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WO2016150336A1 - Système crispr-cas9, et son procédé de préparation et d'utilisation - Google Patents

Système crispr-cas9, et son procédé de préparation et d'utilisation Download PDF

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Publication number
WO2016150336A1
WO2016150336A1 PCT/CN2016/076638 CN2016076638W WO2016150336A1 WO 2016150336 A1 WO2016150336 A1 WO 2016150336A1 CN 2016076638 W CN2016076638 W CN 2016076638W WO 2016150336 A1 WO2016150336 A1 WO 2016150336A1
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WIPO (PCT)
Prior art keywords
cas9
primer pair
crispr
ltr
vif
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Ceased
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PCT/CN2016/076638
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English (en)
Chinese (zh)
Inventor
蒋兴宇
刘野
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National Center for Nanosccience and Technology China
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National Center for Nanosccience and Technology China
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    • 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
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • 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
    • 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
    • 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 present invention relates to the field of gene therapy drugs, and in particular to the field of gene therapy of HIV, and more particularly to a CRISPR-Cas9 system which can be used for the prevention and/or treatment of HIV, a preparation method and use thereof.
  • HIV Human Immunodeficiency Virus
  • the conventional means of preventing infectious diseases are human intervention and vaccines.
  • Human interventions (such as condoms) cannot truly eliminate HIV. They are only a physical coercive measure to limit HIV.
  • the related vaccines have been used for more than 30 years. Exploration and development, there are no mature products available yet. Therefore, the current primary treatment for HIV is the highly effective combination of antiretroviral methods, but it can only delay the development of HIV. Different from the above treatments, gene therapy has always been regarded as the most ideal mode of HIV treatment; because it can theoretically achieve the effect of preventing and eliminating HIV.
  • CRISPR-Cas9 technology has been rapidly promoted and applied for its efficiency and simplicity. It is an adaptive immune defense system formed by bacteria and archaea during long-term evolution and can be used against invading viruses and foreign DNA.
  • the CRISPR-Cas9 system directs the degradation of homologous sequences by integrating fragments of invading phage and plasmid DNA into CRISPR and using the corresponding CRISPR RNAs (sgRNAs).
  • the main components of the system are: (1) sgRNA sequence, responsible for targeting specific gene loci; (2) Cas9 enzyme, responsible for modification and cleavage of DNA at the target site.
  • a CRISPR-Cas9 system comprising an sgRNA that specifically targets a particular gene locus on the HIV genome, the specific gene loci on the HIV genome comprising Gag, Env, One or more of Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev.
  • the particular genetic locus on the HIV genome is selected from one or more of Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5' LTR, 3' LTR and Rev.
  • the specific genetic loci on the HIV genome are Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5' LTR, 3' LTR and Rev.
  • each of the sgRNAs specifically targeting each of the specific gene loci is independently one or more, preferably from 1 to 11, and may be, for example, one, two, or three.
  • Articles, 4, 5, 6, 7, 8, 9, 10 or 11 may be most preferably 4.
  • the length of each of the sgRNAs is independently 16-22 bp, for example, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp or 22 bp, respectively.
  • the CRISPR-Cas9 system further includes Cas9.
  • the Cas9 and the sgRNA specific for a particular gene locus on the HIV genome can be present in a plasmid, respectively.
  • a method of making the CRISPR-Cas9 system of the first aspect of the invention comprising the step of obtaining an sgRNA fragment by PCR amplification.
  • the primer used for the PCR amplification is selected from the group consisting of a Cas9-5'LTR-1 primer pair, a Cas9-5'LTR-2 primer pair, a Cas9-5'LTR-3 primer pair, and a Cas9-5'LTR-4.
  • primer pair base sequence is in the 5'-3' direction as follows:
  • Ggaagctttagagaagtttt and TTCTCTAAAGCTTCCcggtg.
  • the annealing temperatures of the PCR amplification are respectively:
  • Gag 63 ° C
  • Vpu 60 ° C
  • the reaction conditions for the PCR amplification may further include: pre-denaturation at 95 ° C for 3 min (minutes), denaturation at 95 ° C for 30 sec (seconds), annealing for 30 sec (annealing temperature may be respectively used at the above temperature), and extension at 72 ° C for 30 sec to 1 min. 30 cycles, extending at 72 ° C for 8 min after cycling.
  • the PCR amplification reaction system can be: sterile ddH 2 O: 37.5 ⁇ L, 10 ⁇ PCR Buffer (containing MgCl 2 ): 5 ⁇ L, 2.5 mM dNTP: 4 ⁇ L, and corresponding upper and downstream primers each 1 ⁇ L (primer concentration 50 pmol/ ⁇ L) ), template DNA (50 ng/ ⁇ L): 1 ⁇ L, PyrobestTM DNA Polymerase: 0.5 ⁇ L. After gentle shaking and mixing, PCR amplification can be carried out according to the conditions described above.
