WO2024075838A1 - 肺線維症モデル、及び、肺線維症の予防又は治療剤のスクリーニング方法 - Google Patents

肺線維症モデル、及び、肺線維症の予防又は治療剤のスクリーニング方法 Download PDF

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WO2024075838A1
WO2024075838A1 PCT/JP2023/036549 JP2023036549W WO2024075838A1 WO 2024075838 A1 WO2024075838 A1 WO 2024075838A1 JP 2023036549 W JP2023036549 W JP 2023036549W WO 2024075838 A1 WO2024075838 A1 WO 2024075838A1
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pulmonary fibrosis
alveolar
reporter gene
pulmonary
fibroblasts
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充 森本
泰典 榎本
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RIKEN
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a pulmonary fibrosis model and a method for screening a preventive or therapeutic agent for pulmonary fibrosis.
  • the lungs In addition to the epithelium, the lungs contain tissue called the interstitium. Pathological conditions of inflammation and fibrosis that occur primarily in the interstitium of the lungs are collectively called interstitial pneumonia, and cases involving fibrosis of the interstitium of the alveoli are called pulmonary fibrosis.
  • interstitial pneumonia cases involving fibrosis of the interstitium of the alveoli.
  • pulmonary fibrosis The prevalence of pulmonary fibrosis increases with age, there are few effective treatments, and the average survival time after diagnosis is 2 to 3 years, making it a disease with an extremely poor prognosis (see, for example, Non-Patent Documents 1 and 2).
  • an in vitro experimental system that reproduces the molecular mechanisms by which the lungs become fibrotic is required.
  • pulmonary fibrosis model has been a drug-induced pulmonary fibrosis model in which bleomycin is administered to mice to induce the onset of pulmonary fibrosis.
  • pulmonary fibrosis models using cell culture systems have included experimental systems in which epithelial cells are cultured with bleomycin to test for the expression of myofibrosis-inducing factors, and experimental systems in which fibroblasts are cultured in the presence of molecules with myofibrogenic potential such as TGF- ⁇ to test for differentiation into myofibroblasts.
  • US Patent No. 5,999, 10, 2003 describes an ex vivo pulmonary fibrosis model using co-cultured lung fibroblasts and pre-alveolar type 1 transitional state (PATS) cells for use in drug screening.
  • PATS pre-alveolar type 1 transitional state
  • US Patent No. 5,999, 10, 2003 describes that mutated lung blast organoids (LBOs) can be used to screen for potential drugs to treat pulmonary fibrosis.
  • LBOs mutated lung blast organoids
  • Epithelial cells are often used as systematic cultured cell lines, which have different properties from cells in the body. It is necessary to develop a model that is closer to actual pulmonary fibrosis than conventional models by reproducing multicellular tissue using cells that are similar to those in the lungs of the living body.
  • the present invention aims to provide a pulmonary fibrosis model and a method for screening preventive or therapeutic agents for pulmonary fibrosis.
  • a pulmonary fibrosis model comprising a co-culture system containing alveolar organoids and pulmonary fibroblasts that express a reporter gene when they undergo myofibrosis, wherein when the alveolar organoids are injured, the pulmonary fibroblasts undergo myofibrosis and express the reporter gene.
  • the pulmonary fibrosis model described in [1] wherein the alveolar organoid is formed by three-dimensional culture of type II alveolar epithelial cells.
  • the reporter gene is a gene encoding a fluorescent protein linked downstream of the ACTA2 gene promoter.
  • a method for screening a preventive or therapeutic agent for pulmonary fibrosis comprising the steps of culturing the pulmonary fibrosis model according to any one of [1] to [5] in the presence of a test substance, damaging the alveolar organoid, and detecting the expression of the reporter gene, wherein a significant decrease in the expression level of the reporter gene compared to that in the absence of the test substance indicates that the test substance is an active ingredient of a preventive or therapeutic agent for pulmonary fibrosis.
  • the present invention provides a pulmonary fibrosis model and a method for screening preventive or therapeutic agents for pulmonary fibrosis.
  • FIG. 1 shows representative microscopic images of alveolar organoids in Experimental Example 1.
