WO2022010049A1 - Composition for cardiomyocyte toxicity assay of candidate drug for sars-cov-2 virus, using human pluripotent stem cell-derived cardiomyocytes, and cardiomyocyte toxicity assay method using same - Google Patents

Composition for cardiomyocyte toxicity assay of candidate drug for sars-cov-2 virus, using human pluripotent stem cell-derived cardiomyocytes, and cardiomyocyte toxicity assay method using same Download PDF

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WO2022010049A1
WO2022010049A1 PCT/KR2020/016771 KR2020016771W WO2022010049A1 WO 2022010049 A1 WO2022010049 A1 WO 2022010049A1 KR 2020016771 W KR2020016771 W KR 2020016771W WO 2022010049 A1 WO2022010049 A1 WO 2022010049A1
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cov
sars
virus
human
cell
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문성환
박순정
고윤영
반기원
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주식회사 티앤알바이오팹
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    • 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

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  • the present invention was made with the support of the Ministry of Health and Welfare under project number HI20C0184, the research and management agency for the project is the Health Industry Promotion Agency, the research project name is “Advanced medical technology development”, and the research project name is “pluripotent stem cell-derived cardiomyocyte maturation” Development of convergence and commercialization technology for maturation”, organized by T&R Biofab, research period on April 23, 2020. ⁇ 2022.12.31.
  • the present invention was made under project number HD20A0339 under the support of the Korea Centers for Disease Control and Prevention. Construction of drug response characteristics of cardiomyocytes derived from pluripotent stem cell lines using ⁇ 2021.12.31.
  • the present invention was made under project number 20009748 under the support of the Ministry of Trade, Industry and Energy.
  • the research project title is “Development of a three-dimensional cardiac microenvironment-simulating chip for cardiac toxicity evaluation of human stem cell-derived cardiomyocyte-based drugs”, organized by CHA Medical Science University Industry-Academic Cooperation Foundation, and research period is April 2020. 01. ⁇ 2023.12.31.
  • the present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, specifically, to human embryonic stem cells and human reverse
  • the present invention relates to a method for accurately measuring in vitro cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using differentiated stem cell-derived cardiomyocytes.
  • SARS-CoV-2 virus is an RNA virus belonging to Coronaviridae , and is known to be spread through droplets or contact.
  • the fatality rate of SARS-CoV-2 virus known so far is about 3.4%, and although the fatality level varies by country or age, mainly elderly patients, patients with weakened immune function, and patients with underlying diseases develop severe disease or die. are doing
  • coronavirus disease When infected with the coronavirus disease (COVID-19), various respiratory infections ranging from mild to severe such as fever, fever, malaise, cough, dyspnea and pneumonia appear in patients, and in addition to sputum, sore throat, headache, hemoptysis and nausea, diarrhea, etc. Symptoms are also present.
  • the present inventors treated human embryonic stem cells and human immunized stem cell-derived cardiomyocytes with a candidate drug for SARS-CoV-2 virus, and then measured the viability of cardiomyocytes. As a result, it was confirmed that the composition for evaluating the myocardial cell toxicity of the candidate drug for SARS-CoV-2 virus according to the present invention and the method for evaluating the myocardial cell toxicity using the same were significantly superior in accuracy compared to the existing toxicity evaluation method.
  • Another object of the present invention is to provide a composition for evaluating cardiomyocyte toxicity of a candidate drug targeted for SARS-CoV-2 virus, comprising human pluripotent stem cell-derived cardiomyocytes.
  • Another object of the present invention is to provide a method for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus.
  • Another object of the present invention is to provide the use of a composition comprising human pluripotent stem cell-derived cardiomyocytes to evaluate cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus.
  • the present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, and the composition according to the present invention and method using the same It is possible to accurately evaluate the cardiomyocyte toxicity of the target drug.
  • the inventors of the present invention have developed four candidate drugs for the treatment of COVID-19, hydroxychloroquine, chloroquine, remdesivir, and favipiravir, on cardiomyocytes derived from human embryonic stem cells and human immunized stem cells. Cell viability was analyzed after treatment with (favipiravir). And, as a result of comparing the toxicity evaluation results of the drugs with the cytotoxicity evaluation results using the existing Vero E6 cells, it was found that the cardiomyocyte toxicity evaluation method according to the present invention can more sensitively and accurately evaluate the cardiomyocyte toxicity of the target drug. Confirmed.
  • One aspect of the present invention is a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, including human pluripotent stem cell-derived cardiomyocytes.
  • evaluation refers to confirming the presence or characteristics of a pathological condition, and for the purpose of the present invention, evaluation may mean diagnosing, predicting, searching, or confirming whether or not cardiomyocyte toxicity occurs and is likely to occur.
  • the term “candidate drug” refers to a drug that is judged to have a therapeutic effect on a COVID-19 disease or a drug that is expected to have a therapeutic effect for a COVID-19 disease.
  • the human pluripotent stem cells may be selected from the group consisting of human embryonic stem cells and human immunized stem cells.
  • stem cell refers to a cell having the ability to differentiate into two or more different types of cells while having the ability to self-replicate as an undifferentiated cell.
  • the stem cell may be an autologous or allogeneic stem cell, may be derived from any type of animal including humans and non-human mammals, may be an adult-derived stem cell, and may be an embryo-derived stem cell. It may be a stem cell.
  • the stem cells may be selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs) and mesenchymal stem cells derived from induced pluripotent stem cells, but are limited thereto. it is not
  • adult stem cell is a cell extracted from umbilical cord blood, adult bone marrow, blood, etc., and refers to a cell just before differentiation into a cell of a specific organ, and can develop into a tissue within the body when necessary. It refers to a cell in an undifferentiated state with the ability to
  • adult stem cells are adult stem cells of human, animal or animal tissue origin, mesenchymal stromal cells derived from human, animal or animal tissue, and derivation of human, animal or animal tissue origin. It may be selected from the group consisting of mesenchymal stem cells derived from pluripotent stem cells, but is not limited thereto.
  • human, animal or animal tissue may be selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta, but is not limited thereto.
  • the stem cells of various human or animal tissue origin are selected from the group consisting of hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory neural stem cells, and testicular stem cells.
  • the present invention is not limited thereto.
  • embryonic stem cell is a cell extracted during embryonic development, and the inner cell mass is extracted from the blastocyst embryo just before the fertilized egg is implanted in the mother's uterus and cultured in vitro. it means done
  • Embryonic stem cells refer to cells with self-renewal ability that are pluripotent or totipotent capable of differentiating into cells of all tissues of an individual, and in a broad sense is meant to include embryoid bodies derived from embryonic stem cells.
  • the stem cells may include, but are not limited to, embryonic stem cells derived from all types of human, monkey, pig, horse, cow, sheep, dog, cat, mouse, rabbit, and the like.
  • iPSC induced pluripotent stem cell
  • the artificial redifferentiation process is carried out by the introduction of a non-viral-mediated dedifferentiation factor using a virus-mediated or non-viral vector using a retrovirus, a lentivirus and a Sendai virus, a protein and cell extract, etc., or a stem cell extract, It may include a dedifferentiation process by a compound or the like.
  • Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, have a similar cell shape, have similar gene and protein expression, have pluripotency in vitro and in vivo, and form teratoma. And, when inserted into the blastocyst of a mouse, a chimera mouse is formed, and germline transmission of the gene is possible.
  • SARS-CoV-2 virus is an RNA virus belonging to Coronaviridae that causes COVID-19 disease.
  • various respiratory infections ranging from mild to severe such as fever, fever, malaise, cough, shortness of breath and pneumonia It refers to a virus that appears in patients and causes other symptoms such as sputum, sore throat, headache, hemoptysis, nausea, and diarrhea.
  • the composition for evaluation may further include a composition for measuring the viability of cardiomyocytes, for example, may further include an MTT assay composition, but is not limited thereto.
  • MTT assay is a colorimetric assay for evaluating cellular metabolic activity, and measures the number of viable cells through a reduction reaction between NAD(P)H-dependent cellular oxidase and tetrazolium dye, A method used to measure the cytotoxicity of potentially medicinal and toxic substances.
  • the composition may comprise a tetrazolium dye.
  • the tetrazolium dye is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (2,3-bis-(2-methoxy-4-nitro-5) -sulfophenyl)-2H-tetrazolium-5-carboxanilide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), It may be selected from the group consisting of water-soluble tetrazolium salts (WSTs), for example, MTS, but is not limited thereto.
  • WSTs water-soluble tetrazolium salts
  • the number of human pluripotent stem cell-derived cardiomyocytes included in the composition is 1 ⁇ 10 2 to 5 ⁇ 10 6 cell/ml, 3 ⁇ 10 2 to 1 ⁇ 10 6 cell/ml, 5 ⁇ 10 2 to 1 ⁇ 10 6 cell/ml, 7 ⁇ 10 2 to 1 ⁇ 10 6 cell/ml, 1 ⁇ 10 3 to 1 ⁇ 10 6 cell/ml, 5 ⁇ 10 3 to 1 ⁇ 10 6 cell/ml , 7 ⁇ 10 3 to 1 ⁇ 10 6 cell/ml, 1 ⁇ 10 4 to 1 ⁇ 10 6 cell/ml, 1 ⁇ 10 4 to 5 ⁇ 10 5 cell/ml, 1 ⁇ 10 4 to 3 ⁇ 10 5 cell /ml, 1 ⁇ 10 4 to 1 ⁇ 10 5 cell/ml and 1 ⁇ 10 4 to 7 ⁇ 10 4 cell/ml, for example, may be 5 ⁇ 10 4 cell/ml, but is limited thereto it is not
  • Another aspect of the present invention is a kit for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising human pluripotent stem cell-derived cardiomyocytes.
