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WO2025197865A1 - Technique de production à haut débit de vecteur viral adéno-associé - Google Patents

Technique de production à haut débit de vecteur viral adéno-associé

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Publication number
WO2025197865A1
WO2025197865A1 PCT/JP2025/010284 JP2025010284W WO2025197865A1 WO 2025197865 A1 WO2025197865 A1 WO 2025197865A1 JP 2025010284 W JP2025010284 W JP 2025010284W WO 2025197865 A1 WO2025197865 A1 WO 2025197865A1
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protein
helper
aav
nucleic acid
vector
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Japanese (ja)
Inventor
世志幸 宮川
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Nippon Medical School Foundation
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Nippon Medical School Foundation
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
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    • 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
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the present invention relates to a helper factor capable of inducibly expressing an adeno-associated virus (AAV) vector, which can increase the production yield of the vector, and to a method for producing an AAV vector using the helper factor.
  • AAV adeno-associated virus
  • Viral vectors can express specific target genes in target cells or tissues by infecting them. Because they utilize the infectious properties of viruses, viral vectors have high gene transfer and expression efficiencies, making them capable of transferring genes into blood cells and primary cultured cells, which are difficult to transfer using other gene transfer methods. They are also capable of in vivo gene transfer into many animals, including humans, rats, and mice.
  • AAV vectors are non-pathogenic, can transfer genes into terminally differentiated, non-dividing cells such as neurons, and their viral particles are physicochemically stable and can be purified and concentrated. These factors make them one of the gene therapy vectors currently seeing the most medical application. Gene therapy using AAV vectors is being performed worldwide for a variety of intractable diseases, and its therapeutic effectiveness has been proven in numerous clinical studies, so it is expected that further clinical applications will be made in the future.
  • AAV vectors are produced by transiently transfecting producer cells with plasmid DNA containing AAV components and adenovirus-derived helper factors that enhance vector production.
  • Patent Document 1 Previous attempts have been made to create producer cells for AAV vectors, including methods for controlling the expression of E1A and E1B (Patent Document 1), inactivating Rep78 and Rep68 through mutation (Patent Document 2), and expression control systems using Rep gene recombination (Patent Document 3). However, these methods have not been directed toward constructing an expression control system capable of inducible expression of helper factors.
  • the helper plasmid (pHelper) currently used for AAV vector production is composed of gene fragments containing E2A, E4, and VA-RNA, excluding E1A and E1B, from the E1A, E1B, E2A, VA, and E4 gene regions responsible for the adenovirus helper function required for AAV replication in 293 cells derived from human fetal kidney tissue.
  • an objective of the present invention is to develop a helper factor expression system that contains a new helper factor capable of increasing the amount of AAV vector produced and that is capable of inducible expression.
  • the present inventors attempted to identify a sequence required for helper function from an adenovirus genome fragment and to construct an inducible expression cassette using the same.
  • the proteins encoded by the 22k gene, 100k gene, and 33k gene present in the genome fragment containing the adenovirus E2A gene each have helper functions.
  • the proteins encoded by the orf2, orf3, and orf6-7 genes other than the orf6 gene present in the genome fragment containing the adenovirus E4 gene each have helper functions.
  • helper factor expression was suppressed in the absence of an inducer, but that helper factor expression could be induced in a concentration-dependent manner with the addition of an inducer. Furthermore, they confirmed that AAV vector production could be induced in a concentration-dependent manner with the addition of an inducer, resulting in increased vector production compared to conventional methods.
  • an expression cassette used for producing an adeno-associated virus (AAV) vector comprising a nucleic acid containing a helper factor capable of increasing the production yield of the AAV vector, and a heterologous promoter operably linked to the helper factor, wherein the nucleic acid comprises any one of the following nucleic acids (1) to (4): (1) A nucleic acid comprising the coding region of adenovirus E2A protein. (2) A nucleic acid comprising the coding region of one or more proteins selected from adenovirus 22k protein, 100k protein, 33k protein, E4orf2 protein, E4orf3 protein, and E4orf6/7 protein.
  • AAV adeno-associated virus
  • a nucleic acid comprising the coding region of adenovirus E4orf6 protein is represented by A nucleic acid comprising template DNA of adenovirus VA-RNA.
  • a helper plasmid or helper virus used in producing an adeno-associated virus (AAV) vector comprising the expression cassette according to any one of [1] to [3].
  • a method for producing an AAV vector comprising the step of transfecting a virus-producing cell with the helper plasmid or helper virus described in [4], a transfer plasmid containing an expression cassette for a gene of interest between the ITRs at both ends of the adenovirus genome, and a packaging plasmid containing the Rep gene and Cap gene of the adeno-associated virus.
  • An AAV vector-producing cell characterized in that the expression cassette according to any one of [1] to [3] is stably maintained within the cell.
