WO2022021660A1 - Vecteur de co-expression de mir-16 et mir-30c, son procédé de construction et son utilisation - Google Patents

Vecteur de co-expression de mir-16 et mir-30c, son procédé de construction et son utilisation Download PDF

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WO2022021660A1
WO2022021660A1 PCT/CN2020/127155 CN2020127155W WO2022021660A1 WO 2022021660 A1 WO2022021660 A1 WO 2022021660A1 CN 2020127155 W CN2020127155 W CN 2020127155W WO 2022021660 A1 WO2022021660 A1 WO 2022021660A1
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mir
expression vector
sequence
neural stem
vector
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孙婷婷
李天鹏
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枣庄学院
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron

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  • the invention provides a combined expression vector of miR-16 and miR-30c, which is applied to the treatment of Alzheimer's stem cell proliferation or neurogenesis, and belongs to the field of biomedicine.
  • the proliferation ability of adult neural stem cells has been continuously reduced, especially recent studies have found that the occurrence of neurological diseases, especially neurodegenerative diseases, is closely related to the ability of adult neurogenesis. Enhancing neurogenesis has important application value in the diagnosis and treatment of neurological diseases.
  • miR-16 has the effect of inhibiting proliferation in various tumor cells, and it is also an inducer of early apoptosis, which can cause cell apoptosis.
  • miR-30c has a positive effect on cell proliferation and can promote neurogenesis in the subventricular zone of the nervous system and the dentate gyrus of the hippocampus.
  • the present invention provides a combined expression vector of miR-16 and miR-30c, and uses miR-16 and miR-30c as a method for regulating the proliferation of neural stem cells, specifically:
  • the present invention provides a miR-16 and miR-30c joint expression vector, comprising a miR-16 down-regulation sequence, a miR-30c over-expression sequence and a plasmid vector; the miR-16 down-regulation sequence is miR-16-KD, and the miR-16 down-regulation sequence is miR-16-KD.
  • the miR-30 overexpression sequence miR-30c-OE, its coding sequence is SEQ ID NO: 1 and SEQ ID NO: 2.
  • the plasmid vector is a pLV-shRNA2 vector
  • the expression vector is a dual-promoter expression vector, specifically a dual-promoter lentiviral vector.
  • the combined expression vector is used to promote the proliferation of neural stem cells.
  • the combined expression vector is used to promote neurogenesis.
  • the nerve cells acted by the combined expression vector are subventricular zone nerve cells and hippocampal nerve cells.
  • the combined expression vector is used in the preparation of neural stem cell migration drugs.
  • the combined expression vector is used in the application of Alzheimer's disease early detection reagents.
  • the miR-16 down-regulation vector is a sponge vector sequence complementary to the miR-16 mature sequence 8 times in series, which can capture the mature miR-16 sequence in cells;
  • the miR-30c overexpression vector is a constitutively expressed precursor sequence of miR-30c.
  • miR-16 and miR-30c were differentially expressed with development, and their expressions were negatively correlated with age.
  • the miR-16 down-regulation vector and miR-30c over-expression vector were constructed, and neural stem cells were transduced in vitro, and it was found that the down-regulation of miR-16 and the over-expression of miR-30c increased the proliferation of neural stem cells;
  • neural stem cell markers and neural precursor markers it was found that inhibiting the expression of miR-16 and promoting the expression of miR-30c can keep stem cells stem cells and inhibit their differentiation into neural precursors.
  • miR-16 and miR-30c can effectively increase the proliferation of neural stem cells in Alzheimer's mice and prevent subsequent differentiation. At the same time, it can also improve neurogenesis, which is useful for early detection and treatment of Alzheimer's disease significant.
