Cell for producing recombinant herpes simplex virus vector, and method for producing recombinant herpes simplex virus vector

Abstract

The present invention pertains to cells for producing a recombinant herpes simplex virus (HSV) vector. The cells retain a specific first base sequence. When the first base sequence includes a specific twenty eighth base sequence, the cells retain a specific thirtieth base sequence.

Description

Cells for producing recombinant herpes simplex virus vectors and method for producing recombinant herpes simplex virus vectors

[0001] The present invention relates to cells for producing recombinant herpes simplex virus vectors and methods for producing recombinant herpes simplex virus vectors.

[0002] Gene therapy involves administering genes or cells into the body with the aim of treating or preventing disease, and the market for gene therapy has been expanding in recent years. Among gene therapies, treatments using recombinant viral vectors have demonstrated high efficacy and safety in clinical settings, and in fact, some recombinant viral vector medicines have already been applied clinically.

[0003] Among various recombinant viral vectors, the majority of clinical trials have been conducted using recombinant adeno-associated viral vectors (recombinant AAV vectors) because they are available in many laboratories, etc. On the other hand, recombinant herpes simplex viral vectors (recombinant HSV vectors) are sometimes preferable to recombinant AAV vectors as recombinant viral vectors for gene therapy depending on the purpose, because they have excellent immune evasion ability, allowing for multiple administrations, and can carry a large foreign gene.

[0004] Various techniques have been proposed for producing recombinant HSV vectors. For example, Patent Document 1 discloses a herpesvirus vector packaging system for packaging a herpesvirus vector in the absence of a helper virus, comprising: a. at least one first vector that is capable of forming one or more DNA segments capable of expressing herpesvirus structural proteins when introduced into cells capable of supporting herpesvirus DNA replication, but that lacks a herpesvirus cleavage / packaging site-containing sequence (herpesvirus DNA segment); and b. a packaging vector that includes a herpesvirus packaging site-containing sequence and a DNA replication origin recognized by herpesvirus DNA replication proteins and enzymes.

[0005] Furthermore, for example, Patent Document 2 discloses a herpes simplex virus (HSV) vector that does not express toxic HSV genes in non-complementing cells, contains a genome containing one or more introduced genes, and is capable of expressing the introduced genes in non-complementing cells for at least 28 days.

[0006] Special table No. 11-510053 Publication No. 2016-525349

[0007] On the other hand, conventional methods for producing recombinant viral vectors require purified viral genomes, but purifying the HSV genome requires several days and expensive equipment. Furthermore, the HSV genome is large, approximately 150 kbp, and cannot be frozen, making it difficult to handle. Furthermore, the currently widely used cells for producing recombinant HSV vectors (Vero cells) have very poor gene transfer efficiency, and the organism from which the cells are derived is designated as an endangered species under the Washington Convention, resulting in restrictions on import and export. For these reasons, even for experts, the production and use of recombinant HSV vectors is extremely difficult and limited, and is also expensive.

[0008] Therefore, an object of the present invention is to provide cells for producing a recombinant HSV vector easily and at low cost, and a method for producing a recombinant HSV vector using the same.

[0009] The present invention includes, for example, the following inventions: [1] A mammalian-derived cell for producing a recombinant herpes simplex virus (HSV) vector, the cell retaining a first base sequence, the first base sequence satisfying the following (1), (2), (3), (4), and (5): (1) comprising base sequences 2 to 27 that express proteins 1 to 26, (2) lacking base sequences encoding proteins 27 to 28, (3) lacking base sequences encoding protein 29, (4) lacking base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, and (5) comprising a 28th base sequence consisting of the base sequence shown in SEQ ID NO: 7 or a 29th base sequence consisting of the base sequence shown in SEQ ID NO: 8, the first to 26th proteins having 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 76 to 101, respectively, and having the same quality of function as proteins consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 101, The 27th to 29th proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively, and have the same functions as proteins consisting of the amino acid sequences shown in SEQ ID NOs: 12 to 14, and the cell, when the first base sequence includes the 28th base sequence, retains a 30th base sequence that expresses a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and having the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence. [2] The cell according to [1], wherein the first base sequence further satisfies the following (6): (6) comprising at least one base sequence selected from all of the base sequences of 31 to 52 that express the proteins of 30 to 51, and the base sequences of 53 to 84 that express the proteins of 52 to 83, and wherein the proteins of 30 to 83 have 90% or more sequence identity with the amino acid sequences of SEQ ID NOs: 102 to 155, respectively, and have the same quality of function as the proteins consisting of the amino acid sequences of SEQ ID NOs: 102 to 155. [3] The cell according to [2], comprising all of the base sequences of 53 to 84.[4] The cell according to any one of [1] to [3], wherein the first base sequence has 95% or more sequence identity with the base sequence shown in SEQ ID NO: 1. [5] The cell according to any one of [1] to [4], wherein the first base sequence further comprises the 30th base sequence and has 95% or more sequence identity with the base sequence shown in SEQ ID NO: 11. [6] The cell according to any one of [1] to [5], wherein the first base sequence comprises the 28th base sequence. [7] The cell according to any one of [1] to [6], wherein the 30th base sequence is a base sequence that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 9. [8] A method for producing a recombinant HSV vector, comprising the steps of: introducing an amplicon plasmid into a mammalian cell; and expressing all of proteins 1 to 26, all of proteins 30 to 51, and at least one protein from proteins 52 to 83, wherein the cell retains a 85th base sequence, and the 85th base sequence satisfies the following (A), (B), (C), (D), and (E): (A) comprising at least one of nucleotide sequences 2 to 27 and 31 to 84 that express proteins 1 to 26 and 30 to 83; (B) lacking a nucleotide sequence encoding proteins 27 and 28; (C) lacking a nucleotide sequence encoding protein 29; (D) lacking the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; and (E) The proteins satisfy the above condition: (A) contain a 28th base sequence consisting of the base sequence shown in SEQ ID NO: 7, or a 29th base sequence consisting of the base sequence shown in SEQ ID NO: 8; (B) the proteins 1 to 26 and 30 to 83 have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 76 to 155, respectively, and have the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 155; (C) the proteins 27 to 29 have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively, and have the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 12 to 14;A method for producing a recombinant HSV vector that expresses a gene of interest, wherein the amplicon plasmid comprises a gene of interest, an 86th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 15, and an 87th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and the cell, when the 85th nucleotide sequence comprises the 28th nucleotide sequence, retains a 30th nucleotide sequence that expresses a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and has the ability to bind to chromatin and the ability to bind to the 28th or 29th nucleotide sequence. [9] A method for producing a recombinant HSV vector that expresses a gene of interest, wherein the amplicon plasmid comprises a gene of interest, an 86th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 15, and an 87th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and the cell, when the 85th nucleotide sequence comprises the 28th nucleotide sequence, retains a 30th nucleotide sequence that expresses a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and has the ability to bind to chromatin and the ability to bind to the 28th or 29th nucleotide sequence.

[10] A method for producing a recombinant HSV vector that expresses a gene of interest, wherein the step of expressing the protein comprises expressing proteins 1 to 26 and proteins 30 to 83.

[11] A method for producing a recombinant HSV vector that expresses a gene of interest, wherein the step of expressing the protein comprises expressing at least one of proteins 27 to 29.

[12] The method according to any one of [8] to

[11] , wherein the 87th base sequence is the base sequence shown in SEQ ID NO: 5.

[13] A kit for producing a recombinant HSV vector, comprising a mammal-derived cell and one or more first plasmids, wherein the cell retains the 89th base sequence, and the 89th base sequence satisfies the following (i), (ii), (iii), (iv), and (v): (i) comprising at least one of the 2nd to 27th base sequences and the 31st to 52nd base sequences that express the 1st to 26th and 30th to 51st proteins, (ii) lacking the 27th to 28th base sequences, (iii) lacking the 29th base sequence, (iv) lacking the 5th and 6th base sequences, and (v) comprising the 28th base sequence consisting of the 7th base sequence or the 89th base sequence consisting of the 89th base sequence. At least one of the first plasmids comprises an 86th base sequence consisting of the base sequence shown in SEQ ID NO: 15 and an 87th base sequence consisting of the base sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6,the first plasmid contains a base sequence not contained in the 89th base sequence among the 2nd to 27th base sequences and the 31st to 52nd base sequences, and either or both of the 89th base sequence and the first plasmid contain at least one of the 53rd to 84th base sequences that express the 52nd to 83rd proteins, the 1st to 26th and 30th to 83rd proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 76 to 155, respectively, and are proteins having the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 155, the 27th to 29th proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively, and are proteins having the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 12 to 14, and the cell, when the 89th base sequence contains the 28th base sequence, A kit comprising a 30th base sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:9 and expressing a protein having the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence.

[14] The kit according to

[13] , wherein the 89th base sequence comprises at least one of the 53rd to 84th base sequences, and the first plasmid comprises a base sequence not contained in the 89th base sequence among the 2nd to 27th base sequences and the 31st to 84th base sequences.

[15] The kit according to

[13] or

[14] , wherein the cell is the cell according to any of [1] to [7].

[16] The kit according to

[13] to

[15] , wherein the 87th base sequence is the base sequence shown in SEQ ID NO:5.

[17] The kit according to any of

[13] to

[16] , wherein the first plasmid consists of a base sequence having 90% or more sequence identity with the 26th base sequence.

[18] The kit according to any one of

[13] to

[17] , further comprising one or more second plasmids, wherein the second plasmids comprise a base sequence for expressing at least one of the proteins of Nos. 27 to 29.

[19]

[18] The kit according to

[18] , wherein the second plasmid contains a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 75 and expresses a protein having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12.

[20] The kit according to

[18] or

[19] , wherein the second plasmid contains a plasmid consisting of a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 70.

[21] A mammal-derived cell for producing a recombinant herpes simplex virus (HSV) vector, wherein the cell harbors the genomic DNA of the recombinant HSV, the genomic DNA of the recombinant HSV contains the nucleotide sequences of all essential genes of the recombinant HSV and a nucleotide sequence for being retained in the cell, and is deficient in the nucleotide sequence of an immediate early gene and a nucleotide sequence for packaging into the recombinant HSV.

[22] The cell according to

[21] , wherein the genomic DNA of the recombinant HSV further contains the nucleotide sequence of at least one non-essential gene of the recombinant HSV.

[23] A method for producing a recombinant HSV vector, comprising the steps of: introducing an amplicon plasmid into a mammalian cell; and expressing all of the proteins encoded by the essential genes of the recombinant HSV and at least one of the proteins encoded by the non-essential genes of the recombinant HSV, wherein the cell retains the genomic DNA of the recombinant HSV, and the genomic DNA of the recombinant HSV contains the nucleotide sequence of at least one of the essential and non-essential genes of the recombinant HSV and a nucleotide sequence for being retained in the cell, and is deficient in the nucleotide sequence of an immediate early gene and a nucleotide sequence for packaging into the recombinant HSV, and the amplicon plasmid contains the gene of interest, the nucleotide sequence for packaging into the recombinant HSV, and the nucleotide sequence of a replication origin of the recombinant HSV.

[24] The method for producing a recombinant HSV vector according to

[23] , wherein the cell is the cell according to

[21] or

[22] .

[25] A kit for producing a recombinant HSV vector, comprising a mammalian cell and one or more first plasmids,

[26] The kit according to

[25] , wherein the cell retains the genomic DNA of the recombinant HSV, the genomic DNA of the recombinant HSV contains the base sequence of at least one of the essential gene of the recombinant HSV and the non-essential gene of the recombinant HSV, and a base sequence for being retained in the cell, and is deficient in the base sequence of an immediate early gene and a base sequence for packaging into the recombinant HSV, at least one of the first plasmids contains the base sequence for packaging into the recombinant HSV and the base sequence of a replication origin of the recombinant HSV, and the first plasmid contains the base sequence of the essential gene of the recombinant HSV and the non-essential gene of the recombinant HSV that is not contained in the genomic DNA of the recombinant HSV.

[0010] The present invention also encompasses, for example, the following inventions. [2-1] The cell according to any one of [1] to [4], wherein the first base sequence includes the base sequence of SEQ ID NO: 29. [2-2] The cell according to [2-1], wherein the first base sequence has 95% or more sequence identity with the base sequence shown in SEQ ID NO: 16. [2-3] The production method according to any one of [8] to [9] and

[11] to

[12] , wherein the cell is the cell according to [2-1] or [2-2]. [2-4] The kit according to any one of

[13] to

[20] , wherein the cell is the cell according to [2-1] or [2-2].

[0011] The present invention further encompasses, for example, the following inventions: [3-1] Use (application) of a mammalian cell for producing a recombinant HSV vector, wherein the cell retains the 85th nucleotide sequence, and the 85th nucleotide sequence satisfies the following (A), (B), (C), (D), and (E): (A) comprising at least one of the 2nd to 27th nucleotide sequences and the 31st to 84th nucleotide sequences that express the 1st to 26th and 30th to 83rd proteins, (B) lacking the nucleotide sequence encoding the 27th to 28th proteins, (C) lacking the nucleotide sequence encoding the 29th protein, (D) lacking the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6, and (E) comprising the 28th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO:7 or the 29th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO:8, the proteins 1 to 26 and 30 to 83 have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 76 to 155, respectively, and have the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 155; the proteins 27 to 29 have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively, and have the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 12 to 14; the amplicon plasmid contains a target gene, an 86th base sequence consisting of the base sequence shown in SEQ ID NO: 15, and an 87th base sequence consisting of the base sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6; and when the 85th base sequence contains the 28th base sequence, the cell holds a 30th base sequence that expresses a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and having the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence. [3-2] The use (application) according to [3-1], wherein the cell is a cell according to any one of [1] to [7]. [3-3] The use (application) according to [3-1] or [3-2], wherein the 87th base sequence is the base sequence shown in SEQ ID NO: 5.[3-4] A use (application) of a mammalian cell for producing a recombinant herpes simplex virus (HSV) vector, wherein the cell retains the genomic DNA of the recombinant HSV, the genomic DNA of the recombinant HSV contains the nucleotide sequence of at least one of the essential and non-essential genes of the recombinant HSV and a nucleotide sequence for being retained in the cell, and is deficient in the nucleotide sequence of an immediate early gene and a nucleotide sequence for packaging into the recombinant HSV, and the amplicon plasmid contains a gene of interest, a nucleotide sequence for packaging into the recombinant HSV, and a nucleotide sequence of a replication origin of the recombinant HSV. [3-5] The use (application) of [3-4], wherein the cell is the cell of

[21] or

[22] . [3-6] A method for producing a gene therapy composition containing the recombinant HSV vector, comprising producing a recombinant HSV vector by the production method of any of [8] to

[12] and [2-3]. [3-7] A gene therapy method comprising: producing a recombinant HSV vector by the production method according to any one of [8] to

[12] ,

[23] to

[24] , and [2-3]; and introducing the target gene into a subject using the recombinant HSV vector.

[0012] According to the present invention, it is possible to provide cells for producing herpes simplex virus vectors easily and at low cost, and a method for producing herpes simplex virus vectors using the same.

[0013] 1 is a schematic diagram showing the wild-type HSV-1 genome (line 1) and the recombinant HSV genome (line 2) prepared in Example 1.

[0034] FIG. 1 is a schematic diagram showing an outline of the method for establishing cells harboring a recombinant HSV-1 genome.

[0035] FIG. 1 is a fluorescent microscope image of HEK293 cells and 293 / HSV cells.

[0036] FIG. 1 is a vector map of pA-ICP0-GFP.

[0037] FIG. 1 is a diagram showing the results of Western blot analysis of the expression of ICP0, ICP27, and gB in each cell.

[0038] FIG. 1 is a fluorescent microscope image of 293 / HSV cells mock-transfected or transfected with pA-ICP0-GFP and pICP27 plasmids.

[0039] FIG. 1 is a fluorescent microscope image of Vero cells infected with recombinant HSV using the culture supernatant of mock-transfected or transfected 293 / HSV cells transfected with each plasmid.

[0039] FIG. 1 is a fluorescent microscope image of GFP expression when Vero cells were infected with recombinant HSV vectors using the prepared vector stocks and their dilutions. 1 is a graph showing the results of a quantitative analysis by flow cytometry of GFP expression when Vero cells are infected with the prepared vector stock. 2 is a graph showing the results of a flow cytometry analysis of GFP expression when Vero cells are infected with the prepared vector stock. 3 is a graph showing the titer of the vector stock when Vero cells are infected with the prepared vector stock.

[0014] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0015] [Cells for Producing a Recombinant Herpes Simplex Virus (HSV) Vector] Mammal-derived cells for producing a recombinant HSV vector according to this embodiment (hereinafter also simply referred to as "cells according to this embodiment") (cells according to the first embodiment) retain a specific first base sequence, and when the first base sequence includes a specific 28th base sequence, the cells retain a specific 30th base sequence.

