WO2023283749A1 - Mini-promoter pcalm1 and application thereof - Google Patents

Mini-promoter pcalm1 and application thereof Download PDF

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WO2023283749A1
WO2023283749A1 PCT/CN2021/101456 CN2021101456W WO2023283749A1 WO 2023283749 A1 WO2023283749 A1 WO 2023283749A1 CN 2021101456 W CN2021101456 W CN 2021101456W WO 2023283749 A1 WO2023283749 A1 WO 2023283749A1
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pcalm1
promoter
associated virus
mini
recombinant adeno
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PCT/CN2021/101456
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路中华
林剑邦
李田
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深圳市恩辑生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus

Definitions

  • the invention belongs to the technical field of neural engineering, and in particular relates to a mini-promoter pCALM1 and its application.
  • Adeno-associated virus is a non-enveloped, single-stranded DNA virus.
  • Recombinant adeno-associated virus rAAV
  • Recombinant adeno-associated virus is a recombinant virus modified on the basis of wild-type AAV.
  • Recombinant adeno-associated virus is a widely used gene delivery vector in the field of neuroscience and clinical gene therapy of nervous system diseases due to its advantages of long-term stable expression of genes in mammals, low immunogenicity, and wide host range.
  • rAAV serotypes applicable to the nervous system include: type 1, type 2, type 5, type 6, type 8, type 9, DJ, PHP.B, PHP.eB, PHP.S, Retro , rh10 and more than a dozen.
  • rAAV serotypes have different affinities, infection efficiencies, and diffusion capabilities for different brain regions. Therefore, we can choose different rAAV serotypes as gene delivery vehicles according to the research objects and therapeutic targets. However, given the complexity of the CNS, these serotypes are far from meeting the current need for gene delivery specificity.
  • Another approach is to use tissue-specific promoters in the rAAV genome. Promoters are essential cis-acting elements in the regulation of gene expression. Under the regulation of expression-specific promoters, foreign genes are generally only expressed in certain cell types or brain regions. The use of specific promoters in rAAV to drive gene expression can improve the efficiency of gene expression and reduce the immune response generated during rAAV infection.
  • rAAV is a reliable viral vector for delivering therapeutic genetic elements to the central nervous system, but two important factors hinder its development and application in gene therapy strategies.
  • the maximum packaging capacity of rAAV is 5.2kb, which greatly limits the packaging of large therapeutic gene elements in a single AAV vector;
  • the low targeting of rAAV is also a problem that limits the development of rAAV viral tools.
  • the currently commonly used neuron-specific promoter hSyn has a size of 485bp, which further limits the packaging capacity of functional proteins under the premise of a single AAV vector.
  • the present invention provides a mini-promoter pCALM1 and its application.
  • the mini-promoter pCALM1 provided by the present invention is only 120bp in length, and can be used in recombinant adeno-associated virus to enable high-efficiency expression of foreign genes in neuron cells, and has broad application prospects in the fields of gene editing and gene therapy.
  • a mini-promoter pCALM1 the length of the mini-promoter pCALM1 is 120bp, and the nucleotide sequence is as shown in SEQ ID NO.1.
  • the length of the truncated mini-promoter pCALM1-1-12 is 110bp
  • the nucleotide sequence is a part of SEQ ID NO.1
  • the specific nucleotide sequence information is as SEQ ID NO.2-SEQ ID NO. 13.
  • the present invention also provides a recombinant adeno-associated virus vector, including the mini-promoter pCALM1 or truncated mini-promoter pCALM1-1-12.
  • the recombinant adeno-associated virus vector includes one of pAAV-pCALM1(-1 ⁇ 12)-fluorescent protein, pAAV-pCALM1-functional protein or pAAV-pCALM1-therapeutic protein;
  • the fluorescent protein is eYFP or tdTomato; the functional protein is an optogenetically related protein or a chemically genetically related protein.
  • the present invention also provides a recombinant adeno-associated virus, including the adeno-associated virus vector.
  • the present invention also provides a kit comprising the mini-promoter pCALM1 or the recombinant adeno-associated vector or the recombinant adeno-associated virus.
  • the present invention also provides an application of the mini-promoter pCALM1 in preparing and/or manipulating nerve cells and/or gene therapy reagents.
  • the present invention also provides a gene therapy vector, including said recombinant adeno-associated virus.
  • the present invention also provides an application of the gene therapy vector in gene editing and gene therapy.
  • pAAV-pCALM1-fluorescent protein enables specific cell infection tracking.
  • pAAV-pCALM1-functional protein the functional protein includes optogenetic related protein or chemical genetic related protein
  • pAAV-pCALM1-therapeutic protein can be used in gene therapy.
  • the recombinant adeno-associated virus vector pAAV-pCALM1-eYFP uses pAAV-hSyn-eYFP as the backbone vector, and replaces hSyn with the pCALM1 promoter in the vector.
  • the specific preparation process is as follows: the promoter pCALM1 is cloned into the pUC-19 vector The pUC19-pCALM1 vector was obtained; the pAAV-hSyn-eYFP vector was treated with restriction enzymes MluI-HF and BamHI-HF, and the pUC19-pCALM1 vector was treated with restriction enzymes MluI-HF and BamHI-HF at the same time; The digested product was recovered, purified and connected to obtain the recombinant adeno-associated virus vector pAAV-pCALM1-eYFP.
  • the recombinant adeno-associated virus AAV-PHP.B-pCALM1-eYFP and AAV-PHP.B-pCALM1-tdTomato provided by the present invention can specifically infect neuron cells and express eYFP or tdTomato with high fluorescence intensity.