  • the present invention provides an expression cassette capable of inducibly expressing a helper factor that can increase the production yield of an AAV vector.
  • 1 shows a scheme for a test to verify the helper activity of E2A.
  • 1 shows a comparison of helper activity between pHelper-E2A and pCBh-E2A based on the amount of AAV vector produced.
  • 1 shows a scheme for a test to verify the helper activity of E4orf6.
  • 1 shows a comparison of helper activity between pHelper-E4 and pCBh-E4orf6 based on the amount of AAV vector produced. Construction of expression plasmids for proteins (100kJ, 22kJ, 33kJ) contained in the E2A genome fragment is shown.
  • Construction of an expression plasmid for the orf group contained in the E4 genome fragment (orf1-7, orf2-7, orf3-7, orf4-7, orf6, orf6-7) is shown. This shows a comparison of the helper activity of pHelper-E4 and expression plasmids for the orf group contained in the E4 genome fragment (orf1-7, orf2-7, orf3-7, orf4-7, orf6, orf6-7) based on the amount of AAV vector produced.
  • polycistronic expression plasmids (pCBh-E4orf2367, pCBh-E4orf367) for the orf groups contained in the E4 genome fragment (orf2-7, orf3-7, orf6-7) is shown.
  • This shows a comparison of helper activity based on the amount of AAV vector produced by pHelper-E4, pCBh-E4orf6, pCBh-E4orf2367, and pCBh-E4orf367.
  • 1 shows a scheme for a test to verify the helper activity of VA-RNA.
  • 1 shows a comparison of helper activity between pHelper-VA and pCBh-VA based on the amount of AAV vector produced.
  • FIG. 1 shows a scheme for a test to verify the helper activity of optimized helper factor (E2Aopti, E4opti) expression plasmids (pE2Aopti, pE4opti).
  • This shows a comparison of the helper activity of a conventional helper factor expression plasmid (pHelper) and optimized helper factor (E2Aopti, E4opti) expression plasmids (pE2Aopti, pE4opti) based on the amount of AAV vector produced.
  • pHelper helper factor expression plasmid
  • E2Aopti, E4opti optimized helper factor expression plasmids
  • FIG. 1 shows a scheme for the verification test of helper factor expression and helper activity using a polycistronic inducible expression system (pTRE-E2Aopti-E4opti+pTRE-VA) of optimized helper factors (E2Aopti, E4opti, VA-RNA).
  • the expression levels (relative mRNA expression levels) of helper factors (E2A, E4) by the polycistronic inducible expression system (pTRE-E2Aopti-E4opti+pTRE-VA) of optimized helper factors (E2Aopti, E4opti, VA-RNA) are shown.
  • the expression level (relative mRNA expression level) of the helper factor (VA-RNA) by the polycistronic inducible expression system (pTRE-E2Aopti-E4opti+pTRE-VA) of optimized helper factors (E2Aopti, E4opti, VA-RNA) is shown.
  • This figure shows a comparison of helper activity based on AAV vector production volume between a conventional helper factor expression vector (pHelper) and a polycistronic inducible expression system (pTRE-E2Aopti-E4opti+pTRE-VA) of optimized helper factors (E2Aopti, E4opti, VA).
  • FIG. 1 shows a scheme for a test to verify the helper activity of a plasmid (pTRE-E2Aopti-E4opti-VA) carrying a polycistronic inducible expression cassette for optimized helper factors (E2Aopti, E4opti, VA-RNA).
  • Figure 1 shows a comparison of the helper activity of a plasmid (pTRE-E2Aopti-E4opti-VA) carrying a polycistronic inducible expression cassette for optimized helper factors (E2Aopti, E4opti, VA-RNA) and the helper activity of a conventional helper factor expression plasmid (pHelper) based on AAV vector production.
  • Adeno-associated virus is a single-stranded DNA virus of approximately 4.7 kb belonging to the Parvoviridae family. It has an icosahedral capsid with a diameter of 20-30 nm and infects a wide host range because it recognizes heparan sulfate proteoglycans, a ubiquitous component of cell membranes.
  • the AAV genome structure contains inverted terminal repeats (ITRs) at both ends, which contain Rep (a regulatory protein responsible for replication and transcription) and Cap (three capsid proteins: VP1, VP2, and VP3).
  • AAV AAV type 1 virus
  • AAV2 AAV type 2 virus
  • AAV3 AAV type 3 virus
  • AAV4 AAV type 4 virus
  • AAV type 5 virus AAV5
  • AAV type 6 AAV6
  • AAV type 7 AAV7
  • AAV8 AAV8
  • AAV type 9 AAV9
  • AAV type 10 AAV10
  • AAV type 11 AAV11
  • AAV type 12 AAV12
  • AAV13 AAV type 13
  • Adeno-associated virus vectors (hereinafter referred to as "AAV vectors") can basically be produced without host infection by a helper virus by transiently co-transfecting HEK 293 cells with three plasmids: a transfer vector plasmid containing the target gene between the AAV ITRs, a packaging plasmid expressing Rep and Cap, and a helper plasmid expressing E2A, E4, and VA-RNA.
