WO2023102735A1 - 一种特异性靶向星形胶质细胞的重组腺相关病毒及其应用 - Google Patents

一种特异性靶向星形胶质细胞的重组腺相关病毒及其应用 Download PDF

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WO2023102735A1
WO2023102735A1 PCT/CN2021/136105 CN2021136105W WO2023102735A1 WO 2023102735 A1 WO2023102735 A1 WO 2023102735A1 CN 2021136105 W CN2021136105 W CN 2021136105W WO 2023102735 A1 WO2023102735 A1 WO 2023102735A1
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associated virus
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林坤章
韩增鹏
骆能松
徐富强
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Shenzhen Institute of Advanced Technology of CAS
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  • the invention belongs to the field of biotechnology, and in particular relates to a recombinant adeno-associated virus specifically targeting astrocytes and an application thereof.
  • the brain network is composed of various nerve cells.
  • Nerve cells mainly include neurons and glial cells (neuroglia), which play different roles in the brain.
  • the number of glial cells is dozens of times more than that of neurons, and can generally be divided into microglia, astrocytes, and oligodendrocytes.
  • glial cells mainly include neurons and glial cells (neuroglia), which play different roles in the brain.
  • the number of glial cells is dozens of times more than that of neurons, and can generally be divided into microglia, astrocytes, and oligodendrocytes.
  • astrocytes are related to various brain diseases (Nature.2014; 509(7499):189-194.). Therefore, it is necessary to develop tools that specifically target astrocytes to analyze the structure and function of astrocytes and provide theoretical basis and technical support for the treatment of brain diseases.
  • transgenic animals such as mice, rats, dogs, pigs, and monkeys
  • transgenic animals have long reproductive cycles, high feeding costs, and cannot flexibly carry target genes for manipulation of nerve cells and treatment of diseases. Therefore, it is of great significance to continue to develop methods that can specifically target astrocytes.
  • the object of the present invention is to provide a recombinant adeno-associated virus specifically targeting astrocytes and its application.
  • the first aspect of the present invention provides a recombinant adeno-associated virus core plasmid, which comprises inverted terminal repeat sequence ITR, GfaABC1D promoter, foreign gene, transcriptional regulatory element WPRE and transcriptional termination sequence SV40polyA.
  • the exogenous gene includes a reporter gene
  • the reporter gene is EGFP.
  • nucleotide sequence of the recombinant adeno-associated virus core plasmid is shown in SEQ ID NO: 3.
  • the second aspect of the present invention provides a recombinant adeno-associated virus vector system, comprising the adeno-associated virus core plasmid, the recombinant adeno-associated virus packaging plasmid and the adeno-associated virus element helper plasmid.
  • the recombinant adeno-associated virus packaging plasmid includes the Rep gene of type 2 adeno-associated virus and the Cap gene of type 11 adeno-associated virus;
  • the recombinant adeno-associated virus packaging plasmid uses pAAV-RC2/1 as the backbone;
  • the adenovirus element helper plasmid is pAd-Helper.
  • nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is shown in SEQ ID NO:4.
  • the third aspect of the present invention provides a recombinant adeno-associated virus, which is obtained by co-transfecting a packaging cell line with the adeno-associated virus core plasmid, recombinant adeno-associated virus packaging plasmid and adenovirus element helper plasmid.
  • the packaging cell line is selected from HEK-293 cells, HEK-293T cells or HEK-293FT cells;
