CN116064671A - Application of gene delivery system in delivering genes to microglia - Google Patents
Application of gene delivery system in delivering genes to microglia Download PDFInfo
- Publication number
- CN116064671A CN116064671A CN202210959274.8A CN202210959274A CN116064671A CN 116064671 A CN116064671 A CN 116064671A CN 202210959274 A CN202210959274 A CN 202210959274A CN 116064671 A CN116064671 A CN 116064671A
- Authority
- CN
- China
- Prior art keywords
- protein
- rna
- adeno
- gene
- small
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Images
Classifications
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the field of biotechnology, and provides application of a gene delivery system for delivering genes to microglia or application of the gene delivery system in preparation of reagents for delivering genes to microglia, wherein the gene delivery system comprises an adeno-associated viral vector, the adeno-associated viral vector is rAAV11, and the adeno-associated viral vector also comprises genes encoding marker proteins, promoters and neuron expression inhibition elements. The invention discovers for the first time that the rAAV11 vector can express the target gene in microglia, and based on the rAAV11 vector, the rAAV11 vector can carry the target gene for marking and controlling microglia and can also be used as a vector for gene therapy.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an application of a gene delivery system in delivering genes to microglia.
Background
Microglia have important neuroprotective functions, but are also associated with neurodegenerative diseases and play an increasingly important role in the central nervous system. However, the mechanism of action of microglia in neurological diseases is not yet clear, severely hampering clinical diagnosis and gene therapy of neurological diseases, among which reasons include the lack of tools to effectively and specifically target such cells, and furthermore, the analysis of microglia by researchers has been largely dependent on transgenic animals. However, transgenic animals such as mice, rats, dogs, pigs, monkeys, etc., have a long breeding cycle, high feeding costs, and are not flexible to carry genes of interest for neuronal manipulation and disease treatment. Therefore, there is a need to develop tool viral vectors that can target microglial cells efficiently and specifically.
In view of this, the present invention provides the use of a gene delivery system to deliver genes to microglia.
Disclosure of Invention
In order to solve the problem that the prior art lacks effective and specific targeting microglia, and a transgenic animal cannot flexibly carry a target gene, the invention provides an application of delivering the gene to microglia by a delivery system.
Use of a gene delivery system comprising an adeno-associated viral vector, said adeno-associated viral vector being rAAV11, comprising a gene encoding a marker protein, a promoter and a neuronal expression suppression element, for delivering a gene to microglial cells, or for the preparation of a reagent for delivering a gene to microglial cells.
Further, the marker protein is selected from a fluorescent protein selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, cerulean, venus, eGFP, eCFP, eYFP, eBFP, dsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP and/or an enzyme-reacted chromogenic protein selected from one or more of HRP, firefly luciferase and Renilla luciferase;
the promoter is selected from one or more of CD68, IBA1, CAG, CMV, hUbC, TRE, CBA, PGK, E-SARE, C-fos and RAM;
the neuron expression inhibition element is miR9T.
It is an object of the present invention to provide a method for labeling microglial cells.
The labeling method of microglial cells comprises the following steps:
s1: stereotactic injection of adeno-associated viral vectors into the dorsal hippocampus of mice;
s2: the distribution of marker proteins in the dorsal hippocampus was observed,
wherein the adeno-associated virus vector is rAAV11, and comprises a gene encoding a marker protein, a promoter and a neuron expression inhibition element.
Further, the marker protein is selected from a fluorescent protein selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, cerulean, venus, eGFP, eCFP, eYFP, eBFP, dsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP and/or an enzyme-reacted chromogenic protein selected from one or more of HRP, firefly luciferase and Renilla luciferase;
the promoter is selected from one or more of CD68, IBA1, CAG, CMV, hUbC, TRE, CBA, PGK, E-SARE, C-fos and RAM;
the neuron expression inhibition element is miR9T.
It is an object of the present invention to provide the use of an adeno-associated viral vector for expressing a protein or functional RNA of interest in microglia, or for preparing a reagent for expressing a protein or functional RNA of interest in microglia.