  • AD Alzheimer's disease mouse group
  • shRNA miR-16 and miR-30c combined action group
  • Neural stem cells were labeled with GFAP and PH3 for immunofluorescence detection, scale bar, 50 ⁇ m.
  • the reaction mixture system LA Taq (5U/ ⁇ l), 2 ⁇ l, 10 ⁇ LA Taq buffer 2 ⁇ l, dNTP (2.5mM) 8 ⁇ l, pSUPER.retro-GFP/Neo plasmid 500ng, primer mix primer mixture (10 ⁇ M) 1 ⁇ l, ddH 2 O make up to 20 ⁇ l.
  • Thermal reaction system pre-denaturation at 95°C for 5 min, 30 cycles at 94°C for 30s, 57.5°C for 40s, and 72°C for 1 min; finally, extension at 72°C for 10 min, and incubation at 4°C for 60 min.
  • Reverse transcription mixed system 0.25 ⁇ l of dNTPs, 1 ⁇ l of 5 ⁇ Reverse Transcription Buffer, 0.5 ⁇ l of Reverse Transcription primer (2nM), 1.25 ⁇ l of Total RNA (250ng), 0.5 of M-MLV (40U/ ⁇ l), 0.25 ⁇ l of RNase inhibitor and RNase Free ddH 2 O 1.25 ⁇ l.
  • Reverse transcription thermal reaction system total RNA was pre-denatured at 70°C for 10min to remove the secondary structure of RNA, cooled on ice for 2min, then the reverse transcription mixture was reacted at 42°C for 1h, and heated at 70°C for 15min to extinguish and reverse recorder.
  • Amplification reaction mixture LA Taq (5U/ ⁇ l), 5 ⁇ l, 10 ⁇ LA Taq buffer 2.5 ⁇ l, dNTP (2.5mM) 8 ⁇ l, reverse transcription product 500ng, primer mixture (10 ⁇ M) 1 ⁇ l, ddH 2 O supplemented to 25 ⁇ l .
  • the thermal reaction conditions are as follows:
  • Example 1-3 Coating and titer determination of lentivirus containing double promoter pLV-U6-miR-30c-OE-H1-miR-16-KD-ZsGreen1 expression vector
  • the double-promoter pLV-U6-miR-30c-OE-H1-miR-16-KD-ZsGreen1 expression vector we need to detect the function of the two small RNAs under the combined action, so we obtained the slow expression vector of the expression vector. virus fluid.
  • the extracted pLV-U6-miR-30c-OE-H1-miR-16-KD-ZsGreen1 expression vector and the auxiliary vectors psPAX2 and PMD2.G were mixed and dissolved in the ratio (molecular weight) of 1:1:1.
  • Opti-MEM 10ml Opti-MEM was incubated at room temperature for 5min, then mixed with Opti-MEM medium containing Lipofactamine3000, incubated at room temperature for 25min, and transfected into exponential phase HEK293T cells (10cm culture dish, 2.5 ⁇ 10 6 cells). After 6 h of transfection, it was replaced with complete culture medium, and the virus-containing supernatant was harvested 48-72 h after transfection, filtered with a 45 ⁇ m filter, and then centrifuged at 24,000 rpm for 120 min for concentration.
  • viral titers need to be determined prior to brain stereotaxic.
  • the virus titer was accurately determined by flow cytometry, that is, the virus was first diluted to four concentrations of 1/10, 1/100, 1/1000 and 1/10000, and added to HEK293T cells that had been inoculated for 6 hours.
  • Single-cell suspension was prepared by lysis with 0.25% trypsin-EDTA (500 ⁇ l, 6-well plate), centrifuged to remove the supernatant (12,000 rpm, 5 min), and fixed with ethanol at -20 °C for 2 h, then washed with PBS and resuspended , the percentage of ZsGreen1 positive cells was determined by flow cytometry.
  • Example 2-1 In vitro experiments to detect the effect of the carrier on the proliferation of neural stem cells
  • Sodium pentobarbital solution (45mg/kg) was used for intraperitoneal injection to anesthetize AD pregnant mice at E14.5 days, the abdomen was cut open, the embryos were taken out from the uterine horn, and the hippocampal brain region was dissected under the stereo microscope.
  • Papain (50U) was subjected to tissue digestion at 37°C for 1 h to obtain a single cell suspension, which was cultured in Neurobasal-A medium, in which Neurobasal-A was supplemented with B27 (final concentration 20ng/ml), L-glutamine ( The final concentration of 2mM, bFGF (final concentration of 20ng/ml) and EGF (final concentration of 20ng/ml).
  • the nerve cell spheres were gently pipetted with a Pasteur pipette to form a single-cell suspension, passaged, and inoculated into poly -L-ornithine and fibronectin-coated cell plates.
