WO2021042944A1 - Thérapie génique d'adn minicercle ciblant les muscles - Google Patents

Thérapie génique d'adn minicercle ciblant les muscles Download PDF

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WO2021042944A1
WO2021042944A1 PCT/CN2020/108361 CN2020108361W WO2021042944A1 WO 2021042944 A1 WO2021042944 A1 WO 2021042944A1 CN 2020108361 W CN2020108361 W CN 2020108361W WO 2021042944 A1 WO2021042944 A1 WO 2021042944A1
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gene
vector
fix
fviii
seq
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PCT/CN2020/108361
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谌平
谢亦武
陈志英
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深圳新诺微环生物科技有限公司
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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
    • 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
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • 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)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • 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
    • 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
    • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a minicircle (MC) DNA carrier, more specifically a muscle-targeted minicircle DNA gene therapy.
  • MC minicircle
  • Protein drugs (including hormones and peptides) have been widely used in the clinical treatment of a variety of monogenic disorders and chronic diseases.
  • protein/hormone/polypeptide drugs have poor thermal stability and inconvenient preparation and storage; and their half-life in vivo is limited, requiring repeated administration.
  • gene therapy can continuously express the target gene product in the body, and it is convenient to achieve the purpose of long-term treatment.
  • the essence of gene therapy is to deliver the gene vector carrying the target gene to the target organ/tissue of the body.
  • the host cell's transcription/translation system is used to continuously express the therapeutic gene product.
  • Muscle skeletal muscle
  • Gene carriers and corresponding gene delivery methods are the key to gene therapy.
  • Gene vectors include viral vectors and non-viral vectors. Viral vectors can actively enter host cells and are highly transfected. They are the mainstream gene vectors, and the representative of them is Adeno-associated Virus (AAV) vectors. On the contrary, non-viral vectors represented by plasmids cannot actively enter host cells, and transfection in vivo is limited.
  • AAV Adeno-associated Virus
  • the solution most similar to the present invention is gene therapy based on AAV vectors or plasmid vectors.
  • the viral vector represented by the AAV vector has a strong immunogenicity of the virus shell, which is likely to cause a severe immune response and has a high safety risk.
  • AAV vector gene capacity is limited, the total packaging capacity is 4.7kb, except for the inherent ITR sequence and promoter, polyA and other regulatory sequences, the length of the target gene generally cannot exceed 3.5kb.
  • the target gene is large, the AAV vector cannot be packaged.
  • the biggest disadvantage of plasmid vectors is the low transfection efficiency in vivo and the difficulty of sustained expression.
  • Minicircle (MC) DNA has no immunogenicity, no risk of gene integration, and is not limited by the size of the target gene. It overcomes the defect that plasmids cannot be expressed continuously, and cooperates with muscle-targeted delivery technology. , Can express therapeutic gene products efficiently in muscle for a long time.
  • the invention relates to a minicircle (MC) DNA vector and a muscle-targeted minicircle DNA gene therapy.
  • the present invention provides a recombinant gene vector expressing a therapeutic gene product, wherein the recombinant gene vector contains the coding gene or the target gene of the gene product.
  • the gene product is selected from protein, polypeptide or nucleic acid fragment.
  • the protein or polypeptide is selected from antibodies, functional proteins, cytokines, enzymes or hormones, or the nucleic acid fragments are selected from gene fragments, mRNA, siRNA, shRNA or miRNA.
  • the recombinant gene vector is selected from a non-viral vector or a viral vector.
  • the non-viral vector is selected from standard plasmids or other circular expression cassettes, or the viral vector is selected from retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated Virus (AAV) Carrier.
  • AAV adeno-associated Virus
  • the functional protein is selected from coagulation factor IX (FIX), coagulation factor VIII (FVIII) or variants thereof.
  • FIX is selected from full-length, FIX-Padua mutant or FIX-KLW mutant.
  • FVIII is selected from a full-length, B-domain deleted (B-domain deleted) coagulation factor VIII (BDD-FVIII) mutant or a BDD-FVIII-FD mutant.
  • the non-viral vector is selected from a minicircle DNA vector, or the viral vector is selected from an adeno-associated virus vector.
  • the amino acid sequence of FIX is shown in SEQ ID NO: 3
  • the amino acid sequence of FIX-Padua is shown in SEQ ID NO: 4
  • the amino acid sequence of FIX-KLW is shown in SEQ ID NO:
  • the amino acid sequence of the BDD-FVIII is shown in SEQ ID NO: 7
  • the amino acid sequence of the BDD-FVIII-FD is shown in SEQ ID NO: 8.
  • the FIX has an amino acid sequence that is at least 90%, 95%, 98%, or 99% homologous to the sequence shown in SEQ ID NO: 3, and has the same function as the previous FIX;
  • the FIX- Padua has an amino acid sequence with at least 90%, 95%, 98% or 99% homology with the sequence shown in SEQ ID NO: 4, and has the same function as the previous FIX;
  • the FIX-KLW has the same function as SEQ ID NO:
  • the sequence shown in 5 has an amino acid sequence with at least 90%, 95%, 98% or 99% homology, and has the same function as the previous FIX;
  • the BDD-FVIII has at least 90% of the sequence shown in SEQ ID NO: 7 , 95%, 98%, or 99% homology of the amino acid sequence, and has the same function as the previous BDD-FVIII;
  • the BDD-FVIII-FD has at least 90%, 95% of the sequence shown in SEQ ID NO: 8 , 98%
  • the nucleotide sequence of the FIX encoding gene is shown in SEQ ID NO: 9
  • the nucleotide sequence of the FIX-Padua encoding gene is shown in SEQ ID NO: 10
  • the FIX-KLW The nucleotide sequence of the coding gene is shown in SEQ ID NO: 11
  • the nucleotide sequence of the BDD-FVIII coding gene is shown in SEQ ID NO: 12
  • the nucleotide sequence of the BDD-FVIII-FD coding gene The sequence is shown in SEQ ID NO: 13.
  • the FIX encoding gene has a nucleotide sequence that is at least 90%, 95%, 98%, or 99% homologous to the sequence shown in SEQ ID NO: 9, and the encoded FIX has a nucleotide sequence that is at least 90%, 95%, 98%, or 99% homologous to the sequence shown in SEQ ID NO: 9.
  • the FIX-Padua encoding gene has a nucleotide sequence with at least 90%, 95%, 98% or 99% homology with the sequence shown in SEQ ID NO: 10, and the encoded FIX-Padua has The same function as the previous FIX-Padua;
