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WO2020121366A1 - Procédé de désactivation de cellules stellaires hépatiques activées - Google Patents

Procédé de désactivation de cellules stellaires hépatiques activées Download PDF

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WO2020121366A1
WO2020121366A1 PCT/JP2018/045242 JP2018045242W WO2020121366A1 WO 2020121366 A1 WO2020121366 A1 WO 2020121366A1 JP 2018045242 W JP2018045242 W JP 2018045242W WO 2020121366 A1 WO2020121366 A1 WO 2020121366A1
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Prior art keywords
tcf21
gene
hepatic stellate
stellate cells
protein
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Japanese (ja)
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豊 稲垣
泰博 中野
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Tokai University Educational System
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Tokai University Educational System
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Priority to PCT/JP2018/045242 priority Critical patent/WO2020121366A1/fr
Priority to PCT/JP2019/017361 priority patent/WO2020121546A1/fr
Publication of WO2020121366A1 publication Critical patent/WO2020121366A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates to a method for deactivating activated hepatic stellate cells.
  • Organ fibrosis is a condition in which extracellular matrix components such as collagen are excessively deposited in tissues as represented by liver cirrhosis, resulting in organ dysfunction.
  • stellate cells are the main collagen-producing cells, and when inflammation occurs in the liver, they are activated and transformed into myofibroblast-like activated stellate cells that excessively produce collagen.
  • suppression of inflammation suppression of inflammation, inhibition of stellate cell activation (transformation into myofibroblast-like cells), suppression of collagen production by activated stellate cells, and Numerous attempts have been made, including the degradation of collagen fibers secreted and deposited in liver tissue.
  • Non-patent papers 1 to 6 various transcriptional regulatory factors that regulate the activation of hepatic stellate cells have been identified (Non-patent papers 1 to 6) and it was also reported that administration of vitamin A inhibits the activation of hepatic stellate cells (Non-patent literature). Reference 7) has not yet reached clinical application.
  • the col1a1 gene encoding the ⁇ 1 chain of type I collagen, which is a typical extracellular matrix component expressed in fibrotic tissue upon activation of hepatic stellate cells, and ⁇ smooth muscle actin, a marker of myofibroblasts
  • the expression of the Acta2 gene which encodes for, is increased.
  • expression of the Col1a1 gene and Acta2 gene was decreased in about half of the activated stellate cells, resulting in quiescent stellate cells. It was clarified that it was deactivated to a similar trait (Non-Patent Document 8).
  • Non-Patent Document 9 rat rats with liver cirrhosis-dependent liver cancer, administration of autotaxin inhibitors and LPAR1 inhibitors suppressed the expression of Col1a1 gene and Acta2 gene in hepatic stellate cells, improved liver fibrosis, and It has been reported that the number of cancer nodules was also significantly reduced.
  • Tcf21 is a protein that regulates the development of the kidney and has been suggested to be involved in diabetic nephropathy and renal fibrosis (Non-Patent Document 10). In addition, it is known that deficiency of Tcf21 suppresses fibrosis after drug-induced myocardial injury (Non-Patent Document 11).
  • the transcription factor Tcf21 can deactivate activated hepatic stellate cells.
  • the object of the present invention is to provide a method for deactivating activated hepatic stellate cells.
  • the present inventors have found that when the expression level of the transcription control factor Tcf21 is increased in activated hepatic stellate cells, the hepatic stellate cells are deactivated, and arrived at the present invention.
  • the present invention is as follows.
  • the present invention provides a method for deactivating activated hepatic stellate cells, which comprises a step of introducing the Tcf21 gene and/or Tcf21 protein into activated hepatic stellate cells.
  • the present invention also provides a method for producing deactivated hepatic stellate cells, which comprises a step of introducing the Tcf21 gene and/or Tcf21 protein into activated hepatic stellate cells.
  • the production method includes a step of measuring the expression level of the Col1a1 gene, Acta2 gene, or Gfap gene in the hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein have been introduced.
  • the present invention also provides a method for screening deactivated hepatic stellate cells, which comprises a step of introducing the Tcf21 gene and/or Tcf21 protein into activated hepatic stellate cells.
  • the screening method includes a step of measuring the expression level of the Col1a1 gene, Acta2 gene, or Gfap gene in the hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein have been introduced.
  • the present invention also provides a pharmaceutical composition containing the Tcf21 gene and/or Tcf21 protein for the prevention or treatment of liver failure due to hepatitis, cirrhosis, or liver cancer, or any of these diseases.
  • the present invention also provides a pharmaceutical composition for liver antifibrosis, which comprises the Tcf21 gene and/or Tcf21 protein.
  • the present invention also provides a pharmaceutical composition for improving liver function, which comprises the Tcf21 gene and/or Tcf21 protein.
  • a method for deactivating activated hepatic stellate cells can be provided.
  • a method for producing deactivated hepatic stellate cells a method for screening deactivated hepatic stellate cells, and a pharmaceutical composition.
  • the graph which shows the relative expression level of Tcf21 gene, Col1a1 gene, Acta2 gene, and Gfap gene with respect to a non-introduced cell (Control) in the Tcf21 gene introduction cell concerning one mode of the present invention.
  • a section image of a liver tissue section after staining with Sirius red fast green photograph as a substitute for a drawing
  • liver tissue of a Tcf21 gene-transfected mouse relative to non-transfected mouse liver tissue
  • Sirius The graph which shows the result of having calculated the relative area of the red staining positive (azuki) part.
  • 3 is a graph showing the relative expression levels of the Col1a1 gene, Acta2 gene, and Gfap gene in the liver tissue of a Tcf21 gene-transfected mouse with respect to the non-transfected mouse liver tissue (Control) according to one embodiment of the present invention.
