이드는 구체적으로, 3차원 뇌오가노이드 또는 2차원 신경세포로 분화된 조직일 수 있다. As used in the present invention, "organoid" refers to a cell mass having a structure, cell components, and functions similar to those of living (human) organs. The organoid of the present invention may include a two-dimensional cell tissue cultured on a plate as well as a three-dimensional cell mass. Specifically, the organoid may be a tissue differentiated into a three-dimensional brain organoid or a two-dimensional nerve cell.
상기 오가노이드는 상기 세포주를 배양하는 단계를 포함하는 오가노이드 제조방법을 통해 제조될 수 있다.The organoid may be prepared through a method for preparing an organoid comprising the step of culturing the cell line.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.
재료 및 방법Materials and Methods
인간 배아줄기세포 배양Human embryonic stem cell culture
인간배아줄기세포주 (hESC, SNUES31)는 한국 서울대학교병원 의학연구센터인 '인구의학연구소(Institute of Reproductive Medicine and Population)'에서 제공되었다. hESC는 10μg/mL의 미토마이신-C-처리 마우스 배아 섬유아세포에서 배양되었고, hESC 배지 (20% knockout serum replacement, 1% minimum essential media-nonessential amino acids (MEM-NEAA) (Invitrogen), 1% Glutamax (Invitrogen), 20 ng/mL basic fibroblast growth factor (bFGF), 7μL/L β-mercaptoethanol in DMEM/F-12)에서 유지되었다. 피더 프리 hESC (feeder-free hESC) 배양을 위해 hESC들은 1 mg/mL dispase® (Invitrogen)를 이용하여 피더 세포로부터 분리되었고, Essential-8 medium (Invitrogen) on Geltrex (Invitrogen)-coated culture plate에서 배양되었다. hESC들은 매 4일마다 0.5 mM EDTA solution를 이용하여 작은 클러스터로 계대 배양되었다. Human embryonic stem cell lines (hESC, SNUES31) were provided by the 'Institute of Reproductive Medicine and Population', a medical research center at Seoul National University Hospital in Korea. hESCs were cultured in mitomycin-C-treated mouse embryonic fibroblasts at 10 μg/mL, hESC medium (20% knockout serum replacement, 1% minimum essential media-nonessential amino acids (MEM-NEAA) (Invitrogen), 1% Glutamax (Invitrogen), 20 ng/mL basic fibroblast growth factor (bFGF), 7 μL/L β-mercaptoethanol in DMEM/F-12). For feeder-free hESC culture, hESCs were isolated from feeder cells using 1 mg/mL dispase® (Invitrogen), and cultured in Essential-8 medium (Invitrogen) on Geltrex (Invitrogen)-coated culture plate. became hESCs were subcultured into small clusters using 0.5 mM EDTA solution every 4 days.
FAD hESC 세포주의 제조Preparation of FAD hESC cell lines
K670M/N671L (Swedish), I716V (Florida) and V717I (London) 돌연변이 (APPSweFlLon)를 포함하는 전장 인간 아밀로이드 전구체 단백질 (human amyloid precursor protein, APP) 및 M146L and L286V 돌연변이 (PSEN1M146L/L286V)를 포함하는 presenilin-1 (PSEN1)을 암호화하는 구조체는 8개월령 5xFAD 마우스 (The Jackson Laboratory)의 뇌 조직으로부터 획득되었다. Full-length human amyloid precursor protein (APP) containing K670M/N671L (Swedish), I716V (Florida) and V717I (London) mutations (APPSweFlLon) and presenilin containing M146L and L286V mutations (PSEN1M146L/L286V) The construct encoding -1 (PSEN1) was obtained from brain tissue of an 8-month-old 5xFAD mouse (The Jackson Laboratory).
먼저, RNeasy Plus RNA extraction kit (Qiagen)를 이용하여 RNA를 추출하였다. iScriptTM cDNA synthesis kit (Bio-Rad)를 이용하여 역전사를 수행하였다. APPSweFlLon 및 PSEN1M146L/L286V cDNA는 KOD-PlusNeo (Toyobo)를 이용하여 제한 효소로 PCR 증폭되었다. 복제에 사용된 프라이머는 다음과 같다:First, RNA was extracted using the RNeasy Plus RNA extraction kit (Qiagen). Reverse transcription was performed using the iScript™ cDNA synthesis kit (Bio-Rad). APPSweFlLon and PSEN1M146L/L286V cDNAs were PCR-amplified with restriction enzymes using KOD-PlusNeo (Toyobo). The primers used for replication were as follows:
APP-BamH I-F, 5'- GAATGGATCCATGCTGCCCGGTTTGGCACTG-3' 및 APP-BamH I-F, 5'-GAATGGATCCATGCTGCCCGGTTTGGCACTG-3' and
APP-Mlu I-R, 5'- GAATACGCGTCTAGTTCTGCATCTGCTCAAA-3'; APP-Mlu I-R, 5'-GAATACGCGTCTAGTTCTGCATCTGCTCAAA-3';
PSEN1-BamH I-F, GAATGGATCCATGACAGAGTTACCTGCACCG-3' 및PSEN1-BamH I-F, GAATGGATCCATGACAGAGTTACCTGCACCG-3' and
PSEN1-Mlu I-R, GAATACGCGTCTAGATATAAAATTGATGGAA-3'.PSEN1-Mlu I-R, GAATACGCGTCTAGATATAAAATTGATGGAA-3'.
