WO2024031889A1 - 一种基因递送系统将基因递送到小胶质细胞的应用 - Google Patents

一种基因递送系统将基因递送到小胶质细胞的应用 Download PDF

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WO2024031889A1
WO2024031889A1 PCT/CN2022/137741 CN2022137741W WO2024031889A1 WO 2024031889 A1 WO2024031889 A1 WO 2024031889A1 CN 2022137741 W CN2022137741 W CN 2022137741W WO 2024031889 A1 WO2024031889 A1 WO 2024031889A1
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rna
adeno
associated virus
virus vector
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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 specifically relates to the application of a gene delivery system to deliver genes to microglia.
  • Microglia have important neuroprotective functions, but are also associated with neurodegenerative diseases and play an increasingly important role in the central nervous system.
  • the mechanism of action of microglia in neurological diseases is still unclear, which seriously hinders clinical diagnosis and gene therapy of neurological diseases.
  • reasons include the lack of tools to effectively and specifically target this type of cells.
  • scientific research Workers' analysis of microglia mainly relies on genetically modified animals.
  • genetically modified animals such as mice, rats, dogs, pigs, and monkeys, have long breeding cycles, high feeding costs, and cannot flexibly carry target genes for nerve cell manipulation and disease treatment. Therefore, it is necessary to develop tool viral vectors that can effectively and specifically target microglia.
  • the present invention provides an application of a gene delivery system to deliver genes to microglia.
  • the present invention provides an application of a delivery system to deliver genes to microglia.
  • the present invention provides an application of a delivery system to deliver genes to microglia.
  • the gene delivery system comprising an adeno-associated virus vector, the adeno-associated virus vector being rAAV11,
  • the adeno-associated virus vector contains a gene encoding a marker protein, a promoter and a neuron expression inhibitory element.
  • the marker protein is selected from a fluorescent protein and/or an enzyme reaction chromogenic protein
  • the fluorescent protein is selected from the group consisting of BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, One or more of DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP
  • the enzyme reaction chromogenic protein is selected from one of HRP, Firefly luciferase and Renilla luciferase species or species;
  • the promoter is selected from one or more of CD68, IBA1, CAG, CMV, hUbC, TRE, CBA, PGK, E-SARE, C-fos and RAM;
  • the neuronal expression inhibitory element is miR9T.
  • One object of the present invention is to provide a method for labeling microglia.
  • the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector contains a gene encoding a marker protein, a promoter and a neuron expression inhibitory element.
  • the marker protein is selected from a fluorescent protein and/or an enzyme reaction chromogenic protein
  • the fluorescent protein is selected from the group consisting of BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, One or more of DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP
  • the enzyme reaction chromogenic protein is selected from one of HRP, Firefly luciferase and Renilla luciferase species or species;
  • the promoter is selected from one or more of CD68, IBA1, CAG, CMV, hUbC, TRE, CBA, PGK, E-SARE, C-fos and RAM;
  • the neuronal expression inhibitory element is miR9T.
  • An object of the present invention is to provide an application of an adeno-associated virus vector to express a target protein or functional RNA in microglia, or to prepare a reagent for expressing a target protein or functional RNA in microglia. application.
  • adeno-associated virus vectors to express target proteins or functional RNA in microglia, or application in preparing reagents for expressing target proteins or functional RNA in microglia, where the adeno-associated virus vector is rAAV11, the adeno-associated virus vector contains a gene encoding a target protein or functional RNA, a promoter and a neuron expression inhibitory element.
  • the target protein is selected from marker proteins and/or active proteins
  • the active protein is selected from activating microglia proteins, inhibiting microglia proteins, calcium ion signal probe proteins, and small molecule signal probe proteins. , one or more of apoptosis-mediating proteins, disease-related mutant proteins, normal proteins under physiological conditions, cytokines, antiviral factors, viral infection co-receptors, recombinases and gene editing tool proteins;
  • the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle-localized RNA and Barcode RNA for RNA sequencing or in situ hybridization analysis.
  • One object of the present invention is to provide a method for expressing target proteins or functional RNA in microglia.
