WO2018170761A1 - Virus adéno-associé recombinant pour l'inactivation des expressions de mir-140, mir-152 et mir-185 - Google Patents

Virus adéno-associé recombinant pour l'inactivation des expressions de mir-140, mir-152 et mir-185 Download PDF

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Publication number
WO2018170761A1
WO2018170761A1 PCT/CN2017/077603 CN2017077603W WO2018170761A1 WO 2018170761 A1 WO2018170761 A1 WO 2018170761A1 CN 2017077603 W CN2017077603 W CN 2017077603W WO 2018170761 A1 WO2018170761 A1 WO 2018170761A1
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WIPO (PCT)
Prior art keywords
mir
tud
associated virus
recombinant adeno
recombinant
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PCT/CN2017/077603
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English (en)
Chinese (zh)
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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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Priority to PCT/CN2017/077603 priority Critical patent/WO2018170761A1/fr
Publication of WO2018170761A1 publication Critical patent/WO2018170761A1/fr

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  • the present invention belongs to the field of molecular biology and biomedicine technology, and particularly relates to a recombinant adeno-associated virus which knocks down the expression of miR-140, mi R-152 and miR-185.
  • 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-140 is closely related to the development of various diseases, such as bone and joint diseases, liver diseases, pituitary adenomas, testicular development, head and neck tumors, ovarian and breast diseases. miR-140 can inhibit the proliferation and invasion and metastasis of hepatocellular carcinoma by targeting TG FBR1 and other gene expression. miR-140 is highly expressed in articular cartilage and plays a vital role in the pathogenesis of osteoarthritis.
  • miR-152 is a kind of Functional miRNAs, studies have found that miR-152 is associated with methylation, such as methyltransferase DNMT1 content and enzyme activity, miR-152 can be methylated by endometrial cancer DNA into a silent gene, and its Related to the development of cancer, it is a tumor suppressor microRNA, which is associated with many diseases such as preeclampsia, trophoblastic tumor, bladder cancer, gastrointestinal cancer, ovarian cancer, etc.
  • miR-185 is a 22 nt miRNA, localization Human chromosome 22ql 1.21 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, and liver cancer, and it also has a methylation phase.
  • the inhibition of miRNA may be through direct targeted expression DNMT1 level of methylation of the genome, and thus the regulation of the methylation status of certain modification genes, gene expression.
  • the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer. technical problem
  • 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.
  • Adeno-associated virus is a non-pathogenic human parvovirus that can infect a wide variety of cells, including mitotic and non-dividing cells, and is characterized by its safety, long-lasting, high-efficiency, and high specificity.
  • researchers are highly regarded and favored and are widely used in the field of biology.
  • the use of AAV as a gene therapy vector has potential advantages.
  • the technical solution adopted by the present invention is: a knockdown of miR-140, miR-152 and mi
  • the recombinant adeno-associated virus expressed by R-185 was constructed by the following methods:
  • the Tud RNA sequences specific for miR-140, 1 ⁇ 11-152 and 1 ⁇ 11-18 5 were designed to construct the recombinant plasmid pAKD-Tud-140-152. -185, and performing DNA sequencing analysis;
  • the Tud RNA sequence specific for miR-140, miR-152 and miR-185 in step a is: 5'- GCCCAAGATGATCCTAGCGCCACCTTTTT -3,.
  • step a a Kpn I and Bg III endonuclease cleavage site sequence and a transcription termination sequence are added to the selected target sequence to synthesize a complete Tud DNA sequence:
  • Tud DNA-F 5'-
  • the present invention constructs a recombinant adeno-associated virus rAAV-T ud-140-152-185 which knocks down miR-140, miR-152 and miR-185, and can stably express the target protein and high biosafety for a long time.
  • the serotype 8 adeno-associated virus (AAV8) is a transduction vector that stably expresses Tud RNA in vivo for a long time, thereby knocking down the expression of miR-140, miR-152 and miR-185.
  • FIG. 1 is a schematic diagram showing the results of quantitative PCR detection of miRNA expression levels in each group of cells, wherein a. miR-140 expression, b. miR-152 expression, c. miR-185 expression.
  • Example 1 Construction of a recombinant adeno-associated virus expressed by miR-140, miR-152 and miR-185
  • the cleavage site sequence and transcription termination sequence of Kpn I and Bgl II endonucleases were commissioned by Shanghai Yingjun Biotechnology Co., Ltd. to synthesize the complete Tud DNA sequence ij ij.
  • Tud-140-152-185 The Tud DNA template was annealed, and after annealing, a double-stranded oligonucleotide fragment having a Kpn I and Bgl II restriction sites at both ends was formed, which was labeled Tud-140-152-185.
  • the pAKD-shRNA plasmid vector was obtained by linear digestion with Kpn I and Bgl II, and an appropriate amount of the product was detected by 1% agarose gel electrophoresis and recovered.
  • the double-stranded DNA was ligated into the restriction vector according to the ligation reaction system, ligated and incubated at 4 ° C overnight, and transformed into competent E. coli DH5oc, which was plated and incubated overnight. Single colonies were picked for cultivation and sent to Shanghai Yingjun Biotechnology for sequencing. The bacteria with the correct sequencing results were cultured, and the pAKD-Tud-140-152-185 plasmid was extracted using the Promega plasmid extraction kit (endotoxin).
  • Virus packaging, purification and titer determination was obtained by linear digestion with Kpn I and Bgl II, and an appropriate amount of the product was detected by 1% agarose gel electrophoresis and recovered.
  • the double-stranded DNA was ligated
  • AAV-293 cell culture is carried out by a conventional method, and when the cells reach 70-80% fusion, the calcium phosphate method is used for virus transfection, and rAAV8 expressing Tud-140-152-185 is obtained, and virus collection is performed. Concentrate and purify.
  • the virus was titrated by a quantitative PCR method.
  • the number of viral particles of rAAV is determined by detecting the genomic copy number of the rAAV vector in the viral genome, and the titer unit is expressed in vg/ml, that is, the number of viral genomes per ml (Vims Genome).
  • the virus sample to be tested was digested with DNase and RNase at 37 ° C for 2 ⁇ 3 h, and the virus DNA was extracted and denatured in a boiling water bath for 5 min, immediately placed on ice for 2 min, and the virus sample and the standard product were diluted to different concentrations.
  • the primers used were: upstream: 5'-CCTTTCCGGGACTTTCGCTTT-3', downstream: 5,-GCAGAATCCAGGTGGCAAC A-3.
  • the reaction conditions were: denaturation at 94 ° C for 15 s, annealing at 52 ° C for 30 s, and extension at 72 ° C for 30 s, for a total of 40 cycles.
  • a standard curve was drawn based on the standard and the virus sample titer was calculated.
  • the average virus titer was calculated to be 1.05x1012 vg/ml.
  • Example 2 AAV transfection ACHN cells
  • ACHN cells were inoculated into a six-well plate, 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 1000 times with DMEM complete medium to remove the medium in the 6-well plate. , add virus-containing DMEM complete medium (containing 10% fetal bovine serum), 24h
  • the miRNAs of normal ACHN cells and TuD-140-152-185 cells were extracted using the miRcute miRNA extraction and isolation kit, followed by S-Poly(T) hsa-miR-140 qPCR-assay primer set, S-Poly ( T) hsa-miR-152 qPCR-assay primer set and S-Poly(T) hsa-miR-185 qPCR-assay primer set kit for reverse transcription and tailing of miRNA to obtain the corresponding cDNA.
  • the cDNA of each of the two cells was used as a template.
  • the expression levels of miR-140, miR-152 and miR-185 were detected by real-time PCR.
  • the present invention constructs a recombinant adeno-associated virus rAAV-T ud-140-152-185 which knocks down miR-140, miR-152 and miR-185, and has a long-term stable expression of the target protein and high biosafety.
  • the adeno-associated virus (AAV8) is a transduction vector that stably expresses Tud RNA in vivo for a long time, thereby knocking down the expression of miR-140, miR-152 and miR-185.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un virus adéno-associé recombinant, capable d'exprimer un ARN Tud pour miR-140, miR-152 et miR-185, ce qui permet d'inactiver les expressions de miR-140, miR-152 et miR-185.
PCT/CN2017/077603 2017-03-22 2017-03-22 Virus adéno-associé recombinant pour l'inactivation des expressions de mir-140, mir-152 et mir-185 WO2018170761A1 (fr)