  • FIG. 2 shows representative microscopic images of alveolar organoids in Experimental Example 2.
  • FIG. 3 is an image showing the results of Western blotting in Experimental Example 2.
  • FIG. 4 is a schematic diagram showing an experimental schedule of Experimental Example 3.
  • FIG. 5 shows representative microscopic images and a graph illustrating the results of Experimental Example 3.
  • FIG. 6 is a schematic diagram showing an experimental schedule of Experimental Example 4.
  • FIG. 7 shows representative microscopic images and a graph illustrating the results of Experimental Example 4.
  • FIG. 8 shows representative microscopic images and a graph illustrating the results of Experimental Example 6.
  • FIG. 9 shows representative microscopic images of alveolar organoids in Experimental Example 8.
  • FIG. 10 shows representative microscopic images and a graph illustrating the results of Experimental Example 9.
  • human genes and human proteins are represented by capital letters.
  • Mouse genes are represented by capital letters for the first letter and lowercase letters thereafter.
  • Mouse proteins are represented by capital letters.
  • human genes, mouse genes, human proteins, and mouse proteins may be represented without strict distinction.
  • the present invention provides a pulmonary fibrosis model comprising a co-culture system comprising alveolar organoids and pulmonary fibroblasts that express a reporter gene upon myofibrosis, wherein injury to the alveolar organoids results in myofibrosis of the pulmonary fibroblasts and expression of the reporter gene.
  • the inventors have developed a co-culture system of alveolar organoids, which are epithelial multicellular tissues similar to those in living organisms, and myofibrosis reporter lung fibroblasts that visualize myofibrosis using a fluorescent reporter.
  • the pulmonary fibrosis model of this embodiment can reproduce the epithelial-mesenchymal interaction that is a characteristic of pulmonary fibrosis.
  • the pulmonary fibrosis model of this embodiment is closer to actual pulmonary fibrosis than conventional models, and can reflect pulmonary fibrosis whose pathology is complex cell-cell interactions.
  • mini-organs organoids that contain not only stem cells but also differentiated cells in artificially created cell masses and partially retain the functions of the tissues from which they originate, using a method of culturing stem cells and progenitor cells in three dimensions (organoid culture). Cultured organoids are expected to be used in drug discovery applications such as drug screening, and as a source of regenerative medicine.
  • the alveolar organoid is preferably formed by three-dimensional culture of alveolar epithelial type 2 cells (AT2 cells), which are alveolar epithelial stem cells.
  • AT2 cells are preferably derived from mammals. Examples of mammals include humans, monkeys, mice, rats, and pigs.
  • Three-dimensional cultivation of mammalian-derived AT2 cells on a three-dimensional support promotes proliferation and self-organization, allowing the creation of alveolar organoids that reproduce the cellular composition and morphology of the alveolar epithelium, a multicellular tissue. While it was not possible to maintain or proliferate AT2 cells using conventional two-dimensional culture, the three-dimensional culture technique makes it possible to maintain and proliferate AT2 cells.
  • Extracellular matrix can be used as a three-dimensional support.
  • Extracellular matrix is a substance that serves as a scaffold for cells during cell culture.
  • Commercially available products of extracellular matrix include Matrigel (registered trademark) and collagen.
  • Extracellular matrix components include, for example, components contained in basement membranes and glycoproteins present in intercellular spaces.
  • Components contained in basement membranes include, for example, type IV collagen, laminin, heparan sulfate proteoglycan, entactin, etc.
  • Glycoproteins present in intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, heparin sulfate, etc. These may be used alone or in combination of two or more.
  • AT1 cells type I alveolar epithelial cells
  • alveolar organoids tissue structure similar to that of alveolar epithelium.
  • lung fibroblasts that express a reporter gene when transformed into muscle fibrosis can be lung fibroblasts in which a reporter gene is linked downstream of the Actin Alpha 2, Smooth Muscle (ACTA2) gene promoter.
  • ACTA2 Actin Alpha 2, Smooth Muscle
  • the pulmonary fibroblasts that express a reporter gene upon myofibrosis may be pulmonary fibroblasts into which a construct linking a reporter gene downstream of the ACTA2 gene promoter has been introduced as a transgene.