  • the molecular ratio of the adeno-associated virus core plasmid, recombinant adeno-associated virus packaging plasmid and adenovirus element helper plasmid is 1:1:1.
  • the recombinant adeno-associated virus packaging plasmid includes the Rep gene of type 2 adeno-associated virus and the Cap gene of type 11 adeno-associated virus;
  • the recombinant adeno-associated virus packaging plasmid uses pAAV-RC2/1 as the backbone;
  • nucleotide sequence of the recombinant adeno-associated virus packaging plasmid is shown in SEQ ID NO: 4;
  • the adenovirus element helper plasmid is pAd-Helper.
  • the fourth aspect of the present invention provides the recombinant adeno-associated virus core plasmid, the recombinant adeno-associated virus vector system or the application of the recombinant adeno-associated virus in specifically targeting astrocytes.
  • the invention uses pAAV2/11 as a gene transfer carrier for the first time to realize the specific targeting of astrocytes, which provides better tools and technical support for neuroscience research, disease model establishment and gene therapy, etc., and has wide application value and market prospects.
  • Figure 1 is a map of the recombinant adeno-associated virus core plasmid expression vector.
  • Figure 2 shows the signal of recombinant adeno-associated virus infection in the dorsal hippocampus.
  • DAPI represents the signal of staining the nucleus, which is blue fluorescence
  • GFAP represents the signal of staining the astrocyte marker GFAP, which is red fluorescence
  • EGFP represents rAAV2/11-GFaABC1D - EGFP-WPRE-SV40polyA labeled cell signal, as green fluorescence, co-labeled with red fluorescence.
  • each original reagent material can be obtained commercially, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the instrument manufacturer.
  • Example 1 Construction of recombinant adeno-associated virus core plasmid
  • the recombinant adeno-associated virus packaging plasmid pAAV-RC2/11 expression vector was used to package the recombinant adeno-associated virus, and the recombinant adeno-associated virus core plasmid pAAV-GFaABC1D-EGFP-WPRE-SV40polyA was combined with the pAAV-RC2/11 serotype AAV capsid plasmid, adeno
  • the viral element helper plasmid pAd-Helper was co-transfected into HEK-293T cells according to the number of plasmid molecules 1:1:1.
  • the supernatant and cell pellet were collected, concentrated and purified by iodixanol gradient centrifugation, and finally The titer of recombinant adeno-associated virus was detected by SYBR Green qPCR method, and the titer of rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA virus was finally obtained as 6.0 ⁇ 10 12 VG/mL.
  • the recombinant adeno-associated virus packaging plasmid pAAV-RC2/11 has a nucleotide sequence as shown in SEQ ID NO:4.
  • Example 3 In vivo application of recombinant adeno-associated virus
  • the prepared rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA (100nL/mouse) virus was injected into 8-10 week-old C57BL/6 mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) through brain stereotaxic injection
  • the dorsal hippocampal region of the dorsal hippocampus was perfused 3 weeks later, and the brain tissues of the mice were fixed with DEPC-treated PFA solution for 4 hours and then dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours.
  • the dehydrated brain tissues were Fully embedded with tissue embedding medium and sliced into 40 ⁇ m thick brain slices with a cryostat.
  • FIG. 1 shows the signal of rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA recombinant adeno-associated virus infection in the dorsal hippocampal region.
  • the nucleotide sequence of the present invention is as follows:

