WO2021193408A1 - Modèle de troubles congénitaux du cycle de l'urée et son utilisation - Google Patents

Modèle de troubles congénitaux du cycle de l'urée et son utilisation Download PDF

Info

Publication number
WO2021193408A1
WO2021193408A1 PCT/JP2021/011317 JP2021011317W WO2021193408A1 WO 2021193408 A1 WO2021193408 A1 WO 2021193408A1 JP 2021011317 W JP2021011317 W JP 2021011317W WO 2021193408 A1 WO2021193408 A1 WO 2021193408A1
Authority
WO
WIPO (PCT)
Prior art keywords
hepatocytes
urea cycle
cells
congenital
gene
Prior art date
Application number
PCT/JP2021/011317
Other languages
English (en)
Japanese (ja)
Inventor
健二 長船
真希 小▲高▼
Original Assignee
国立大学法人京都大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Priority to JP2022510408A priority Critical patent/JPWO2021193408A1/ja
Publication of WO2021193408A1 publication Critical patent/WO2021193408A1/fr

Links

Images

Classifications

    • 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
    • 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
    • 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 screens or evaluates a disease model using hepatocytes derived from induced pluripotent stem (iPS) cells derived from a patient with congenital urea cycle disorder and a therapeutic agent for congenital urea cycle disorder using the disease model. Regarding the method.
  • iPS induced pluripotent stem
  • ASS argininosuccinate synthase
  • NASH non-alcoholic steatohepatitis
  • Non-Patent Document 1 hepatocytes induced to differentiate from iPS cells derived from patients with neonatal (type I) citrullinemia, which is a related disease, were used to partially reproduce the pathophysiology of urea cycle abnormalities.
  • Non-Patent Document 2 hepatocytes induced to differentiate from iPS cells derived from the same patient and hepatocytes induced to differentiate from iPS cells derived from healthy subjects.
  • the present invention provides a disease model that can be used for pathological analysis and drug discovery of congenital urea cycle disorders such as adult citrullinemia, preferably congenital urea cycle disorders accompanied by fatty liver and NASH.
  • the task is to do.
  • iPS cells were established from the somatic cells of patients with adult citrullinemia, and ammonium chloride was added to the hepatocytes obtained by inducing differentiation from the iPS cells to evaluate ammonia uptake and urea production.
  • ammonium chloride was added to the hepatocytes obtained by inducing differentiation from the iPS cells to evaluate ammonia uptake and urea production.
  • PPAR peroxisome proliferator activation receptor
  • the present inventors prepared a gene repair strain in which the mutation of the causative gene was repaired in iPS cells derived from the patient with adult citrullinemia, induced hepatocytes from this gene repair strain, and described the above. Ammonia uptake, urea production, and fat accumulation were evaluated. Then, the measurement results in hepatocytes obtained by inducing differentiation from iPS cells derived from the above-mentioned adult citrullinemia patient were compared with the measurement results in hepatocytes obtained by inducing differentiation from a gene repair strain.
  • the gist of the present invention is as follows.
  • a method of screening or evaluating a therapeutic agent for congenital urea cycle disorders which comprises the step of measuring the urea cycle activity and / or fat accumulation of cells.
  • the method according to [1] wherein the congenital urea cycle disorder is adult citrullinemia.
  • step (I) The step of culturing iPS cells in a medium containing Activin A, GSK ((glycogen synthase kinase))-3 ⁇ inhibitor and HDAC (histone deacetylase) inhibitor, (Ii) A step of culturing the cells obtained in step (i) in a medium containing dimethyl sulfoxide (DMSO), BMP (bone morphogenetic protein) 4 and FGF (fibroblast growth factor) 2, and (iii) step. The step of culturing the cells obtained in (ii) in a medium containing HGF (hepatocellular growth factor) and oncostatin M.
  • DMSO dimethyl sulfoxide
  • BMP bone morphogenetic protein
  • FGF fibroblast growth factor
  • the pathological conditions of urea cycle abnormality and lipid metabolism abnormality can be stably reproduced, and by using this, congenital urea including adult citrullinemia can be reproduced. It is possible to develop therapeutic agents for cycle disorders, fatty liver, and NASH with high accuracy.
  • the pathological condition of the above-mentioned diseases can be elucidated by using the pathological condition model produced by the present invention.
  • noise can be reduced and the pathological condition can be accurately reproduced. The accuracy of the evaluation of is also improved.
  • Photomicrograph showing the results of staining for evaluating albumin (ALB) expression, LDL uptake, and glycogen accumulation in hepatocytes induced to differentiate from iPS cells derived from healthy subjects or adult citrullinemia patients.
  • the graph which shows the result of having evaluated the ammonia removal ability and the urea production ability in the hepatocyte differentiation induction from the iPS cell derived from the healthy person or the adult type citrullinemia patient.
  • HepG2 cells were shown as controls.
  • PA indicates palmitic acid.
  • Fat accumulation by palmitate in the presence or absence of clofibrate in hepatocytes induced to differentiate from iPS cells derived from healthy individuals or adult citrullinemia patients and hepatocytes induced to differentiate from gene-repaired iPS cells The graph which shows the result of evaluation by TG quantification.
  • EA Elaidic acid
  • OA oleic acid
  • PA palmitic acid
  • Ammonia in hepatocytes obtained by inducing differentiation from iPS cells derived from adult citrullinemia patients and in hepatocytes obtained by inducing differentiation from gene repair strains The graph which shows the result of having evaluated the removal ability.