  • the method of expressing target protein or functional RNA in microglia includes the following steps:
  • Adeno-associated viral vectors were injected into the dorsal hippocampus of mice.
  • the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector contains a gene encoding a target protein or functional RNA, a promoter and a neuron expression inhibitory element.
  • the target protein is selected from marker proteins and/or active proteins
  • the active protein is selected from activating microglia proteins, inhibiting microglia proteins, calcium ion signal probe proteins, and small molecule signal probe proteins. , one or more of apoptosis-mediating proteins, disease-related mutant proteins, normal proteins under physiological conditions, cytokines, antiviral factors, viral infection co-receptors, recombinases and gene editing tool proteins;
  • the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle-localized RNA and Barcode RNA for RNA sequencing or in situ hybridization analysis.
  • One object of the present invention is to provide an application of an adeno-associated virus vector in the preparation of drugs for treating diseases caused by microglial abnormalities.
  • adeno-associated virus vector in the preparation of drugs for treating diseases caused by abnormal microglial cells.
  • the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector contains genes encoding therapeutic proteins or functional RNA, promoter subunits and neurons express inhibitory elements.
  • the target protein is selected from marker proteins and/or active proteins
  • the active protein is selected from microglial activating proteins, microglial inhibiting proteins, normal proteins under physiological conditions, cytokines, and antiviral factors. and one or more gene editing tool proteins,
  • the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA and small guide RNA.
  • the present invention has discovered a new use of the adeno-associated virus rAAV11 vector.
  • the rAAV11 vector can express the target gene in microglia. Based on this, the rAAV11 vector can carry the target gene for labeling and controlling microglia, and can also be used as a Gene therapy vectors.
  • Viral vectors can also express therapeutic genes for gene therapy, which cannot be achieved with transgenic animals.
  • Figure 1 shows the adeno-associated virus vector map.
  • Figure 2 shows the combination of rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH Labeling results of polyA injection into the dorsal hippocampus.
  • the present invention provides a new use of adeno-associated virus vector rAAV11, that is, using the rAAV11 vector carrying the target gene to infect microglia, thereby achieving the delivery of the target gene to microglia.
  • rAAV11 adeno-associated virus vector carrying the target gene to infect microglia
  • This method provides a new means for studying the structure and function of microglia, and has broad application value and broad market prospects in the fields of microglia-related disease model establishment and gene therapy.
  • the present invention provides a method for developing genes to deliver genes to microglia using rAAV11 vectors.
  • Adeno-associated virus vector rAAV11 can label microglia.
  • rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA was injected into the dorsal hippocampus of C57BL/6J strain mice, and green fluorescent protein was observed in the dorsal hippocampus. Labeled microglial cell bodies, the results indicate that rAAV11 can infect microglia in the dorsal hippocampus via stereotaxic injection.
  • the adeno-associated virus vector rAAV11 is a recombinant virus obtained through three-plasmid packaging. See AAV11 permits efficient retrograde targeting of projection neurons, Han et al. bioRxiv 2022.01.13.476170; doi: https://doi.org/10.1101/2022.01.13.476170.
  • Example 1 Construction of adeno-associated virus core plasmid
  • pAAV-CD68-hM4D(Gi)-mCherry purchased from Addgene, No.: 75033
  • clone the CD68 promoter and replace pAAV-hSyn-EGFP-WPRE-hGH polyA (Brinkers (Shenzhen) Biotechnology Co., Ltd.
  • pAAV-CD68-EGFP-WPRE-hGH polyA use pLV.PGK.GFP.miR9T (purchased from Addgene, No.: 115973) as a template to clone the WPRE-4xmiR9T element and replace pAAV-CD68-EGFP-WPRE-hGH WPRE element on polyA to obtain adeno-associated virus core plasmid pAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA.
  • the obtained adeno-associated virus core plasmid pAAV-CD68-EGFP-WPRE-4xmiR9T-hGH was constructed
  • the map of the polyA expression vector is shown in Figure 1, and its gene sequence is shown in SEQ ID NO.1.
