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WO2017219170A1 - Construction et application d'un vecteur lentiviral pour l'inhibition spécifique de l'expression de miarn-29a humain et de mir-424 humain - Google Patents

Construction et application d'un vecteur lentiviral pour l'inhibition spécifique de l'expression de miarn-29a humain et de mir-424 humain Download PDF

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WO2017219170A1
WO2017219170A1 PCT/CN2016/086336 CN2016086336W WO2017219170A1 WO 2017219170 A1 WO2017219170 A1 WO 2017219170A1 CN 2016086336 W CN2016086336 W CN 2016086336W WO 2017219170 A1 WO2017219170 A1 WO 2017219170A1
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sequence
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vector
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毛侃琅
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    • 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 the construction and application of a lentiviral vector which specifically inhibits the expression of human mi RNA-29a and miR-424.
  • 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. Correlation, its expression level is an important reference for evaluating the benign and malignant gliomas. It is also associated with diseases such as atherosclerosis and liver fibrosis. It has important potential application value for the treatment of various tumors; miR-424 is found in recent years.
  • miRNA that affects the biological effects and development of tumor cells by acting on target genes by acting on target genes in a variety of tumors, exerting effects similar to oncogenes, tumor suppressor genes, or promoting, Inhibit the invasion and metastasis of tumors.
  • miR-424 is a multifunctional miR NA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-oc; since the miR-424 promoter region has CpG Island, which is also associated with methylation-induced gene silencing.
  • miRNA silencing is the presentation of synthetic oligonucleotides into cells, with endogenous miRNAs.
  • the miRNA reduces the inhibition of the target gene, in order to achieve the 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.
  • anti-miR and antagomiR are transient transfection techniques, and the interference effect cannot be stably maintained, while the miRNA sponge effect is far from optimal. There are no reports of homologous optimization of miR-29a and miR-424 interference to enhance its effect.
  • 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-424, and constructing a lentiviral vector capable of stably maintaining interference effects, in view of 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-424 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-424 lentiviral vector which stably maintains the interference effect, and its nucleotide sequence ⁇ IJ is as SEQ ID NO.: 2 is shown.
  • the present invention constructs and synthesizes the gene interference sequence of the corresponding TuD RNA of miR-29a and miR-424, and is ligated to the lentiviral vector pLKO.l-puro, and the resulting vector has the same interference with miR-29a.
  • the specific integration steps are as follows:
  • the sequence information of miR-29a and miR-424 provided in miRBase was designed to design the same TuD RNA oligonucleotide sequence for mi R-29a and miR-424, and its sequence is as SEQ ID NO.: 1 Show, commissioned Shanghai Biotech to synthesize the sequence by means of primers.
  • S20 The synthesized sequence is two complementary single-stranded DNAs.
  • the two single-stranded DNAs were dissolved in ddH20, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.
  • S30 the extraction vector pLK0.1-puro, after digestion with Ag I and Eco RI enzyme for 16 h, the enzymatically digested vector was recovered by MinElute Reaction Cleanup Kit, and the previous step was obtained by using T4 DNA ligase.
  • RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-29a-424, 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 on 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-424 which were required for the present invention.
  • the homologous interference miR-29a and miR-424 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-424 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-424 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-424 qPCR-assay primer set
  • the miRNA reverse transcription and fluorescence quantification kit was purchased from Shenzhen Anran Biotechnology Co., Ltd.
  • the TuD RNA oligonucleotide sequence targeting miR-29a and miR-424 was designed, and its sequence is SEQ ID. ⁇ .: 1, commissioned by Shanghai Biotech to synthesize by means of 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, treated at 95 ° C for 5 min, and allowed to cool to room temperature by allowing them to stand at 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-424, 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-Tud-29a-424 lentiviral recombinant vector.