다음으로 pLV-mcherry, pSico-PGK-Puro 및 pCDH-EF1α-MCS-IRES-RFP (System Bioscience) 플라스미드를 편집하여 렌티바이러스 듀얼 프로모터 플라스미드 벡터 CAG-MCS-PGK-Puro and CAG-MCS-PGK-mCherry를 제작하였다. pLV-mCherry는 Pantelis Tsoulfas (Addgene plasmid # 36084; http://n2t.net/addgene:36084 ; RRID:Addgene_36084)에서 제공받았고, pSico-PGK puro는 Tyler Jacks (Addgene plasmid # 11586 ; http://n2t.net/addgene:11586 ; RRID:Addgene_11586)으로부터 제공받았다. 그리고, APPSweFlLon, PSEN1M146L/L286V, APPSweFlLon-IRES-PSEN1M146L/L286V을 삽입하였고, 이는 APPSweFlLon or PSEN1M146L/L286V의 증폭된 유전자들, 또는 mCherry 유전자를 MCS에 도입하여 제조하였고, 네 가지의 구조체 CAG-APPSweFlLon-IRES-PSEN1M146L/L286V-PGK-Puro (CAPPP), CAG-APPSweFlLon-PGKPuro(CAPP), CAG-PSEN1M146L/L286V-PGK-mCherry(CPPmC) and CAG-mCherry-PGK-Puro (CmCPP)를 얻었다. 새롭게 제작된 벡터들은 모두 서열 분석 (Enzynomics, 서울, 한국)으로 확인되었다. CAPPP, CAPP, CPPmC or CmCPP 렌티바이러스 제작을 위해 각 플라스미드는 바이러스 패키징 플라스미드[VSV-G 발현 엔벨롭 플라스미드 (envelop plasmid) 및 gag, pol 및 rev 유전자를 포함하는 플라스미드로서 이는 Dr. Yibing Qyang (Yale Cardiovascular Research Center, Yale School of Medicine, CT, USA)에게서 제공받았다]와 함께 X-tremeGene HP DNA transfection reagent (Roche)를 이용하여 100mm 조직 배양 플레이트에서 배양된 HEK293T(ATCC)세포에 형질도입 하였고, 37℃, 5% CO2에서 배양되었다. 배양액은 형질도입 후 24시간 뒤에 바꿔주었고, 10mL의 바이러스 포함 배양액을 매일 3일간 수거하였다. 채취한 30mL의 각 배양액들은 55,200g 및 4℃ 에서 2 시간 조건의 초고속원심분리기(ultracentrifugation) (Hitachi)를 통해 200μL로 농축되었다. 다음으로, 12-웰 세포 배양 플레이트 (corning)에서 피더 없이 배양된 hESCs의 배양액을 20 μL의 농축 바이러스와 2μg/mL polybrene (hexadimethrine bromide, sigma-aldrich)를 포함하는 Essential-8 배양액으로 바꿔줌으로서 바이러스 감염을 통한 CmCPP, CPPmC 또는 CAPPP 유전자 도입을 유도하였다. 배양액은 매 3일마다 바꿔주었다. CAPPP 유전자가 도입된 hESC는 5μg/ml puromycin (Invitrogen) 처리 후 선별하였다. 이 세포주는 CAPPP hESC주로 명명하였다. CmCPP 및 CPPmC 바이러스로 유전자가 도입된 hESC는 단일세포로 분리 후, 500개의 세포를 6-웰 플레이트 STO-plated well(corning)에 접종하였다. 배양 7일 후, 단일 세포 유래 mCherry 발현 콜로니들을 수작업으로 분리 후, Matrigel- 코팅된 12-웰 조직 배양 플레이트로 옮겨 피더 프리 상태에서 배양하였다. 이 세포주는 각각 CmCPP 및 CPPmC hESC주로 명명하였다. 세포가 안정화 된 후, CPPmC hESC는 20 μL의 농축된 CAPP 바이러스와 2μg/mL polybrene을 함유하는 Essential-8 medium으로 배양액을 교체하여 24시간 배양 함으로써, 바이러스 감염을 통한 CAPP 유전자 도입을 유도하였다. 이 후 5μg/ml 퓨로마이신 (puromycin)을 이용하여 CAPP 유전자가 도입된 CPPmC hESC를 선별하였다. CPPmC와 CAPP 렌티바이러스로 이중 감염된 hESC 세포주는 CACPP hESC 주로 명명하였다. 각각의 hESC는 형광현미경 (DM IL LED Fluo, Leica)을 이용하여 이미징되었다. Next, edit the pLV-mcherry, pSico-PGK-Puro and pCDH-EF1α-MCS-IRES-RFP (System Bioscience) plasmids to create the lentiviral dual promoter plasmid vectors CAG-MCS-PGK-Puro and CAG-MCS-PGK-mCherry was produced. pLV-mCherry was provided by Pantelis Tsoulfas (Addgene plasmid # 36084; http://n2t.net/addgene:36084 ; RRID:Addgene_36084) and pSico-PGK puro was provided by Tyler Jacks (Addgene plasmid # 11586; http://n2t) .net/addgene:11586 ; RRID:Addgene_11586). And, APP SweFlLon , PSEN1 M146L/L286V , APP SweFlLon -IRES-PSEN1 M146L/L286V was inserted, which was prepared by introducing the amplified genes of APP SweFlLon or PSEN1 M146L/L286V , or the mCherry gene into MCS, and four Constructs of CAG-APP SweFlLon -IRES-PSEN1 M146L/L286V -PGK-Puro (CAPPP), CAG-APP SweFlLon -PGKPuro(CAPP), CAG-PSEN1 M146L/L286V -PGK-mCherry(CPPmC) and CAG-mCherry-PGK -Puro (CmCPP) was obtained. All newly constructed vectors were confirmed by sequence analysis (Enzynomics, Seoul, Korea). For CAPPP, CAPP, CPPmC or CmCPP lentivirus construction, each plasmid is a viral packaging plasmid [VSV-G expression envelope plasmid and a plasmid containing gag, pol and rev genes, which are prepared by Dr. HEK293T (ATCC) cells cultured in 100 mm tissue culture plates were transfected using X-tremeGene HP DNA transfection reagent (Roche) together with Yibing Qyang (provided by Yale Cardiovascular Research Center, Yale School of Medicine, CT, USA)]. was introduced, and incubated at 37° C., 5% CO 2 . The culture medium was changed 24 hours after transduction, and 10 mL of the virus-containing culture medium was collected every day for 3 days. Each 30mL of the collected culture solution was concentrated to 200μL by ultracentrifugation (Hitachi) at 55,200g and 4℃ for 2 hours. Next, by changing the culture medium of hESCs cultured without a feeder in a 12-well cell culture plate (corning) to an Essential-8 culture medium containing 20 μL of concentrated virus and 2 μg/mL polybrene (hexadimethrine bromide, sigma-aldrich). Transduction of CmCPP, CPPmC or CAPPP genes via viral infection was induced. The culture medium was changed every 3 days. hESCs introduced with the CAPPP gene were selected after treatment with 5 μg/ml puromycin (Invitrogen). This cell line was designated as the CAPPP hESC