  • Urea in hepatocytes obtained by inducing differentiation from iPS cells derived from adult citrullinemia patients and in hepatocytes obtained by inducing differentiation from gene repair strains A graph showing the result of evaluating the production volume.
  • the screening method or evaluation method for a therapeutic agent for congenital urea cycle abnormality of the present invention is a step of contacting hepatocytes induced to differentiate from iPS cells derived from a patient with congenital urea cycle abnormality with a drug candidate substance (test substance). It is characterized by measuring the urea cycle activity and / or fat accumulation of the hepatocytes.
  • the congenital urea cycle disorder is not particularly limited as long as it is a disease that causes a congenital abnormality in the urea cycle in the liver, and examples thereof include diseases that cause hyperammonemia. Specifically, N-acetylglutamic acid. Synthetic enzyme (NAGS) deficiency, carbamylphosphate synthase (CPS1) deficiency, ornithine transcarbamylase (OTC) deficiency, citrullinemia, argininosuccinate, arginineemia, hyperornitinemia / hyperammonemia -Homocitrulinuria syndrome (HHH syndrome) is mentioned, of which citrullinemia is more preferable.
  • NAGS N-acetylglutamic acid. Synthetic enzyme
  • CPS1 carbamylphosphate synthase
  • OTC ornithine transcarbamylase
  • citrullinemia argininosuccinate
  • arginineemia hyperornitinemia
  • the congenital urea cycle disorder is preferably a disease accompanied by non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • NAFLD includes nonalcoholic fatty liver (NAFL) and nonalcoholic steato-hepatitis (NASH).
  • Urea cycle disorders are generally congenital disorders caused by genetic abnormalities.
  • the causative gene for adult citrullinemia is the SLC25A13 (Solute Carrier Family 25 Member 13) gene that encodes citrine
  • the gene mutation that causes adult citrullinemia is, for example, Kobayashi K et.al., Nat Genet. It has been reported in 22 159-163 1999 and PLOS ONE September 2013, Volume 8, Issue 9, e74544.
  • Specific examples include mutations such as 851del4 and IVS11 + 1G> A.
  • the somatic cells used for producing iPS cells have a gene mutation in at least one allele of the causative gene.
  • the causative gene in hereditary urea cycle dysfunction is considered to be a recessive mutation, so it is preferable that the somatic cells used for producing iPS cells have gene mutations in both alleles.
  • the mutation may be homozygous with the same type of mutation or heterozygous with different types of mutation.
  • somatic cells derived from the patients may be used as they are, or the mutant SLC25A13 gene is introduced into normal somatic cells and used. You may use it.
  • the somatic cells are not particularly limited, but include not only mature somatic cells but also fetal (pup) and neonatal (pup) somatic cells, and are any of primary cultured cells, passaged cells, and established cells. Is also included.
  • the somatic cells include, for example, tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, tissue precursor cells, blood cells (peripheral blood cells, umbilical cord blood cells, etc.), and lymph.
  • Spheres epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.), brain cells, lung cells , Differentiated cells such as renal cells and fat cells are exemplified.
  • the iPS cells can be produced, for example, by introducing a reprogramming factor into somatic cells.
  • the reprogramming factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, ECAT15.
  • Genes or gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 are exemplified, and these reprogramming factors may be used alone or in combination. Is also good.
  • the combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010
  • a known method can be used for inducing differentiation of iPS cells into hepatocytes.
  • a method as described in WO2016 / 104717 may be used, but the following steps (i) to A method including (iii) can be used.
  • Steps (i) to (iii) are preferably carried out by adhesive culture.
  • Adhesive culture means that cells are cultured in a state of being adhered to a culture substrate, and for example, they are cultured in a coated culture dish.
  • the coating agent is preferably an extracellular matrix, and examples thereof include substances such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrin and laminin, or fragments thereof.
  • extracellular matrices may be used in combination and may be preparations from cells such as BD Matrigel TM.
  • the extracellular matrix is preferably laminin or a fragment thereof.
  • laminin is a protein having a heterotrimer structure having one ⁇ chain, one ⁇ chain, and one ⁇ chain, and is an extracellular matrix protein in which isoforms having different subunit chain compositions are present. ..
  • Laminin is a combination of 5 ⁇ -chains, 4 ⁇ -chains and 3 ⁇ -chain heterotrimers and has about 15 isoforms.
  • the ⁇ chain is ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4 or ⁇ 5
  • the ⁇ chain is ⁇ 1, ⁇ 2, ⁇ 3 or ⁇ 4
  • the ⁇ chain is ⁇ 1, ⁇ 2 or ⁇ 3.
  • Laminin is more preferably laminin 511 consisting of ⁇ 5, ⁇ 1 and ⁇ 1 (Nat Biotechnol 28, 611-615 (2010)).
  • Laminin may be a fragment, and is not particularly limited as long as it has integrin-binding activity.
  • E8 fragment laminin 511E8 (EMBO J.), which is a fragment obtained by digestion with elastase. , 3: 1463-1468, 1984, J. Cell Biol., 105: 589-598, 1987, WO 2011/043405).
  • Laminin 511E8 is commercially available and can be purchased from, for example, Nippi Co., Ltd.
  • the medium used in each step can be prepared by adding cytokines and drugs necessary for each step to the basal medium used for culturing animal cells.
  • the basal medium for example, IMDM medium, Medium 199 medium, Eagle's Minimium Essential Medium (EMEM) medium, ⁇ MEM medium, Dulvecco's Modern Medium (DMEM) medium, Ham's F12 (F12) medium, Ham's F12 (F12) medium , Fisher's medium, and a mixed medium thereof and the like are included.