  • the FIX-KLW encoding gene has a nucleotide sequence that is at least 90%, 95%, 98% or 99% homologous to the sequence shown in SEQ ID NO: 11, and the encoding is obtained
  • the FIX-KLW has the same function as the previous FIX-KLW;
  • the BDD-FVIII coding gene has nucleotides with at least 90%, 95%, 98% or 99% homology with the sequence shown in SEQ ID NO: 12 Sequence, and the encoded BDD-FVIII has the same function as the previous BDD-FVIII;
  • the BDD-FVIII-FD coding gene has at least 90%, 95%, 98% or 99%
  • This application also provides a method for preparing the aforementioned recombinant gene vector, the specific steps include:
  • the recombinant gene vector contains the coding gene or the target gene of the gene product.
  • the gene product is selected from protein, polypeptide or nucleic acid fragment.
  • the protein or polypeptide is selected from antibodies, functional proteins, cytokines, enzymes or hormones, or the nucleic acid fragments are selected from gene fragments, mRNA, siRNA, shRNA or miRNA.
  • the recombinant gene vector is selected from a non-viral vector or a viral vector.
  • the non-viral vector is selected from a standard plasmid or other circular expression cassettes, or the viral vector is selected from a retroviral vector, a lentiviral vector, an adenovirus vector and an adeno-associated virus vector.
  • the functional protein is selected from coagulation factor IX (FIX), coagulation factor VIII (FVIII) or their variants
  • the non-viral vector is selected from minicircle DNA vectors
  • the viral vector is selected from Adeno-associated virus vector.
  • FIX is selected from full length, FIX-Padua mutant or FIX-KLW mutant
  • FVIII is selected from full length, BDD-FVIII mutant or BDD-FVIII-FD mutant.
  • the application also provides a host cell, which contains the aforementioned recombinant gene vector.
  • the host cell includes a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.
  • the application also provides a pharmaceutical composition, which comprises the aforementioned recombinant gene carrier and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be made into a pharmaceutical preparation according to conventional methods. During the preparation process, it is preferable to mix or dilute the recombinant gene carrier with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier When the carrier serves as a diluent, it can be solid, semi-solid or liquid.
  • the preparation is selected from the form of tablets, pills, powders, capsules, suspensions, emulsions, solutions, aerosols, injection solutions and the like.
  • Suitable carriers, excipients or diluents include water, lactose, glucose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, polyvinylpyrrolidone, methyl hydroxybenzoate, propyl hydroxybenzoate, talc , Magnesium stearate and mineral oil.
  • the formulation may also include fillers, anticoagulants, lubricants, moisturizers, flavoring agents, emulsifiers, preservatives, and the like.
  • the application also provides the use of the aforementioned recombinant gene vector, the host cell, or the pharmaceutical composition in the preparation of a medicine for the treatment of diseases.
  • the disease is selected from inherited gene defect disease or chronic disease.
  • the genetic disease is selected from type A or type B hemophilia.
  • This application also provides a method for treating a patient’s disease, the method comprising: (1) preparing a recombinant gene vector as described before; or implementing the preparation method as described before to prepare a recombinant gene vector; (2) administering by local delivery
  • An effective dose of the recombinant gene vector for muscle is highly transgene expressed in muscle (such as skeletal muscle) to produce a gene product sufficient to achieve a therapeutic effect.
  • the positive effects of the present invention include: the use of microcircle DNA vector combined with muscle targeted delivery technology can efficiently express therapeutic gene products in muscles for a long time, thereby overcoming the shortcomings of limited half-life of gene products (especially protein drugs) and unsustainable curative effect. .
  • the blood coagulation effect was consistent with that of wild-type mice. It can be seen that the minicircle DNA vector of the present invention has a good prospect for treating hereditary gene defects or chronic diseases, and provides new clinical ideas.
  • Figure 1 Minicircle DNA empty vector pMC.DTS-CMVmax, containing attB and attP recombination sites, 32 tandem repeat I-SceI restriction sites (I-SceI*32), kana resistance gene, SV40 DNA nuclear target DNA nuclear targeting sequence (DTS), CMV promoter/enhancer, chimeric intron, multiple cloning site (MCS), bovine growth hormone (bGH) poly A signal.
  • I-SceI*32 32 tandem repeat I-SceI restriction sites
  • kana resistance gene kana resistance gene
  • DTS SV40 DNA nuclear target DNA nuclear targeting sequence
  • CMV promoter/enhancer CMV promoter/enhancer
  • chimeric intron multiple cloning site
  • MCS multiple cloning site
  • bGH bovine growth hormone
  • Figure 2 Map of FIX factor minicircle DNA vector.
  • FIG. 3 BDD-FVIII factor minicircle DNA vector map.
  • FIG. 4 Mice were injected intramuscularly with the FIX factor minicircle DNA vector or BDD-FVIII minicircle DNA, and the plasma FIX factor or BDD-FVIII factor expression level was detected by ELISA.
  • Figure 5 One month after intramuscular injection of the FIX factor minicircle DNA vector, a tail-clip assay was used to detect the coagulation effect of bleeding in hemophilia B mice and wild-type mice.
  • the minicircle DNA parent plasmid is transformed into genetic engineering E coli bacteria ZYCY10P3S2T (Nature Biotechnology 2010, 28:1287-9).
  • ZYCY10P3S2T expresses ⁇ C31 recombinase and an endonuclease that recognizes the I-SceI site.
  • the minicircle DNA parent plasmid undergoes DNA recombination at the attB/attP recombination site to form two small circular DNA molecules: i) Minicircle DNA (only containing the target gene expression box and 36-bp AttR site); ii) a small circle composed of plasmid backbone DNA (containing I-SceI restriction site).
  • minicircle formed by the plasmid backbone DNA and the unrecombined residual minicircle DNA parent plasmid are linearized under the action of I-SceI endonuclease, and then degraded by DNase, leaving only the minicircle DNA in the ZYCY10P3S2T bacteria.
  • C57BL/6 mice were injected intramuscularly with FIX minicircle DNA and BDD-FVIII minicircle DNA.
  • ELISA kit detects the expression of plasma FIX factor and BDD-FVIII factor (see Figure 3).
  • FIX and BDD-FVIII A single intramuscular injection of the microcircle (MC.FIX and MC.BDD-FVIII), FIX and BDD-FVIII continue to express in mice for more than 40 days, the plasma FIX concentration is maintained at about 30ng/mL, and the plasma BDD-FVIII is maintained at normal 2-3% of the level.
  • mice were injected intramuscularly, the grouping situation is shown in the table below:
  • C57/BL6 normal mice with normal coagulation function
  • F9-KO FIX knockout mice with C57/BL6 genetic background (hemophilia B mice)