  • they may be pulmonary fibroblasts into which a reporter gene has been introduced into the ACTA2 locus by genome editing.
  • pulmonary fibroblasts into which a gene fragment encoding a 2A self-cleaving peptide and a reporter gene have been introduced in this order downstream of the ACTA2 gene at the ACTA2 locus may be mentioned.
  • Examples of 2A self-cleaving peptides include T2A, P2A, E2A, and F2A.
  • NCBI accession numbers for the cDNA of mouse ACTA2 gene include NM_007392, etc.
  • NCBI accession numbers for the cDNA of human ACTA2 gene include NM_001141945.3, NM_001320855.2, etc.
  • reporter genes include genes encoding luminescent enzymes such as luciferase, genes encoding color-producing enzymes such as alkaline phosphatase, and genes encoding fluorescent proteins. Among these, genes encoding fluorescent proteins are preferably used. In other words, the reporter gene may be a gene that encodes a fluorescent protein linked downstream of the ACTA2 gene promoter.
  • Fluorescent proteins are not particularly limited, and examples include red fluorescent protein DsRed, green fluorescent protein GFP, blue fluorescent protein EBFP, and yellow fluorescent protein YFP.
  • the pulmonary fibrosis model of this embodiment when the alveolar organoids are damaged, the pulmonary fibroblasts undergo myofibrosis and the reporter gene is expressed.
  • damage that can be caused to the alveolar organoids include DNA damage.
  • Methods for inflicting DNA damage on alveolar organoids include, but are not limited to, a method of adding a DNA damage inducer to the culture medium for alveolar organoids.
  • DNA damage inducers include, but are not limited to, bleomycin (CAS number: 11056-06-7).
  • DNA damage can be caused to alveolar organoids by adding bleomycin (CAS number: 11056-06-7) to the culture medium for alveolar organoids.
  • the present invention provides a screening method for a preventive or therapeutic agent for pulmonary fibrosis, comprising the steps of culturing the above-mentioned pulmonary fibrosis model in the presence of a test substance, damaging the alveolar organoid, and detecting the expression of the reporter gene, and the expression level of the reporter gene is significantly reduced compared to that in the absence of the test substance, indicating that the test substance is an active ingredient of a preventive or therapeutic agent for pulmonary fibrosis.By the screening method of this embodiment, a preventive or therapeutic agent for pulmonary fibrosis can be screened.
  • the pulmonary fibrosis model, alveolar organoids, and pulmonary fibroblasts that express a reporter gene when they become myofibrotic are the same as those described above.
  • test substances there are no particular limitations on the test substances, and examples include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, antibody libraries, peptide libraries, etc.
  • the step of damaging the alveolar organoids may be a step of inflicting DNA damage on the alveolar organoids.
  • the method of inflicting DNA damage on the alveolar organoids is not particularly limited, but may include, for example, a method of adding a DNA damage inducer to the culture medium for the alveolar organoids.
  • An example of the DNA damage inducer is bleomycin.
  • the step of damaging the alveolar organoids and the step of culturing the pulmonary fibrosis model in the presence of the test substance may be performed in any order.
  • the alveolar organoids may be cultured for several days in the presence of the test substance, and then the alveolar organoids may be damaged, and then pulmonary fibroblasts that express a reporter gene when they become myofibrotic may be added and co-cultured.
  • the alveolar organoids may be damaged, and then pulmonary fibroblasts that express a reporter gene when they become myofibrotic may be added and co-cultured, and the test substance may be added after the reporter gene has been expressed.
  • the detection of reporter gene expression may be performed by a method appropriate for the reporter gene used.
  • the reporter gene is a fluorescent protein
  • the lung fibroblasts may be irradiated with excitation light and the fluorescence may be measured.
  • the reporter gene is a luminescent enzyme
  • a luminescent substrate may be added and the luminescence may be measured.
  • the reporter gene is a chromogenic enzyme
  • a chromogenic substrate may be added and the color may be measured.
  • test substance is considered to be a candidate for a preventive or therapeutic agent for pulmonary fibrosis.
  • mice (Jackson Laboratory) and Rosa26 mTmG mice (Jackson Laboratory) were crossed to obtain Sftpc CreERT2 : Rosa26 mTmG mice.
