WO2018049980A1 - Méthode de construction d'un vecteur de thérapie génique servant à l'immunodéficience congénitale, et utilisation associée - Google Patents

Méthode de construction d'un vecteur de thérapie génique servant à l'immunodéficience congénitale, et utilisation associée Download PDF

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WO2018049980A1
WO2018049980A1 PCT/CN2017/099603 CN2017099603W WO2018049980A1 WO 2018049980 A1 WO2018049980 A1 WO 2018049980A1 CN 2017099603 W CN2017099603 W CN 2017099603W WO 2018049980 A1 WO2018049980 A1 WO 2018049980A1
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vector
promoter
recombinant vector
il2rg
seq
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程田林
仇子龙
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苏州兰希亚生物科技有限公司
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    • 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
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a recombinant human interleukin 2 receptor subunit ⁇ and a method for constructing the expression vector thereof, and belongs to the technical field of genetic engineering.
  • Immunodeficiency disease refers to a disease in which any part of the immune system is abnormally functioned or absent, resulting in immune dysfunction. According to the pathogenesis of diseases, immunodeficiency diseases can be divided into primary immunodeficiency disease (PIDD) and secondary immunodeficiency disease (SIDD). Most of these PIDDs are genetic diseases and are more common in children under one year of age. According to different genetic mutations, primary immunodeficiency disease (PIDD) can be subdivided into a variety of sub-categories, and more than 300 have been reported including X-linked, autosomal recessive, and autosomal dominant. Genetic disease.
  • PIDD primary immunodeficiency disease
  • Gene therapy refers to the introduction of a target gene into a specific cell type to cure the disease.
  • the conventional way of gene therapy is to isolate the patient's hematopoietic stem cells, and to introduce the target gene by means of a viral vector such as retrovirus to restore the expression of the damaged gene, repair the completed hematopoietic stem cells or Hematopoietic progenitor cells are reintroduced into the patient's body to restore the patient's own immune system (Alain Fischer et. al. Nature reviews disease primers, 2015).
  • X-SCID X-linked severe immunity Disease-deficient disease
  • ADA-SCID adenosine deaminase-deficient severe immunodeficiency disease
  • WAS Wiskott-Aldrich syndrome
  • CCD chronic granuloma
  • ⁇ RV gamma retrovirus
  • ⁇ RV viral vectors can successfully achieve the introduction of foreign genes in clinical studies, all patients except ADA-SCID have symptoms such as leukemia or myelodysplasia, which is closely related to the safety of the viral vectors used. .
  • the researchers spent a great deal of effort in the transformation of viral vectors and constructed a viral vector with a higher safety factor, self-inactivation of retrovirus (SIN- ⁇ RV) and self-inactivating lentivirus (SIN-LV, Self-inactivating). Lentiviral vector).
  • SIN- ⁇ RV self-inactivation of retrovirus
  • SIN-LV self-inactivating lentivirus
  • Lentiviral vector Lentiviral vector
  • ⁇ RV viral vector For another primary immunodeficiency disease X-SCID, a ⁇ RV viral vector has also been applied thereto. In 1999-2006, a total of 20 patients received ⁇ RV viral vector-mediated gene therapy, and 18 of them survived to date. However, due to the safety of the ⁇ RV viral vector, five patients developed T-cell acute lymphoblastic leukemia (T-ALL) within a few years after treatment. After that, in 2010, researchers began to use X-SCID gene therapy with safer SIN- ⁇ RV and SIN-LV viral vectors. The results of clinical trials with SIN- ⁇ RV viral vector were published in 2014.
  • T-ALL T-cell acute lymphoblastic leukemia
  • the present invention designs and uses self-inactivating lentivirus (SIN-LV) to construct IL2RG vectors for innate Gene therapy for sexually immunodeficient diseases.
  • non-SCID-type primary immunodeficiency diseases are also underway. Compared with SCID-like diseases, non-SCID primary immunodeficiency disease is difficult to carry out hematopoietic stem cell transplantation, so gene therapy is more urgent.
  • the non-SCID primary immunodeficiency disease represented by WAS and CGD also encountered safety problems in early ⁇ RV viral vector-mediated gene therapy, but with the improvement of viral vectors, WAS and CGD are currently underway. In the clinical trials of gene therapy, the safer SIN- ⁇ RV and SIN-LV viral vectors were used, and the preliminary clinical results showed that the overall condition of the patients treated was significantly improved.