  • kit according to the present invention contains human pluripotent stem cell-derived cardiomyocytes in the same way as the composition of the present invention described above, descriptions of common contents between the two are omitted in order to avoid excessive complexity of the present specification.
  • Another aspect of the present invention is a method for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising the following steps.
  • a treatment step of treating human pluripotent stem cell-derived cardiomyocytes with a candidate drug targeting SARS-CoV-2 virus is provided.
  • the concentration of the candidate drug may be gradually increased and the cells are treated, for example, the candidate drug is 0.0051 ⁇ M, 0.015 ⁇ M, 0.046 ⁇ M, 0.14 ⁇ M, 0.41 ⁇ M, 1.2 ⁇ M, 3.7 ⁇ M,
  • the cells may be treated with different concentrations of 11 ⁇ M, 33 ⁇ M, and 100 ⁇ M, but the present invention is not limited thereto.
  • the method may further include a measuring step of measuring the viability of cardiomyocytes.
  • the measuring step may be to evaluate the viability of cells through a colorimetric cell viability assay, for example, an MTT assay.
  • the measuring step may further include calculating the CC50 value of the candidate drug based on the measured cell viability.
  • the CC50 value means a concentration at which about 50% of cells exhibit cytotoxicity, that is, a concentration at which 50% of cardiomyocytes treated with a candidate drug die.
  • the method may further include an evaluation step of evaluating the cardiomyocyte toxicity of the SARS-CoV-2 virus target drug candidate based on the measured survival rate.
  • the evaluation step may be to determine that the candidate drug has cardiomyocyte toxicity when the measured CC50 value is 150 uM, 140 uM, 130 uM, 120 uM, 110 uM, or 100 uM concentration or less.
  • Another aspect of the present invention is the use of a composition comprising human pluripotent stem cell-derived cardiomyocytes to evaluate cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus.
  • the present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, and the composition according to the present invention and method using the same It is possible to accurately evaluate the myocardial cell toxicity of the target drug, so it can be usefully used in discovering new drug candidates for COVID-19 disease.
  • hESC-CM human embryonic stem cell
  • hiPSC-CM human inversely differentiated stem cell-derived cardiomyocytes
  • FIG. 1B is a microscopic view of human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention.
  • hESC-CM human embryonic stem cell-derived cardiomyocytes
  • hiPSC-CM human retrodifferentiated stem cell-derived cardiomyocytes
  • FIG. 2A is a graph showing the cTnT gene expression levels in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention.
  • 2B is a graph showing the ACE2 gene expression levels of human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human immunized stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention.
  • 2C is a photograph showing the results of immunofluorescence staining of human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention.
  • hESC-CM human embryonic stem cell-derived cardiomyocytes
  • hiPSC-CM human retrodifferentiated stem cell-derived cardiomyocytes
  • Figure 3a shows that Vero E6 cells were infected with SARS-CoV-2 and administered with hydroxychloroquine at various concentrations (100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015). After treatment with ⁇ M and 0.0051 ⁇ M), it is a graph showing the results of measuring cell viability through a colorimetric cell viability assay.
  • Figure 3b shows that Vero E6 cells were infected with SARS-CoV-2 and treated with chloroquine at various concentrations (100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015 ⁇ M and 0.0051 ⁇ M). It is a graph showing the results of measuring cell viability through colorimetric cell viability analysis after treatment with .
  • Figure 3c shows that Vero E6 cells were infected with SARS-CoV-2 and treated with remdesivir at various concentrations (100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015 ⁇ M and 0.0051 ⁇ M).
  • ⁇ M is a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
  • Figure 3d shows that Vero E6 cells were infected with SARS-CoV-2 and administered favipiravir at various concentrations (100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015 ⁇ M and 0.0051 ⁇ M).
  • ⁇ M is a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
  • Figure 4a shows hydroxychloroquine at various concentrations (100 ⁇ M, 33 ⁇ M) in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention; , 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015 ⁇ M, and 0.0051 ⁇ M), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
  • hESC-CM human embryonic stem cell-derived cardiomyocytes
  • hiPSC-CM human retrodifferentiated stem cell-derived cardiomyocytes
  • Figure 4b shows chloroquine in various concentrations (100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015 ⁇ M, and 0.0051 ⁇ M), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
  • Figure 4c shows the concentration of remdesivir in various concentrations (100 ⁇ M, 33 ⁇ M) in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human inversely differentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention; , 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015 ⁇ M, and 0.0051 ⁇ M), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
  • hESC-CM human embryonic stem cell-derived cardiomyocytes
  • hiPSC-CM human inversely differentiated stem cell-derived cardiomyocytes
  • FIG. 4D shows faviparavir at various concentrations (100 ⁇ M, 33 ⁇ M) in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention. , 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, 0.015 ⁇ M, and 0.0051 ⁇ M), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
  • the present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising human pluripotent stem cell-derived cardiomyocytes.
  • Example 1 Production of cardiomyocytes derived from human pluripotent stem cells
  • the human embryonic stem cell (hESC) cell line H9 was maintained and cultured in an undifferentiated state for 3 days in Stem MACS TM iPSC BREW XF medium. After that, the stem cells cultured as shown in FIG. 1a were sequentially treated with the low molecular weight compounds CHIR 99021 and C59 for 2 days, respectively, and differentiation was induced in AD-MEM/B27 (+insulin) medium for 4 days, as shown in FIG. 1b. Human embryonic stem cell-derived cardiomyocytes (hESC-CM) in which beating was observed were obtained. Then, the cardiomyocytes were purified to a high purity of 95% or more using a Glucose(-)/Lactate medium.
  • hESC-CM Human embryonic stem cell-derived cardiomyocytes
  • hiPSC human induced pluripotent stem cell
  • Example 2 Analysis of gene expression in cardiomyocytes derived from human pluripotent stem cells
  • qRT-PCR For qRT-PCR to confirm gene expression level, undifferentiated stem cells and differentiated cardiomyocytes in a culture dish were washed twice with DPBS, 1 ml of TRIZOL solution was added to dissolve the cell membrane, and then transferred to a 1.5 ml tube. 200 ⁇ l of chloroform was added to the tube and centrifuged for 20 minutes at 13,000 rpm in a centrifuge maintained at 4 °C. After the uppermost transparent solution was transferred to a new tube, the same volume of 2-propanol was added, and centrifuged again at 13,000 rpm for 20 minutes in a centrifuge maintained at 4 °C.
  • Total RNA isolated from cells by the above method and total RNA recovered from human heart tissue per 1 ⁇ g of total RNA were mixed with 2 ⁇ l of random primer and RT buffer, 0.8 ⁇ l of dNTP, and 1 ⁇ l of ribonuclease, and then added with tertiary distilled water. The volume was made up to 20 ⁇ l and placed in a PCR tube. Thereafter, using PCR equipment, cDNA synthesis was induced by reacting at 25° C. for 10 minutes, 37° C. for 120 minutes, and at 85° C. for 5 minutes.
  • step 1 Prepare 1 ⁇ l of the synthesized cDNA in a PCR tube with 10 ⁇ l of 2X SYBR Mixture, 8 ⁇ l of tertiary distilled water (DW), 1 ul of 10 pM primer and a total volume of 20 ⁇ l in a PCR tube, and then use qRT-PCR equipment and repeat 45 times in step 1 at 95 °C for 600 seconds, at step 2 at 95 °C for 10 seconds, at 56 °C for 10 seconds, at 72 °C for 10 seconds, then step 3 at 95 °C for 10 seconds, at 65 °C for 60 seconds at 97 °C The gene expression was compared by reacting for 1 second.
  • DW tertiary distilled water
  • cardiomyocytes differentiated from undifferentiated stem cells were washed twice with DPBS, 4% Paraformaldehyde was added, and the cells were reacted at room temperature for 10 minutes to fix the cells.
  • DPBS 0.03% Triton
  • Triton X-100 Triton X-100
  • DPBS 5% NGS
  • ACE2 and cTnT protein expression was confirmed in both hESC-CM and hiPSC-CM cells as shown in FIG. 2c.
  • both hESC-CM and hiPSC-CM cells exhibited the characteristics of normal cardiomyocytes. It was confirmed that both -CM and hiPSC-CM cells could be target cells for SARS-CoV-2 infection.
  • Example 3 Evaluation of antiviral efficacy against four candidate drugs for treatment of COVID-19 in Vero E6 cells infected with COVID-19
  • CPE Colorimetric cell viability and in vitro antiviral activity and cytopathic effects (CPE) of potential COVID-19 drugs, hydroxychloroquine, chloroquine, remdesivir and favipiravir It was measured through a colorimetric cell viability assay.
  • Each potential COVID-19 drug was administered to Vero E6 cells infected with 0.1 MOI of SARS-CoV-2 (COVID19) at 100 ⁇ M, 33 ⁇ M, 11 ⁇ M, 3.7 ⁇ M, 1.2 ⁇ M, 0.41 ⁇ M, 0.14 ⁇ M, 0.046 ⁇ M, Various concentrations of 0.015 ⁇ M and 0.0051 ⁇ M were treated. 72 hours after infection, the cells were reacted with the reaction product of CellTiter 96®AQueous One Solution Cell Proliferation Assay (MTS, Promega), and the absorbance was measured at 490 nm using a plate reader to analyze cell viability. Then, the above process was independently performed two or more times to measure the average cell viability.
  • MTS CellTiter 96®AQueous One Solution Cell Proliferation Assay
  • Example 4 Cytotoxicity evaluation of 4 candidate drugs for treatment of COVID-19 using human pluripotent stem cell-derived cardiomyocytes
  • cytotoxicity of hESC-CM and hiPSC-CM cells was evaluated by cell viability through a colorimetric cell viability assay.
