WO2022166413A1 - Promoteur pcalm2 et son application - Google Patents

Promoteur pcalm2 et son application Download PDF

Info

Publication number
WO2022166413A1
WO2022166413A1 PCT/CN2021/138035 CN2021138035W WO2022166413A1 WO 2022166413 A1 WO2022166413 A1 WO 2022166413A1 CN 2021138035 W CN2021138035 W CN 2021138035W WO 2022166413 A1 WO2022166413 A1 WO 2022166413A1
Authority
WO
WIPO (PCT)
Prior art keywords
pcalm2
associated virus
recombinant adeno
promoter
protein
Prior art date
Application number
PCT/CN2021/138035
Other languages
English (en)
Chinese (zh)
Inventor
王静怡
陈晔菲
朱钰媛
林剑邦
张建情
徐冬冬
李梦奇
柏艳阳
路中华
Original Assignee
中国科学院深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院深圳先进技术研究院 filed Critical 中国科学院深圳先进技术研究院
Publication of WO2022166413A1 publication Critical patent/WO2022166413A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • 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
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • 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
    • C12N15/63—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
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the technical field of neural engineering, in particular to a promoter pCALM2 and its application.
  • the forebrain belongs to the highest part of the brain and is the most complex and important nerve center in mammals. It is mainly composed of two parts: the telencephalon and the diencephalon.
  • the telencephalon mainly includes the entire cerebral cortex, limbic system and basal ganglia
  • the diencephalon mainly includes the thalamus and hypothalamus. It is involved in the regulation of many important physiological functions including movement, sensation, perception, autonomic nervous system, endocrine, feeding, learning, memory, cognition and so on.
  • the analysis of forebrain neural circuits and their functions can help us better understand the high-level functions of the brain.
  • the study of the forebrain can also help us better understand the pathogenesis of neurological diseases, and then find more effective treatments.
  • adeno-associated virus and lentivirus among which recombinant adeno-associated virus (rAAV) is a gene vector transformed on the basis of non-pathogenic wild-type AAV. Due to its high performance, wide host cell range, strong diffusivity, and long time to express genes in vivo, rAAV is regarded as one of the most promising vectors for gene research and gene therapy.
  • the key to the successful application of rAAV in neuroscience research and clinical gene therapy is to improve the spatiotemporal specificity and expression level of gene expression.
  • One of the important methods is to select rAAV viruses of different serotypes. Different serotypes of viruses have different affinities to different tissues and show certain organ targeting specificity.
  • the reported AAV serotypes applicable to the nervous system are: type 1, type 2, type 5, type 6, type 8, type 9, DJ, PHP.B, PHP.eB, PHP.S, Retro, rh10, etc. dozens of.
  • these serotypes are far from meeting the current needs for specificity of gene delivery and gene manipulation.
  • tissue-specific promoters in the rAAV genome.
  • the promoter is a necessary cis-acting element in the process of gene expression regulation, and the promoter element of the viral vector determines the specificity of viral expression.
  • the purpose of the present invention is to provide a promoter pCALM2 with high forebrain expression and its application.
  • the said promoter pCALM2 can be used in recombinant adeno-associated virus, so that the exogenous gene can be highly expressed in the forebrain, and has broad application prospects in the field of neural circuit tracing and gene therapy.
  • the present invention provides a promoter pCALM2, and the nucleotide sequence of the promoter pCALM2 is shown in SEQ ID NO: 1.
  • the promoter pCALM22 is a partial sequence of the calmodulin 2 (CALM2) gene, and the sequence selects the partial sequence near the transcription initiation site of the CALM2 gene and the partial sequence of the first exon, a total of 3020 sequences.
  • CALM2 calmodulin 2
  • promoter pCALM2 is highly expressed in the forebrain.
  • the present invention provides a recombinant adeno-associated virus vector, comprising the above-mentioned promoter pCALM2.
  • the recombinant adeno-associated virus vector includes pAAV-pCALM2-fluorescent protein, pAAV-pCALM2-functional protein or pAAV-pCALM2-therapeutic protein.