  • Recombinant adeno-associated virus rAAV11 was obtained using known methods. For details, see AAV11 permits efficient retrograde targeting of projection neurons, Han et al. bioRxiv 2022.01.13.476170; doi: https://doi.org/10.1101/2022.01.13.476170, using three plasmids
  • the packaging system uses the method of packaging adeno-associated virus.
  • the rAAV11 vector is packaged, and concentrated and purified using iodixanol gradient centrifugation. Finally, the SYBR Green qPCR method is used to detect the titer of the recombinant adeno-associated virus, and the obtained titer is 1.0 ⁇ 10 13 vg/mL.
  • Example 3 In vivo test of recombinant adeno-associated virus labeled microglia
  • rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH The polyA (200 nL/mouse) virus was injected into the dorsal hippocampus of 8-10 week old C57BL/6 mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) through brain stereotaxic injection, and was taken out by perfusion 3 weeks later.
  • mice brain tissue was fixed with DEPC (diethyl pyrocarbonate)-treated PFA (paraformaldehyde) solution for 4 hours and then dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours, and the dehydrated brain was The tissue was fully embedded with tissue embedding agent and cut into brain slices with a thickness of 40 ⁇ m using a freezing microtome. Brain sections containing the dorsal hippocampal region were immunohistochemically stained with Iba1 antibody, mounted and imaged using a slide scanner microscope. The in vivo detection results are shown in Figure 2 of the instruction manual.
  • Iba1 represents the signal after staining the microglia marker Iba1, which is red fluorescence
  • EGFP represents rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA
  • the labeled cell signal is green fluorescence. It can be seen from Figure 2 of the instruction manual that rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH
  • the polyA-labeled green fluorescence signal co-labeled with the red signal of the microglia marker Iba1, indicating rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA recombinant adeno-associated virus can specifically target microglia.
  • the nucleotide sequence of the present invention is as follows:

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Abstract

本发明涉及生物技术领域，提供基因递送系统将基因递送到小胶质细胞的应用，或者在制备递送基因到小胶质细胞的试剂中的应用，所述基因递送系统包含腺相关病毒载体，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中还包含编码标记蛋白的基因、启动子和神经元表达抑制元件。本发明首次发现了腺相关病毒rAAV11载体能够将目的基因表达在小胶质细胞中，基于此rAAV11载体可以携带目的基因用于标记、操控小胶质细胞，也可以作为基因治疗的载体。

Description

一种基因递送系统将基因递送到小胶质细胞的应用 技术领域