  • 3 is a graph showing Alanine transaminase (ALT) values and Aspartate transaminase (AST) values in serum of AAV6-Tcf21-administered mice and non-administered mice (Control) according to one embodiment of the present invention.
  • ALT Alanine transaminase
  • AST Aspartate transaminase
  • relative expression level of the Col1a1 gene and Acta2 gene (vertical axis) relative to non-expressing cells (Control) in cells forcibly expressing a gene involved in hepatic stellate cell differentiation (horizontal axis)
  • Vertical axis relative expression level of the Col1a1 gene and Acta2 gene relative to non-expressing cells
  • Control in cells forcibly expressing a gene involved in hepatic stellate cell differentiation
  • Hepatic stellate cells are one of the non-parenchymal cells that make up the liver, and are vitamin A-storing cells that exist in the sinusoidal space (Dysse's space). When hepatitis becomes chronic, hepatic stellate cells are activated to release vitamin A and transform into ⁇ -smooth muscle actin-positive myofibroblast-like cells to produce excess collagen fibers. As a result, liver fibrosis progresses, and cirrhosis occurs when left untreated. The accumulation of collagen fibers can be confirmed by, for example, Sirius red/Fast green staining.
  • the present invention is based on the fact that when the expression level of Tcf21 in activated hepatic stellate cells is increased, the hepatic stellate cells are deactivated and fibrosis is suppressed. Deactivated hepatic stellate cells have traits similar to stationary hepatic stellate cells.
  • the expression level of the Tcf21 gene is larger when the hepatic stellate cells are in the deactivated state (close to the stationary phase) than when they are in the activated state.
  • the expression level of the Col1a1 gene is low
  • the expression level of the Acta2 gene is low
  • the expression level of the Glial fibrillary acidic protein (Gfap) gene is high. That is, the increased expression level of Tcf21 in activated hepatic stellate cells deactivates hepatic stellate cells, and the deactivation decreases the expression level of the Col1a1 gene and decreases the expression level of the Acta2 gene.
  • the expression level of the Tcf21 gene in the deactivated hepatic stellate cell is preferably 10 when the expression level in the activated hepatic stellate cell is 1, when a cell population of 1 ⁇ 10 5 cells or more is targeted. Or more, more preferably 50 or more, still more preferably 100 or more.
  • the upper limit is not particularly limited, but is 1,000 or less, for example.
  • the expression level of the Col1a1 gene in the deactivated hepatic stellate cells when targeting a cell population of 1 ⁇ 10 5 or more cells, the expression level in the activated hepatic stellate cells is 1, and preferably 0.8. Or less, more preferably 0.7 or less, still more preferably 0.6 or less.
  • the lower limit is not particularly limited, but is 0.1 or more, for example.
  • the expression level of the Acta2 gene in the deactivated hepatic stellate cell is preferably 0.8 when the expression level in the activated hepatic stellate cell is 1, when the cell population of 1 ⁇ 10 5 or more cells is targeted.
  • the lower limit is not particularly limited, but is 0.1 or more, for example.
  • the expression level of the Gfap gene in the deactivated hepatic stellate cells when targeting a cell population having a cell number of 1 ⁇ 10 5 or more, the expression level in the activated hepatic stellate cells is 1, and preferably 1.5. Or more, more preferably 2 or more, and further preferably 2.5 or more.
  • the upper limit is not particularly limited, but is 20 or less, for example.
  • the cell size is smaller when the hepatic stellate cells are in the deactivated state than when they are in the activated state, based on the cell population level. .. That is, an increase in the expression level of Tcf21 in activated hepatic stellate cells deactivates the hepatic stellate cells, and the deactivated hepatic stellate cells can be identified by reducing the average cell size based on the cell population level. .. The reduction in average cell size can be identified by measuring the average area of hepatic stellate cells observed under a microscope.
  • the average cell area of deactivated hepatic stellate cells when targeting a cell population of 1 ⁇ 10 4 or more cells, the average cell area of activated hepatic stellate cells is 1, preferably 0.8 or less. , More preferably 0.7 or less, still more preferably 0.6 or less.
  • the lower limit is not particularly limited, but is 0.1 or more, for example.
  • the liver tissue is The area of fibrosis shown by Sirius red staining positive (adzuki color) when stained with Fast Green is small. That is, hepatic stellate cells are deactivated due to an increase in the expression level of Tcf21 in activated hepatic stellate cells, and the deactivation can be identified by a reduction in the fibrotic area positive for Sirius red staining.
  • the area of the Sirius red staining positive part in the fibrotic liver tissue in which the hepatic stellate cells are not deactivated is 1 and the hepatic star
  • the area of the liver tissue in which the cells are deactivated is preferably 0.7 or less, more preferably 0.6 or less, and further preferably 0.5 or less.
  • the lower limit is not particularly limited, but is 0.1 or more, for example.
  • the Tcf21 gene is added. It is possible to use a known molecular biology technique such as preparing an expression vector, an adeno-associated virus, or the like, and introducing the vector into activated hepatic stellate cells for forced expression.
  • the type and characteristics of the expression vector and virus are not particularly limited as long as they can increase the expression level of the Tcf21 gene or Tcf21 protein when introduced into activated hepatic stellate cells.
  • the Tcf21 gene to be introduced into activated hepatic stellate cells is not limited as long as it can deactivate activated hepatic stellate cells, but depending on the animal species, for example, in the case of mouse, SEQ ID NO: 1 (NCBI accession No.: NC_000076. 6) In the case of human, a gene having the nucleotide sequence shown in SEQ ID NO: 4 (NCBI accession No.: NG_032121.1) can be mentioned. As long as the Tcf21 gene has one or more of decreasing the expression level of the Col1a1 gene, decreasing the expression level of the Acta2 gene, and increasing the expression level of the Gfap gene, SEQ ID NO: 1 or 4 is used. DNAs that hybridize under stringent conditions with DNAs having complementary sequences to the base sequences described are included.