  • the mini-promoter pCALM1 provided by the present invention has the following advantages:
  • the mini-promoter pCALM1 and its truncated promoters pCALM1-1-12 provided by the present invention are 120bp and 110bp in length respectively, and have high expression efficiency in neuron cells, and can realize exogenous High-efficiency gene expression can improve the specificity of expression after virus infection, which has broad application prospects in the fields of gene editing and gene therapy;
  • the mini-promoter pCALM1 and the truncated promoter pCALM1-1-12 provided by the present invention have no cytotoxicity in bacteria and mammalian cells;
  • the recombinant adeno-associated virus vector of the present invention adopts pCALM1 or the truncated promoter pCALM1-1-12 as the promoter, has greater packaging capacity, and has high expression efficiency in excitatory and inhibitory neurons;
  • the recombinant adeno-associated virus of the present invention can infect neuron cells in the whole brain, including the cortex, hippocampus and midbrain, and can drive high-level expression of eYFP.
  • Fig. 1 is the construction flowchart of pAAV-pCALM1-eYFP vector of the present invention
  • Fig. 2 is a graph showing the expression efficiency of AAV-PHP.B-pCALM1-eYFP of the present invention in the mouse brain.
  • Fig. 3 is a diagram showing the expression efficiency of AAV-PHP.B-pCALM1-1( ⁇ 12)-eYFP of the present invention in mouse brain.
  • the pAAV-hSyn-eYFP vector can be purchased from Addgene; the restriction enzymes MluI-H and BamHI-HF can be purchased from NEB; the full medium is DMEM medium, 10% fetal bovine serum, 1% double antibody can be purchased from ThermoFisher and HyClone respectively; the packaging plasmid AAV-PHP.B was prepared by Lu Zhonghua Research Group, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and the helper plasmid pHelper can be purchased from Nova lifetech Inc, item number PVT2101; The chloroquine can be purchased from SigmaAldrich Company.
  • Embodiment 1 pCALM1 fragment synthesis
  • the pCALM1 promoter is a partial sequence of the human calmodulin 1 (CALM1) gene, and the sequence selects the sequence of the core promoter region from -80 to +40 of the transcription initiation site of the CALM1 gene, and a total of 120bp of the sequence is used as Final promoter sequence, the nucleotide sequence of described promoter pCALM1 is shown in SEQ ID NO.1. MluI and BamHI restriction sites were added to both ends of the promoter pCALM1, and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the YFP gene was selected as the carrier backbone to connect the pCALM1 promoter.
  • the specific construction process is shown in Figure 1, that is, the preparation The process is: use restriction endonucleases MluI-HF and BamHI-HF to treat the pAAV-hSyn-eYFP vector, and simultaneously use restriction endonucleases MluI-HF and BamHI-HF to treat the pCALM1 fragment synthesized in Example 1, and combine the two
  • the enzyme digestion systems were placed at 37°C for 3 hours.
  • the enzyme digestion systems of the pAAV-hSyn-eYFP vector and the pCALM1 fragment are shown in Table 1 and Table 2 respectively. After recovering the two enzyme digestion products, use the ligation master mix (2x ligation premix, TAKARA) was ligated at 16°C for 30 minutes. The ligation system was shown in Table 3. After the single clone was sequenced to verify the sequence, the successfully ligated pAAV-pCALM1-eYFP vector was obtained.
  • Ligation master mix 2x ligation premix 5 ⁇ L pAAV-eYFP linked backbone 0.5 ⁇ L pCALM1 linker fragment 4.5 ⁇ L
  • the virus was prepared by a three-plasmid co-transfection method. Before virus preparation, the plasmids required for packaging viruses need to be extracted using QIAGEN Plasmid Plus Midi Kit (QIAGEN Company, Cat. No. 12943), including recombinant adeno-associated virus vector pAAV-pCALM1-eYFP, packaging plasmid AAV-PHP.B and auxiliary Plasmid pHHelper.
  • Cell preparation spread 293T cells in a petri dish containing full medium (DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin double antibody), culture in an incubator, and set the culture parameters to 5% CO 2 , Cultivate at 37°C for 12-24h.
  • DMEM 10% fetal bovine serum, 1% penicillin-streptomycin double antibody
  • transfection reagent pipette 5.25mL ultrapure water, 75 ⁇ g packaging plasmid, 75 ⁇ g recombinant plasmid, 75 ⁇ g helper plasmid and 800 ⁇ L 2M calcium chloride solution, mix gently; then add an equal volume of 2 ⁇ HBS to it, vortex Let stand for 30 minutes.
  • Transfection of cells Add 20 ⁇ L of 25 mM chloroquine to each culture dish containing cells, and then add all the transfection reagents prepared above, and continue to culture at 36°C and 5% CO 2 .
  • the transfected cells were collected in a centrifuge tube and centrifuged at 3000rpm for 30min. After the supernatant was discarded, 2 mL of cell lysate (100 mM Tris-HCL, 150 mM NaCl, pH 8.0) was added, and then the cells were lysed by four rounds of repeated freezing and thawing. and a 37°C water bath for 10 minutes each for rapid freezing and thawing, and briefly vortexed after each thawing to facilitate the realization of lysis, and finally obtain a cell lysate containing the virus.
  • cell lysate 100 mM Tris-HCL, 150 mM NaCl, pH 8.0
  • Purify the virus centrifuge the above cell disruption solution at 11500rpm for 30min, discard the supernatant, add 200 ⁇ l HBS and mix well, add 200 ⁇ L chloroform and centrifuge at 12000rpm for 5min, take the supernatant, add 100 ⁇ L, 2.5mM NaCl and 100 ⁇ L, 40% PEG8000, vortex Swirl to mix well, and let stand overnight in a 4°C refrigerator. Centrifuge the aforementioned overnight sample at 12,000 rpm for 30 min, discard the supernatant, add 30 ⁇ L of HBS, 0.5 ⁇ L of nuclease, and let stand for 30 min. Subsequently, 30 ⁇ L of chloroform was added, and centrifuged at 12000 rpm for 5 min. Store in a -80°C refrigerator after purification.