  • Sftpc is a marker for type II alveolar epithelial cells (AT2 cells).
  • AT2 cells of Sftpc CreERT2 : Rosa26 mTmG mice express green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Sftpc CreERT2 :Rosa26 mTmG mice were euthanized and the lungs were removed. The removed lungs were minced and placed in 4 mL of protease solution, placed on a rotator, and incubated at 37°C for 30 minutes. The tissue/cells were then dissociated by pipetting, and phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) was added. The cells were then filtered through 100 ⁇ m and 40 ⁇ m strainers. The cells were then suspended in PBS containing 3% FBS and centrifuged at 400 ⁇ g for 5 minutes.
  • PBS phosphate-buffered saline
  • FBS fetal bovine serum
  • GFP + cells (AT2 cells) were sorted and collected.
  • the collected AT2 cells were suspended in culture medium and mixed with an equal volume of growth factor reduced Matrigel (registered trademark, Corning).
  • 20 ⁇ L droplets containing 20,000 cells were placed on the bottom of a 24-well plate.
  • the droplets were incubated at 37° C. for 20 minutes to solidify the gel.
  • 400 ⁇ L of culture medium was added to the well.
  • the composition of the culture medium is shown in Table 1 below. In Table 1, "F.C.” indicates the final concentration.
  • the medium was changed every 3 days.
  • Y27632 (10 ⁇ M) was added to the medium for the first 3 days after sorting or passaging to increase cell viability.
  • the left side of Figure 1 is a representative bright-field image of the obtained alveolar organoids.
  • the scale bar is 1 mm.
  • the center of Figure 1 is an image of GFP fluorescence taken in the same field of view as the left side of Figure 1.
  • the scale bar is 1 mm.
  • the right side of Figure 1 is a fluorescent microscope image showing the results of staining a slice of alveolar organoid with anti-SFTPC antibody, anti-RAGE antibody, and DAPI.
  • the scale bar is 20 ⁇ m.
  • Figure 2 is a fluorescent microscope image showing the results of staining a slice of alveolar organoid with anti- ⁇ H2AX antibody and DAPI. The scale bar is 20 ⁇ m.
  • Figure 3 is an image showing the results of examining the expression of ⁇ H2AX and GAPDH by Western blotting of alveolar organoids. In Figure 3, “BLM” indicates bleomycin, “+” indicates that it was added, and “-” indicates that it was not added.
  • Alveolar organoids were prepared from AT2 cells derived from wild-type mice. Alveolar organoids were prepared in the same manner as in Experimental Example 1, except that EPCAM + MHCII + CD31 - CD45 - cells were sorted and collected from the lungs derived from wild-type mice and used as AT2 cells.
  • Pulmonary fibroblasts were collected from Acta2-DsRed genetically modified mice (Jackson Laboratory). These pulmonary fibroblasts (hereinafter sometimes referred to as "Acta2-DsRed fibroblasts") express the red fluorescent protein DsRed when differentiated into myofibroblasts. Therefore, when fibrosis factors are present in the culture medium, they differentiate into myofibroblasts and emit red fluorescence.
  • FIG. 5 shows representative microscopic images of (myo)fibroblasts co-cultured with alveolar organoids (scale bar, 100 ⁇ m), a graph showing the percentage of DsRed + myofibroblasts among fibroblasts, and a graph showing the size (relative value) of DsRed + myofibroblasts.
  • the DsRed + myofibroblasts generated from fibroblasts co-cultured with bleomycin-treated alveolar organoids were larger than those co-cultured with control organoids (alveolar organoids not treated with bleomycin), indicating that differentiation of fibroblasts into myofibroblasts was induced by bleomycin-treated AT2 lineage cells.
  • [Experimental Example 4] (Study of the sensitivity of the Acta2-DsRed reporter) 6 is a schematic diagram illustrating the experimental schedule of this experimental example. 1 pg/mL to 1,000 pg/mL TGF- ⁇ was added to the culture medium of Acta2-DsRed fibroblasts, and the fluorescence of DsRed was detected. As the TGF- ⁇ , a mixture of recombinant TGF- ⁇ 1, TGF- ⁇ 2, and TGF- ⁇ 3 was used. TGF- ⁇ is a strong fibrosis promoting factor.