line. hESCs introduced with CmCPP and CPPmC viruses were separated into single cells, and then 500 cells were inoculated into a 6-well plate STO-plated well (corning). After 7 days of culture, single-cell-derived mCherry-expressing colonies were manually isolated, transferred to a Matrigel-coated 12-well tissue culture plate, and cultured in a feeder-free state. These cell lines were named CmCPP and CPPmC hESC lines, respectively. After the cells were stabilized, CPPmC hESCs were cultured for 24 hours by replacing the culture medium with Essential-8 medium containing 20 µL of concentrated CAPP virus and 2 µg/mL polybrene, thereby induced CAPP gene introduction through virus infection. Thereafter, CPPmC hESCs into which the CAPP gene was introduced were selected using 5 μg/ml puromycin. The hESC cell line double-infected with CPPmC and CAPP lentivirus was named CACPP hESC line. Each hESC was imaged using a fluorescence microscope (DM IL LED Fluo, Leica).
뇌 오가노이드 배양조건 Brain organoid culture conditions
뇌 오가노이드 (Cerebral organoids, COs)는 변형된 프로토콜 7을 이용하여 hESC로부터 제조하였다. Cerebral organoids (COs) were prepared from hESCs using modified protocol 7.
간략하게, 피더 없이 배양된 hESC는 0.5mM EDTA에서 4분간 배양하여 단일세포로 분해하였고, Accutase®에서 4분간 배양하였다. 그리고 세포들은 9x104 cells/cm2 밀도로 4 ng/ml bFGF 및 50 μM Y27632, a Rho-associated protein kinase inhibitor (Tocris)를 함유하는 low bFGF hESC 배양액을 이용하여 96-well ultra low-attachment U-bottom plate (Corning)에서 배양하였다. 해당일을 day0으로 하고, day2에 배양액은 5 ng/ml bFGF 및 50 μM Y27632를 함유하는 hESC 배양액으로 바꿔주었다. day4에 bFGF 및 Y27632가 없는 hESC 배양액으로 바꿔주었다. day6에 각 배상체 (embryo bodies, EBs)는 500 μl 신경분화 유도 배양액 (neural induction medium) [1% N2 supplement (Invitrogen), 1% Glutamax (Invitrogen), MEM-NEAA, 1μg/ml Heparin (Sigma-aldrich) in DMEM/F-12]을 함유하는 24-well ultra low-attachment plate로 이동시켰다. day8에서, 500 μl의 neural induction medium을 추가하였다. day10에서, 신경상피 조직 (neuroepithelial tissues)은 3mm 홈이 있는 Parafilm에 20 μl matrigel (BD Bioscience) droplets으로 옮겼고, 37℃에서 1시간 방치하였다. matrigel droplet은 비타민A가 없는 5ml 뇌오가노이드(CO) 분화 배양액 [0.5% N2 supplement, B27 supplement minus vitamin A (Invitrogen), 2.5 μg/mL human insulin (Roche), 1% Glutamax, 0.5% MEMNEAA, 3.5μl/l β-mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium (Invitrogen)]을 함유하는 60mm 조직 배양 디쉬(corning)으로 옮겨졌다. 배양액은 매일 바꿔주었고, day 14에서 뇌오가노이드를 포함하는 matrigel droplet은 125ml 크기의 바이오리엑터에 뇌오가노이드 분화 배양액 [0.5% N2 supplement, B27 supplement (Invitrogen), 2.5 μg/mL human insulin 1%, Glutamax, 0.5% MEM-NEAA, 3.5 μl/l β-mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium]과 함께 옮겨주었다. 그 이후 배양액은 7일에 한 번씩 바꿔주었다. Briefly, hESCs cultured without a feeder were digested into single cells by incubation in 0.5 mM EDTA for 4 min, and incubated in Accutase® for 4 min. And the cells were 96-well ultra low-attachment U- at a density of 9x10 4 cells/cm 2 using low bFGF hESC culture medium containing 4 ng/ml bFGF and 50 μM Y27632, a Rho-associated protein kinase inhibitor (Tocris). Cultured on a bottom plate (Corning). The corresponding day was designated as day 0, and on day 2, the culture medium was changed to a hESC culture medium containing 5 ng/ml bFGF and 50 μM Y27632. On day 4, it was changed to hESC culture without bFGF and Y27632. On day 6, each embryoid body (EBs) was treated with 500 μl neural induction medium [1% N2 supplement (Invitrogen), 1% Glutamax (Invitrogen), MEM-NEAA, 1 μg/ml Heparin (Sigma-) aldrich) in DMEM/F-12] and transferred to a 24-well ultra low-attachment plate. On day 8, 500 μl of neural induction medium was added. On day 10, neuroepithelial tissues were transferred to 20 μl matrigel (BD Bioscience) droplets on a 3 mm grooved Parafilm, and left at 37° C. for 1 hour. The matrigel droplet contains 5ml brain organoid (CO) differentiation culture medium without vitamin A [0.5% N2 supplement, B27 supplement minus vitamin A (Invitrogen), 2.5 μg/mL human insulin (Roche), 1% Glutamax, 0.5% MEMNEAA, 3.5 μl/l β-mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium (Invitrogen)] was transferred to a 60 mm tissue culture dish (corning). The culture medium was changed every day, and on day 14, matrigel droplets containing brain organoids were placed in a 125 ml bioreactor with a brain organoid differentiation culture medium [0.5% N2 supplement, B27 supplement (Invitrogen), 2.5 μg/mL human insulin 1%, Glutamax, 0.5% MEM-NEAA, 3.5 μl/l β-mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium] was transferred together. After that, the culture medium was changed once every 7 days.