  • the medium may contain serum (eg, fetal bovine serum (FBS)) or may be serum-free.
  • albumin transferase, KnockOut Serum Replacement (KSR) (serum substitute during ES cell culture) (ThermoFisherScientific), N2 supplement (ThermoFisherScientific), B27 supplement (ThermoFisherScient) , Insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol and the like, may contain one or more serum substitutes, lipids, amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), It may also contain one or more substances such as non-essential amino acids (NEAAs), vitamins, growth factors, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and their equivalents.
  • a medium pre-optimized for stem cell culture, such as ReproFF2 may be used.
  • step (I) Culturing iPS cells in a medium containing Activin A, GSK-3 ⁇ inhibitor and HDAC inhibitor
  • the GSK-3 ⁇ inhibitor used in step (i) is GSK-3 ⁇ .
  • the function is not particularly limited as long as it can inhibit the kinase activity, and for example, BIO (also known as GSK-3 ⁇ inhibitor IX; 6-bromoinsilvin-3'-oxym), which is an indylvin derivative, and a maleimide derivative.
  • BIO also known as GSK-3 ⁇ inhibitor IX; 6-bromoinsilvin-3'-oxym
  • SB216763 3- (2,4-dichlorophenyl) -4- (1-methyl-1H-indol-3-yl) -1H-pyrrole-2,5-dione
  • GSK-3 ⁇ inhibitor VII ( ⁇ , 4-dibromoacetophenone), cell membrane permeabilizing phosphorylated peptide L803-mts (also known as GSK-3 ⁇ peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH 2 ) (SEQ ID NO: 3) )
  • CHIR99021 (Nature (2008) 453: 519-523), which has high selectivity. These compounds are available from, for example, Stemgent, Calbiochem, Biomol and the like. CHIR99021 is a preferred GSK-3 ⁇ inhibitor used in this step.
  • the concentration of the GSK-3 ⁇ inhibitor used in this step can be appropriately selected by those skilled in the art depending on the GSK-3 ⁇ inhibitor used, and is, for example, 0.01 ⁇ M to 100 ⁇ M, preferably 0.1 ⁇ M to 10 ⁇ M. More preferably, it is 0.5 ⁇ M to 3 ⁇ M, and particularly preferably 0.5 ⁇ M to 1.5 ⁇ M.
  • Activin A used in step (i) includes Activin A derived from humans and other animals, but human Activin A is preferable, and the human Activin A is, for example, NCBI (National Center for Biotechnology Information). Accession numbers: NP_002183.1, AAA59168.1, AAH07858.1 or UniProt (UniProtKB / Swiss-Prot), examples of proteins having the amino acid sequence of accession number P08476.2.
  • Activin A includes fragments and functional variants as long as it has differentiation-inducing activity.
  • Activin A a commercially available product may be used, or a protein purified from cells or a protein produced by genetic recombination may be used.
  • the concentration of Activin A in the medium used in this step is 1 ng / ml to 1000 ng / ml, preferably 10 ng / ml to 500 ng / ml, more preferably 50 ng / ml to 200 ng / ml. Yes, more preferably 90 ng / ml to 100 ng / ml.
  • HDAC inhibitors used in step (i) include, for example, valproic acid (VPA), tricostatin A, NaB, Vorinostat, NCC-149, NCH-47, NCH-51, MS-275, FK228, Apicidin, and MGCD-0103 etc. can be mentioned.
  • VPA valproic acid
  • tricostatin A tricostatin A
  • NaB Vorinostat
  • NCC-149 NCH-47, NCH-51, MS-275, FK228, Apicidin, and MGCD-0103 etc.
  • MGCD-0103 MGCD-0103
  • concentration of NaB used in this step is 10 ⁇ M to 10 mM, preferably 100 ⁇ M to 1 mM, and more preferably 0.2 mM to 0.5 mM.
  • the medium used in step (i) may contain a ROCK inhibitor.
  • the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK), and is, for example, Y-27632 (eg, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000)). Narumiya et al., Methods Enzymol. 325,273-284 (2000)), Fasudil / HA1077 (eg Uenata et al., Nature 389: 990-994 (1997)), H-1152 (eg Sasaki et al) ., Pharmacol. Ther.
  • ROCK Rho-kinase
  • a preferred ROCK inhibitor used in this step is Y-27632.
  • the concentration of the ROCK inhibitor used in this step can be appropriately selected by those skilled in the art depending on the ROCK inhibitor used. For example, when Y-27632 is used as the ROCK inhibitor, it is 0.1 ⁇ M to 100 ⁇ M, preferably 0.1 ⁇ M. It is 1 ⁇ M to 50 ⁇ M, more preferably 5 ⁇ M to 20 ⁇ M.
  • the culture temperature is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time in step (i) is, for example, 1 to 10 days, preferably 3 to 6 days, and more preferably 4 to 5 days.
  • step (Ii) A step of culturing the cells obtained in the step (i) in a medium containing DMSO, BMP4 and FGF2.
  • the DMSO used in the step (ii) is commercially available from Sigma-Aldrich and the like. You can use what you have.
  • the concentration of DMSO used in this step in the medium is 0.01% to 10%, preferably 0.1% to 5%, more preferably 0.5% to 2%, and particularly preferably about 1%.
  • the BMP4 used in the step (ii) is preferably human BMP4, and examples of human BMP4 include proteins having an amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: AAH20546.1. As long as BMP4 has differentiation-inducing activity, its fragments and functional variants are included. BMP4 may be commercially available, or a protein purified from cells or a protein produced by genetic recombination may be used. You may.
  • the concentration of BMP4 used in this step is from 1 ng / ml to 100 ng / ml, preferably from 5 ng / ml to 50 ng / ml, more preferably from 10 ng / ml to 30 ng / ml.
  • the FGF2 (basic FGF: bFGF) used in the step (ii) is preferably human FGF2, and the human FGF2 has, for example, the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: ABO43041.1. Examples include proteins. Fragments and functional variants of FGF2 are included as long as they have differentiation-inducing activity. Commercially available FGF2 may be used, or a protein purified from cells or a protein produced by genetic recombination may be used. You may.