  • the stringent conditions include, for example, conditions of washing under the conditions of 0.1 ⁇ SDS, 0.1 ⁇ SSC and 68° C.
  • the method for measuring the expression of the Tcf21 gene, the Col1a1 gene, the Acta2 gene, the Gfap gene and their gene products is not particularly limited, and examples thereof include a method of measuring the expression level of a known mRNA or protein.
  • the expression level of mRNA can be examined by, for example, RT-PCR method, quantitative PCR method, microarray method, or Northern blot method.
  • the expression level of the gene product protein can be examined by, for example, Western blotting, ELISA, or the like.
  • the nucleotide sequence of the coding region of the Tcf21 gene is, for example, SEQ ID NO: 2 (NCBIaccessionNo.: NM_011545.2) in the case of mouse and SEQ ID NO: 5 (NCBI accessionNo.: NM_198392.2) in the case of human.
  • SEQ ID NO: 2 NCBIaccessionNo.: NM_011545.2
  • SEQ ID NO: 5 NCBI accessionNo.: NM_198392.2
  • Examples include the cDNA sequences described (sequences corresponding to the sequences obtained by removing the untranslated region from the transcript (mRNA) of the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4, respectively). It is possible to design or obtain primers and probes for expression analysis.
  • the base sequence may have 90% or more, preferably 95% or more, and more preferably 98% or more identity (homology) with the base sequence of SEQ ID NO: 2 or 5.
  • the primer, the probe and the like have 90% or more, preferably 95% or more, more preferably 98% or more identity (homology) with the complementary sequence of the base sequence as long as it can specifically bind to the base sequence. May be included.
  • the anti-Tcf21 protein antibody used for Western blotting, ELISA, etc. a commercially available antibody can be used, and in the case of mouse, the Tcf21 protein consisting of the amino acid sequence represented by SEQ ID NO: 3 (NP_035675.1).
  • an antibody prepared by using the amino acid sequence of a part thereof, in the case of human being, the Tcf21 protein consisting of the amino acid sequence represented by SEQ ID NO: 6 (NP_938206.1) or a part of the amino acid sequence thereof as an immunogen may be used. it can.
  • the Tcf21 protein has a function of reducing the expression level of the Col1a1 gene, a function of decreasing the expression level of the Acta2 gene, or a function of increasing the expression level of the Gfap gene, it is a sequence It may have 80% or more, preferably 85% or more, more preferably 90% or more identity (homology) with the amino acid sequence of No. 3 or 6.
  • the antibody or the like has an amino acid sequence having 80% or more, preferably 85% or more, and more preferably 90% or more identity (homology) with the amino acid sequence, as long as it can specifically bind to the amino acid sequence, or A part thereof may be prepared as an immunogen.
  • activated hepatic stellate cells are deactivated by the increase of Tcf21 protein
  • a known molecular biology such as preparing Tcf21 protein and introducing it into activated hepatic stellate cells using liposomes, exosomes, etc.
  • the activated hepatic stellate cells can also be deactivated by using a dynamic method.
  • the Tcf21 protein may be introduced into activated hepatic stellate cells when the Tcf21 gene is not introduced into activated hepatic stellate cells, or when the Tcf21 gene is introduced into activated hepatic stellate cells. May be done. In the latter case, the Tcf21 protein may be introduced before or after the Tcf21 gene is introduced.
  • the Tcf21 protein to be introduced into activated hepatic stellate cells is not limited as long as it can deactivate activated hepatic stellate cells.
  • SEQ ID NO: 3 for mouse SEQ ID NO: for human
  • the Tcf21 protein having the amino acid sequence represented by 6 can be mentioned.
  • any one or more of decreasing the expression level of Col1a1 gene, decreasing the expression level of Acta2 gene and increasing the expression level of Gfap gene can be used as long as it is SEQ ID NO: 3 or SEQ ID NO: 3.
  • It may be a protein consisting of an amino acid sequence having 80% or more, preferably 90% or more, and more preferably 95% or more identity (homology) with the amino acid sequence of 6.
  • the Tcf21 protein may be modified.
  • the modification include amidation, addition of a lipid chain (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, Examples thereof include, but are not limited to, phosphorylation at tyrosine residue and the like), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.
  • the Tcf21 protein may have an amino acid sequence added so that it can be selectively delivered to the liver or hepatic stellate cells.
  • an amino acid sequence include the amino acid sequences described in Nature Communications 3:951 doi: 10.1038/ncomms1952.2012 Kondo E and the like.
  • a recombinant expression vector can be constructed using the DNA encoding the Tcf21 gene, introduced into a host for expression, purified, and produced. Any known molecular biology method can be used.
  • One embodiment of the present invention is a method for deactivating activated hepatic stellate cells, which comprises a step of introducing the Tcf21 gene and/or Tcf21 protein into activated hepatic stellate cells.
  • the active hepatic stellate cell of the present invention may be an active hepatic stellate cell in a state that has not been collected in the target liver, or may be an active hepatic stellate cell immediately after being collected from the target liver, It may be a primary culture cell or a culture cell line of activated hepatic stellate cells.
  • the marker gene for activated hepatic stellate cells for example, genes involved in fibrosis such as Col1a1 gene and Acta2 gene can be used.
  • genes involved in fibrosis such as Col1a1 gene and Acta2 gene can be used.
  • genes that are highly expressed in quiescent stellate cells such as Tcf21 gene and Gfap gene can be used.