신경세포 분화nerve cell differentiation
신경세포 분화를 유도하기 위해 피더 프리 hESC 세포주는 5-8분동안 Accutase®와 배양함으로써 단일세포로 분리하였고, 5 x 104 cells/cm2 밀도로 10 μM Y27632, a Rhoassociated protein kinase inhibitor 가 첨가된 mTeSRTM1이 포함된 Matrigel-coated 24-well plates에 접종하였다. 이 때를 day-4로 정하였다. 그 다음날 Y27632이 없는 mTeSRTM1로 배양액을 바꿔주었고, 추가적으로 2일간 매일 배양액을 바꿔주었다. 신경세포 분화를 유도하기 위해 mTeSRTM1 배양액은 신경세포 유도 배양액 (1:1 mixture of DMEM/F12 medium and neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 10 μg/ml human Insulin, 1% Glutamax, 1% NEAA and 3.5 μl/l β-mercaptoethanol)으로 바꿔주었고 (day0) 매일 10일동안 배양액을 바꿔주었다. day11에 분화하는 세포들을 Accutase®로 단일 세포 분리 후, 1.5x105 cells/cm2 밀도로 Matrigel coated 24-well plates에 다시 접종하였다. day13에, 배양액은 100 nM LDN193189, selective BMP type I receptor inhibitor, 10 μM SB431542, a selective TGF-β receptor inhibitor 및 2 μM XAV939, a tankyrase inhibitor가 첨가된 신경세포 배양액으로 교체하였고 매 7일마다 배양액을 교체하였다. day19에 배양액은 신경세포 성숙 배양액 (neuronal maturation medium) (neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 1% Glutamax and 25 ng/ml BDNF) 으로 바꿔주었고, 이후 매일 바꿔주었다. day25에, 세포들은 Accutase®로 단일세포 분리 후, 5 x 104 cells/cm2 밀도로 재접종하였다. 약물 처리의 경우, 1 μM β-secretase inhibitor IV (BSI) 또는 10 nM compound E (CE)를 함유하는 신경세포 성숙 배양액을 이용하였다 (도 12). To induce neuronal differentiation, feeder-free hESC cell lines were separated into single cells by incubation with Accutase® for 5-8 minutes, and 10 μM Y27632, a Rhoassociated protein kinase inhibitor was added at a density of 5 x 10 4 cells/cm 2 Inoculated on Matrigel-coated 24-well plates containing mTeSR TM 1. This time was designated as day-4. The next day, the culture medium was changed to mTeSR TM 1 without Y27632, and the culture medium was changed daily for additional 2 days. In order to induce neuronal differentiation, the mTeSR TM 1 culture medium is a neuronal cell induction medium (1:1 mixture of DMEM/F12 medium and neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 10 μg/ml human Insulin, 1% Glutamax, 1% NEAA and 3.5 μl/l β-mercaptoethanol) were replaced (day 0) and the culture medium was changed every day for 10 days. Cells differentiated on day 11 were separated into single cells with Accutase®, and then inoculated again on Matrigel-coated 24-well plates at a density of 1.5x10 5 cells/cm 2 . On day 13, the culture medium was replaced with a neuronal cell culture medium supplemented with 100 nM LDN193189, selective BMP type I receptor inhibitor, 10 μM SB431542, a selective TGF-β receptor inhibitor and 2 μM XAV939, a tankyrase inhibitor, and the culture medium was washed every 7 days. replaced. On day 19, the culture medium was changed to a neuronal maturation medium (neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 1% Glutamax and 25 ng/ml BDNF), and thereafter changed daily. On day 25, cells were re-inoculated at a density of 5 x 10 4 cells/cm 2 after single cell isolation with Accutase®. For drug treatment, a neuronal maturation culture medium containing 1 μM β-secretase inhibitor IV (BSI) or 10 nM compound E (CE) was used ( FIG. 12 ).