  • the present invention provides expression cassettes for helper factors that can be used in place of the above-mentioned helper plasmids expressing E2A, E4, and VA-RNA in the production of AAV vectors.
  • helper factor refers to a gene for a factor that, when expressed in AAV vector-producing cells, can assist in the amplification of viruses that cannot grow on their own and increase the amount of AAV vector produced by the AAV vector-producing cells.
  • the term "expression cassette” refers to a nucleic acid construct containing the elements necessary for expressing a target product. Specifically, it refers to a nucleic acid construct containing at least a promoter and a nucleic acid for expressing the target product, operably linked to the promoter.
  • a nucleic acid containing a coding region for a target protein is linked downstream of a mammalian promoter. It is preferable, but not essential, to use a promoter with a polyA sequence added downstream of the promoter. As described below, various promoters of heterologous origin can be used. Nucleic acids containing coding regions for one or more proteins may also be linked downstream of the promoter.
  • a sequence that enables polycistronic expression may be used. Examples include an internal ribosome entry site (IRES) and a 2A self-cleaving peptide.
  • IRS internal ribosome entry site
  • a nucleic acid containing a region encoding a non-coding RNA may also be used.
  • a variety of heterologous polyA sequences can also be used downstream.
  • the expression cassette of the present invention is a nucleic acid construct used in the production of an AAV vector, and comprises a nucleic acid containing a helper factor that can increase the production yield of the AAV vector, and a heterologous promoter operably linked to the helper factor.
  • the nucleic acid containing a helper factor contained in the expression cassette of the present invention includes the following nucleic acids (1) to (4): (1) A nucleic acid containing a coding region of an adenovirus E2A protein. (2) A nucleic acid containing a coding region of one or more proteins selected from the group consisting of an adenovirus 22k protein, a 100k protein, a 33k protein, an E4orf2 protein, an E4orf3 protein, and an E4orf6/7 protein. (3) A nucleic acid containing a coding region of an adenovirus E4orf6 protein. (4) A nucleic acid containing a template DNA of an adenovirus VA-RNA.
  • coding region refers to the portion of the genome that encodes a protein (open reading frame (ORF)).
  • ORF open reading frame
  • the coding region does not include promoters, ribosome binding sites, transcription terminators, introns, etc. Stop codons (TAG, TGA, or TAA) are not translated into amino acids, but are included in the coding region.
  • the nucleic acid containing the helper factors (1) to (4) above can be derived from adenovirus serotypes 1 to 52 or modified forms thereof.
  • the helper factors may be natural or may be artificially mutated.
  • the base sequence of a nucleic acid comprising the coding region of adenovirus type 5 E2A protein is shown in SEQ ID NO: 1.
  • the nucleic acid comprising the coding region of adenovirus E2A protein may be a nucleic acid comprising a base sequence having 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the base sequence of SEQ ID NO: 1, or a nucleic acid in which several bases (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) have been substituted, inserted, added, and/or deleted in the base sequence of SEQ ID NO: 1, as long as it has the activity of increasing the production yield of an AAV vector.
  • codon-optimized nucleic acids may be used to optimize protein expression in mammalian cells.
  • nucleotide sequence of a nucleic acid containing the coding region for the 22k protein of adenovirus type 5 is shown in SEQ ID NO: 2
  • nucleotide sequence of a nucleic acid containing the coding region for the 100k protein is shown in SEQ ID NO: 3
  • nucleotide sequence of a nucleic acid containing the coding region for the 33k protein is shown in SEQ ID NO: 4.
  • the nucleic acids containing the coding regions for the adenovirus 22k protein, 100k protein, and 33k protein may be nucleic acids containing nucleotide sequences with 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 2, 3, and 4, respectively, as long as they have the activity of increasing the production yield of AAV vectors, or nucleic acids in which several nucleotides (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) have been substituted, inserted, added, and/or deleted in the nucleotide sequences of SEQ ID NOs: 2, 3, and 4.
  • codon-optimized nucleic acids may be used to optimize the expression of each protein in mammalian cells.
  • the nucleotide sequence of a nucleic acid containing the coding region of adenovirus type 5 E4orf2 protein is shown in SEQ ID NO: 5
  • the nucleotide sequence of a nucleic acid containing the coding region of E4orf3 protein is shown in SEQ ID NO: 6
  • the nucleotide sequence of a nucleic acid containing the coding region of E4orf6 protein is shown in SEQ ID NO: 7
  • the nucleotide sequence of a nucleic acid containing the coding region of E4orf6/7 protein is shown in SEQ ID NO: 8.