本发明属于生物技术领域，具体涉及一种基因递送系统将基因递送到小胶质细胞的应用。
 背景技术
小胶质细胞具有重要的神经保护功能，但也跟神经退行性疾病存在关联，在中枢神经系统中扮演着越来越重要的角色。然而，小胶质细胞在神经系统疾病中的作用机制尚不清楚，严重阻碍了神经系统疾病的临床诊断和基因治疗，其中原因包括有效和特异靶向这类细胞的工具的缺乏，此外，科研工作者对小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，有必要开发能够有效地且特异性靶向小胶质细胞的工具病毒载体。
鉴于此，本发明提供了一种基因递送系统将基因递送到小胶质细胞的应用。
 技术问题
为了解决现有技术中缺乏有效和特异靶向小胶质细胞，转基因动物不能灵活地携带目的基因的问题，本发明提供一种因递送系统将基因递送到小胶质细胞的应用。
 技术解决方案
本发明提供一种因递送系统将基因递送到小胶质细胞的应用。
基因递送系统将基因递送到小胶质细胞的应用，或者在制备递送基因到小胶质细胞的试剂中的应用，所述基因递送系统包含腺相关病毒载体，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码标记蛋白的基因、启动子和神经元表达抑制元件。
进一步地，所述标记蛋白选自荧光蛋白和/或酶反应显色蛋白，所述荧光蛋白选自BFP、CFP、GFP、YFP、RFP、iRFP、Cerulean、Venus、eGFP、eCFP、eYFP、eBFP、DsRed、dTomato、tdTomato、mCherry、mKate、mApple、mBanana、mCitrine、mOrange、mPlum、tagRFP和tagBFP中的一种或多种，所述酶反应显色蛋白选自HRP、Firefly luciferase和Renilla luciferase中的一种或多种；
所述启动子选自CD68、IBA1、CAG、CMV、hUbC、TRE、CBA、PGK、E-SARE、C-fos和RAM中的一种或多种；
所述神经元表达抑制元件为miR9T。
本发明的一个目的是提供一种小胶质细胞的标记方法。
小胶质细胞的标记方法包括以下步骤：
S1：将腺相关病毒载体立体定位注射至小鼠的背侧海马中；
S2：观察背侧海马中标记蛋白的分布情况，
其中，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码标记蛋白的基因、启动子和神经元表达抑制元件。
进一步地，所述标记蛋白选自荧光蛋白和/或酶反应显色蛋白，所述荧光蛋白选自BFP、CFP、GFP、YFP、RFP、iRFP、Cerulean、Venus、eGFP、eCFP、eYFP、eBFP、DsRed、dTomato、tdTomato、mCherry、mKate、mApple、mBanana、mCitrine、mOrange、mPlum、tagRFP和tagBFP中的一种或多种，所述酶反应显色蛋白选自HRP、Firefly luciferase和Renilla luciferase中的一种或多种；
所述启动子选自CD68、IBA1、CAG、CMV、hUbC、TRE、CBA、PGK、E-SARE、C-fos和RAM中的一种或多种；
所述神经元表达抑制元件为miR9T。
本发明的一个目的是提供一种腺相关病毒载体的在小胶质细胞中表达目的蛋白或功能性RNA的应用，或者在制备在小胶质细胞中表达目的蛋白或功能性RNA的试剂中的应用。
腺相关病毒载体的在小胶质细胞中表达目的蛋白或功能性RNA的应用，或者在制备在小胶质细胞中表达目的蛋白或功能性RNA的试剂中的应用，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码目的蛋白或功能性RNA的基因、启动子和神经元表达抑制元件。
进一步地，所述目的蛋白选自标记蛋白和/或活性蛋白，所述活性蛋白选自激活小胶质细胞蛋白、抑制小胶质细胞蛋白、钙离子信号探针蛋白、小分子信号探针蛋白、介导凋亡蛋白、疾病相关突变蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子、病毒感染辅助受体、重组酶和基因编辑工具蛋白中的一种或多种；
所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA、小向导RNA、细胞器定位RNA和用于RNA测序或原位杂交分析的Barcode RNA中的一种或多种。
本发明的一个目的是提供一种小胶质细胞中表达目的蛋白或功能性RNA的方法。
小胶质细胞中表达目的蛋白或功能性RNA的方法包括以下步骤：
将腺相关病毒载体注射至小鼠的背侧海马，
其中，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码目的蛋白或功能性RNA的基因、启动子和神经元表达抑制元件。
进一步地，所述目的蛋白选自标记蛋白和/或活性蛋白，所述活性蛋白选自激活小胶质细胞蛋白、抑制小胶质细胞蛋白、钙离子信号探针蛋白、小分子信号探针蛋白、介导凋亡蛋白、疾病相关突变蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子、病毒感染辅助受体、重组酶和基因编辑工具蛋白中的一种或多种；
所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA、小向导RNA、细胞器定位RNA和用于RNA测序或原位杂交分析的Barcode RNA中的一种或多种。
本发明的一个目的是提供一种腺相关病毒载体在制备治疗小胶质细胞异常导致的疾病的药物中的应用。
腺相关病毒载体在制备治疗小胶质细胞异常导致的疾病的药物中的应用，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码治疗用蛋白或功能性RNA的基因、启动子和神经元表达抑制元件。