  • the dilution ratio of the solution of the recombinant lentivirus is 1, 10, 100, 1000, 10000, 1 00000, 1000000, 10000000 and 100000000
  • the solution of the recombinant lentivirus was serially diluted with a medium, and then 100 gradient-diluted solutions of the recombinant lentivirus and 10 (L perforated plate in a cell culture medium in a multiwell plate) Mixed transfection in different wells, 24 h after transfection, aspirate the medium and replace with 50 (L containing 5 U DNasel in fresh medium, incubate at 37 °C for 30 min to remove residual plasmid DNA that may attach to the cell surface Then, the medium was changed to 1 mL of normal medium, and the cultivation was continued 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-424 cell line.
  • Normal 16HBE cells and TuD-29a-424 cells were inoculated into 6-well plates (about 300,000 per well), and the cells were cultured for about 24 hours to a degree of fusion of 80%.
  • the miRNAs of these cells were extracted using the miRcute miRNA extraction and isolation kit, followed by S-Poly(T) hsa-miR-29a qPCR-assay primer set and S-Poly(T) hsa-miR-424 qPCR-assay primer set kit.
  • the miRNA is reverse transcribed and tailed to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2 As a template, real-time PCR was used to detect the expression levels of miR-29a and miR-424. The experiment was repeated 3 times, and 3 parallel samples were set per well to snord.
  • the homologous interference miR-29a and miR-424 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 la construction et l'application d'un vecteur lentiviral permettant d'inhiber spécifiquement l'expression de miARN-29a humain et de miR-424 humain. Le vecteur lentiviral comprend une séquence fondamentale, une séquence de gène de résistance, une séquence de sites multiples de clonage, une séquence de promoteur, et une séquence oligonucléotidique ciblant miR-29a et miR-424 d'un vecteur d'expression de pLKO.1-puro. Le site multiple de clonage comprend un site de découpe de l'enzyme Age I et un site de découpe de l'enzyme EcoR I, la séquence oligonucléotidique ciblant miR-29a et miR-424 est insérée dans le site multiple de clonage dans l'orientation sens. Un vecteur d'expression lentiviral de pLKO-Tud-29a-424 présente les avantages d'une efficacité de transfection élevée, d'une petite quantité requise, et d'une inhibition spécifique, stable, efficace et continue de l'expression de miARN-29a humain et de miR-424 humain, et peut servir d'outil puissant pour la préparation de médicaments destinés à traiter des maladies associées à l'expression anormale de miARN-29a et de miR-424.
PCT/CN2016/086336 2016-06-19 2016-06-19 Construction et application d'un vecteur lentiviral pour l'inhibition spécifique de l'expression de miarn-29a humain et de mir-424 humain Ceased WO2017219170A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133901A2 (fr) * 2010-04-23 2011-10-27 University Of Massachusetts Traitement à base d'aav pour des troubles liés au cholestérol
CN102703507A (zh) * 2012-05-18 2012-10-03 深圳市疾病预防控制中心 特异抑制肝细胞CYP2E1基因表达的shRNA慢病毒表达载体及其构建方法与应用
CN102939090A (zh) * 2010-04-01 2013-02-20 法兰克福大学 miRNA-29表达拮抗剂及其在预防和治疗主动脉瘤中的用途
WO2013192486A1 (fr) * 2012-06-21 2013-12-27 Miragen Therapeutics Inhibiteurs de la famille mir-15 de micro-arn
CN104685056A (zh) * 2012-06-21 2015-06-03 米拉根医疗股份有限公司 包含锁核酸基序的基于寡核苷酸的抑制剂
WO2015164786A1 (fr) * 2014-04-25 2015-10-29 University Of Massachusetts Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102939090A (zh) * 2010-04-01 2013-02-20 法兰克福大学 miRNA-29表达拮抗剂及其在预防和治疗主动脉瘤中的用途
WO2011133901A2 (fr) * 2010-04-23 2011-10-27 University Of Massachusetts Traitement à base d'aav pour des troubles liés au cholestérol
CN102703507A (zh) * 2012-05-18 2012-10-03 深圳市疾病预防控制中心 特异抑制肝细胞CYP2E1基因表达的shRNA慢病毒表达载体及其构建方法与应用
WO2013192486A1 (fr) * 2012-06-21 2013-12-27 Miragen Therapeutics Inhibiteurs de la famille mir-15 de micro-arn
CN104685056A (zh) * 2012-06-21 2015-06-03 米拉根医疗股份有限公司 包含锁核酸基序的基于寡核苷酸的抑制剂
WO2015164786A1 (fr) * 2014-04-25 2015-10-29 University Of Massachusetts Vecteurs de virus adéno-associés recombinants utiles pour réduire une immunité contre des produits transgéniques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HARAGUCHI, T. ET AL.: "Vectors Expressing Efficient RNA Decoys Achieve the Long-Term Suppression of Specific Microrna Activity In Mammalian Cells", NUCLEIC ACIDS RESEARCH, vol. 37, no. 6, 17 February 2009 (2009-02-17), pages E43-1 - E43-13, XP002656483 *
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 204 - 208, XP055447857, DOI: doi:10.3969/j.issn.1004-616x.2014.03.010 *

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