  • the nucleic acids containing the coding regions of adenovirus E4orf2 protein, E4orf3 protein, E4orf6 protein, and E4orf6/7 protein may be nucleic acids containing a nucleotide sequence that has 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 5, 6, 7, and 8, or nucleic acids in which several nucleotides (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) have been substituted, inserted, added, and/or deleted from the nucleotide sequences of SEQ ID NOs: 5, 6, 7, and 8, as long as they have the activity of increasing the production yield of AAV vectors. Codon-optimized nucleic acids may also be used to optimize the expression of each protein in mammalian cells.
  • the base sequence of a nucleic acid containing the template DNA of adenovirus type 5 VA-RNA is shown in SEQ ID NO: 9.
  • the nucleic acid containing the template DNA of adenovirus VA-RNA may be a nucleic acid containing a base sequence that has 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the base sequence of SEQ ID NO: 9, or a nucleic acid in which several bases (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) have been substituted, inserted, added, and/or deleted from the base sequence of SEQ ID NO: 9, as long as it has the activity of increasing the production yield of an AAV vector. It may also be a nucleic acid containing the template DNA of a VA-RNA-derived microRNA contained within the same sequence (Proc Natl Acad Sci U S A. 1980 May; 77(5): 2424-2428).
  • nucleic acids When producing an AAV vector, all of the following nucleic acids are introduced into virus-producing cells: (1) a nucleic acid containing the coding region of the adenovirus E2A protein; (2) a nucleic acid containing the coding region of one or more proteins selected from the adenovirus 22k protein, 100k protein, 33k protein, E4orf2 protein, E4orf3 protein, and E4orf6/7 protein; (3) a nucleic acid containing the coding region of the adenovirus E4orf6 protein; and (4) a nucleic acid containing the template DNA of the adenovirus VA-RNA.
  • the nucleic acids containing the above-mentioned helper factors used in the present invention may be separated into the same expression cassette or separate expression cassettes. For example, they may be separated into an expression cassette using nucleic acids containing the coding regions for the adenoviral E2A protein, 22k protein, and 100k protein, an expression cassette using nucleic acids containing the coding regions for the adenoviral E4orf2 protein, E4orf3 protein, E4orf6 protein, and E4orf6/7 protein, and an expression cassette using nucleic acid containing template DNA for adenoviral VA-RNA.
  • the combination of nucleic acids contained in the expression cassettes is not limited.
  • the helper factors expressed by the same or separate expression cassettes of the present invention use a combination of nucleic acids containing the helper factors specified in (1) to (4) above.
  • the nucleic acid specified in (2) above is a nucleic acid containing the coding region of one or more proteins selected from the group consisting of the 22k protein, the 100k protein, the 33k protein, the E4orf2 protein, the E4orf3 protein, and the E4orf6/7 protein.
  • helper activity can be further improved by using a nucleic acid containing the coding region of the 22k protein and the 100k protein, or a nucleic acid containing the coding region of the E4orf2 protein, the E4orf3 protein, and the E4orf6/7 protein, or a nucleic acid containing the coding region of the 22k protein, the 100k protein, the E4orf2 protein, the E4orf3 protein, and the E4orf6/7 protein.
  • nucleic acids containing the helper factors (1) to (4) above are not limited, but preferred combinations include nucleic acids containing the coding regions of E2A protein, E4orf6 protein, and 22k protein, nucleic acids containing the coding regions of E2A protein, E4orf6 protein, 22k protein, and 100k protein, nucleic acids containing the coding regions of E2A protein, E4orf6 protein, 22k protein, 100k protein, and 33k protein, nucleic acids containing the coding regions of E2A protein, E4orf6 protein, and E4orf3 protein, nucleic acids containing the coding regions of E2A protein, E4orf6 protein, and E4orf
  • suitable nucleic acids include nucleic acids containing the coding regions for E2A protein, E4orf6 protein, E4orf2 protein, E4orf3 protein, and E4orf6/7 protein, and nucleic acids containing the coding regions for E2A protein, 100k protein
  • a heterologous promoter is operably linked to the above-mentioned helper factor.
  • a heterologous promoter refers to a promoter other than the intrinsic promoter that is naturally present upstream of each of the above-mentioned helper factors and controls (regulates) their transcription; in the present invention, a heterologous promoter is artificially introduced by genetic engineering methods.
  • an intrinsic promoter is the opposite of a heterologous promoter; it refers to a promoter that is present in the genome of a non-genetically engineered host and under whose control a gene is originally expressed; in other words, a promoter that is originally operably linked to the gene.
  • control sequence and a coding sequence are arranged in such a manner that the control sequence can control the expression of the target protein encoded by the coding sequence.
  • control sequence can control the expression of the target protein encoded by the coding sequence.
  • a heterologous promoter other than the native promoter of the helper factor is arranged with the helper factor in a state in which the helper factor can be expressed under the control of the heterologous promoter.
  • the heterologous promoter may be a constitutive promoter or an inducible promoter, as long as it is a promoter that can express the above-mentioned helper factor in mammalian cells.