[0016] <Herpes Simplex Virus (HSV)> HSV is a herpesvirus whose natural host is humans. HSV particles are roughly spherical with a diameter of approximately 200 nm and consist of the main basic structures of an envelope, tegument, and capsid from the outside. HSV also has a linear double-stranded DNA genome of approximately 150 kbp, which is encapsulated in an icosahedral capsid. Currently, 83 protein-encoding genes are known on the HSV genome, and it is expected that additional genes will be discovered in the future. HSV genes are broadly classified into three types based on their expression time: immediate early genes (IE genes, α genes), early genes (Early genes: E genes, β genes), and late genes (Late genes: L genes, γ genes). When HSV infects a cell, the VP16 protein, a component of the viral particle, binds to the Oct-1 and HCF-1 proteins in the cell to form a transcription complex, which then binds to the promoter of the IE gene and promotes transcription of the IE gene. The timing of HSV gene expression is controlled by the IE gene, and transcription of the E gene and L gene is under the control of the IE gene. Previous research has shown that deletion of the IE gene eliminates or reduces gene expression of the E gene and L gene.

[0017] The IE genes (α genes) include, for example, genes encoding proteins (gene expression regulators) involved in the expression of the E gene (β gene) and the L gene (γ gene). Five IE genes are known: ICP0, ICP4, ICP22, ICP27, and ICP47 (Fields Virology, 7th ed., Howley, Peter M., Knipe, David M. & Cohen, Jeffrey L., WOLTERS KLUWER HEALTH, 2021 / 10, ISBN: 9781975112578).

[0018] As used herein, "immediate early gene (IE gene, α gene)" refers to a gene that expresses a protein having the same function as the protein expressed by the immediate early gene. That is, the "immediate early gene" may be wild-type or mutant. A mutant gene may be a gene that expresses a protein that has 90% to less than 100%, 91% to less than 100%, 92% to less than 100%, 93% to less than 100%, 94% to less than 100%, 95% to less than 100%, 96% to less than 100%, 97% to less than 100%, 98% to less than 100%, or 99% to less than 100% sequence identity to the protein expressed by the wild-type gene. Sequence identity will be described later.

[0019] E genes (β genes) include many genes encoding proteins that control DNA synthesis, such as proteins necessary for DNA replication and enzymes involved in deoxyribonucleotide metabolism. More specific examples of E genes include UL9, UL29, and UL30 (Field's Virology, 7th Edition, Chapter 60).

[0020] As used herein, "early genes (E genes, β genes)" refer to genes that express proteins with the same functions as proteins expressed by early genes. That is, "early genes" may be wild-type or mutant. Mutant genes may be genes that express proteins with sequence identity of 90% to less than 100%, 91% to less than 100%, 92% to less than 100%, 93% to less than 100%, 94% to less than 100%, 95% to less than 100%, 96% to less than 100%, 97% to less than 100%, 98% to less than 100%, or 99% to less than 100% to the proteins expressed by wild-type genes. Sequence identity will be described later.

[0021] The L gene (γ gene) contains many genes encoding structural proteins of the virus particle, such as envelope protein, capsid protein, and tegument protein. More specific examples of the L gene include UL27, UL19, and UL48 (Field's Virology, 7th Edition, Chapter 60).

[0022] As used herein, "late genes (L genes, γ genes)" refer to genes that express proteins with the same functions as proteins expressed by late genes. That is, "late genes" may be wild-type or mutant. A mutant gene may be a gene that expresses a protein with sequence identity of 90% to less than 100%, 91% to less than 100%, 92% to less than 100%, 93% to less than 100%, 94% to less than 100%, 95% to less than 100%, 96% to less than 100%, 97% to less than 100%, 98% to less than 100%, or 99% to less than 100% to the protein expressed by a wild-type gene. Sequence identity will be described later.

[0023] <Cells for Producing Recombinant HSV Vectors> Generally, when HSV infects a cell and the HSV genome enters the cell, HSV genes are expressed, causing the production of virus particles and lytic infection of the virus, resulting in cell death and making it impossible to maintain the cell. Even if a toxic HSV gene is not expressed from the HSV genome, a genome or plasmid or the like that does not have a replication origin that functions in the cell type will not be replicated during cell division unless it is inserted into the genome of the cell, and will decrease and eventually disappear.

[0024] As used herein, "cells for producing a recombinant HSV vector" refers to cells that can be induced to produce a recombinant HSV vector at any time. Furthermore, these cells retain a recombinant HSV genome, are capable of cell proliferation before induction of recombinant HSV vector production, and after cell division, daughter cells stably inherit the recombinant HSV genome without losing it, and maintain a state in which HSV gene expression is suppressed or eliminated. The induction of viral vector production will be described below.

[0025] As used herein, the term "recombinant HSV vector" refers to a recombinant HSV vector produced using genomic DNA that has been genetically modified by substitution, insertion, addition, and / or deletion, etc., in the base sequence shown in SEQ ID NO: 17, which is the wild-type herpes simplex virus type 1 (hereinafter also referred to as "HSV-1"). Furthermore, as used herein, "retaining a recombinant HSV genome" means that the recombinant HSV genome is replicated within the cell and is not lost from the cell even after cell division.

[0026] The cells according to this embodiment are not particularly limited as long as they are mammal-derived cells that can be used to produce a recombinant HSV vector, and may be, for example, cultured cells (also referred to as "mammal-derived cells") derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, etc., with human-derived cultured cells being preferred. Specific examples of mammal-derived cells include HEK293 cells (human-derived), HeLa cells (human-derived), HT1080 cells (human-derived), and Vero cells (derived from African green monkeys).

[0027] <First base sequence> The first base sequence is a genomic DNA that has been genetically modified by substitution, insertion, addition, and / or deletion, etc., in the base sequence shown in SEQ ID NO: 17, which is the wild-type HSV-1 genome, and satisfies the following (1), (2), (3), (4), and (5): (1) It contains base sequences 2 to 27 that express proteins 1 to 26. (2) It lacks base sequences that encode proteins 27 to 28. (3) It lacks base sequences that encode protein 29. (4) It lacks base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6. (5) It contains the 28th base sequence consisting of the base sequence shown in SEQ ID NO: 7, or the 29th base sequence shown in SEQ ID NO: 8.

[0028] The first to 26th proteins in (1) have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 76 to 101, respectively, and have the same functions as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 101.

[0029] The genes encoding proteins 1 to 26 are classified as essential genes of HSV-1. As used herein, "essential genes of HSV-1" refers to genes that, when removed from the wild-type HSV-1 genome, render HSV-1 unable to grow or significantly reduce its growth rate. In other words, if a gene that renders HSV-1 unable to grow or significantly reduces its growth rate can enable HSV-1 to grow or restores the significantly reduced growth rate by introducing the gene into an HSV-1 genome from which a gene that renders HSV-1 unable to grow or significantly reduces its growth rate has been removed, then the gene can be said to be an essential gene of HSV-1. Therefore, as used herein, "essential genes of HSV-1" encompasses not only wild-type genes but also mutant genes. The mutant gene may be a gene that expresses a protein having 90% to less than 100%, 91% to less than 100%, 92% to less than 100%, 93% to less than 100%, 94% to less than 100%, 95% to less than 100%, 96% to less than 100%, 97% to less than 100%, 98% to less than 100%, or 99% to less than 100% sequence identity to the protein expressed by the wild-type gene. Sequence identity will be described later. Essential HSV-1 genes are those involved in viral DNA replication, viral gene transcription, genes encoding capsid proteins, genes encoding viral DNA packaging proteins, and the expression of several envelope glycoproteins.

[0030] Here, "the same quality of function" means that the properties are qualitatively the same as those of the control protein, for example, from a physiological perspective, but the degree of function or quantitative factors such as the molecular weight of the protein may be different. As described above, the degree of function is not particularly limited, but it may be determined that the protein has "the same quality of function" when it exerts, for example, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more of the qualitatively same function as the control protein.

[0031] The functions of proteins 1 to 26 or the genes encoding them are described in, for example, Fields Virology, 7th ed., and are publicly known. Therefore, for example, a protein having the known function of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 76 is considered to be a "protein having the same function" as a protein consisting of the amino acid sequence set forth in SEQ ID NO: 76. The same applies to proteins having the same function as a protein consisting of the amino acid sequence set forth in SEQ ID NOs: 77 to 101. Furthermore, whether a protein has the same function as a protein consisting of the amino acid sequence set forth in SEQ ID NOs: 76 to 101 can be determined by known methods. Therefore, those skilled in the art can appropriately determine whether a protein has the same function as a protein consisting of the amino acid sequence set forth in SEQ ID NOs: 76 to 101. For example, if HSV-1 can grow when an HSV-1 genome lacking the gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 76 is introduced into a cell expressing the protein, it can be determined that the protein expressed in the cell has the same function as a protein consisting of the amino acid sequence set forth in SEQ ID NO: 76.

[0032] The second base sequence is a base sequence that expresses a first protein. The base sequence that expresses a protein may include, for example, a base sequence that encodes the protein and a regulatory sequence operably linked to the base sequence. That is, the second base sequence includes, for example, a base sequence that encodes the first protein and a regulatory sequence operably linked to the base sequence.

[0033] The first protein is a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 76 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 76. The first protein is the UL1 protein of HSV-1. An example of a nucleotide sequence encoding the first protein is the nucleotide sequence shown in SEQ ID NO: 156.

[0034] Regulatory sequences are sequences that control the expression of proteins in host cells (e.g., promoters, enhancers, ribosome binding sequences, transcription termination sequences, poly A addition signal sequences, etc.), and can be selected appropriately depending on the type of host cell.

[0035] The third base sequence is a base sequence that expresses a second protein. The second protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 77 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 77. The second protein is the UL5 protein of HSV-1. An example of a base sequence that encodes the second protein is the base sequence shown in SEQ ID NO: 157.

[0036] The fourth base sequence is a base sequence that expresses a third protein. The third protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 78 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 78. The third protein is the UL6 protein of HSV-1. An example of a base sequence encoding the third protein is the base sequence shown in SEQ ID NO: 158.

[0037] The fifth base sequence is a base sequence that expresses a fourth protein. The fourth protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 79 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 79. The fourth protein is the UL8 protein of HSV-1. An example of a base sequence that encodes the fourth protein is the base sequence shown in SEQ ID NO: 159.

[0038] The sixth base sequence is a base sequence that expresses a fifth protein. The fifth protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 80 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 80. The fifth protein is the UL9 protein of HSV-1. An example of a base sequence encoding the fifth protein is the base sequence shown in SEQ ID NO: 160.

[0039] The seventh base sequence is a base sequence that expresses a sixth protein. The sixth protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 81 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 81. The sixth protein is the UL15 protein of HSV-1. An example of a base sequence that encodes the sixth protein is the base sequence shown in SEQ ID NO: 161.

[0040] The eighth base sequence is a base sequence that expresses the seventh protein. The seventh protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 82 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 82. The seventh protein is the UL17 protein of HSV-1. An example of a base sequence that encodes the seventh protein is the base sequence shown in SEQ ID NO: 162.

[0041] The ninth base sequence is a base sequence that expresses the eighth protein. The eighth protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 83 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 83. The eighth protein is the UL18 protein of HSV-1. An example of a base sequence that encodes the eighth protein is the base sequence shown in SEQ ID NO: 163.

[0042] The tenth base sequence is a base sequence that expresses the ninth protein. The ninth protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 84 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 84. The ninth protein is the UL19 protein of HSV-1. An example of a base sequence encoding the ninth protein is the base sequence shown in SEQ ID NO: 164.

[0043] The eleventh base sequence is a base sequence that expresses the tenth protein. The tenth protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 85 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 85. The tenth protein is the UL22 protein of HSV-1. An example of a base sequence encoding the tenth protein is the base sequence shown in SEQ ID NO: 165.

[0044] The twelfth base sequence is a base sequence that expresses the eleventh protein. The eleventh protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 86 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 86. The eleventh protein is the UL25 protein of HSV-1. An example of a base sequence that encodes the eleventh protein is the base sequence shown in SEQ ID NO: 166.

[0045] The thirteenth base sequence is a base sequence that expresses the twelfth protein. The twelfth protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 87 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 87. The twelfth protein is the UL26 protein of HSV-1. An example of a base sequence encoding the twelfth protein is the base sequence shown in SEQ ID NO: 167.

[0046] The 14th base sequence is a base sequence that expresses the 13th protein. The 13th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 88 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 88. The 13th protein is the UL27 protein of HSV-1. An example of a base sequence that encodes the 13th protein is the base sequence shown in SEQ ID NO: 168.

[0047] The 15th base sequence is a base sequence that expresses the 14th protein. The 14th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 89 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 89. The 14th protein is the UL28 protein of HSV-1. An example of a base sequence that encodes the 14th protein is the nucleotide sequence shown in SEQ ID NO: 169.

[0048] The 16th base sequence is a base sequence that expresses the 15th protein. The 15th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 90 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 90. The 15th protein is the UL29 protein of HSV-1. An example of a base sequence encoding the 15th protein is the nucleotide sequence shown in SEQ ID NO: 170.

[0049] The 17th base sequence is a base sequence that expresses the 16th protein. The 16th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 91 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 91. The 16th protein is the UL30 protein of HSV-1. An example of a base sequence that encodes the 16th protein is the nucleotide sequence shown in SEQ ID NO: 171.

[0050] The 18th base sequence is a base sequence that expresses the 17th protein. The 17th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 92 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 92. The 17th protein is the UL32 protein of HSV-1. An example of a base sequence that encodes the 17th protein is the nucleotide sequence shown in SEQ ID NO: 172.

[0051] The 19th base sequence is a base sequence that expresses the 18th protein. The 18th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 93 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 93. The 18th protein is the UL33 protein of HSV-1. An example of a base sequence encoding the 18th protein is the nucleotide sequence shown in SEQ ID NO: 173.

[0052] The 20th base sequence is a base sequence that expresses the 19th protein. The 19th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 94 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 94. The 19th protein is the UL36 protein of HSV-1. An example of a base sequence encoding the 19th protein is the base sequence shown in SEQ ID NO: 174.

[0053] The 21st base sequence is a base sequence that expresses the 20th protein. The 20th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 95 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 95. The 20th protein is the UL37 protein of HSV-1. An example of a base sequence encoding the 20th protein is the base sequence shown in SEQ ID NO: 175.

[0054] The 22nd base sequence is a base sequence that expresses the 21st protein. The 21st protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 96 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 96. The 21st protein is the UL38 protein of HSV-1. An example of a base sequence encoding the 21st protein is the nucleotide sequence shown in SEQ ID NO: 176.

[0055] The 23rd base sequence is a base sequence that expresses the 22nd protein. The 22nd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 97 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 97. The 22nd protein is the UL42 protein of HSV-1. An example of a base sequence encoding the 22nd protein is the nucleotide sequence shown in SEQ ID NO: 177.

[0056] The 24th base sequence is a base sequence that expresses the 23rd protein. The 23rd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 98 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 98. The 23rd protein is the UL48 protein of HSV-1. An example of a base sequence encoding the 23rd protein is the nucleotide sequence shown in SEQ ID NO: 178.

[0057] The 25th base sequence is a base sequence that expresses the 24th protein. The 24th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 99 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 99. The 24th protein is the UL52 protein of HSV-1. An example of a base sequence encoding the 24th protein is the nucleotide sequence shown in SEQ ID NO: 179.

[0058] The 26th base sequence is a base sequence that expresses the 25th protein. The 25th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 100 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 100. The 25th protein is the a22 (ICP22, US1) protein of HSV-1. An example of a base sequence encoding the 25th protein is the nucleotide sequence shown in SEQ ID NO: 180.

[0059] The 27th base sequence is a base sequence that expresses the 26th protein. The 26th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 101 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 101. The 26th protein is the HSV-1 US6 protein. An example of a base sequence that encodes the 26th protein is the nucleotide sequence shown in SEQ ID NO: 181.

[0060] The above-mentioned proteins 1 to 26 may have a sequence identity of 90% or more to the amino acid sequences shown in SEQ ID NOs: 76 to 101, respectively, and the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or even 100%.

[0061] As used herein, sequence identity refers to the percentage (%) of matching bases or residues when the base sequences or amino acid sequences being compared are aligned (multiple alignment). Multiple alignment refers to an alignment of base sequences with appropriate gaps inserted so that corresponding base or amino acid sequence portions are aligned to make the base sequences or amino acid sequences comparable to each other. For multiple alignment, known multiple alignment creation programs can be used. For example, Clustal W, Clustal X, BLAST programs, etc. can be suitably used.

[0062] The first base sequence may further satisfy the following condition (6): (6) The first base sequence further includes at least one base sequence selected from all of the base sequences of the 31st to 52nd bases that express the 30th to 51st proteins and the 53rd to 84th base sequences that express the 52nd to 83rd proteins.

[0063] The above-mentioned proteins Nos. 30 to 83 have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 102 to 155, respectively, and have the same functions as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 102 to 155.

