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WO2025206262A1 - VARIANT DE PROTÉINE REP, GÈNE rep ET UTILISATION DUDIT GÈNE REP - Google Patents

VARIANT DE PROTÉINE REP, GÈNE rep ET UTILISATION DUDIT GÈNE REP

Info

Publication number
WO2025206262A1
WO2025206262A1 PCT/JP2025/012668 JP2025012668W WO2025206262A1 WO 2025206262 A1 WO2025206262 A1 WO 2025206262A1 JP 2025012668 W JP2025012668 W JP 2025012668W WO 2025206262 A1 WO2025206262 A1 WO 2025206262A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
rep
amino acid
acid residue
rep protein
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.)
Pending
Application number
PCT/JP2025/012668
Other languages
English (en)
Japanese (ja)
Inventor
太一 村口
一生 土井
勇介 森
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.)
Fujifilm Corp
Original Assignee
Fujifilm Corp
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 Fujifilm Corp filed Critical Fujifilm Corp
Publication of WO2025206262A1 publication Critical patent/WO2025206262A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • 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
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to an REP protein having a specific amino acid mutation, and a rep gene encoding the REP protein.
  • the present invention also relates to an expression vector, a vector kit for producing a recombinant adeno-associated virus vector, cells, and a method for producing a recombinant adeno-associated virus vector.
  • Adeno-associated virus is a linear, single-stranded DNA virus belonging to the Parvoviridae family.
  • the wild-type AAV genome contains a replication regulatory gene (rep gene) and a capsid structural gene (cap gene), flanked by inverted terminal repeats (ITRs) for viral replication and packaging.
  • AAV vectors are capable of transducing genes into both proliferating and non-proliferating cells, and are particularly capable of long-term expression in non-dividing cells.
  • AAV is also considered non-pathogenic and has low immunogenicity. For these reasons, AAV vectors are increasingly being used clinically as gene therapy vectors.
  • AAV is a non-enveloped virus that grows in the presence of a helper virus such as adenovirus or herpesvirus.
  • AAV replication has traditionally been achieved by co-infecting host cells with adenovirus.
  • the genes responsible for the adenovirus helper function have been identified, and plasmids carrying these genes are also used.
  • a plasmid containing the rep and cap genes, an adenovirus helper plasmid, and a plasmid containing the gene of interest (GOI) to be introduced can be simultaneously transfected into HEK293 cells, allowing the virus to be packaged into recombinant AAV (rAAV).
  • Non-Patent Document 1 identifies REP protein mutations by screening a plasmid in which REP protein has been randomly synthesized. Patent Document 1 describes inactivating REP protein by introducing a mutation into it in order to avoid its toxicity. Non-Patent Document 2 describes mutating M225, the initiation methionine of Small REP protein, in order to avoid its toxicity.
  • Patent Document 2 describes an REP protein with amino acid substitutions in the DNA-binding domain.
  • An objective of the present invention is to provide a mutant REP protein that can improve low production of adeno-associated virus (AAV) vectors.
  • a further objective of the present invention is to provide a rep gene encoding the mutant REP protein, an expression vector, a vector kit for producing a recombinant adeno-associated virus vector, cells, and a method for producing a recombinant adeno-associated virus vector.
  • ⁇ 2> In the amino acid sequence of the REP protein, a serine residue corresponding to the 102nd amino acid residue of the AAV2-derived REP protein; a glutamic acid residue corresponding to amino acid residue 114 of the AAV2-derived REP protein; The mutant REP protein according to ⁇ 1>, wherein one or more of the glutamic acid residue corresponding to the 125th amino acid residue of the AAV2-derived REP protein and the valine residue corresponding to the 134th amino acid residue of the AAV2-derived REP protein are substituted with a basic amino acid residue.
  • ⁇ 3> The mutant REP protein according to ⁇ 1> or ⁇ 2>, wherein the basic amino acid residue is a lysine residue.
  • the present invention makes it possible to improve the low production of adeno-associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • a part of the rep gene including the amino acid at the mutated site can be partially synthesized and replaced with a rep gene encoding the corresponding part derived from a wild-type REP protein that does not contain the mutation by genetic engineering techniques.
  • a gene encoding the mutant REP protein can be prepared by PCR using primer sequences that encode the mutated amino acids.
  • Adeno-associated virus refers to a small, replication-defective, non-enveloped virus containing single-stranded DNA in the Parvoviridae and Dependoparvovirus families.
  • Serotype 2 is one of the serotypes that has been widely studied for a long time and is known to have an extremely broad host range.
  • Serotype 1 AAV1
  • serotype 5 AAV5
  • serotype 6 AAV6 are serotypes with higher tissue tropism.
  • AAV1 is said to have high gene transfer efficiency in muscle, liver, respiratory tract, central nervous system, etc.; AAV5 in the central nervous system, liver, retina, etc.; and AAV6 in the heart, muscle, liver, etc.
  • An adeno-associated virus gene refers to a gene composed of one or more nucleic acid sequences derived from one or more adeno-associated virus serotypes.
  • Adeno-associated virus genes are preferably genes involved in AAV replication and packaging (specifically, rep genes) and genes encoding AAV structural proteins (specifically, cap genes).
  • rep gene refers to a region of the AAV genome encoding viral replication proteins collectively required for viral genome replication, as known to those skilled in the art, or functional homologs thereof, such as the human herpesvirus 6 (HHV-6) rep gene (known to mediate AAV-2 DNA replication).
  • HHV-6 human herpesvirus 6
  • the coding region of the rep gene includes at least the genes encoding AAV REP78 and REP68 ("long form REP proteins”) and REP52 and REP40 ("short form REP proteins”), or functional homologs thereof.
  • the coding region of the rep gene used in the present invention may be derived from any AAV serotype, but is preferably derived from AAV1, AAV2, AAV5, AAV6, or AAV8, more preferably from AAV2, AAV5, AAV6, or AAV8, and even more preferably from AAV2.
  • Those derived from AAV2 include REP78, REP68, REP52, REP40, and ITR.
  • An expression vector carrying the rep gene may contain a promoter for expressing the rep gene.