进一步地，所述目的蛋白选自标记蛋白和/或活性蛋白，所述活性蛋白选自激活小胶质细胞蛋白、抑制小胶质细胞蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子和基因编辑工具蛋白中的一种或多种，
所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA和小向导RNA中的一种或多种。
 有益效果
本发明的有益效果：
1、本发明发现了腺相关病毒rAAV11载体的新用途，rAAV11载体能够将目的基因表达在小胶质细胞中，基于此rAAV11载体可以携带目的基因用于标记、操控小胶质细胞，也可以作为基因治疗的载体。
2、利用病毒载体实现目的基因特异性表达比传统的转基因方法更灵活，应用更方便，成本更低。
3、病毒载体同时可表达治疗基因用于基因治疗，这是不能用转基因动物实现的。
 附图说明
图1为腺相关病毒载体图谱。
图2为将rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA注射到背侧海马的标记结果。
 本发明的实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
基于目前现有技术中存在的缺陷，本发明提供了一种腺相关病毒载体rAAV11的新用途，即利用携带目的基因的rAAV11载体感染小胶质细胞，从而实现将目的基因递送到小胶质细胞的方法。该方法为研究小胶质细胞的结构与功能研究提供了新手段，且在小胶质细胞相关的疾病模型建立和基因治疗等领域具有广泛的应用价值和广阔的市场前景。
本发明提供了利用rAAV11载体开发了一种递送基因到小胶质细胞的方法。腺相关病毒载体rAAV11能够标记小胶质细胞，将rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA注射至C57BL/6J品系小鼠的背侧海马中，在背侧海马中观察到被绿色荧光蛋白标记的小胶质细胞胞体，结果表明rAAV11可以通过立体定位注射感染背侧海马的小胶质细胞。
在本发明中，腺相关病毒载体rAAV11为通过三质粒包装获得的重组病毒。参见AAV11 permits efficient retrograde targeting of projection neurons，Han et al. bioRxiv 2022.01.13.476170; doi: https://doi.org/10.1101/2022.01.13.476170。
实施例 1 ：腺相关病毒核心质粒的构建
以pAAV-CD68-hM4D(Gi)-mCherry（购自Addgene，编号：75033）为模板，克隆CD68启动子并替换pAAV-hSyn-EGFP-WPRE-hGH polyA（布林凯斯（深圳）生物技术有限公司）上的hSyn启动子，获得pAAV-CD68-EGFP-WPRE-hGH polyA；以pLV.PGK.GFP.miR9T（购自Addgene，编号：115973）为模板，克隆WPRE-4xmiR9T元件并替换pAAV-CD68-EGFP-WPRE-hGH polyA上的WPRE元件，获得腺相关病毒核心质粒pAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA。构建获得的腺相关病毒核心质粒pAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA表达载体的图谱如图1所示，其基因序列如SEQ ID NO .1所示。
实施例 2 ：重组腺相关病毒的制备
采用已知方法获得重组腺相关病毒rAAV11，具体参见AAV11 permits efficient retrograde targeting of projection neurons，Han et al. bioRxiv 2022.01.13.476170; doi: https://doi.org/10.1101/2022.01.13.476170，采用三质粒包装系统包装腺相关病毒方法，对rAAV11载体进行病毒包装，并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，获得滴度为1.0×10 13 vg/mL。
实施例 3 ：重组腺相关病毒标记小胶质细胞的活体测试
将rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA（200 nL/只）病毒通过脑立体定位注射方式注射于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（焦碳酸二乙酯）处理过的PFA（多聚甲醛）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40 μm厚度的脑片。将包含背侧海马区域的脑片利用Iba1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。活体检测结果如说明书图2所示，其中，“Iba1”代表对小胶质细胞标志物Iba1进行染色后的信号，为红色荧光；“EGFP”代表rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA标记的细胞信号，为绿色荧光。从说明书图2中可以看出rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA标记的绿色荧光信号与小胶质细胞标志物Iba1的红色信号共标，表明rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA重组腺相关病毒能够特异性靶向小胶质细胞。
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
tattcttttgatttataagggattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgtgatatcaaactgcctgtttgggcttctcatttcttacctccccttccctctcccacctgctactgggtgcatctctgctccccccttccccagcagatggttacctttgggctgttgctttcttgtcaccatctgagttctcagacgctggaaagccatgttctcggctctgtgaatgacaatgctgactggagtgctgcccctctgtaaagggctgggtgtggatggtcacaagcccctcacatgcctcagccaagaggaagtagtacaggggtcagcccagaggtccaggggaaaggagtggaaaccgatttccccaccaagggaggggcctgtacctcagctgttcccatagctacttgccacaactgccaagcaagtttcgctgagtttgacacatggatccctgtggatcaactgccctaggactccgtttgcacccatgtgacactgttgactttgccctgacgaagcagggccaacagtcccctaacttaattacaaaaactaatgactaagagagaggtggctagagctgaggcccctgagtcaggctgtgggtgggatcatctccagtacaggaagtgagactttcatttcctcctttccaagagagggctgagggagcagggttgagcaactggtgcagacagcctagctggactttgggtgaggcggttcagccatatcgtcgacgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaagcttaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctagagtcatacagctagataaccaaagacgtatcatacagctagataaccaaagagctagagtcatacagctagataaccaaagacgtatcatacagctagataaccaaagagctagagcctcttccgcgtcttcgccttcccgggtcgagctcggtaccctcgagagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggc。

Claims (10)

  1. 一种基因递送系统将基因递送到小胶质细胞的应用，或者在制备递送基因到小胶质细胞的试剂中的应用，其特征在于，所述基因递送系统包含腺相关病毒载体，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码标记蛋白的基因、启动子和神经元表达抑制元件。
  2. 如权利要求1所述的应用，其特征在于，
    所述标记蛋白选自荧光蛋白和/或酶反应显色蛋白，所述荧光蛋白选自BFP、CFP、GFP、YFP、RFP、iRFP、Cerulean、Venus、eGFP、eCFP、eYFP、eBFP、DsRed、dTomato、tdTomato、mCherry、mKate、mApple、mBanana、mCitrine、mOrange、mPlum、tagRFP和tagBFP中的一种或多种，所述酶反应显色蛋白选自HRP、Firefly luciferase和Renilla luciferase中的一种或多种；
    所述启动子选自CD68、IBA1、CAG、CMV、hUbC、TRE、CBA、PGK、E-SARE、C-fos和RAM中的一种或多种；
    所述神经元表达抑制元件为miR9T。
  3. 一种小胶质细胞的标记方法，其特征在于，包括以下步骤：
    S1：将腺相关病毒载体立体定位注射至小鼠的背侧海马中；
    S2：观察背侧海马中标记蛋白的分布情况，
    其中，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码标记蛋白的基因、启动子和神经元表达抑制元件。
  4. 如权利要求3所述的标记方法，其特征在于，
    所述标记蛋白选自荧光蛋白和/或酶反应显色蛋白，所述荧光蛋白选自BFP、CFP、GFP、YFP、RFP、iRFP、Cerulean、Venus、eGFP、eCFP、eYFP、eBFP、DsRed、dTomato、tdTomato、mCherry、mKate、mApple、mBanana、mCitrine、mOrange、mPlum、tagRFP和tagBFP中的一种或多种，所述酶反应显色蛋白选自HRP、Firefly luciferase和Renilla luciferase中的一种或多种；
    所述启动子选自CD68、IBA1、CAG、CMV、hUbC、TRE、CBA、PGK、E-SARE、C-fos和RAM中的一种或多种；
    所述神经元表达抑制元件为miR9T。
  5. 一种腺相关病毒载体的在小胶质细胞中表达目的蛋白或功能性RNA的应用，或者在制备在小胶质细胞中表达目的蛋白或功能性RNA的试剂中的应用，其特征在于，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码目的蛋白或功能性RNA的基因、启动子和神经元表达抑制元件。
  6. 如权利要求5所述的应用，其特征在于，
    所述目的蛋白选自标记蛋白和/或活性蛋白，所述活性蛋白选自激活小胶质细胞蛋白、抑制小胶质细胞蛋白、钙离子信号探针蛋白、小分子信号探针蛋白、介导凋亡蛋白、疾病相关突变蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子、病毒感染辅助受体、重组酶和基因编辑工具蛋白中的一种或多种；
    所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA、小向导RNA、细胞器定位RNA和用于RNA测序或原位杂交分析的Barcode RNA中的一种或多种。
  7. 一种小胶质细胞中表达目的蛋白或功能性RNA的方法，其特征在于，包括以下步骤：将腺相关病毒载体注射至小鼠的背侧海马，