  • the miR-16 down-regulation vector and miR-30c over-expression vector were transfected into AD with nucleofector kit according to the kit instructions In neural stem cells derived from pregnant mice.
  • PH3 and GFAP immunostaining and DAPI counterstaining of nuclei were followed by imaging under a fluorescence microscope to detect the effect of miR-16 and miR-30c combined vector on neural stem cell proliferation.
  • the ruler tool of Image J software calculates the diameter of neurospheres and the number of PH3 (10 neurospheres are selected for measurement and calculation in each group).
  • the results show that the combined effect of miR-16 and miR-30c group
  • the proliferation diameter of neurospheres in vitro was 1.48 times that of the control group ( Figure 1).
  • the number of neurosphere proliferative cells in vitro in the combined miR-16 and miR-30c group increased 1.72 times compared with the control group ( Figure 2).
  • Example 2-2 Microinjection of lentiviruses carrying miR-16 downregulation and miR-30c overexpression into neural stem cell proliferation areas of the brain using stereotaxic specificity in the brain: the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus .
  • mice were divided into two groups: Alzheimer's disease mouse group and lentivirus-infected mouse group (shRNA group), with 15 mice in each group.
  • the mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (45 mg/kg).
  • the mice were immobilized (binaural and maxillary immobilization) and the mouse head was pushed from all directions without movement.
  • hippocampal dentate gyrus (subventricular zone: AP+0.86mm, ML-0.8mm, DV-3.8mm; hippocampal dentate gyrus: AP-1.75mm, ML0.75mm, DV-3.8mm).
  • the number of neural stem cells and the proliferation of neural stem cells were detected using serial brain slices.
  • mice were anesthetized 4 weeks after lentivirus injection with sodium pentobarbital solution by intraperitoneal injection, followed by perfusion, gradient sucrose dehydration and continuous sagittal freezing. Section preparation (20 ⁇ m), two serial sections were selected for immunofluorescence staining, one of which was used to detect the number of neural stem cells (co-incubation of mouse anti-Nestin, goat anti-GFAP and rabbit anti-Ki67 on the blocked sections), and the other Neural proliferation was assayed in pairs (mouse anti-GFAP and rabbit anti-Ki67 or mouse anti-GFAP and rabbit anti-PH3) and incubated overnight at 4°C.
  • the proliferation of neural stem cells in the dentate gyrus and subventricular zone of the hippocampus was detected by using Image J software to count the number of ki67 cells in 15 laser confocal images of serial brain slices of each mouse (6 mice in each group), Finally, the ratio of the number of ki67 cells in the shRNA treatment group and the AD group was calculated as the multiplication of the neural stem cells in the shRNA group compared with the AD group ( Figure 3).
  • the stemness of the neural stem cells is reflected by the number of neural stem cells, and the co-targeted cells of GFAP, ki67 and Nestin are neural stem cells.
  • Image J software was used to count the number of GFAP/ki67/Nestin co-labeled cells in 15 laser confocal images of serial brain sections of each mouse (6 mice in each group), and finally the shRNA-treated group and AD were calculated.
  • the ratio of the number of neural stem cells in the group was the multiple of the neural stem cells in the shRNA group compared with the AD group ( Figure 4).

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Abstract

L'invention concerne un vecteur de co-expression de miR-16 et miR-30c, qui comprend une séquence de régulation à la baisse de miR-16, une séquence de surexpression de miR-30 c et un vecteur plasmidique. La séquence de régulation à la baisse de miR-16 est miR-16-KD et sa séquence de codage est SEQ ID NO: 1, et la séquence de surexpression de miR-30 est miR-30 c-OE et sa séquence de codage est SEQ ID NO : 1. L'invention concerne également l'utilisation du vecteur d'expression.
PCT/CN2020/127155 2020-07-28 2020-11-06 Vecteur de co-expression de mir-16 et mir-30c, son procédé de construction et son utilisation WO2022021660A1 (fr)

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