  • the concentrations of the four drug candidates for COVID19 treatment were gradually increased to 0.0051 ⁇ M, 0.015 ⁇ M, 0.046 ⁇ M, 0.14 ⁇ M, 0.41 ⁇ M, 1.2 ⁇ M, 3.7 ⁇ M, 11 ⁇ M, 33 ⁇ M, and 100 ⁇ M, respectively.
  • the cells were reacted with increasing increments.
  • the cell viability was evaluated using the CellTiter 96®AQueous One Solution Cell Proliferation Assay (MTS, Promega) through colorimetric cell viability assay for drug-induced cytotoxicity. , which are shown by dose-response curve analysis in FIGS. 4A to 4D .
  • hydroxychloroquine showed a pharmacotoxic response in hESC-CM cells at a concentration of 100 ⁇ M, and showed an estimated CC50 value ranging from 84.6 ⁇ M to 31.7 ⁇ M after 24 and 48 hours of drug treatment.
  • hiPSC-CM cells showed an estimated CC50 value in the range of 49.1 ⁇ M to 55.4 ⁇ M after 24 and 48 hours of drug treatment.
  • Chloroquine did not show cardiomyocyte toxicity, with a CC50 value of 100 ⁇ M or higher in all hESC-CM cells after 24 hours of drug treatment, but the CC50 value increased to 84.9 ⁇ M after 48 hours.
  • hiPSC-CM showed no cardiomyocyte toxicity after 24 and 48 hours of drug treatment.
  • remdesivir and favipiravir showed significant cardiomyocyte toxicity in both hESC-CM and hiPSC-CM cells. Specifically, remdesivir showed CC50 values of 18.0 ⁇ M and 11.8 ⁇ M after 24 and 48 hours of drug treatment in hESC-CM cells, respectively, and in hiPSC-CM cells, CC50 values of 11.8 ⁇ M at 24 and 48 hours. and 5.4 ⁇ M, which was confirmed to have significant cardiomyocyte toxicity.
  • favipiravir showed a CC50 value ranging from 9.7 ⁇ M to 34.8 ⁇ M at 24 and 48 hours after drug treatment in hESC-CM, and in hiPSC-CM, a CC50 value of 21.3 ⁇ M after 24 and 48 hours after drug treatment. and 10.7 ⁇ M, indicating significant cardiomyocyte toxicity.
  • the present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, specifically, to human embryonic stem cells and human reverse
  • the present invention relates to a method for accurately measuring in vitro cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using differentiated stem cell-derived cardiomyocytes.

Abstract

The present invention relates to a composition for cardiomyocyte toxicity assay of a candidate drug for the SARS-CoV-2 virus, using human pluripotent stem cell-derived cardiomyocytes, and a cardiomyocyte toxicity assay method using same. The composition and the method using same, according to the present invention, enable the accurate assessment of cardiomyocyte toxicity of a drug being assessed, and thus may be usefully employed in discovering a new drug candidate for the COVID-19 disease.

Description

인간 전분화능줄기세포 유래 심근세포를 이용한 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물 및 이를 이용한 심근세포 독성 평가 방법Composition for evaluating cardiomyocyte toxicity of candidate drug for SARS-CoV-2 virus using cardiomyocytes derived from human pluripotent stem cells and method for evaluating cardiomyocyte toxicity using the same
본 발명은 보건복지부의 지원 하에서 과제번호 HI20C0184에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 보건산업진흥원, 연구사업명은 “첨단의료기술개발”, 연구과제명은 “전분화능줄기세포 유래 심근세포 성숙화(maturation)를 위한 융복합 실용화 기술 개발”, 주관기관은 티앤알바이오팹, 연구기간은 2020.04.23. ~ 2022.12.31이다.The present invention was made with the support of the Ministry of Health and Welfare under project number HI20C0184, the research and management agency for the project is the Health Industry Promotion Agency, the research project name is “Advanced medical technology development”, and the research project name is “pluripotent stem cell-derived cardiomyocyte maturation” Development of convergence and commercialization technology for maturation”, organized by T&R Biofab, research period on April 23, 2020. ~ 2022.12.31.
또한 본 발명은 질병관리본부의 지원 하에서 과제번호 HD20A0339에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 질병관리본부, 연구사업명은 “국가보건의료연구인프라”, 연구과제명은 “국가줄기세포은행 자원을 활용한 전분화능줄기세포주 유래 심근세포의 약물반응 특성정보 구축”, 주관기관은 티앤알바이오팹, 연구기간은 2020.03.16. ~ 2021.12.31이다.In addition, the present invention was made under project number HD20A0339 under the support of the Korea Centers for Disease Control and Prevention. Construction of drug response characteristics of cardiomyocytes derived from pluripotent stem cell lines using ~ 2021.12.31.
또한 본 발명은 산업통상자원부의 지원 하에서 과제번호 20009748에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 한국산업기술평가관리원, 연구사업명은 “3D 생체 조직칩 기반 신약개발 플랫폼 구축 사업(다부처) - 3D 생체 조직칩 제품화”, 연구과제명은 “인간 줄기세포 유래 심근세포 기반 약물의 심장 독성평가용 3차원 심장 미세환경 모사 칩 개발”, 주관기관은 차의과학대학교 산학협력단, 연구기간은 2020.04.01. ~ 2023.12.31이다.In addition, the present invention was made under project number 20009748 under the support of the Ministry of Trade, Industry and Energy. - Commercialization of 3D living tissue chip”, the research project title is “Development of a three-dimensional cardiac microenvironment-simulating chip for cardiac toxicity evaluation of human stem cell-derived cardiomyocyte-based drugs”, organized by CHA Medical Science University Industry-Academic Cooperation Foundation, and research period is April 2020. 01. ~ 2023.12.31.
본 특허출원은 2020년 7월 9일에 대한민국 특허청에 제출된 대한민국 특허출원 제10-2020-0084634호에 대하여 우선권을 주장하며, 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.This patent application claims priority to Korean Patent Application No. 10-2020-0084634 filed with the Korean Intellectual Property Office on July 9, 2020, the disclosure of which is incorporated herein by reference.
본 발명은 인간 전분화능줄기세포 유래 심근세포를 이용한 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물 및 이를 이용한 심근세포 독성 평가 방법에 관한 것으로, 구체적으로 인간 배아줄기세포 및 인간 역분화줄기세포 유래의 심근세포를 이용하여 SARS-CoV-2 바이러스 대상 후보 약물의 체외 심근세포 독성을 정확하게 측정할 수 있는 방법에 관한 것이다.The present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, specifically, to human embryonic stem cells and human reverse The present invention relates to a method for accurately measuring in vitro cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using differentiated stem cell-derived cardiomyocytes.
세계는 현재 심각한 급성 호흡기 증후군-코로나 바이러스 (SARS-CoV-2)에 의해 유발되는 코로나 바이러스 질병 (COVID-19) 전염병으로부터 전례 없는 질환 극복 문제에 직면해 있다. SARS-CoV-2 바이러스는 Coronaviridae에 속하는 RNA 바이러스로, 현재까지는 비말 또는 접촉을 통해 전파되는 것으로 알려져 있다. 현재까지 알려진 SARS-CoV-2 바이러스의 치명률은 약 3.4% 정도로, 국가별 또는 연령별 치명률 수준은 상이하나, 주로 고령, 면역기능이 저하된 환자, 기저질환을 가진 환자가 주로 중증으로 발병하거나, 사망하고 있다.The world currently faces the challenge of overcoming an unprecedented disease from the coronavirus disease (COVID-19) epidemic caused by the severe acute respiratory syndrome-coronavirus (SARS-CoV-2). SARS-CoV-2 virus is an RNA virus belonging to Coronaviridae , and is known to be spread through droplets or contact. The fatality rate of SARS-CoV-2 virus known so far is about 3.4%, and although the fatality level varies by country or age, mainly elderly patients, patients with weakened immune function, and patients with underlying diseases develop severe disease or die. are doing
코로나 바이러스 질병 (COVID-19) 감염 시 발열, 발열, 권태감, 기침, 호흡곤란 및 폐렴 등 경증에서 중증까지 다양한 호흡기감염증이 환자에게서 나타나고, 그 외에도 가래, 인후통, 두통, 객혈과 오심, 설사 등의 증상도 함께 나타나고 있다. When infected with the coronavirus disease (COVID-19), various respiratory infections ranging from mild to severe such as fever, fever, malaise, cough, dyspnea and pneumonia appear in patients, and in addition to sputum, sore throat, headache, hemoptysis and nausea, diarrhea, etc. Symptoms are also present.
다만, 현재는 이러한 SARS-CoV-2 바이러스에 대한 특이적인 항바이러스제가 알려지거나 개발되지 않은 상태이며, 수액 보충, 해열제 등의 보존적 치료를 통한 대증 치료 만이 유일한 치료 수단으로 알려져있다.However, at present, antiviral agents specific for this SARS-CoV-2 virus have not been known or developed, and symptomatic treatment through conservative treatment such as fluid supplementation and antipyretics is known as the only treatment means.
이러한 상황에서 최근 질환 극복을 위해, 응급 사용 승인을 받은 렘데시비르 (Remdesivir) 약물을 포함하여 여러 후보 약물이 COVID-19 질환의 잠재적 치료 옵션으로 제안되고 있는 상황이다.In this situation, to overcome the recent disease, several candidate drugs, including the drug Remdesivir, which have been approved for emergency use, are being proposed as potential treatment options for COVID-19 disease.