  • the subject is not particularly limited as long as it has hepatic stellate cells, for example, mammals such as humans, mice, rats, dogs, cats, rabbits, cows, horses, goats, sheep and pigs, and chickens and the like. Examples include birds.
  • a mammal such as human, mouse, dog, cat, cow, horse, pig, etc., more preferably human, mouse, dog, cat, etc., further preferably human or mouse, most preferably human. Is. Further, the age and sex (male or female) of the individual does not matter.
  • primary culture cells or cultured cell lines of activated hepatic stellate cells also include cells in which resting hepatic stellate cells have been activated for testing.
  • primary culture activated hepatic stellate cells that were activated by culturing quiescent stellate cells on a culture dish, or cultured activated hepatic stellate cells that were forced to express a marker gene for active hepatic stellate cells Examples include hepatic stellate cell lines.
  • a marker gene of activated hepatic stellate cells is incorporated into a plasmid or a viral vector for introducing into mammalian cells, and the cells are obtained by transfection into cells by a usual method such as lipofection. Cells and the like. Transfection may be transient or stable.
  • the amount of Tcf21 gene and/or Tcf21 protein introduced may be the amount of the activated hepatic stellate cells immediately after being collected from the target liver. Also, in the case of targeting primary culture activated hepatic stellate cells or cultured activated hepatic stellate cell lines, the transfer amount usually used for gene transfer or protein transfer to cells may be used. In the case of introducing the Tcf21 gene and/or Tcf21 protein into activated hepatic stellate cells in the target liver that have not been collected, the pharmaceutical composition described below is considered to be equivalent to the pharmaceutical composition described below. The description of the object is incorporated.
  • Deactivated hepatic stellate cells can be produced by the above-described method for deactivating activated hepatic stellate cells. Therefore, another embodiment of the present invention is a method for producing deactivated hepatic stellate cells, which comprises a step of introducing the Tcf21 gene and/or Tcf21 protein into activated hepatic stellate cells. Further, when the active hepatic stellate cells are the active hepatic stellate cells in the target liver that have not been collected, after introducing the Tcf21 gene and/or Tcf21 protein, a part thereof or All may be removed in vitro.
  • the production method preferably includes a step of measuring the expression level of the Col1a1 gene, Acta2 gene, or Gfap gene in the hepatic stellate cell into which the Tcf21 gene and/or Tcf21 protein has been introduced. It is also preferable to include a step of measuring the expression level of the Tcf21 gene in the hepatic stellate cell into which the Tcf21 gene has been introduced. In addition, a step of measuring the amount of Tcf21 protein in the hepatic stellate cell into which the Tcf21 protein has been introduced may be included.
  • the expression level of the Tcf21 gene, the Col1a1 gene, the Acta2 gene, or the Gfap gene in the hepatic stellate cells into which the Tcf21 gene and/or the Tcf21 protein have been introduced and the method for measuring the expression level are as described above.
  • the expression level of the Tcf21 protein can also be examined by Western blotting, ELISA, etc., as described above.
  • the production method preferably includes a step of measuring an average cell area in hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein has been introduced.
  • the average cell area in hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein has been introduced is as described above.
  • the production method includes a step of staining liver tissue with Sirius red fast green after the step of introducing the Tcf21 gene and/or Tcf21 protein. Further, it is preferable that the production method includes a step of measuring a fibrotic area positive for Sirius red staining after the staining step.
  • the sirius red fast green staining and the sirius red staining-positive fibrotic area in the liver tissue in which the hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein have been introduced are present are as described above.
  • Another aspect of the present invention is a method for screening deactivated hepatic stellate cells, which comprises a step of introducing the Tcf21 gene and/or Tcf21 protein into activated hepatic stellate cells.
  • the deactivated hepatic stellate cell screened by the present method may be a cell that is a candidate for the deactivated hepatic stellate cell.
  • the active hepatic stellate cells are the active hepatic stellate cells in the target liver that have not been collected, after introducing the Tcf21 gene and/or Tcf21 protein, a part thereof or All may be removed in vitro.
  • the screening method preferably includes the step of measuring the expression level of the Col1a1 gene, the Acta2 gene, or the Gfap gene in the hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein have been introduced. It is also preferable to include a step of measuring the expression level of the Tcf21 gene in the hepatic stellate cell into which the Tcf21 gene has been introduced. In addition, a step of measuring the amount of Tcf21 protein in the hepatic stellate cell into which the Tcf21 protein has been introduced may be included.
  • the expression level of the Tcf21 gene, the Col1a1 gene, the Acta2 gene, or the Gfap gene in the hepatic stellate cells into which the Tcf21 gene and/or the Tcf21 protein have been introduced and the method for measuring the expression level are as described above.
  • the expression level of Tcf21 protein is also as described above.
  • the screening method preferably includes a step of measuring an average cell area in the hepatic stellate cell into which the Tcf21 gene and/or Tcf21 protein has been introduced.
  • the average cell area in hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein has been introduced is as described above.
  • the screening method preferably includes a step of staining liver tissue with Sirius red fast green after the step of introducing the Tcf21 gene and/or Tcf21 protein. Further, the screening method preferably includes a step of measuring a fibrotic area positive for Sirius red staining after the staining step.
  • the sirius red fast green staining and the sirius red staining-positive fibrotic area in the liver tissue in which the hepatic stellate cells into which the Tcf21 gene and/or Tcf21 protein have been introduced are present are as described above.
  • a known cell selection method can be used for the selection of the deactivated hepatic stellate cells. For example, a method in which a reporter gene that expresses a fluorescent protein is ligated to a vector containing the Tcf21 gene, and after introduction into cells, the fluorescence intensity thereof is used as an index to select deactivated hepatic stellate cells can be mentioned. In addition, a method may be mentioned in which a Tcf21 protein fused with a fluorescent protein is introduced into cells and then the deactivated hepatic stellate cells are selected using the fluorescence intensity as an index. As a specific example of a method for selecting deactivated hepatic stellate cells using fluorescence intensity as an index, the FACS technique and the like can be mentioned.