  • the concentration of FGF2 used in this step is from 1 ng / ml to 100 ng / ml, preferably from 5 ng / ml to 50 ng / ml, more preferably from 10 ng / ml to 20 ng / ml.
  • the culture temperature is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time of the step (ii) is, for example, 1 to 10 days, preferably 3 to 8 days, and more preferably 5 to 7 days.
  • the step of culturing the cells obtained in step (ii) in a medium containing HGF and OsM The HGF used in step (iii) is preferably human HGF, and the human HGF is, for example, Examples include proteins having the amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: AAA64297.1. HGF includes fragments and functional variants as long as it has differentiation-inducing activity. Commercially available HGF may be used, or a protein purified from cells or a protein produced by genetic recombination may be used.
  • the concentration of HGF used in this step is from 1 ng / ml to 100 ng / ml, preferably from 5 ng / ml to 50 ng / ml, more preferably from 10 ng / ml to 30 ng / ml.
  • the OsM used in step (iii) is preferably human OsM, and examples of human OsM include proteins having an amino acid sequence of NCBI (National Center for Biotechnology Information) accession number: AAH11589.1. As long as OsM has differentiation-inducing activity, fragments and functional variants thereof are included. OsM may be commercially available, or a protein purified from cells or a protein produced by genetic recombination may be used. You may.
  • the concentration of OsM used in this step is from 1 ng / ml to 100 ng / ml, preferably from 5 ng / ml to 50 ng / ml, more preferably from 10 ng / ml to 30 ng / ml.
  • the culture temperature is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2-containing air.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture time in step (i) is, for example, 1 to 14 days, preferably 5 to 10 days, and more preferably 7 to 9 days.
  • Hepatocytes induced to differentiate from iPS cells as described above can be used for screening and evaluation of therapeutic agents for congenital urea cycle disorders.
  • test substance can be used, for example, cell extract, cell culture supernatant, microbial fermentation product, marine organism-derived extract, plant extract, purified protein or crude protein, and the like. Peptides, non-peptide compounds, synthetic small molecule compounds, and natural compounds are exemplified.
  • the test substance is also (1) a biological library method, (2) a synthetic library method using deconvolution, (3) a "one-bead one-compound” library method, And (4) can be obtained using any of the many approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography sorting.
  • Biological library methods using affinity chromatography screening are limited to peptide libraries, but the other four approaches are applicable to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des). . 12: 145-67).
  • Examples of methods for synthesizing molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al.
  • Compound libraries include solutions (see Houghten (1992) Bio / Techniques 13: 412-21) or beads (Lam (1991) Nature 354: 82-4), chips (Fodor (1993) Nature 364: 555-6). ), Bacteria (US Pat. No. 5,223,409), Spores (US Pat. No. 5,571,698, No. 5,403,484, and No.
  • an ammonia source such as ammonium chloride can be added to the culture solution of hepatocytes to measure ammonia uptake and urea production, but the test substance is brought into contact with the test substance. After incubating for a certain period of time (for example, 1 to 72 hours), an ammonia source was added, ammonia uptake and urea production were measured, and the results were measured without contact with the test substance. An aspect of comparison with the case where the measurement is performed after contacting with only the solvent can be mentioned.
  • the test substance is brought into contact with the test substance for a certain period of time (for example, from 1 hour). Incubate for 72 hours), then measure changes in ammonia uptake and urea production, and measure the results without contact with the test substance or after contact with the solvent only. There is also an aspect of comparison with the case where it is performed.
  • concentration of the ammonia source such as ammonium chloride added is, for example, 2 mM to 10 mM.
  • Hepatocytes induced to differentiate from iPS cells derived from patients with congenital urea cycle disorders have lower ammonia uptake and lower urea production than hepatocytes induced to differentiate from healthy human-derived iPS cells.
  • the amount of ammonia uptake increases or the amount of urea produced increases when the test substance is brought into contact with the test substance, it is evaluated that the test substance improves the urea cycle abnormality, or it is congenital. It can be selected as a candidate substance for a therapeutic agent for sexual urea cycle disorder. It can also be evaluated by comparing the effect of the test substance with the effect of the positive control having the effect of improving the urea cycle abnormality.
  • Positive controls include arginine.
  • the amount of ammonia uptake and the amount of urea produced can be measured by, for example, the amount of ammonia remaining in the medium and the amount of urea secreted into the medium by known means.
  • fatty acids such as palmitic acid, elaidic acid, and oleic acid can be added to the culture solution of hepatocytes, and the amount of fat accumulated can be measured. After contacting and incubating for a certain period of time (for example, 48 to 72 hours), fatty acids were added and fat accumulation was measured, and the results were measured without contact with the test substance or only in the solvent. An aspect of comparison with the case where the measurement is performed after the contact with the above can be mentioned.
