WO2023283749A1 - Mini-promoteur pcalm1 et son application - Google Patents

Mini-promoteur pcalm1 et son application 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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English (en)
Chinese (zh)
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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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Priority to PCT/CN2021/101456 priority Critical patent/WO2023283749A1/fr
Publication of WO2023283749A1 publication Critical patent/WO2023283749A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

L'invention concerne un mini-promoteur pCALM1 et une application de celui-ci. Le promoteur est une séquence partielle du gène de la calmoduline 1 humaine, une séquence de la région centrale du promoteur de -80 à +40 d'un site d'initiation de la transcription du gène CALM1, un total de 120 pb, étant sélectionnée dans cette séquence ; l'information de séquence nucléotidique de celle-ci étant telle que représentée dans SEQ ID NO : 1. Il peut être utilisé dans le virus adéno-associé recombiné afin que les gènes exogènes puissent être exprimés dans les neurones du cerveau entier, et peut être utilisé dans les domaines de l'édition génique et de la thérapie génique.
PCT/CN2021/101456 2021-07-13 2021-07-13 Mini-promoteur pcalm1 et son application WO2023283749A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112680443A (zh) * 2020-12-10 2021-04-20 中国科学院深圳先进技术研究院 一种启动子pCalm1及其应用
CN112725342A (zh) * 2021-02-02 2021-04-30 中国科学院深圳先进技术研究院 一种启动子pCALM2及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112680443A (zh) * 2020-12-10 2021-04-20 中国科学院深圳先进技术研究院 一种启动子pCalm1及其应用
CN112725342A (zh) * 2021-02-02 2021-04-30 中国科学院深圳先进技术研究院 一种启动子pCALM2及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE ANONYMOUS : "Homo sapiens Sharpr-MPRA regulatory region 5900 (LOC112272566) on chromosome 14 ", XP093024358, retrieved from NCBI *
SHI DONGQUAN; NI HAIJIAN; DAI JIN; QIN JIANGHUI; XU YONG; ZHU LUNQING; YAO CHEN; SHAO ZHENXING; CHEN DONGYANG; XU ZHIHONG; YI LONG: "Lack of association between the CALM1 core promoter polymorphism (-16C/T) and susceptibility to knee osteoarthritis in a Chinese Han population", BMC MEDICAL GENETICS, BIOMED CENTRAL, LONDON, GB, vol. 9, no. 1, 22 October 2008 (2008-10-22), GB , pages 91, XP021042802, ISSN: 1471-2350, DOI: 10.1186/1471-2350-9-91 *

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