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WO2018170757A1 - Adénovirus ad-29a-185-424-tud recombinant, et construction et application associées - Google Patents

Adénovirus ad-29a-185-424-tud recombinant, et construction et application associées Download PDF

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Publication number
WO2018170757A1
WO2018170757A1 PCT/CN2017/077599 CN2017077599W WO2018170757A1 WO 2018170757 A1 WO2018170757 A1 WO 2018170757A1 CN 2017077599 W CN2017077599 W CN 2017077599W WO 2018170757 A1 WO2018170757 A1 WO 2018170757A1
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WO
WIPO (PCT)
Prior art keywords
mir
tud
adenovirus
recombinant
phbad
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PCT/CN2017/077599
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English (en)
Chinese (zh)
Inventor
毛吉炎
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深圳市博奥康生物科技有限公司
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Application filed by 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2017/077599 priority Critical patent/WO2018170757A1/fr
Publication of WO2018170757A1 publication Critical patent/WO2018170757A1/fr

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  • the present invention belongs to the field of genetic engineering, and relates to the construction of an adenovirus, and more particularly to a recombinant adenovirus which interferes with the expression of miR-29a, miR-185 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.
  • miR-185 is a length 22nt miRNA, located in human chromosome 22ql l.21, plays an important role as a tumor suppressor gene in the development and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, liver cancer, etc.
  • the tumor suppressor miRNA can directly affect the methylation level of the whole genome by directly targeting the expression of DNMT1, thereby regulating the methylation status of some genes and affecting gene expression;
  • miR-424 is a miRNA discovered in recent years. It affects the biological effects and occurrence of tumor cells by acting on target genes in various tumors and participating in the signal pathway of target gene regulation.
  • miR-424 is a multifunctional miRNA, which is involved in cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and T NF-ot; since the miR-424 promoter region has CpG Island, which is also associated with methylation-induced gene silencing.
  • 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 present invention provides a recombinant Ad-29a-185-424-Tud adenovirus containing a Tud RNA which interferes with the expression of iR-29a, miR-185 and miR-424, the Tud RNA has SEQ
  • the shuttle plasmid used to construct the recombinant Ad-29a-185-424-Tud adenovirus is pHBAd-U6-
  • the Tud RNAs that interfere with the expression of miR-29a, miR-185 and miR-424 are ligated under the U6 promoter of the shuttle plasmid.
  • framework plasmid used in the construction of the recombinant adenovirus of the present invention is pHBAd-BHG, and the packaging cell is 293 cells.
  • Ad-29a-185-424-Tud adenovirus of the present invention is constructed by designing a Tud RNA of the nucleotide sequence shown in SEQ ID No. 1, and chemically synthesizing the corresponding SEQ ID No 2 and SEQ ID No.
  • the Tud RNA complementary single-stranded target gene fragment shown in 3 is annealed to obtain a double-stranded expression template of the target gene fragment, and ligated with the BAHHI and EcoRI double-digested vector pHBAd-U6-GFP to construct an adenoviral shuttle plasmid, and then The adenovirus shuttle plasmid was co-transfected with the backbone plasmid pHBAd-BHG into 293 cells to obtain recombinant adenovirus A d-29a-185-424-Tud.
  • Ad-29a-185-424-Tud adenovirus constructed by the present invention can be used as a tool for blocking/reducing the expression of miR-29a, miR-185 and miR-424.
  • 1 is the expression level of miRNA, wherein a. miR-29a expression, b. miR-185 expression, c. miR-424 expression.
  • Tud DNA-F/Tud DNA-R 1/1 ⁇ l
  • Procedure 95 ° C for 5 min, cooled to 25 ° C at a rate of 0.1 ° C / s, and then stored at 4 ° C.
  • the linearized pHBAd-U6-GFP vector was recovered using a PCR reaction recovery kit (Axygen).
  • the annealing product of the above Tud RNA expression sequence was ligated to the BamHI and EcoRI sites of the vector pHBAd-U6-GFP with T4 ligase to construct an adenoviral shuttle plasmid, which was regulated by the U6 promoter.
  • Tud RNA and carrier-coupled reaction system 1 ⁇ annealing product; 100-200
  • Ngx linearized pHBAd-U6-GFP vector 2 ⁇ 1 T4 DNA Ligase Buffer; ⁇ ⁇ 4 DNA Ligase; diluted to ⁇ 20 to a total volume of 20 ⁇ 1.
  • the ligation reaction system was allowed to stand at 4 ° C overnight.
  • the constructed adenovirus shuttle plasmid was transformed into competent cells Jml07, resistant: ampicillin; cultured overnight at 37 °C.
  • the transformed Tud RNA was picked by a plate, and the bacteria were shaken at 37 ° C for 200 hours, and the bacterial solution was sequenced.
  • the sequencing result was consistent with the target sequence, and the plasmid containing pHBAd-T U d-29a-185-424 was obtained. Enterobacter.