  • AAV titer determination dye method fluorescent quantitative kit (TAKARA company) was used to measure the titer of the aforementioned purified virus, and the titer result was 1.0 ⁇ 10 13 vg/mL.
  • Example 4 Specific expression of recombinant adeno-associated virus in mouse whole brain neurons
  • the purified recombinant adeno-associated virus obtained in Example 3 was injected into the unilateral lateral ventricle of a 9-10 week old C57 mouse (Bregma: +0.02mm; R: -0.80mm; D: 2.40mm) by stereotaxic injection. ), to study the expression characteristics of pCALM1 in the whole brain. Three weeks later, the mice were perfused with 4% PFA to take the whole brain, stained by immunohistochemical method, and the results of the brain slices were observed. The experimental results are shown in Figure 2.
  • eYFP has a high expression level in neurons in the whole brain of mice, indicating that the PHP.B serotype virus can cross the brain-cerebrospinal fluid barrier and infect cells in the whole brain.
  • pCALM1 is transcriptionally active throughout the brain and can drive high-level expression of eYFP in the cortex, hippocampus, and midbrain. It is worth noting that under a high-power microscope, it can be observed that pCALM1 only drives the expression of eYFP in neurons, but not in glial cells, which can prove that the promoter pCALM1 has neuron specificity and high expression level.
  • Example 5 The specific expression of truncated pCALM1 recombinant adeno-associated virus in mouse whole brain neurons
  • truncation attempts on pCALM1 In order to further reduce the length of the promoter, we performed truncation attempts on pCALM1.
  • the truncation strategy is as follows: remove the 1-10 bp of pCALM1 to obtain pCALM1-1 with a length of 110 bp; remove the 11-20 bp of pCALM1 to obtain pCALM1-2 with a length of 110 bp.
  • pCALM1-1--12 a total of 12 truncated pCALM1 promoters were obtained, named pCALM1-1--12 respectively.
  • the truncated promoter was cloned according to the method of Example 1-3 to obtain pAAV-pCALM1(-12)-eYFP, and the corresponding recombinant adeno-associated virus AAV was prepared.
  • the corresponding AAV was injected into the unilateral lateral ventricle of the mouse by stereotaxic method, and after 3 weeks, the samples were taken, the sections were stained, and the results of the brain slices were observed.
  • the experimental results are shown in Figure 3, most of the truncated pCALM1 retains the characteristics of pCALM1, that is, it can specifically drive the expression of eYFP in neuronal cells in the whole brain.

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Abstract

A mini-promoter pCALM1 and an application thereof are provided. The promoter is a partial sequence of the human calmodulin 1 gene, a core promoter region sequence from -80 to +40 of a transcription initiation site of the CALM1 gene, a total of 120 bp, being selected in this sequence; the nucleotide sequence information thereof being as shown in SEQ ID NO.1. It can be used in recombinant adeno-associated virus so that exogenous genes can be expressed in neurons in the whole brain, and can be used in the fields of gene editing and gene therapy.

Description

一种迷你启动子pCALM1及其应用A Mini Promoter pCALM1 and Its Application 技术领域technical field
本发明属于神经工程技术领域，具体涉及一种迷你启动子pCALM1及其应用。The invention belongs to the technical field of neural engineering, and in particular relates to a mini-promoter pCALM1 and its application.
背景技术Background technique
腺相关病毒(AAV)是一种无包膜的单链DNA病毒。重组腺相关病毒(rAAV)是在野生型AAV基础上改造而成的重组型病毒。重组腺相关病毒由于其具有能介导基因在哺乳动物体内长期稳定表达、免疫原性低、宿主范围广等优点，是神经科学领域中和临床神经系统疾病基因治疗中广泛使用的基因递送载体。Adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus. Recombinant adeno-associated virus (rAAV) is a recombinant virus modified on the basis of wild-type AAV. Recombinant adeno-associated virus is a widely used gene delivery vector in the field of neuroscience and clinical gene therapy of nervous system diseases due to its advantages of long-term stable expression of genes in mammals, low immunogenicity, and wide host range.
在应用rAAV于神经科学研究及临床治疗的过程中，提高目的基因表达的时空特异性和表达水平是基因递送的关键。调控外源基因表达的策略直接影响了基因递送的效率和安全性。对于神经系统疾病的基因治疗而言，若要干预不同的神经系统疾病，就要对全脑或不同亚型的神经元进行操纵。其中一个重要的方法是选择不同血清型的rAAV病毒。目前已有的报道中，适用于神经系统的rAAV血清型包括：1型、2型、5型、6型、8型、9型、DJ、PHP.B、PHP.eB、PHP.S、Retro、rh10等十几种。不同血清型的病毒对不同的脑区具有不同的亲和性、感染效率及扩散能力，因此，我们可以根据研究对象及治疗靶点，选择不同的rAAV血清型作为基因递送载体。然而，鉴于中枢神经系统的复杂性，这些血清型远远无法满足当前基因递送特异性的需求。另一种方法是在rAAV基因组中应用组织特异性的启动子。启动子是基因表达调控过程中必要的顺式作用元件。在具有表达特异性启动子的调控下，外源基因一般只在某些特定的细胞类型或脑区中表达。在rAAV中使用特异性启动子来驱动基因表达，能提供基因表达的效率，降低rAAV感染过程中产生的免疫反应。In the process of applying rAAV in neuroscience research and clinical treatment, improving the spatiotemporal specificity and expression level of target gene expression is the key to gene delivery. Strategies to regulate the expression of exogenous genes directly affect the efficiency and safety of gene delivery. For gene therapy of neurological diseases, to intervene in different neurological diseases, it is necessary to manipulate the whole brain or different subtypes of neurons. One of the important methods is to select rAAV viruses of different serotypes. In the existing reports, rAAV serotypes applicable to the nervous system include: type 1, type 2, type 5, type 6, type 8, type 9, DJ, PHP.B, PHP.eB, PHP.S, Retro , rh10 and more than a dozen. Different serotypes of viruses have different affinities, infection efficiencies, and diffusion capabilities for different brain regions. Therefore, we can choose different rAAV serotypes as gene delivery vehicles according to the research objects and therapeutic targets. However, given the complexity of the CNS, these serotypes are far from meeting the current need for gene delivery specificity. Another approach is to use tissue-specific promoters in the rAAV genome. Promoters are essential cis-acting elements in the regulation of gene expression. Under the regulation of expression-specific promoters, foreign genes are generally only expressed in certain cell types or brain regions. The use of specific promoters in rAAV to drive gene expression can improve the efficiency of gene expression and reduce the immune response generated during rAAV infection.