  • homeostatic promoters include virus-derived vectors such as the CBh promoter, cytomegalovirus (CMV)-derived promoters (e.g., the CMV immediate early promoter), human immunodeficiency virus (HIV)-derived promoters (e.g., the HIV LTR), Rous sarcoma virus (RSV)-derived promoters (e.g., the RSV LTR), mouse mammary tumor virus (MMTV)-derived promoters (e.g., the MMTV LTR), Moloney murine leukemia virus (MoMLV)-derived promoters (e.g., the MoMLV LTR), SV40-derived promoters (e.g., the SV40 early promoter), Epstein-Barr virus (EB
  • An inducible promoter is a promoter that can induce expression of a nucleic acid operably linked to it only in the presence of an inducer that drives the promoter, typically a drug-inducible promoter.
  • drug-inducible promoters include tetracycline-inducible promoters, Cumate operator sequences, and lambda operator sequences (e.g., 12 ⁇ lambda Op), with tetracycline-inducible promoters being preferred.
  • Tetracycline-inducible promoters include promoters that induce gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline) or reverse tetracycline-controlled transactivators (rtTA).
  • the TRE promoter (a mammalian cell promoter with a Tet response sequence consisting of multiple consecutive tetO sequences) is preferably used.
  • the TRE promoter it is preferable to simultaneously express a fusion protein of tetR and VP16AD or a fusion protein (rtTA) of reverse tetR (rtetR) and P16AD in the same cell.
  • rtTA fusion protein
  • the fusion protein does not bind to the TRE and transcription does not occur.
  • the fusion protein binds to the TRE promoter and transcription occurs, allowing the desired helper factor to be transiently expressed while the drug is added.
  • genome editing technology may be used as an inducible expression system for helper factors.
  • inducible expression systems and repression systems that utilize the genome editing tool CRISPR/Cas9 system, such as an inducible expression system that uses transcription activation/inactivation by a protein formed by fusing inactive Cas9 with a transcription activator such as VP64 and a transcription repressor such as KRAB.
  • Conditional expression systems that utilize recombinases are also similar to inducible expression systems.
  • a typical example is an expression system that uses a LoxP-Stop-LoxP (LSL) cassette, where the LSL cassette is removed by introducing the recombinase Cre recombinase into cells, allowing the promoter to conditionally transcribe the gene.
  • LSL LoxP-Stop-LoxP
  • the above expression cassette may be incorporated into a plasmid used to produce an AAV vector and used as a helper plasmid.
  • plasmid used here, but mammalian expression plasmids are preferred, such as pBApo-CMV DNA and pIRES Bicistronic Expression Vector (manufactured by Takara Bio Inc.).
  • the plasmid may be selected appropriately depending on the type of cell to be introduced, and may be a cosmid, phage vector, phagemid vector, BAC vector, YAC vector, MAC vector, HAC vector, etc.
  • helper plasmid of the present invention When multiple expression cassettes are carried in the helper plasmid of the present invention, their arrangement is not particularly limited, but they are preferably arranged so that they are transcribed in the same direction, and include, in the direction of the reading frame of transcription, a promoter region, an expression cassette of the present invention, a transcription terminator region, and a self-cleaving peptide sequence (e.g., a P2A sequence).
  • the above-mentioned expression cassette may be incorporated into a viral vector used in the production of an AAV vector and used as a helper virus.
  • the helper virus used here is not particularly limited, but examples include non-enveloped viruses such as adenovirus, enterovirus, parvovirus, papovavirus, human papillomavirus, rotavirus, coxsackievirus, sapovirus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, circovirus, and simian virus; herpes viruses such as retrovirus, lentivirus, Sendai virus, and herpes simplex virus; and enveloped viruses such as vaccinia virus, measles virus, baculovirus, influenza virus, leukemia virus, and Sindbis virus and poxvirus.
  • non-enveloped viruses such as adenovirus, enterovirus, parvovirus, papovavirus, human
  • the helper plasmid of the present invention can further include, as needed, a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, or a reporter gene sequence such as a fluorescent protein, ⁇ -glucuronidase (GUS), or FLAG.
  • a drug resistance gene e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.
  • a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene
  • a reporter gene sequence such as a fluorescent protein, ⁇ -glucuronidase (GUS), or FLAG.
  • the AAV vector of the present invention is produced by co-transfecting virus-producing cells with the above-mentioned helper plasmid or helper virus, a vector plasmid (transfer plasmid) containing an expression cassette for a gene of interest between the ITRs at both ends of the adenovirus genome, and a plasmid (packaging plasmid) containing the AAV Rep and Cap genes, to create cells capable of producing virus, and collecting the AAV vector of interest produced in the culture supernatant of the cells.