  • the recombinant adenoviral vector plasmid pHBAd-Tud-29 a-185-424 and the backbone plasmid pHBAd-BHG were used, and Lipofectamine 2000 (purchased from Invitrogen) was used.
  • Dyeing reagents for transfection The specific steps are:
  • pHB Ad-Tud-29a-185-424, lO g backbone plasmid pHBAd-BHG, was diluted with 300 ⁇ DMEM medium and allowed to stand at room temperature for 5 min.
  • the 50 ml centrifuge tube was repeatedly freeze-thawed three times in a liquid nitrogen and a 37 ° C constant temperature water bath, centrifuged at 3000 rpm for 5 min, and the virus-containing supernatant was collected, and the precipitate was discarded.
  • the supernatant is the first generation of Ad-29a-185-424-T U d (P1), which is used as a virus for subsequent amplification of a large number of viruses.
  • [0047] Take 2 ml from the P1 generation virus supernatant and infect a cell of 100 mm cell culture dish (cell density is guaranteed over 90%). The remaining virus supernatant was placed in an externally-circulated cryotube, kept at -80 ° C, and retained as a poisonous species. After the virus is expanded for two days, all the cells are detached from the bottom surface and can be taken for poisoning. The cells are taken together with the culture solution into a 50 ml centrifuge tube, frozen and thawed three times according to the previous freeze-thaw method, and the supernatant is taken for the next generation amplification or Store at 80 ° C. Each generation of virus amplification and drug collection will be repeated as such. [0048] Four T75 flasks were inoculated with 400,000 293 cells per T75 culture flask, and cultured overnight, until the cells grew to 90%.
  • P2 generation virus (except for a small amount of virus-retaining species) was inoculated into the culture flask for 24 hours. 60% of the cytopathic lesions were observed under the microscope, and the cells were completely lesioned 48 hours later.
  • Harvest the diseased cell suspension centrifuge at 2000 rpm for 5 min, discard the supernatant, add 4 ml ST buffer (culture medium + 10% serum + 2.5% glycerol), vortex and mix, freeze at -80 ° C and 37 ° C After three times of centrifugation, centrifugation at 3000 rpm for 5 min, the supernatant was taken as a third generation virus (P3).
  • the WRL-68 cells were seeded into a six-well plate at a density of 20,000 cells/well, and the Ad-29a-185-424-Tud adenovirus was infected at 37° after the cells were grown to 60% confluence. Incubate for 2 h in a C 5% C02 incubator, change the medium, and continue to culture for 36 hours.
  • the miRNAs of these cells were extracted using the miRcute miRNA extraction and isolation kit, and then S-Poly(T) hsa-miR-29a qPCR-assay primer set, S-Poly(T) hsa-miR-185 qPCR-assay primer Set and S-Poly(T) hsa-miR-424 qPCR-assay primer seti box to reverse transcribe and tail the miRNA to obtain the corresponding cDNA. Take 2 kinds of cells of cDNA 2
  • the expression levels of miR-29a, miR-185 and miR-424 were detected by real-time PCR, and the experiment was repeated 3 times with 3 parallel samples per well, with snord 44 as an internal reference.
  • Fig. 2 it was found that the expression level of miR-29a in TuD-29a-185-424 cells was 57% lower than that of WRL-68 cells, and the expression level of miR-185 was 51% lower than that of WRL-68 cells.
  • the expression level of miR-424 was 65 ⁇ 3 ⁇ 4 lower than that of WRL-68 cells. The difference was statistically significant (p ⁇ 0.01), indicating that the TuD-29a-185-424 cell line was successfully constructed.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un adénovirus Ad-29a-185-424-Tud recombinant et son procédé de préparation. L'adénovirus contient un ARNTud, inhibant les expressions des miR-29a, miR-185 et miR-424 humains, d'une séquence nucléotidique telle que représentée par SEQ ID NO. 1. Le procédé de préparation de l'adénovirus consiste : à relier l'ARNTud à un promoteur U6 d'un plasmide navette, pHBAd-U6-GFP, pour préparer un plasmide navette d'adénovirus, et transfecter des cellules 293 avec le plasmide navette d'adénovirus et un plasmide squelette, pHBAd-BHG, pour obtenir l'adénovirus Ad-29a-185-424-Tud recombinant.
PCT/CN2017/077599 2017-03-22 2017-03-22 Adénovirus ad-29a-185-424-tud recombinant, et construction et application associées WO2018170757A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
CN105349502A (zh) * 2015-12-09 2016-02-24 山西医科大学 Tnfr1基因重组腺病毒及其构建

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
CN105349502A (zh) * 2015-12-09 2016-02-24 山西医科大学 Tnfr1基因重组腺病毒及其构建

Non-Patent Citations (3)

* 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, XP055538526, ISSN: 0305-1048 *
WU, KEMIN: "Mechanism and Role of miRNA-424-5p in Pancreatic Cancer", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 1 May 2014 (2014-05-01) *
ZHAO, MING: "MicroRNA-137 and MicroRNA-29a Expression and the Clinical Significance in Non-Small Cell Lung Cancer", MEDICINE & PUBLIC HEALTH , CHINA MASTER'S THESES FULL-TEXT DATABASE, 15 December 2014 (2014-12-15), ISSN: 1674-0246 *

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