rAAV是递送治疗性基因元件至中枢神经系统的可靠病毒载体，但有两个重要因素阻碍了其在基因治疗策略中的开发应用。首先，rAAV的最大包装容量为5.2kb，极大限制了大的治疗性基因元件在单一AAV载体中的包装；其次，rAAV的低靶向性也是限制发展rAAV病毒工具的难题。目前常用的神经元特异性启动 子hSyn大小为485bp，这一启动子长度在单一AAV载体的前提下进一步限制了功能蛋白的包装容量。rAAV is a reliable viral vector for delivering therapeutic genetic elements to the central nervous system, but two important factors hinder its development and application in gene therapy strategies. First, the maximum packaging capacity of rAAV is 5.2kb, which greatly limits the packaging of large therapeutic gene elements in a single AAV vector; second, the low targeting of rAAV is also a problem that limits the development of rAAV viral tools. The currently commonly used neuron-specific promoter hSyn has a size of 485bp, which further limits the packaging capacity of functional proteins under the premise of a single AAV vector.
发明内容Contents of the invention
针对现有技术普遍存在的缺陷，本发明提供了一种迷你启动子pCALM1及其应用。本发明提供的迷你启动子pCALM1长度仅为120bp，能适用在重组腺相关病毒中，使外源基因得以在神经元细胞中高效表达，在基因编辑和基因治疗领域具有广泛的应用前景。Aiming at the common defects in the prior art, the present invention provides a mini-promoter pCALM1 and its application. The mini-promoter pCALM1 provided by the present invention is only 120bp in length, and can be used in recombinant adeno-associated virus to enable high-efficiency expression of foreign genes in neuron cells, and has broad application prospects in the fields of gene editing and gene therapy.
为了达到上述目的，本发明采用的技术方案为：In order to achieve the above object, the technical scheme adopted in the present invention is:
一种迷你启动子pCALM1，所述迷你启动子pCALM1的长度为120bp，核苷酸序列如SEQ ID NO.1所示。A mini-promoter pCALM1, the length of the mini-promoter pCALM1 is 120bp, and the nucleotide sequence is as shown in SEQ ID NO.1.
Figure PCTCN2021101456-appb-000001
Figure PCTCN2021101456-appb-000001
优选地，所述截短迷你启动子pCALM1-1～12的长度为110bp，核苷酸序列为SEQ ID NO.1的一部分，具体核苷酸序列信息如SEQ ID NO.2～SEQ ID NO.13所示。Preferably, the length of the truncated mini-promoter pCALM1-1-12 is 110bp, the nucleotide sequence is a part of SEQ ID NO.1, and the specific nucleotide sequence information is as SEQ ID NO.2-SEQ ID NO. 13.
Figure PCTCN2021101456-appb-000002
Figure PCTCN2021101456-appb-000002
Figure PCTCN2021101456-appb-000003
Figure PCTCN2021101456-appb-000003
本发明还提供了一种重组腺相关病毒载体，包括所述的迷你启动子pCALM1或截短的迷你启动子pCALM1-1～12。The present invention also provides a recombinant adeno-associated virus vector, including the mini-promoter pCALM1 or truncated mini-promoter pCALM1-1-12.
优选地，所述重组腺相关病毒载体包括pAAV-pCALM1(-1～12)-荧光蛋白、pAAV-pCALM1-功能性蛋白或pAAV-pCALM1-治疗性蛋白中的一种；Preferably, the recombinant adeno-associated virus vector includes one of pAAV-pCALM1(-1~12)-fluorescent protein, pAAV-pCALM1-functional protein or pAAV-pCALM1-therapeutic protein;
优选地，所述荧光蛋白为eYFP或tdTomato；所述功能性蛋白为光遗传相关蛋白或化学遗传相关蛋白。Preferably, the fluorescent protein is eYFP or tdTomato; the functional protein is an optogenetically related protein or a chemically genetically related protein.
本发明还提供了一种重组腺相关病毒，包括所述的腺相关病毒载体。The present invention also provides a recombinant adeno-associated virus, including the adeno-associated virus vector.
本发明还提供了一种试剂盒，包括所述的迷你启动子pCALM1或所述的重组腺相关载体或所述的重组腺相关病毒。The present invention also provides a kit comprising the mini-promoter pCALM1 or the recombinant adeno-associated vector or the recombinant adeno-associated virus.
本发明还提供了一种所述的迷你启动子pCALM1在制备和/或操控神经细胞和/或基因治疗试剂中的应用。The present invention also provides an application of the mini-promoter pCALM1 in preparing and/or manipulating nerve cells and/or gene therapy reagents.
本发明还提供了一种基因治疗载体，包括所述的重组腺相关病毒。The present invention also provides a gene therapy vector, including said recombinant adeno-associated virus.