  • the promoter for expressing the rep gene may be natural or artificial, and is not particularly limited. However, examples of promoters naturally contained in the rep gene include the p5 and p19 promoters.
  • the cap gene refers to a region in the AAV genome that encodes viral capsid proteins known to those skilled in the art. Examples of these capsid proteins are AAV capsid proteins VP1, VP2, and VP3.
  • the cap gene used in the present invention may be derived from any AAV serotype or may be an artificial gene into which a partial mutation has been introduced, for example, derived from AAV2, AAV5, AAV6, or AAV8.
  • the cap gene may be a wild-type gene, but a gene that has been modified by base substitution, deletion, insertion, or addition, etc., may also be used, as long as it exhibits its original function.
  • the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
  • the base sequence of the modified cap gene preferably exhibits 85% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity with the base sequence of the wild-type cap gene.
  • the culture method is not particularly limited, and may be batch culture, fed-batch culture, shaking culture, agitation culture, static culture, or adherent culture.
  • the culture vessel may be a flask or a bioreactor, but is not particularly limited thereto.
  • the shape of the culture vessel is not particularly limited.
  • the size of the culture vessel is such that culture can be carried out at the culture scale described below.
  • the culture may be carried out with shaking agitation.
  • the agitation speed is generally 50 rpm to 200 rpm, preferably 80 to 200 rpm.
  • the agitation culture may be a rotation agitation culture using blades, propellers, paddles, or the like in the reactor.
  • a three-blade propeller or two-blade paddle can be used.
  • the size of the blades, propellers, or paddles is determined depending on the size of the culture tank.
  • a sparger can be used to introduce oxygen-containing gas into the culture solution. It is preferable to adjust the dissolved oxygen concentration in the culture solution by introducing oxygen-containing gas into the culture solution.
  • the dissolved oxygen concentration in the culture solution can be set as appropriate and is not particularly limited, but is generally 10-150%, preferably 15-120%, and more preferably 20-100%, assuming that the saturated dissolved oxygen concentration in air at 1 atmosphere and at 37°C is 100%.
  • the pore size of the sparger is not particularly limited, but is preferably 5 ⁇ m to 100 ⁇ m, and more preferably 10 ⁇ m to 50 ⁇ m.
  • the amount of oxygen-containing gas passed through is not particularly limited, but is generally 0.001 to 1.0 vvm, and preferably 0.005 to 0.5 vvm.
  • VVM stands for volume per volume per minute.
  • a membrane separation process may be performed in which the cell suspension withdrawn from the culture tank is passed through a separation membrane to separate it into a cell-containing liquid and a permeate.
  • the cell suspension withdrawn from the culture tank is separated into a cell-containing liquid having a higher cell concentration than the cell suspension and a permeate having a lower cell concentration than the cell suspension.
  • Examples of materials for the membrane used in the membrane separation treatment step include polyethersulfone, modified polyethersulfone, and mixed cellulose ester.
  • the pore size of the membrane used in the membrane separation treatment step is preferably 0.1 ⁇ m to 0.4 ⁇ m, more preferably 0.15 ⁇ m to 0.3 ⁇ m.
  • Plasmids containing mutant rep genes were prepared by adding base mutations to the above SEQ ID NOS: 2 to 5. Primers with the above SEQ ID NOS were combined and subjected to PCR using high-precision enzymes such as the PrimeSTAR® Mutagenesis Basal Kit (Takarabio). After transformation into an Escherichia coli strain (DH5 ⁇ strain: Takarabio) or the like, the plasmid was extracted. The entire gene sequence was then determined by the Sanger method. The base sequences of each plasmid containing the mutant rep2 gene are listed in the sequence listing below.
  • RepCap plasmid (pRC) cap5 rep2 (N139R mutation): SEQ ID NO: 17 RepCap plasmid (pRC) cap5 rep2 (S102K mutation): SEQ ID NO: 18 RepCap plasmid (pRC) cap5 rep2 (E114K mutation): SEQ ID NO: 19 RepCap plasmid (pRC) cap5 rep2 (E125K mutation): SEQ ID NO: 20 RepCap plasmid (pRC) cap5 rep2 (V134K mutation): SEQ ID NO: 21 RepCap plasmid (pRC) cap2 rep2 (S102K mutation): SEQ ID NO: 22 RepCap plasmid (pRC) cap6 rep2 (S102K mutation): SEQ ID NO: 23 RepCap plasmid (pRC) cap8 rep2 (S102K mutation): SEQ ID NO: 24
  • S102K mutation indicates that S (serine residue) corresponding to the 102nd amino acid residue is substituted with K (lysine residue).
  • HEK293 cells Adherent cells: AAV5 ⁇ Cell preparation> On the day before transfection, HEK293 cells were seeded in a 12-well plate (Thermo) at 3 x 105 cells/well. The culture medium used was DMEM supplemented with Tet System Approved FBS (Takarabio) at a final concentration of 10%.
  • the plasmids were added in the following amounts: pGOI (GOI plasmid): 0.4 ⁇ g pRC (plasmid containing Rep and Cap5) or pRC with base mutation (plasmid containing mutant Rep and Cap5): 0.4 ⁇ g pHelper (Helper plasmid): 0.4 ⁇ g
  • pGOI GOI plasmid
  • pRC plasmid containing Rep and Cap5
  • pRC with base mutation Plas containing mutant Rep and Cap5
  • pHelper Helper plasmid
  • ⁇ Transfection and culture> The entire amount of the plasmid-transfection reagent complex prepared above was added to the cell culture medium, and the cells were cultured at 37°C, 8% CO 2 , and 150 rpm for 72 hours.
  • the genomic titer was improved compared to when no mutations were present.
  • HEK293 cells adherent cells AAV2, 6, 8
  • the cells were prepared in the same manner as in Example 1.
  • ⁇ Preparation of transfection complex> According to the manufacturer's protocol, 4 ⁇ L of the transfection reagent TransIT®-293 Reagent was added to 130 ⁇ L of culture medium, and then the three plasmids were added so that the total amount was 1.2 ⁇ g (0.4 ⁇ g of each plasmid).
  • pGOI GOI plasmid
  • pRC plasmid containing Rep and Cap2/6/8
  • pRC with S102K base mutation Plasmid containing mutant Rep and Cap2/6/8
  • pHelper Helper plasmid
  • Example 2 The following steps were carried out under the same conditions as in Example 1. The results of measuring the genome titer for Experimental Example 2 are shown in FIG.
  • transfection reagent PEIpro was added to Balan CD medium at a volume equivalent to 7.5% of the culture volume (1.8 mL for a 24 mL culture) to give a total volume of 3 ⁇ L/mL of the culture volume (72 ⁇ L for a 24 mL culture).
  • the entire contents of the tube containing the transfection reagent were transferred to the tube containing the plasmid, mixed gently to homogenize, and allowed to stand for 15 minutes.
  • AAV8 (SEQ ID NO: 28)