  • helper plasmid or helper virus a vector plasmid (transfer plasmid) containing an expression cassette for a gene of interest between the ITRs at both ends of the adenovirus genome
  • a plasmid packetaging plasmid
  • adeno-associated viruses include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), and AAV type 10 (AAV10), and any of these can be used to produce AAV vectors.
  • Other examples include artificially produced AAV vectors such as AAV-DJ and AAV-PHP.B.
  • this technology can also be applied to packaging and amplifying artificial nucleic acids within AAV capsids.
  • virus-producing cells refer to cells capable of producing viruses, into which the elements necessary for forming virus particles have been introduced in such a way that virus particles are formed and produced intracellularly.
  • Virus-producing cells used in the present invention include all mammalian cells, including humans, such as somatic cells that make up a living organism, precursor cells, and cancer cells, cells (cell lines) that have been isolated from a living organism and acquired immortalization ability and are stably maintained ex vivo, and cells that have been isolated from a living organism and artificially genetically modified. There are no particular limitations on the origin of the cells, as long as they are mammalian, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses.
  • human cells include HEK293 cells, HEK293T cells derived from HEK293 cells, HEK293S cells, HEK293F cells, HEK293FT cells, HEK293FTM cells, HEK293SG cells, HEK293SGGD cells, HEK293H cells, HEK293E cells, HEK293MSR cells, and VPC2.0 cells.
  • adenovirus e.g., Adeno-X 293 Cell Line
  • AAV e.g., AAVpro 293T Cell Line
  • human cells include G3T-hi cells (Takara Bio), HeLa cells (ATCC CCL-2), MOLT-4 cells (ATCC CRL-1582), human lung cancer-derived A549 cells, human fibrosarcoma HT-1080 cells, human retinal tissue-derived cells such as PER.C6 cells, human tissue-derived mesenchymal stem cells, cells derived from human uterine contents, placenta, and fetal tissue, and human liver-derived cells.
  • Non-human primate cells include Vero cells, COS-1 cells (ATCC CRL-1650), and COS-7 cells (ATCC CRL-1651), while rodent cells include BHK cells, CHO cells (ATCC CCL-61), and HePa1-6 cells (ATCC CRL-1830).
  • target genes include human therapeutic genes and marker genes for evaluating gene transfer efficiency or expression stability, such as genes encoding GFP (Green Fluorescent Protein), ⁇ -galactosidase, and luciferase.
  • the above-mentioned plasmid can be transfected into virus-producing cells using any method that can introduce DNA into animal cells, such as electroporation, calcium phosphate, lipofection, or DEAE-dextran, all of which are well known to those skilled in the art.
  • the medium used for culturing virus-producing cells may be a medium commonly used for culturing animal cells.
  • a basal medium containing components necessary for cell survival and proliferation may be used.
  • D-MEM Dulbecco's Modified Eagle's Medium
  • Dulbecco's Modified Eagle's Medium:Nutient Mixture F-12 (D-MEM/F-12) medium Glasgow MEM (G-MEM) medium, etc.
  • suitable medium examples include Basal Medium Eagle (BME) medium, Minimum Essential Medium (MEM) medium, Eagle's minimal essential medium (EMEM) medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, RPMI 1640 medium, Medium 199 medium, ⁇ MEM medium, Ham's medium, Fischer's medium, and mixtures thereof.
  • the medium may also contain growth factors (FGF, EGF, etc.), interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 supplement, N2 supplement, antibiotics (penicillin, streptomycin, etc.), etc.
  • the medium may be serum-containing or serum-free, as needed. To prevent contamination with components derived from different animal species, it is preferable to use serum-free or serum derived from the same animal species as the cells being cultured. Serum substitutes such as albumin may also be used.
  • Virus-producing cells into which a plasmid has been introduced can be cultured under standard culture conditions.
  • the culture temperature is not particularly limited, but is, for example, 30 to 40°C, preferably 35 to 37°C.
  • the CO2 concentration is, for example, 1 to 10%, preferably 5 to 6%.
  • the culture time is not particularly limited, but is, for example, 24 to 120 hours, preferably 48 to 96 hours.
  • the culture supernatant or cells are collected to obtain the AAV vector.
  • the AAV vector is produced in the form of the supernatant, a filtrate obtained by filtering the supernatant, an extract obtained by disrupting the cells, or a concentrate or purified product concentrated or purified by known methods, and is stored by an appropriate method, for example, by freezing, until use.
  • the present invention also provides an adeno-associated virus (AAV) vector-producing cell characterized in that the expression cassette used in the production of the AAV vector is stably maintained within the cell.
  • AAV vector-producing cells refers to virus-producing cells in which the expression cassette necessary for AAV vector production is stably maintained within the cell, and is different from virus-producing cells in which the expression cassette has been transiently introduced.
  • AAV vector-producing cells may be cells in which the introduced gene has been integrated into the host genome of the cell and is maintained permanently without being diluted even with cell division, or cells in which the introduced gene can be autonomously replicated with cell division and continuously expressed without being integrated into the host genome of the cell, for example, by using an episomal vector.
