WO2020037882A1 - 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 - Google Patents

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 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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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

本公开提供了一种具有同时降低甘油三酯和胆固醇作用的干细胞的制备方法，其特征在于，该方法包括如下步骤：S1、将敲除载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述敲除载体的序列如SEQ ID NO.3所示；S2、将干细胞的悬液与所述待转染的AAV病毒颗粒的悬液以进行转染，得到转染后的培养物；S3、将所述转染后的培养物极限稀释后进行单克隆化培养，并筛选出敲除了PCSK9基因和APOC3基因的单克隆细胞株。本公开还提供了如上所述的制备方法制备得到的干细胞及其在制备用于治疗心血管疾病的药物中的用途。通过上述技术方案，本公开提供了一种新的防治心血管疾病的方案，有效地降低了心血管疾病的发病率。

Description

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 技术领域
本公开涉及医药生物技术领域，具体地，涉及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。
背景技术
心血管疾病根据世界卫生组织的报道是全球的头号死因。2008年有约1730万人死于心血管疾病，预计到2030年，死于心血管疾病的人数将增至2330万人。目前被证明防治心血管疾病发病的最有效的方法是降低血脂含量，尤其是降低低密度胆固醇(LDL-C)含量。他汀类药物(statins)被有效的应用在心血管疾病的防治中，尽管如此，服用他汀类药物并不能完全防治心血管疾病：病人只会降低30～40％的发病率，并且有部分病人群体对他汀类药物治疗方案没有疗效反应。因此，亟需寻找新的治疗心血管疾病方案。
发明内容
本公开的目的是克服心血管疾病防治效果较差的缺陷，进一步降低心血管疾病的发病率。
为了实现上述目的，本公开提供了一种具有同时降低甘油三酯和胆固醇作用的干细胞的制备方法，其特征在于，该方法包括如下步骤：S1、将敲除载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述敲除载体的序列如SEQ ID NO.3所示；S2、将干细胞的悬液与所述待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S3、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出敲除了PCSK9基因和APOC3基因的单克隆细胞株。
另一方面，本公开还提供了一种干细胞，该干细胞为如上所述的制备方法制备得到的干细胞。
另一方面，本公开还提供了一种干细胞，该干细胞中的PCSK9基因和APOC3基因被定向敲除。
再一方面，本公开还提供了一种干细胞在制备用于治疗心血管疾病的药物中的用途，所述干细胞为如上所述的制备方法制备得到的干细胞或如上所述的干细胞。
通过上述技术方案，本发明在干细胞中将PCSK9基因和APOC3基因进行了双敲除，双敲除之后得到的干细胞通过注射的方式进入受试者后能够定植在肝脏中，并且有效地降低血液中甘油三酯和胆固醇水平，由此可以产生对心血管疾病的防治效果，从而提供了一种新的防治心血管疾病的方案，有效地降低了心血管疾病的发病率。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
本公开提供了一种具有同时降低甘油三酯和胆固醇作用的干细胞的制备方法，其特征在于，该方法包括如下步骤：S1、将敲除载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述敲除载体的序列如SEQ ID NO.3所示；S2、将干细胞的悬液与所述待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S3、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出敲除了PCSK9基因和APOC3基因的单克隆细胞株。
