WO2020037882A1 - Cellule souche capable de réduire simultanément l'effet du triglycéride et du cholestérol ainsi que son procédé de préparation et son utilisation - Google Patents

Cellule souche capable de réduire simultanément l'effet du triglycéride et du cholestérol ainsi que son procédé de préparation et son utilisation Download PDF

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WO2020037882A1
WO2020037882A1 PCT/CN2018/119844 CN2018119844W WO2020037882A1 WO 2020037882 A1 WO2020037882 A1 WO 2020037882A1 CN 2018119844 W CN2018119844 W CN 2018119844W WO 2020037882 A1 WO2020037882 A1 WO 2020037882A1
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stem cells
transfected
aav
virus
stem cell
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李程
丁秋蓉
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杭州观梓健康科技有限公司
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N2750/14011—Parvoviridae
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Definitions

  • the present disclosure relates to the field of medical biotechnology, and in particular, to a stem cell having simultaneous effects of reducing triglyceride and cholesterol, and a preparation method and use thereof.
  • Cardiovascular disease is the number one cause of death worldwide, according to the World Health Organization. Approximately 17.3 million people died of cardiovascular disease in 2008, and it is expected that the number of deaths from cardiovascular disease will increase to 23.3 million by 2030. At present, it has been proved that the most effective method for preventing and treating cardiovascular diseases is to reduce blood lipid content, especially to reduce low density cholesterol (LDL-C) content.
  • LDL-C low density cholesterol
  • Statins are effectively used in the prevention and treatment of cardiovascular disease. However, taking statins cannot completely prevent cardiovascular disease: patients will only reduce the incidence of 30-40%, and some patient groups There is no response to statin therapy. Therefore, there is an urgent need to find new treatments for cardiovascular disease.
  • the purpose of the present disclosure is to overcome the disadvantage of poor prevention and treatment of cardiovascular diseases and further reduce the incidence of cardiovascular diseases.
  • the present disclosure provides a method for preparing stem cells having a function of simultaneously reducing triglyceride and cholesterol, which is characterized in that the method includes the following steps: S1, packaging a knockout vector into AAV virus, and forming a AAV virus particles were transfected; the sequence of the knockout vector is shown in SEQ ID NO. 3; S2, the suspension of stem cells and the suspension of AAV virus particles to be transfected for 4-30 hours S3, the transfected culture is obtained by limiting dilution. The transfected culture is subjected to limiting dilution and then subjected to monoclonal culture, and the PCSK9 gene and the APOC3 gene knockout monoclonal cell strain are selected by PCR and sequencing.
  • the present disclosure also provides a stem cell, which is a stem cell prepared by the preparation method described above.
  • the present disclosure also provides a stem cell in which the PCSK9 gene and the APOC3 gene are targeted for knockout.
  • the present disclosure also provides the use of stem cells in the manufacture of a medicament for treating cardiovascular disease, the stem cells being stem cells prepared by the preparation method described above or stem cells described above.
  • the present invention double-knocks out the PCSK9 gene and the APOC3 gene in stem cells.
  • the stem cells obtained after the double-knockout can be colonized in the liver after being injected into the subject, and effectively reduce the blood
  • the levels of triglyceride and cholesterol can thus prevent and cure cardiovascular diseases, thus providing a new program for preventing and curing cardiovascular diseases, and effectively reducing the incidence of cardiovascular diseases.
  • the present disclosure provides a method for preparing stem cells with the function of simultaneously reducing triglyceride and cholesterol, which is characterized in that the method includes the following steps: S1, packaging a knockout vector into AAV virus to form AAV virus particles to be transfected
  • the sequence of the knockout vector is shown in SEQ ID No. 3; S2, the suspension of the stem cells and the suspension of the AAV virus particles to be transfected for 4-30 hours to obtain transfection S3.
  • the transfected culture is subjected to limiting dilution, and then subjected to monoclonal culture, and the PCSK9 gene and the APOC3 gene knockout monoclonal cell strain are selected by PCR and sequencing.