  • the present invention provides, in another aspect, a pharmaceutical composition containing the Tcf21 gene and/or Tcf21 protein (hereinafter sometimes referred to as “pharmaceutical composition of the present invention”).
  • a pharmaceutical composition for preventing or treating liver failure due to hepatitis, cirrhosis, or liver cancer, or any of these diseases a pharmaceutical composition for antifibrotic liver, and liver function improvement Pharmaceutical compositions for use.
  • the pharmaceutical composition of the present invention contains the Tcf21 gene and/or Tcf21 protein.
  • the Tcf21 gene in this embodiment the previously mentioned embodiment of the Tcf21 gene is used, and specific embodiments include an expression vector containing the Tcf21 gene and an adeno-associated virus.
  • the type and characteristics of the expression vector or virus are not particularly limited as long as they can increase the expression level of the Tcf21 gene when introduced into activated hepatic stellate cells.
  • the specific embodiment of the Tcf21 protein the aforementioned embodiment of the Tcf21 protein is incorporated.
  • the pharmaceutical composition of the present invention is usually used by formulating a liquid or solid pharmaceutical carrier that is physiologically acceptable.
  • the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and specific examples thereof include solutions, suspensions, emulsions and injections. Further, upon formulation, addition of an excipient, a binder, a disintegrating agent, a lubricant, a stabilizer, a flavoring agent, a diluent, a surfactant, a solvent for injection, etc. which are usually used as a formulation carrier. Agents, liposomes, exosomes, etc. as means for delivery to target cells can be used.
  • the dose of the pharmaceutical composition of the present invention can be appropriately selected depending on the dosage form, usage, target age, sex, body weight, type of disease, degree of disease, symptom, administration route, administration schedule, dosage form and the like.
  • the amount to be administered as the Tcf21 gene is, for example, an amount converted into the amount of adeno-associated virus genome in terms of body weight ratio, preferably 1 ⁇ 10 10 viral genome/kg or more, more preferably 1 ⁇ 10 11 viral genome/kg. Or more, more preferably 1 ⁇ 10 12 viral genome/kg or more, while preferably 1 ⁇ 10 16 viral genome/kg or less, more preferably 1 ⁇ 10 15 viral genome/kg or less, further preferably 1 ⁇ 10 14 viral genome/kg or less.
  • the amount when administered as Tcf21 protein is, for example, preferably 0.1 mg/kg or more, more preferably 1 mg/kg or more, still more preferably 10 mg/kg or more in terms of body weight ratio, while preferably 1,000 mg/kg or less, more preferably 500 mg/kg or less, still more preferably 100 mg/kg or less.
  • the route of administration may be, but is not limited to, oral administration, rectal injection, subcutaneous injection, intramuscular injection, peripheral vein injection, hepatic artery injection, intraperitoneal injection and the like.
  • the administration timing of the pharmaceutical composition of the present invention is not particularly limited, and the administration timing can be appropriately selected according to the method for preventing or treating the target disease. It may also be administered prophylactically or used for maintenance therapy.
  • the dosage form is preferably determined according to the dosage form, the age and sex of the patient, other conditions, the degree of symptoms of the patient, and the like.
  • the pharmaceutical composition of the present invention can be administered once a day or divided into a plurality of times, or may be administered once every several days or weeks. Furthermore, irregular administration may be performed while evaluating the therapeutic effect.
  • the pharmaceutical composition of the present invention has an antifibrotic effect on the liver, it can be preferably used for the prevention or treatment of diseases that can be prevented or treated by antifibrosis of the liver. Both “treatments” also include the control and improvement of disease progression. Specific examples of its application include liver failure due to hepatitis, cirrhosis, or liver cancer, or any of these diseases. The liver failure may be acute liver failure or chronic liver failure.
  • the subject to which the pharmaceutical composition of the present invention has been administered has a smaller value of ALT or AST in serum, which is an index of liver function, than the subject to which it has not been administered. Therefore, the pharmaceutical composition of the present invention is useful as a pharmaceutical composition for improving liver function.
  • the ALT of the subject to which the pharmaceutical composition of the present invention has been administered is preferably 0.7 or less, more preferably 0.6 or less, still more preferably 0.5 or less, with 1 being the ALT of the subject not administered.
  • the lower limit is not particularly limited, but is 0.1 or more, for example.
  • the AST of the subject to which the pharmaceutical composition of the present invention is administered is preferably 0.7 or less, more preferably 0.6 or less, still more preferably 0.5 or less, where 1 is the AST of the subject not administered.
  • the lower limit is not particularly limited, but is 0.1 or more, for example.
  • the pharmaceutical composition of the present invention may be administered alone, or other pharmaceutical composition or medicament, for preventing or treating a disease which can be prevented or treated by antifibrosis of the liver or improvement of liver function. You may use together with a pharmaceutical composition or a pharmaceutical.
  • Another aspect of the present invention is the use of the Tcf21 gene and/or Tcf21 protein in the manufacture of a pharmaceutical composition for liver antifibrosis or a pharmaceutical composition for improving liver function.
  • Another aspect of the present invention is the use of the Tcf21 gene and/or Tcf21 protein for antifibrosis of the liver or improvement of liver function.
  • Another aspect of the present invention is a Tcf21 gene and/or Tcf21 protein used for antifibrosis of liver or improvement of liver function.
  • another embodiment of the present invention is the Tcf21 gene and/or Tcf21 protein used for the prevention or treatment of diseases that can be prevented or treated by antifibrosis of the liver or improvement of liver function.