  • the "AAV vector-producing cells" of the present invention stably maintain within the cells an expression cassette capable of inducibly expressing a helper factor that can increase the production amount of the AAV vector. Therefore, the AAV-producing cells of the present invention do not constantly express the AAV components used in AAV vector production or toxic proteins derived from the helper factors, which have been a problem in the past, during cell maintenance and culture. This prevents cell death, interference with cell proliferation, and no changes in cell function, including AAV production ability.
  • Adeno-associated virus (AAV) vector-producing cells are produced by gene transfer of the expression cassette used to produce the AAV vector into cells.
  • Gene transfer methods can be chemical methods such as the liposome method or calcium phosphate method, physical methods such as electroporation, or methods using viral vectors.
  • the expression cassette can be introduced using a plasmid vector (e.g., a conventional plasmid vector or an episomal vector), or it can be introduced (knocked in) using the transposon method or genome editing method.
  • a viral vector When using a viral vector, it can be introduced by infection with, for example, a retroviral vector, lentiviral vector, adeno-associated virus vector, adenovirus vector, Sendai virus vector, vaccinia virus vector, baculovirus vector, etc. After gene transfer, clones in which the expression cassette is stably maintained in the cells can be selected by drug selection or limiting dilution.
  • the level of AAV vector production is indicated using the titer of the AAV vector.
  • the titer of the AAV vector can be expressed either as the number of genomes obtained from properly formed AAV vector particles in a certain amount of sample (genomic titer), or as the experimentally measured ability of the AAV vector to infect cells (infectious titer).
  • “genomic titer” refers to the amount of viral vector
  • infectious titer refers to the efficiency (ability) of infection of specific cells.
  • the genome titer can be measured by a method of measuring the copy number of the viral vector, and the infectious titer can be measured, for example, by a method of infecting target cells with the viral vector and detecting the expression of the introduced gene.
  • AAV vector type 1 was constructed using pAAV-AcGFP (a plasmid in which AcGFP was cloned from pAcGFP-N1 (Takara Bio) into pAAV-mcs (Agilent Technologies)), pAAV2/1 (Takara Bio), and each helper plasmid to be tested for helper activity.
  • pAAV-AcGFP a plasmid in which AcGFP was cloned from pAcGFP-N1 (Takara Bio) into pAAV-mcs (Agilent Technologies)
  • pAAV2/1 Takara Bio
  • the above mixture of plasmids for AAV1 production was adjusted in OPTI-MEM to a DNA:PEI ratio of 1:2, and allowed to react at room temperature for 20 minutes. After the reaction, the plasmid mixture was transfected into 80% or more confluent AAV293 cells or AAV293-rtTA cells in D-MEM containing 10% FBS for 6 hours, and then cultured in serum-free D-MEM. If necessary, 10-1000ng/ml of doxycycline was added as an inducer.
  • Genomic DNA of the AAV vector was extracted to measure viral titer. Specifically, the culture supernatant was collected and treated with Benzonase at 37°C for 1 hour, after which viral genomic DNA was extracted and purified using the DNeasy Blood & Tissue kit (QIAGEN). Viral titers were measured in triplicate by qPCR.
  • qPCR was performed using primers targeting AcGFP (Forward: 5'-ATCACATGAAGCAGCACGAC-3' (SEQ ID NO: 10), Reverse: 5'-TAGTTGCCGTCATCCTCGAA-3' (SEQ ID NO: 11), TaqMAN: 5'Fam-AGCCCTCAGGCATGGCGCTC-3'TAMRA (SEQ ID NO: 12)).
  • Plasmid Construction pHelper-E2A, pHelper-E4, and pHelper-VA were constructed from the pHelper plasmid (Takara Bio) by restriction enzyme digestion and self-ligation.
  • pCBh-E2A, pCBh-E4orf6, and pCBh-VA were constructed by PCR amplification of the E2A, E4orf6, and VA-RNA regions using the pHelper plasmid (Takara Bio) as a template and ligating them to the pCBh promoter.
  • pCBh-E2A-22k-100k (E2Aopti) was constructed by PCR amplification of the E2A, 22k, and 100k coding regions to confer the 2A peptide, then ligating them to the pCBh promoter using overlapping PCR.
  • pCBh-E4orf1-7, pCBh-E4orf2-7, pCBh-E4orf3-7, pCBh-E4orf4-7, and pCBh-E4orf6-7 were constructed by PCR amplification of E4orf1-7, E4orf2-7, E4orf3-7, E4orf4-7, and E4orf6-7, respectively, followed by ligation to the pCBh promoter.
  • pCBh-E4-2367 (E4opti) and pCBh-E4-367 were constructed by PCR amplification of E4orf2, E4orf3, and E4orf6-7, respectively, to confer the 2A peptide, then ligated by overlapping PCR and ligated to the pCBh promoter.