[0064] The genes encoding the 30th to 83rd proteins are classified as non-essential HSV-1 genes. As used herein, "non-essential HSV-1 genes" refers to HSV genes other than the essential genes. Non-essential HSV-1 genes play important roles, for example, in nucleic acid metabolism, viral pathogenesis, and infection control (e.g., countering the antiviral response of host cells). Therefore, when a mammalian-derived cell according to this embodiment satisfies condition (6), the proliferation efficiency or infectivity of a recombinant HSV vector produced using the cell is improved. A gene that is not an essential HSV gene and that improves the proliferation efficiency or infectivity of a recombinant HSV vector can be considered a non-essential HSV-1 gene. Therefore, as used herein, "non-essential HSV-1 genes" encompasses not only wild-type genes but also mutant genes. The mutant gene may be a gene that expresses a protein having 90% to less than 100%, 91% to less than 100%, 92% to less than 100%, 93% to less than 100%, 94% to less than 100%, 95% to less than 100%, 96% to less than 100%, 97% to less than 100%, 98% to less than 100%, or 99% to less than 100% sequence identity to a protein expressed by a wild-type gene. On the other hand, the base sequence of bases 53 to 84 that expresses proteins of bases 52 to 83 is a base sequence that is known to be sometimes deleted from HSV vectors to reduce the proliferation or replication ability of HSV from the standpoint of safety.

[0065] Wild-type HSV-1 has all of the genes encoding the proteins consisting of the amino acid sequences shown in SEQ ID NOS: 76 to 101 (proteins 1 to 26), the genes encoding the proteins consisting of the amino acid sequences shown in SEQ ID NOS: 102 to 155 (proteins 30 to 83), and the genes encoding the proteins consisting of the amino acid sequences shown in SEQ ID NOS: 12 to 14 (proteins 27 to 29) described below.

[0066] The functions of the 30th to 83rd proteins or the genes encoding them are described in, for example, Fields Virology, 7th ed., and are publicly known. Therefore, for example, a protein having the function of the publicly known 30th protein is considered to be a "protein having the same function" as the 30th protein. The same applies to proteins having the same function as the 31st to 83rd proteins. Furthermore, whether a protein has the same function as the 30th to 83rd proteins can be determined by publicly known methods. For example, if an HSV-1 genome lacking the gene encoding the corresponding wild-type protein is introduced into cells expressing the mutant protein to be analyzed, and the proliferation or infectivity of HSV-1 is improved, it can be determined that the protein expressed in the cells has the same function as the deleted wild-type protein. Furthermore, for example, by inoculating an animal model with wild-type HSV-1 or modified HSV-1 expressing the mutant protein to be analyzed and comparing the mortality rate or skin lesions with those of an animal model inoculated with wild-type HSV-1, it is possible to determine whether the mutant protein has the same quality of function as the wild-type protein.

[0067] The 31st base sequence is a base sequence that expresses the 30th protein. The 30th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 102 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 102. The 30th protein is the UL2 protein of HSV-1. An example of a base sequence encoding the 30th protein is the nucleotide sequence shown in SEQ ID NO: 182.

[0068] The 32nd base sequence is a base sequence that expresses the 31st protein. The 31st protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 103 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 103. The 31st protein is the UL7 protein of HSV-1. An example of a base sequence encoding the 31st protein is the nucleotide sequence shown in SEQ ID NO: 183.

[0069] The 33rd base sequence is a base sequence that expresses the 32nd protein. The 32nd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 104 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 104. The 32nd protein is the UL8.5 protein of HSV-1. An example of a base sequence encoding the 32nd protein is the nucleotide sequence shown in SEQ ID NO: 184.

[0070] The 34th base sequence is a base sequence that expresses the 33rd protein. The 33rd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 105 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 105. The 33rd protein is the UL11 protein of HSV-1. An example of a base sequence encoding the 33rd protein is the nucleotide sequence shown in SEQ ID NO: 185.

[0071] The 35th base sequence is a base sequence that expresses the 34th protein. The 34th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 106 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 106. The 34th protein is the UL12 protein of HSV-1. An example of a base sequence encoding the 34th protein is the nucleotide sequence shown in SEQ ID NO: 186.

[0072] The 36th base sequence is a base sequence that expresses the 35th protein. The 35th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 107 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 107. The 35th protein is the UL12.5 protein of HSV-1. An example of a base sequence encoding the 35th protein is the nucleotide sequence shown in SEQ ID NO: 187.

[0073] The 37th base sequence is a base sequence that expresses the 36th protein. The 36th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 108 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 108. The 36th protein is the UL14 protein of HSV-1. An example of a base sequence encoding the 36th protein is the nucleotide sequence shown in SEQ ID NO: 188.

[0074] The 38th base sequence is a base sequence that expresses the 37th protein. The 37th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 109 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 109. The 37th protein is the UL15.5 protein of HSV-1. An example of a base sequence encoding the 37th protein is the nucleotide sequence shown in SEQ ID NO: 189.

[0075] The 39th base sequence is a base sequence that expresses the 38th protein. The 38th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 110 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 110. The 38th protein is the UL16 protein of HSV-1. An example of a base sequence encoding the 38th protein is the nucleotide sequence shown in SEQ ID NO: 190.

[0076] The 40th base sequence is a base sequence that expresses the 39th protein. The 39th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 111 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 111. The 39th protein is the UL20 protein of HSV-1. An example of a base sequence encoding the 39th protein is the nucleotide sequence shown in SEQ ID NO: 191.

[0077] The 41st base sequence is a base sequence that expresses the 40th protein. The 40th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 112 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 112. The 40th protein is the UL20.5 protein of HSV-1. An example of a base sequence encoding the 40th protein is the base sequence shown in SEQ ID NO: 192.

[0078] The 42nd base sequence is a base sequence that expresses the 41st protein. The 41st protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 113 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 113. The 41st protein is the UL26.5 protein of HSV-1. An example of a base sequence encoding the 41st protein is the nucleotide sequence shown in SEQ ID NO: 193.

[0079] The 43rd base sequence is a base sequence that expresses the 42nd protein. The 42nd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 114 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 114. The 42nd protein is the UL27.5 protein of HSV-1. An example of a base sequence encoding the 42nd protein is the nucleotide sequence shown in SEQ ID NO: 194.

[0080] The 44th base sequence is a base sequence that expresses the 43rd protein. The 43rd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 115 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 115. The 43rd protein is the UL31 protein of HSV-1. An example of a base sequence encoding the 43rd protein is the base sequence shown in SEQ ID NO: 195.

[0081] The 45th base sequence is a base sequence that expresses the 44th protein. The 44th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 116 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 116. The 44th protein is the UL34 protein of HSV-1. An example of a base sequence encoding the 44th protein is the nucleotide sequence shown in SEQ ID NO: 196.

[0082] The 46th base sequence is a base sequence that expresses the 45th protein. The 45th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 117 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 117. The 45th protein is the UL40 protein of HSV-1. An example of a base sequence encoding the 45th protein is the base sequence shown in SEQ ID NO: 197.

[0083] The 47th base sequence is a base sequence that expresses the 46th protein. The 46th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 118 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 118. The 46th protein is the UL49.5 protein of HSV-1. An example of a base sequence encoding the 46th protein is the base sequence shown in SEQ ID NO: 198.

[0084] The 48th base sequence is a base sequence that expresses the 47th protein. The 47th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 119 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 119. The 47th protein is the UL50 protein of HSV-1. An example of a base sequence encoding the 47th protein is the nucleotide sequence shown in SEQ ID NO: 199.

[0085] The 49th base sequence is a base sequence that expresses the 48th protein. The 48th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 120 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 120. The 48th protein is the UL53 protein of HSV-1. An example of a base sequence encoding the 48th protein is the nucleotide sequence shown in SEQ ID NO: 200.

[0086] The 50th base sequence is a base sequence that expresses the 49th protein. The 49th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 121 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 121. The 49th protein is the US1.5 protein of HSV-1. An example of a base sequence encoding the 49th protein is the nucleotide sequence shown in SEQ ID NO: 201.

[0087] The 51st base sequence is a base sequence that expresses the 50th protein. The 50th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 122 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 122. The 50th protein is the HSV-1 US2 protein. An example of a base sequence that encodes the 50th protein is the base sequence shown in SEQ ID NO: 202.

[0088] The 52nd base sequence is a base sequence that expresses the 51st protein. The 51st protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 123 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 123. The 51st protein is the HSV-1 US3.5 protein. An example of a base sequence encoding the 51st protein is the base sequence shown in SEQ ID NO: 203.

[0089] The 53rd base sequence is a base sequence that expresses the 52nd protein. The 52nd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 124 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 124. The 52nd protein is an HSV-1 ORF-P protein. An example of a base sequence encoding the 52nd protein is the base sequence shown in SEQ ID NO: 204.

[0090] The 54th nucleotide sequence is a nucleotide sequence that expresses the 53rd protein. The 53rd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 125 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 125. The 53rd protein is an ORF-O protein of HSV-1. An example of a nucleotide sequence encoding the 53rd protein is the nucleotide sequence shown in SEQ ID NO: 205.

[0091] The 55th nucleotide sequence is a nucleotide sequence that expresses the 54th protein. The 54th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 126 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 126. The 54th protein is the a34.5 (RL1) protein of HSV-1. An example of a nucleotide sequence encoding the 54th protein is the nucleotide sequence shown in SEQ ID NO: 206.

[0092] The 56th base sequence is a base sequence that expresses the 55th protein. The 55th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 127 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 127. The 55th protein is the UL3 protein of HSV-1. An example of a base sequence encoding the 55th protein is the base sequence shown in SEQ ID NO: 207.

[0093] The 57th base sequence is a base sequence that expresses the 56th protein. The 56th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 128 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 128. The 56th protein is the UL4 protein of HSV-1. An example of a base sequence encoding the 56th protein is the base sequence shown in SEQ ID NO: 208.

[0094] The 58th base sequence is a base sequence that expresses the 57th protein. The 57th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 129 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 129. The 57th protein is the UL10 protein of HSV-1. An example of a base sequence encoding the 57th protein is the nucleotide sequence shown in SEQ ID NO: 209.

[0095] The 59th base sequence is a base sequence that expresses the 58th protein. The 58th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 130 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 130. The 58th protein is the UL13 protein of HSV-1. An example of a base sequence encoding the 58th protein is the nucleotide sequence shown in SEQ ID NO: 210.

[0096] The 60th base sequence is a base sequence that expresses the 59th protein. The 59th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 131 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 131. The 59th protein is the UL21 protein of HSV-1. An example of a base sequence encoding the 59th protein is the base sequence shown in SEQ ID NO: 211.

[0097] The 61st base sequence is a base sequence that expresses the 60th protein. The 60th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 132 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 132. The 60th protein is the UL23 protein of HSV-1. An example of a base sequence encoding the 60th protein is the base sequence shown in SEQ ID NO: 212.

[0098] The 62nd base sequence is a base sequence that expresses the 61st protein. The 61st protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 133 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 133. The 61st protein is the UL24 protein of HSV-1. An example of a base sequence encoding the 61st protein is the base sequence shown in SEQ ID NO: 213.

[0099] The 63rd base sequence is a base sequence that expresses the 62nd protein. The 62nd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 134 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 134. The 62nd protein is the UL35 protein of HSV-1. An example of a base sequence encoding the 62nd protein is the base sequence shown in SEQ ID NO: 214.

[0100] The 64th nucleotide sequence is a nucleotide sequence that expresses the 63rd protein. The 63rd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 135 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 135. The 63rd protein is the UL39 protein of HSV-1. An example of a nucleotide sequence encoding the 63rd protein is the nucleotide sequence shown in SEQ ID NO: 215.

[0101] The 65th nucleotide sequence is a nucleotide sequence that expresses the 64th protein. The 64th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 136 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 136. The 64th protein is the UL41 protein of HSV-1. An example of a nucleotide sequence encoding the 64th protein is the nucleotide sequence shown in SEQ ID NO: 216.

[0102] The 66th nucleotide sequence is a nucleotide sequence that expresses the 65th protein. The 65th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 137 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 137. The 65th protein is the UL43 protein of HSV-1. An example of a nucleotide sequence encoding the 65th protein is the nucleotide sequence shown in SEQ ID NO: 217.

[0103] The 67th nucleotide sequence is a nucleotide sequence that expresses the 66th protein. The 66th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 138 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 138. The 66th protein is the UL44 protein of HSV-1. An example of a nucleotide sequence encoding the 66th protein is the nucleotide sequence shown in SEQ ID NO: 218.

[0104] The 68th nucleotide sequence is a nucleotide sequence that expresses the 67th protein. The 67th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 139 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 139. The 67th protein is the UL45 protein of HSV-1. An example of a nucleotide sequence encoding the 67th protein is the nucleotide sequence shown in SEQ ID NO: 219.

[0105] The 69th nucleotide sequence is a nucleotide sequence that expresses the 68th protein. The 68th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 140 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 140. The 68th protein is the UL46 protein of HSV-1. An example of a nucleotide sequence encoding the 68th protein is the nucleotide sequence shown in SEQ ID NO: 220.

[0106] The 70th base sequence is a base sequence that expresses the 69th protein. The 69th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 141 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 141. The 69th protein is the UL47 protein of HSV-1. An example of a base sequence encoding the 69th protein is the base sequence shown in SEQ ID NO: 221.

[0107] The 71st base sequence is a base sequence that expresses the 70th protein. The 70th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 142 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 142. The 70th protein is the UL49 protein of HSV-1. An example of a base sequence encoding the 70th protein is the base sequence shown in SEQ ID NO: 222.

[0108] The 72nd nucleotide sequence is a nucleotide sequence that expresses the 71st protein. The 71st protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 143 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 143. The 71st protein is the UL51 protein of HSV-1. An example of a nucleotide sequence encoding the 71st protein is the nucleotide sequence shown in SEQ ID NO: 223.

[0109] The 73rd base sequence is a base sequence that expresses the 72nd protein. The 72nd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 144 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 144. The 72nd protein is the UL55 protein of HSV-1. An example of a base sequence encoding the 72nd protein is the base sequence shown in SEQ ID NO: 224.

[0110] The 74th nucleotide sequence is a nucleotide sequence that expresses the 73rd protein. The 73rd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 145 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 145. The 73rd protein is the UL56 protein of HSV-1. An example of a nucleotide sequence encoding the 73rd protein is the nucleotide sequence shown in SEQ ID NO: 225.

[0111] The 75th nucleotide sequence is a nucleotide sequence that expresses the 74th protein. The 74th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 146 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 146. The 74th protein is the HSV-1 US3 protein. An example of a nucleotide sequence encoding the 74th protein is the nucleotide sequence shown in SEQ ID NO: 226.

[0112] The 76th nucleotide sequence is a nucleotide sequence that expresses the 75th protein. The 75th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 147 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 147. The 75th protein is the HSV-1 US4 protein. An example of a nucleotide sequence encoding the 75th protein is the nucleotide sequence shown in SEQ ID NO: 227.

[0113] The 77th nucleotide sequence is a nucleotide sequence that expresses the 76th protein. The 76th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 148 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 148. The 76th protein is the HSV-1 US5 protein. An example of a nucleotide sequence encoding the 76th protein is the nucleotide sequence shown in SEQ ID NO: 228.

[0114] The 78th nucleotide sequence is a nucleotide sequence that expresses the 77th protein. The 77th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 149 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 149. The 77th protein is the HSV-1 US7 protein. An example of a nucleotide sequence encoding the 77th protein is the nucleotide sequence shown in SEQ ID NO: 229.

[0115] The 79th nucleotide sequence is a nucleotide sequence that expresses the 78th protein. The 78th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 150 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 150. The 78th protein is the HSV-1 US8 protein. An example of a nucleotide sequence encoding the 78th protein is the nucleotide sequence shown in SEQ ID NO: 230.

[0116] The 80th nucleotide sequence is a nucleotide sequence that expresses the 79th protein. The 79th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 151 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 151. The 79th protein is the HSV-1 US8.5 protein. An example of a nucleotide sequence encoding the 79th protein is the nucleotide sequence shown in SEQ ID NO: 231.

[0117] The 81st nucleotide sequence is a nucleotide sequence that expresses the 80th protein. The 80th protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 152 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 152. The 80th protein is the UL9 protein of HSV-1. An example of a nucleotide sequence encoding the 80th protein is the nucleotide sequence shown in SEQ ID NO: 232.

[0118] The 82nd nucleotide sequence is a nucleotide sequence that expresses the 81st protein. The 81st protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 153 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 153. The 81st protein is the US10 protein of HSV-1. An example of a nucleotide sequence encoding the 81st protein is the nucleotide sequence shown in SEQ ID NO: 233.

[0119] The 83rd base sequence is a base sequence that expresses the 82nd protein. The 82nd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 154 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 154. The 82nd protein is the HSV-1 US11 protein. An example of a base sequence encoding the 82nd protein is the nucleotide sequence shown in SEQ ID NO: 234.

[0120] The 84th nucleotide sequence is a nucleotide sequence that expresses the 83rd protein. The 83rd protein has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 155 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 155. The 83rd protein is the HSV-1 a47 (ICP47, US12) protein. An example of a nucleotide sequence encoding the 83rd protein is the nucleotide sequence shown in SEQ ID NO: 235.