本发明通过选择AAV病毒来将敲除载体转染入细胞中，最终使得 sgRNA、Cas9蛋白和敲除载体共同通过CRISPR基因编辑来使得PCSK9基因和APOC3基因得以共敲除。
其中，PCSK9基因是指NCBI GENE ID为255738的基因；APOC3基因是指NCBI GENE ID为345的基因。
其中，优选地，步骤S2中，将干细胞的悬液与所述待转染的AAV病毒颗粒的悬液以进行8-24小时的转染，得到转染后的培养物。在该优选情况下，能够进一步增加在干细胞中进行基因敲除的效率。
其中，优选
地，步骤S2中，待转染的AAV病毒颗粒的悬液的加入量使得待转染的AAV病毒颗粒的MOI值为10 4-10 6。在该优选情况下，能够进一步增加在干细胞中进行基因敲除的效率。MOI值是感染时病毒与细胞数量的比值。
其中，所述干细胞可以为间充质干细胞或诱导多能干细胞。
优选地，所述间充质干细胞为骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、宫血间充质干细胞及牙髓间充质干细胞。
所述AAV病毒的血清型可以为AAV-1病毒、AAV-2病毒、AAV-3病毒、AAV-4病毒、AAV-5病毒、AAV-6病毒、AAV-7病毒、AAV-8病毒、AAV-9病毒、AAV-DJ病毒及AAV-DJ/8病毒，优选为AAV-8病毒、AAV-6病毒、AAV-1病毒或AAV9病毒，更优选所述AAV病毒的血清型为AAV9病毒，在该优选情况下，能够进一步增加在干细胞中进行基因敲除的效率。
另一方面，本公开还提供了一种干细胞，该干细胞为如上所述的制备方法制备得到的干细胞。
另一方面，本公开还提供了一种干细胞，该干细胞中的PCSK9基因和APOC3基因被定向敲除。
其中，在PCSK9基因和APOC3基因二者都被定向敲除的情况下，干细胞能够获得同时降低甘油三酯和胆固醇的功能。
再一方面，本公开还提供了一种干细胞在制备用于治疗心血管疾病的药物中的用途，所述干细胞为如上所述的制备方法制备得到的干细胞或如上所述的干细胞。
其中，所述心血管疾病可以包括高脂血症、脑卒中、心肌梗死、冠心病、心绞痛和血栓中的至少一种。
以下通过实施例进一步详细说明本发明：
实施例1
试剂来源：常规引物由生工生物工程(上海)股份有限公司合成；DMEM/F12培养基、DMEM培养基、Opti-MEM、Hyclone胎牛血清、胰酶购于赛默飞世尔科技(中国)有限公司；SpeI和HindIII限制性内切酶购于NEB(北京)有限公司；PrimerStar高保真DNA聚合酶购于宝生物工程(大连)有限公司；Benzonase、碘克沙醇购于默克化工技术(上海)有限公司；2×SYBR PCR mix购于东洋纺(上海)生物科技有限公司；Matrigel购于BD Biosciences(货号354277)。
1、sgRNA设计以及合成
(1)通过在线sgRNA设计工具(http://crispr.mit.edu/)在APOC3基因ENST00000375345.3转录本3号外显子上设计靶向识别的APOC3sgRNA，优选结果如下：APOC3sgRNA:5’-cagtgcatccttggcggtcttgg-3’(SEQ ID NO.1)。同时，针对PCSK9基因ENST00000302118.5转录本1号外显子上设计靶向识别的PCSK9sgRNA。优选结果如下：PCSK9sgRNA:5’-ggtgctagccttgcgttccgagg-3’(SEQ ID NO.2)。其中1-20位的序列为识别基序，其余的序列为tracrRNA。
(2)该sgRNA由苏州吉玛基因股份有限公司合成并在该sgRNA序列的5’端和3’端末尾分的三个碱基的二号位和三号位上别添加O-Me、 phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)分别针对APOC3和PCSK9基因，从基因组扩增识别靶序列片段(APOC3:622bp；PCSK9:1027bp)，引物由生工生物工程(上海)股份有限公司合成。
APOC3-Test-FW:5’-tactccttctggcagacccagc-3’，(SEQ ID NO.4)；
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)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于上海近岸科技有限公司)，10×缓冲液2μL配置成20μL反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装***选择及质粒构建