그러나 제안된 COVID-19 질환 치료 후보 약물은 안전성 프로파일, 특히 약물 유발 심독성에 관한 제한된 정보로 인해 시판 후 승인이 철회되는 주요 원인 중 하나이다. 따라서 COVID-19 약리 요법과 관련된 잠재적 심혈관 부작용을 조사하는 것이 중요한 이슈로 부각되고 있다.However, the proposed drug candidate for the treatment of COVID-19 disease is one of the main reasons for the withdrawal of post-market approval due to limited information about its safety profile, particularly drug-induced cardiotoxicity. Therefore, investigating the potential cardiovascular side effects associated with pharmacotherapy for COVID-19 is emerging as an important issue.
이에 본 발명자들은 인간 배아줄기세포 및 인간 역분화줄기세포 유래 심근세포에 SARS-CoV-2 바이러스 대상 후보 약물을 처리한 후, 심근세포의 생존율을 측정하였다. 그 결과, 본 발명에 따른 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물 및 이를 이용한 심근세포 독성 평가 방법은 기존의 독성 평가방법에 비해 그 정확도가 월등히 우수한 것을 확인하였다.Therefore, the present inventors treated human embryonic stem cells and human immunized stem cell-derived cardiomyocytes with a candidate drug for SARS-CoV-2 virus, and then measured the viability of cardiomyocytes. As a result, it was confirmed that the composition for evaluating the myocardial cell toxicity of the candidate drug for SARS-CoV-2 virus according to the present invention and the method for evaluating the myocardial cell toxicity using the same were significantly superior in accuracy compared to the existing toxicity evaluation method.
이에, 본 발명의 목적은 인간 전분화능 줄기세포 유래 심근세포를 포함하는, SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 키트를 제공하는 것이다.Accordingly, it is an object of the present invention to provide a kit for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, including cardiomyocytes derived from human pluripotent stem cells.
본 발명의 다른 목적은 인간 전분화능 줄기세포 유래 심근세포를 포함하는, SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for evaluating cardiomyocyte toxicity of a candidate drug targeted for SARS-CoV-2 virus, comprising human pluripotent stem cell-derived cardiomyocytes.
본 발명의 또 다른 목적은 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가 방법을 제공하는 것이다.Another object of the present invention is to provide a method for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus.
본 발명의 또 다른 목적은 인간 전분화능 줄기세포 유래 심근세포를 포함하는 조성물의 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가 용도를 제공하는 것이다.Another object of the present invention is to provide the use of a composition comprising human pluripotent stem cell-derived cardiomyocytes to evaluate cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus.
본 발명은 인간 전분화능줄기세포 유래 심근세포를 이용한 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물 및 이를 이용한 심근세포 독성 평가 방법에 관한 것으로, 본 발명에 따른 조성물 및 이를 이용한 방법은 대상 약물의 심근세포 독성을 정확하게 평가하는 것이 가능하다.The present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, and the composition according to the present invention and method using the same It is possible to accurately evaluate the cardiomyocyte toxicity of the target drug.
이에 본 발명의 발명자들은 인간 배아줄기세포 및 인간 역분화줄기세포 유래의 심근세포에 COVID19 치료용 후보 약물 4종인 하이드록시클로로퀸 (hydroxychloroquine), 클로로퀸 (chloroquine), 렘데시비르 (remdesivir) 및 파비피라비어 (favipiravir)를 처리한 후 세포 생존율을 분석하였다. 그리고, 상기 약물들의 독성평가 결과를 기존의 Vero E6 세포를 이용한 세포 독성평가 결과와 비교한 결과, 본 발명에 따른 심근세포 독성 평가방법이 더욱 민감하고 정확하게 대상 약물의 심근세포 독성을 평가할 수 있는 것을 확인하였다.Accordingly, the inventors of the present invention have developed four candidate drugs for the treatment of COVID-19, hydroxychloroquine, chloroquine, remdesivir, and favipiravir, on cardiomyocytes derived from human embryonic stem cells and human immunized stem cells. Cell viability was analyzed after treatment with (favipiravir). And, as a result of comparing the toxicity evaluation results of the drugs with the cytotoxicity evaluation results using the existing Vero E6 cells, it was found that the cardiomyocyte toxicity evaluation method according to the present invention can more sensitively and accurately evaluate the cardiomyocyte toxicity of the target drug. Confirmed.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는, 인간 전분화능 줄기세포 유래 심근세포를 포함하는, SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물이다.One aspect of the present invention is a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, including human pluripotent stem cell-derived cardiomyocytes.
본 명세서 상의 용어, “평가”는 병리 상태의 존재 또는 특징을 확인하는 것으로, 본 발명의 목적상, 평가는 심근세포 독성의 발생 여부 및 발생 가능성 여부를 진단, 예측, 검색 또는 확인하는 것을 의미할 수 있다.As used herein, the term “assessment” refers to confirming the presence or characteristics of a pathological condition, and for the purpose of the present invention, evaluation may mean diagnosing, predicting, searching, or confirming whether or not cardiomyocyte toxicity occurs and is likely to occur. can
본 명세서 상의 용어, “후보 약물”은 COVID-19 질환의 치료 효과가 있다고 판단되는 약물 또는 COVID-19 질환의 치료 효과가 있을 것으로 예상되는 약물을 의미한다.As used herein, the term “candidate drug” refers to a drug that is judged to have a therapeutic effect on a COVID-19 disease or a drug that is expected to have a therapeutic effect for a COVID-19 disease.
본 발명의 일 구현예에서, 인간 전분화능 줄기세포는 인간배아줄기세포 및 인간 역분화줄기세포로 구성된 그룹에서 선택되는 것일 수 있다.In one embodiment of the present invention, the human pluripotent stem cells may be selected from the group consisting of human embryonic stem cells and human immunized stem cells.
본 명세서상의 용어 “줄기세포 (Stem cell)”는 미분화된 세포로서 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 의미한다.As used herein, the term “stem cell” refers to a cell having the ability to differentiate into two or more different types of cells while having the ability to self-replicate as an undifferentiated cell.
본 발명의 일 구현예에서, 줄기 세포는 자가 또는 동종 유래 줄기세포일 수 있고, 인간 및 비인간 포유류를 포함한 임의 유형의 동물 유래일 수 있으며, 성체로부터 유래된 줄기세포일 수 있고, 배아로부터 유래된 줄기세포일 수 있다. 예를 들어, 줄기세포는 배아 줄기세포, 성체 줄기세포, 유도만능줄기세포 (induced pluripotent stem cell; iPSC) 및 유도만능 줄기세포 유래 중간엽 줄기세포로 이루어진 군으로부터 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the stem cell may be an autologous or allogeneic stem cell, may be derived from any type of animal including humans and non-human mammals, may be an adult-derived stem cell, and may be an embryo-derived stem cell. It may be a stem cell. For example, the stem cells may be selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSCs) and mesenchymal stem cells derived from induced pluripotent stem cells, but are limited thereto. it is not
본 명세서상의 용어 “성체 줄기세포 (adult stem cell)”는 제대혈이나 성인의 골수, 혈액 등에서 추출되는 세포로 구체적 장기의 세포로 분화되기 직전의 세포를 의미하며, 필요한 때에 신체 내 조직으로 발달할 수 있는 능력을 보유한 미분화 상태의 세포를 의미한다.As used herein, the term “adult stem cell” is a cell extracted from umbilical cord blood, adult bone marrow, blood, etc., and refers to a cell just before differentiation into a cell of a specific organ, and can develop into a tissue within the body when necessary. It refers to a cell in an undifferentiated state with the ability to
본 발명의 일 구현예에서, 성체 줄기세포는 인간, 동물 또는 동물 조직 기원의 성체 줄기세포, 인간, 동물 또는 동물 조직 유래 중간엽 줄기세포 (mesenchymal stromal cell) 및 인간, 동물 또는 동물 조직 기원의 유도만능줄기세포로부터 유래된 중간엽 줄기세포로 이루어진 군으로부터 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, adult stem cells are adult stem cells of human, animal or animal tissue origin, mesenchymal stromal cells derived from human, animal or animal tissue, and derivation of human, animal or animal tissue origin. It may be selected from the group consisting of mesenchymal stem cells derived from pluripotent stem cells, but is not limited thereto.
본 발명에 있어서 인간, 동물 또는 동물 조직은 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 및 태반으로 이루어진 군으로부터 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, human, animal or animal tissue may be selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta, but is not limited thereto.
본 발명에 있어서 인간 또는 동물의 다양한 조직 기원의 줄기세포는 조혈모세포, 유선 줄기세포, 장 줄기세포, 혈관내피 줄기세포, 신경 줄기세포, 후각신경 줄기세포 및 정소 줄기세포로 이루어진 군에서 선택되는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the stem cells of various human or animal tissue origin are selected from the group consisting of hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory neural stem cells, and testicular stem cells. However, the present invention is not limited thereto.
본 명세서상의 용어 “배아 줄기세포 (embryonic stem cell)”는 배아의 발생과정에서 추출한 세포로, 수정란이 모체의 자궁에 착상하기 직전인 포배기 배아에서 내세포괴 (inner cell mass)를 추출하여 체외에서 배양한 것을 의미한다.As used herein, the term “embryonic stem cell” is a cell extracted during embryonic development, and the inner cell mass is extracted from the blastocyst embryo just before the fertilized egg is implanted in the mother's uterus and cultured in vitro. it means done
배아 줄기세포는 개체의 모든 조직의 세포로 분화할 수 있는 다능성 (多能性, pluripotent)이거나 전능성 (全能性, totipotent)이 있는 자가재생능 (selfrenewal)을 갖는 세포를 의미하며, 넓은 의미로는 배아 줄기세포로부터 유래한 배아체 (embryoid bodies)도 포함하는 것을 의미한다.Embryonic stem cells refer to cells with self-renewal ability that are pluripotent or totipotent capable of differentiating into cells of all tissues of an individual, and in a broad sense is meant to include embryoid bodies derived from embryonic stem cells.