Use of an adeno-associated viral vector, which is rAAV11, comprising a gene encoding a protein or functional RNA of interest, a promoter, and a neuronal expression suppression element, for expressing the protein or functional RNA of interest in microglial cells, or for the preparation of a reagent for expressing the protein or functional RNA of interest in microglial cells.
Further, the protein of interest is selected from a marker protein and/or an active protein selected from one or more of activating microglial cell protein, inhibiting microglial cell protein, calcium ion signaling probe protein, small molecule signaling probe protein, mediating apoptosis protein, disease-related mutein, normal protein under physiological conditions, cytokine, antiviral factor, viral infection accessory receptor, recombinase and gene editing tool protein;
the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle localization RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.
It is an object of the present invention to provide a method for expressing a protein of interest or a functional RNA in microglia.
The method for expressing the target protein or the functional RNA in the microglial cell comprises the following steps:
adeno-associated viral vectors were injected into the dorsal hippocampus of mice,
wherein the adeno-associated viral vector is rAAV11, and comprises a gene encoding a target protein or functional RNA, a promoter and a neuron expression inhibition element.
Further, the protein of interest is selected from a marker protein and/or an active protein selected from one or more of activating microglial cell protein, inhibiting microglial cell protein, calcium ion signaling probe protein, small molecule signaling probe protein, mediating apoptosis protein, disease-related mutein, normal protein under physiological conditions, cytokine, antiviral factor, viral infection accessory receptor, recombinase and gene editing tool protein;
the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle localization RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.
An object of the present invention is to provide an application of an adeno-associated virus vector in preparing a medicament for treating diseases caused by microglial cell abnormality.
Use of an adeno-associated viral vector, which is rAAV11, comprising a gene encoding a therapeutic protein or a functional RNA, a promoter, and a neuronal expression suppression element, in the manufacture of a medicament for the treatment of a disease caused by microglial abnormalities.
Further, the target protein is selected from a marker protein and/or an active protein selected from one or more of an activated microglial cell protein, an inhibited microglial cell protein, a normal protein under physiological conditions, a cytokine, an antiviral factor and a gene editing tool protein,
the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA and small guide RNA.
Compared with the prior art, the invention has the following advantages:
1. the invention discovers the new application of the rAAV11 vector, the rAAV11 vector can express the target gene in microglia, and the rAAV11 vector can carry the target gene for marking and controlling microglia based on the rAAV11 vector and can also be used as a vector for gene therapy.
2. The virus vector is utilized to realize the specific expression of the target gene, which is more flexible than the traditional transgenic method, more convenient to apply and lower in cost.
3. Viral vectors can simultaneously express therapeutic genes for gene therapy, which cannot be achieved with transgenic animals.
Drawings
FIG. 1 is a map of an adeno-associated viral vector.
FIG. 2 is a labeling result of rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA injected into dorsal hippocampus.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawings, but are not to be construed as limiting the scope of the invention.
Based on the defects existing in the prior art, the invention provides a new application of an adeno-associated virus vector rAAV11, namely a method for delivering a target gene to microglial cells by infecting microglial cells by using the rAAV11 vector carrying the target gene. The method provides a new means for researching the structure and the function of microglial cells, and has wide application value and broad market prospect in the fields of microglial cell related disease model establishment, gene therapy and the like.
The present invention provides methods of delivering genes to microglia using rAAV11 vectors. The adeno-associated virus vector rAAV11 can mark microglia, rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA is injected into dorsal hippocampus of a mouse with a C57BL/6J strain, microglia marked by green fluorescent protein are observed in the dorsal hippocampus, and the result shows that rAAV11 can infect microglia of the dorsal hippocampus through stereotactic injection.
In the present invention, the adeno-associated viral vector rAAV11 is a recombinant virus obtained by 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
Cloning CD68 promoter and replacing hSyn promoter on pAAV-hSyn-EGFP-WPRE-hGH polyA (Brinz Biotechnology Co., ltd.) with pAAV-CD68-hM4D (Gi) -mCherry (available from Addgene, accession number 75033) as template to obtain pAAV-CD68-EGFP-WPRE-hGH polyA; cloning the WPRE-4xmiR9T element and replacing the WPRE element on pAAV-CD68-EGFP-WPRE-hGH polyA by using pLV.PGK.GFP.miR9T (purchased from Addgene, number: 115973) as a template to obtain the adeno-associated virus core plasmid pAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA. The map of the constructed adeno-associated virus core plasmid pAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA expression vector is shown in figure 1, and the gene sequence is shown in SEQ ID NO. 1.
