WO2019000151A1 - Virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-arn - Google Patents

Virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-arn Download PDF

Info

Publication number
WO2019000151A1
WO2019000151A1 PCT/CN2017/089931 CN2017089931W WO2019000151A1 WO 2019000151 A1 WO2019000151 A1 WO 2019000151A1 CN 2017089931 W CN2017089931 W CN 2017089931W WO 2019000151 A1 WO2019000151 A1 WO 2019000151A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
tud
associated virus
recombinant adeno
expression
Prior art date
Application number
PCT/CN2017/089931
Other languages
English (en)
Chinese (zh)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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/CN2017/089931 priority Critical patent/WO2019000151A1/fr
Publication of WO2019000151A1 publication Critical patent/WO2019000151A1/fr

Links

Classifications

    • 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
    • C12N15/11—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

Definitions

  • the present invention belongs to the field of molecular biology and biomedical technology, and in particular relates to a recombinant adeno-associated virus for synchronously inhibiting the expression of three microRNAs.
  • 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-140 plays an oncogene role in some tumors, and plays a tumor suppressor role in other tumors, and is associated with multiple tumor chemotherapy resistance; miR-185 is a 22nt in length
  • the miRNA located at the 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, and liver cancer, and it is also associated with methylation.
  • Anti-tumor miRNA can 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 development of tumor cells by acting on target genes and participating in the signal transduction pathway of target genes in a variety of tumors. , Tumor suppressor gene, or promoting, inhibition of tumor invasion and metastasis.
  • miR-424 is a multifunctional miRNA, 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-ot; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing.
  • miR-140, miR-185 and miR-424 peers work synergistically with other drugs to provide new treatments for cancer. Epigenetic ideas.
  • 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.
  • a recombinant adeno-associated virus for synchronously inhibiting the expression of three microRNAs is constructed by the following methods:
  • b recombinant plasmid pAKD-Tud-140-185-424, packaging plasmid pAAV-RC and helper plasmid pHelper were co-transfected into AAV-293 cells with calcium phosphate method, and recombinant adeno-associated virus rAAV-Tud-140- was harvested.
  • 185-424, the titer of the virus was determined by quantitative PCR.
  • the specific Tud RNA sequence for miR-140, miR-185 and miR-424 in step a is: 5'- TGCCCAAGATGATCCTAGCGCCACCTTTTT -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-Tu d-140-185-424 for simultaneously inhibiting the expression of three microRNAs, and adopts a type 8 which can stably express a target protein for a long period of time and is highly biosafety.
  • 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-185 and miR-424.
  • 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-185 expression, c. miR-424 expression.
  • Example 1 Construction of a recombinant adeno-associated virus that inhibits the expression of miR-140, miR-185 and miR-424
  • Tud RNA sequences specific for miR-140, miR-185 and miR-424 were designed: 5' -
  • 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-140-185-424.
  • 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-140-185-424 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 lig
  • 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-140-185-424 can be 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 1.13x1012 vg/ml.
  • T24 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 T24 cells and TuD-140-185-424 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-185 qPCR-assay primer set and S-Poly(T) hsa-miR-424 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, 11 11 -185 and 1 ⁇ 1 -424 were detected by real-time PCR.
  • the present invention constructs a recombinant adeno-associated virus rAAV-Tu d-140-185-424 for synchronously inhibiting the expression of three microRNAs, and adopts an adeno-associated virus type 8 which can stably express the target protein for a long period of time and is highly biosafety.
  • 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-185 and miR-424.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-ARN. Le virus adéno-associé recombinant rAAV-Tud-140-185-424 peut être exprimé de manière stable in vivo pendant une longue période, jouant ainsi un rôle dans le knock-down des expressions de miR-140, miR-185 et miR-424.
PCT/CN2017/089931 2017-06-26 2017-06-26 Virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-arn WO2019000151A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/089931 WO2019000151A1 (fr) 2017-06-26 2017-06-26 Virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-arn

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/089931 WO2019000151A1 (fr) 2017-06-26 2017-06-26 Virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-arn