  • the preparation method may further comprise the step of separately constructing each of the amplified sgRNA fragments into a plasmid vector.
  • the step of constructing the vector may include: recovering the PCR amplification product according to a conventional method using a PCR recovery kit manufactured by Quigen, and then separately digesting the PCR fragment with SalI, and double-digesting the plasmid vector p1.0 with SalI and EcoRV. Ligation, transformation of Top10 or DH5 ⁇ competent state, screening positive clones by SalI and EcoRV digestion. The positive clone can be sequenced, for example, sent to Invitrogen for sequencing.
  • kits comprising the present The CRISPR-Cas9 system of the first aspect of the invention or the CRISPR-Cas9 system prepared according to the method of the second aspect of the invention, or the expression cassette or recombinant vector expressing the CRISPR-Cas9 system.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and the CRISPR-Cas9 system of the first aspect of the invention or according to the second aspect of the invention
  • the CRISPR-Cas9 system prepared by the method described.
  • the CRISPR-Cas9 system of the first aspect of the invention or the CRISPR-Cas9 system prepared according to the method of the second aspect of the invention may be in an effective amount or in a therapeutically effective amount in the pharmaceutical composition.
  • a fifth aspect of the invention there is provided an application of the CRISPR-Cas9 system according to the first aspect of the invention or the CRISPR-Cas9 system or the method prepared according to the method of the second aspect of the invention
  • a method of preventing and/or treating an HIV infection comprising administering to a subject in need thereof a therapeutically effective amount of a CRISPR-Cas9 system according to the first aspect of the invention or according to The CRISPR-Cas9 system prepared by the method of the second aspect of the invention or the pharmaceutical composition of the fourth aspect of the invention.
  • the CRISPR-Cas9 system can be administered by a highly efficient gene delivery system, for example using a gene delivery vector. More preferably, the CRISPR-Cas9 system can be administered by nanocarrier delivery.
  • a CRISPR-Cas9 system for preventing and/or treating HIV infection, the CRISPR-Cas9 system being the CRISPR-Cas9 system of the first aspect of the invention or according to the invention
  • the CRISPR-Cas9 system prepared by the method described in the second aspect.
  • the present invention has at least but not limited to the following beneficial effects:
  • the CRISPR-Cas9 system of the present invention optimizes the design of sgRNAs targeting 11 gene loci (Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR, Rev) on the HIV genome.
  • sgRNAs targeting 11 gene loci Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR, Rev
  • the inhibition rate is as high as 96%, and the inhibition rate is comparable to that of peptide anti-HIV drugs.
  • FIG. 1 shows the function of the CRISPR-Cas9 system of the present invention.
  • the inventors designed sgRNAs for 11 loci on the HIV genome, and then prepared the CRISPR-Cas9 system, which can effectively inhibit the production of HIV, and its inhibition rate is equivalent to that of peptide anti-HIV drugs. Effect.
  • the present invention has been completed on this basis.
  • Examples 1-11 are used to illustrate a CRISPR-Cas9 system comprising sgRNAs that specifically target specific gene loci on the HIV genome, and methods for their preparation, which are Gag, Env, Pol, Tat, Nef, respectively. , Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev (which can be respectively referred to as Examples 1 to 11).
  • Primer sequence (5'-3') Primer name serial number Gacaagatatccttggtttt Cas9-5'LTR-1 SEQ ID NO: 1 CAAGGATATCTTGTCcggtg Cas9-5'LTR-1 SEQ ID NO: 2 Cctatgagcctgcagtttt Cas9-5'LTR-2 SEQ ID NO: 3 TGCAGGCTCATAGGcggtg Cas9-5'LTR-2 SEQ ID NO: 4 Gcttttgcctgtagtttt Cas9-5'LTR-3 SEQ ID NO: 5
  • Reaction system Sterile ddH 2 O: 37.5 ⁇ L, 10 ⁇ PCR Buffer (containing MgCl 2 ): 5 ⁇ L, 2.5 mM dNTP: 4 ⁇ L, corresponding 1 ⁇ L of each of the upstream and downstream primers (primer concentration 50 pmol/ ⁇ L), template DNA (50 ng / ⁇ L): 1 ⁇ L, PyrobestTM DNA Polymerase: 0.5 ⁇ L. After gently shaking and mixing, PCR amplification was carried out under the conditions shown in Table 2.
  • Pre-denaturation transsexual annealing extend cycle extend Gag 95 ° C, 3 min 95°C, 30sec 63°C, 30sec 72 ° C, 1 min 30 72 ° C, 8 min Env 95 ° C, 3 min 95°C, 30sec 63°C, 30sec 72°C, 30sec 30 72 ° C, 8 min Pol 95 ° C, 3 min 95°C, 30sec 58°C, 30sec 72°C, 30sec 30 72 ° C, 8 min Tat 95 ° C, 3 min 95°C, 30sec 51 ° C, 30 sec 72 ° C, 1 min 30 72 ° C, 8 min Vif 95 ° C, 3 min 95°C, 30sec 51 ° C, 30 sec 72°C, 30sec 30 72 ° C, 8 min Nef 95 ° C, 3 min 95°C, 30sec 64°C, 30sec 72°C, 30sec 30 72 ° C, 8 min Vpu 95 ° C, 3 min 95°C, 30sec 60 ° C, 30 sec 72°
  • PCR amplification kits were used to recover PCR amplification products according to the conventional method. Then, the PCR fragments were digested with SalI, and the plasmid vector p1.0 was double-digested with SalI and EcoRV. The two were ligated to transform Top10 or DH5 ⁇ . Positive clones were screened by SalI and EcoRV and sent to Invitrogen for sequencing.