본 발명에 있어서 줄기세포는 인간, 원숭이, 돼지, 말, 소, 양, 개, 고양이, 생쥐, 토끼 등의 모든 유래의 배아 줄기세포를 포함할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the stem cells may include, but are not limited to, embryonic stem cells derived from all types of human, monkey, pig, horse, cow, sheep, dog, cat, mouse, rabbit, and the like.
본 명세서상의 용어 “유도만능줄기세포 (induced pluripotent stem cell; iPSC)”는 분화된 세포들로부터 인위적인 역분화 과정을 통해 다능성 분화능을 가지도록 유도된 세포들을 의미하며, “역분화줄기세포”와 동일한 의미로 사용될 수 있다.As used herein, the term “induced pluripotent stem cell (iPSC)” refers to cells induced to have pluripotent differentiation potential from differentiated cells through an artificial retrodifferentiation process, and “induced pluripotent stem cell” and can be used with the same meaning.
인위적인 역분화 과정은 레트로바이러스, 렌티바이러스 및 센다이바이러스를 이용한 바이러스-매개 또는 비바이러스성 벡터 이용, 단백질 및 세포 추출물 등을 이용하는 비바이러스-매개 역분화 인자의 도입에 의해 수행되거나, 줄기세포 추출물, 화합물 등에 의한 역분화 과정을 포함할 수 있다.The artificial redifferentiation process is carried out by the introduction of a non-viral-mediated dedifferentiation factor using a virus-mediated or non-viral vector using a retrovirus, a lentivirus and a Sendai virus, a protein and cell extract, etc., or a stem cell extract, It may include a dedifferentiation process by a compound or the like.
유도만능줄기세포는 배아 줄기세포와 거의 동일한 특성을 가지며, 구체적으로는, 유사한 세포 모양을 가지고, 유전자, 단백질 발현이 유사하며, in vitro 및 in vivo에서 전분화능을 가지고, 테라토마 (teratoma)를 형성하며, 생쥐의 배반포 (blastocyst)에 삽입시켰을 때, 키메라 (chimera) 생쥐를 형성하고, 유전자의 생식선 전이 (germline transmission)가 가능하다.Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, have a similar cell shape, have similar gene and protein expression, have pluripotency in vitro and in vivo, and form teratoma. And, when inserted into the blastocyst of a mouse, a chimera mouse is formed, and germline transmission of the gene is possible.
본 명세서 상의 용어 “SARS-CoV-2 바이러스”는 COVID-19 질환을 유발하는 Coronaviridae에 속하는 RNA 바이러스로, 감염 시 발열, 발열, 권태감, 기침, 호흡곤란 및 폐렴 등 경증에서 중증까지 다양한 호흡기 감염증이 환자에게서 나타나고, 그 외에도 가래, 인후통, 두통, 객혈과 오심, 설사 등의 증상도 함께 유발하는 바이러스를 의미한다.As used herein, the term “SARS-CoV-2 virus” is an RNA virus belonging to Coronaviridae that causes COVID-19 disease. During infection, various respiratory infections ranging from mild to severe such as fever, fever, malaise, cough, shortness of breath and pneumonia It refers to a virus that appears in patients and causes other symptoms such as sputum, sore throat, headache, hemoptysis, nausea, and diarrhea.
본 발명에 있어서 평가용 조성물은 심근세포의 생존율을 측정하는 조성물을 더 포함할 수 있고, 예를 들어, MTT 어세이 조성물을 더 포함할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the composition for evaluation may further include a composition for measuring the viability of cardiomyocytes, for example, may further include an MTT assay composition, but is not limited thereto.
본 명세서 상의 용어 “MTT 어세이”는 세포 대사 활동을 평가하기 위한 비색 분석이며, NAD(P)H-의존성 세포 산화 효소와 테트라졸륨 염료와의 환원 반응을 통해 생존 세포의 수를 측정하는 것으로, 잠재적인 의약 및 독성 물질의 세포 독성을 측정하는데 사용되는 방법을 의미한다.As used herein, the term “MTT assay” is a colorimetric assay for evaluating cellular metabolic activity, and measures the number of viable cells through a reduction reaction between NAD(P)H-dependent cellular oxidase and tetrazolium dye, A method used to measure the cytotoxicity of potentially medicinal and toxic substances.
본 발명의 일 구현예에서, 조성물은 테트라졸륨 염료를 포함할 수 있다.In one embodiment of the present invention, the composition may comprise a tetrazolium dye.
본 발명에 있어서 테트라졸륨 염료는 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), WSTs (water-soluble tetrazolium salts)으로 구성되는 군에서 선택된 것일 수 있고, 예를 들어, MTS 일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the tetrazolium dye is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), XTT (2,3-bis-(2-methoxy-4-nitro-5) -sulfophenyl)-2H-tetrazolium-5-carboxanilide), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), It may be selected from the group consisting of water-soluble tetrazolium salts (WSTs), for example, MTS, but is not limited thereto.
본 발명의 일 구현예에서, 조성물에 포함되는 인간 전분화능 줄기세포 유래 심근세포의 수는 1×102 내지 5×106 cell/ml, 3×102 내지 1×106 cell/ml, 5×102 내지 1×106 cell/ml, 7×102 내지 1×106 cell/ml, 1×103 내지 1×106 cell/ml, 5×103 내지 1×106 cell/ml, 7×103 내지 1×106 cell/ml, 1×104 내지 1×106 cell/ml, 1×104 내지 5×105 cell/ml, 1×104 내지 3×105 cell/ml, 1×104 내지 1×105 cell/ml 및 1×104 내지 7×104 cell/ml일 수 있고, 예를 들어, 5×104 cell/ml 일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the number of human pluripotent stem cell-derived cardiomyocytes included in the composition is 1×10 2 to 5×10 6 cell/ml, 3×10 2 to 1×10 6 cell/ml, 5 ×10 2 to 1×10 6 cell/ml, 7×10 2 to 1×10 6 cell/ml, 1×10 3 to 1×10 6 cell/ml, 5×10 3 to 1×10 6 cell/ml , 7×10 3 to 1×10 6 cell/ml, 1×10 4 to 1×10 6 cell/ml, 1×10 4 to 5×10 5 cell/ml, 1×10 4 to 3×10 5 cell /ml, 1×10 4 to 1×10 5 cell/ml and 1×10 4 to 7×10 4 cell/ml, for example, may be 5×10 4 cell/ml, but is limited thereto it is not
본 발명의 다른 양태는 인간 전분화능 줄기세포 유래 심근세포를 포함하는, SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 키트이다.Another aspect of the present invention is a kit for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising human pluripotent stem cell-derived cardiomyocytes.
본 발명에 따른 키트는 상술한 본 발명의 조성물과 동일하게 인간 전분화능 줄기세포 유래 심근세포를 포함하므로, 이 둘 사이에 공통되는 내용은 본 명세서의 과도한 복잡성을 회피하기 위하여 그 기재를 생략한다.Since the kit according to the present invention contains human pluripotent stem cell-derived cardiomyocytes in the same way as the composition of the present invention described above, descriptions of common contents between the two are omitted in order to avoid excessive complexity of the present specification.
본 발명의 또 다른 양태는, 다음 단계를 포함하는 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가 방법이다.Another aspect of the present invention is a method for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising the following steps.
인간 전분화능 줄기세포 유래 심근세포에 SARS-CoV-2 바이러스 대상 후보 약물을 처리하는 처리 단계.A treatment step of treating human pluripotent stem cell-derived cardiomyocytes with a candidate drug targeting SARS-CoV-2 virus.
본 발명에 있어서 처리 단계 후보 약물의 농도가 점차 증가되면서 세포에 처리되는 것일 수 있고, 예를 들어, 후보 약물은 0.0051 μM, 0.015 μM, 0.046 μM, 0.14 μM, 0.41 μM, 1.2 μM, 3.7 μM, 11 μM, 33 μM, 100 μM로 농도를 달리하여 세포에 처리되는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, in the treatment step, the concentration of the candidate drug may be gradually increased and the cells are treated, for example, the candidate drug is 0.0051 μM, 0.015 μM, 0.046 μM, 0.14 μM, 0.41 μM, 1.2 μM, 3.7 μM, The cells may be treated with different concentrations of 11 μM, 33 μM, and 100 μM, but the present invention is not limited thereto.
본 발명의 일 구현예에서, 방법은 심근세포의 생존율을 측정하는 측정 단계를 더 포함하는 것일 수 있다.In one embodiment of the present invention, the method may further include a measuring step of measuring the viability of cardiomyocytes.
본 발명에 있어서 측정 단계는 비색 세포 생존력 분석 (colorimetric cell viability assay), 예를 들어, MTT 어세이를 통해 세포의 생존율을 평가하는 것일 수 있다.In the present invention, the measuring step may be to evaluate the viability of cells through a colorimetric cell viability assay, for example, an MTT assay.
본 발명의 일 구현예에서, 측정 단계는 측정된 세포 생존율을 기반으로 후보 약물의 CC50 값을 산출하는 산출 단계를 더 포함할 수 있다.In one embodiment of the present invention, the measuring step may further include calculating the CC50 value of the candidate drug based on the measured cell viability.
본 발명에 있어서 CC50 값은 세포의 50% 정도가 세포독성을 나타내는 농도를 의미하는 것으로, 즉, 후보 약물이 처리된 심근세포의 50%가 사망하는 농도를 나타낸다.In the present invention, the CC50 value means a concentration at which about 50% of cells exhibit cytotoxicity, that is, a concentration at which 50% of cardiomyocytes treated with a candidate drug die.