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le problème à résoudre par la présente invention est de fournir : un variant de protéine REP qui peut améliorer la faible productivité d'un vecteur VAA; un gène rep qui code pour le variant de protéine REP; un vecteur d'expression; un kit de vecteur pour produire un vecteur de virus adéno-associé recombinant; une cellule; et un procédé de production d'un vecteur de virus adéno-associé recombinant. La présente invention concerne un variant de protéine REP, un ou plusieurs résidus d'acides aminés parmi le 102ème résidu d'acide aminé, le 114ème résidu d'acide aminé, le 125ème résidu d'acide aminé et le 134ème résidu d'acide aminé de la protéine REP étant substitués par un résidu d'acide aminé basique dans la séquence d'acides aminés de la protéine REP de virus adéno-associé.
PCT/JP2025/012668 2024-03-29 2025-03-28 VARIANT DE PROTÉINE REP, GÈNE rep ET UTILISATION DUDIT GÈNE REP Pending WO2025206262A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2024057612 2024-03-29
JP2024-057612 2024-03-29

Publications (1)

Publication Number Publication Date
WO2025206262A1 true WO2025206262A1 (fr) 2025-10-02

Family

ID=97218599

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2025/012668 Pending WO2025206262A1 (fr) 2024-03-29 2025-03-28 VARIANT DE PROTÉINE REP, GÈNE rep ET UTILISATION DUDIT GÈNE REP

Country Status (1)

Country Link
WO (1) WO2025206262A1 (fr)

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