本发明还提供了一种所述的基因治疗载体在基因编辑及基因治疗中的应用。The present invention also provides an application of the gene therapy vector in gene editing and gene therapy.
pAAV-pCALM1-荧光蛋白可以实现特异性细胞感染示踪。pAAV-pCALM1-功能性蛋白(所述功能性蛋白包括光遗传相关蛋白或化学遗传相关蛋白)可以用于制备激活或者抑制特定神经元的重组腺相关病毒。pAAV-pCALM1-治疗性蛋白可以用于基因治疗。pAAV-pCALM1-fluorescent protein enables specific cell infection tracking. pAAV-pCALM1-functional protein (the functional protein includes optogenetic related protein or chemical genetic related protein) can be used to prepare recombinant adeno-associated virus that activates or inhibits specific neurons. pAAV-pCALM1-therapeutic protein can be used in gene therapy.
本发明提供的重组腺相关病毒载体pAAV-pCALM1-eYFP采用pAAV-hSyn-eYFP作为骨架载体，向该载体中替换hSyn为pCALM1启动子，具体制备过程为：将启动子pCALM1克隆于pUC-19载体得到pUC19-pCALM1载体；采用限制性内切酶MluI-HF和BamHI-HF处理pAAV-hSyn-eYFP载体，同时用限制性内切酶MluI-HF和BamHI-HF处理pUC19-pCALM1载体；对两种酶切产物进行回收、纯化、连接，得到重组腺相关病毒载体pAAV-pCALM1-eYFP。The recombinant adeno-associated virus vector pAAV-pCALM1-eYFP provided by the present invention uses pAAV-hSyn-eYFP as the backbone vector, and replaces hSyn with the pCALM1 promoter in the vector. The specific preparation process is as follows: the promoter pCALM1 is cloned into the pUC-19 vector The pUC19-pCALM1 vector was obtained; the pAAV-hSyn-eYFP vector was treated with restriction enzymes MluI-HF and BamHI-HF, and the pUC19-pCALM1 vector was treated with restriction enzymes MluI-HF and BamHI-HF at the same time; The digested product was recovered, purified and connected to obtain the recombinant adeno-associated virus vector pAAV-pCALM1-eYFP.
本发明提供的重组腺相关病毒AAV-PHP.B-pCALM1-eYFP、AAV-PHP.B-pCALM1-tdTomato可以特异性地感染神经元细胞，并表达高荧光强度的eYFP或tdTomato。The recombinant adeno-associated virus AAV-PHP.B-pCALM1-eYFP and AAV-PHP.B-pCALM1-tdTomato provided by the present invention can specifically infect neuron cells and express eYFP or tdTomato with high fluorescence intensity.
与现有技术相比，本发明提供的迷你启动子pCALM1具有如下优势：Compared with the prior art, the mini-promoter pCALM1 provided by the present invention has the following advantages:
(1)本发明提供的迷你启动子pCALM1及其截短的启动子pCALM1-1～12,其长度分别为120bp和110bp，且在神经元细胞中具有高效表达效率，在神经元可以实现外源基因高效表达，提高病毒感染后的表达特异性，其在基因编辑和基因治疗领域具有广泛的应用前景；(1) The mini-promoter pCALM1 and its truncated promoters pCALM1-1-12 provided by the present invention are 120bp and 110bp in length respectively, and have high expression efficiency in neuron cells, and can realize exogenous High-efficiency gene expression can improve the specificity of expression after virus infection, which has broad application prospects in the fields of gene editing and gene therapy;
(2)本发明提供的迷你启动子pCALM1及截短的启动子pCALM1-1～12，在细菌和哺乳动物细胞中无细胞毒性；(2) The mini-promoter pCALM1 and the truncated promoter pCALM1-1-12 provided by the present invention have no cytotoxicity in bacteria and mammalian cells;
(3)本发明重组腺相关病毒载体采用pCALM1或截短的启动子pCALM1-1～12作为启动子，具有更大的包装能力，在兴奋性及抑制性神经元均 具有高效表达效率；(3) The recombinant adeno-associated virus vector of the present invention adopts pCALM1 or the truncated promoter pCALM1-1-12 as the promoter, has greater packaging capacity, and has high expression efficiency in excitatory and inhibitory neurons;
(4)本发明重组腺相关病毒可以在全脑范围内感染神经元细胞，包括皮层、海马及中脑都能驱动eYFP高水平表达。(4) The recombinant adeno-associated virus of the present invention can infect neuron cells in the whole brain, including the cortex, hippocampus and midbrain, and can drive high-level expression of eYFP.
附图说明Description of drawings
图1是本发明pAAV-pCALM1-eYFP载体的构造流程图；Fig. 1 is the construction flowchart of pAAV-pCALM1-eYFP vector of the present invention;
图2是本发明AAV-PHP.B-pCALM1-eYFP在小鼠大脑中的表达效率图。Fig. 2 is a graph showing the expression efficiency of AAV-PHP.B-pCALM1-eYFP of the present invention in the mouse brain.
图3是本发明AAV-PHP.B-pCALM1-1(～12)-eYFP在小鼠大脑中的表达效率图。Fig. 3 is a diagram showing the expression efficiency of AAV-PHP.B-pCALM1-1(~12)-eYFP of the present invention in mouse brain.
具体实施方式detailed description
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步的说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商获得的常规产品。In order to further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific implementation manners described here are only used to explain the present invention, rather than to limit the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that can be obtained through formal channels.
所述pAAV-hSyn-eYFP载体可购自Addgene公司；所述限制性内切酶MluI-H和BamHI-HF可购自NEB公司；所述全培养基为DMEM培养基、10％胎牛血清、1％双抗，可分别购自ThermoFisher和HyClone公司；所述包装质粒AAV-PHP.B为中国科学院深圳先进技术研究院路中华研究组制备，辅助质粒pHelper可购自Nova lifetech Inc，货号PVT2101；所述氯奎可购自SigmaAldrich公司。The pAAV-hSyn-eYFP vector can be purchased from Addgene; the restriction enzymes MluI-H and BamHI-HF can be purchased from NEB; the full medium is DMEM medium, 10% fetal bovine serum, 1% double antibody can be purchased from ThermoFisher and HyClone respectively; the packaging plasmid AAV-PHP.B was prepared by Lu Zhonghua Research Group, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and the helper plasmid pHelper can be purchased from Nova lifetech Inc, item number PVT2101; The chloroquine can be purchased from SigmaAldrich Company.