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Abstract

本发明公开了一种靶向星形胶质细胞的重组腺相关病毒及其应用，所述重组腺相关病毒的骨架为pAAV2/11，由腺相关病毒核心质粒、重组腺相关病毒包装质粒和腺病毒元件辅助质粒共转染包装细胞系获得，所述重组腺相关病毒核心质粒包含两端反向末端重复序列ITR、GfaABC1D启动子、外源基因、转录调节元件WPRE和转录终止序列SV40 polyA，所述重组腺相关病毒包装质粒包括2型腺相关病毒的Rep基因和11型腺相关病毒的Cap基因。本发明首次将pAAV2/11作为基因转移载体实现星形胶质细胞的特异性靶向，为神经科学研究、疾病模型建立和基因治疗等提供了更好的工具和技术支撑，具有广泛的应用价值和市场前景。

Description

一种特异性靶向星形胶质细胞的重组腺相关病毒及其应用 技术领域
本发明属于生物技术领域，具体涉及一种特异性靶向星形胶质细胞的重组腺相关病毒及其应用。
背景技术
大脑网络由各种各样的神经细胞组成，神经细胞主要包括神经元和胶质细胞(neuroglia)两大类，分别在大脑中扮演不同的角色。神经胶质细胞的数量比神经元多几十倍，一般可分为小胶质细胞、星形胶质细胞以及少突胶质细胞等。随着神经科学的发展，人们发现不同类型的神经细胞具有不同的网络结构连接与功能，其发生变异可引起不同的大脑疾病和行为障碍。越来越多的证据表明星形胶质细胞跟各种脑疾病相关(Nature.2014；509(7499):189-194.)。因此，有必要开发特异性靶向星形胶质细胞的工具，用于解析星形胶质细胞的结构与功能，为脑疾病的治疗提供理论依据和技术支撑。
目前，科研工作者对星形胶质细胞的解析主要依赖于转基因动物。然而，转基因动物，如小鼠、大鼠、狗、猪和猴子等，具有繁殖周期长、饲养成本高以及不能灵活地携带目的基因进行神经细胞的操控与疾病的治疗。因此，继续发展能够特异性靶向星形胶质细胞的方法具有十分重要的意义。
发明内容
为了解决现有技术中的不足，本发明的目的是提供一种特异性靶向星形胶质细胞的重组腺相关病毒及其应用。
本发明第一方面提供一种重组腺相关病毒核心质粒，其包含两端反向末端重复序列ITR、GfaABC1D启动子、外源基因、转录调节元件WPRE和转录终止序列SV40polyA。
进一步地，所述外源基因包括报告基因；
优选地，所述报告基因为EGFP。
进一步地，所述重组腺相关病毒核心质粒的核苷酸序列如SEQ ID NO：3所示。
本发明第二方面提供一种重组腺相关病毒载体系统，包括所述的腺相关病毒核心质粒、重组腺相关病毒包装质粒和腺病毒元件辅助质粒。
进一步地，所述重组腺相关病毒包装质粒包括2型腺相关病毒的Rep基因和11型腺相关病毒的Cap基因；
优选地，所述重组腺相关病毒包装质粒以pAAV-RC2/1为骨架；
优选地，所述腺病毒元件辅助质粒为pAd-Helper。
进一步地，所述重组腺相关病毒包装质粒的核苷酸序列如SEQ ID NO：4所示。
本发明第三方面提供一种重组腺相关病毒，由所述的腺相关病毒核心质粒、重组腺相关病毒包装质粒和腺病毒元件辅助质粒共转染包装细胞系获得。
进一步地，所述包装细胞系选自HEK-293细胞、HEK-293T细胞或HEK-293FT细胞；
优选地，所述的腺相关病毒核心质粒、重组腺相关病毒包装质粒和腺病毒元件辅助质粒的分子数为1:1:1。
进一步地，所述重组腺相关病毒包装质粒包括2型腺相关病毒的Rep基因和11型腺相关病毒的Cap基因；
优选地，所述重组腺相关病毒包装质粒以pAAV-RC2/1为骨架；
优选地，所述重组腺相关病毒包装质粒的核苷酸序列如SEQ ID NO：4所示；
优选地，所述腺病毒元件辅助质粒为pAd-Helper。
本发明第四方面提供所述的重组腺相关病毒核心质粒、所述的重组腺相关病毒载体系统或所述的重组腺相关病毒在特异性靶向星形胶质细胞中的应用。
本发明的有益效果为；
本发明首次将pAAV2/11作为基因转移载体实现星形胶质细胞的特异性靶向，为神经科学研究、疾病模型建立和基因治疗等提供了更好的工具和技术支撑，具有广泛的应用价值和市场前景。
附图说明
图1为重组腺相关病毒核心质粒表达载体图谱。
图2为重组腺相关病毒感染背侧海马区域的信号。其中，“DAPI”代表对细胞核进行染色的信号，为蓝色荧光；“GFAP”代表对星形胶质细胞标志物GFAP进行染色后的信号，为红色荧光；“EGFP”代表rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA标记的细胞信号，为绿色荧光，与红色荧光共标。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条 件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1：重组腺相关病毒核心质粒的构建