  • pTRE-E2Aopti-E4opti and pTRE-VA were constructed by artificial DNA synthesis.
  • pTRE-E2Aopti-E4opti-VA was prepared by cloning the pTRE-E2Aopti-E4opti and pTRE-VA expression cassettes into the p15-amp plasmid (prepared by artificial DNA synthesis).
  • E2A, E4, and VA-RNA gene expression levels were measured in triplicate using qRT-PCR. qPCR was performed using primers targeting E2A, E4, and VA-RNA, and the amount of S18rRNA was measured as an endogenous control for correction. The primer sequences used for PCR are shown below.
  • Example 1 (1) Analysis of the helper activity of expression cassettes for proteins contained in the E2A genomic fragment.
  • AAV vectors were constructed using pCBh-E2A, pCBh-100k, pCBh-22k, and pCBh-33k, along with pHelper-E4, pHelper-VA, pAAV-AcGFP, and pAAV2/1 (Fig. 5).
  • Three days after transfection, culture supernatants were collected and AAV vector production was measured by quantitative PCR.
  • the results demonstrated that the genes encoding the 100k and 22k proteins, when combined with E2A, enhanced the helper activity, and their combination further enhanced the helper activity (Fig. 6). Furthermore, the combination of these with the gene encoding the 33k protein further enhanced the helper activity (Fig. 6).
  • AAV vectors were constructed using pCBh-E2A-22k-100k, pHelper-E4, pHelper-VA, pAAV-AcGFP, and pAAV2/1. Three days after transfection, culture supernatants were collected and AAV vector production was measured by quantitative PCR. As a result, it was confirmed that the helper activity of pCBh-E2A-22k-100k was greater than or equal to that of the original pHelper-E2A (FIG. 8).
  • AAV vectors were constructed using these plasmids along with pHelper-E2A, pHelper-VA, pAAV-AcGFP, and pAAV2/1. Three days after transfection, culture supernatants were collected and AAV vector production was measured by quantitative PCR. The results demonstrated that E4orf1-7, E4orf2-7, and E4orf3-7 all possessed helper activity equivalent to that of the original E4 genomic fragment (Figure 10).
  • Example 2 (1) Analysis of helper activity of optimized helper factor expression cassettes pCBh-E2A-22k-100k (pCBh-E2Aopti: SEQ ID NO: 25) and pCBh-E4orf2367 (pCBh-E4opti: SEQ ID NO: 26), which were constructed by combining factors considered to be optimal among the factors whose helper activity was confirmed in Example 1, were used in combination with pCBh-VA to verify their helper activity.
  • pAAV-AcGFP and pAAV2/1 AAV vectors were constructed using these plasmids in combination with pAAV-AcGFP and pAAV2/1 ( Figure 15).
  • the present invention can be used in the fields of gene cell therapy, vaccines, and the production of viral vectors that serve as tools for gene function analysis. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

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Abstract

La présente invention a pour but de mettre au point un nouveau système d'expression de facteurs auxiliaires permettant d'augmenter la quantité de production d'un vecteur viral adéno-associé (AAV) et de rendre possible l'expression inductible.　La présente invention concerne une cassette d'expression destinée à être utilisée dans la production d'un vecteur AAV. La cassette d'expression inclut un acide nucléique contenant un facteur auxiliaire pouvant augmenter la quantité de production d'un vecteur AAV, et un promoteur hétérologue fonctionnellement lié au facteur auxiliaire, l'acide nucléique incluant les acides nucléiques (1) à (4) suivants. (1) Un acide nucléique comportant une région codante pour une protéine E2A d'un adénovirus; (2) un acide nucléique comportant des régions codantes pour une ou plusieurs protéines choisies parmi une protéine 22k, une protéine 100k, une protéine 33k, une protéine E4 orf2, une protéine E4 orf3 et une protéine E4 orf6/7 d'un adénovirus; (3) un acide nucléique comportant une région codante pour une protéine E4 orf6 d'un adénovirus; et (4) un acide nucléique comportant un ADN matrice pour l'ARN-VA d'un adénovirus.
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Title
ALLEN, J. M. ET AL.: "Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6", MOL. THER., vol. 1, 2000, pages 88 - 95, XP093276869, DOI: 10.1006/mthe.1999.0010 *
COLLACO, R. F. ET AL.: "A helper virus-free packaging system for recombinant adeno- associated virus vectors", GENE, vol. 238, 1999, pages 397 - 405, XP002200459, DOI: 10.1016/S0378-1119(99)00347-9 *
MATSUSHITA, T. ET AL.: "Adeno-associated virus vectors can be efficiently produced without helper virus", GENE THERAPY, vol. 5, no. 7, 1998, pages 938 - 945, XP037773443, DOI: 10.1038/sj.gt.3300680 *

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