    其中，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码目的蛋白或功能性RNA的基因、启动子和神经元表达抑制元件。
  8. 如权利要求7所述的方法，其特征在于，
    所述目的蛋白选自标记蛋白和/或活性蛋白，所述活性蛋白选自激活小胶质细胞蛋白、抑制小胶质细胞蛋白、钙离子信号探针蛋白、小分子信号探针蛋白、介导凋亡蛋白、疾病相关突变蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子、病毒感染辅助受体、重组酶和基因编辑工具蛋白中的一种或多种；
    所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA、小向导RNA、细胞器定位RNA和用于RNA测序或原位杂交分析的Barcode RNA中的一种或多种。
  9. 一种腺相关病毒载体在制备治疗小胶质细胞异常导致的疾病的药物中的应用，其特征在于，所述腺相关病毒载体为rAAV11，所述腺相关病毒载体中包含编码治疗用蛋白或功能性RNA的基因、启动子和神经元表达抑制元件。
  10. 如权利要求9所述的应用，其特征在于，
    所述目的蛋白选自标记蛋白和/或活性蛋白，所述活性蛋白选自激活小胶质细胞蛋白、抑制小胶质细胞蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子和基因编辑工具蛋白中的一种或多种，
    所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA和小向导RNA中的一种或多种。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101883859A (zh) * 2007-10-05 2010-11-10 吉尼松公司 使用外周注射aav载体向运动神经元进行广泛基因投送
US20150051267A1 (en) * 2013-08-16 2015-02-19 Purdue Research Foundation BICISTRONIC GENE TRANSFER TOOLS FOR DELIVERY OF miRNAS AND PROTEIN CODING SEQUENCES
CN111621507A (zh) * 2012-09-28 2020-09-04 北卡罗来纳-查佩尔山大学 靶向少突胶质细胞的aav载体
CN114196636A (zh) * 2021-12-09 2022-03-18 中国科学院深圳先进技术研究院 腺相关病毒载体在逆向标记外周组织或器官一级神经网络中的应用
CN114516901A (zh) * 2022-03-11 2022-05-20 上海勉亦生物科技有限公司 一种神经系统高亲和性的aav载体及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021011368A1 (en) * 2019-07-12 2021-01-21 Dmitry Dmitrievich Genkin Treatment of diseases associated with protein misfolding by nervous system expression of an enzyme which has a deoxyribonuclease (dnase) activity
US12442017B2 (en) * 2020-03-02 2025-10-14 National University Corporation Gunma University Microglial selective gene expression vector

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101883859A (zh) * 2007-10-05 2010-11-10 吉尼松公司 使用外周注射aav载体向运动神经元进行广泛基因投送
CN111621507A (zh) * 2012-09-28 2020-09-04 北卡罗来纳-查佩尔山大学 靶向少突胶质细胞的aav载体
US20150051267A1 (en) * 2013-08-16 2015-02-19 Purdue Research Foundation BICISTRONIC GENE TRANSFER TOOLS FOR DELIVERY OF miRNAS AND PROTEIN CODING SEQUENCES
CN114196636A (zh) * 2021-12-09 2022-03-18 中国科学院深圳先进技术研究院 腺相关病毒载体在逆向标记外周组织或器官一级神经网络中的应用
CN114516901A (zh) * 2022-03-11 2022-05-20 上海勉亦生物科技有限公司 一种神经系统高亲和性的aav载体及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAN ZENGPENG, LUO NENGSONG, MA WENYU, LIU XIAODONG, CAI YUXIANG, KOU JIAXIN, WANG JIE, LI LEI, PENG SIQI, XU ZIHONG, ZHANG WEN, QI: "AAV11 enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, UK, vol. 14, no. 1, 26 June 2023 (2023-06-26), UK, pages 3792 , XP093140119, ISSN: 2041-1723, DOI: 10.1038/s41467-023-39554-7 *

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