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WO2017219168A1 - Vecteur lentiviral destiné à inactiver l'expression de miarn-29a et de mir-152 et application associée - Google Patents

Vecteur lentiviral destiné à inactiver l'expression de miarn-29a et de mir-152 et application associée Download PDF

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Publication number
WO2017219168A1
WO2017219168A1 PCT/CN2016/086334 CN2016086334W WO2017219168A1 WO 2017219168 A1 WO2017219168 A1 WO 2017219168A1 CN 2016086334 W CN2016086334 W CN 2016086334W WO 2017219168 A1 WO2017219168 A1 WO 2017219168A1
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mir
sequence
cells
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vector
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毛侃琅
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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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N15/867—Retroviral vectors

Definitions

  • the present invention relates to the field of gene editing and epigenetics, and in particular to a lentiviral vector for knockdown of miRNA-29a and miR-152 and uses thereof.
  • MicroRNAs are a class of endogenous, non-coding RNAs found in eukaryotes, typically between 22 and 25 nt in size. miRNAs are widely distributed in plants, animals, and multicellular organisms, and can Play an important regulatory role, and in the study of human miRNAs, it is found that the expression of miRNA in normal tissues and tumor tissues is significantly different, some miRNAs are lowly expressed in tumor tissues, and some are highly expressed in tumor tissues. This suggests that miRNAs play a crucial role in tumorigenesis.
  • miR-29a is a small RNA closely related to cell proliferation. It is involved in many diseases and can act as a tumor suppressor gene in a variety of tumors. It has the ability to grow and invade cells such as human gastric cancer and bladder cancer.
  • miR-152 is a kind of Multi-functional miRNA, the study found that miR-152 is related to methylation, such as the methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and its It is involved in the development of various cancers. It is a tumor suppressor microRNA, which is associated with many diseases such as pre-eclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, and ovarian cancer. By controlling the expression of miR-29a and miR-152, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer. technical problem
  • miRNA silencing is the presentation of synthetic oligonucleotides into cells, with endogenous miRNAs.
  • a heteroduplex causes the miRNA to reduce the inhibition of the target gene to achieve regulation of gene function.
  • Silencing of miRNAs is currently difficult to achieve.
  • Commonly used methods for silencing miRNA are anti- miR, antagomiR, miRNA sponge, etc., wherein anti-miR and antagomiR are In the transient transfection technology, the interference effect can not be maintained stably, and the miRNA sponge effect is far from optimal. At present, there is no report on the optimization of miR-29a and miR-152 interference.
  • Tough Decoy RNA is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.
  • the object of the present invention is to provide a lentiviral vector for constructing homologous interference miR-29a and miR-152 and constructing a lentiviral vector capable of stably maintaining interference effects, aiming at the deficiencies in the prior art. Applied to the field of gene editing.
  • the gene interference sequence of the corresponding TuD RNA against miR-29a and miR-152 was designed and synthesized, and the nucleotide sequence thereof is shown in SEQ ID NO.: 1.
  • This sequence was ligated to the lentiviral vector pLKO.l-puro to obtain a lentiviral vector pLKO-T U d-29a-152 lentiviral vector which stably maintains the interference effect, and its nucleotide sequence ⁇ IJ is as SEQ ID NO.: 2 is shown.
  • the present invention is designed to synthesize a gene interference sequence targeting the corresponding TuD RNA of miR-29a and miR-152, and is ligated to the lentiviral vector pLKO.l-puro to form a vector capable of interfering with miR-29a.
  • the specific integration steps are as follows:
  • the sequence information of miR-29a and miR-152 provided in miRBase was designed to design the same TuD RNA oligonucleotide sequence for mi R-29a and miR-152, and its sequence is as SEQ ID NO.: 1 Show, commissioned Shanghai Biotech to synthesize the sequence by means of primers.
  • the synthesized sequence is two complementary single-stranded DNAs.
  • the two single-stranded DNAs were dissolved in ddH 2 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-29a-152, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Five single colonies were picked from the plates and added to 5 tubes containing ampicillin in liquid LB medium for 8 hours at 37 °C. The bacteria were sent to Shanghai Biotech for sequencing. Sequencing was performed in the correct strain and extracted with a small amount of endotoxin-free extraction kit. The extracted plasmid was the plasmid for miR-29a and miR-152 which was required for the present invention.
  • the homologous interference miR-29a and miR-152 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.
  • FIG. 1 is a schematic view showing the structure of a pLKO-TuD-29a-152 lentiviral expression vector according to an embodiment
  • FIG. 2 is a flow diagram showing the steps required to transform the lentiviral expression vector shown in FIG. 1 into the lentiviral vector of the present invention
  • Example 3 is a miRNA expression level of 16HBE cells and TuD-29a-152 cells in Example 6, wherein
  • the lentiviral plasmid pLKO.l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from the United States ATCC; S-Poly(T) hsa-miR- 29a qPCR-assay primer set and S-Poly(T) hsa-miR-152 qPCR-assay primer set
  • the miRNA reverse transcription and fluorescence quantification kit was purchased from Shenzhen Anran Biotechnology Co., Ltd.
  • TuD RNA oligonucleotide sequence targeting miR-29a and miR-152 was designed and its sequence was as shown in SEQ ID NO.: l, and was commissioned by Shanghai Biotech to synthesize by gene synthesis.
  • the synthesized sequence is two complementary single-stranded DNA.
  • the two single-stranded DNAs were dissolved in ddH20, mixed at an equimolar ratio, and treated at 95 ° C for 5 min, and then allowed to stand at room temperature to allow them to naturally cool to room temperature.
  • the vector pLK0.1-puro was extracted and digested with Age I and Eco RI for 16 h, and the digested vector was recovered with MinElute Reaction Cleanup Kit, and then T4 DNA was used.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-29a-152, and finally the ligation product was transformed into competent E. coli ToplO and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Five single colonies were picked from the plate and added to 5 test tubes of liquid LB medium containing ampicillin for 8 h at 37 °C. The bacteria were sent to Shanghai Biotech for sequencing. The sequencing results were completely correct. This is the pLKO-T U d-29a-152 lentiviral recombinant vector.
  • the dilution ratio of the recombinant lentivirus solution is 1, 10, 100, 1000, 10000, 10,000, 1000000, 10000000, and 100000000, and the solution gradient of the recombinant lentivirus is used in the medium. Diluted, then 100 gradient dilutions of the recombinant lentivirus solution and 10 (L perforated plate)
  • the cell culture medium was mixed and transfected in different wells of the multi-well plate. 24 h after the start of transfection, the medium was aspirated and replaced with 50 (L containing 5 U DNasel in fresh medium, and cultured at 37 ° C for 30 min to remove possible adhesion. Remaining plasmid DNA on the cell surface, then changing the medium to 1 mL of normal medium, and continuing to culture for 48 hours;
  • the medium in each well of the multiwell plate was aspirated, 50 (L-trypsin-EDTA solution was added to digest the cells, reacted at 37 ° C for 1 minute, and then the medium was added to terminate the digestion reaction. After the cells are purged, the cells of each well are collected by centrifugation, the total RNA of each well is extracted, and then the total cDNA of each well is reverse-transcribed; and the total cDNA of each of the obtained cells is separately fluorescent.
  • Quantitative PCR was performed to obtain the ct value of each well of the cells, and the experimental group with the smallest difference from the ⁇ value of the control group but exceeding 2 was selected to obtain the dilution factor, and the lentivirus titer was calculated according to the following formula:
  • T 20000 x R, where T is the lentivirus titer, T is in units of TU/mL, and R is the dilution factor.
  • the lentivirus titer of the package is greater than 10000000 TU/mL, indicating that the packaging of the lentivirus is successful.
  • 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 50% after 12 hours.
  • the virus solution was taken separately, and the virus was diluted 10 times with DMEM complete medium, and then polyglycolamine was added.
  • the medium in the 6-well plate was removed, and the virus-containing DMEM complete medium (containing 10% fetal bovine serum) was added. After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used.
  • the cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.0 g/ml.
  • the cell line obtained by screening was named TuD-29a-152 cell line.
  • the homologous interference miR-29a and miR-152 TuD RNA sequences designed by the invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the commonly used single-stranded miRNA sponge, and the same ⁇ Targeting two targets can better achieve the interference of two miRNAs and improve the efficiency of miRNA function research.