实施例1 pCALM1片段合成Embodiment 1 pCALM1 fragment synthesis
pCALM1启动子是人源钙调蛋白1(CALM1)基因的部分序列，所述序列选取了CALM1基因的转录起始位点的-80到+40位的核心启动子区序列，共120bp的序列作为最终启动子序列，所述启动子pCALM1的核苷酸序列如SEQ ID NO.1所示。将所述启动子pCALM1两端分别加上MluI和BamHI酶切位点， 经苏州金唯智生物科技有限公司合成。The pCALM1 promoter is a partial sequence of the human calmodulin 1 (CALM1) gene, and the sequence selects the sequence of the core promoter region from -80 to +40 of the transcription initiation site of the CALM1 gene, and a total of 120bp of the sequence is used as Final promoter sequence, the nucleotide sequence of described promoter pCALM1 is shown in SEQ ID NO.1. MluI and BamHI restriction sites were added to both ends of the promoter pCALM1, and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.
实施例2 构建重组腺相关病毒载体pAAV-pCALM1-eYFPExample 2 Construction of recombinant adeno-associated virus vector pAAV-pCALM1-eYFP
以eYFP作为启动子的报告基因，在构建重组载体时，选用了含有YFP基因的重组腺相关载体pAAV-hSyn-eYFP作为载体骨架来连接pCALM1启动子，具体构造流程如图1所示，即制备过程为：采用限制性内切酶MluI-HF和BamHI-HF处理pAAV-hSyn-eYFP载体，同时用限制性内切酶MluI-HF和BamHI-HF处理实施例1合成的pCALM1片段，将两种酶切体系分别置于37℃下酶切3小时，pAAV-hSyn-eYFP载体及pCALM1片段的酶切体系分别如表1、表2所示，然后回收两种酶切产物后，采用连接预混液(2x ligation premix，TAKARA)在16℃下进行连接30分钟，连接体系如表3所示，挑单克隆测序验证序列后，得到连接成功的pAAV-pCALM1-eYFP载体。For the reporter gene using eYFP as the promoter, when constructing the recombinant vector, the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the YFP gene was selected as the carrier backbone to connect the pCALM1 promoter. The specific construction process is shown in Figure 1, that is, the preparation The process is: use restriction endonucleases MluI-HF and BamHI-HF to treat the pAAV-hSyn-eYFP vector, and simultaneously use restriction endonucleases MluI-HF and BamHI-HF to treat the pCALM1 fragment synthesized in Example 1, and combine the two The enzyme digestion systems were placed at 37°C for 3 hours. The enzyme digestion systems of the pAAV-hSyn-eYFP vector and the pCALM1 fragment are shown in Table 1 and Table 2 respectively. After recovering the two enzyme digestion products, use the ligation master mix (2x ligation premix, TAKARA) was ligated at 16°C for 30 minutes. The ligation system was shown in Table 3. After the single clone was sequenced to verify the sequence, the successfully ligated pAAV-pCALM1-eYFP vector was obtained.
表1 pAAV-hSyn-eYFP载体酶切体系Table 1 pAAV-hSyn-eYFP vector restriction enzyme digestion system
试剂Reagent 用量Dosage
酶切缓冲液10x CutSmart BufferDigestion buffer 10x CutSmart Buffer 5μL5μL
MluI-HFMluI-HF 1μL1μL
BamHI-HFBamHI-HF 1μL1μL
pAAV-hSyn-eYFPpAAV-hSyn-eYFP 1μg1μg
ddH 2O ddH 2 O 补至50μLMake up to 50μL
表2 pCALM1片段酶切体系Table 2 pCALM1 fragment digestion system
试剂Reagent 用量Dosage
酶切缓冲液10x CutSmart BufferDigestion buffer 10x CutSmart Buffer 5μL5μL
MluI-HFMluI-HF 1μL1μL
BamHI-HFBamHI-HF 1μL1μL
pCALM1片段pCALM1 fragment 1μg1μg
ddH 2O ddH 2 O 补至50μLMake up to 50μL
表3连接体系Table 3 connection system
试剂Reagent 用量Dosage
连接预混液2x ligation premixLigation master mix 2x ligation premix 5μL5μL
pAAV-eYFP连接骨架pAAV-eYFP linked backbone 0.5μL0.5μL
pCALM1连接片段pCALM1 linker fragment 4.5μL4.5μL
实施例3 重组腺相关病毒AAV-PHP.B-pCALM1-eYFP的制备Example 3 Preparation of recombinant adeno-associated virus AAV-PHP.B-pCALM1-eYFP
采用三质粒共转染法制备所述病毒。在病毒制备前，需要将包装病毒所需质粒采用QIAGEN Plasmid Plus Midi Kit(QIAGEN公司，货号12943)进行中提，包括重组腺相关病毒载体pAAV-pCALM1-eYFP、包装质粒AAV-PHP.B和辅助质粒pHelper。The virus was prepared by a three-plasmid co-transfection method. Before virus preparation, the plasmids required for packaging viruses need to be extracted using QIAGEN Plasmid Plus Midi Kit (QIAGEN Company, Cat. No. 12943), including recombinant adeno-associated virus vector pAAV-pCALM1-eYFP, packaging plasmid AAV-PHP.B and auxiliary Plasmid pHHelper.