본 발명의 일 구현예에서, 방법은 측정된 상기 생존율을 통해 상기 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성을 평가하는 평가 단계를 더 포함할 수 있다.In one embodiment of the present invention, the method may further include an evaluation step of evaluating the cardiomyocyte toxicity of the SARS-CoV-2 virus target drug candidate based on the measured survival rate.
본 발명에 있어서, 평가 단계는 측정된 CC50 값이 150 uM, 140uM, 130uM, 120 uM, 110 uM, 또는 100 uM 농도 이하인 경우, 후보 대상약물이 심근세포 독성을 가지는 것으로 판단하는 것일 수 있다. In the present invention, the evaluation step may be to determine that the candidate drug has cardiomyocyte toxicity when the measured CC50 value is 150 uM, 140 uM, 130 uM, 120 uM, 110 uM, or 100 uM concentration or less.
본 발명의 또 다른 양태는, 인간 전분화능 줄기세포 유래 심근세포를 포함하는 조성물의 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가 용도이다.Another aspect of the present invention is the use of a composition comprising human pluripotent stem cell-derived cardiomyocytes to evaluate cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus.
본 발명은 인간 전분화능줄기세포 유래 심근세포를 이용한 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물 및 이를 이용한 심근세포 독성 평가 방법에 관한 것으로, 본 발명에 따른 조성물 및 이를 이용한 방법은 대상 약물의 심근세포 독성을 정확하게 평가하는 것이 가능하여, COVID-19 질환의 신약 후보 물질을 발굴하는데 있어 유용하게 활용될 수 있다.The present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, and the composition according to the present invention and method using the same It is possible to accurately evaluate the myocardial cell toxicity of the target drug, so it can be usefully used in discovering new drug candidates for COVID-19 disease.
도 1a는 본 발명의 일 실시예에 따른 인간 배아줄기세포 (human embryonic stem cell; hESC) 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)를 생산하는 과정을 보여주는 그래프이다.1a is a process for producing human embryonic stem cell (hESC)-derived cardiomyocytes (hESC-CM) and human inversely differentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention; This is a graph showing
도 1b는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)를 현미경으로 관찰한 사진이다.1B is a microscopic view of human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention.
도 2a는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)의 cTnT 유전자 발현량을 나타내는 그래프이다.FIG. 2A is a graph showing the cTnT gene expression levels in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention.
도 2b는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)의 ACE2 유전자 발현량을 나타내는 그래프이다.2B is a graph showing the ACE2 gene expression levels of human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human immunized stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention.
도 2c는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)의 면역형광염색 결과를 나타내는 사진이다. (DAPI, cTnT, ACE2)2C is a photograph showing the results of immunofluorescence staining of human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention. (DAPI, cTnT, ACE2)
도 3a는 Vero E6 세포에 SARS-CoV-2를 감염시키고 하이드록시클로로퀸 (hydroxychloroquine)을 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석 (colorimetric cell viability assay)을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.Figure 3a shows that Vero E6 cells were infected with SARS-CoV-2 and administered with hydroxychloroquine at various concentrations (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015). After treatment with μM and 0.0051 μM), it is a graph showing the results of measuring cell viability through a colorimetric cell viability assay.
도 3b는 Vero E6 세포에 SARS-CoV-2를 감염시키고 클로로퀸을 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.Figure 3b shows that Vero E6 cells were infected with SARS-CoV-2 and treated with chloroquine at various concentrations (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM and 0.0051 μM). It is a graph showing the results of measuring cell viability through colorimetric cell viability analysis after treatment with .
도 3c는 Vero E6 세포에 SARS-CoV-2를 감염시키고 렘데시비르를 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.Figure 3c shows that Vero E6 cells were infected with SARS-CoV-2 and treated with remdesivir at various concentrations (100 µM, 33 µM, 11 µM, 3.7 µM, 1.2 µM, 0.41 µM, 0.14 µM, 0.046 µM, 0.015 µM and 0.0051 µM). μM), and is a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
도 3d는 Vero E6 세포에 SARS-CoV-2를 감염시키고 파비피라비어를 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.Figure 3d shows that Vero E6 cells were infected with SARS-CoV-2 and administered favipiravir at various concentrations (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM and 0.0051 μM). μM), and is a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
도 4a는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)에 하이드록시클로로퀸을 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.Figure 4a shows hydroxychloroquine at various concentrations (100 μM, 33 μM) in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention; , 11 µM, 3.7 µM, 1.2 µM, 0.41 µM, 0.14 µM, 0.046 µM, 0.015 µM, and 0.0051 µM), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
도 4b는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)에 클로로퀸을 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.Figure 4b shows chloroquine in various concentrations (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM, and 0.0051 μM), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
도 4c는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)에 렘데시비르를 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.Figure 4c shows the concentration of remdesivir in various concentrations (100 μM, 33 μM) in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human inversely differentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention; , 11 µM, 3.7 µM, 1.2 µM, 0.41 µM, 0.14 µM, 0.046 µM, 0.015 µM, and 0.0051 µM), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
도 4d는 본 발명의 일 실시예에 따른 인간 배아줄기세포 유래 심근세포 (hESC-CM) 및 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)에 파비파라비어를 다양한 농도 (100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM)로 처리한 후, 비색 세포 생존율 분석을 통해 세포 생존율을 측정한 결과를 나타내는 그래프이다.FIG. 4D shows faviparavir at various concentrations (100 μM, 33 μM) in human embryonic stem cell-derived cardiomyocytes (hESC-CM) and human retrodifferentiated stem cell-derived cardiomyocytes (hiPSC-CM) according to an embodiment of the present invention. , 11 µM, 3.7 µM, 1.2 µM, 0.41 µM, 0.14 µM, 0.046 µM, 0.015 µM, and 0.0051 µM), a graph showing the results of measuring cell viability through colorimetric cell viability analysis.
본 발명은 인간 전분화능 줄기세포 유래 심근세포를 포함하는, SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물에 관한 것이다.The present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising human pluripotent stem cell-derived cardiomyocytes.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
실시예 1: 인간 전분화능 줄기세포 유래 심근세포 생산Example 1: Production of cardiomyocytes derived from human pluripotent stem cells
인간배아줄기세포 (human embryonic stem cell; hESC) 세포주 H9을 Stem MACSTM iPSC BREW XF 배지에서 3일간 미분화 상태로 유지배양 하였다. 이 후, 도 1a와 같이 배양된 줄기세포에 저분자화합물 CHIR 99021 와 C59를 순차적으로 각각 2일간 처리하고, AD-MEM/B27 (+insulin) 배지에서 4일간 분화를 유도하여 도 1b와 같이 응수축 (beating)이 관찰되는 인간배아줄기세포 유래 심근세포 (hESC-CM)를 수득하였다. 이어서, 상기 심근세포를 Glucose(-)/Lactate 배지를 이용하여 95% 이상의 고순도로 정제하였다. The human embryonic stem cell (hESC) cell line H9 was maintained and cultured in an undifferentiated state for 3 days in Stem MACS TM iPSC BREW XF medium. After that, the stem cells cultured as shown in FIG. 1a were sequentially treated with the low molecular weight compounds CHIR 99021 and C59 for 2 days, respectively, and differentiation was induced in AD-MEM/B27 (+insulin) medium for 4 days, as shown in FIG. 1b. Human embryonic stem cell-derived cardiomyocytes (hESC-CM) in which beating was observed were obtained. Then, the cardiomyocytes were purified to a high purity of 95% or more using a Glucose(-)/Lactate medium.
그리고, 상기와 동일한 방법으로 인간 역분화줄기세포 (human induced pluripotent stem cell; hiPSC) 세포주 CMC11의 분화를 유도하여 인간 역분화줄기세포 유래 심근세포 (hiPSC-CM)를 수득하였으며, 마찬가지로 도 1b와 같이 심근세포에서 응수축이 관찰되었다.And, by inducing the differentiation of human induced pluripotent stem cell (hiPSC) cell line CMC11 in the same manner as above, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were obtained. Constriction was observed in cardiomyocytes.
실시예 2: 인간 전분화능줄기세포 유래 심근세포에서의 유전자 발현 분석Example 2: Analysis of gene expression in cardiomyocytes derived from human pluripotent stem cells
유전자 발현량 확인을 위한 qRT-PCR 진행을 위해, 배양접시의 미분화 줄기세포 및 분화된 심근세포를 DPBS로 2회 세척하고 1 ml의 TRIZOL 용액을 첨가하여 세포막을 융해시킨 뒤 1.5 ml 튜브로 옮겼다. 튜브에 200 μl의 클로로포름을 첨가하고 4 ℃가 유지되는 원심분리기에서 13,000 rpm으로 20 분 동안 원심 분리하였다. 가장 상층부의 투명한 용액을 새로운 튜브에 옮긴 뒤 동일한 용량의 2-프로판올을 첨가한 뒤, 4 ℃가 유지되는 원심분리기에서 13,000 rpm으로 20 분 동안 다시 원심 분리하였다. 이어서, 상층액을 제거한 후 1 ml의 70% DEPC-Ethanol을 첨가하고 4 ℃가 유지되는 원심분리기에서 12,000 rpm으로 5 분 동안 원심 분리하였다. 상층액을 완전히 제거한 뒤, DEPC-water로 침전물을 풀어준 뒤 나노드롭을 이용하여 총 RNA의 농도를 측정하였다.For qRT-PCR to confirm gene expression level, undifferentiated stem cells and differentiated cardiomyocytes in a culture dish were washed twice with DPBS, 1 ml of TRIZOL solution was added to dissolve the cell membrane, and then transferred to a 1.5 ml tube. 200 μl of chloroform was added to the tube and centrifuged for 20 minutes at 13,000 rpm in a centrifuge maintained at 4 °C. After the uppermost transparent solution was transferred to a new tube, the same volume of 2-propanol was added, and centrifuged again at 13,000 rpm for 20 minutes in a centrifuge maintained at 4 °C. Then, after removing the supernatant, 1 ml of 70% DEPC-Ethanol was added and centrifuged at 12,000 rpm for 5 minutes in a centrifuge maintained at 4°C. After the supernatant was completely removed, the precipitate was released with DEPC-water, and the concentration of total RNA was measured using nanodrops.