Example 2: preparation of recombinant adeno-associated virus
Recombinant adeno-associated virus rAAV11, see in particular AAV11 permits efficient retrograde targeting of projection neurons, han et al biorxiv 2022.01.13.476170, was obtained using known methods; doi https/(doi. Org/10.1101/2022.01.13.476170), packaging the adenovirus-associated virus by a three-plasmid packaging system, packaging the rAAV11 vector, concentrating and purifying by iodixanol gradient centrifugation, and detecting recombinant adenovirus titer by SYBR Green qPCR to obtain 1.0X10-10 titer 13 vg/mL。
Example 3: in vivo testing of recombinant adeno-associated virus-labeled microglia
rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA (200 nL/virus) was injected into the dorsal hippocampal region of 8-10 week old C57BL/6 mice (purchased from Hunan Stokes Leachi laboratory animal Co., ltd.) by brain stereotactic injection, brains were perfused after 3 weeks, mouse brain tissue was fixed with DEPC (diethyl pyrocarbonate) -treated PFA (paraformaldehyde) solution for 4 hours, dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours, and the dehydrated brain tissue was fully embedded with tissue embedding agent and cut into brain pieces of 40 μm thickness with a frozen microtome. Brain patches containing dorsal hippocampal areas were immunohistochemically stained with Iba1 antibody, followed by patch and imaging thereof using a slide scanner microscope. The living body detection result is shown in the specification and figure 2, wherein 'Iba 1' represents a signal after staining the microglial cell marker Iba1, and the signal is red fluorescence; "EGFP" represents the rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA-labeled cell signal, and is green fluorescent. From the specification, as can be seen in FIG. 2, the green fluorescent signal of the rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA label is co-labeled with the red fluorescent signal of the microglial cell marker Iba1, which indicates that the rAAV-CD68-EGFP-WPRE-4xmiR9T-hGH polyA recombinant adeno-associated virus can specifically target microglial cells.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
The nucleotide sequence of the invention is as follows:
SEQ ID NO:1
tattcttttgatttataagggattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcgtgatatcaaactgcctgtttgggcttctcatttcttacctccccttccctctcccacctgctactgggtgcatctctgctccccccttccccagcagatggttacctttgggctgttgctttcttgtcaccatctgagttctcagacgctggaaagccatgttctcggctctgtgaatgacaatgctgactggagtgctgcccctctgtaaagggctgggtgtggatggtcacaagcccctcacatgcctcagccaagaggaagtagtacaggggtcagcccagaggtccaggggaaaggagtggaaaccgatttccccaccaagggaggggcctgtacctcagctgttcccatagctacttgccacaactgccaagcaagtttcgctgagtttgacacatggatccctgtggatcaactgccctaggactccgtttgcacccatgtgacactgttgactttgccctgacgaagcagggccaacagtcccctaacttaattacaaaaactaatgactaagagagaggtggctagagctgaggcccctgagtcaggctgtgggtgggatcatctccagtacaggaagtgagactttcatttcctcctttccaagagagggctgagggagcagggttgagcaactggtgcagacagcctagctggactttgggtgaggcggttcagccatatcgtcgacgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaaagcttaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctagagtcatacagctagataaccaaagacgtatcatacagctagataaccaaagagctagagtcatacagctagataaccaaagacgtatcatacagctagataaccaaagagctagagcctcttccgcgtcttcgccttcccgggtcgagctcggtaccctcgagagatctacgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacggggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtccttctgattttgtaggtaaccacgtgcggaccgagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggc
Claims (10)
1. use of a gene delivery system for delivering a gene to microglial cells, or for the preparation of a reagent for delivering a gene to microglial cells, wherein the gene delivery system comprises an adeno-associated viral vector, which is rAAV11, comprising a gene encoding a marker protein, a promoter and a neuronal expression suppression element.