细胞制备：将293T细胞铺在含有全培养基(DMEM、10％胎牛血清、1％青霉素-链霉素双抗)培养皿中，于培养箱中培养，设置培养参数为5％的CO 2、37℃下培养12-24h。 Cell preparation: spread 293T cells in a petri dish containing full medium (DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin double antibody), culture in an incubator, and set the culture parameters to 5% CO 2 , Cultivate at 37°C for 12-24h.
制备转染试剂：吸取5.25mL超纯水、75μg包装质粒、75μg重组质粒、75μg辅助质粒和800μL的2M氯化钙溶液，轻轻混匀；然后向其中加入等体积2×HBS，涡旋后静置30分钟。Prepare transfection reagent: pipette 5.25mL ultrapure water, 75μg packaging plasmid, 75μg recombinant plasmid, 75μg helper plasmid and 800μL 2M calcium chloride solution, mix gently; then add an equal volume of 2×HBS to it, vortex Let stand for 30 minutes.
转染细胞：向每个含细胞的培养皿中加入20μL的25mM氯奎，然后将上述配制好的转染试剂全部加入后，继续于36℃、5％的CO 2下培养。 Transfection of cells: Add 20 μL of 25 mM chloroquine to each culture dish containing cells, and then add all the transfection reagents prepared above, and continue to culture at 36°C and 5% CO 2 .
收取病毒：培养72小时后，将转染后的细胞收集于离心管以3000rpm离心30min。弃上清后加入2mL细胞裂解液(100mM Tris-HCL,150mM NaCl，pH8.0)，随后经过四轮反复冻融法裂解细胞，所述反复冻融法是通过每轮分别用-80℃冰箱和37℃水浴锅各10分钟速冻和融化，每次融化后进行简短涡旋，以促进裂解实现的，最终得到含有病毒的细胞破碎液。Collection of virus: After 72 hours of culture, the transfected cells were collected in a centrifuge tube and centrifuged at 3000rpm for 30min. After the supernatant was discarded, 2 mL of cell lysate (100 mM Tris-HCL, 150 mM NaCl, pH 8.0) was added, and then the cells were lysed by four rounds of repeated freezing and thawing. and a 37°C water bath for 10 minutes each for rapid freezing and thawing, and briefly vortexed after each thawing to facilitate the realization of lysis, and finally obtain a cell lysate containing the virus.
纯化病毒：将上述细胞破碎液以11500rpm离心30min，弃上清后加入200μl HBS混匀，加入200μL氯仿以12000rpm离心5min，取上清，加入100μL、2.5mM NaCl和100μL、40％的PEG8000，涡旋混匀，4℃冰箱静置过夜。将前述过夜样品以12000rpm离心30min，弃上清，加入30μL的HBS、0.5μL核酸酶，静置30min。随后加入30μL氯仿，以12000rpm离心5min。纯化完成后于-80℃冰箱保存。Purify the virus: centrifuge the above cell disruption solution at 11500rpm for 30min, discard the supernatant, add 200μl HBS and mix well, add 200μL chloroform and centrifuge at 12000rpm for 5min, take the supernatant, add 100μL, 2.5mM NaCl and 100μL, 40% PEG8000, vortex Swirl to mix well, and let stand overnight in a 4°C refrigerator. Centrifuge the aforementioned overnight sample at 12,000 rpm for 30 min, discard the supernatant, add 30 μL of HBS, 0.5 μL of nuclease, and let stand for 30 min. Subsequently, 30 μL of chloroform was added, and centrifuged at 12000 rpm for 5 min. Store in a -80°C refrigerator after purification.
测定滴度：用AAV滴度测定染料法荧光定量试剂盒(TAKARA公司)对前述纯化病毒进行滴度测定，滴度结果为1.0×10 13vg/mL。 Determination of titer: AAV titer determination dye method fluorescent quantitative kit (TAKARA company) was used to measure the titer of the aforementioned purified virus, and the titer result was 1.0×10 13 vg/mL.
实施例4 重组腺相关病毒在小鼠全脑神经元的特异性表达Example 4 Specific expression of recombinant adeno-associated virus in mouse whole brain neurons
将实施例3获得的纯化后的重组腺相关病毒，采用立体定位法注射至9-10周龄的C57小鼠单侧侧脑室(Bregma:+0.02mm；R:-0.80mm；D:2.40mm)，在全脑的范围内对pCALM1表达特性进行研究。三周后，将小鼠用4％的PFA灌流取全脑，利用免疫组化方法切片染色，并观察脑片结果。实验结果如图2所示，eYFP在小鼠全脑范围内的神经元中均有很高的表达水平，表明PHP.B血清型病毒能跨越脑-脑脊液屏障，感染全脑的细胞。pCALM1在全脑范围内，均具有转录活性，能够在皮层、海马和中脑驱动eYFP的高水平表达。值得注意的是，在高倍显微镜下可以观察到pCALM1只驱动eYFP在神经元中的表达，而不在胶质细胞中表达，由此可以证明启动子pCALM1具有神经元特异性及高效表达水平。The purified recombinant adeno-associated virus obtained in Example 3 was injected into the unilateral lateral ventricle of a 9-10 week old C57 mouse (Bregma: +0.02mm; R: -0.80mm; D: 2.40mm) by stereotaxic injection. ), to study the expression characteristics of pCALM1 in the whole brain. Three weeks later, the mice were perfused with 4% PFA to take the whole brain, stained by immunohistochemical method, and the results of the brain slices were observed. The experimental results are shown in Figure 2. eYFP has a high expression level in neurons in the whole brain of mice, indicating that the PHP.B serotype virus can cross the brain-cerebrospinal fluid barrier and infect cells in the whole brain. pCALM1 is transcriptionally active throughout the brain and can drive high-level expression of eYFP in the cortex, hippocampus, and midbrain. It is worth noting that under a high-power microscope, it can be observed that pCALM1 only drives the expression of eYFP in neurons, but not in glial cells, which can prove that the promoter pCALM1 has neuron specificity and high expression level.