면역세포화학 (Immunocytochemistry)Immunocytochemistry
Matrigel-coated coverslip의 세포 클러스터는 3.2% 파라포름알데하이드 포스페이트 버퍼 용액(PBS) (Wako)으로 4℃에서 2시간 동안 고정하였다. 그리고 세포는 PBST (0.1% [vol/vol] Triton X-100 in PBS)로 투과처리 하였고, 10% normal goat serum (Vector)로 블록 하였다. 세포들은 2% normal goat serum을 함유하는 PBST에 일차 항체를 희석하여 4℃ 에서 밤새 방치하였다. 사용한 일차 항체는 다음과 같다: Cell clusters on Matrigel-coated coverslip were fixed with 3.2% paraformaldehyde phosphate buffer solution (PBS) (Wako) at 4°C for 2 hours. Then, cells were permeabilized with PBST (0.1% [vol/vol] Triton X-100 in PBS) and blocked with 10% normal goat serum (Vector). Cells were diluted with the primary antibody in PBST containing 2% normal goat serum and left overnight at 4°C. The primary antibodies used were:
Oct4 (mouse, Santa Cruz sc- 365509, 1:100),Oct4 (mouse, Santa Cruz sc- 365509, 1:100),
nanog (rabbit, Cell Signaling 3580,1:100), nanog (rabbit, Cell Signaling 3580,1:100),
SSEA4 (mouse, Santa Cruz sc-21704,1:100), SSEA4 (mouse, Santa Cruz sc-21704,1:100),
Tra-1-60 (mouse, Santa Cruz sc-21705,1:100), Tra-1-60 (mouse, Santa Cruz sc-21705,1:100),
PHF-1 (mouse, Biolegend 836101, 1:500)PHF-1 (mouse, Biolegend 836101, 1:500)
PBST로 3회 세척 후 세포들은 상온에서 3시간 조건에서 2차 항체화 함께 방치하였다. PBST로 3회 세척한 후, Vectashield mounting medium (Vector Laboratories)를 이용하여 슬라이드글라스에 커버슬립을 부착하고 컨포컬 현미경 (Leica TCS SP5 II, Leica)을 사용하여 이미지화하였다.After washing 3 times with PBST, the cells were left with secondary antibody formation at room temperature for 3 hours. After washing three times with PBST, a coverslip was attached to a slide glass using Vectashield mounting medium (Vector Laboratories) and imaged using a confocal microscope (Leica TCS SP5 II, Leica).
조직학 및 면역형광분석 (Histology and immunofluorescence)Histology and immunofluorescence
뇌오가노이드는 3.2% 파라포름알데이드 포스페이트 버퍼 용액으로 4℃에서 2시간동안 고정하였고, 15% 및 30% 수크로스 용액으로 순차적으로 뇌 오가노이드가 가라앉을 때까지 침지하였다. 뇌오가노이드 샘플은 OCT compound (Leica) 내에 위치시키고 Leica CM1850 cryostat (Leica)를 이용하여 20μm 두께로 동결절단하였다 (cryosectioned). 면역화학염색을 위해, 조직 절편은 상온에서 1시간 동안 10% normal goat serum을 포함하는 PBST (0.1% [vol/vol] Triton X-100 in PBS)에 침지하였다. 조직절편은 또한 2% normal goat serum을 함유하는 PBST에서 1차 항체와 함께 4℃ 에서 밤새 방치되었다. 사용된 1차 항체는 다음과 같다:Brain organoids were fixed with a 3.2% paraformaldehyde phosphate buffer solution at 4° C. for 2 hours, and sequentially immersed in 15% and 30% sucrose solutions until the brain organoids subsided. Brain organoid samples were placed in OCT compound (Leica) and cryosectioned (cryosectioned) to a thickness of 20 μm using a Leica CM1850 cryostat (Leica). For immunochemical staining, tissue sections were immersed in PBST (0.1% [vol/vol] Triton X-100 in PBS) containing 10% normal goat serum at room temperature for 1 hour. Tissue sections were also incubated overnight at 4°C with primary antibody in PBST containing 2% normal goat serum. The primary antibodies used were:
PHF-1 (mouse, Biolegend 836101, 1:500), PHF-1 (mouse, Biolegend 836101, 1:500),
TUJ1 (rabbit, Cell Signaling 5666, 1:100),TUJ1 (rabbit, Cell Signaling 5666, 1:100),
SOX2 (rabbit, Cell Signaling 3579, 1:100), SOX2 (rabbit, Cell Signaling 3579, 1:100),
TUJ1 (mouse, R&D systems MAB1195, 1:200), TUJ1 (mouse, R&D systems MAB1195, 1:200),
TBR2 (mouse, R&D systems MAB6166, 1:100), TBR2 (mouse, R&D systems MAB6166, 1:100),
DCX (rabbit, Cell Sgnaling 4604, 1:100), DCX (rabbit, Cell Sgnaling 4604, 1:100),
PAX6 (rabbit, Biolegend 901301, 1:200), PAX6 (rabbit, Biolegend 901301, 1:200),
MAP2 (rabbit, Cell Signaling 4542, 1:100), MAP2 (rabbit, Cell Signaling 4542, 1:100),
MAP2 (mouse, abcam ab11267, 1:300) 및 MAP2 (mouse, abcam ab11267, 1:300) and
N-cadherin (rabbit, Santa Cruz sc-7939, 1:100)N-cadherin (rabbit, Santa Cruz sc-7939, 1:100)
PBST로 3회 세척 후, 세포들은 상온에서 3시간 동안 2차 항체와 함께 배양하였다. 2차 항체는 Donkey Alexa Fluor 488 and 647 conjugates (Life technologies, 1:500)을 사용하였다. PBST로 3회 세척 후, Vectashield mounting medium (Vector Laboratories)를 이용하여 슬라이드에 커버슬립을 부착하고 컨포컬 현미경 (Leica TCS SP5 II, Leica)을 사용하여 이미지화하였다.After washing 3 times with PBST, the cells were incubated with the secondary antibody for 3 hours at room temperature. As a secondary antibody, Donkey Alexa Fluor 488 and 647 conjugates (Life technologies, 1:500) were used. After washing 3 times with PBST, coverslips were attached to slides using Vectashield mounting medium (Vector Laboratories) and imaged using a confocal microscope (Leica TCS SP5 II, Leica).