  • the fluorescent protein includes eYFP, tdTomato;
  • the functional protein comprises an optogenetically related protein or a chemogenetically related protein.
  • pAAV-pCALM2-fluorescent protein fluorescent proteins including eYFP, tdTomato
  • pAAV-pCALM2-functional protein the functional protein includes optogenetically related protein or chemogenetically related protein
  • pAAV-pCALM2-therapeutic protein can be applied in gene therapy.
  • the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP adopts pAAV-hSyn-eYFP as the backbone vector, and replaces hSyn with the pCALM2 promoter in this vector to obtain the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP.
  • the preparation method of the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP includes the following steps: cloning the promoter pCALM2 into the pUC-57 vector to obtain the pUC57-pCALM2 vector; using restriction enzymes MluI-HF and KpnI-
  • the pAAV-hSyn-eYFP vector was treated with HF, and the pUC57-pCALM2 vector was treated with the restriction enzymes MluI-HF and KpnI-HF at the same time; the two products were recovered, purified and connected to obtain the recombinant adeno-associated virus vector pAAV- pCALM2-eYFP.
  • the present invention provides a recombinant adeno-associated virus, including the recombinant adeno-associated virus vector described above.
  • the recombinant adeno-associated virus includes AAV-retro-pCALM2-fluorescent protein, AAV-retro-pCALM2-functional protein or AAV-retro-pCALM2-therapeutic protein;
  • the fluorescent protein includes eYFP, tdTomato;
  • the functional protein comprises an optogenetically related protein or a chemogenetically related protein.
  • Recombinant adeno-associated virus AAV-retro-pCALM2-eYFP and AAV-retro-pCALM2-tdTomato can effectively retrograde cortical to striatal neural circuit and express eYFP or tdTomato with high fluorescence intensity.
  • the present invention provides a kit comprising the above-mentioned promoter pCALM2, any of the above-mentioned recombinant adeno-associated virus vectors or any of the above-mentioned recombinant adeno-associated viruses.
  • the present invention provides a kind of the above-mentioned promoter pCALM2, any of the above-mentioned recombinant adeno-associated virus vector, any of the above-mentioned recombinant adeno-associated virus or the above-mentioned kit in the preparation of neural Applications in circuit tracing and/or manipulation of neural cells and/or gene therapy reagents.
  • the present invention provides a neural network tracer comprising pAAV-pCALM2-fluorescent protein and/or recombinant adeno-associated virus comprising pAAV-pCALM2-fluorescent protein;
  • the fluorescent protein includes eYFP, tdTomato;
  • the recombinant adeno-associated virus includes AAV-retro-pCALM2-eYFP, AAV-retro-pCALM2-tdTomato.
  • the present invention provides an application of the above-mentioned neural network tracer in the tracing of cortical-striatal neural circuits.
  • the present invention has the following beneficial effects:
  • the promoter pCALM2 of the present invention has high expression efficiency in the forebrain, can realize the high-efficiency expression of exogenous genes in the forebrain, and improve the expression specificity after virus infection. application prospects;
  • the promoter pCALM2 of the present invention has no cytotoxicity in bacteria and mammalian cells
  • the recombinant adeno-associated virus vector of the present invention adopts pCALM2 as the promoter, and has high expression efficiency in the forebrain;
  • the recombinant adeno-associated virus of the present invention can efficiently reversely label the neural circuit from cortex to striatum in the brain, with high labeling efficiency and strong infectivity.
  • Fig. 1 is the structural flow chart of pAAV-pCALM2-eYFP carrier of the present invention
  • Fig. 2 is a graph showing the expression efficiency of AAV-retro-pCALM2-eYFP of the present invention in cortex and striatum.
  • the pCALM2 promoter is a partial sequence of the calmodulin 2 (CALM2) gene, and the sequence is selected from the partial sequence near the transcription initiation site of the CALM2 gene and the partial sequence of the first exon, totaling 3020 sequences.
  • the nucleotide sequence of the promoter pCALM2 is shown in SEQ ID NO: 1.
  • the promoter pCALM2 was artificially synthesized and then cloned into the pUC-57 vector to obtain the pUC57-pCALM2 vector.