实施例5 截短pCALM1重组腺相关病毒在小鼠全脑神经元的特异性表达Example 5 The specific expression of truncated pCALM1 recombinant adeno-associated virus in mouse whole brain neurons
为了进一步缩小启动子的长度，我们对pCALM1进行了截短的尝试。截短的策略如下：去掉pCALM1的第1-10bp，得到长度为110bp的pCALM1-1；去掉pCALM1的第11-20bp，得到长度为110bp的pCALM1-2。以此类推，共得到12个截短的pCALM1启动子，分别命名为pCALM1-1--12。In order to further reduce the length of the promoter, we performed truncation attempts on pCALM1. The truncation strategy is as follows: remove the 1-10 bp of pCALM1 to obtain pCALM1-1 with a length of 110 bp; remove the 11-20 bp of pCALM1 to obtain pCALM1-2 with a length of 110 bp. By analogy, a total of 12 truncated pCALM1 promoters were obtained, named pCALM1-1--12 respectively.
将截短启动子按照实施例1-3的方法克隆获得pAAV-pCALM1(-12)-eYFP，并制备相应的重组腺相关病毒AAV。将相应AAV根据实施例4的方法采用立体定位法注射至小鼠单侧侧脑室，并于3周后取材、切片染色并观察脑片结果。实验结果如图3所示，其中大多数截短pCALM1保留了pCALM1的特征，即能够在全脑范围内特异性的驱动eYFP在神经元细胞中的表达。The truncated promoter was cloned according to the method of Example 1-3 to obtain pAAV-pCALM1(-12)-eYFP, and the corresponding recombinant adeno-associated virus AAV was prepared. According to the method of Example 4, the corresponding AAV was injected into the unilateral lateral ventricle of the mouse by stereotaxic method, and after 3 weeks, the samples were taken, the sections were stained, and the results of the brain slices were observed. The experimental results are shown in Figure 3, most of the truncated pCALM1 retains the characteristics of pCALM1, that is, it can specifically drive the expression of eYFP in neuronal cells in the whole brain.
最后应当说明的是，上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的解释，不 脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。Finally, it should be noted that the above description of the embodiments is to facilitate the understanding and use of the invention by those of ordinary skill in the technical field. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to above-mentioned embodiment, those skilled in the art explain according to the present invention, the improvement and modification that do not depart from the scope of the present invention all should be within the protection scope of the present invention.

Claims (10)

  1. 一种迷你启动子pCALM1，其特征在于，所述迷你启动子pCALM1的长度为120bp，核苷酸序列如SEQ ID NO.1所示。A mini-promoter pCALM1 is characterized in that the length of the mini-promoter pCALM1 is 120bp, and the nucleotide sequence is as shown in SEQ ID NO.1.
  2. 如权利要求1所述的迷你启动子pCALM1截短的启动子，其特征在于，所述截短迷你启动子pCALM1的长度为110bp，核苷酸序列为SEQ ID NO.1的一部分，具体核苷酸序列信息如SEQ ID NO.2～SEQ ID NO.13所示。The promoter of mini-promoter pCALM1 truncated as claimed in claim 1, is characterized in that, the length of described truncated mini-promoter pCALM1 is 110bp, and nucleotide sequence is a part of SEQ ID NO.1, specific nucleoside The acid sequence information is shown in SEQ ID NO.2～SEQ ID NO.13.
  3. 一种重组腺相关病毒载体，其特征在于，包括权利要求1或2所述的迷你启动子pCALM1。A recombinant adeno-associated virus vector, characterized in that it comprises the mini-promoter pCALM1 according to claim 1 or 2.
  4. 根据权利要求3所述的重组腺相关病毒载体，其特征在于，所述重组腺相关病毒载体包括pAAV-pCALM1-荧光蛋白、pAAV-pCALM1-功能性蛋白或pAAV-pCALM1-治疗性蛋白中的一种；The recombinant adeno-associated virus vector according to claim 3, wherein the recombinant adeno-associated virus vector comprises one of pAAV-pCALM1-fluorescent protein, pAAV-pCALM1-functional protein or pAAV-pCALM1-therapeutic protein kind;
  5. 如权利要求4所述的重组腺相关病毒载体，其特征在于，所述荧光蛋白为eYFP或tdTomato；所述功能性蛋白为光遗传相关蛋白或化学遗传相关蛋白。The recombinant adeno-associated virus vector according to claim 4, wherein the fluorescent protein is eYFP or tdTomato; the functional protein is an optogenetic related protein or a chemical genetic related protein.
  6. 一种重组腺相关病毒，其特征在于，包括权利要求4或5所述的腺相关病毒载体。A recombinant adeno-associated virus, characterized by comprising the adeno-associated virus vector according to claim 4 or 5.
  7. 一种试剂盒，其特征在于，包括权利要求1或2所述的迷你启动子pCALM1或权利要求4所述的重组腺相关病毒载体或权利要求6所述的重组腺相关病毒。A kit, characterized by comprising the mini-promoter pCALM1 according to claim 1 or 2, the recombinant adeno-associated virus vector according to claim 4 or the recombinant adeno-associated virus according to claim 6.
  8. 一种如权利要求1或2所述的迷你启动子pCALM1在制备和/或操控神经细胞和/或基因治疗试剂中的应用。An application of the mini-promoter pCALM1 as claimed in claim 1 or 2 in preparing and/or manipulating nerve cells and/or gene therapy reagents.
  9. 一种基因治疗载体，其特征在于，包括如权利要求6所述的重组腺相关病毒。A gene therapy vector, characterized in that it comprises the recombinant adeno-associated virus as claimed in claim 6.
  10. 一种如权利要求9所述的基因治疗载体在基因编辑及基因治疗中的应用。An application of the gene therapy vector according to claim 9 in gene editing and gene therapy.
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