웨스턴 블롯 분석Western blot analysis
각각의 뇌오가노이드 또는 신경세포는 얼음에서 lysis buffer (iNtRON biotechnology) 와 함께 초음파 분쇄 (sonication) (Vibra-Cell)를 진행하였다. 세포 용해물은 10-12% 도데실황산나트륨 (SDS)-폴리아크릴아미드겔 전기영동으로 분리하였고, polyvinylidene fluoride transfer membranes (Millipore)로 트랜스퍼를 진행하였다. 블롯은 TBST(10mM Tris-HCl [pH 7.6], 150mM NaCl, 0.1% Tween-20)로 세척하고 1시간 뒤 5% skim milk (Millipore)로 블록 하였고, 1차 항체와 함께 4℃ 에서 밤새 방치하였다. 사용된 1차 항체는 다음과 같다:Each brain organoid or nerve cell was subjected to sonication (Vibra-Cell) with a lysis buffer (iNtRON biotechnology) on ice. Cell lysates were separated by 10-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride transfer membranes (Millipore). The blot was washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween-20), blocked with 5% skim milk (Millipore) after 1 hour, and left overnight at 4° C. together with the primary antibody. . The primary antibodies used were:
anti-phospho-tau (mouse, Biolegend, 836101, 1:1000), anti-phospho-tau (mouse, Biolegend, 836101, 1:1000),
anti-Tau (mouse, Cell Signaling 4019, 1:1000), anti-Tau (mouse, Cell Signaling 4019, 1:1000),
anti-amyloid precursor protein (APP) (moouse Cell Signaling 2450, 1:1000), anti-amyloid precursor protein (APP) (moouse Cell Signaling 2450, 1:1000),
1차 항체는 호스래디쉬 퍼옥시다제 (horseradish peroxidase, HRP)와 결합된 goat anti-rabbit (Santa Cruz sc-2004, 1:10000) or goat anti-mouse (Santa Cruz sc-2005, 1:10,000) IgG 로 검출하였다. 밴드는 enhanced chemiluminescence solution (Thermo Scientific)로 시각화 하였고, ImageQuant LAS 4000 Mini (GE Healthcare)를 이용하여 이미지를 얻었다. The primary antibody was goat anti-rabbit (Santa Cruz sc-2004, 1:10000) or goat anti-mouse (Santa Cruz sc-2005, 1:10,000) conjugated to horseradish peroxidase (HRP). It was detected with IgG. The bands were visualized with an enhanced chemiluminescence solution (Thermo Scientific), and images were acquired using an ImageQuant LAS 4000 Mini (GE Healthcare).
ELISA 분석ELISA analysis
Aβ 1-40 및 Aβ 1-42 수준은 시판되는 human amyloid-β ELISA Kit (Immuno-Biological laboratories Inc.)로 측정되었다. 각각의 뇌오가노이드 (COs)는 얼음에서 lysis buffer와 초음파 처리하여 균질화 하였고, 원심분리 후 상층액을 채취하였다. ELISA는 제조업자의 지침에 따라 수행되었다. ELISA 플레이트 리더 (BioTek, Winooski, VT, USA)를 사용하여 Aβ 1-40 및 Aβ 1-42 ELISA 신호를 정량화 하였다. Aβ 1-40 and Aβ 1-42 levels were measured with a commercially available human amyloid-β ELISA Kit (Immuno-Biological laboratories Inc.). Each brain organoid (COs) was homogenized by ultrasonication with a lysis buffer on ice, and the supernatant was collected after centrifugation. ELISA was performed according to the manufacturer's instructions. Aβ 1-40 and Aβ 1-42 ELISA signals were quantified using an ELISA plate reader (BioTek, Winooski, VT, USA).
통계학적 분석statistical analysis
모든 결과들은 mean ± standard error of mean (S.E.M)로 표시하였다. Student's t-test를 사용하여 유의성을 분석하였다. p<0.05의 값은 통계적으로 유의한 것으로 간주되었다. All results were expressed as mean ± standard error of mean (S.E.M). Significance was analyzed using Student's t-test. Values of p<0.05 were considered statistically significant.