  • the knockout vector is transfected into the cell by selecting AAV virus, and finally the sgRNA, Cas9 protein and the knockout vector are jointly edited by CRISPR to make the PCSK9 gene and APOC3 gene co-knock out.
  • the PCSK9 gene refers to the NCBI gene with ID 255738;
  • the APOC3 gene refers to the NCBI gene with ID 345.
  • step S2 the suspension of the stem cells and the suspension of the AAV virus particles to be transfected are subjected to transfection for 8-24 hours to obtain a transfected culture.
  • the efficiency of gene knockout in stem cells can be further increased.
  • the amount of the suspension of the AAV virus particles to be transfected is such that the MOI value of the AAV virus particles to be transfected is 10 4 -10 6 .
  • the efficiency of gene knockout in stem cells can be further increased.
  • the MOI value is the ratio of the virus to the number of cells at the time of infection.
  • the stem cells may be mesenchymal stem cells or induced pluripotent stem cells.
  • the mesenchymal stem cells are bone marrow mesenchymal stem cells, fat mesenchymal stem cells, umbilical cord mesenchymal stem cells, uterine blood mesenchymal stem cells and dental pulp mesenchymal stem cells.
  • the serotype of the AAV virus may be AAV-1 virus, AAV-2 virus, AAV-3 virus, AAV-4 virus, AAV-5 virus, AAV-6 virus, AAV-7 virus, AAV-8 virus, AAV -9 virus, AAV-DJ virus and AAV-DJ / 8 virus, preferably AAV-8 virus, AAV-6 virus, AAV-1 virus or AAV9 virus, more preferably the serotype of AAV virus is AAV9 virus, In this preferred case, the efficiency of gene knockout in stem cells can be further increased.
  • the present disclosure also provides a stem cell, which is a stem cell prepared by the preparation method described above.
  • the present disclosure also provides a stem cell in which the PCSK9 gene and the APOC3 gene are targeted for knockout.
  • stem cells can acquire the function of reducing triglyceride and cholesterol at the same time.
  • the present disclosure also provides the use of stem cells in the manufacture of a medicament for treating cardiovascular disease, the stem cells being stem cells prepared by the preparation method described above or stem cells described above.
  • the cardiovascular disease may include at least one of hyperlipidemia, stroke, myocardial infarction, coronary heart disease, angina pectoris, and thrombosis.
  • the sgRNA was synthesized by Suzhou Gima Gene Co., Ltd. and added with O-Me and phosphorothioate on the second and third positions of the three bases at the 5 'end and the 3' end of the sgRNA sequence. Modification.
  • the target sequence fragments (APOC3: 622bp; PCSK9: 1027bp) were amplified and identified from the genome, and the primers were synthesized by Biotech Biotechnology (Shanghai) Co., Ltd.
  • APOC3-Test-REV 5’-ctgtcatctctcccgcagcag-3 ’, (SEQ ID NO. 5);
  • PCSK9-Test-FW 5’-gtctgagcctggaggagtgag-3 ’, (SEQ ID NO. 6);
  • PCSK9-Test-REV 5'-ctagagcatgagttctgtgtc-3 ', (SEQ ID NO. 7).
  • the cell to be edited is a umbilical cord mesenchymal stem cell cell line.
  • a packaging system with a serotype of AAV-9 is preferred.
  • the total packaging capacity of AAV-9 is 4.7Kb.
  • the pAAV-Cassette vector sequence is:
  • the vector contains a liver-specific promoter LP1 driven saCas9 expression element, and an insert is inserted, the insert is two human U6 promoters-APOC3 sgRNA-gRNA scaffold-human U6 promoter- The complete Cassette of PCSK9 sgRNA-gRNA Scaffold, a total of 778bp.
  • the sequence is:
  • Cassette of SEQ ID NO. 8 was inserted into the pAAV vector using SpeI and HindIII restriction enzymes (purchased from NEB (Beijing) Co., Ltd.).
  • HEK293T cells were seeded in a number of 5 ⁇ 10 6 per dish into a CORNING culture dish containing 10 mL of complete medium (DMEM + 10% FBS + 1% P / S double antibody) with a diameter of 10 cm. A total of 30 dishes were planted. The cells were cultured in a 37 ° C, 5% CO 2 cell incubator for 24 hours.
  • complete medium DMEM + 10% FBS + 1% P / S double antibody
  • the collected supernatant was centrifuged at 4000 rpm at 4 degrees for 10 minutes, and the impurities were discarded.
  • the supernatant from which impurities were removed was added to an Amicon Ultra-15 column (purchased from Merck Chemical Technology (Shanghai) Co., Ltd.), and the volume was concentrated to 10 to 15 mL after several centrifugations at 4000 rpm and 4 degrees for 30 minutes.
  • HEK293T cells scraped with a cell spatula were blown with an appropriate amount of medium and transferred to a 50 mL centrifuge tube. After centrifugation at 1500 rpm and 4 degrees for 10 min, the supernatant was discarded.
  • a total of 3 mL of cell lysis buffer (150 mM NaCl, 20 mM tris was added to all the pellets (pH 8.0).