[0121] The 30th to 83rd proteins may have 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 102 to 155, respectively. The sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or even 100%.

[0122] When the first base sequence satisfies (6), it is sufficient that the first base sequence further contains at least one of the 53rd to 84th base sequences. Of the above base sequences, the first base sequence may contain, for example, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, or 31 or more base sequences, and preferably contains all of the 32nd to 84th base sequences. In the base sequence (SEQ ID NO: 11) of the recombinant HSV genome prepared in the Examples described below, the 84th base sequence has been deleted. When the first base sequence satisfies (6), it is more preferable that the first base sequence contains the 32nd to 83rd base sequences.

[0123] The 27th protein in (2) is a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12. The nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 12 is the ICP27 gene of HSV-1 (UL54 gene; for example, a nucleotide sequence including the nucleotide sequence shown in SEQ ID NO: 2). The ICP27 protein has RNA binding ability and is known to perform functions such as inhibiting splicing of HSV gene mRNA, 3' processing of HSV gene mRNA, and transporting HSV gene mRNA from the cell nucleus to the cytoplasm (Nat Commun 11, 293 (2020)). ICP27 is thought to regulate the expression of the E gene (β gene) and L gene (γ gene) through these functions.

[0124] That is, a protein having at least one of the functions of the protein consisting of the amino acid sequence shown in SEQ ID NO: 12, such as (a) the ability to bind to RNA, (b) the function of inhibiting splicing of HSV gene mRNA, (c) the function of 3' processing HSV gene mRNA, and (d) the function of transporting HSV gene mRNA from the cell nucleus to the cytoplasm, is considered to be a "protein having the same quality of function" as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12.

[0125] It can be determined by known methods whether or not a protein has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12. Furthermore, for example, if HSV-1 is produced when an HSV-1 genome lacking the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 12 is introduced into a cell that expresses the protein, it can be determined that the protein expressed in the cell has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12.

[0126] The 27th protein may have a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 12, and the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or even 100%. The 27th protein is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 12.

[0127] The ICP27 gene is present in one copy in the wild-type HSV-1 genome (the base sequence shown in SEQ ID NO: 17), and is an essential gene in HSV-1.

[0128] The 28th protein in (2) is a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13 and encodes a protein having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 13. The nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 13 is the ICP0 gene of HSV-1 (a0(RL2); for example, a nucleotide sequence including the nucleotide sequence shown in SEQ ID NO: 3). The ICP0 protein is known as a transactivation protein and is a multifunctional protein. One function of the ICP0 protein is the degradation of host cell proteins. Examples of proteins that can be degraded include some proteins of the PML body and their sumo-modified isoforms, sumo-modified Sp100, etc. As a result of the degradation of these proteins, a protein cluster called nuclear domain 10 (ND10) is dispersed. Since ND10 contains a molecule that inhibits viral gene expression, dispersion of ND10 suppresses the inhibitory effect of viral gene expression, inducing enhanced viral gene expression. Furthermore, the ICP0 protein binds to a corepressor of the RE1 silencing transcription factor (CoREST), a protein that controls gene expression in host cells. This inhibits the formation of a complex between histone deacetylase (HDAC) and CoREST. The complex between HDAC and CoREST causes gene expression suppression through heterochromatinization. As a result, CoREST improves the expression of proteins that suppress gene expression, inducing enhanced viral gene expression. It has also been reported that antiviral activity is suppressed by inhibiting interferon regulatory factor 3 or Toll-like receptor signaling, etc. Through these functions, the ICP0 protein is thought to regulate the expression of the E gene (β gene) and the L gene (γ gene).

[0129] That is, a protein having at least one of the functions of a protein consisting of the amino acid sequence shown in SEQ ID NO: 13, for example, (e) the function of degrading some proteins of the PML body and their sumo-modified isoforms, and sumo-modified Sp100, (f) the function of binding to CoREST, and (g) the function of inhibiting interferon regulatory factor 3 or Toll-like receptor signaling, is considered to be a "protein having the same quality of function" as the protein consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0130] It can be determined by known methods whether or not a protein has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 13. Furthermore, for example, if HSV-1 is produced when an HSV-1 genome lacking the gene encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 13 is introduced into a cell that expresses the protein, it can be determined that the protein expressed in the cell has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0131] The 28th protein may have a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 13, and the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be 100%. The 28th protein is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 13.

[0132] The ICP0 gene is present in two copies in the wild-type HSV-1 genome (the base sequence shown in SEQ ID NO: 17), and is a non-essential gene in HSV-1.

[0133] In (2), "deleted" means that the nucleotide sequence encoding the 27th to 28th proteins lacks the above-mentioned function. Specific embodiments of "deleted" in (2) include an embodiment in which the nucleotide sequence encoding the 27th to 28th proteins is deleted from the first nucleotide sequence, an embodiment in which the nucleotide sequence encoding the 27th to 28th proteins has a mutation and cannot be translated into a normal protein, and an embodiment in which the first nucleotide sequence contains the nucleotide sequence encoding the 27th to 28th proteins but transcription is suppressed so that the proteins are not produced. A preferred embodiment is that the nucleotide sequence encoding the 27th protein is deleted from the first nucleotide sequence. A preferred embodiment is that the nucleotide sequence encoding the 28th protein is a embodiment in which the nucleotide sequence encoding the 28th protein has a mutation and cannot be translated into a normal protein, or an embodiment in which the nucleotide sequence is deleted from the first nucleotide sequence.

[0134] In an embodiment in which the base sequences encoding the 27th and 28th proteins are deleted from the first base sequence, the base sequences encoding the 27th and 28th proteins may be removed from the first base sequence, and a more specific example is that they may be replaced by, for example, another gene.

[0135] More specific examples of embodiments in which a mutation in the nucleotide sequence encoding the 27th to 28th proteins prevents translation into normal proteins include, for example, cases in which the gene has a nonsense mutation, a frameshift mutation, a splicing site mutation, or the like. The nucleotide sequence encoding the 28th protein preferably has a nonsense mutation, and more preferably has a nonsense mutation in the codon at the position corresponding to position 212 in the nucleotide sequence shown in SEQ ID NO: 3. Furthermore, when the nucleotide sequence encoding the 28th protein has a nonsense mutation, it is particularly preferred that the codon at the position corresponding to position 212 in two or three reading frames of the nucleotide sequence shown in SEQ ID NO: 3 has a nonsense mutation.

[0136] A more specific example of an embodiment in which the first base sequence contains the base sequences encoding the 27th and 28th proteins but transcription is suppressed so that the proteins are not produced is, for example, the first base sequence may contain the base sequences encoding the 27th and 28th proteins such that their expression is induced only by a chemical or physical stimulus.

[0137] When the expression of the gene is to be induced only by a chemical stimulus, for example, the Tet on / off system can be used, in which the expression of the gene of interest is induced by doxycycline or tetracycline. The Tet on / off system utilizes the Tet repressor and Tet operator of Escherichia coli, and is a system that can reversibly regulate the expression of the gene of interest by administering a tetracycline derivative such as doxycycline or tetracycline. When the Tet on system is used, the transcription of the gene of interest is activated by the presence of a tetracycline derivative. Conversely, when the Tet off system is used, the transcription of the gene of interest is suppressed by the presence of a tetracycline derivative. When the Tet on / off system is used, the Tet on system is preferably used.

[0138] When using the Tet on system, a tetracycline response element (TRE) and a reverse tetracycline-regulated transactivator (reverse tTA or rtTA) may be used. The TRE consists of a repeat sequence of the Escherichia coli tet operator sequence, and when rtTA or the tTA described below binds, it activates transcription of the target gene from a downstream promoter. The rtTA is a fusion protein of reverse TetR (rTetR), which was created by modifying the amino acids of the Tet repressor, and the VP16 transcription activation domain (VP16AD) derived from herpesvirus. The rtTA binds to the TRE (tet operator sequence) by binding to a tetracycline derivative.

[0139] When the Tet on system is used, a Tet operator sequence and a Tet repressor may be used. When the Tet repressor binds to the Tet operator sequence, transcription of the target gene downstream of the Tet operator sequence is suppressed. When the Tet repressor binds to a tetracycline derivative, it can no longer bind to the Tet operator sequence, activating transcription of the target gene.

[0140] When the Tet on system is utilized using a Tet operator sequence and a Tet repressor, for example, in the first base sequence, the base sequence encoding the proteins 27 and 28 is under the control of a transcription regulatory region consisting of the base sequence shown in SEQ ID NO: 19, and the cell according to this embodiment further harbors the base sequence shown in SEQ ID NO: 20. The fact that the cell according to this embodiment harbors the base sequence shown in SEQ ID NO: 20 may mean that the base sequence has been incorporated into the genome of the cell according to this embodiment, or that the first base sequence contains the base sequence.

[0141] The method for incorporating a specific base sequence into the genome of the cell according to this embodiment is not particularly limited and can be carried out by a method commonly used in this technical field, such as random recombination, homologous recombination, or site-specific recombination.

[0142] When the Tet off system is used, a tetracycline response element (TRE) and a tetracycline-regulated transactivator (tTA) may be used. tTA is a fusion protein of the Tet repressor (TetR) and VP16AD. When tTA binds to a tetracycline derivative, it is no longer able to bind to the TRE (tet operator sequence). In other words, in the absence of a tetracycline derivative, tTA can bind to the TRE, activating transcription of the target gene from the promoter downstream of the TRE.

[0143] When the expression of the gene is to be induced only by a physical stimulus, for example, a photo-activated Cre system can be used, in which the expression of the gene of interest is induced using Cre recombinase, which is activated by light such as red light or blue light. In the photo-activated Cre system, a LoxP cassette is inserted immediately below the start codon of the gene of interest to cause a deletion of the gene. Then, by irradiating the cells with light such as red light or blue light, the photo-activated Cre recombinase is activated, the LoxP cassette inserted into the gene of interest is removed, and the gene of interest is expressed.

[0144] When a photoactivatable Cre system is used, the cell according to this embodiment may harbor a gene that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 21, and further may have the base sequence encoding the protein at positions 27 and 28 in the first base sequence deleted by the insertion of the base sequence shown in SEQ ID NO: 22. "The cell according to this embodiment harbors a gene that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 21" may mean that the genome of the cell according to this embodiment has incorporated a gene that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 21, or that the first base sequence contains the base sequence of the gene.

[0145] The 29th protein in (3) is a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14 and encodes a protein having the same quality of function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 14. The nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 14 is the ICP4 gene of HSV-1 (a4(RS1); for example, a nucleotide sequence including the nucleotide sequence shown in SEQ ID NO: 4). The ICP4 protein has DNA binding ability and has been reported to bind to transcription factor IID (transcription factor II D; TFIID). This induces the expression of the E gene (β gene) and the L gene (γ gene), and is known to suppress the expression of the ICP4 gene itself and the ICP0 gene after infection has progressed.

[0146] In other words, a protein having at least one of the functions of the protein consisting of the amino acid sequence shown in SEQ ID NO: 14, for example, (h) DNA binding function and (i) TFIID binding function, is considered to be a "protein having the same function" as the protein consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0147] It can be determined by known methods whether or not a protein has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 14. Furthermore, for example, if HSV-1 is produced when an HSV-1 genome lacking the nucleotide sequence encoding the protein consisting of the amino acid sequence shown in SEQ ID NO: 14 is introduced into a cell that expresses the protein, it can be determined that the protein expressed in the cell has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0148] The 29th protein may have a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 14, and the sequence identity may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be 100%. The 29th protein is preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0149] The ICP4 gene is present in two copies in the wild-type HSV-1 genome (the base sequence shown in SEQ ID NO: 17), and is an essential gene in HSV-1.

[0150] In (3), "deficient" means that the nucleotide sequence encoding the 29th protein lacks the above-mentioned function. Specific embodiments of "deficient" in (3) include, for example, an embodiment in which the copy number of the nucleotide sequence encoding the 29th protein is reduced, an embodiment in which the nucleotide sequence encoding the 29th protein is deleted from the first nucleotide sequence, an embodiment in which the nucleotide sequence encoding the 29th protein has a mutation and cannot be translated into a normal protein, an embodiment in which the first nucleotide sequence contains the nucleotide sequence encoding the 29 protein but transcription is suppressed so that the protein is not produced, and the embodiment in which the copy number of the nucleotide sequence encoding the 29 protein is reduced or an embodiment in which the nucleotide sequence encoding the 29 protein is deleted from the first nucleotide sequence is preferred.

[0151] A more specific example of an embodiment in which the copy number of the base sequence encoding protein No. 29 is reduced is, for example, a case in which the first base sequence has only one copy of the base sequence encoding protein No. 29. Note that, based on the results of the Examples, it is believed that by deleting one copy of the ICP4 gene, of which two copies are present in the wild-type HSV-1 genome (the base sequence shown in SEQ ID NO: 17), the ICP4 protein is not sufficiently expressed in cells and becomes non-functional.

[0152] More specific examples of the embodiment in which the base sequence encoding the 29th protein is deleted from the first base sequence, and the embodiment in which the base sequence encoding the 29th protein has a mutation and cannot be translated into a normal protein, are the same as those in (2).

[0153] A more specific example of an embodiment in which the first base sequence contains a base sequence encoding the 29th protein but transcription is suppressed so that the protein is not produced is, for example, the first base sequence may contain a base sequence encoding the 29th protein such that expression of the gene is induced only by a chemical or physical stimulus.

[0154] As in the case of (2), when the Teton system is used, for example, in the first base sequence, the base sequence encoding the 29th protein is under the control of a transcriptional regulatory region consisting of the base sequence shown in SEQ ID NO: 19, and the cell according to this embodiment further harbors the base sequence shown in SEQ ID NO: 20. The fact that the cell according to this embodiment harbors the base sequence shown in SEQ ID NO: 20 may mean that the base sequence has been incorporated into the genome of the cell according to this embodiment, or that the first base sequence contains the base sequence.

[0155] When the photoactivatable Cre system is used, the same embodiment as in (2) above can be applied. That is, the cell according to this embodiment harbors a gene that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 21, and furthermore, the base sequence encoding the 29th protein in the first base sequence is deleted by inserting the base sequence shown in SEQ ID NO: 22.

[0156] When the first base sequence satisfies the above (1) to (3), the cell according to this embodiment maintains a state in which expression of the HSV gene is suppressed or eliminated, and the cell according to this embodiment is capable of cell proliferation unless induced to produce the recombinant HSV vector.

[0157] The base sequence shown in SEQ ID NO: 5 in (4) is one of two packaging sequences present in the wild-type HSV-1 genome (the base sequence shown in SEQ ID NO: 17). The packaging sequence is a base sequence necessary for packaging into HSV particles. The base sequence shown in SEQ ID NO: 6 in (4) is a packaging sequence present in a region called the a region of the wild-type HSV-1 genome.

[0158] In (4), "missing" means that the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 lack the above-mentioned function. Specific embodiments of "missing" in (4) include an embodiment in which the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 are deleted from the first base sequence, an embodiment in which the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 have a mutation and do not function normally, and the like. An embodiment in which the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 are deleted from the first base sequence is preferred.

[0159] In an embodiment in which the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 are deleted from the first base sequence, it is sufficient that the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 are removed from the first base sequence, and a more specific example is that they may be replaced by, for example, another gene, etc.

[0160] More specific examples of cases in which the base sequences shown in SEQ ID NO:5 and SEQ ID NO:6 have a mutation and do not function normally include cases in which a portion of the base sequences shown in SEQ ID NO:5 and SEQ ID NO:6 has been removed or substituted, or cases in which a base sequence of 5 or more bases, 10 or more bases, or 20 or more bases has been inserted into the base sequences shown in SEQ ID NO:5 and SEQ ID NO:6.

[0161] When the first base sequence satisfies the above (4), the cells of this embodiment can inhibit packaging of the first base sequence (recombinant HSV genome) into HSV particles when recombinant HSV vector production is induced.

[0162] (5) The first base sequence includes the 28th base sequence or the 29th base sequence. The 28th base sequence is a base sequence consisting of the base sequence shown in SEQ ID NO: 7. The 29th base sequence is a base sequence consisting of the base sequence shown in SEQ ID NO: 8.

[0163] The nucleotide sequence shown in SEQ ID NO:7 is a minimized nucleotide sequence of the OriP sequence, which is the origin of replication of the Epstein-Barr virus (EBV) genome. The EBV EBNA1 protein binds to the EBV OriP sequence and binds the EBV genome to the host cell chromosome via the OriP sequence. By utilizing this function, a plasmid expressing EBNA1 and having the EBV OriP sequence can be integrated into the host cell chromosome via EBNA1 and stably maintained in the host cell as an episome (Mol Ther. 2008 Sep;16(9):1525-38.). As shown in the Examples below, by inserting the minimized EBV OriP sequence into the first nucleotide sequence and maintaining and expressing the EBV EBNA1 gene in a cell, the recombinant HSV genome is maintained in the cell, even in daughter cells after cell division. The EBNA1 gene will be described later.