  • Figure 7 shows representative microscopic images of (myo)fibroblasts (scale bar 100 ⁇ m) and a graph showing the area (relative value) of DsRed + regions.
  • "Gel” indicates empty Matrigel (registered trademark).
  • "**" indicates a significant difference at p ⁇ 0.01
  • "***” indicates a significant difference at p ⁇ 0.001
  • "N.S.” indicates no significant difference.
  • Acta2-DsRed fibroblasts were able to detect 1 pg/mL of TGF- ⁇ .
  • Acta2-DsRed fibroblasts were shown to increase DsRed fluorescence in a dose-dependent manner by the addition of TGF- ⁇ in the range of 1 to 100 pg/mL.
  • the conventional method for detecting TGF- ⁇ has a detection limit of 3-16 pg/mL, and the Acta2-DsRed reporter developed in this study was shown to be more sensitive than the conventional method.
  • Pathway analysis revealed that the p53 signaling pathway was significantly enriched in bleomycin-treated alveolar organoids.
  • Alveolar organoids were cultured for 24 hours in the presence of Nutlin-3a (2 ⁇ M), a compound that activates the p53 signaling pathway instead of bleomycin. The alveolar organoids were then co-cultured with Acta2-DsRed fibroblasts.
  • FIG. 8 is a representative microscopic image (scale bar 1 mm) of (myo)fibroblasts co-cultured with alveolar organoids, and a graph showing the area (relative value) of DsRed + regions.
  • AOs indicates Matrigel (registered trademark) containing alveolar organoids.
  • CTRL indicates the result of a control not treated with a compound
  • BLM indicates the result of alveolar organoids treated with bleomycin (100 ⁇ M, 24 hours)
  • Nutlin-3a indicates the result of alveolar organoids treated with Nutlin-3a (2 ⁇ M, 24 hours).
  • “*” indicates a significant difference at p ⁇ 0.05
  • ** indicates a significant difference at p ⁇ 0.01
  • N.S. indicates no significant difference.
  • HTII-280 + human AT2 cells were sorted and collected from human normal lung tissue.
  • Human normal lung tissue was collected from lung tissue surgically removed from an early stage lung cancer patient.
  • Human alveolar organoids were prepared in the same manner as in Experimental Example 1, except that the collected human AT2 cells were used as AT2 cells.
  • FIG. 9 is a fluorescent microscopy image showing the results of immunostaining.
  • the scale bar is 20 ⁇ m.
  • Example 9 (Co-culture system of human alveolar organoids and pulmonary fibroblasts) Human alveolar organoids were incubated for 24 hours in the presence or absence of bleomycin (100 ⁇ M) and co-cultured with pulmonary fibroblasts.
  • Figure 10 is a representative microscopic image (scale bar 1 mm) of (myo)fibroblasts co-cultured with human alveolar organoids, and a graph showing the area (relative value) of DsRed + regions.
  • hAOs indicates Matrigel (registered trademark) containing human alveolar organoids
  • CTRL indicates the result of the control not treated with bleomycin
  • BBM indicates the result of human alveolar organoids treated with bleomycin.
  • “*” indicates a significant difference at p ⁇ 0.05.
  • AT2 cells which are mouse alveolar epithelial stem cells, are isolated and three-dimensionally cultured in a three-dimensional support for cells (gelled collagen, etc.).
  • the spherical spheres (alveolar organoids) obtained on the sixth day of culture are treated with bleomycin, and on the seventh day of culture, lung fibroblasts (Acta2-DsRed fibroblasts) are added in the presence or absence of a test substance and co-cultured.
  • test substance After co-culturing for a specified period, reporter expression in the Acta2-DsRed fibroblasts is observed. If the expression level of the fluorescent reporter is significantly reduced compared to the absence of the test substance, the test substance is considered to be a candidate for an active ingredient in a preventive or therapeutic agent for pulmonary fibrosis.
  • the present invention provides a pulmonary fibrosis model and a method for screening preventive or therapeutic agents for pulmonary fibrosis.

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