  • the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the eYFP gene is selected as the vector backbone to connect the pCALM2 promoter, and the steps are as follows:
  • the pAAV-hSyn-eYFP vector was treated with restriction enzymes MluI-HF and KpnI-HF, while the pUC57-pCALM2 vector was treated with restriction enzymes MluI-HF and KpnI-HF at 37°C.
  • Enzyme digestion for 3h the digestion system is shown in Table 1 and Table 2, after the recovery of the digestion product, the linking premix (2 ⁇ ligation premix, TAKARA) was used for ligation at 16 ° C for 30min, the system was shown in Table 3, and the connection was obtained.
  • Successful pAAV-hSyn-eYFP vector was treated with restriction enzymes MluI-HF and KpnI-HF, while the pUC57-pCALM2 vector was treated with restriction enzymes MluI-HF and KpnI-HF at 37°C.
  • the three-plasmid co-transfection method was used to prepare the virus.
  • the plasmids required for packaging the virus need to be extracted, including the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP, the packaging plasmid AAV-retro-RCB and the helper plasmid. Specific steps are as follows:
  • 293T cells were plated in culture dishes containing complete medium (10% fetal bovine serum, 1% double antibody), and cultured in an incubator.
  • transfection reagent Pipette 5.25 mL of ultrapure water, 75 ⁇ g of packaging plasmid, 75 ⁇ g of recombinant plasmid, 75 ⁇ g of helper plasmid, and 800 ⁇ L of calcium chloride solution, and mix gently. To the aforementioned reagents, an equal volume of 2 ⁇ HBS was added, vortexed and left to stand for 30 min.
  • Transfected cells Add 20 ⁇ L of chloroquine to each plate of cells, add transfection reagent, and culture.
  • Virus collection 72 hours later, the transfected cells were collected in a centrifuge tube and centrifuged at 3000 rpm for 30 min. After the supernatant was discarded, the cell lysate was added, and the cells were then lysed by repeated freezing and thawing. Obtain virus-containing cell disruption fluid.
  • Virus purification Centrifuge the above cell disruption solution at 11500rpm for 30min, discard the supernatant, add 200 ⁇ L of HBS and mix, add 200 ⁇ L of chloroform and centrifuge at 12000rpm for 5min, take the supernatant, add 100 ⁇ L of 2.5mM NaCl and 100 ⁇ L of 40% PEG8000, and mix by vortexing , 4 °C refrigerator overnight. The aforementioned overnight samples were centrifuged at 12,000 rpm for 30 min, the supernatant was discarded, 30 ⁇ L of HBS and 0.5 ⁇ L of nuclease were added, and the mixture was allowed to stand for 30 min. Then 30 ⁇ L of chloroform was added and centrifuged at 12000 rpm for 5 min. After purification, it was stored in -80°C refrigerator.
  • titer of the purified virus was determined by AAV titer dye method fluorescence quantitative kit (TAKARA company), and the titer was 1.53 ⁇ 10 13 VG/mL.
  • Example 4 Recombinant adeno-associated virus labeling mouse cortex-striatal neural circuit
  • the purified recombinant adeno-associated virus was injected into the right striatum of mice (AP: +0.62mm, ML: +1.75mm, D/V: -3.5mm).
  • the recombinant adeno-associated virus capsid is AAV-retro, which has the ability of retrograde infection and can retrogradely trace cortical neurons upstream of the striatum.
  • the mice were perfused and the brains were taken out, sliced and stained by immunohistochemistry, and the results of the brain slices were observed.
  • the experimental results are shown in Figure 2, eYFP has a high expression efficiency in the mouse forebrain.
  • the present invention is verified in mice, which proves that the promoter pCALM2 has a high expression level in the forebrain.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Pathology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un promoteur pCALM2 et son application. La séquence nucléotidique du promoteur pCALM2 est telle que représentée dans SEQ ID NO: 1. Le promoteur pCALM2 peut être appliqué à un virus adéno-associé recombinant, peut permettre à un gène exogène d'être exprimé efficacement dans le prosencéphale et présente une perspective d'application large dans les domaines du traçage de boucles neuronales et de la thérapie génique.
PCT/CN2021/138035 2021-02-02 2021-12-14 Promoteur pcalm2 et son application WO2022166413A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110142184.5A CN112725342B (zh) 2021-02-02 2021-02-02 一种启动子pCALM2及其应用
CN202110142184.5 2021-02-02