  • the selected viral vector is a safer SIN-LV vector, and the IL2RG overexpressing SIN-LV viral vector is constructed, and the promoter in the vector is modified to obtain a SIN-LV-IL2RG vector with high promoter activity. Has important research and clinical value.
  • the invention provides a recombinant vector, characterized in that the recombinant vector comprises a viral vector and at least a portion of an IL2RG encoding polynucleotide.
  • the viral vector is selected from a lentiviral vector that is inactivated by itself; preferably the viral vector is selected from the group consisting of FUGW; preferably, the promoter of the viral vector is selected from the group consisting of an EF1a promoter, a CAG promoter, a hUbi promoter, and a CBh promoter.
  • the sequence of the viral vector is as set forth in SEQ ID NO: 2; preferably the sequence of the recombinant vector is selected from the group consisting of SEQ ID NOs: 3, 4, 5 and 6.
  • a recombinant vector as described above which is in the cloning position of the FUGW vector
  • a DNA fragment in which the IL2RG gene is ligated, and preferably the sequence of the IL2RG gene is shown in SEQ ID NO: 1.
  • the invention provides a preparation method of a recombinant vector as described above, characterized in that at least a part of the IL2RG encoding polynucleotide is ligated into a multiple cloning site of a viral vector to construct a recombinant vector, and then the positive recombinant vector is transferred.
  • the host cell is cultured to obtain the recombinant vector;
  • the viral vector is selected from a lentiviral vector which is inactivated by itself; preferably, the viral vector is selected from FUGW;
  • the promoter of the viral vector is selected from the group consisting of EF1a promoter, CAG a promoter, a hUbi promoter, a CBh promoter; preferably the sequence of the viral vector is set forth in SEQ ID NO: 2; preferably the sequence of the IL2RG gene is set forth in SEQ ID NO: 1; preferably the recombinant vector
  • the sequences were selected from the sequences in the group consisting of SEQ ID NOs: 3, 4, 5 and 6.
  • a BamHI and MfeI cleavage site is introduced at the end of the at least part of the IL2RG-encoding polynucleotide, preferably the cleavage site is introduced by a primer;
  • the primer sequences described are: GGTACCGAGCTCGGATCCGC as shown in SEQ ID NO: 7 and CCCAATTGGCGGGTTTATCACTTATCGTCGTC as shown in SEQ ID NO: 8.
  • connection employs a T4 ligase.
  • the invention also provides a virus comprising a recombinant vector as described above.
  • the invention also provides a host cell comprising a recombinant vector as described above.
  • the host cell is selected from the group consisting of 293T cells, hematopoietic stem cells, and hematopoietic precursor cells.
  • the present invention also provides a composition comprising the recombinant vector of any one of claims 1 to 3 or the virus of claim 10 or the host cell of claim 11 or 12 At least one.
  • the invention still further provides the use of a recombinant vector as described above or a virus as described above or a host cell as described above or a composition as described above for the preparation of a medicament for the treatment of primary immunodeficiency disease.
  • the primary immunodeficiency disease is selected from the group consisting of: antibody deficiency disease, T cell deficiency disease, T cell and B cell combined deficiency disease, phagocytic cell deficiency disease and non-systemic deficiency disease.
  • the medicament further comprises a pharmaceutically acceptable diluent, excipient or carrier for administration.
  • the present invention has the beneficial effects of constructing an IL2RG overexpressing SIN-LV viral vector by using a more safe SIN-LV vector, and over-expressing the IL2RG-infected rat model (IL2RG knockout rat model) by SIN-LV,
  • IL2RG knockout rat model IL2RG knockout rat model
  • Figure 1 shows a SIN-LV-IL2RG vector map of the promoter hHbi.
  • Figure 2 shows the immunoblotting profile after transfection of 293T cells with the SIN-LV-IL2RG vector with the promoter hHbi.
  • Figure 3 shows a SIN-LV-IL2RG vector map of the promoter EF1a.
  • Figure 4 shows a SIN-LV-IL2RG vector map of the promoter as CAG.
  • Figure 5 shows a SIN-LV-IL2RG vector map of the promoter CBh.
  • Figure 6 shows the immunoblotting profile after transfection of 293T cells with the promoter-engineered SIN-LV-IL2RG vector.