  • another embodiment of the present invention comprises the administration of the Tcf21 gene and/or Tcf21 protein, a liver anti-fibrotic pharmaceutical composition or a liver function improving pharmaceutical composition to an application subject, wherein It is a fibrosis method. Further, another embodiment of the present invention comprises the step of administering the Tcf21 gene and/or Tcf21 protein, the pharmaceutical composition for liver antifibrosis or the pharmaceutical composition for improving liver function to an application subject, to antifibrotic liver. Alternatively, it is a method for preventing or treating a disease that can be prevented or treated by improving liver function.
  • Tcf21 gene and/or Tcf21 gene in the manufacture of a pharmaceutical composition for the prevention or treatment of liver failure based on hepatitis, cirrhosis, or liver cancer, or any of these diseases.
  • another embodiment of the present invention is a Tcf21 gene and/or Tcf21 protein used for the prevention or treatment of liver failure caused by hepatitis, cirrhosis, liver cancer, or any of these diseases.
  • the step of administering the Tcf21 gene and / or Tcf21 protein to the subject, or hepatitis, cirrhosis, or liver cancer according to one aspect of the present invention, or any of these diseases
  • a method for preventing or treating liver failure based on hepatitis, cirrhosis, or liver cancer, or any of these diseases, which comprises a step of administering a pharmaceutical composition for prevention or treatment of liver failure based on is there.
  • Example 1 Isolation of hepatic stellate cells
  • Somnopentyl (Kyoritsu Pharmaceutical Co., Ltd.) (75 ⁇ L) was intraperitoneally administered under deep anesthesia to open the abdomen to expose the portal vein.
  • Surfloflash 22G (Terumo) was inserted into the portal vein, and the cannula tube was connected there.
  • a peristaltic pump (ATTO)
  • the following perfusates 1 to 3 were sequentially sent at 50 mL per flow rate of 6 mL/min to digest the liver.
  • GBSS buffer Calcium-free Gey's balanced salt solutions buffer
  • GBSS buffer Calcium-free Gey's balanced salt solutions buffer
  • EGTA Dojindo
  • Perfusate 2 Prolongase E 30 in GBSS buffer 100 mL Solution in which mg (Millipore) is dissolved
  • Perfusion solution 3 Solution in which 20 mg of Collagenase (Fujifilm Wako Pure Chemical Industries) is dissolved in 100 mL of GBSS buffer.
  • the cell suspension that was made into single cells by these operations was collected in a 50 mL tube (Corning) while being washed with GBSS buffer, and centrifuged at 4°C for 5 minutes at 2,500 rpm. The supernatant was removed with an aspirator, and the cell pellet was suspended in a mixed solution of 50 mL of GBSS buffer and 20 ⁇ L of DNase solution until the pellet was completely broken, and centrifuged at 4°C for 5 minutes at 2,500 rpm for 5 minutes. The same operation was repeated twice.
  • the cells obtained were suspended and suspended in GBSS buffer so that the total volume was 7 mL, and this cell suspension was lysed with GBSS buffer in a 15 mL tube (Corning) with 30% Nycodenz AG. (Proteogenix) solution was mixed with 3 mL. On this solution, 2 mL of 8.8% Nycodenz AG solution was gently overlaid so as to form a layer, and 3 mL of GBSS buffer solution was overlaid in the same manner. This was centrifuged at 4° C. at 3,300 rpm for 15 minutes. After centrifugation, hepatic stellate cells arranged at the interface between Nycodenz AG solution and GBSS buffer were collected using a 1 mL pipette. The obtained hepatic stellate cells were seeded on a 60 mm dish containing 5 mL of the culture medium at 3.5 ⁇ 10 4 cells/cm 2 .
  • Hepatic stellate cells were cultured at 37° C. under a 5% carbon dioxide concentration.
  • the culture solution was prepared by dissolving 4.75 g of Dulbecco's modified Eagle medium (Nissui) powder in 500 mL of pure water and then sterilizing under high pressure steam.
  • the culture supernatant was removed by an aspirator, 2 mL of TrypLExpress (Invitrogen) was added to this, and the mixture was allowed to undergo an enzymatic reaction while stirring for 10 minutes at room temperature, and adhered cells were detached from the bottom of the dish. This was collected in a 15 mL tube and centrifuged at 4°C for 5 minutes at 2,500 rpm. The supernatant was removed by an aspirator, and the cell pellet was resuspended by stirring well in the culture medium, seeded on a new dish and cultured. Two days after this passage treatment, the above passage operation was repeated to activate the hepatic stellate cells.
  • TrypLExpress Invitrogen
  • AAV6 capable of overexpressing Tcf21 in activated hepatic stellate cells was prepared as follows using adeno-associated virus 6 (hereinafter, AAV6) of AAVpro Helper Free System (Takara Bio).
  • AAV6 adeno-associated virus 6
  • PAAV-CMV-Tcf21-IRES- was incorporated into the pAAV-CMV vector included in the same system by incorporating the Tcf21 cDNA prepared from adult mouse hepatic stellate cells and pMYs-IRES-GFP retrovirus vector-derived IRES-GFP. GFP was made.
  • pAAV-CMV-IRES-GFP containing only IRES-GFP was simultaneously prepared.
  • Tcf21 cDNA was synthesized using the oligo DNA described below as a primer.
  • ⁇ Tcf21-Forward primer ATGTCCACTGGCTCCCTCAGCGATGTAGAA (NCBI accession No. NM_011545.2) (SEQ ID NO: 7)
  • ⁇ Tcf21-Reverse primer TCAGGATGCTGTAGTTCCACACAAG (same as above) (SEQ ID NO: 8)
  • AAVpro 293T Cell Line (Takara Bio) was subcultured and expanded, and finally 40 mm 100 mm collagen-coated dishes (AGC technograss) were inoculated.