Publications (1)

Publication Number Publication Date
WO2022166413A1 true WO2022166413A1 (fr) 2022-08-11

Family

ID=75595496

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/138035 WO2022166413A1 (fr) 2021-02-02 2021-12-14 Promoteur pcalm2 et son application

Country Status (2)

Country Link
CN (1) CN112725342B (fr)
WO (1) WO2022166413A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112725342B (zh) * 2021-02-02 2024-05-17 中国科学院深圳先进技术研究院 一种启动子pCALM2及其应用
WO2023283749A1 (fr) * 2021-07-13 2023-01-19 深圳市恩辑生物科技有限公司 Mini-promoteur pcalm1 et son application
CN113667671B (zh) * 2021-08-20 2023-08-29 深圳市恩辑生物科技有限公司 一种迷你启动子pRTN1及其应用
CN113652427B (zh) * 2021-08-20 2023-08-29 深圳市恩辑生物科技有限公司 一种迷你启动子pATP1B1及其应用

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

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6509190B2 (en) * 1997-11-12 2003-01-21 The Trustees Of Columbia University In The City Of New York DNA regulatory element for the expression of transgenes in neurons of the mouse forebrain
CN106687588B (zh) * 2014-06-11 2021-06-08 国立研究开发法人科学技术振兴机构 钙指示基因
US10676715B2 (en) * 2017-03-28 2020-06-09 The Board Of Trustees Of The Leland Stanford Junior University Assembly of functionally integrated human forebrain spheroids and methods of use thereof
PT3684423T (pt) * 2017-09-20 2023-06-09 4D Molecular Therapeutics Inc Cápsides variantes de vírus adeno-associado e métodos de utilização das mesmas
CN110452925A (zh) * 2019-08-02 2019-11-15 中国科学院武汉物理与数学研究所 一种基于磁共振成像的活体示踪大脑神经连接的方法及其应用
CN112143712B (zh) * 2020-09-30 2022-11-01 中国科学院深圳先进技术研究院 一种重组腺相关病毒及其制备方法和在抗体检测的应用

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 (9)

* Cited by examiner, † Cited by third party
Title
BASTING TYLER, XU JIAXI, MUKERJEE SNIGDHA, EPLING JOEL, FUCHS ROBERT, SRIRAMULA SRINIVAS, LAZARTIGUES ERIC: "Glutamatergic neurons of the paraventricular nucleus are critical contributors to the development of neurogenic hypertension", THE JOURNAL OF PHYSIOLOGY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 596, no. 24, 1 December 2018 (2018-12-01), GB , pages 6235 - 6248, XP055957386, ISSN: 0022-3751, DOI: 10.1113/JP276229 *
DATABASE GenBank NCBI; ANONYMOUS : "Mus musculus CaMII gene for calmodulin, exon 1 and flanking region", XP055957368 *
KARKERA J D, FRIEDBERG F: "Sequence of the human CALM II calmodulin gene promoter region", MOLECULAR BIOLOGY REPORTS, SPRINGER NETHERLANDS, NL, vol. 25, no. 3, 1 July 1998 (1998-07-01), NL , pages 193 - 195, XP055957379, ISSN: 0301-4851, DOI: 10.1023/A:1016561809397 *
KORTVELY ELOD, GULYA KAROLY: "Calmodulin, and various ways to regulate its activity", LIFE SCIENCE, PERGAMON PRESS, OXFORD, GB, vol. 74, no. 9, 1 January 2004 (2004-01-01), GB , pages 1065 - 1070, XP055957372, ISSN: 0024-3205, DOI: 10.1016/j.lfs.2003.07.026 *
MATSUO, K. ; IKESHIMA, H. ; SHIMODA, K. ; UMEZAWA, A. ; HATA, J.I. ; MAEJIMA, K. ; NOJIMA, H. ; TAKANO, T.: "Expression of the rat calmodulin gene II in the central nervous system: a 294-base promoter and 68-base leader segment mediates neuron-specific gene expression in transgenic mice", MOLECULAR BRAIN RESEARCH, ELSEVIER SCIENCE BV, AMSTERDAM, NL, vol. 20, no. 1-2, 1 October 1993 (1993-10-01), NL , pages 9 - 20, XP023999215, ISSN: 0169-328X, DOI: 10.1016/0169-328X(93)90106-Y *
PAPALE, A. ET AL.: "Viral vector approaches to modify gene expression in the brain.", JOURNAL OF NEUROSCIENCE METHODS, vol. 185, no. 1, 15 December 2009 (2009-12-15), XP026748078, DOI: 10.1016/j.jneumeth.2009.08.013 *
ROOPASHRI HOLEHONNUR;JONATHAN A LUONG;DUSHYANT CHATURVEDI;ANTHONY HO;SRIHARI K LELLA;MATTHEW P HOSEK;JONATHAN E PLOSKI: "Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala", BMC NEUROSCIENCE, BIOMED CENTRAL, LONDON, GB, vol. 15, no. 1, 18 February 2014 (2014-02-18), GB , pages 28, XP021177935, ISSN: 1471-2202, DOI: 10.1186/1471-2202-15-28 *
SATO KEIICHIRO, HAMASAKI YUMI, FUKUI KIYOSHIRO, ITO KAZUKI, MIYAMICHI KAZUNARI, MINAMI MASABUMI, AMANO TAIJU: "Amygdalohippocampal Area Neurons That Project to the Preoptic Area Mediate Infant-Directed Attack in Male Mice", THE JOURNAL OF NEUROSCIENCE, SOCIETY FOR NEUROSCIENCE, US, vol. 40, no. 20, 13 May 2020 (2020-05-13), US , pages 3981 - 3994, XP055957387, ISSN: 0270-6474, DOI: 10.1523/JNEUROSCI.0438-19.2020 *
TOUTENHOOFD, S.L. ET AL.: "Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALM3.", CELL CALCIUM., vol. 23, no. 5, 31 May 1998 (1998-05-31), XP002580659 *