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PCT/CN2017/077603 WO2018170761A1 (fr) 2017-03-22 2017-03-22 Virus adéno-associé recombinant pour l'inactivation des expressions de mir-140, mir-152 et mir-185

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PCT/CN2017/077603 WO2018170761A1 (fr) 2017-03-22 2017-03-22 Virus adéno-associé recombinant pour l'inactivation des expressions de mir-140, mir-152 et mir-185

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Citations (1)

* 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

Patent Citations (1)

* 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

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
B SONG ET AL.,: "Mechanism of chemoresistance mediated by miR-140 in human osteo- sarcoma and colon cancer cells", ONCOGENE, vol. 28, no. 46, 7 September 2009 (2009-09-07), pages 4065 - 4074, XP055022150, ISSN: 0950-9232 *
DATABASE Nucleotide 11 December 2014 (2014-12-11), YU M.: "Homo sapiens microRNA 185 (MIR185), microRNA", XP055540744, retrieved from NCBI Database accession no. NR_029706 *
DATABASE Nucleotide 21 May 2015 (2015-05-21), YAO Y.: "Homo sapiens microRNA 152 (MIR152), microRNA", XP055540739, retrieved from NCBI Database accession no. NR_029687 *
DATABASE Nucleotide 21 May 2015 (2015-05-21), YUAN L: "Homo sapiens microRNA 140 (MIR140), microRNA", XP055608377, retrieved from NCBI Database accession no. NR_029681 *
LINWENSI ZHU: "The Structure and Clinical Roles of MicroRNA in Colorectal Cancer", GASTROENTEROLOGY RESEARCH AND PRACTICE, 31 December 2016 (2016-12-31), XP055540703, ISSN: 1687-630X *

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