使用BglII和NheI(New England Biolabs)限制性内切酶酶切载体pZac2.1-GfaABC1D-rM3D-mCherry-SV40polyA(购自Addgene,编号：92285)，用胶回收试剂盒(Omega公司)回收GfaABC1D启动子片段，用T4连接酶(购于TaKaRa公司)连入同样经BglII和NheI双酶切的腺相关病毒载体pAAV-hSyn-EGFP-WPRE-SV40polyA(布林凯斯(深圳)生物技术有限公司)，连接产物转化大肠杆菌Stbl3感受态细胞，置于35℃培养箱过夜，挑取单克隆进行菌落PCR鉴定，鉴定引物为GfaABC1D-F(SEQ ID NO.1)和GfaABC1D-R(SEQ ID NO.2)，对阳性菌落进行扩大培养和质粒提取，质粒用BglII和NheI双酶切验证和测序，测序正确的质粒命名为pAAV-GFaABC1D-EGFP-WPRE-SV40polyA。构建获得的重组腺相关病毒核心质粒pAAV-GFaABC1D-EGFP-WPRE-SV40polyA表达载体的图谱如图1所示，其基因序列如SEQ ID NO.3所示。本发明所有PCR使用引物合成和测序均由生工生物工程(上海)股份有限公司完成。
实施例2：重组腺相关病毒的制备
利用重组腺相关病毒包装质粒pAAV-RC2/11表达载体包装重组腺相关病毒，将重组腺相关病毒核心质粒pAAV-GFaABC1D-EGFP-WPRE-SV40polyA与pAAV-RC2/11血清型AAV衣壳质粒、腺病毒元件辅助质粒pAd-Helper按质粒分子数1：1：1共转染HEK-293T细胞，转染72小时后分别收集上清和细胞沉淀，用碘克沙醇梯度离心法进行浓缩和纯化，最后用SYBR Green qPCR法检测重组腺相关病毒滴度，最终获得rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA病毒的滴度为6.0×10 12VG/mL。
重组腺相关病毒包装质粒pAAV-RC2/11具有如SEQ ID NO：4所示的核苷酸序列。
实施例3：重组腺相关病毒的活体应用
将制备的rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA(100nL/只)病毒通过脑立体定位方式注射于8-10周龄C57BL/6小鼠(购自湖南斯莱克景达实验动物有限公司)的背侧海马区域，3周后灌流取脑，小鼠脑组织经DEPC处理过的PFA溶液固定4小时后再用DEPC处理过的30％蔗糖-PBS溶液脱水48小时，将脱水后的脑组织用组织包埋剂充分包埋并用冰冻切片机切成40μm厚度的脑片。将包含背侧海马区域的脑片利用GFAP抗体进行免疫组化染色，之后贴片并使用玻片扫描仪显微镜对其进行成像。活体检测结果可以看出， rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA标记的绿色荧光信号与星形胶质细胞标志物GFAP的红色信号共标，表明rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA重组腺相关病毒能够特异性靶向星形胶质细胞。图2为rAAV2/11-GFaABC1D-EGFP-WPRE-SV40polyA重组腺相关病毒感染背侧海马区域的信号。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
本发明的核苷酸序列如下：
SEQ ID NO：1
Figure PCTCN2021136105-appb-000001
SEQ ID NO：2
Figure PCTCN2021136105-appb-000002
SEQ ID NO：3
Figure PCTCN2021136105-appb-000003
Figure PCTCN2021136105-appb-000004
Figure PCTCN2021136105-appb-000005
SEQ ID NO： 4
Figure PCTCN2021136105-appb-000006
Figure PCTCN2021136105-appb-000007
Figure PCTCN2021136105-appb-000008