  • the TZM-BL cell line was selected and previously infected with HIV for 6 hours, and the CRISPR-Cas9 system of the present invention was delivered into the cells using a nanocarrier system to direct the expression of CRISPR-Cas9 in the cells.
  • the cell group in which only HIV was infected alone was set as a negative control, and the cell group in which neither HIV nor CRISPR-Cas9 was added as a blank control; the cell group to which HIV and the marketed anti-HIV drug T20 were added was set as a positive control.
  • the test results are shown in Table 3.
  • CRISPR-Cas9 System Inhibition rate CRISPR-Cas9(Env) 0.96 CRISPR-Cas9(Pol) 0.96 CRISPR-Cas9(Gag) 0.96 CRISPR-Cas9(3’LTR) 0.92 CRISPR-Cas9(5’LTR) 0.93 CRISPR-Cas9(Vif) 0.96 CRISPR-Cas9(Rev) 0.95 CRISPR-Cas9(Tat) 0.96 CRISPR-Cas9(Nef) 0.90 CRISPR-Cas9(Vpr) 0.88 CRISPR-Cas9(Vpu) 0.73 Positive control 0.90 Negative control 0 Blank control 0
  • the CRISPR-Cas9 system of the present invention has an inhibition rate of HIV of 73% to 96%, indicating that the CRISPR-Cas9 system of the present invention can inhibit the production of HIV in cells; the positive control is a marketed anti-antibody.
  • the HIV drug has an inhibition rate of 90%.
  • the CRISPR-Cas9 system of the present invention achieves comparable effects to commercially available anti-HIV drugs and peptide anti-HIV drugs.
  • the present invention also provides a kit comprising the CRISPR-Cas9 system of the present invention or the CRISPR-Cas9 system prepared according to the method of the present invention, or an expression cassette or recombinant vector expressing the CRISPR-Cas9 system.
  • reagents required for PCR amplification, vector construction, and the like including but not limited to amplification buffers, primers, template DNA, enzymes, and the like, can also be included in the kit.
  • instructions for use and/or use/analysis software may also be included in the kit.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a CRISPR-Cas9 system of the invention or a CRISPR-Cas9 system prepared according to the methods of the invention.
  • the CRISPR-Cas9 system can be an effective amount or a therapeutically effective amount in the pharmaceutical composition.
  • an effective amount refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is suitable for use in humans and/or animals (eg, mammals and birds) without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, has reasonable benefits / risk ratio substance.
  • “Pharmaceutically acceptable carrier” means a carrier for administration, and may include various excipients, diluents and the like.
  • the pharmaceutical composition of the present invention may contain a safe and effective amount of the CRISPR-Cas9 system of the present invention as an active ingredient together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical composition of the present invention can be prepared as an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a diluent, and the like as needed.
  • an oral preparation tablette, oral liquid
  • transdermal agent a transdermal agent
  • a diluent a diluent
  • the aqueous solution of the material is usually prepared in a conventional manner.
  • the pharmaceutical composition is more suitably manufactured under sterile conditions.
  • the effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, The route of medicine, etc.
  • the active ingredient of the present invention is administered at a dose of about 0.00001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • Pharmaceutically acceptable carriers of the invention include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide materials, cellulose, nanogels, or combinations thereof .
  • the choice of carrier should generally be matched to the mode of administration, as is well known to those of ordinary skill in the art.
  • the invention also provides the use of the pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of HIV infection.
  • the invention also provides a method of preventing and/or treating HIV infection.
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of a CRISPR-Cas9 system of the invention or a CRISPR-Cas9 system prepared according to the methods of the invention, or a pharmaceutical composition of the invention.
  • the present invention illustrates the process of the present invention by the above-described embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. It will be apparent to those skilled in the art that any modifications of the present invention, equivalent substitutions of the materials selected for the present invention, and the addition of the auxiliary ingredients, the selection of the specific means, etc., are all within the scope of the present invention.

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Abstract

L'invention concerne un système CRISPR-Cas9, ainsi que son procédé de préparation et d'utilisation. Le système comprend des ARNsg au niveau de loci de gènes spécifiques sur un génome du VIH cible spécifique, et les loci de gènes spécifiques sur le génome du VIH comprennent un ou plusieurs éléments sélectinnés parmi Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR et Rev.
PCT/CN2016/076638 2015-03-23 2016-03-17 Système crispr-cas9, et son procédé de préparation et d'utilisation Ceased WO2016150336A1 (fr)

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CN201510127880.3A CN104726449A (zh) 2015-03-23 2015-03-23 一种用于预防和/或治疗HIV的CRISPR-Cas9系统及其制备方法和用途

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