  • fatty acids such as palmitic acid, elaidic acid, and oleic acid are added to the hepatocytes and cultured to accumulate fat in the hepatocytes, and then the test substance is brought into contact with the test substance for a certain period (for example, 48 hours to 72 hours). ) Incubate, then measure the change in fat accumulation, and compare the results with the case of measurement without contact with the test substance or the case of measurement after contact with only the solvent. Can be mentioned.
  • the concentration of fatty acids such as palmitic acid, elaidic acid, and oleic acid added is, for example, 1 mM to 5 mM.
  • Hepatocytes induced to differentiate from iPS cells derived from patients with congenital urea cycle abnormalities have increased lipid accumulation compared to hepatocytes induced to differentiate from iPS cells derived from healthy subjects, but when the test substance is brought into contact with them,
  • the test substance is evaluated to improve lipid abnormalities in urea cycle abnormalities, and candidates for therapeutic agents to improve non-alcoholic fatty liver disease status in congenital urea cycle abnormalities. It can be selected as a substance.
  • the effect of the test substance can be evaluated by comparing it with the effect of a positive control having an effect of improving dyslipidemia.
  • Positive controls include PPAR ⁇ agonists such as clofibrate.
  • the amount of fat accumulated in hepatocytes can be measured using a commercially available staining reagent or quantitative reagent.
  • the gene mutation repair iPS cell means an iPS cell in which the mutation site of the causative gene of a patient with congenital urea cycle abnormality is replaced with a normal sequence, and the mutation site is replaced with a normal sequence. It means iPS cells that have the same genetic information as patients with congenital urea cycle disorders.
  • Gene mutation repair iPS cells can be prepared by known gene repair techniques.
  • genome editing techniques such as CRISPR / Cas9, TALEN, and ZFN can be used to replace the gene responsible for urea cycle abnormalities in patient-derived iPS cells with the normal form.
  • CRISPR / Cas9 it can be carried out by introducing an oligonucleotide having a normal sequence together with Cas9 and gRNA into a patient-derived iPS cell and substituting the mutant sequence.
  • hereditary urea cycle disorders are caused by recessive mutations, so gene repair requires repairing at least one of the alleles, but both alleles may be repaired.
  • Fibroblasts were collected from two adult citrusemia (citrin deficiency) patients who received informed outlets, and fibroblasts were used as somatic cells.
  • Patient 1 was OCT4, SOX2, KLF4, C-Myc.
  • Patient 2 introduced OCT4, SOX2, KLF4, LMYC, LIN28 and p53 shRNA with an episomal vector to establish iPS cells.
  • Patient 1 and Patient 2 each had 851del4 + IVS11 + 1G> A and 851del4 + 851del4 as mutations in the SLC25A13 gene, and that the obtained iPS cells also had these mutations.
  • Expression of undifferentiated markers, karyotype analysis, and trigerm differentiation tests confirmed that the established iPS cells retain the same properties as human ES cells.
  • a gRNA-containing sequence (GAAATTAATACGACTCACTATAGgtctgctaaggtcgtctgtaGTTTTAGAGCTAGAAATAGCAAG: SEQ ID NO: 2) was designed. 2. The cleavage efficiency of the designed gRNA was confirmed. Using the gRNA confirmed in 3.2, Cas9 and a single-stranded oligo (ssODN) serving as donor DNA were introduced into iPS cells derived from disease patients. 4. The repair efficiency was confirmed by a method using a restriction enzyme. 5. A single-stranded oligo (ssODN) for repair is introduced into iPS cells derived from diseased patients. 6.
  • the colonies were picked up, the target region was PCR amplified, and the PCR product was purified and then repaired by sequence.
  • As the one-sided allele repair strain a strain in which the four bases of the defective portion were repaired on only one side was selected. Using the same Cas9 and the single-stranded oligo (ssODN) that serves as the donor DNA, the above steps 3 to 6 were performed again on the one-sided allele repair strain, and the defective 4-base portion was repaired on the other side that had not been repaired. The strain was selected as a bilateral allele repair strain.
  • ⁇ Induction of differentiation into hepatocytes > 1.
  • Cultivate undifferentiated human iPS cells to a cell density similar to that used for subculture.
  • the medium After culturing at 9.37 ° C. at 5% CO 2 for 1 day, the medium is removed and replaced with a medium containing the differentiation-inducing factor of Stage 1. (In the case of 96 well plate, 100 ⁇ L / well.) Replace with medium containing Stage 1 differentiation-inducing factor every day until the 5th day of culture. 10. After culturing for 5 days after the start of induction, the medium is removed and replaced with a medium containing the differentiation-inducing factor of Stage 2. (For a 96-well plate, use 100 ⁇ L / well.) Replace with a medium containing Stage 2 differentiation-inducing factor every day from the start of culture to the 11th day. 11.
  • the medium After culturing for 11 days after the start of induction, the medium is removed and replaced with a medium containing the differentiation-inducing factor of Stage 3. (For a 96-well plate, use 100 ⁇ L / well.) Replace with a medium containing Stage 3 differentiation-inducing factor every 2 days from the start of culture to the 19th day.
  • KO-DMEM (Thermo Fisher Scientific) 10% KSR 1mM L-glutamine 1% (vol / vol) nonessential amino acids 0.1mM 2-mercaptoethanol 1% (vol / vol) DMSO 500 U / ml P / S 20 ng / ml BMP4, 10 ng / ml FGF2
  • the medium On the 17th day, the medium is removed and replaced with a medium containing 20 ng / ml HGF and 20 ng / ml OsM supplemented with 2 mM NH 4 Cl. (In the case of a 24-well plate, it should be 500 ⁇ L / well.) In the test to see the effect of arginine, 10 mM arginine was added at the same time as 2 mM NH 4 Cl.