Claims (10)

  1. 一种重组腺相关病毒核心质粒，其特征在于，所述重组腺相关病毒核心质粒包含两端反向末端重复序列ITR、GfaABC1D启动子、外源基因、转录调节元件WPRE和转录终止序列SV40 polyA。
  2. 根据权利要求1所述的重组腺相关病毒核心质粒，其特征在于，所述外源基因包括报告基因；
    优选地，所述报告基因为EGFP。
  3. 根据权利要求1所述的重组腺相关病毒核心质粒，其特征在于，所述重组腺相关病毒核心质粒的核苷酸序列如SEQ ID NO：3所示。
  4. 一种重组腺相关病毒载体系统，其特征在于，包括权利要求1所述的腺相关病毒核心质粒、重组腺相关病毒包装质粒和腺病毒元件辅助质粒。
  5. 根据权利要求4所述的重组腺相关病毒载体系统，其特征在于，所述重组腺相关病毒包装质粒包括2型腺相关病毒的Rep基因和11型腺相关病毒的Cap基因；
    优选地，所述重组腺相关病毒包装质粒以pAAV-RC2/1为骨架；
    优选地，所述腺病毒元件辅助质粒为pAd-Helper。
  6. 根据权利要求5所述的重组腺相关病毒包装质粒，其特征在于，所述重组腺相关病毒包装质粒的核苷酸序列如SEQ ID NO：4所示。
  7. 一种重组腺相关病毒，其特征在于，由权利要求1所述的腺相关病毒核心质粒、重组腺相关病毒包装质粒和腺病毒元件辅助质粒共转染包装细胞系获得。
  8. 根据权利要求7所述的重组腺相关病毒，其特征在于，所述包装细胞系选自HEK-293细胞、HEK-293T细胞或HEK-293FT细胞；
    优选地，权利要求1所述的腺相关病毒核心质粒、重组腺相关病毒包装质粒和腺病毒元件辅助质粒的分子数为1:1:1。
  9. 根据权利要求7所述的重组腺相关病毒，其特征在于，所述重组腺相关病毒包装质粒包括2型腺相关病毒的Rep基因和11型腺相关病毒的Cap基因；
    优选地，所述重组腺相关病毒包装质粒以pAAV-RC2/1为骨架；
    优选地，所述重组腺相关病毒包装质粒的核苷酸序列如SEQ ID NO：4所示；
    优选地，所述腺病毒元件辅助质粒为pAd-Helper。
  10. 权利要求1-3任一项所述的重组腺相关病毒核心质粒、权利要求4-6任一项所述的重组腺相关病毒载体系统或权利要求7-9任一项所述的重组腺相关病毒在特异性靶向星形胶质细胞中的应用。
PCT/CN2021/136105 2021-12-07 2021-12-07 一种特异性靶向星形胶质细胞的重组腺相关病毒及其应用 Ceased WO2023102735A1 (zh)

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CN105916990A (zh) * 2013-08-02 2016-08-31 巴塞罗那自治大学 用于转导脂肪组织的腺相关病毒载体
CN106884014A (zh) * 2015-12-16 2017-06-23 北京五加和分子医学研究所有限公司 腺相关病毒反向末端重复序列突变体及其应用
CN111808175A (zh) * 2020-09-01 2020-10-23 和元生物技术（上海）股份有限公司 重组腺相关病毒颗粒及其应用
CN112501209A (zh) * 2020-12-07 2021-03-16 和元生物技术（上海）股份有限公司 外源基因可控表达的腺相关病毒包装方法
CN113025655A (zh) * 2019-12-24 2021-06-25 上海吉凯基因医学科技股份有限公司 一种基因载体

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105916990A (zh) * 2013-08-02 2016-08-31 巴塞罗那自治大学 用于转导脂肪组织的腺相关病毒载体
CN106884014A (zh) * 2015-12-16 2017-06-23 北京五加和分子医学研究所有限公司 腺相关病毒反向末端重复序列突变体及其应用
CN113025655A (zh) * 2019-12-24 2021-06-25 上海吉凯基因医学科技股份有限公司 一种基因载体
CN111808175A (zh) * 2020-09-01 2020-10-23 和元生物技术（上海）股份有限公司 重组腺相关病毒颗粒及其应用
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