[0164] The base sequence shown in SEQ ID NO: 8 is a human S / MAR (scaffold attachment factor-A (SAF-A) / Nuclear Matrix Attachment Region (MAR)) sequence. The S / MAR sequence is a sequence present in the chromosomal DNA of eukaryotes, and is thought to bind to the nuclear matrix via binding with SAF-A. By utilizing this function, it is known that a plasmid having an S / MAR sequence can bind to the host cell chromosome (the S / MAR sequence of the host cell chromosome) via SAF-A expressed by the host cell, and is stably maintained in the host cell as an episome (Mol Ther. 2008 Sep;16(9):1525-38.). Furthermore, mammalian-derived cells have a base sequence that endogenously expresses the SAF-1 protein. Therefore, by inserting a human S / MAR sequence into the first base sequence, the recombinant HSV genome is retained in the cell even in daughter cells after cell division. The SAF-A gene will be described later.

[0165] Furthermore, because EBNA1 can also bind to S / MAR sequences (J Virol. 2004 Nov;78(21):11487-505), it is believed that even if a human S / MAR sequence is used in place of the EBV OriP sequence, the above-mentioned function of the EBV EBNA1 protein and OriP sequence to maintain the viral genome in host cells can be similarly exerted.

[0166] The first base sequence includes the 28th base sequence or the 29th base sequence, and preferably includes the 28th base sequence.

[0167] The first base sequence may further include a gene that expresses a fluorescent protein, a selectable marker gene, a base sequence recognized by a probe, and a base sequence that expresses a recombinase gene (e.g., a gene encoding Cre recombinase).

[0168] The cells according to this embodiment may retain the first base sequence. For example, the cells may retain the first base sequence as is, or may retain the first base sequence cloned into a bacterial artificial chromosome (BAC). When the cells according to this embodiment retain the first base sequence as is, the first base sequence may be linear or circular, but a circular form is preferred from the perspective of longer-term retention. The wild-type HSV-1 genome exists as a circular genome in infected cells. When the cells according to this embodiment retain the first base sequence cloned into a BAC, the cells may be genetically modified using pYEbac102 (J. Virol. 2003 Jan;77(2):1382-91.) in which the wild-type HSV-1 genome (the base sequence represented by SEQ ID NO: 17) has been inserted into the BAC, and the first base sequence may be retained in the cells.

[0169] The base sequence shown in SEQ ID NO: 1 is the base sequence of the recombinant HSV genome prepared in the Examples described below (SEQ ID NO: 11) minus the base sequence of the EBNA1 gene (SEQ ID NO: 18), and is the HSV genome. The first base sequence satisfies the above (1) to (5) and may have a sequence identity of 95% or more with the base sequence shown in SEQ ID NO: 1, and the sequence identity may be, for example, 95.1% or more, 95.2% or more, 95.3% or more, 95.4% or more, 95.5% or more, 95.6% or more, 95.7% or more, 95.8% or more, 95.9% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.6% or more, 96.7% or more, 96.8% or more, 96.9% or more, 97% or more, 97.1% or more, 97.2% or more, 97.3% or more, 97.4% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9 ...9% or more, 97.9% or more, 9 4% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, or 99.99% or more, or 99.999% or more, or may be 100%. The sequence identity between the base sequence shown in SEQ ID NO: 1 and the base sequence shown in SEQ ID NO: 11 is approximately 98.7%.

[0170] <30th base sequence> The 30th base sequence expresses a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and having the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence. The protein consisting of the amino acid sequence shown in SEQ ID NO: 9 is the EBV EBNA1 protein described above. The protein expressed by the 30th base sequence may have 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, or may even be 100%. The 30th base sequence is preferably a gene that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 9.

[0171] The 30th base sequence includes, for example, a base sequence encoding a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and having the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence, and a regulatory sequence operably linked to the base sequence.

[0172] The protein expressed by the 30th base sequence has the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence. Because the EBV EBNA1 protein binds to the EBV OriP sequence and the chromosomes of the host cell, the recombinant HSV genome is maintained in the cells of this embodiment even after cell division. Furthermore, because EBNA1 can also bind to an S / MAR sequence, the recombinant HSV genome is maintained in the cells of this embodiment even after cell division, even when an S / MAR sequence is used instead of the OriP sequence. The protein expressed by the 30th base sequence only needs to have the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence so as to be able to exert these functions.

[0173] The regulatory sequence is a sequence (e.g., promoter, enhancer, ribosome binding sequence, transcription termination sequence, poly A addition signal sequence, etc.) that controls the expression of a protein in a host cell, which has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and has the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence, and can be selected appropriately depending on the type of host cell.

[0174] An example of the 30th base sequence is the base sequence shown in SEQ ID NO: 23. In this base sequence, a base sequence (SEQ ID NO: 18) encoding the EBNA1 protein is linked in an expressible manner under the control of a promoter (pEF1α) of the human polypeptide chain elongation factor (EF1α) gene, and further downstream thereof, the poly(A) addition signal sequence of the EBNA1 gene is included.

[0175] When the first base sequence includes the 28th base sequence, the first base sequence may further include the 30th base sequence. In this case, the first base sequence may have a sequence identity of 95% or more to the base sequence shown in SEQ ID NO: 11, and the sequence identity is 95.1% or more, 95.2% or more, 95.3% or more, 95.4% or more, 95.5% or more, 95.6% or more, 95.7% or more, 95.8% or more, 95.9% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.6% or more, 96.7% or more, 96.8% or more, 96.9% or more, 97% or more, 97.1% or more, 97.2% or more, 97.3% or more, 97.4% or more, 97.5% or more Preferably, the purity is 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, more preferably 99.99% or more, even more preferably 99.999% or more, and particularly preferably 100%.

[0176] <SAF-A Protein> The SAF-A protein may be, for example, a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10 and having the ability to bind to the 29th base sequence. A protein consisting of the amino acid sequence shown in SEQ ID NO: 10 is a human SAF-A protein. The SAF-A protein may have 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10, or may even have 100% identity. The SAF-A gene is preferably a gene that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0177] The SAF-A protein has the ability to bind to the base sequence 29. The human SAF-A protein binds to the 29th base sequence (S / MAR sequence) in the first base sequence and to the S / MAR in the chromosomal DNA of the host cell, and therefore the recombinant HSV genome is maintained in the cell according to this embodiment even after cell division.

[0178] The base sequence of the SAF-A gene includes, for example, a base sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10 and encodes a protein that has the ability to bind to the 29th base sequence, and a regulatory sequence operably linked to the base sequence.

[0179] The base sequence of the SAF-A gene may be, for example, a base sequence (SEQ ID NO: 24) encoding the human SAF-A protein linked in an expressible manner under the control of a promoter of the human SAF-A gene, and further comprising a poly(A) addition signal sequence of the human SAF-A gene downstream thereof.

[0180] When the first base sequence includes the 29th base sequence, the first base sequence may have a sequence identity of 99% or more to the base sequence shown in SEQ ID NO: 16, and the sequence identity is preferably 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, more preferably 99.99% or more, even more preferably 99.999% or more, and particularly preferably 100%.

[0181] The nucleotide sequence shown in SEQ ID NO: 16 is a recombinant HSV genome in which a nucleotide sequence (SEQ ID NO: 11) encoding an EBNA1 protein (SEQ ID NO: 18) is linked in an expressible manner under the control of a promoter (pEF1α) of the human polypeptide chain elongation factor (EF1α) gene in the nucleotide sequence of the recombinant HSV genome prepared in the Examples described below, and further downstream, the nucleotide sequence from a nucleotide sequence (SEQ ID NO: 23) containing the poly A addition signal sequence of the EBNA1 gene to a nucleotide sequence (SEQ ID NO: 7) in which the OriP sequence has been minimized is replaced with the nucleotide sequence of a human S / MAR sequence (SEQ ID NO: 8).

[0182] When the first base sequence satisfies the above (5) and the cell according to this embodiment retains the 30th base sequence (the base sequence of the EBNA1 gene) or the base sequence of the SAF-A gene, the cell according to this embodiment can stably inherit the first base sequence (recombinant HSV genome) without loss even in daughter cells after cell division.

[0183] The cell according to this embodiment may have incorporated into its genomic DNA a base sequence that expresses a base sequence encoding proteins 27 and 28, which is under the control of a transcriptional regulatory region consisting of the base sequence shown in SEQ ID NO: 19, and may further have incorporated into its genomic DNA the base sequence shown in SEQ ID NO: 20.

[0184] The cell according to this embodiment may have incorporated into its genomic DNA a base sequence that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 21, and may further have incorporated into it a base sequence that expresses base sequences that encode proteins 27 and 28 that are deleted due to the insertion of the base sequence shown in SEQ ID NO: 22.

[0185] The cell according to this embodiment may have incorporated into its genomic DNA a base sequence that expresses a base sequence encoding the 29th protein, which is under the control of a transcriptional regulatory region consisting of the base sequence shown in SEQ ID NO: 19, and may further have incorporated into it the base sequence shown in SEQ ID NO: 20.

[0186] The cell according to this embodiment may have incorporated into its genomic DNA a base sequence that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO: 21, and may further have incorporated into it a base sequence that expresses a base sequence encoding the 29th protein that is missing due to the insertion of the base sequence shown in SEQ ID NO: 22.

[0187] A cell according to another embodiment (a cell according to a second embodiment) carries the genomic DNA of a particular recombinant HSV, wherein the genomic DNA of the recombinant HSV contains the nucleotide sequences of all essential genes of the recombinant HSV and the nucleotide sequences required for the recombinant HSV to be retained in the cell, but lacks the nucleotide sequences of the immediate early genes and the nucleotide sequences required for packaging into the recombinant HSV.

[0188] The "nucleotide sequence for being retained in a cell" refers to a nucleotide sequence that allows the cell according to this embodiment to retain the nucleotide sequence. Furthermore, the "nucleotide sequence for being packaged into a recombinant HSV" refers to a nucleotide sequence that allows the cell to be packaged into a recombinant HSV.

[0189] The genomic DNA of the recombinant HSV in the cell according to the second embodiment may further contain the base sequence of at least one non-essential gene of the recombinant HSV, and preferably further contains the base sequences of all of the non-essential genes of the recombinant HSV.

[0190] Specific embodiments of the cell according to the second embodiment include the specific embodiments of the cell according to the first embodiment, the "genomic DNA of recombinant HSV" includes the specific embodiment of the "first base sequence," the "essential gene of recombinant HSV" includes the specific embodiment of the "second to twenty-seventh base sequences," the "base sequence of a non-essential gene of recombinant HSV" includes the specific embodiment of the "thirty-first to eighty-fourth base sequence," the "base sequence for being retained in a cell" includes the specific embodiment of the "twenty-ninth base sequence," the "base sequence of an immediate early gene" includes the specific embodiment of the "base sequence encoding the twenty-seventh to twenty-eighth proteins" and the "base sequence encoding the twenty-ninth protein," and the "base sequence for being packaged into recombinant HSV" includes the specific embodiment of the "base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6."

[0191] The genomic DNA of the recombinant HSV may be, for example, the nucleotide sequence 1. The nucleotide sequence of the essential gene of the recombinant HSV may be, for example, the nucleotide sequence of nucleotides 2 to 27. The nucleotide sequence of the non-essential gene of the recombinant HSV may be, for example, the nucleotide sequence of nucleotides 31 to 84. The nucleotide sequence for retention in the cell may be, for example, the nucleotide sequence 29. The nucleotide sequence of the immediate early gene may be, for example, the nucleotide sequence encoding the proteins 27 to 28 and the nucleotide sequence encoding the 29 protein. The nucleotide sequences for packaging into the recombinant HSV may be, for example, the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0192] [Method for Producing a Recombinant Herpes Simplex Virus (HSV) Vector] The method for producing a recombinant herpes simplex virus (HSV) vector that expresses a gene of interest according to this embodiment (hereinafter also referred to as the "production method according to this embodiment") (the production method according to the first embodiment) comprises the steps of introducing an amplicon plasmid into a specific mammal-derived cell (introduction step), and expressing at least one protein selected from all of proteins 1 to 26, all of proteins 30 to 51, and proteins 52 to 83 (expression step A).

[0193] The amplicon plasmid comprises a target gene, the 86th base sequence consisting of the base sequence shown in SEQ ID NO: 15, and the 87th base sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6. The recombinant HSV vector expresses the target gene contained in the amplicon plasmid. "The recombinant HSV vector expresses the target gene" means that the recombinant HSV vector of this embodiment is introduced into an infected cell, causing the target gene to be expressed in the infected cell.

[0194] The production method according to this embodiment may further include a step of culturing the cells that have undergone the introduction step and the expression step A to produce recombinant HSV particles (recombinant HSV vectors) encapsulating the amplicon plasmid (production step), a step of preparing a solution containing the HSV particles (recombinant HSV vectors) produced in the production step (preparation step), and a step of purifying the HSV particles (recombinant HSV vectors) produced in the production step or purifying the HSV particles (recombinant HSV vectors) from the solution containing the HSV particles (recombinant HSV vectors) prepared in the preparation step (purification step).

[0195] The method for producing a recombinant HSV vector according to this embodiment uses specific mammalian-derived cells, and therefore, it is only necessary to carry out at least the introduction step and the expression step A, making it possible to produce a recombinant HSV vector easily and at low cost.

[0196] <Mammalian-derived cells> The mammalian-derived cells used in the production method according to this embodiment have a specific 85th base sequence, and when the 85th base sequence includes the specific 28th base sequence, the mammalian-derived cells also have a specific 30th base sequence.

[0197] The cells used in the production method according to this embodiment may be the mammal-derived cells immediately after production, or may be cells that have been cultured for at least one week, at least three weeks, at least one month, or at least two months. The mammal-derived cells used in the expression step A may be cells that have been cultured for no more than six months. Furthermore, the mammal-derived cells used in the production method according to this embodiment may be the cells according to this embodiment described above.

[0198] <85th base sequence> The 85th base sequence is a genomic DNA that has been genetically modified by substitution, insertion, addition, and / or deletion, etc., in the base sequence shown in SEQ ID NO: 17, which is the wild-type HSV-1 genome, and satisfies the following (A), (B), (C), (D), and (E): (A) It contains at least one of the base sequences of 2 to 27 and the base sequences of 31 to 84 that express the proteins of 1 to 26 and 30 to 83; (B) It lacks the base sequence encoding the proteins of 27 to 28; (C) It lacks the base sequence encoding the 29th protein; (D) It lacks the base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; and (E) It contains the 28th base sequence consisting of the base sequence shown in SEQ ID NO: 7, or the 29th base sequence consisting of the base sequence shown in SEQ ID NO: 8.

[0199] The nucleotide sequences of 2 to 27 and 31 to 84, which express the proteins of 1 to 26 and 30 to 83 in (A), are as described in "Cells for producing a recombinant herpes simplex virus (HSV) vector." Furthermore, (B) to (E) are as described in (2) to (5) in "Cells for producing a recombinant herpes simplex virus (HSV) vector." Furthermore, the nucleotide sequence of 30 is also as described in "Cells for producing a recombinant herpes simplex virus (HSV) vector." The 85th nucleotide sequence may be the first nucleotide sequence described above.

[0200] <Introduction step> In the introduction step, the amplicon plasmid is introduced (transfected) into the mammalian-derived cells, whereby the amplicon plasmid is packaged into viral particles during the viral particle formation process, thereby producing the desired recombinant HSV-1 vector.

[0201] The amplicon plasmid contains a gene of interest, the 86th base sequence consisting of the base sequence shown in SEQ ID NO: 15, and the 87th base sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6. As used herein, "amplicon plasmid" refers to a nucleic acid that contains a gene of interest, is packaged in HSV particles, is replicated within the cell, and is retained in daughter cells after cell division.

[0202] The gene of interest is not particularly limited as long as it is a desired gene to be carried in a viral vector. As used herein, "gene of interest" refers to a DNA sequence capable of expressing a protein of interest. The gene of interest according to this embodiment may include, for example, a region (transcription region) that is transcribed into an RNA molecule (e.g., mRNA) in a cell, an expression regulatory region located upstream and / or downstream of the transcription region, and an untranslated region (5'UTR, 3'UTR). The transcription region may be an open reading frame (ORF) including not only exons but also introns, or may be cDNA.

[0203] The expression regulatory region may be, for example, a transcription regulatory region or a translation regulatory region. Examples of the transcription regulatory region include a promoter, an enhancer, and a silencer. Examples of the translation regulatory region include a ribosome binding region.

[0204] The base sequence shown in SEQ ID NO: 15 is the OriS sequence, which is the origin of replication of the HSV-1 genome. The amplicon plasmid contains this sequence, allowing it to be replicated in the mammalian-derived cells and retained in daughter cells after cell division.

[0205] The 87th base sequence is a packaging sequence deleted from the 85th base sequence. When the amplicon plasmid contains this base sequence, the 86th base sequence is not packaged into HSV particles in the mammalian-derived cells, and only the amplicon vector is packaged into recombinant HSV particles.