  • vector refers to a self-replicating DNA molecule, including bacterial plasmids, bacteriophages, and animal and plant viruses, that transfer a DNA fragment (the gene of interest) to a recipient cell in genetically engineered recombinant DNA technology.
  • viral vector utilizes a molecular mechanism in which a virus transmits its genome into other cells for infection, occurs in a whole in vivo or in vitro, and can also be applied to include but not Limited to basic research, gene therapy or vaccine development.
  • FUGW vector is also referred to as a FUGW plasmid, which includes, but is not limited to, a vector engineered by a promoter, wherein the promoter includes, but is not limited to, an EF1a promoter, a CAG promoter, a hUbi promoter, a CBh promoter.
  • Ubi and UbI have the same meaning and are a type of promoter in two forms: one is the human ubiquitin promoter, UbC; the other is the plant ubiquitin promoter Ubi. Therefore, in the field of human diseases, "Ubi” has the same meaning as "UbC”.
  • the sequence of the recombinant human interleukin 2 receptor subunit ⁇ gene (IL2RG) of the present invention is artificially synthesized according to the IL2RG gene sequence (shown as SEQ ID NO: 1) provided in the GenBank database.
  • Recombinant human interleukin 2 receptor subunit ⁇ gene recombinant method for constructing a lentiviral vector the steps of which include:
  • IL2RG human interleukin 2 receptor subunit gamma gene
  • dNTPs 2.5 mM each: 5 ⁇ L
  • DNA template (50 ng/ ⁇ L): 1 ⁇ L;
  • IL2RG primer forward and reverse 10 ⁇ M: 1.5 ⁇ L each
  • Double distilled water 33 ⁇ L.
  • the PCR product was purified by DNA gelatinization, and digested with BamHI and MfeI (endase from NEB), FUGW vector (transformed vector modified by promoter, including but not limited to: EF1a promoter, CAG promoter, The hUbi promoter and the CBh promoter were digested with BamHI and EcoRI (endases were all from NEB).
  • BamHI and MfeI enzyme from NEB
  • FUGW vector transformed vector modified by promoter, including but not limited to: EF1a promoter, CAG promoter, The hUbi promoter and the CBh promoter were digested with BamHI and EcoRI (endases were all from NEB).
  • the digested product was recovered by tapping, and ligated with T4 ligase (from NEB), the IL2RG digested product and the FUGW digested product).
  • the ligation product was transformed by E. coli competent TOP10, and the monoclonal was selected, and the positive clones were selected by PCR identification, sequencing and identification. Positive cloning vectors are:
  • the promoter is hUbi SIN-LV-IL2RG vector, the vector sequence is shown in SEQ ID NO: 3, and the vector map is shown in Figure 1;
  • the promoter is SF1a SIN-LV-IL2RG vector, the vector sequence is shown in SEQ ID NO: 4, and the vector map is shown in Figure 3;
  • the promoter is a SIN-LV-IL2RG vector of CAG, the vector sequence is shown in SEQ ID NO: 5, and the vector map is shown in Figure 4;
  • the promoter is CBh SIN-LV-IL2RG vector, the vector sequence is shown in SEQ ID NO: 6, and the vector map is shown in Figure 5;
  • the promoters were transfected into 293 cells with the EF1a promoter, CAG promoter, hUbi promoter, and CBh promoter SIN-LV-IL2RG expression vector, and the total protein was extracted 48 hours later for Western Blot analysis. Untransfected vector), EF1a promoter, CAG promoter, hUbi promoter, CBh promoter SIN-LV-IL2RG expression vector transfected cells expressed IL2RG, and EF1a promoter, hUbi promoter The expression of IL2RG in the cells transfected with SIN-LV-IL2RG expression vector was higher (Fig. 6), indicating that the above promoters have a better correlation with the expression of the target gene in the cells, and the protein expression effect is also better.
  • Mammalian cell expression vectors must contain control elements such as prokaryotic sequences, promoters, enhancers, selectable marker genes, terminators, and polynucleotide signals.
  • control elements such as prokaryotic sequences, promoters, enhancers, selectable marker genes, terminators, and polynucleotide signals.
  • IL2RG IL2RG was expressed, but compared with other promoters, SIN-LV-IL2RG of EF1a promoter and hUbi promoter had the best effect.
  • the SIN-LV overexpressing IL2RG-infected rat model (IL2RG knockout rat model) can be used to accurately assess the effectiveness of gene therapy X-SCID by analyzing the symptom changes in the rat model.
  • the GMP-grade SIN-LV virus can be further prepared, and clinical gene therapy can be carried out on the basis of fully assessing the safety and effectiveness of the relevant gene therapy, achieving a breakthrough in the domestic X-SCID gene therapy.