실험결과Experiment result
FAD 뇌오가노이드에서 Aβ 및 tau phosphorylation의 병리학적 표현형 발현을 확인하였다. Aβ 1-40 및 1-42 수준을 70일령 대조군 및 FAD 뇌오가노이드를을 사용하여 측정, 비교하였다. 도 2에서 확인되는 바와 같이, Aβ 1-40 및 1-42의 수준은 CmCPP 대조군 뇌오가노이드 와 비교하여 CAPPP 및 CACPP 뇌오가노이드에서 유의하게 증가하였다. 또한, 웨스턴 블롯 분석을 통해 CAPPP 및 CACPP 뇌오가노이드에서 tau phosphorylation(phosphorylated at Ser396, Ser404)의 유의적인 증가를 확인하였다 (도 3). 이러한 결과는 면역화학분석에서도 확인되었고 (도 4 및 5), Aβ 응집은 CACPP 및 CAPPP 뇌오가노이드에서 확인되었다. 이러한 뇌오가노이드는 광범위한 PHF-1 발현을 보여주었다. The pathological phenotypic expression of Aβ and tau phosphorylation was confirmed in FAD brain organoids. Aβ 1-40 and 1-42 levels were measured and compared using 70-day-old controls and FAD brain organoids. As confirmed in FIG. 2 , the levels of Aβ 1-40 and 1-42 were significantly increased in CAPPP and CACPP brain organoids compared to CmCPP control brain organoids. In addition, a significant increase in tau phosphorylation (phosphorylated at Ser396, Ser404) was confirmed in CAPPP and CACPP brain organoids through Western blot analysis (FIG. 3). These results were also confirmed by immunochemical analysis ( FIGS. 4 and 5 ), and Aβ aggregation was confirmed in CACPP and CAPPP brain organoids. These brain organoids showed extensive PHF-1 expression.
약물 개발을 위한 수단으로서 FAD 뇌오가노이드 모델의 유용성을 평가하기 위해, 도 6과 같이 CACPP 뇌오가노이드를 BACE1 inhibitor IV (BSI), a β-secretase inhibitor 또는 compound E (CE), a γ-secretase inhibitor 로 처리하였다. BSI 또는 CE 처리는 CACPP 뇌오가노이드에서 Aβ 1-40 및 1-42의 수준을 현저하게 낮추었다(도 7). 웨스턴 블롯 분석은 CACPP 뇌오가노이드 의 p-tau 발현 또한 BSI 또는 CE에 의해 감소하였다(도 8). 이러한 결과는 종래 잘 알려진 이론적 결과 (tau phosphorylation은 FAD 돌연변이에 의한 Aβ의 과도한 발현에 의해 유도됨)가 본 발명의 FAD 뇌오가노이드 모델에서 재현되고 동시에 상기 모델이 약물 개발 연구에 유용한 수단으로 활용될 수 있음을 암시한다. In order to evaluate the usefulness of the FAD brain organoid model as a means for drug development, as shown in FIG. 6, CACPP brain organoids were treated with BACE1 inhibitor IV (BSI), a β-secretase inhibitor or compound E (CE), a γ-secretase It was treated with inhibitor. BSI or CE treatment significantly lowered the levels of Aβ 1-40 and 1-42 in CACPP brain organoids ( FIG. 7 ). Western blot analysis showed that p-tau expression of CACPP brain organoids was also decreased by BSI or CE (FIG. 8). These results suggest that the well-known theoretical results (tau phosphorylation is induced by excessive expression of Aβ by FAD mutation) are reproduced in the FAD brain organoid model of the present invention, and at the same time, the model will be utilized as a useful means for drug development research. imply that it can
또한, FAD 뇌오가노이드 모델이 FAD hPSCs로부터 생성될 수 있다면, FAD 신경세포 또한 FAD hPSCs으로부터 생산될 수 있고, FAD 신경세포도 2차원 FAD 모델로서 활용될 수 있다는 가설을 설정하였다. CmCPP 대조군 및 CACPP FAD hPSCs가 신경세포로 분화되었고, FAD 신경세포에서 Aβ 및 tau phosphorylation 즉 AD의 병리학적 표현형 분석을 실시하였다. ELISA분석은 Aβ 1-40 및 1-42의 수준이 CmCPP 대조군에 비교하여 CACPP 신경세포에서 유의적으로 증가한 것을 보여준다(도 9). 그리고 웨스턴 블롯 분석 결과 p-tau 발현 수준이 CACPP 신경세포에서 유의하게 증가한 것을 확인하였고 (도 10). CACPP 신경세포에서의 광범위한 PHF-1 발현 또한 면역염색으로 확인되었다 (도 11).In addition, if the FAD brain organoid model can be generated from FAD hPSCs, we hypothesized that FAD neurons can also be produced from FAD hPSCs, and that FAD neurons can also be utilized as a two-dimensional FAD model. CmCPP control and CACPP FAD hPSCs were differentiated into neurons, and Aβ and tau phosphorylation in FAD neurons, ie, pathological phenotyping of AD, was performed. ELISA analysis showed that the levels of Aβ 1-40 and 1-42 were significantly increased in CACPP neurons compared to the CmCPP control ( FIG. 9 ). And as a result of Western blot analysis, it was confirmed that the expression level of p-tau was significantly increased in CACPP neurons ( FIG. 10 ). Extensive expression of PHF-1 in CACPP neurons was also confirmed by immunostaining ( FIG. 11 ).
그리고 상기 신경세포들에 대한 BSI 또는 CE의 영향을 확인하였다 (도 12). 약물 스크리닝을 위한 수단으로서 FAD 신경세포 모델의 유용성을 확인하기 위한 실험에서, Aβ 1-40 및 1-42의 수준이 CACPP 신경세포에서 BSI 또는 CE의 처리에 의해 유의적으로 억제되었다 (도 13). 웨스턴 블롯 및 면역염색에서도 p-tau 수준이 BSI 또는 CE에 의해 유의적으로 감소되었다 (도 14 및 15).And the effect of BSI or CE on the neurons was confirmed (FIG. 12). In an experiment to confirm the usefulness of the FAD neuron model as a means for drug screening, the levels of Aβ 1-40 and 1-42 were significantly inhibited by treatment with BSI or CE in CACPP neurons ( FIG. 13 ). . Western blot and immunostaining also significantly reduced p-tau levels by BSI or CE ( FIGS. 14 and 15 ).