  • the resuspended cells were repeatedly freeze-thawed three times in a -80 ° C alcohol bath and a 37 ° C water bath.
  • the concentrated supernatant and the freeze-thawed cell suspension were mixed and 1 M MgCl 2 was added to a final concentration of 1 mM.
  • Add Benzonase (purchased from Merck Chemicals (Shanghai) Co., Ltd.) to a final concentration of 25 U / mL, and mix for 40 minutes at 37 ° C after mixing. Take out the 50mL centrifuge tube, centrifuge at 4 °C, 4000rpm for 20min, and take the supernatant.
  • the virus was purified by iodixanol density gradient centrifugation (purchased from Merck Chemical Technology (Shanghai) Co., Ltd.). Configure iodixanol gradient 17%: 5mL 10 ⁇ PBS, 0.05mL 1M MgCl 2 , 0.125mL 1M KCl, 10mL 5M NaCl, 12.5mL Optiprep, and make up to 50mL with water. 25%: 5mL 10 ⁇ PBS, 0.05mL 1M MgCl 2 , 0.125mL 1M KCl, 20mL Optiprep, 0.1mL 0.5% phenol red, make up to 50mL with water.
  • Beckman L-80XP floor ultracentrifuge 70Ti fixed-angle rotor, acceleration 6, deceleration 9,600,000 rpm, 4 degrees centrifugation for 2 hours. Pipet a 40% concentration layer of iodixanol with a flat-tip syringe, transfer to an Amicon Ultra-15 column, add 10 mL of PBS at 4000 rpm and centrifuge at 4 ° C for 20 minutes, and repeat 3 times. The virus was concentrated by centrifugation to 1 mL.
  • AAV9 was tested for titer by qPCR.
  • the primers were designed according to the saCas9 sequence so that the length of the qPCR product was 191bp.
  • qPCR primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AAV-saCas9-F 5’-cagattcgatgtctatctgg-3 ’(SEQ ID NO.9)
  • AAV-saCas9-R 5'-cattgatcttaatcaggtcg-3 '(SEQ ID NO. 10).
  • AAV9 knockout vector standards were prepared and diluted 1:10 in a gradient. Dilution starts from concentrations below 10ng / ⁇ L, which are 10ng / ⁇ L, 1ng / ⁇ L, 0.1ng / ⁇ L, 0.01ng / ⁇ L, 0.001ng / ⁇ L, 0.0001ng / ⁇ L, and 0.00001ng / ⁇ L, respectively.
  • Virus samples were pretreated using DNase (purchased from Bao Bioengineering (Dalian) Co., Ltd.).
  • a 2 ⁇ SYBR PCR mix purchased from Toyobo (Shanghai) Biotechnology Co., Ltd. was used to configure the qPCR reaction system.
  • Roche 480II real-time PCR system was used for quantitative PCR. Based on the Ct value, a standard curve was drawn and the titer of AAV9 was calculated.
  • the size of the clone is equivalent to a coin under a ten-fold objective lens 12 to 14 days after the electric transfer. Do not allow the clones to continue to grow or intersect.
  • Add 120 ⁇ L of DMEM / F12 complete medium to each well of a 96-well plate and label plate O. Observe through a microscope in a clean bench, adjust the P200 pipette to 45 ⁇ L, scrape the clones with a pipette tip with a filter element, collect the cells with a pipette, and transfer to the wells of a 96-well plate.
  • Example 1 The method according to Example 1 was performed, except that the Casette of the pAAV-Cassette vector did not include the human-derived U6 promoter-APOC3sgRNA-gRNA Scaffold fragment, that is, only the PCSK9 gene was knocked out, and the APOC3 gene was not knocked out.
  • Example 1 The method according to Example 1 was performed, except that the Cassette of the pAAV-Cassette vector did not include the human U6 promoter-PCSK9 sgRNA-gRNA Scaffold fragment, that is, only the APOC3 gene was knocked out, and the PCSK9 gene was not knocked out.
  • the Cassette of the pAAV-Cassette vector did not include the human U6 promoter-PCSK9 sgRNA-gRNA Scaffold fragment, that is, only the APOC3 gene was knocked out, and the PCSK9 gene was not knocked out.
  • Example 1 The effects of umbilical cord mesenchymal stem cells obtained in Example 1 and Comparative Examples 1 and 2 on the total blood cholesterol level and triglyceride level in human liver mice (relative to umbilical cord mesenchymal stem cells that were not knocked out)
  • Human-derived mice were constructed by liver cell transplantation, and umbilical cord mesenchymal stem cells obtained from Example 1 and Comparative Examples 1 and 2 and umbilical cord mesenchymal stem cells without knockout were used to inject tail veins into liver-humanized mice, respectively. (Injection dose is 1 ⁇ 10 6 cells / 0.1mL). The effects on the total cholesterol level and triglyceride level in the blood were examined.
  • the results are shown in Table 1, which shows that the umbilical cord mesenchymal stem cells obtained in the example are relatively
  • the knocked-out umbilical cord mesenchymal stem cells can significantly reduce total cholesterol levels (a 43.5% decrease) and triglyceride levels (a 37.3% decrease) in the blood of liver-humanized mice, while knocking out the APOC3 gene alone or None of the PCSK9 genes could achieve the same effect.