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Abstract

L'invention concerne une thérapie génique d'ADN minicercle ciblant les muscles. En particulier, l'invention concerne un vecteur d'ADN minicercle pour une expression efficace à long terme d'un produit génique thérapeutique dans les muscles. Le vecteur d'ADN minicercle est combiné à une technologie d'administration ciblée sur les muscles, et peut exprimer efficacement le produit génique thérapeutique dans les muscles pendant une longue période, ce qui permet de surmonter les défauts d'une période de demi-vie limitée de médicaments protéiques et d'un effet thérapeutique non durable. Le vecteur d'ADN minicercle peut être utilisé pour traiter des défauts génétiques héréditaires communs ou des maladies chroniques, et présente les avantages d'être sûr, efficace et peu coûteux.
PCT/CN2020/108361 2019-09-03 2020-08-11 Thérapie génique d'adn minicercle ciblant les muscles WO2021042944A1 (fr)

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CN201910825998.1A CN110684798A (zh) 2019-09-03 2019-09-03 肌肉靶向的微环dna基因治疗

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CN113336841B (zh) * 2021-06-02 2022-09-23 中国医学科学院血液病医院(中国医学科学院血液学研究所) F8蛋白变体及利用其制备的基因治疗载体
WO2024007978A1 (fr) * 2022-07-07 2024-01-11 深圳新诺微环生物科技有限公司 Peptide lieur, protéine fviii contenant un peptide lieur ou un variant de celui-ci et leur utilisation

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