  • the medium After culturing until the 19th day, the medium is collected, and the collected medium is secreted with urea amount, ammonia amount or albumin according to the protocol of Urea Assay Kit (BioAssay Systems), Ammonia Test Wako (Wako) or Albumin ELISA Quantitation Set (Bethyl Laboratories). Quantify the amount.
  • the medium is removed at the 11th day and replaced with a Stage 3 medium containing 20 ng / ml HGF and 20 ng / ml OsM. (For a 96-well plate, use 100 ⁇ L / well.) Replace with a medium containing Stage 3 differentiation-inducing factor every 2 days from the start of culture to the 17th day.
  • the medium is removed and replaced with a medium containing 20 ng / ml HGF and 20 ng / ml OsM supplemented with 1 mM palmitic acid.
  • a medium containing 20 ng / ml HGF and 20 ng / ml OsM supplemented with 1 mM palmitic acid In the case of 96 well plate, set to 100 ⁇ L / well. ) In the test to see the effect of clofibrate, clofibrate was added at the same time as palmitic acid and cultured.
  • the medium was removed at the 17th day, and 20 ng / ml HGF, 20 ng / ml OsM were included.
  • the medium was replaced with a medium supplemented with 1 mM EA, 1 mM OA or 1 mM PA, the medium was removed on the 19th day, and triglyceride was quantified according to the protocol of Triglyceride Assay Kit (BioAssay Systems).
  • the medium is removed at the 11th day and replaced with a Stage 3 medium containing 20 ng / ml HGF and 20 ng / ml OsM. (In the case of a 24-well plate, use 500 ⁇ L / well.) From the start of culturing to the 17th day, replace with a medium containing Stage 3 differentiation-inducing factor every 2 days.
  • the medium On the 17th day, the medium is removed, and the medium containing 20 ng / ml HGF and 20 ng / ml OsM is replaced with a medium containing 1 mM palmitic acid. (In the case of 24 well plate, it is 500 ⁇ L / well.)
  • the hepatocytes derived from iPS cells derived from patients with citrullinemia and the hepatocytes derived from iPS cells derived from healthy subjects were used to evaluate the ability to remove ammonia and the ability to produce urea.
  • the ability to remove ammonia and the ability to produce urea were decreased in hepatocytes derived from patients as compared with hepatocytes derived from healthy subjects.
  • the amount of triglyceride accumulated was evaluated using hepatocytes derived from iPS cells derived from patients with citrulinemia and hepatocytes derived from iPS cells derived from healthy subjects, and is shown in FIG. As described above, the amount of triglyceride accumulated in hepatocytes derived from patients was increased as compared with that of hepatocytes derived from healthy subjects.
  • hepatocytes were induced to differentiate from a gene mutation repair strain (repairing mutations in one-sided allele), and the amount of TG accumulated was examined. The amount of triglyceride accumulated decreased.
  • the amount of triglyceride accumulated in hepatocytes decreased after repair as compared with before repair.
  • the triglyceride-reducing effect of clofibrate was observed both before and after repair.
  • the amount of triglyceride in hepatocytes showed an increasing tendency due to the addition of elidinic acid, oleic acid or palmitic acid, and the degree of the triglyceride level was in hepatocytes induced to differentiate from iPS cells derived from adult citrullinemia patients. Although remarkable, the amount of triglyceride accumulated decreased in hepatocytes induced to differentiate from gene-repaired iPS cells (repairing mutations in one-sided alleles). Similar results were observed 4 days (Day 21) and 6 days (Day 23) after the addition of elaidic acid, oleic acid or palmitic acid.
  • hepatocytes induced to differentiate from iPS cells derived from adult citrulinemia patients and hepatocytes induced to differentiate from gene-repaired iPS cells in which one-sided alleles have been repaired or gene-repaired iPS cells in which both-sided alleles have been repaired.
  • FIGS. 10 and 11 hepatocytes induced to differentiate from patient-derived iPS cells and gene-repaired iPS cells in which one side of the allele was repaired induced differentiation.
  • the hepatocytes produced had lower ammonia-removing ability and urea-producing ability than hepatocytes induced to differentiate from gene-repaired iPS cells in which bilateral alleles were repaired.
  • hepatocytes induced to differentiate from patient-derived iPS cells had lower ammonia-removing ability and urea-producing ability than hepatocytes induced to differentiate from gene-repaired iPS cells in which one side of the allele was repaired. ..
  • the amount was measured, as shown in FIG. 12, the amount of albumin secreted was about the same.
  • hepatocytes induced to differentiate from patient-derived iPS cells and gene-repaired iPS cells reproduce the phenotype of congenital urea cycle abnormalities such as urea cycle abnormalities and lipid metabolism abnormalities while maintaining the functions of hepatocytes. I found that I could do it.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de criblage ou d'évaluation d'un médicament thérapeutique pour des troubles congénitaux du cycle de l'urée, ledit procédé comprenant une étape de mise en contact d'une substance médicamenteuse candidate avec des hépatocytes, lesquels ont été obtenus par induction de la différenciation de cellules souches pluripotentes induites (iPS) obtenues à partir de cellules somatiques provenant d'un patient atteint de troubles congénitaux du cycle de l'urée, et la mesure de l'activité du cycle de l'urée et/ou de l'accumulation de graisse dans les hépatocytes.
PCT/JP2021/011317 2020-03-23 2021-03-19 Modèle de troubles congénitaux du cycle de l'urée et son utilisation WO2021193408A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022510408A JPWO2021193408A1 (fr) 2020-03-23 2021-03-19

