WO2019033246A1 - Arn tud pour la co-invalidation génique de trois miarn et utilisation associée - Google Patents

Arn tud pour la co-invalidation génique de trois miarn et utilisation associée Download PDF

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Publication number
WO2019033246A1
WO2019033246A1 PCT/CN2017/097429 CN2017097429W WO2019033246A1 WO 2019033246 A1 WO2019033246 A1 WO 2019033246A1 CN 2017097429 W CN2017097429 W CN 2017097429W WO 2019033246 A1 WO2019033246 A1 WO 2019033246A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
tud
vector
pglv3
preparation
Prior art date
Application number
PCT/CN2017/097429
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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/CN2017/097429 priority Critical patent/WO2019033246A1/fr
Publication of WO2019033246A1 publication Critical patent/WO2019033246A1/fr

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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
    • 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

Definitions

  • miR -185 is a 22 nt 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.
  • 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 RNA is constructed on a lentiviral vector to obtain a recombinant vector containing the Tud RNA; and the recombinant vector containing the Tud RNA is applied to 16HBE cells to inhibit the expression of miR-152, miR-185 and miR-424;
  • the 5' end of the sense strand template is added with GATCC, which is complementary to the sticky end formed by BamHI digestion; the AATTC is added to the 5' end of the antisense strand template, which is complementary to the sticky end formed by EcoRI digestion.
  • the cDNA of each of the two cells was used as a template.
  • the expression levels of miR-152, miR-185 and miR-424 were detected by real-time PCR, and the experiment was repeated three times. Three parallel samples were set per well, and snord 44 was used as an internal reference. The results are shown in Figure 2. It can be seen that the expression level of miR-152 in TuD-152-185-424 cells is 58 ⁇ 3 ⁇ 4 lower than that in 16HBE cells, and the expression level of miR-185 is 49% lower than that in 16HBE cells, miR-424. The expression level was 68% lower than that of 16HBE cells. The difference was statistically significant ( ⁇ 0.01), indicating that the TuD-152-185-424 cell line was successfully constructed.

Abstract

L'invention concerne un ARN Tud pour l'inactivation génique des miARN-29a, miARN-152, et miARN-424. L'invention concerne également un procédé de connexion d'une séquence nucléotidique codant pour l'ARN TuD à un vecteur navette lentiviral pour préparer un vecteur recombinant et son utilisation pharmaceutique.
PCT/CN2017/097429 2017-08-14 2017-08-14 Arn tud pour la co-invalidation génique de trois miarn et utilisation associée WO2019033246A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/097429 WO2019033246A1 (fr) 2017-08-14 2017-08-14 Arn tud pour la co-invalidation génique de trois miarn et utilisation associée

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/097429 WO2019033246A1 (fr) 2017-08-14 2017-08-14 Arn tud pour la co-invalidation génique de trois miarn et utilisation associée

Publications (1)

Publication Number Publication Date
WO2019033246A1 true WO2019033246A1 (fr) 2019-02-21

Family

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Family Applications (1)

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PCT/CN2017/097429 WO2019033246A1 (fr) 2017-08-14 2017-08-14 Arn tud pour la co-invalidation génique de trois miarn et utilisation associée

Country Status (1)

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WO (1) WO2019033246A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101368213A (zh) * 2008-10-13 2009-02-18 南京大学 血清微小核糖核酸试剂盒及其在乙肝早期诊断中的应用
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
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
CN103080334A (zh) * 2010-06-04 2013-05-01 复旦大学 用于诊断早期结直肠癌及高级腺瘤的微rna生物标记及方法
CN104531700A (zh) * 2014-11-11 2015-04-22 西北工业大学 抑制小鼠MACF1基因表达的shRNA序列及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101368213A (zh) * 2008-10-13 2009-02-18 南京大学 血清微小核糖核酸试剂盒及其在乙肝早期诊断中的应用
WO2010138263A2 (fr) * 2009-05-28 2010-12-02 University Of Massachusetts Nouveaux virus adéno-associés (aav) et leurs utilisations
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
CN103080334A (zh) * 2010-06-04 2013-05-01 复旦大学 用于诊断早期结直肠癌及高级腺瘤的微rna生物标记及方法
CN104531700A (zh) * 2014-11-11 2015-04-22 西北工业大学 抑制小鼠MACF1基因表达的shRNA序列及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHU LINWENSI, FANG JINGYUAN: "The Structure and Clinical Roles of MicroRNA in Colorectal Cancer", GASTROENTEROLOGY RESEARCH AND PRACTICE, vol. 2016, 31 December 2016 (2016-12-31), pages 1 - 6, XP055540703, ISSN: 1687-630X *

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