2. The use according to claim 1, wherein,
the marker protein is selected from fluorescent protein and/or enzyme reaction chromogenic protein, the fluorescent protein is selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, cerulean, venus, eGFP, eCFP, eYFP, eBFP, dsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP, and the enzyme reaction chromogenic protein is selected from one or more of HRP, firefly luciferase and Renilla luciferase;
the promoter is selected from one or more of CD68, IBA1, CAG, CMV, hUbC, TRE, CBA, PGK, E-SARE, C-fos and RAM;
the neuron expression inhibition element is miR9T.
3. A method for labeling microglial cells, comprising the steps of:
s1: stereotactic injection of adeno-associated viral vectors into the dorsal hippocampus of mice;
s2: the distribution of marker proteins in the dorsal hippocampus was observed,
wherein the adeno-associated virus vector is rAAV11, and comprises a gene encoding a marker protein, a promoter and a neuron expression inhibition element.
4. The marking method according to claim 3, wherein,
the marker protein is selected from fluorescent protein and/or enzyme reaction chromogenic protein, the fluorescent protein is selected from one or more of BFP, CFP, GFP, YFP, RFP, iRFP, cerulean, venus, eGFP, eCFP, eYFP, eBFP, dsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP and tagBFP, and the enzyme reaction chromogenic protein is selected from one or more of HRP, firefly luciferase and Renilla luciferase;
the promoter is selected from one or more of CD68, IBA1, CAG, CMV, hUbC, TRE, CBA, PGK, E-SARE, C-fos and RAM;
the neuron expression inhibition element is miR9T.
5. Use of an adeno-associated viral vector comprising a gene encoding a protein or functional RNA of interest, a promoter and a neuronal expression suppression element in microglial cells for the expression of the protein or functional RNA of interest or for the preparation of a reagent for the expression of the protein or functional RNA of interest in microglial cells, wherein the adeno-associated viral vector is rAAV 11.
6. The use according to claim 5, wherein,
the target protein is selected from a marker protein and/or an active protein, and the active protein is selected from one or more of an activated microglial cell protein, an inhibited microglial cell protein, a calcium ion signal probe protein, a small molecule signal probe protein, a mediated apoptosis protein, a disease-related mutant protein, a normal protein under physiological conditions, a cytokine, an antiviral factor, a viral infection auxiliary receptor, a recombinase and a gene editing tool protein;
the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle localization RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.
7. A method for expressing a protein of interest or a functional RNA in microglia comprising the steps of:
adeno-associated viral vectors were injected into the dorsal hippocampus of mice,
wherein the adeno-associated viral vector is rAAV11, and comprises a gene encoding a target protein or functional RNA, a promoter and a neuron expression inhibition element.
8. The method of claim 7, wherein,
the target protein is selected from a marker protein and/or an active protein, and the active protein is selected from one or more of an activated microglial cell protein, an inhibited microglial cell protein, a calcium ion signal probe protein, a small molecule signal probe protein, a mediated apoptosis protein, a disease-related mutant protein, a normal protein under physiological conditions, a cytokine, an antiviral factor, a viral infection auxiliary receptor, a recombinase and a gene editing tool protein;
the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle localization RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.
9. An application of an adeno-associated virus vector in preparing a medicament for treating diseases caused by microglial cell abnormality, which is characterized in that the adeno-associated virus vector is rAAV11, and the adeno-associated virus vector comprises a gene for encoding therapeutic protein or functional RNA, a promoter and a neuron expression inhibition element.