Also Published As

Publication number Publication date
CN112725342A (zh) 2021-04-30
CN112725342B (zh) 2024-05-17

Similar Documents

Publication Publication Date Title
WO2022166413A1 (fr) Promoteur pcalm2 et son application
JP7069088B2 (ja) アデノ随伴ウイルス変異体及びその使用方法
JP6516713B2 (ja) アデノ随伴ウイルス（ａａｖ）の同源系統群（クレイド）、配列、それらを含有するベクターおよびそれらの用途
JP2024045227A (ja) パルボウイルスベクターの高められた送達のための改変キャプシドタンパク質
JP3943048B2 (ja) 組織の細胞ｄｎａからの組込みウイルスの直接レスキュー及び増幅の方法
US20220220453A1 (en) Novel aav capsids and compositions containing same
CN111566220A (zh) 制备病毒载体的手段和方法及其用途
CN111876432B (zh) 一组肝靶向新型腺相关病毒的获得及其应用
CN104877971B (zh) 一种可以缓解小鼠心衰症状的携带mg53基因的腺相关病毒载体
CN115806596A (zh) 腺相关病毒突变体及其应用
CN112680443B (zh) 一种启动子pCalm1及其应用
US20230002788A1 (en) Aav3b variants with improved production yield and liver tropism
CN112899276B (zh) 迷你启动子pHSP90AA1及其应用
WO2023103662A1 (fr) Mutant de virus adéno-associé approprié pour une infection spécifique de cellules u87-mg
WO2023283749A1 (fr) Mini-promoteur pcalm1 et son application
CN112695032B (zh) 一种启动子pLRRK2及其应用
CN113667671B (zh) 一种迷你启动子pRTN1及其应用
CN113652427B (zh) 一种迷你启动子pATP1B1及其应用
CN113584032B (zh) 一种迷你启动子pAPP及其应用
BR112021015281A2 (pt) Ácidos nucleicos recombinantes contendo sequências promotoras de alfa-herpesvírus
CN105969804B (zh) 一种携带scc抗原基因的重组腺相关病毒载体及其构建方法与应用
WO2024199478A1 (fr) Variants de protéines de capside aav9 et leurs utilisations
WO2023102736A1 (fr) Plasmide d'encapsidation de virus adéno-associé recombiné, virus adéno-associé recombiné et son application
CN115991743A (zh) 一种高效感染ht-22细胞的腺相关病毒突变体
CN118853859A (zh) 一种aav外源基因表达水平检测方法及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21924406

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21924406

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 12/01/2024)