  • PRC6 vector, pHelper vector, pAAV-CMV-Tcf21-IRES-GFP vector or control pAAV-CMV-IRES-GFP vector included in AAVproHelper Free System are mixed in Opti-MEM (Gibco) and PEI Max ( Polysciences) was added to prepare vector plasmid liposomes.
  • This liposome was added to the medium of AAVpro293T Cell Line and cultured at 37°C. After 72 hours, remove the infected cells from the dish using a cell scraper, collect in a 50 mL tube, centrifuge at 1,000 rpm for 5 minutes at 4°C to remove the medium, and use the AAVpro Purification Kit (Takara Bio). The adeno-associated virus of interest was purified from infected cells. The concentration of the purified adeno-associated virus was calculated as the amount of virus genome contained in the purified solution using the AAV qPCR rapid titer measurement kit (Takara Bio) and StepOnePlus real-time PCR system (Applied Biosystems).
  • AAV6 produced based on the pAAV-CMV-Tcf21-IRES-GFP vector was referred to as AAV6-Tcf21, and AAV6 produced based on the control pAAV-CMV-IRES-GFP vector.
  • AAV6-control was designated as AAV6-control and was used in the following experiment.
  • the virus mixture prepared by the above method was adjusted to 25,000 virus genome/cell and AAV6-Tcf21 or AAV6-control prepared above was added to the cell culture medium. 96 hours later, the medium in the dish was removed by suction with an aspirator, washed with phosphate buffered saline and removed by suction, and the Buffer RLT plus (2-mercaptoethanol (BioRad)) included in the RNeasy plus mini kit (Qiagen) was removed. 350 ⁇ L was sprinkled onto the lysed cells and the lysed cells were collected in a 1.5 mL tube.
  • RNA concentration in the aqueous solution was measured using a NanoDrop ultratrace spectrometer (Thermo Fisher Scientific).
  • RNA for each RNA sample was added to an 8-tube (Nippon Genetics) and diluted with pure water to a volume of 6 ⁇ L. This was incubated at 65°C for 5 minutes and then rapidly cooled on ice.
  • 2 ⁇ L of 4 x DN Master Mix (gDNA Remover added) included in ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo) was added, and after gently stirring, incubated at 37°C for 5 minutes.
  • 2 ⁇ L of 5 ⁇ RT Master Mix II was added, lightly agitated to homogenize, and then reacted at 37°C for 15 minutes, 50°C for 5 minutes, and 98°C for 5 minutes to synthesize cDNA. ..
  • This reverse transcription reaction was performed using Takara PCR Thermal cycler (Takara Bio).
  • the cDNA obtained by the above method was mixed with SYBR Green real-time PCR master mix (Applied Biosystems) and oligo DNA primers for amplifying each target gene described below and added to a 96-well plate (Applied Biosystems).
  • SYBR Green real-time PCR master mix Applied Biosystems
  • oligo DNA primers for amplifying each target gene described below were added to a 96-well plate (Applied Biosystems).
  • the primers of the Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene described below were used.
  • ⁇ Tcf21-Forward primer, GGCTCCAACTGCGAGAACGGGT NCBI accession No.
  • a PCR reaction was performed on the sample in the 96-well plate by repeating a cycle of reacting at 95°C for 1 second and 60°C for 20 seconds using the StepOnePlus real-time PCR system (Applied Biosystems) 40 times.
  • the relative expression level of each gene was measured based on the Gapdh gene expression level.
  • the Mann-Whitney Utest was used for the significance test between groups.
  • Example 2 Preparation of experimental liver fibrosis mouse
  • Six-week-old male C57Bl/6J mice (CLEA Japan, Inc.) were used.
  • Carbon tetrachloride (Fujifilm Wako Pure Chemical Industries, Ltd.) and olive oil were mixed at a volume ratio of 1:3, and a 4-fold diluted solution was subcutaneously injected every 3 days to the back of the mouse at 1 mL/kg body weight. Fibrosis was created.
  • Example 2 preparation of adeno-associated virus and administration to liver fibrosis mice
  • the same operation as in Example 1 was performed to prepare AAV6-Tcf21 and AAV6-control.
  • Twenty-four hours after the administration of the adeno-associated virus the 21st administration of carbon tetrachloride was performed, and further 4 administrations of carbon tetrachloride were performed (total administration number: 25 times, 75 days).
  • liver tissue collection and liver tissue collection 72 hours after the 25th administration of carbon tetrachloride, the mice were subjected to laparotomy under anesthesia with isoflurane, and blood was collected from the inferior vena cava and liver tissues were collected.
  • the collected liver tissue was cut into small pieces and fixed in a 10% formalin solution in which formalin (Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted with pure water for 16 hours. After that, the tissue is transferred to an embedding cassette (MURAZUMI) and, using an automatic fixed embedding device (Sakura Finetech Japan), it is immersed in 100% ethanol (Fujifilm Wako Pure Chemical Industries) at room temperature for 2 hours with stirring.
  • MURAZUMI an embedding cassette
  • the liver tissue was dehydrated by repeating immersion in 100% ethanol for a total of 7 times. Next, transfer to xylene (Fujifilm Wako Pure Chemical Industries) and similarly soak for 2 hours with stirring, which is repeated 3 times in total, and then heat to 65°C to liquefy histoprep 568/paraffin (Fujifilm Wako Pure Chemicals). It was soaked for 2 hours with stirring in Kojunkaku Co., Ltd. to allow paraffin to permeate the liver tissue. Paraffin blocks were prepared using Tissue Tech TEC Plus Cryo Console (Sakura Fine Tech Japan).