상기 방법으로 세포로부터 분리된 total RNA와 인간 심장조직으로부터 회수된 총 RNA 1 μg 당 2 μl의 Random primer 및 RT buffer, 0.8 μl의 dNTP, 1 μl의 리보뉴클레이스와 섞고 3차 증류수를 첨가하여 총 부피를 20 μl로 만들어 PCR 튜브에 넣었다. 이 후, PCR 장비를 이용하여, 25 ℃에서 10분, 37 ℃에서 120분, 85 ℃에서 5분간 반응시켜 cDNA 합성을 유도하였다.Total RNA isolated from cells by the above method and total RNA recovered from human heart tissue per 1 μg of total RNA were mixed with 2 μl of random primer and RT buffer, 0.8 μl of dNTP, and 1 μl of ribonuclease, and then added with tertiary distilled water. The volume was made up to 20 μl and placed in a PCR tube. Thereafter, using PCR equipment, cDNA synthesis was induced by reacting at 25° C. for 10 minutes, 37° C. for 120 minutes, and at 85° C. for 5 minutes.
합성된 cDNA 1 μl를 10 μl의 2X SYBR Mixture, 8 μl의 3차 증류수 (D.W) 및 1 ul의 10 pM 프라이머와 총 부피 20 μl의 용액을 PCR 튜브에 넣어 준비한 후, qRT-PCR 장비를 이용하여 1단계 95 ℃에서 600 초, 2단계 95 ℃에서 10 초, 56 ℃에서 10 초, 72 ℃에서 10 초의 방법으로 45회 반복 후 3단계 95 ℃에서 10 초, 65 ℃에서 60초 97 ℃에서 1 초간 반응시켜 유전자 발현을 비교하였다.Prepare 1 μl of the synthesized cDNA in a PCR tube with 10 μl of 2X SYBR Mixture, 8 μl of tertiary distilled water (DW), 1 ul of 10 pM primer and a total volume of 20 μl in a PCR tube, and then use qRT-PCR equipment and repeat 45 times in step 1 at 95 °C for 600 seconds, at step 2 at 95 °C for 10 seconds, at 56 °C for 10 seconds, at 72 °C for 10 seconds, then step 3 at 95 °C for 10 seconds, at 65 °C for 60 seconds at 97 °C The gene expression was compared by reacting for 1 second.
qRT-PCR 분석결과, 음성 대조군인 미분화 인간 전분화능줄기세포와 양성 대조군인 인간의 심장조직에 비해 hESC-CM 및 hiPSC-CM 세포에서 심근세포 특이 유전자인 심장 트로포닌 T (cTnT) 유전자의 높은 발현량을 확인하였다. 또한, SARS-CoV-2 (COVID19)의 기능적 수용체인 안지오텐신 전환 효소-2 (ACE2)도 hESC-CM 및 hiPSC-CM 세포에서 모두 발현량이 높은 것을 확인하였다. (도 2a 및 2b 참조)As a result of qRT-PCR analysis, higher expression of cardiac troponin T (cTnT) gene, a cardiomyocyte-specific gene, in hESC-CM and hiPSC-CM cells compared to undifferentiated human pluripotent stem cells as a negative control and human heart tissue as a positive control amount was confirmed. In addition, it was confirmed that the expression level of angiotensin converting enzyme-2 (ACE2), a functional receptor of SARS-CoV-2 (COVID19), was also high in both hESC-CM and hiPSC-CM cells. (See Figures 2a and 2b)
면역형광염색을 통한 cTnT와 ACE2의 유전자발현 분석을 위해, 미분화 줄기세포로부터 분화된 심근세포를 DPBS로 2회 세척한 뒤 4% Paraformaldehyde를 첨가하고 상온에서 10 분 동안 반응시켜 세포를 고정하였다. Triton X-100이 0.03% 농도로 첨가된 DPBS (0.03% Triton)로 2회 세척한 뒤, 5% Normal goat serum이 첨가된 DPBS (5% NGS)를 첨가하고 상온에서 30 분 동안 반응시켜 블로킹을 진행하였다. 5% NGS를 이용하여 1차 항체 cTnT와 ACE2를 1:100의 비율로 희석한 뒤 이 용액을 배양접시에 첨가하고 하룻밤동안 4 ℃에서 냉장 보관하였다. 1차 항체 염색이 완료된 세포는 0.03% Triton으로 2회 세척하고, 5% NGS에 2차 항체를 1:500의 비율로 희석하여 배양접시에 첨가한 뒤 상온에서 90분 간 반응시켰다. 2차 항체 반응이 완료된 세포는 0.03% Triton으로 2회 세척한 뒤, 0.03% Triton에 DAPI 용액을 1:1000의 비율로 희석하여 배양접시에 첨가하고 상온에서 10분 간 반응시키고, 반응이 완료된 배양접시는 0.03% Triton으로 2회 세척 후 형광현미경 하에서 관찰하였다.For cTnT and ACE2 gene expression analysis through immunofluorescence staining, cardiomyocytes differentiated from undifferentiated stem cells were washed twice with DPBS, 4% Paraformaldehyde was added, and the cells were reacted at room temperature for 10 minutes to fix the cells. After washing twice with DPBS (0.03% Triton) supplemented with Triton X-100 at a concentration of 0.03%, DPBS (5% NGS) supplemented with 5% normal goat serum was added and reacted at room temperature for 30 minutes to prevent blocking. proceeded. After diluting the primary antibodies cTnT and ACE2 in a ratio of 1:100 using 5% NGS, this solution was added to a culture dish and refrigerated at 4°C overnight. After the primary antibody staining was completed, the cells were washed twice with 0.03% Triton, diluted with a secondary antibody in 5% NGS at a ratio of 1:500, added to a culture dish, and reacted at room temperature for 90 minutes. After the secondary antibody reaction was completed, the cells were washed twice with 0.03% Triton, diluted with a DAPI solution in 0.03% Triton at a ratio of 1:1000, added to a culture dish, reacted at room temperature for 10 minutes, and the reaction was completed. The dish was washed twice with 0.03% Triton and observed under a fluorescence microscope.
면역형광염색 결과, 도 2c와 같이 hESC-CM 및 hiPSC-CM 세포 모두에서 ACE2와 cTnT 단백질 발현을 확인하였다.As a result of immunofluorescence staining, ACE2 and cTnT protein expression was confirmed in both hESC-CM and hiPSC-CM cells as shown in FIG. 2c.
따라서, 상기 세포들에서 높은량으로 발현되는 cTnT 유전자가 관찰됨에 따라 hESC-CM 및 hiPSC-CM 세포는 모두 정상적인 심근세포의 특성을 나타내는 것이 확인되었으며, 또한, 높은량으로 발현되는 ACE2 유전자를 통해 hESC-CM 및 hiPSC-CM 세포는 모두 SARS-CoV-2 감염의 표적 세포가 될 수 있음이 확인되었다.Therefore, as the cTnT gene expressed in high amounts in the cells was observed, it was confirmed that both hESC-CM and hiPSC-CM cells exhibited the characteristics of normal cardiomyocytes. It was confirmed that both -CM and hiPSC-CM cells could be target cells for SARS-CoV-2 infection.
실시예 3: COVID19 감염된 Vero E6 세포에서의 COVID19 치료용 후보 약물 4종에 대한 항바이러스 효능 평가Example 3: Evaluation of antiviral efficacy against four candidate drugs for treatment of COVID-19 in Vero E6 cells infected with COVID-19
잠재적인 COVID-19 약물, 하이드록시클로로퀸 (hydroxychloroquine), 클로로퀸 (chloroquine), 렘데시비르 (remdesivir) 및 파비피라비어 (favipiravir)의 체외 항바이러스 활성 및 세포 독성 (cytopathic effects, CPE)을 비색 세포 생존력 분석 (colorimetric cell viability assay)을 통해 측정하였다.Colorimetric cell viability and in vitro antiviral activity and cytopathic effects (CPE) of potential COVID-19 drugs, hydroxychloroquine, chloroquine, remdesivir and favipiravir It was measured through a colorimetric cell viability assay.
0.1 MOI의 SARS-CoV-2 (COVID19)로 감염된 Vero E6 세포에 각각의 잠재적인 COVID-19 약물을 100 μM, 33 μM, 11 μM, 3.7 μM, 1.2 μM, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM 및 0.0051 μM의 다양한 농도로 처리하였다. 감염 72 시간 후, CellTiter 96®AQueous One Solution Cell Proliferation Assay (MTS, Promega)의 반응물을 세포와 반응시킨 후, 플레이트 리더기를 사용하여 490nm에서 흡광도를 측정해 세포 생존력을 분석하였다. 그리고, 상기의 과정을 2회 이상 독립적으로 실시하여 평균적인 세포의 생존율을 측정하였다.Each potential COVID-19 drug was administered to Vero E6 cells infected with 0.1 MOI of SARS-CoV-2 (COVID19) at 100 µM, 33 µM, 11 µM, 3.7 µM, 1.2 µM, 0.41 µM, 0.14 µM, 0.046 µM, Various concentrations of 0.015 μM and 0.0051 μM were treated. 72 hours after infection, the cells were reacted with the reaction product of CellTiter 96®AQueous One Solution Cell Proliferation Assay (MTS, Promega), and the absorbance was measured at 490 nm using a plate reader to analyze cell viability. Then, the above process was independently performed two or more times to measure the average cell viability.