(1)待编辑细胞为脐带间充质干细胞细胞株，根据AAV组织亲和性对照表，优选血清型为AAV-9的包装***。
(2)AAV-9的总包装容量为4.7Kb。pAAV-Cassette载体序列为：
Figure PCTCN2018119844-appb-000001
Figure PCTCN2018119844-appb-000002
Figure PCTCN2018119844-appb-000003
Figure PCTCN2018119844-appb-000004
Figure PCTCN2018119844-appb-000005
该载体包含了肝脏特异性启动子LP1驱动的saCas9表达元件，并且***有***片段，所述***片段为两个串联表达的人源U6启动子-APOC3 sgRNA-gRNA Scaffold-人源U6启动子-PCSK9 sgRNA-gRNA Scaffold的完整的Cassette,共778bp。序列为：
Figure PCTCN2018119844-appb-000006
使用SpeI和HindIII限制性内切酶(购于NEB(北京)有限公司)将SEQ ID NO.8的Cassette***到pAAV载体上。
4、AAV病毒包装及纯化
(1)在病毒包装前一天将HEK293T细胞按照每皿5×10 6的数量种植到直径10cm的含有10mL完全培养基(DMEM+10％FBS+1％P/S双抗)的CORNING培养皿中，共种植30皿。在37度、5％CO 2的细胞培养箱中培养 24小时。
(2)病毒包装当天检查HEK293T细胞汇合度是否达到80％。每皿按照以下步骤单独配置转染体系：将10μg的pAAV-Cassette，10μg的pHelper，10μg的pAAV9-RC混合后使用无血清的DMEM调整体积到910μL后经漩涡振荡器混匀，加入90μL的PEI(购于Polysciences Asia Pacific,Inc.)后再次使用漩涡振荡器混匀，静置15分钟。将CORNING培养皿中的完全培养基替换成9mL无血清培养基，再将之前经过静置的DNA-PEI复合物均匀滴加到培养皿中，轻柔晃匀后置于37度、5％CO 2的细胞培养箱中培养6小时。转染6h后将CORNING培养皿中的无血清培养基替换成完全培养基，于37度、5％CO 2的细胞培养箱中继续培养。
(3)在转染60小时之后，分别收获上清和细胞。
将收集得到的上清经4000rpm 4度离心10分钟后弃去杂质。将去除杂质的上清加入Amicon Ultra-15超离柱中(购于默克化工技术(上海)有限公司)，经过若干次4000rpm、4度离心30分钟将体积浓缩至10到15mL。将用细胞刮刀刮下的HEK293T细胞用适量培养基吹匀并转移至50mL离心管中，经1500rpm、4度离心10min后弃上清，所有沉淀总共加3mL细胞裂解缓冲液(150mM NaCl,20mM tris pH8.0)使其重悬。将重悬细胞在-80℃酒精浴和37℃水浴中反复冻融三次。将浓缩的上清和冻融的细胞悬液混匀，添加1M MgCl 2至终浓度为1mM。添加Benzonase(购于默克化工技术(上海)有限公司)至终浓度为25U/mL，混匀后37℃反应40min。取出50mL离心管，4℃，4000rpm离心20min，取上清。
(4)采用碘克沙醇密度梯度离心法纯化病毒(购于默克化工技术(上海)有限公司)。配置碘克沙醇梯度17％：5mL 10×PBS,0.05mL 1M MgCl 2,0.125mL 1M KCl,10mL 5M NaCl,12.5mL Optiprep，加水补足到50mL。25％：5mL 10×PBS,0.05mL 1M MgCl 2,0.125mL 1M KCl,20mL Optiprep,0.1mL  0.5％phenol red，加水补足到50mL。40％：5mL 10×PBS,0.05mL 1M MgCl 2,0.125mL 1M KCl,33.3mL Optiprep，加水补足到50mL。60％：0.05mL 1M MgCl 2,0.125mL 1M KCl,50mL Optiprep,0.025mL 0.5％phenol red。向超速离心管中由下至上依次缓慢加入3.5mL 60％、3.5mL 40％、4mL 25％、4mL 17％的碘克沙醇。将浓缩的上清和细胞裂解液缓慢加在离心管最上层，用细胞裂解缓冲液补满离心管。使用贝克曼L-80XP落地超速离心机、70Ti定角转子，加速6，减速9，60000rpm 4度离心2小时。用平头注射器吸取40％浓度层碘克沙醇，转移至Amicon Ultra-15超离柱中，加入10mL PBS 4000rpm 4℃离心20分钟，重复3次。将病毒离心浓缩至1mL。