  • FIG. 2 A photograph of liver tissue stained with Sirius red and fast green is shown on the left side of FIG.
  • AAV6-Tcf21-administered mice (the right photograph in the tissue photograph of FIG. 2) showed azuki bean color in comparison with the control mouse (the left photograph of the tissue photograph of FIG. 2) which was administered AAV6-control.
  • the accumulation of collagen fibers visualized in Fig. 2 was significantly suppressed (p ⁇ 0.01) (graph on the right in Fig. 2), and suppression of liver fibrosis by Tcf21 treatment was observed.
  • Example 3 Purification of liver tissue RNA
  • a part of the liver tissue collected in Example 2 above was collected in a 1.5 mL tube, and Buffer RLT plus (2-mercaptoethanol (BioRad) included in the RNeasy plus mini kit was adjusted to 1% thereof. Addition) 350 ⁇ L was added, and the tissue was disrupted and suspended in the solution using a homogenizer. Next, the same operation as in Example 1 was performed to purify total RNA.
  • RNA sample 500 ng was added to an 8-tube (Nippon Genetics) and diluted with pure water so that the liquid volume was 6 ⁇ L. Thereafter, the same operation as in Example 1 was performed to synthesize cDNA. The same operation as in Example 1 was performed to perform a quantitative analysis of gene expression. Statistical significance test was performed using Mann-Whitney U test.
  • Example 4 (Serum separation) The mouse blood collected in Example 2 above was allowed to stand at room temperature for 20 minutes, and then centrifuged at 4° C. at 3,000 rpm for 15 minutes using a cooling centrifuge (Eppendorf). The obtained supernatant was transferred to a new tube and stored at -30°C until measurement.
  • Example 1 (Isolation and activation induction of hepatic stellate cells) Hepatic stellate cells were isolated by the same procedure as in Example 1. The isolated hepatic stellate cells were seeded in a 24-well plate at 3.5 ⁇ 10 4 cells/cm 2 . After inoculation, the cells were cultured for 5 days at 37° C. under a concentration of 5% carbon dioxide to activate hepatic stellate cells.
  • Cebpb-Forward primer ATGCACCGCCTGCTGGCCTGGGACG (NCBI accession No. NM_009883.4) (SEQ ID NO: 19) Cebpb-Reverse primer, CTAGCAGTGGCCCGCCGAGGCCAGC (same as above) (SEQ ID NO: 20) Epas1-Forward primer, ATGACAGCTGACAAGGAGAAAAAAA (NCBI accession No. NM_010137.3) (SEQ ID NO: 21) Epas1-Reverse primer, TCAGGTGGCCTGGTCCAGAGCTCTG (same as above) (SEQ ID NO: 22) Fosb-Forward primer, ATGTTTCAAGCTTTTCCCGGAGACT (NCBI accession No.
  • Nfib-Reverse primer Nfib-Reverse primer
  • TCAGTTGCTTGTCTCCGCTTGAAGG Nfib-Reverse primer
  • ATGGACTCCAAAGAATCCTTAGCTC NCBI accession No. NM_001361209.1
  • Nr3c1-Reverse primer TCATTTCTGATGAAACAGAAGCTTTTTG (same as above)
  • SEQ ID NO: 38 Ppara-Forward primer, ATGGTGGACACAGAGAGCCCCATCT (NCBI accession No.
  • the culture medium of hepatic stellate cells was replaced with a new culture medium, and the liposomes prepared by the above method were added to each, and the cells were cultured for 24 hours. After 24 hours, the medium was replaced with a new culture medium, and the culture was further continued for 24 hours.
  • FIG. 5 shows changes in expression of fibrosis marker genes when each gene was overexpressed.
  • Col1a1 gene expression was significantly suppressed by overexpression of Nr3c1, Ppara, Tcf21, Gata4, Lhx2 (*: p ⁇ 0.05, **: p ⁇ 0.01), compared to control pcDNA3 (Control).
  • the suppressive effect of Tcf21 was most remarkable.
  • the expression of Acta2 gene was also significantly decreased by the transfection of Nr3c1, Tcf21, and Gata4 expression vectors (*: p ⁇ 0.05, **: p ⁇ 0.01), and Tcf21 was the most prominent suppressor of expression like Col1a1 gene. Showed the effect. From these results, it was clarified that Tcf21 showed the strongest inhibitory effect on hepatic fibrosis among all 16 kinds of transcription factors examined in comparison.
  • the present invention is useful as a technique for preventing or treating liver diseases, a technique for liver antifibrosis, and a technique for improving liver function.

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Abstract

La présente invention a pour objet de fournir un procédé de désactivation de cellules stellaires hépatiques activées. A cet effet, l'invention a trait à un procédé de désactivation de cellules stellaires hépatiques activées, ledit procédé comprenant une étape consistant à introduire un gène Tcf21 et/ou une protéine Tcf21 dans les cellules stellaires hépatiques activées.
PCT/JP2018/045242 2018-12-10 2018-12-10 Procédé de désactivation de cellules stellaires hépatiques activées Ceased WO2020121366A1 (fr)

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WO2023223520A1 (fr) * 2022-05-19 2023-11-23 Ｉｓｍ株式会社 Système de criblage pour inducteur de désactivation pour cellules stellaires hépatiques activées

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JP2022171486A (ja) * 2021-04-30 2022-11-11 学校法人東海大学 筋線維芽細胞を脱活性化するためのＴｃｆ２１タンパク質の部分ペプチド
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WO2023171448A1 (fr) * 2022-03-11 2023-09-14 学校法人東海大学 Composition pharmaceutique pour la prévention ou le traitement d'une maladie à prévenir ou à traiter par désactivation de myofibroblastes

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