[0206] The amplicon plasmid may, for example, contain at least one of the 2nd to 27th nucleotide sequences and the 31st to 84th nucleotide sequences that is not contained in the 85th nucleotide sequence. Furthermore, the amplicon plasmid may, for example, contain at least one of the 27th to 29th nucleotide sequences that express the 27th to 29th proteins. The 27th to 29th nucleotide sequences may be contained in the amplicon plasmid so that expression of the corresponding genes is induced only by chemical or physical stimulation, and the same aspects as those using the above-mentioned Tet on / off system and photoactivatable Cre system can be applied.

[0207] The amplicon plasmid may also contain elements typically found in vectors used in molecular cloning. Examples of such elements include selection markers and multicloning sites. The amplicon plasmid may also be genetically modified to stabilize expression of the target gene. Such modifications may include, for example, inserting the ICP0 gene or an insulator sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 25. The ICP0 gene and the insulator sequence have been reported to inhibit gene expression silencing and prolong gene expression (Molecular Therapy, Vol. 17, No. 4, 707-715, Apr. 2009; Molecular Therapy - Methods & Clinical Development, Vol. 21, No. 11, 399-412, 2021). The modification may also involve removing bacterial-derived sequences, such as Ori sequences, from the amplicon plasmid. This has been reported to stabilize the expression of the target gene from the amplicon plasmid (J Virol. 2006 Apr;80(7):3293-300.).

[0208] The amplicon plasmid may be, for example, one that uses a nucleic acid consisting of the base sequence shown in SEQ ID NO: 26 as a backbone and has a target gene introduced into the nucleic acid.

[0209] In the introduction step, one or more plasmids other than the amplicon plasmid (hereinafter also referred to as "helper plasmids") may be introduced into the mammalian-derived cells. The helper plasmid may contain, for example, a gene or a base sequence that assists in the production of the recombinant HSV vector. Examples of such genes or base sequences include genes or base sequences that promote the production of the recombinant HSV vector or that improve the proliferation ability or infectivity of the recombinant HSV vector.

[0210] The helper plasmid may, for example, contain the 85th base sequence and a base sequence not contained in the amplicon plasmid, among the 2nd to 27th base sequences and the 31st to 84th base sequences. The helper plasmid may also contain, for example, at least one of the 27th to 29th base sequences that express the 27th to 29th proteins. The 27th to 29th base sequences contained in the helper plasmid may be those contained in the amplicon plasmid, or may not be those contained in the amplicon plasmid.

[0211] The helper plasmid may contain, for example, a nucleotide sequence that expresses a protein (protein No. 27) that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 75 and has the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12. The nucleotide sequence shown in SEQ ID NO: 75 is a nucleotide sequence in which a nucleotide sequence (SEQ ID NO: 2) encoding the ICP27 protein (SEQ ID NO: 12) is linked in an expressible manner under the control of the promoter of the ICP27 gene present in the wild-type HSV-1 genome (the nucleotide sequence shown in SEQ ID NO: 17). Proteins having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12 are as described above. The sequence identity with the nucleotide sequence shown in SEQ ID NO: 75 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be 100%.

[0212] The helper plasmid may comprise a plasmid consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 70. The plasmid may comprise a nucleotide sequence that expresses at least one protein selected from the group consisting of proteins 27 to 29, and the sequence identity with the nucleotide sequence shown in SEQ ID NO: 70 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be 100%.

[0213] Depending on the plasmid introduced into the mammal-derived cell in the introduction step, the introduction step and the expression step A can be carried out simultaneously. For example, the 86th base sequence in the mammal-derived cell used includes at least one base sequence selected from the 2nd to 27th base sequences that express the 1st to 26th proteins and the 53rd to 84th base sequences that express the 52nd to 83rd proteins, and the plasmid introduced in the introduction step includes at least one base sequence selected from the 31st to 52nd base sequences that express the 30th to 51st proteins and the 27th to 29th proteins. Another example is the 86th base sequence in the mammal-derived cell used includes the 2nd to 27th base sequences and the 31st to 84th base sequences, and the plasmid introduced in the introduction step includes at least one base sequence selected from the 27th to 29th proteins.

[0214] The introduction step can be performed by a general method used by those skilled in the art when producing viral vectors, such as a method using a gene introduction reagent such as a cationic lipid (e.g., lipofectamine), polyethyleneimine, or calcium phosphate, a liposome method, or an electroporation method.

[0215] The introduction step may be any step as long as it involves introducing the amplicon plasmid into the mammalian-derived cells, and may be performed before the production step, before the expression step A, or after the expression step A and before the production step. The introduction step may be performed on cells before culture, may be performed simultaneously with cell culture, or may be performed on cells after culture. When the introduction step is performed on cells after culture, it may be performed, for example, on cells after 30 minutes or more, after 1 hour or more, after 2 hours or more, or after 3 hours or more after culture, or may be performed on cells within 6 hours, after 5 hours, or after 4 hours after culture.

[0216] <Expression Step A> In the expression step A, all of the proteins 1 to 26, all of the proteins 30 to 51, and at least one of the proteins 52 to 83 are expressed in the mammalian-derived cells. The proteins 1 to 26 and 30 to 83 include all of the proteins expressed from the β gene (E gene) and γ gene (L gene) necessary for the production of HSV-1 particles, and therefore, the expression step A makes it possible to produce a recombinant HSV-1 vector that can grow in host cells and has high production efficiency.

[0217] The expression step A can be carried out by introducing into cells an expression vector that expresses the desired protein, mRNA having a base sequence that encodes the desired protein, or the desired protein, or, if the expression of the desired protein is controlled by an inducer, by allowing the inducer to be present.

[0218] In the expression step A, it is sufficient that at least one of the 52nd to 83rd proteins is expressed. For example, of the above proteins, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, or 31 or more proteins may be expressed, and it is preferable to express all of the 52nd to 83rd proteins.

[0219] As described above, transcription of the β gene (E gene) and γ gene (L gene) is under the control of the α gene (IE gene). Furthermore, as will be understood from the examples described below, when the 85th base sequence in the mammal-derived cell contains at least one base sequence in its wild form among the 2nd to 27th base sequences and the 31st to 52nd base sequences that express the 1st to 26th and 30th to 51st proteins, and the 53rd to 84th base sequences that express the 52nd to 83rd proteins, expression of these proteins is induced by expressing at least one protein selected from the group consisting of the ICP27 protein (27th protein), the ICP0 protein (28th protein), and the ICP4 protein (29th protein), which are α genes (IE genes). That is, the expression step A can be carried out by a step of expressing at least one of the 27th to 29th proteins (expression step B), or may include the expression step B.

[0220] <Expression Step B> In the expression step B, at least one protein selected from the group consisting of a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12 and having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12 (protein No. 27), a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13 and having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 13 (protein No. 28), and a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14 and having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 14 (protein No. 29) is expressed in the mammalian cell. Proteins Nos. 27 to 29 are as described above. As a result, in a mammal-derived cell that retains at least one base sequence selected from the base sequences of the second to second nucleotide sequences and the base sequences of the third nucleotide sequence that express the first to second nucleotide sequences and the third nucleotide sequence that express the third nucleotide sequence, and at least one base sequence selected from the base sequences of the third nucleotide sequence and the fourth nucleotide sequence that express the fifth nucleotide sequence, all of the first to second nucleotide sequences, all of the third nucleotide sequences and at least one of the fifth nucleotide sequences can be expressed.

[0221] The proteins to be expressed in the expression step B are not particularly limited, but are preferably the 27th protein and the 28th protein.

[0222] In the case where the nucleotide sequence encoding the 27th to 29th proteins is deleted from the 86th nucleotide sequence in the mammal-derived cell, or where there is a mutation in the nucleotide sequence encoding the 27th to 29th proteins and translation into normal proteins is not possible, the expression step B can be carried out by introducing into the cell an expression vector that expresses the nucleotide sequence encoding the 27th to 29th proteins, mRNA of the nucleotide sequence encoding the 27th to 29th proteins, or the 27th to 29th proteins. Furthermore, for example, in the case where the 86th nucleotide sequence contains the nucleotide sequence encoding the 27th to 29th proteins but transcription is suppressed so that no proteins are produced, the expression step B may be carried out by activating the transcription.

[0223] For example, when the Tet on system is used in the mammalian-derived cells, the expression step B may involve culturing the cells in the presence of a tetracycline derivative. When the Tet off system is used in the mammalian-derived cells, the expression step B may involve culturing the cells in the absence of a tetracycline derivative. When the photoactivatable Cre system is used in the mammalian-derived cells, the expression step B may involve irradiating the cells with light.

[0224] <Production Step> The production step can be carried out by culturing the mammalian-derived cells that have undergone the expression step and the introduction step under general cell culture conditions that are commonly used by those skilled in the art when producing viral vectors. The cell culture conditions in the production step can be set appropriately depending on the type of cells used, etc. The culture temperature may be 20°C to 40°C, or 30°C to 37°C. The pH of the medium may be 6 to 8, or 7.2 to 7.4. The culture time may be 1 to 2 days, or 3 to 5 days. The medium used for culture may be, for example, Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), etc. The medium may be a serum-supplemented medium or a serum-free medium. The medium may also be a mixed medium comprising two types of medium.

[0225] The culture substrate on which the mammalian-derived cells that have undergone the expression and introduction steps are cultured is not particularly limited, and may be, for example, one coated with an extracellular matrix, etc. In this case, effects such as suppressing detachment of the cells from the dish and enhancing production of the recombinant HSV vector can be obtained.

[0226] <Preparation Step> The preparation step involves preparing a solution containing the HSV particles (recombinant HSV vector) produced in the production step. The preparation step may include, for example, obtaining a culture supernatant of the mammal-derived cells in the production step, or obtaining a lysate of the mammal-derived cells that have undergone the production step. The lysate of the mammal-derived cells that have undergone the production step may contain the culture supernatant of the mammal-derived cells in the production step. The lysate containing the culture supernatant of the mammal-derived cells in the production step can be obtained by performing the cell lysis treatment described below using the cell culture solution that has undergone the production step.

[0227] The mammal-derived cells that have undergone the production process can be disrupted by known cell disruption treatments. The mammal-derived cells that have undergone the production process can be disrupted, for example, by physical disruption treatment or chemical disruption treatment. Examples of physical disruption treatments include freeze-thawing, as well as methods using ultrasound, a homogenizer, a bead mill, and a French press. Examples of chemical disruption treatments include methods using surfactants or enzymes.

[0228] <Purification Step> The purification step is a general purification method carried out by those skilled in the art when producing a viral vector, and can purify the HSV particles produced in the production step or the HSV particles (recombinant HSV vector) contained in the solution prepared in the preparation step. Examples of such methods include sucrose density gradient centrifugation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, precipitation using PEG or the like, and appropriate combinations of these.

[0229] The recombinant HSV vector produced by the production method according to this embodiment can be used, for example, as a basic research tool, gene therapy, viral vector vaccine, and the like.

[0230] A production method according to another embodiment (a production method according to a second embodiment) comprises the steps of introducing an amplicon plasmid into a mammalian cell (introduction step A') and expressing all of the proteins encoded by the essential genes of the recombinant HSV and at least one of the proteins encoded by the non-essential genes of the recombinant HSV (expression step A'). The cell contains the genomic DNA of the recombinant HSV, and the genomic DNA of the recombinant HSV contains the nucleotide sequence of at least one of the essential and non-essential genes of the recombinant HSV and a nucleotide sequence for being retained in the cell, and is deficient in the nucleotide sequence of an immediate-early gene and a nucleotide sequence for packaging into the recombinant HSV. The amplicon plasmid also contains the gene of interest, the nucleotide sequence for packaging into the recombinant HSV, and the nucleotide sequence of the replication origin of the recombinant HSV.

[0231] Specific aspects of the production method according to the second embodiment include specific aspects of the production method according to the first embodiment; the "genomic DNA of recombinant HSV" includes a specific aspect of the "85th base sequence"; the "essential gene of recombinant HSV" includes a specific aspect of the "2nd to 27th base sequences"; the "base sequence of a non-essential gene of recombinant HSV" includes a specific aspect of the "31st to 84th base sequence"; the "base sequence for retention in cells" includes a specific aspect of the "29th base sequence"; the "base sequence of an immediate early gene" includes specific aspects of the "27th to 28th base sequences" and the "29th base sequence"; and the "base sequence for packaging into recombinant HSV" includes specific aspects of the "base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6".

[0232] The "mammalian-derived cells" in the production method according to the second embodiment may be the cells according to the first or second embodiment. The genomic DNA of the recombinant HSV may be, for example, the 85th base sequence. The nucleotide sequence of the essential gene of the recombinant HSV may be, for example, the 2nd to 27th base sequences. The nucleotide sequence of the non-essential gene of the recombinant HSV may be, for example, the 31st to 84th base sequences. The nucleotide sequence for retention in the cell may be, for example, the 29th base sequence. The nucleotide sequence of the immediate early gene may be, for example, the 27th to 28th protein encoding protein and the 29th protein encoding protein. The nucleotide sequences for packaging into the recombinant HSV may be, for example, the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0233] [Kit for Producing a Recombinant Herpes Simplex Virus (HSV) Vector] A kit for producing a recombinant HSV vector according to this embodiment (hereinafter also referred to as "the kit according to this embodiment") (the kit according to the first embodiment) comprises the specific mammal-derived cells described above and one or more first plasmids.

[0234] <Mammal-derived cells> The mammal-derived cells used in the production method according to this embodiment have a specific base sequence of 89, and when the 89th base sequence includes the specific base sequence of 28, they also have a specific base sequence of 30. The mammal-derived cells used in the production method according to this embodiment may be the cells according to this embodiment described above.

[0235] <89th Nucleotide Sequence> The 89th nucleotide sequence is a genomic DNA that has been genetically modified by substitution, insertion, addition, and / or deletion, etc., in the nucleotide sequence shown in SEQ ID NO: 17, which is the wild-type HSV-1 genome, and satisfies the following (i), (ii), (iii), (iv), and (v): (i) it comprises at least one of the nucleotide sequences of 2 to 27 and the nucleotide sequences of 31 to 52 that express the proteins of 1 to 26 and 30 to 51; (ii) it lacks the nucleotide sequence encoding the proteins of 27 to 28; (iii) it lacks the nucleotide sequence encoding the 29th protein; (iv) it lacks the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; and (v) it comprises the 28th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 7, or the 29th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 8.

[0236] (i) is the same as (A) of the 85th base sequence in the "Method for producing a recombinant herpes simplex virus (HSV) vector," except that it "contains at least one of the 2nd to 27th base sequences and the 31st to 52nd base sequences, which express the 1st to 26th and 30th to 51st proteins." (ii) to (v) are the same as (B) to (E) in the "Method for producing a recombinant herpes simplex virus (HSV) vector." The 30th base sequence is also the same as the 30th base sequence described in the "Method for producing a recombinant herpes simplex virus (HSV) vector."

[0237] Furthermore, the 89th base sequence may include at least one of the 53rd to 84th base sequences that express the 52nd to 83rd proteins. That is, the 89th base sequence may or may not include at least one of the 53rd to 84th base sequences. When the 89th base sequence includes at least one of the 53rd to 84th base sequences, it may include, for example, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, or 31 or more base sequences from the above base sequences, and preferably includes all of the 53rd to 84th base sequences. The 53rd to 84th base sequences are as described above.

[0238] <Plasmid> At least one of the first plasmids contains an 87th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 15, and an 88th nucleotide sequence consisting of the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6. Hereinafter, this plasmid will also be referred to as "plasmid 1-1." Plasmid 1-1 is the above-mentioned amplicon plasmid from which the gene of interest has been removed, and the same aspects as those described above can be applied. That is, when plasmid 1-1 is used to produce a recombinant HSV-1 vector, the nucleotide sequence of the gene of interest can be inserted into the plasmid, and the plasmid can be used as the above-mentioned amplicon plasmid.

[0239] The number of first plasmids may be, for example, 1, 2 or more, or 3 or more.

[0240] Furthermore, the first plasmid contains, of the 2nd to 27th nucleotide sequences and the 31st to 52nd nucleotide sequences, nucleotide sequences that are not contained in the 86th nucleotide sequence. As a result, the 2nd to 27th nucleotide sequences and the 31st to 52nd nucleotide sequences are each contained in either the 86th nucleotide sequence in the mammal-derived cell or the nucleotide sequence of the plasmid. In other words, by introducing the first plasmid into the mammal-derived cell, all of the 1st to 26th and 30th to 51st proteins can be expressed in the mammal-derived cell.

[0241] When there are a plurality of first plasmids, of the second to twenty-seventh base sequences and the thirty-first to eighty-fourth base sequences, the base sequences not contained in the 86th base sequence may be contained in the 1-1 plasmid or in a plasmid other than the 1-1 plasmid.

[0242] The first plasmid may contain at least one of the 53rd to 84th base sequences that express the 52nd to 83rd proteins. That is, the first plasmid may or may not contain at least one of the 53rd to 84th base sequences. When the first plasmid contains at least one of the 53rd to 84th base sequences, it may contain, for example, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, or 31 or more base sequences from the above base sequences. Furthermore, when the 89th base sequence contains at least one of the 53rd to 84th base sequences, the first plasmid may contain a base sequence from the 2nd to 27th base sequences and the 31st to 84th base sequences that is not contained in the 89th base sequence. The 53rd to 84th base sequences are as described above.

