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WO2019237381A1 - Vecteur de modification pour édition du gène alps5 humain et son procédé de préparation et son application - Google Patents

Vecteur de modification pour édition du gène alps5 humain et son procédé de préparation et son application Download PDF

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Publication number
WO2019237381A1
WO2019237381A1 PCT/CN2018/091711 CN2018091711W WO2019237381A1 WO 2019237381 A1 WO2019237381 A1 WO 2019237381A1 CN 2018091711 W CN2018091711 W CN 2018091711W WO 2019237381 A1 WO2019237381 A1 WO 2019237381A1
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WO
WIPO (PCT)
Prior art keywords
alps5
vector
human
gene
editing
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2018/091711
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English (en)
Chinese (zh)
Inventor
毛吉炎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Biocan Technologies Co Ltd
Original Assignee
Shenzhen Biocan Technologies Co Ltd
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 Shenzhen Biocan Technologies Co Ltd filed Critical Shenzhen Biocan Technologies Co Ltd
Priority to PCT/CN2018/091711 priority Critical patent/WO2019237381A1/fr
Publication of WO2019237381A1 publication Critical patent/WO2019237381A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a modified vector for editing human ALPS5 gene, a preparation method and application thereof.
  • the human anti-cancer immune mechanism includes two aspects of cellular immunity and humoral immunity. However, it is mainly based on cellular immunity.
  • the effector cells that play a major role in the cellular immune mechanism are T cells, of which CD8 + T cells are the most important effect in tumor immunity. Perform the cell.
  • T-cell activation requires dual signal recognition.
  • the tumor antigen first binds to MHC molecules on antigen-presenting cells or target cells, and then binds to T-lymphocyte surface antigen recognition receptors to provide the first signal for T-cell activation; CD28 on the T-cell surface Binding to B7 molecules on the surface of antigen presenting cells or on the surface of target cells provides a second signal.
  • the T cells are fully activated, and the activated T cells first proliferate, generating a large number of antigen-specific T cells, which migrate to the tumor site to play a killing role.
  • negative regulatory molecules such as ALPS5 began to be upregulated.
  • ALPS5 is a transmembrane protein and belongs to the CD28 family.
  • the ligand of ALPS5 is a member of the B7 family. It competes with CD28 molecules and binds to B7-1 and B7-2. It inhibits the proliferation of activated T cells and negatively regulates immunity. Under physiological conditions, it can prevent the immune from being enlarged. It maintains a dynamic balance between health and disease, but is often used by tumor cells to achieve the purpose of immune escape. Therefore, the research on the role of ALPS5 in tumorigenesis and development can promote the development of tumor therapy. However, the lack of targeted knockout of ALPS5 gene expression in the prior art has caused certain obstacles to the progress of related research.
  • a first object of the present invention is to provide a modified vector for human ALPS5 gene editing.
  • a second object of the present invention is to provide a method for preparing a modified vector for human ALPS5 gene editing.
  • the present invention provides a targeted sgRNA for human ALPS5 gene editing, and its sequence is 5'- AATTTCTACTCTTGTAGATCTGATCCCAGATATGTATTACAC -3 ’.
  • the invention also provides a method for preparing the modified vector for editing human ALPS5 gene, which includes the following steps:
  • step (3) ligating the sgRNA obtained in step (1) to a linearized core vector with T4 DNA ligase;
  • the ligation product is transformed into competent E. coli Stbl3. After a large amount of culture, the recombinant vector is extracted and commissioned for sequencing. The correct sequencing result is the modified vector for human ALPS5 gene editing.
  • the core vector is pLVX-ascpf1-puro.
  • the modified vector for human ALPS5 gene editing provided by the present invention has strong specificity, can edit human ALPS5 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of ALPS5 gene.
  • Figure 1 is a vector map of pLVX-AsCpf1-puro
  • FIG. 1 T7 Endonuclease I test results of Jurkat cells in the control group and the experimental group, wherein: 1-experimental group, 2-control group.
  • Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System provides crRNA design rules and gRNA sequences that target the ALPS5 gene, design crRNAs that target the ALPS5 gene, and according to the actual situation of the pLVX-AsCpf1-puro vector in its 5 ' The sticky ends of BamHI and EcoRI digestion sites were added at the 3 'and 3' ends, respectively.
  • the forward sequence was 5'-GATCCTAATTTCTACTCTTGTAGATCTGTCATCTGGCACGTAAGGCCG -3 '
  • the reverse sequence is 5'-GCGGCCTTACGTGCCAGATGACAGATCTACAAGAGTAGAAATTC-3'
  • these two sequences were synthesized. After dissolution, 5 ⁇ L of each was mixed, heated at 95 ° C for 5 minutes, and then naturally cooled to room temperature to form a double-stranded DNA with sticky ends of BamHI and EcoRI.
  • T4 DNA ligase was used to ligate a and b products.
  • the ligated product was then transformed into E. coli Stbl3 and identified by sequencing.
  • the recombinant vector contained in the correct strain was the pLVX-AsCpf1-puro-ALPS5 vector.
  • the correct strain was sequenced and identified in Example 1 and placed in an LB liquid medium having an ampicillin concentration of 100 ⁇ g / ml, and cultured with shaking at 250 rpm and 37 ° C. for 12-16 h. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the endotoxin-free pLVX-AsCpf1-puro-ALPS5 plasmid.
  • 293T cells were cultured and transfected after two passages of growth and culture: pLVX-AsCpf1-puro-ALPS5 vector was taken and transfected with the packaging plasmid and transfection reagent provided by the integrase-deficient Lenti-X HTX lentiviral packaging system Stained in 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ⁇ 95%. The staining time was the starting point. The supernatants were harvested after 48 h and 72 h, filtered through a 0.45 ⁇ m filter and stored at -80 ° C.
  • Untreated Jurkat cells (control group) and lentivirus-treated Jurkat cells (experimental group) were seeded into six-well plates. After the cells were full, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to expand The gene editing site is expected to be amplified, and the PCR product is recovered by electrophoresis.
  • the modified vector for human ALPS5 gene editing provided by the present invention has strong specificity, can edit human ALPS5 gene through the CRISPR / Cpf1 system very efficiently, and can be used in research and development of drugs related to abnormal expression of ALPS5 gene.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un vecteur de modification pour l'édition du gène ALPS5 humain et son procédé de préparation et son application. L'édition du gène ALPS5 humain à un niveau cellulaire à l'aide d'un système CRISPR/Cpf1, permet d'obtenir des cellules d'édition de gène ALPS5.
PCT/CN2018/091711 2018-06-16 2018-06-16 Vecteur de modification pour édition du gène alps5 humain et son procédé de préparation et son application Ceased WO2019237381A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091711 WO2019237381A1 (fr) 2018-06-16 2018-06-16 Vecteur de modification pour édition du gène alps5 humain et son procédé de préparation et son application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091711 WO2019237381A1 (fr) 2018-06-16 2018-06-16 Vecteur de modification pour édition du gène alps5 humain et son procédé de préparation et son application

Publications (1)

Publication Number Publication Date
WO2019237381A1 true WO2019237381A1 (fr) 2019-12-19

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PCT/CN2018/091711 Ceased WO2019237381A1 (fr) 2018-06-16 2018-06-16 Vecteur de modification pour édition du gène alps5 humain et son procédé de préparation et son application

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WO (1) WO2019237381A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107815468A (zh) * 2016-08-31 2018-03-20 北京百奥赛图基因生物技术有限公司 人源化基因改造动物模型的制备方法及应用
CN107847524A (zh) * 2015-03-27 2018-03-27 哈佛学院校长同事会 经过修饰的t细胞及其制备和使用方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107847524A (zh) * 2015-03-27 2018-03-27 哈佛学院校长同事会 经过修饰的t细胞及其制备和使用方法
CN107815468A (zh) * 2016-08-31 2018-03-20 北京百奥赛图基因生物技术有限公司 人源化基因改造动物模型的制备方法及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZETSCHE, B. ET AL.: "Cpfl is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-C System", CELL, vol. 163, no. 3, 22 October 2015 (2015-10-22), pages 759 - 771, XP055553375, ISSN: 0092-8674, DOI: 10.1016/j.cell.2015.09.038 *

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