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2020-051532 2020-03-23
JP2020051532 2020-03-23

Publications (1)

Publication Number Publication Date
WO2021193408A1 true WO2021193408A1 (fr) 2021-09-30

Family

ID=77890282

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2021/011317 WO2021193408A1 (fr) 2020-03-23 2021-03-19 Modèle de troubles congénitaux du cycle de l'urée et son utilisation

Country Status (2)

Country Link
JP (1) JPWO2021193408A1 (fr)
WO (1) WO2021193408A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140134143A1 (en) * 2012-05-22 2014-05-15 Loma Linda University Vectors and methods for the efficient generation of integration/transgene-free induced pluripotent stem cells from peripheral blood cells
WO2016104717A1 (fr) * 2014-12-26 2016-06-30 国立大学法人京都大学 Procédé d'induction d'hépatocytes
WO2019071276A1 (fr) * 2017-10-06 2019-04-11 Camp4 Therapeutics Corporation Méthodes et compositions pour le traitement de troubles du cycle de l'urée, en particulier d'une déficience en otc
JP2019521693A (ja) * 2016-07-28 2019-08-08 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 非ヒト霊長類人工多能性幹細胞由来肝細胞およびその使用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140134143A1 (en) * 2012-05-22 2014-05-15 Loma Linda University Vectors and methods for the efficient generation of integration/transgene-free induced pluripotent stem cells from peripheral blood cells
WO2016104717A1 (fr) * 2014-12-26 2016-06-30 国立大学法人京都大学 Procédé d'induction d'hépatocytes
JP2019521693A (ja) * 2016-07-28 2019-08-08 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 非ヒト霊長類人工多能性幹細胞由来肝細胞およびその使用
WO2019071276A1 (fr) * 2017-10-06 2019-04-11 Camp4 Therapeutics Corporation Méthodes et compositions pour le traitement de troubles du cycle de l'urée, en particulier d'une déficience en otc