10. The use according to claim 9, wherein,
the target protein is selected from a marker protein and/or an active protein, the active protein is selected from one or more of activating microglial cell protein, inhibiting microglial cell protein, normal protein under physiological condition, cytokine, antiviral factor and gene editing tool protein,
the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA and small guide RNA.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210959274.8A CN116064671A (en) | 2022-08-10 | 2022-08-10 | Application of gene delivery system in delivering genes to microglia |
PCT/CN2022/137741 WO2024031889A1 (en) | 2022-08-10 | 2022-12-08 | Use of gene delivery system in delivering gene to microglial cells |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210959274.8A CN116064671A (en) | 2022-08-10 | 2022-08-10 | Application of gene delivery system in delivering genes to microglia |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116064671A true CN116064671A (en) | 2023-05-05 |
Family
ID=86170535
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210959274.8A Pending CN116064671A (en) | 2022-08-10 | 2022-08-10 | Application of gene delivery system in delivering genes to microglia |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN116064671A (en) |
WO (1) | WO2024031889A1 (en) |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2058401A1 (en) * | 2007-10-05 | 2009-05-13 | Genethon | Widespread gene delivery to motor neurons using peripheral injection of AAV vectors |
EP2900686B1 (en) * | 2012-09-28 | 2020-06-10 | The University of North Carolina At Chapel Hill | Aav vectors targeted to oligodendrocytes |
US20150051267A1 (en) * | 2013-08-16 | 2015-02-19 | Purdue Research Foundation | BICISTRONIC GENE TRANSFER TOOLS FOR DELIVERY OF miRNAS AND PROTEIN CODING SEQUENCES |
CN114196636A (en) * | 2021-12-09 | 2022-03-18 | 中国科学院深圳先进技术研究院 | Application of adeno-associated virus vector in reverse labeling of peripheral tissue or organ primary neural network |
CN114516901B (en) * | 2022-03-11 | 2023-01-17 | 上海勉亦生物科技有限公司 | AAV vector with high affinity for nervous system and application thereof |
-
2022
- 2022-08-10 CN CN202210959274.8A patent/CN116064671A/en active Pending
- 2022-12-08 WO PCT/CN2022/137741 patent/WO2024031889A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
WO2024031889A1 (en) | 2024-02-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zerucha et al. | A highly conserved enhancer in the Dlx5/Dlx6Intergenic region is the site of cross-regulatory interactions betweenDlx genes in the embryonic forebrain | |
JPWO2003041496A1 (en) | Transgenic animals | |
CN104202970A (en) | Transgenic animals with customizable traits | |
Harel-Markowitz et al. | Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone | |
JP5971661B2 (en) | DNA having neural activity-dependent promoter activity and vector containing the same | |
EP1007714A1 (en) | Regulatory sequences capable of conferring expression of a heterologous dna sequence in endothelial cells in vivo and uses thereof | |
JPH07505534A (en) | Transcriptional control elements for increasing gene expression in myoblasts | |
EP1867716B1 (en) | Model animal in which state of disease condition is observable in real time, gene construct for achieving the same and use of the same | |
US20070130629A1 (en) | Renal Regulatory Elements and Methods of Use Thereof | |
JP5250810B2 (en) | Screening for substances that enhance utrophin gene expression | |
WO2024021431A1 (en) | Use of gene delivery system in retrograde delivery of gene from brain to spinal neuron | |
CN116064671A (en) | Application of gene delivery system in delivering genes to microglia | |
CN115323001A (en) | Gene delivery system for targeting retina and application thereof | |
US20080109914A1 (en) | Differential labeling of cells | |
WO2022232348A1 (en) | Angiotensin-converting enzyme ii (ace2) transgenic animal and uses thereof | |
US20240108756A1 (en) | Compositions and methods for using alternative splicing to control specificity of gene therapy | |
Morgan et al. | 3.6 kb of the 5′ flanking DNA activates the mouse tyrosine hydroxylase gene promoter without catecholaminergic‐specific expression | |
US6303370B1 (en) | Tissue-specific regulatory elements | |
JPH05505727A (en) | Binary gene system to control transgene expression in transgenic animals | |
US20230220402A1 (en) | Use of an orphan motif to increase expression of a heterologous transgene | |
EP2176423A1 (en) | In vivo expression analysis using ultrasound-induced transfection of reporter constructs | |
AU2001269862B2 (en) | Renal regulatory elements and methods of use thereof | |
US8771667B2 (en) | Tctex-1 regulatory sequence as stem cell marker | |
CN110747227A (en) | Blue light induced and activated Cre recombination optimization system and application thereof | |
EP2179045A2 (en) | Cartilage-specific expression |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |