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WO2019037053A1 - Arn court en épingle à cheveux du gène aitr humain et applications correspondantes - Google Patents

Arn court en épingle à cheveux du gène aitr humain et applications correspondantes Download PDF

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Publication number
WO2019037053A1
WO2019037053A1 PCT/CN2017/098910 CN2017098910W WO2019037053A1 WO 2019037053 A1 WO2019037053 A1 WO 2019037053A1 CN 2017098910 W CN2017098910 W CN 2017098910W WO 2019037053 A1 WO2019037053 A1 WO 2019037053A1
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WO
WIPO (PCT)
Prior art keywords
aitr
shrna
gene
expression
cells
Prior art date
Application number
PCT/CN2017/098910
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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/CN2017/098910 priority Critical patent/WO2019037053A1/fr
Publication of WO2019037053A1 publication Critical patent/WO2019037053A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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 genetic engineering technology, and relates to the construction and application of a shRNA expression sequence. Specifically, the present invention contemplates the synthesis of shRNA against the nucleotide sequence of the AITR gene on the surface of human T cells, which can inhibit the expression of the human AITR gene after being transferred.
  • AITR is the 18th member of the tumor necrosis factor receptor (TNFR) superfamily and is a thymus-derived C D4+
  • AITR/AITRL A surface molecule on CD25+Treg cells whose ligand is AITRL. Studies have shown that AITR/AITRL has many important biological activities, including cell proliferation, differentiation and survival. Because AITR is mainly expressed on resting Treg cells, anti-AITR antibody (DTA-1) abolishes the immunosuppressive effects of Treg cells.
  • AITR/AITRL system is involved in the regulation of Treg cells, and has a good clinical transformation prospect.
  • lack of vectors for specifically inhibiting the expression of AITR gene in the prior art makes the related research not well developed.
  • shRNA a small hairpin RNA
  • RISC RNA-induced silencing complex
  • the present invention constructs a shRNA, and the sense strand template sequence of the shRNA is as shown in the sequence listing SEQ NO.
  • the antisense strand template sequence is shown in SEQ NO. 2 of the Sequence Listing.
  • the present invention designs a pair of AITR-shRNA targeting the AITR gene according to the mRNA sequence of the human AITR gene in the GenBank database and the primer design principle of the shRNA, and commissions Shanghai Biotech to synthesize the AITR. -shRNA.
  • the oligonucleotide of the above-designed AITR-shRNA is routinely annealed and then double-stranded, double-digested and ligated to the pSilencer 3.1-H1 hygro RNAi expression vector, and the ligation product is transformed into Escherichia coli. Single colonies were picked for PCR and sequencing, and positive clones and plasmids were obtained.
  • the present invention transduces the pSilencer3.1-H1 hygro RNAi expression vector containing AITR-shRNA into the Jurkat cell line, and the siRNA efficiency of AITR mRNA is 66.1 ⁇ 3 ⁇ 4.
  • the AITR-shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit Jurkat cell AI.
  • TR gene expression can be used as a powerful tool for the preparation of drugs for the treatment of AITR gene expression-related diseases.
  • FIG. 1 is a schematic diagram showing the results of AITR gene expression by real-time PCR detection of Jurkat cells transduced with an AITR-shRNA expression vector.
  • Jurkat cells were purchased from the Cell Resource Center of the Shanghai Institute of Biological Sciences, and the RNeasy Mini Kit was purchased from Qiagen.
  • the endotoxin-free plasmid extraction kit was purchased from Tiangen Biochemical (Beijing).
  • the complete medium described below is a cell culture medium supplemented with 10% fetal bovine serum.
  • the "AA or NA" sequence was searched for and the 19-base sequence at the 3' end was recorded as a potential interference target site. Ensure that the target sequence has a GC content of 30% to 60% and is not on the 5' and 3' non-coding regions. NCBI BLAST confirmed that the selected sequences have no homology to other genes.
  • the target sequence obtained in this example is 5'-CGCGCTGCTGCCGGGTTCA
  • the sense strand sequence ⁇ ij of AITR-shRNA is shown as SEQ ID No: 1
  • the antisense strand sequence is SEQ ID No: 2 Shown.
  • Jurkat cells were cultured, and 5,000,000 cells in good growth state were taken, and the cells were collected by centrifugation, and then resuspended in 500 L of PBS with 20 g.
  • the AITR-shRNA expression vector was mixed and added to the electric shock cup.
  • the electroporation was performed using the Invitrogen Neon electrotransfer system.
  • the electroporation procedure was: 2.1 KV, 25 ⁇ , pulse shock once; the cells were transferred to a 6 cm dish containing 5 mL DMEM complete medium. Medium, gently shake the dish to mix the cells, and detect the expression of AITR gene after 48 hours.
  • Example 4 Fluorescence quantitative PCR was used to detect the amount of AITR gene expression.
  • Jurkat cells transfected with normal Jurkat cells and AITR-shRNA expression vector were used to extract total RNA from each group of cells using RNeasy Mini Kit, using PrimeScrip RT reagent
  • Kit reverse-transcribes mRNA into cDNA, and then cDNA is diluted with 90 ⁇ M of RNase-Free dH20 and stored at -20 °C for later detection.
  • GAPDH was used as an internal reference, and real-time quantitative PCR (QPCR) was used to detect the relative expression of AITR.
  • the reaction conditions were set: 95 ° C for 10 s, 1 cycle; 95 ° C 5 s, 60 ° C
  • the AITR-shRNA provided by the invention has high transduction efficiency and can efficiently and specifically inhibit Jurkat cell AI.
  • TR gene expression can be used as a powerful tool for the preparation of drugs for the treatment of AITR gene expression-related diseases.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un ARN court en épingle à cheveux (shARN) pour restreindre l'expression d'un gène AITR. Les séquences d'acide nucléique codantes du shARN sont représentées dans les séquences SEQ ID NO : 1 et 2. L'invention concerne également des applications du shARN dans la préparation de réactifs pour abaisser les ARNm d'AITR de cellules, des applications dans la préparation de réactifs pour restreindre l'expression de la protéine AITR de cellules et des applications dans la préparation de médicaments pour des maladies liées à des anomalies d'expression du gène AITR.
PCT/CN2017/098910 2017-08-24 2017-08-24 Arn court en épingle à cheveux du gène aitr humain et applications correspondantes WO2019037053A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098910 WO2019037053A1 (fr) 2017-08-24 2017-08-24 Arn court en épingle à cheveux du gène aitr humain et applications correspondantes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/098910 WO2019037053A1 (fr) 2017-08-24 2017-08-24 Arn court en épingle à cheveux du gène aitr humain et applications correspondantes

Publications (1)

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WO2019037053A1 true WO2019037053A1 (fr) 2019-02-28

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PCT/CN2017/098910 WO2019037053A1 (fr) 2017-08-24 2017-08-24 Arn court en épingle à cheveux du gène aitr humain et applications correspondantes

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1809589A (zh) * 2003-05-23 2006-07-26 Wyeth公司 Gitr配体和gitr配体相关分子和抗体及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1809589A (zh) * 2003-05-23 2006-07-26 Wyeth公司 Gitr配体和gitr配体相关分子和抗体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE abstract Nucleotide 6 June 2016 (2016-06-06), ANONYMOUS: "PREDICTED: Homo sapiens TNF receptor superfamily member 18 (TNFRSF18), transcript variant X1, mRNA.", XP055579061, retrieved from NCBI GenBank Database accession no. XM_017002722 *
RONCHETTI S. ET AL.: "Glucocorticoid-Induced TNFR family Related gene (GITR) enhances dendritic cell activity", IMMUNOLOGY LETTERS, vol. 135, no. 1-2, 30 March 2011 (2011-03-30), pages 24 - 33, XP055579070, ISSN: 0165-2478, DOI: 10.1016/j.imlet.2010.09.008 *

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