Publications (1)

Publication Number Publication Date
WO2019000151A1 true WO2019000151A1 (fr) 2019-01-03

Family

ID=64741932

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/089931 WO2019000151A1 (fr) 2017-06-26 2017-06-26 Virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-arn

Country Status (1)

Country Link
WO (1) WO2019000151A1 (fr)

Citations (3)

* 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
CN102264898A (zh) * 2008-10-23 2011-11-30 国立大学法人东京大学 微小rna的功能抑制方法
CN102851291A (zh) * 2006-04-03 2013-01-02 桑塔里斯制药公司 包含抗微小rna 反义寡核苷酸的药物组合物

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102851291A (zh) * 2006-04-03 2013-01-02 桑塔里斯制药公司 包含抗微小rna 反义寡核苷酸的药物组合物
CN102264898A (zh) * 2008-10-23 2011-11-30 国立大学法人东京大学 微小rna的功能抑制方法
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SU, QUANQIU ET AL.: "Research progress on the relationship between miR-185 and tumor", JOURNAL OF MODERN MEDICINE & HEALTH, vol. 31, no. 3, 15 February 2015 (2015-02-15), pages 380 - 383, ISSN: 1009-5519 *

Similar Documents

Publication Publication Date Title
CN107502608B (zh) 用于敲除人ALDH2基因的sgRNA、ALDH2基因缺失细胞株的构建方法及应用
JP7432521B2 (ja) 新規小分子活性化ｒｎａ
CN106399313B (zh) 一种抗PRRSV的microRNA样病毒小RNA序列及其用途和检测方法
CN112760320B (zh) 有效抑制猪流行性腹泻病毒复制的外源性人工miRNA及其用途
CN107760718B (zh) 绵羊cnksr2基因双荧光素酶报告基因载体及构建方法和应用
CN109402269B (zh) 一种检测miRNA-217-5p的试剂在制备鸭肠道应激检测试剂中的用途
WO2019000151A1 (fr) Virus adéno-associé recombinant pour inhiber simultanément les expressions de trois micro-arn
WO2018165929A1 (fr) Vecteur d'expression inhibant le micro-arn double brin, son procédé de mise au point et son application
CN111808858B (zh) 一种siRNA序列及其靶标在提高PEDV毒价中的应用
WO2018170761A1 (fr) Virus adéno-associé recombinant pour l'inactivation des expressions de mir-140, mir-152 et mir-185
WO2019000143A1 (fr) Virus adéno-associé recombinant inactivant l'expression de miarn
WO2018170760A1 (fr) Virus adéno-associé recombinant pour l'inhibition ciblée des expressions de mir-140, mir-148a et mir-424
CN110564743A (zh) 一种六盘山黄牛circR-UQCC1基因及其过表达载体、构建方法和应用
CN111228292B (zh) 人tpt1/tctp基因在制备抗肿瘤药物中的应用
CN106591311B (zh) 核酸及其用途
CN103045598A (zh) 一种表达猪microRNA-378的方法及专用DNA片段
WO2018170759A1 (fr) Adénovirus recombinant ad-140-148a-185-tud, construction et application de celui-ci
WO2018170757A1 (fr) Adénovirus ad-29a-185-424-tud recombinant, et construction et application associées
WO2018170623A1 (fr) Méthode de réduction des expressions de miarn-152 et de miarn-424
Cao et al. Computer-based siRNA design to hepatitis B virus and its expression vector construction
WO2018170758A1 (fr) Adénovirus ad-140-148a-152-tud recombinant, et construction et application de celui-ci
WO2018170753A1 (fr) Arn tud pour l'inactivation de miarn-29a, de miarn-152, et de miarn-185, et application de celui-ci
CN116173059A (zh) 人aurkb基因在制备抗肿瘤药物中的用途
WO2018170624A1 (fr) Méthode de réduction des expressions de mir-185 et de mir-424
CN116832162A (zh) Cd244在作为抗非洲猪瘟的基因编辑靶点中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17915652

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17915652

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17915652

Country of ref document: EP

Kind code of ref document: A1