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GUREVICH IGOR, BURTON SARAH A., MUNN CHRISTIE, OHSHIMA MAKIKO, GOEDLAND MADELYN E., CZYSZ KATHERINE, RAJESH DEEPIKA: "iPSC-derived hepatocytes generated from NASH donors provide a valuable platform for disease modeling and drug discovery", BIOLOGY OPEN, vol. 9, no. 12, 16 December 2020 (2020-12-16), pages 1 - 9, XP055861212, DOI: 10.1242/bio.055087 *
ISHIDA, Y ET AL.: "A case of neonatal intrahepatic cholestasis caused by citrin deficiency: NICCD", J. TOKYO MED. UNIV., vol. 65, no. 3, 2007, pages 290 - 293 *
KIM YEJI, CHOI JUNG-YUN, LEE SANG-HEE, LEE BEOM-HEE, YOO HAN-WOOK, HAN YONG-MAHN: "Malfunction in Mitochondrial β-Oxidation Contributes to Lipid Accumulation in Hepatocyte-Like Cells Derived from Citrin Deficiency-Induced Pluripotent Stem Cells", STEM CELLS AND DEVELOPMENT, MARY ANN LIEBERT, INC. PUBLISHERS, US, vol. 25, no. 8, 15 April 2016 (2016-04-15), US , pages 636 - 647, XP055861189, ISSN: 1547-3287, DOI: 10.1089/scd.2015.0342 *
TANAKA T., NAGAO M., TSUTSUMI H.: "Application of Mutation Analysis for the Previously Uncertain Cases of Adult-Onset Type II Citrullinemia(CTLN2) and Their Clinical Profiles", TOHOKU J. EXP. MED., vol. 198, no. 2, 1 October 2002 (2002-10-01), pages 89 - 97, XP055861199 *

Also Published As

Publication number Publication date
JPWO2021193408A1 (fr) 2021-09-30

Similar Documents

Publication Publication Date Title
US9074188B2 (en) Cardiomyocyte- and/or cardiac progenitor cell-proliferating agent and method for proliferating cardiomyocytes and/or cardiac progenitor cells
EP2582795B1 (fr) Procédé de sélection de cellules souches pluripotentes induites humaines
Bharadwaj et al. Conserved chromosome 2q31 conformations are associated with transcriptional regulation of GAD1 GABA synthesis enzyme and altered in prefrontal cortex of subjects with schizophrenia
US20220380730A1 (en) Method for culturing myogenic cells, cultures obtained therefrom, screening methods, and cell culture medium
JP6673586B2 (ja) 異所性骨化の予防・治療剤及びそのスクリーニング方法
WO2015083582A1 (fr) Agent prophylactique et thérapeutique pour maladies fgfr3, et son procédé de dépistage
JP2022537278A (ja) 帯電表面を使用して誘導多能性幹細胞から複数の系統を産生するための方法
JP7253692B2 (ja) 肝細胞誘導方法
JPWO2020022261A1 (ja) 新規腎前駆細胞マーカーおよびそれを利用した腎前駆細胞の濃縮方法
EP3219808B1 (fr) Procédé de criblage utilisant des neurones induits
Wang et al. BMP4 preserves the developmental potential of mESCs through Ube2s-and Chmp4b-mediated chromosomal stability safeguarding
EP4056683A1 (fr) Organoïde cérébral et utilisation correspondante
WO2021193408A1 (fr) Modèle de troubles congénitaux du cycle de l'urée et son utilisation
JP2019136042A (ja) Ｆｇｆｒ３病の予防および治療剤ならびにそのスクリーニング方法
KR20240055838A (ko) 유도된 만능 줄기 세포 유래된 세포의 조성물 및 이의 사용 방법
WO2018115302A1 (fr) Moyens et procédés de génération d'astrocytes
EP3760728A1 (fr) Procédé d'évaluation de médicament
NL2017078B1 (en) A method for culturing myogenic cells, cultures obtained therefrom, screening methods, and cell culture medium.
WO2018225751A1 (fr) Procédé destiné à maintenir et à amplifier des cellules souches du cancer du côlon et procédé destiné à induire un organoïde du cancer du côlon
US20240034992A1 (en) Dopaminergic neurons comprising mutations and methods of use thereof
WO2021015245A1 (fr) Modèle de maladies en lien avec des cils et son utilisation
WO2020090836A1 (fr) Procédé de production de cellules
Geara Dissecting the mechanisms that regulate the quiescence-to-activation transition of skeletal muscle stem cells
JP2023071631A (ja) Ａｌｐｏｒｔ症候群の予防又は治療薬のスクリーニング又は評価方法
De Miguel et al. Toward Induced Pluripotent Stem Cells for Clinical Use: Sources, Methods and Selection

Legal Events

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

Ref document number: 21776953

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022510408

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21776953

Country of ref document: EP

Kind code of ref document: A1