분석 결과, 하이드록시클로로퀸 (CC50: 76.1 μM 및 EC50: 6.8 μM)의 항바이러스 활성은 클로로퀸 (CC50 > 100 μM 및 EC50: 6.3 μM)과 유사 하였으며, 파비피라비어 (CC50 > 100 μM)는 뚜렷한 항바이러스 활성을 나타내지 않았다. 그리고, 렘데시비르는 (CC50 > 100 μM 및 EC50 = 2 μM) 시험된 화합물 중에서 가장 두드러진 항바이러스 활성을 보였다. (도 3a 내지 3d)As a result of the assay, the antiviral activity of hydroxychloroquine (CC50: 76.1 μM and EC50: 6.8 μM) was similar to that of chloroquine (CC50 > 100 μM and EC50: 6.3 μM), and favipiravir (CC50 > 100 μM) had distinct antiviral activity. It showed no viral activity. And, remdesivir (CC50 > 100 μM and EC50 = 2 μM) showed the most prominent antiviral activity among the tested compounds. (FIGS. 3a to 3d)
실시예 4: 인간 전분화능줄기세포 유래 심근세포를 이용한 COVID19 치료용 후보 약물 4종의 세포 독성 평가Example 4: Cytotoxicity evaluation of 4 candidate drugs for treatment of COVID-19 using human pluripotent stem cell-derived cardiomyocytes
hESC-CM 및 hiPSC-CM 세포의 세포 독성을 비색 세포 생존력 분석 (colorimetric cell viability assay)을 통한 세포 생존율로 평가하였다.The cytotoxicity of hESC-CM and hiPSC-CM cells was evaluated by cell viability through a colorimetric cell viability assay.
hESC-CM 및 hiPSC-CM 세포 각각에 COVID19 치료 후보 약물 4종의 농도를 0.0051 μM, 0.015 μM, 0.046 μM, 0.14 μM, 0.41 μM, 1.2 μM, 3.7 μM, 11 μM, 33 μM, 100 μM로 점차 증가시키면서 세포에 반응시켰다. 약물 처리 24 시간 및 48 시간 후 CellTiter 96®AQueous One Solution Cell Proliferation Assay (MTS, Promega)을 사용하여 약물에 의해 유도된 세포 독성을 비색 세포 생존력 분석 (colorimetric cell viability assay)을 통해 세포 생존율을 평가하였고, 이를 도 4a 내지 4d에 용량-반응 곡선 분석 (Dose-response curve analyse)으로 나타내었다.In hESC-CM and hiPSC-CM cells, the concentrations of the four drug candidates for COVID19 treatment were gradually increased to 0.0051 µM, 0.015 µM, 0.046 µM, 0.14 µM, 0.41 µM, 1.2 µM, 3.7 µM, 11 µM, 33 µM, and 100 µM, respectively. The cells were reacted with increasing increments. After 24 and 48 hours of drug treatment, the cell viability was evaluated using the CellTiter 96®AQueous One Solution Cell Proliferation Assay (MTS, Promega) through colorimetric cell viability assay for drug-induced cytotoxicity. , which are shown by dose-response curve analysis in FIGS. 4A to 4D .
분석 결과, 하이드록시클로로퀸은 hESC-CM 세포에서 100 μM 농도에서 약물독성 반응을 보였으며, 약물 처리 24 시간 및 48 시간 후에 84.6 μM 내지 31.7 μM 범위의 추정된 CC50 값을 나타냈다. 그리고, hiPSC-CM 세포에서는 약물 처리 24시간 및 48 시간 후에 49.1 μM 내지 55.4 μM 범위로 추정된 CC50 값을 보여주었다.As a result of the analysis, hydroxychloroquine showed a pharmacotoxic response in hESC-CM cells at a concentration of 100 μM, and showed an estimated CC50 value ranging from 84.6 μM to 31.7 μM after 24 and 48 hours of drug treatment. And, hiPSC-CM cells showed an estimated CC50 value in the range of 49.1 μM to 55.4 μM after 24 and 48 hours of drug treatment.
클로로퀸은 약물 처리 24 시간 후에 hESC-CM 세포 모두에서 CC50 값이 100 μM 이상으로 측정되어 심근세포 독성을 나타내지 않았지만, 48시간 후에는 CC50 값이 84.9 μM로 증가했다. 또한, hiPSC-CM에서는 약물 처리 24시간 및 48시간 후에 심근세포 독성을 나타내지 않았다. Chloroquine did not show cardiomyocyte toxicity, with a CC50 value of 100 μM or higher in all hESC-CM cells after 24 hours of drug treatment, but the CC50 value increased to 84.9 μM after 48 hours. In addition, hiPSC-CM showed no cardiomyocyte toxicity after 24 and 48 hours of drug treatment.
이와 대조적으로, 렘데시비르 및 파비피라비어는 hESC-CM 및 hiPSC-CM 세포 모두에서 유의한 심근세포 독성을 나타냈다. 구체적으로, 렘데시비르는 hESC-CM 세포에서 각각 약물 처리 24 시간 및 48 시간 후에 CC50 값이 18.0 μM 및 11.8 μM 로 나타났으며, hiPSC-CM 세포에서는 24 시간 및 48 시간에 CC50 값이 11.8 μM 및 5.4 μM 로 유의한 심근세포 독성이 있는 것으로 확인되었다. 또한, 파비피라비어는 hESC-CM에서 약물 처리 후 24 시간 및 48 시간에 9.7 μM 내지 34.8 μM 범위의 CC50 값을 보여주었고, hiPSC-CM에서는 약물 처리 후 24 시간 및 48 시간 후에 CC50 값이 21.3 μM 및 10.7 μM으로 나타나 유의한 심근세포 독성이 있는 것으로 나타났다. (도 4a 내지 4d)In contrast, remdesivir and favipiravir showed significant cardiomyocyte toxicity in both hESC-CM and hiPSC-CM cells. Specifically, remdesivir showed CC50 values of 18.0 μM and 11.8 μM after 24 and 48 hours of drug treatment in hESC-CM cells, respectively, and in hiPSC-CM cells, CC50 values of 11.8 μM at 24 and 48 hours. and 5.4 μM, which was confirmed to have significant cardiomyocyte toxicity. In addition, favipiravir showed a CC50 value ranging from 9.7 μM to 34.8 μM at 24 and 48 hours after drug treatment in hESC-CM, and in hiPSC-CM, a CC50 value of 21.3 μM after 24 and 48 hours after drug treatment. and 10.7 μM, indicating significant cardiomyocyte toxicity. (FIGS. 4a to 4d)
본 발명은 인간 전분화능줄기세포 유래 심근세포를 이용한 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물 및 이를 이용한 심근세포 독성 평가 방법에 관한 것으로, 구체적으로 인간 배아줄기세포 및 인간 역분화줄기세포 유래의 심근세포를 이용하여 SARS-CoV-2 바이러스 대상 후보 약물의 체외 심근세포 독성을 정확하게 측정할 수 있는 방법에 관한 것이다.The present invention relates to a composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using human pluripotent stem cell-derived cardiomyocytes, and a method for evaluating cardiomyocyte toxicity using the same, specifically, to human embryonic stem cells and human reverse The present invention relates to a method for accurately measuring in vitro cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus using differentiated stem cell-derived cardiomyocytes.

Claims (7)

  1. 인간 전분화능 줄기세포 유래 심근세포를 포함하는, SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 조성물.A composition for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising human pluripotent stem cell-derived cardiomyocytes.
  2. 제1항에 있어서, 상기 인간 전분화능 줄기세포는 인간배아줄기세포 및 인간 역분화줄기세포로 구성된 그룹에서 선택되는 것인, 조성물.The composition of claim 1, wherein the human pluripotent stem cells are selected from the group consisting of human embryonic stem cells and human immunized stem cells.
  3. 인간 전분화능 줄기세포 유래 심근세포를 포함하는, SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가용 키트.A kit for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising cardiomyocytes derived from human pluripotent stem cells.
  4. 제3항에 있어서, 상기 인간 전분화능 줄기세포는 인간배아줄기세포 및 인간 역분화줄기세포로 구성된 그룹에서 선택되는 것인, 키트.The kit of claim 3, wherein the human pluripotent stem cells are selected from the group consisting of human embryonic stem cells and human immunized stem cells.
  5. 다음 단계를 포함하는 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성 평가 방법:A method for evaluating cardiomyocyte toxicity of a candidate drug for SARS-CoV-2 virus, comprising the steps of:
    인간 전분화능 줄기세포 유래 심근세포에 상기 SARS-CoV-2 바이러스 대상 후보 약물을 처리하는 처리 단계.A processing step of treating human pluripotent stem cell-derived cardiomyocytes with the SARS-CoV-2 virus target drug.
  6. 제5항에 있어서, 상기 방법은 상기 심근세포의 생존율을 측정하는 측정 단계를 더 포함하는 것인, 방법.The method according to claim 5, wherein the method further comprises a measuring step of measuring the viability of the cardiomyocytes.
  7. 제5항에 있어서, 상기 방법은 측정된 상기 생존율을 통해 상기 SARS-CoV-2 바이러스 대상 후보 약물의 심근세포 독성을 평가하는 평가 단계를 더 포함하는 것인, 방법.The method of claim 5, wherein the method further comprises an evaluation step of evaluating cardiomyocyte toxicity of the SARS-CoV-2 virus target drug candidate through the measured survival rate.
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