(5)利用qPCR对AAV9进行滴度检测。根据saCas9序列设计引物，使qPCR产物的长度为191bp。qPCR引物由生工生物工程(上海)股份有限公司合成。
AAV-saCas9-F:5’-cagattcgatgtctatctgg-3’(SEQ ID NO.9)
AAV-saCas9-R:5’-cattgatcttaatcaggtcg-3’(SEQ ID NO.10)。
制备7个AAV9敲除载体标准品，以1：10的比例进行梯度稀释。从10ng/μL以下的浓度开始稀释，分别为10ng/μL、1ng/μL、0.1ng/μL、0.01ng/μL、0.001ng/μL、0.0001ng/μL和0.00001ng/μL。
根据以下公式计算稀释的DNA拷贝数：
Figure PCTCN2018119844-appb-000007
使用DNase对病毒样本进行预处理(购于宝生物工程(大连)有限公司)。使用2×SYBR PCR mix(购于东洋纺(上海)生物科技有限公司)配置qPCR反应体系。使用Roche
Figure PCTCN2018119844-appb-000008
480II实时荧光定量PCR***进行定量PCR。根据Ct值，绘制标准曲线，并计算AAV9的滴度。
5、待编辑细胞的预处理
(1)观察人脐带间充质干细胞细胞株汇合率达到80％后移除含10％ FBS的DMEM/F12培养基(购于ThermoFisher Scientific,Inc.)，用PBS漂洗一次。加入0.05％胰酶(购于ThermoFisher Scientific,Inc.)使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去胰酶。即刻加入新鲜的DMEM/F12完全培养基，用1mL枪扇形吹打培养皿底，使皿/瓶底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，制成干细胞悬液。使用血球计数板对细胞密度进行计数。使用Opti-MEM(14.5mM的ATP、23.6mM的氯化镁)调整细胞密度到5×10 7/mL。
(2)将20μL经过计数并使用Opti-MEM重悬的人脐带间充质干细胞悬液(其中细胞数量为5×10 5个)转移到添加了500ul含10％胎牛血清的DMEM/F12培养基的24孔板中于37度、5％CO 2的细胞培养箱中继续培养。
(4)电转完第5到20分钟内开始按照1×10 5的MOI值轻柔得滴加AAV9，并于电转完20分钟内滴加完毕。
(5)电转完24小时后将旧培养基移除，更换为DMEM/F12完全培养基。
6、单克隆细胞株的培养：
(1)电转完第48小时使用PBS漂洗一次培养皿。加入胰酶使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去胰酶。即刻加入新鲜的DMEM/F12完全培养基，用1mL枪扇形吹打培养皿/瓶底，使皿底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，保证细胞间没有粘连，制成干细胞悬液。使用血球计数板对细胞密度进行计数。按每个10cm培养皿15000个细胞的比例把细胞种植到含DMEM/F12完全培养基的10cm CORNING培养皿中于37度、5％CO 2的细胞培养箱中继续培养。
(2)每24小时进行观察并移除旧的培养基，更换为新的DMEM/F12完全培养基。72小时后可以在镜下观察到克隆的形成。
(3)电转后12到14天在十倍的物镜下可以看到克隆的大小已经相当 于一个硬币。不能让克隆继续变大或者相交。在96孔板上的每个孔中加入120μL DMEM/F12完全培养基，标记板O。在超净台中通过显微镜进行观察，将P200移液器调节至45μL使用带有滤芯的枪头刮碎克隆，用移液器收集细胞并转移到96孔板的小孔中。
(4)克隆挑取之后每24小时后将旧的培养基移除，更换为新的DMEM/F12完全培养基，四天后细胞汇合率达到80％。
(5)在两块96孔板上的每个孔中加入133μL含10％胎牛血清的DMEM/F12完全培养基，分别标记为板A、板B。将板O的每孔使用150μL的PBS漂洗。每孔中加入35μL的胰酶，消化5到10分钟后每孔中添加165μL的DMEM/F12完全培养基。从孔中分别移取66μL的细胞悬液转移到板A和板B的对应小孔中。板A用于基因组提取，板B备用。在板O的每个孔中直接添加165μL的细胞冻存液后至于深低温冰箱存储。