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Abstract

L'invention concerne un procédé de préparation d'une cellule souche capable de réduire simultanément les effets du triglycéride et du cholestérol. Le procédé est caractérisé en ce qu'il comprend les étapes suivantes : S1. L'encapsidation d'un vecteur knock-out en un virus AAV, de façon à former une particule de virus AAV à transfecter, la séquence du vecteur knock-out étant représentée par la SEQ ID NO.3 ; S2. La réalisation d'une transfection sur un liquide de suspension de la cellule souche et un liquide de suspension de la particule de virus AAV à transfecter, de façon à obtenir une culture transfectée ; S3. La réalisation d'une culture monoclonale sur la culture transfectée après un essai de dilution limite, et le criblage d'une souche de cellule monoclonale dans laquelle un gène PCSK9 et un gène APOC3 sont invalidés génétiquement. L'invention concerne en outre la cellule souche préparée par le procédé de préparation et son utilisation dans la préparation de médicaments pour le traitement de maladies cardiovasculaires. Selon la solution technique, l'invention concerne un nouveau schéma de prévention et de traitement des maladies cardiovasculaires, qui diminue efficacement la morbidité de maladies cardiovasculaires.
PCT/CN2018/119844 2018-08-21 2018-12-07 Cellule souche capable de réduire simultanément l'effet du triglycéride et du cholestérol ainsi que son procédé de préparation et son utilisation WO2020037882A1 (fr)

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CN201810955908.6A CN109182379A (zh) 2018-08-21 2018-08-21 一种具有同时降低甘油三酯和胆固醇作用的干细胞及其制备方法和用途

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CN113730439A (zh) * 2021-09-09 2021-12-03 陕西中鸿瑞康健康管理有限公司 能降低甘油三酯的干细胞因子冻干粉及其制备方法和应用
CN118256560B (zh) * 2024-05-31 2024-09-17 中国农业科学院北京畜牧兽医研究所 利用crispr-cas9系统敲除猪胎儿成纤维细胞系中apoc3基因的方法

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