결론적으로, 본 발명의 인간 유래 2차원 신경세포 3차원 뇌오가노이드 시스템은 아밀로이드 β 및 tau 병리학적 특성을 성공적으로 재현할 수 있고, 알츠하이머병(AD)의 병리학적 메커니즘 연구 및 약물 스크리닝의 플랫폼으로 사용할 수 있다. 또한, 본 발명의 모델은 알츠하이머병(AD)의 분자 메커니즘 연구를 위한 신규 인간 질병 모델 플랫폼을 제공할 수 있다. 2차원 및 3차원 모델에서 알츠하이머 (AD) 병리학적 표현형을 재현하는 본 발명은 다른 질병의 정밀한 인간 모델의 개발에 기여할 수 있다. In conclusion, the human-derived two-dimensional neuronal three-dimensional brain organoid system of the present invention can successfully reproduce the pathological characteristics of amyloid β and tau, and serves as a platform for drug screening and pathological mechanism study of Alzheimer's disease (AD). can be used In addition, the model of the present invention can provide a novel human disease model platform for the study of molecular mechanisms of Alzheimer's disease (AD). The present invention, which reproduces the Alzheimer's (AD) pathological phenotype in two-dimensional and three-dimensional models, may contribute to the development of precise human models of other diseases.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 제시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the presented embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (10)

  1. a) 176번 아미노산, 670번 아미노산, 671번 아미노산 및 717번 아미노산이 돌연변이된 아밀로이드 전구단백질(APP)을 암호화하는 APP 돌연변이 유전자, 및a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and
    b) 146번 아미노산 및 286번 아미노산이 돌연변이된 프리세닐린 1(PSEN1)을 암호화하는 PSEN1 돌연변이 유전자를 포함하는, 알츠하이머병 관련 유전자 발현 카세트.b) an Alzheimer's disease-related gene expression cassette comprising a PSEN1 mutant gene encoding presenilin 1 (PSEN1) in which amino acids 146 and 286 are mutated.
  2. 제1항에 있어서, The method of claim 1,
    상기 APP 돌연변이 유전자는, 서열번호 1의 서열을 갖는 것인, 발현 카세트.The APP mutant gene, the expression cassette having the sequence of SEQ ID NO: 1.
  3. 제1항에 있어서, The method of claim 1,
    상기 PSEN1 돌연변이 유전자는, 서열번호 3의 서열을 갖는 것인, 발현 카세트.The PSEN1 mutant gene, the expression cassette having the sequence of SEQ ID NO: 3.
  4. 제1항에 있어서,The method of claim 1,
    상기 발현 카세트는 CMV 프로모터(cytomegalovirus promoter(예를 들어, 인간 또는 마우스 CMV immediate-early 프로모터), U6 프로모터, EF1-alpha(elongation factor 1-a) 프로모터, EF1-alpha short(EFS) 프로모터, SV40 프로모터, 아데노바이러스 프로모터(major late promoter), pLλ 프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터, 백시니아 바이러스 7.5K 프로모터, HSV의 tk 프로모터, SV40E1 프로모터, 호흡기 세포융합 바이러스(Respiratory syncytial virus; RSV) 프로모터, 메탈로 티오닌 프로모터(metallothionin promoter), β-액틴 프로모터, 유비퀴틴 C 프로모터, 인간 IL-2(human interleukin-2) 유전자 프로모터, 인간 림포톡신(human lymphotoxin) 유전자 프로모터, 인간 GM-CSF(human granulocyte-macrophage colony stimulating factor) 유전자 프로모터 및 CAG 프로모터로 이루어진 군으로부터 선택되는 어느 하나의 프로모터를 포함하는 발현 카세트. The expression cassette is a CMV promoter (cytomegalovirus promoter (eg, human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter , adenovirus promoter (major late promoter), pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, HSV tk promoter, SV40E1 promoter, Respiratory syncytial virus (RSV) ) promoter, metallotionin promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene promoter, human GM-CSF ( An expression cassette comprising any one promoter selected from the group consisting of human granulocyte-macrophage colony stimulating factor) gene promoter and CAG promoter.
  5. 제1항의 발현 카세트를 포함하는, 재조합 발현 벡터.A recombinant expression vector comprising the expression cassette of claim 1 .
  6. 제5항에 있어서, 상기 재조합 발현 벡터는, 서열번호 5의 서열을 갖는 것인, 재조합 발현 벡터.The recombinant expression vector of claim 5, wherein the recombinant expression vector has the sequence of SEQ ID NO: 5.
  7. 제5항의 재조합 발현 벡터를 이용하여 형질전환된 세포주.A cell line transformed using the recombinant expression vector of claim 5 .
  8. 제7항에 있어서, 상기 세포주는 인간 성체줄기세포(human adult stem cell, hASC), 인간 배아줄기세포 (human embryonic stem cell, hESCs) 및 인간 유도만능줄기세포 (human induced pluripotent stem cell, hiPSC) 중 어느 하나인 세포주.According to claim 7, wherein the cell line is human adult stem cells (human adult stem cells, hASC), human embryonic stem cells (human embryonic stem cells, hESCs) and human induced pluripotent stem cells (human induced pluripotent stem cell, hiPSC) of any cell line.
  9. 제7항의 세포주로부터 유래된 신경세포.A nerve cell derived from the cell line of claim 7.
  10. 제7항의 세포주로부터 유래된 오가노이드.An organoid derived from the cell line of claim 7.
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