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Abstract

L'invention concerne un vecteur lentiviral recombinant exprimant le gène IL2RG et la préparation associée, le vecteur étant un vecteur lentiviral auto-inactivant de type SIN de troisième génération, la séquence du gène IL2RG étant telle que représentée dans la SEQ ID NO : 1. L'invention concerne une méthode de préparation du vecteur et l'utilisation du vecteur dans la préparation d'un médicament servant au traitement de maladies de type immunodéficience primaire.
PCT/CN2017/099603 2016-09-13 2017-08-30 Méthode de construction d'un vecteur de thérapie génique servant à l'immunodéficience congénitale, et utilisation associée WO2018049980A1 (fr)

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CN112522202A (zh) * 2020-09-23 2021-03-19 南京启真基因工程有限公司 制备addi四个基因联合敲除的重症免疫缺陷猪源重组细胞的方法及其专用试剂盒

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CN106350541B (zh) * 2016-09-13 2020-01-17 合肥瑞灵生物科技有限公司 先天性免疫缺陷基因治疗载体构建方法及其用途
CN108504685A (zh) * 2018-03-27 2018-09-07 宜明细胞生物科技有限公司 一种利用CRISPR/Cas9系统同源重组修复IL-2RG缺陷基因的方法
CN110699381A (zh) * 2019-09-17 2020-01-17 合肥瑞灵生物科技有限公司 地中海贫血病基因治疗载体构建方法及其用途
US20230288432A1 (en) * 2020-07-06 2023-09-14 The Board Of Regents Of The University Of Texas System Tdp-43 biosensor cell lines
CN112522258B (zh) * 2020-09-16 2023-08-22 南京启真基因工程有限公司 Il2rg基因和ada基因联合敲除的重组细胞及其在制备免疫缺陷猪模型中的应用

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CN112522202A (zh) * 2020-09-23 2021-03-19 南京启真基因工程有限公司 制备addi四个基因联合敲除的重症免疫缺陷猪源重组细胞的方法及其专用试剂盒
CN112522202B (zh) * 2020-09-23 2022-04-05 南京启真基因工程有限公司 制备addi四个基因联合敲除的重症免疫缺陷猪源重组细胞的方法及其专用试剂盒

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