[0243] The first plasmid may contain a nucleotide sequence for expressing at least one protein selected from the group consisting of a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 12 and having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 12 (protein No. 27), a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 13 and having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 13 (protein No. 28), and a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 14 and having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (protein No. 29). Proteins Nos. 27 to 29 are as described above.

[0244] When the mammal-derived cell is a cell according to this embodiment and the 89th base sequence is a first base sequence satisfying the above (1) to (6), the 1-1th plasmid may be, for example, a plasmid consisting of a base sequence having 90% or more sequence identity to the 89th base sequence shown in SEQ ID NO: 26. The plasmid consisting of the 89th base sequence is pA-ICP0-GFP used in the Examples. The 1-1th plasmid may contain the 88th base sequence shown in SEQ ID NO: 15 and the 88th base sequence, and the sequence identity to the 88th base sequence shown in SEQ ID NO: 26 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may even be 100%.

[0245] In addition to the first plasmid, the kit according to this embodiment may further include one or more second plasmids containing at least one nucleotide sequence encoding proteins 27 to 29. The second plasmids are as described in the "Helper Plasmid" section of the "Method for Producing a Recombinant Herpes Simplex Virus (HSV) Vector" section.

[0246] The number of second plasmids may be, for example, 1, 2 or more, or 3 or more.

[0247] In addition to the mammalian cells, the first plasmid, and the second plasmid, the kit according to this embodiment may further include reagents necessary for producing a viral vector, such as PCR reagents (e.g., DNA polymerase, dNTPs, etc.), nucleic acid purification reagents, transfection reagents, and buffer solutions.

[0248] A kit according to another embodiment (a kit according to a second embodiment) comprises a specific mammalian cell and one or more first specific plasmids. The cell harbors the genomic DNA of the recombinant HSV, and the genomic DNA of the recombinant HSV contains the nucleotide sequence of at least one of the essential gene and the non-essential gene of the recombinant HSV, as well as a nucleotide sequence for being retained in the cell, and is deficient in the nucleotide sequence of an immediate early gene and a nucleotide sequence for packaging into the recombinant HSV. Furthermore, at least one of the first plasmids contains the nucleotide sequence for being packaged into the recombinant HSV and the nucleotide sequence of the replication origin of the recombinant HSV, and also contains the nucleotide sequences of the essential gene and the non-essential gene of the recombinant HSV that are not contained in the genomic DNA of the recombinant HSV.

[0249] Specific aspects of the kit according to the second embodiment include specific aspects of the kit according to the first embodiment; the "genomic DNA of recombinant HSV" includes a specific aspect of the above-mentioned "85th base sequence"; the "essential gene of recombinant HSV" includes a specific aspect of the above-mentioned "2nd to 27th base sequences"; the "base sequence of a non-essential gene of recombinant HSV" includes a specific aspect of the above-mentioned "31st to 84th base sequences"; the "base sequence for retention in cells" includes a specific aspect of the above-mentioned "29th base sequence"; the "base sequence of an immediate early gene" includes specific aspects of the above-mentioned "27th to 28th base sequences" and "29th base sequence"; and the "base sequence for packaging into recombinant HSV" includes specific aspects of the above-mentioned "base sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6".

[0250] The "mammalian-derived cells" in the kit according to the second embodiment may be the cells according to the first or second embodiment. The genomic DNA of the recombinant HSV may be, for example, the 85th base sequence. The nucleotide sequence of the essential gene of the recombinant HSV may be, for example, the 2nd to 27th base sequences. The nucleotide sequence of the non-essential gene of the recombinant HSV may be, for example, the 31st to 84th base sequences. The nucleotide sequence to be retained in the cell may be, for example, the 29th base sequence. The nucleotide sequence of the immediate early gene may be, for example, the 27th to 28th protein encoding protein and the 29th protein encoding protein. The nucleotide sequences to be packaged into the recombinant HSV may be, for example, the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0251] [Use] The use (application) of this embodiment is a use (application) derived from a mammal for producing a recombinant HSV vector, wherein the cell has a specific 85th base sequence, and when the 85th base sequence includes the specific 28th base sequence, the cell also has a specific 30th base sequence.

[0252] The cell in the use (application) according to this embodiment may be the cell according to Embodiment 1. Furthermore, the 87th base sequence may be the base sequence shown in SEQ ID NO:5.

[0253] In another embodiment, the use (application) is derived from a mammal for producing a recombinant HSV vector, wherein the cells harbor the genomic DNA of the recombinant HSV, and the genomic DNA of the recombinant HSV contains the base sequence of at least one of the essential and non-essential genes of the recombinant HSV and a base sequence for being retained in the cell, and is deficient in the base sequence of an immediate early gene and a base sequence for being packaged into the recombinant HSV.

[0254] The cells in the use (application) according to this embodiment may be the cells according to the first or second embodiment.

[0255] [Method for producing a gene therapy composition] The method for producing a gene therapy composition according to this embodiment includes the step of producing a recombinant HSV vector by the production method according to this embodiment. The gene therapy composition according to this embodiment contains the recombinant HSV vector.

[0256] Gene therapy refers to the administration of a gene or cells into which a gene has been introduced into the human body for the purpose of treating a disease. Therefore, the gene therapy composition may be administered directly to a subject in need of gene therapy, or may be used to introduce a gene of interest into cells to be administered to a subject in need of gene therapy.

[0257] In addition to the recombinant HSV vector, the gene therapy composition of this embodiment may contain a pharmaceutically acceptable carrier, such as saline, buffered saline, water, isotonic aqueous buffer, or a combination thereof.

[0258] The gene therapy composition of this embodiment may contain other pharmaceutically acceptable ingredients, such as stabilizers, preservatives, antioxidants, disintegrants, excipients, binders, glidants, lubricants, etc., as long as the ingredients do not impair the effects of the present invention.

[0259] The dosage form of the gene therapy composition according to this embodiment may be, for example, a liquid, a powder (lyophilized powder, dry powder), a capsule, a tablet, or a frozen state.

[0260] [Therapeutic Method] The therapeutic method according to this embodiment is a gene therapy method, and includes producing a recombinant HSV vector by the production method according to this embodiment, and introducing the target gene into a subject using the recombinant HSV vector.

[0261] The method for introducing the target gene into a subject using the recombinant HSV vector is not particularly limited as long as the target gene is introduced into the subject, but may be, for example, administering the recombinant HSV vector to the subject, or introducing the target gene into cells obtained from the subject using the recombinant HSV vector, and administering the cells into which the target gene has been introduced to the subject.

[0262] In the treatment method according to this embodiment, the administration method, administration target, dosage, etc. are the same as those of known gene therapy methods.

[0263] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0264] (Reagents, Kits, Cells, and Media) The reagents, kits, cells, and media used in this example are as follows. Unless otherwise specified, they were used according to the standard protocols recommended by the manufacturers. General molecular biology techniques were performed according to Molecular Cloning, 4th Edition (author: Green, Michael R., publisher: Cold Spring Harbor Laboratory Press, published in 2012).

[0265] (Plasmid DNA) VB221026-1563hjy (artificial gene synthesis, Vector Builder, SEQ ID NO: 27) pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (Addgene, #67974) pEB6CAG (gift from Yoshihiro Miwa, University of Tsukuba, SEQ ID NO: 28) pcDNA™ 3.1 / Zeo (Thermo Fisher Scientific) pUC57 / polyA (pUC57 (artificial synthesis, GeneScript), SEQ ID NO: 29) pBluescript II KS (+) (Stratagene) pEGFP-C2 (Addgene #6083-1) ・pEGFP-N1 (Addgene #6085-1) ・pEP-Kan-S (Addgene #41017)

[0266] (Nucleic acid purification kit) DNA fragment purification: QIAquick PCR Purification kit (Qiagen) Plasmid DNA preparation: QIAprep Spin Miniprep Kit (Qiagen) Transfection-grade plasmid DNA preparation: PureLink™ HiPure Plasmid Maxiprep Kit (Thermo Fisher Scientific)

[0267] (Enzymes) PCR enzyme: Tks Gflex™ DNA Polymerase (Takara Bio Inc.) Gibson assembly: GeneArt Gibson Assembly EX Master Mix (Thermo Fisher Scientific Inc.) In Fusion HD Cloning kit (Takara Bio Inc.) DNA Ligase kit <Mighty Mix> (Takara Bio Inc.)

[0268] (Transfection reagents) Lipofectamine 3000 (trademark) (Thermo Fisher Scientific) Lipofectamine 2000 (trademark of Thermo Fisher Scientific) PEI Max: Polyethyleneimine (PEI) Max (Polysciences)

[0269] (Culture media, etc.) Phosphate buffer solution (PBS, Fujifilm Wako Pure Chemical Industries, Ltd.) Luria Broth (LB medium, MERCK) Dulbecco's modified Eagle's medium (DMEM, Fujifilm Wako Pure Chemical Industries, Ltd.)

[0270] (E. coli cells) E. coli strain DH5α Competent high DH5α for cloning (Toyobo Co., Ltd.) E. coli strain GS1783 cells for RED-mediated recombination (provided by Greg Smith, Northwestern University, Chicago, USA)

[0271] (Mammalian-derived cells) Vero cells (provided by Yasushi Kawaguchi, Institute of Medical Science, The University of Tokyo) Flp-In™ T-Rex™ 293 cell line (ThermoFisher Scientific, product number: R78007, hereinafter referred to as "HEK293 cells")

[0272] (Antibodies) Mouse anti-gB monoclonal antibody (primary antibody, Virusys, #P1105) Mouse anti-ICP0 monoclonal antibody (primary antibody, Santa Cruz Biotechnology, #sc-56985) Mouse anti-ICP4 monoclonal antibody (primary antibody, Abcam, #ab6514) Mouse anti-ICP27 monoclonal antibody (primary antibody, Abcam, #53480) Mouse anti-α-Tubulin monoclonal antibody (primary antibody, Sigma-Aldrich, #T6199-200UL) Peroxidase-conjugated anti-mouse IgG antibody #NA9310V (secondary antibody, GE Healthcare Bio-Sciences) Peroxidase-conjugated anti-rabbit IgG antibody #NA9340V (secondary antibody, GE Healthcare Bio-Sciences) Healthcare Bio-Sciences)

[0273] (Method) <E. coli Host Cells for Performing RED-Mediated Recombination> Host cells for performing RED-mediated recombination and two-step RED-mediated recombination may be any E. coli strain that harbors and expresses the RED recombinase gene cluster on a chromosome or a plasmid. In this example, the E. coli strain GS1783 cells for RED-mediated recombination were used. In these cells, expression of the RED recombinase is controlled by a thermosensitive promoter that induces expression of the RED recombinase at 42°C. Furthermore, expression of the homing endonuclease I-SceI is induced in the presence of L-arabinose. When culturing the GS1783 cell line and its derivatives for the purpose of proliferation, the culture was carried out at 32°C to prevent unintended recombination unless otherwise specified. In the following examples, at each stage, GS1783 cells may be in various states, such as GS1783 cells into which no foreign gene has been introduced, GS1783 cells harboring a bacterial artificial chromosome (BAC) into which the wild-type HSV-1 genome has been inserted, GS1783 cells harboring a BAC into which a recombinant HSV genome has been inserted, and GS1783 cells into which any DNA fragment has been introduced; however, to avoid complication, these will be referred to as GS1783 cells without any particular distinction.

[0274] <RED-Mediated Recombination> RED-mediated recombination is a technique that utilizes the RED recombinase (three genes, gam, bet, and exo) possessed by bacteriophage λ to insert, replace, delete, or introduce point mutations of a foreign gene at any location in the base sequence of a chromosome, bacterial artificial chromosome (BAC), or plasmid present in a bacterial cell. For example, when replacing a target gene sequence in a BAC maintained in E. coli with an ampicillin resistance gene sequence, a linear DNA fragment in which sequences (40 bp to 50 bp) homologous to the base sequences upstream and downstream of the target gene sequence are added to the 5' and 3' ends of the ampicillin resistance gene sequence, respectively, is introduced into E. coli GS1783 cells. When RED recombinase is expressed in the E. coli cells, homologous recombination occurs, and the target sequence in the BAC is replaced with an ampicillin resistance gene. A linear marker gene fragment containing sequences homologous to the upstream and downstream nucleotide sequences of the target gene sequence can be prepared by PCR using primers with each homologous sequence attached to the 5' end. The RED-mediated recombination method can thus replace the target nucleotide sequence in the BAC with any nucleotide sequence; however, if the substituted nucleotide sequence does not contain a selection marker, clones in which the replacement has occurred cannot be selected. Therefore, in this example, a substitution or deletion of the target gene sequence was introduced by two-step RED-mediated recombination mediated by I-SceI-AphA1 (kanamycin resistance gene) as described below.

[0275] <Two-Step RED-Mediated Recombination Method (Biotechnique. 2006 Feb;40(2):191-7.)> By repeating the RED-mediated recombination method twice, any nucleotide sequence present in a cell can be modified by substitution, insertion, deletion, point mutation, or the like. When replacing a target gene sequence with a gene sequence of interest, and the target gene sequence is a gene sequence in a BAC, PCR is first performed using a nucleotide sequence (I-SceI-AphA1) containing homing endonuclease I-SceI and AphA1 (kanamycin resistance gene) as a template to amplify gene fragment 1. This PCR uses a forward primer containing a sequence homologous to the nucleotide sequence 20 bp to 25 bp downstream of the insertion site in the gene of interest, and a reverse primer containing a sequence homologous to the nucleotide sequence 20 bp to 25 bp upstream. Next, the amplified gene fragment 1 is inserted into a desired site in the gene sequence of interest using the homologous sequences added to the primers to prepare plasmid 1. Then, using primers containing sequences homologous to the upstream and downstream nucleotide sequences of the target gene sequence in the BAC (the sequence homologous to the upstream nucleotide sequence is designated A, and the sequence homologous to the downstream nucleotide sequence is designated B), PCR is performed using I-SceI-AphA1 of plasmid 1 and the added homologous sequences as templates to amplify DNA fragment 2. DNA fragment 2 is introduced into Escherichia coli GS1783 cells harboring pYEbac102, which will be described later, and the target gene sequence is removed by replacing it with the DNA fragment through the first stage of RED-mediated recombination. Recombined GS1783 cells are selected with kanamycin and chloramphenicol and cloned. Note that pYEbac102 contains a chloramphenicol resistance gene. GS1783 cells are precultured in 2 mL of LB medium containing kanamycin and chloramphenicol, and 100 μL of the precultured GS1783 cell culture is added to 2 mL of freshly prepared LB medium containing chloramphenicol and cultured with shaking at 32°C for 3 hours. 400 μL of 10% L-arabinose is added to the medium and cultured with shaking at 32°C for an additional hour. Cultivation in the presence of L-arabinose results in the expression of I-SceI, which cleaves the DNA introduced in the first stage. RED recombinase is expressed by culturing with shaking at 42°C for 30 minutes.The second stage, RED-mediated recombination, removes the introduced I-SceI-AphA1 sequence via a homologous sequence (40-50 bp) that combines the sequence added to the primer during amplification of gene fragment 1 with the sequence of the insertion site in the target gene, completing replacement with the target gene sequence. After 1.5 hours of shaking culture at 32°C, the cells are plated onto a kanamycin-containing LB agar plate. Colonies of GS1783 cells are then inoculated onto a kanamycin-containing LB agar plate and an LB agar plate containing kanamycin and chloramphenicol, yielding GS1783 cells in which the AphA1 fragment has been removed and which are kanamycin-nonresistant. When knocking out a target gene sequence, the forward and reverse primers are designed to further contain a homologous base sequence (40-50 bp) to each other. Using these designed primers, a PCR reaction is performed using a base sequence containing I-SceI and AphA1 as a template to amplify a DNA fragment. This DNA fragment is then introduced into E. coli GS1783 cells harboring pYEbac102. When I-SceI is expressed by the second-stage RED-mediated recombination, the I-SceI sequence in the DNA fragment is cleaved, and homologous recombination occurs between the homologous base sequences, removing the I-SceI-AphAI sequence. The method for selecting the target clone is the same as described above. This completes the deletion of the target gene sequence.

[0276] <Method for incorporating a gene sequence contained in E. coli into a plasmid by RED-mediated recombination (Retrieval method)> The RED-mediated recombination method can also be used to excise and transfer any gene sequence present in E. coli, such as a genomic gene sequence, artificial chromosome, or plasmid, and recover it as a circularized plasmid (Nat Protoc. 2009; 4(2): 206-223.). To recover a nucleic acid sequence present in a cell that is to be transferred to a plasmid, a recipient plasmid is amplified by PCR to obtain a linear DNA fragment. In this case, primers are designed so that a sequence homologous to the 40-50 bp at the 5' end of the target sequence is added in an inverted manner to the 5' end of the linear DNA fragment, and a sequence homologous to the 40-50 bp at the 3' end of the target sequence is added in an inverted manner to the 3' end. When the amplified linear DNA fragment is introduced into GS1783 cells, homologous recombination occurs via RED-mediated recombination, resulting in excision of the target sequence, transfer of the target sequence to the linear DNA fragment, and simultaneous circularization. Subsequently, selective cloning is performed using appropriate agents, and the plasmid is extracted and purified to obtain a plasmid containing the target sequence.