7、APOC3和PCSK9基因双敲纯合克隆检测
(1)选取96个克隆以及未编辑人脐带间充质干细胞细胞株作为对照，将A板或B板96孔板中的克隆所获得的基因组用SEQ ID NO.4-7所述的两对引物进行PCR扩增。
(2)将PCR产物进行电泳并测序分析。测序结果显示APOC3和PCSK9基因同时发生移码突变且两个突变位点纯合的克隆为成功进行了双基因敲除编辑的克隆。
对比例1
按照实施例1的方法进行，不同的是pAAV-Cassette载体的Cassette中不包括人源U6启动子-APOC3sgRNA-gRNA Scaffold的片段，即只敲除PCSK9基因，不敲除APOC3基因。
对比例2
按照实施例1的方法进行，不同的是pAAV-Cassette载体的Cassette中不包括人源U6启动子-PCSK9 sgRNA-gRNA Scaffold的片段，即只敲除APOC3基因，不敲除PCSK9基因。
测试实施例1
测试实施例1和对比例1和2得到的脐带间充质干细胞对肝脏人源小鼠血液总胆固醇水平和甘油三酯的水平的影响(相对于未进行敲除的脐带间充质干细胞)
通过肝脏细胞移植构建人源小鼠，并用实施例1和对比例1和2得到的脐带间充质干细胞和未进行敲除的脐带间充质干细胞对肝脏人源化小鼠分别进行尾静脉注射(注射剂量为1×10 6cell/0.1mL)，查看对血液中总胆固醇水平和甘油三酯水平的影响，结果如表1所示，其表明实施例得到的脐带间充质干细胞相对于未进行敲除的脐带间充质干细胞能够同时显著降低肝脏人源化小鼠血液中的总胆固醇水平(下降了43.5％)和甘油三酯水平(下降了37.3％)，而单独敲除APOC3基因或PCSK9基因均不能达到同时降低的效果。
表1：
Figure PCTCN2018119844-appb-000009
以上详细描述了本公开的优选实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (10)

  1. 一种具有同时降低甘油三酯和胆固醇作用的干细胞的制备方法，其特征在于，该方法包括如下步骤：
    S1、将敲除载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述敲除载体的序列如SEQ ID NO.3所示；
    S2、将干细胞的悬液与所述待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；
    S3、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出敲除了PCSK9基因和APOC3基因的单克隆细胞株。
  2. 根据权利要求1所述的制备方法，其中，步骤S2中，将干细胞的悬液与所述待转染的AAV病毒颗粒的悬液以进行8-24小时的转染，得到转染后的培养物。
  3. 根据权利要求1或2所述的制备方法，其中，步骤S4中，待转染的AAV病毒颗粒的悬液的加入量使得待转染的AAV病毒颗粒的MOI值为10 4-10 6。
  4. 根据权利要求1所述的制备方法，其中，所述干细胞为间充质干细胞或诱导多能干细胞。
  5. 根据权利要求4所述的制备方法，其中，所述间充质干细胞为骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、宫血间充质干细胞及牙髓间充质干细胞。
  6. 根据权利要求1或5所述的制备方法，其中，所述AAV病毒的血清 型为AAV-8病毒、AAV-6病毒、AAV-1病毒或AAV9病毒。
  7. 一种干细胞，其特征在于，该干细胞为权利要求1-6中任意一项所述的制备方法制备得到的干细胞。
  8. 一种干细胞，其特征在于，该干细胞中的PCSK9基因和APOC3基因被定向敲除。
  9. 一种干细胞在制备用于治疗心血管疾病的药物中的用途，其特征在于，所述干细胞为权利要求7或8所述的干细胞。
  10. 根据权利要求9所述的用途，其中，所述心血管疾病包括高脂血症、脑卒中、心肌梗死、冠心病、心绞痛和血栓中的至少一种。
PCT/CN2018/119844 2018-08-21 2018-12-07 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 WO2020037882A1 (zh)

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