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Abstract

L'invention concerne un vecteur lentiviral destiné à inactiver l'expression de miARN-29a et de miR-152, comprenant une séquence fondamentale, une séquence de gène de résistance, une séquence de site de clonage multiple, une séquence de promoteur, ainsi qu'une séquence d'oligonucléotides ciblant miR-29a et miR-152 d'un vecteur d'expression de pLKO.1-puro. Un site de clonage multiple comprend un site de coupe d'enzyme Age I et un site de coupe d'enzyme EcoR I; la séquence d'oligonucléotides ciblant miR-29a et miR-152 est insérée dans le site de clonage multiple dans une orientation sens. Le vecteur d'expression lentiviral selon l'invention présente les avantages d'une efficacité de transfection élevée, d'une petite quantité requise et d'une inhibition continue, efficace, stable et spécifique de l'expression humaine de miARN-29a-152, et il peut servir d'instrument puissant dans la préparation de médicaments destinés au traitement de maladies liées à une expression anormale de miARN-29a-152.
PCT/CN2016/086334 2016-06-19 2016-06-19 Vecteur lentiviral destiné à inactiver l'expression de miarn-29a et de mir-152 et application associée Ceased WO2017219168A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102218144A (zh) * 2010-04-13 2011-10-19 江苏命码生物科技有限公司 一种调节生物体内微小核糖核酸含量的方法及其用途
CN105030808A (zh) * 2007-07-31 2015-11-11 得克萨斯系统大学董事会 调控纤维化的微小rna家族及其用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105030808A (zh) * 2007-07-31 2015-11-11 得克萨斯系统大学董事会 调控纤维化的微小rna家族及其用途
CN102218144A (zh) * 2010-04-13 2011-10-19 江苏命码生物科技有限公司 一种调节生物体内微小核糖核酸含量的方法及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LIU TAO ET AL.: "Construction and Application of Lentiviral Vectors Expressing MicroRNA-203 Tough Decoy", TIANJIN MEDICAL JOURNAL, vol. 42, no. 10, 31 October 2014 (2014-10-31), pages 961 - 964 *
XIE, XING ET AL.: "Construction of a Human Bronchial Epithelial hsa-miR-148a-3p Knockdown Cell Line", CARCINOGENESIS, TERATOGENESIS & MUTAGENESIS, vol. 26, no. 3, 31 May 2014 (2014-05-31), pages 205, XP055447857, DOI: doi:10.3969/j.issn.1004-616x.2014.03.010 *
ZHANG, PENG ET AL.: "Effect of miR-29aig Inhibition Expression on Decidua PRL Production of Decidualized Human Uterus Endometrial Stromal Cells", SHANDONG MEDICAL JOURNAL, vol. 56, no. 7, 19 February 2016 (2016-02-19) *
ZHU, CHEN ET AL.: "MiR 152 Regulates the Migration and Invasion of Prostate Cancer Cells through the Regulation of TGF", MEDICINE & PUBLIC HEALTH , CHINA MASTER'S THESES FULL-TEXT DATABASE, 15 December 2013 (2013-12-15), pages 14 *

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