[0277] <Preparation of Competent Cells of GS1783 Cell Line and Derivatives> GS1783 cells or derivatives were cultured overnight at 32°C with shaking in 2 mL of chloramphenicol-containing Luria broth (LB) medium (preculture). 600 μL of the precultured GS1783 cell culture was added to 25 mL of freshly prepared chloramphenicol-containing LB medium and cultured with shaking at 32°C until the optical density (OD) reached 0.6-0.8 at 600°C. The cells were then shaken in warm water at 42°C for 15 minutes to express the RED recombinase. After thorough washing with ice-cold 10% glycerol solution, the cells were suspended in 200 μL of 10% glycerol solution, dispensed in 50 μL aliquots into new collection containers, and stored at -80°C as competent stocks.

[0278] <Flp-FRT recombination> This is a genetic recombination method that utilizes the recombinase flippase (Flp) derived from budding yeast. When a target sequence contains a flippase recognition target (FRT) sequence at one location, Flp inserts a DNA fragment flanked by two FRT sequences in donor DNA (Science. 1991 251(4999):1351-5.). Conversely, when the target sequence contains two FRT sequences but no donor DNA, the base sequence flanked by the FRT sequences can be excised, resulting in a deletion of the target sequence.

[0279] <Preparation and storage of nucleic acids> In this example, nucleic acids amplified and prepared by restriction enzyme methods, PCR methods, etc. were purified using ethanol precipitation or the above-mentioned nucleic acid purification kit for buffer exchange and enzyme removal. Nucleic acids were generally stored in a medical freezer (-30°C). Genomic DNA and BAC were stored in a refrigerator (4°C) to prevent destruction of nucleic acids by freezing and thawing.

[0280] <Introduction of Nucleic Acid into Escherichia coli Cells> Various nucleic acids were introduced into Escherichia coli GS1783 cells by electroporation, and various nucleic acids were introduced into Escherichia coli DH5α cells by heat shock.

[0281] <Cell Culture Method> The HEK293 cells and Vero cells used in this example were maintained in Dulbecco's modified Eagle's medium (DMEM) (Fujifilm Wako Pure Chemical Industries) containing 10% fetal bovine serum (FBS, Biosera, Nuaille). To maintain the 293 / TO-ICP27 cells described below, 10% Tet-system approved FBS (Thermo Fisher Scientific) was used instead of 10% FBS.

[0282] <Preparation of protein solution (lysate)> To confirm the presence or absence of HSV gene expression after transfection, 72 hours after transfection, the cells were washed with PBS and lysed using SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, 5% 2-mercaptoethanol). The resulting lysate was boiled in a 98°C incubator for 20 minutes, cooled to room temperature, and then subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE).

[0283] Western blotting: SDS-PAGE was performed using each protein solution, followed by transfer to a polyvinylidene difluoride membrane. The membrane was immersed in a blocking solution of 5% skim milk in Tris-buffered saline containing Tween 20 for at least 30 minutes to block nonspecific antibody adsorption. The primary antibody was then added and allowed to react for at least 2 hours at room temperature or 4°C. After washing, the membrane was reacted with a peroxidase-conjugated secondary antibody (GE Healthcare Bio-Sciences) at room temperature for at least 1 hour. The membrane was then reacted with a luminescent reagent (ImmunoStar LD, Fujifilm Wako Pure Chemical Industries), and images were captured using a FUSION SOLO S (VILBER).

[0284] <Microscopic observation and photography> Fluorescent images of transfected cells were taken 72 hours after transfection, and fluorescent images of infected cells were taken 18 hours after infection using an Olympus IX71, DP71 fluorescent microscope (Olympus) with a 40x objective.

[0285] <Long-term subculture> 293 / HSV cells were maintained in DMEM containing 10% FBS and 1 μg / mL puromycin.

[0286] <Tet-on / off system> The T-REx™ system is a system (Thermo Fisher Scientific) that uses the tetracycline (Tet) resistance operon encoded by E. coli Tn10 as a regulatory element to control gene expression in mammalian cells with Tet or doxycycline (Dox). In the absence of Tet or Dox, the Tet repressor (TetR) binds to the Tet operator 2 (TetO2) site inserted in the promoter of the target gene, thereby inhibiting transcription of the target gene and thereby suppressing gene expression. In the presence of Tet or Dox, when Tet or Dox binds to TetR, TetR leaves the TetO2 site, allowing expression of the target gene.

[0287] Example 1: Preparation of cells for producing recombinant HSV vector In order to prepare mammalian-derived cultured cells for producing a recombinant HSV vector, we first attempted to prepare a recombinant HSV genome in, for example, HEK293 cells or Vero cells, which has the following characteristics: (1) the recombinant HSV genome itself is retained within the cell and is stably inherited without loss in daughter cells after cell division; (2) before induction of recombinant HSV vector production, HSV gene expression is maintained in a suppressed or extinguished state; and further, during viral vector production, (3) the recombinant HSV genome itself is not encapsulated in the recombinant HSV particles produced.

[0288] <1-1. Deletion of the ICP27 gene in the wild-type HSV-1 genome and introduction of episome assembly function by EBNA1> To prepare the recombinant HSV genome, pYEbac102, in which the wild-type HSV-1 genome (SEQ ID NO: 17) was inserted into a bacterial artificial chromosome (BAC), was used as the wild-type HSV-1 genome template (J Virol. 2003 Jan;77(2):1382-91). It is known that the wild-type HSV-1 genome contains one copy of the ICP27 gene, which is an IE gene, and two copies each of the ICP0 gene and ICP4 gene (Figure 1). Hereinafter, pYEbac102 into which the wild-type HSV-1 genome has been inserted may also be referred to as HSV-1 BAC.

[0289] First, a promoter sequence for the human polypeptide chain elongation factor (EF1α) gene (pEF1α) was obtained to insert a promoter sequence for expressing the EBNA1 protein of Epstein-Barr virus in mammalian cells. Using plasmid VB221026-1563hjy (SEQ ID NO: 27), constructed by gene synthesis (requested from Vector Builder), as a template, PCR was performed using specific forward primer A (SEQ ID NO: 30), which contains a nucleotide sequence homologous to the 40 bp upstream of the start codon of the ICP27 gene, and specific reverse primer B (SEQ ID NO: 31), which contains a nucleotide sequence homologous to the 5' end of the EBNA1-EBNA1pA-miniOriP restriction enzyme fragment (described below), to obtain a DNA fragment (1259 bp, SEQ ID NO: 32) containing pEF1α.

[0290] Next, to enable the recombinant HSV genome to bind to intracellular genomic DNA and replicate when introduced into mammalian cells, pEB6CAG (SEQ ID NO: 28) was digested with restriction enzymes NheI and SpeI to obtain a 4094-bp DNA fragment. The DNA fragment EBNA1-EBNA1pA-miniOriP (SEQ ID NO: 33) contains the EBNA1 gene, the poly(A) addition signal sequence of the EBNA1 gene (EBNA1pA), and the replication origin miniOriP.

[0291] Furthermore, in order to select only cells into which the recombinant HSV genome had been introduced, a DNA fragment containing a puromycin resistance gene transcriptionally controlled by the human phosphoglycerate kinase 1 (hPGK) promoter (phPGK1), a gene encoding a fusion protein of the fluorescent protein BFP, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was constructed as follows. First, to create the template pBS-Puro2ABFP-WPRE, a PCR reaction was performed using pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W as a template, primer E (SEQ ID NO: 34) and primer F (SEQ ID NO: 35), to obtain the Puro2ABFP-WPRE fragment. The resulting Puro2ABFP-WPRE fragment was digested with BamHI and KpnI, inserted into the BamHI and KpnI sites of pBluescript II KS (+), and ligated and circularized using DNA ligase to obtain pBS-hPGK-Puro2ABFP-WPRE. Next, pBS-hPGK-Puro2ABFP-WPRE was digested with XhoI to obtain a linearized pBS-hPGK-Puro2ABFP-WPRE fragment. The AphA1 fragment was amplified by PCR using pEP-Kan-S as a template and primer U (SEQ ID NO: 36) and primer V (SEQ ID NO: 37). The pBS-hPGK-Puro2ABFP-WPRE fragment and the AphA1 fragment were ligated and circularized using GeneArt Gibson Assembly EX Master Mix to obtain pBS-hPGK-AphA1-Puro2ABFP-WPRE.

[0292] Using the pBS-hPGK-AphA1-Puro2ABFP-WPRE prepared above as a template, PCR was performed using a forward primer C (SEQ ID NO: 38) containing a nucleotide sequence homologous to the 3' end of the EBNA1-EBNA1pA-miniOriP restriction enzyme fragment (SEQ ID NO: 33) and a reverse primer D (SEQ ID NO: 39) containing a nucleotide sequence homologous to a region 40 bp downstream of the termination codon of the ICP27 gene to yield an hPGK-AphA1-Puro2ABFP-WPRE fragment (3556 bp, SEQ ID NO: 40).

[0293] The above three DNA fragments (DNA fragments consisting of the nucleotide sequences shown in SEQ ID NOS: 32, 34, and 41) were ligated into a single DNA fragment (pEF1α-EBNA1-EBNA1pA-miniOriP-hPGK-Puro2ABFP-WPRE, SEQ ID NO: 41) (8907 bp) using GeneArt Gibson Assembly EX Master Mix. The ligated DNA fragment was used to transform E. coli strain GS1783 cells (GS1783-HSV) containing pYEbac102. In the first step, the ORF of the ICP27 gene in the wild-type HSV-1 genome contained in pYEbac102 (HSV-1 BAC) was replaced with pEF1α-EBNA1-EBNA1pA-miniOriP-hPGK-Puro2ABFP-WPRE by RED-mediated recombination, and GS1783-HSV was cultured and selected in LB medium containing chloramphenicol and kanamycin. Next, I-SceI expression was induced in the GS1783 cells to cleave the I-SceI-AphA1 fragment, and the I-SceI-AphAI fragment itself was removed by RED-mediated recombination in the second step, completing the deletion and replacement of the ICP27 gene (SEQ ID NO: 2) contained in the HSV-1 BAC.

[0294] Furthermore, in order to replace the BFP gene in the base sequence shown in SEQ ID NO: 41 inserted into the HSV-1 genome by the above procedure with the mRFP gene, a plasmid carrying mRFP with AphA1 inserted was constructed. First, DNA was amplified by PCR using pDEST-12.5'RFP (Nucleic Acids Res. 2014 Sep; 42(15):10037-49.) as a template and primer X (SEQ ID NO: 42) and primer Y (SEQ ID NO: 43) to obtain an mRFP fragment. pEGFP-C2 was digested with NheI and KpnI to obtain a pEGFP-C2 fragment. These two DNA fragments were ligated and circularized using the In Fusion HD Cloning kit. 10 μL of the reaction solution was used to transform Escherichia coli strain DH5α, and selection was performed on an LB plate containing kanamycin. After cloning, pmRFP-C2 was prepared. Using pEP-Kan-S as a template, DNA was amplified by PCR with primer W (SEQ ID NO: 44) and primer Z (SEQ ID NO: 45) to obtain an AphA1 fragment. pmRFP-C2 was digested with PstI to obtain a pmRFP-C2 fragment. These two DNA fragments were ligated and circularized using the In Fusion HD Cloning kit. 10 μL of the reaction solution was used to transform Escherichia coli strain DH5α, and selection was performed on an LB plate containing kanamycin. After cloning, pmRFP-AphA1 was prepared. A DNA fragment amplified by PCR with primer G (SEQ ID NO: 46) and primer H (SEQ ID NO: 47) using pmRFP-AphA1 as a template was introduced into GS1783 cells harboring the above-mentioned HSV-1 genome from which the ICP27 gene had been deleted. Then, by RED-mediated recombination in the first step, the BFP gene in the base sequence shown in SEQ ID NO: 41 inserted into the HSV-1 genome was replaced with the pmRFP-AphA1 fragment. Next, expression of I-SceI was induced in the GS1783 cells to cleave the I-SceI-AphA1 fragment, and the I-SceI-AphAI fragment itself was removed by RED-mediated recombination in the second step, completing the replacement of the BFP gene with the pmRFP gene.

[0295] As a result of the above, the ICP27 gene in the wild-type HSV-1 genome was replaced with pEF1α-EBNA1-EBNA1pA-miniOriP-hPGK-Puro2AmRFP-WPRE (hereinafter also referred to as the "EBNA1-OriP-PuroR-mRFP cassette"). At the same time, the BAC containing the HSV-1 genome was maintained in mammalian-derived cells as an episome together with the genomic DNA of the cells by EBNA1-miniOriP, and a recombinant HSV genome was obtained that was selectable with puromycin and expressed red fluorescent protein (mRFP), allowing easy identification and confirmation of mammalian-derived cells harboring the recombinant HSV genome.

[0296] <1-2. Deletion of the Terminal Region Containing the ICP0 and ICP4 Genes in the HSV-1 Genome> As described above, the wild-type HSV-1 genome contains the terminal region L (TR) in infected cells. Ｌ ) and terminal region S (TR Ｓ ) and exist as a circular genome. Ｌ The ICP0 gene is Ｓ Therefore, in the HSV-1 genome into which the EBNA1-OriP-PuroR-mRFP cassette has been inserted, the TR containing the ICP0 gene and the ICP4 gene is Ｌ and TR Ｓ The deletion was carried out as follows.

[0297] Using pEP-Kan-S as a template, PCR amplification was performed using primer I (SEQ ID NO: 48) and primer J (SEQ ID NO: 49). An I-SceI-AphAI fragment having a sequence homologous to a 40-bp upstream region of terminal region L (the sequence of bases 1 to 9149 of SEQ ID NO: 17) and a 40-bp downstream region of terminal region S (the sequence of bases 145,428 to 152,151 of SEQ ID NO: 17) was obtained and introduced into GS1783 cells. The ICP0 and ICP4 genes were simultaneously replaced with the fragment by RED-mediated recombination in the first step. Next, expression of I-SceI was induced in the GS1783 cells, cleaving the I-SceI-AphA1 fragment, and the I-SceI-AphAI fragment itself was removed by RED-mediated recombination in the second step.

[0298] From the above, the TR of the HSV-1 genome Ｌ , T.R. Ｓ Deletion of the ICP0 gene (one copy) and the ICP4 gene (one copy), each contained in

[0299] <1-3. Disruption of the ICP0 gene contained in the internal region> One of the two copies of the ICP0 gene in the HSV-1 genome is located in the internal region L (IR). Ｌ Therefore, the ICP0 gene remaining in the HSV-1 genome was disrupted by introducing a nonsense mutation according to a previously reported method (WX Cai et al., JOURNAL OF VIROLOGY, 63(11):4579-4589, 1989).

[0300] Using pEP-Kan-S as a template, PCR was performed using primer K (SEQ ID NO: 50) and primer L (SEQ ID NO: 51) for introducing a mutation into the ORF of the ICP0 gene to obtain an I-SceI-AphAI fragment having a sequence homologous to internal region L. Primer K was designed to convert the 212th amino acid in all three reading frames of the ICP0 gene present in internal region L to a stop codon. As described above, this DNA fragment was introduced into GS1783 cells, and a nonsense mutation was introduced into the ICP0 gene present in internal region L by two-step RED-mediated recombination, disrupting it.

[0301] <1-4. Disruption of the internal region (packaging signal) by inserting the Zeocin® resistance gene (ZeoR)> A PCR reaction was performed using pcDNA™ 3.1 / Zeo as a template and primer Q (SEQ ID NO: 52) and primer I (SEQ ID NO: 53) to amplify a DNA fragment containing the Zeocin® resistance gene (containing ZeoR downstream of the prokaryotic promoter pEM7). An inverse PCR reaction was performed using pUC57 / polyA and primer S (SEQ ID NO: 54) and primer T (SEQ ID NO: 55) to amplify a linear plasmid DNA fragment. These two DNA fragments were ligated and circularized using the In Fusion HD Cloning kit. This plasmid DNA was used to transform Escherichia coli DH5α, followed by selection with ampicillin and cloning. The constructed pEM7 / Zeo plasmid was then purified from the E. coli strain. Finally, using the pEM7 / Zeo plasmid as a template, PCR was performed using primer O (SEQ ID NO: 56) having a sequence homologous to the upstream 40 bp of the 125,770-127,000 base sequence or primer P (SEQ ID NO: 57) having a sequence homologous to the downstream 40 bp, to obtain the pEM7-ZeoR fragment. Using the obtained DNA fragment, the packaging sequence present in the a region containing the packaging signal in the HSV-1 genome (the 125,770-127,000 base sequence of SEQ ID NO: 17) was deleted by replacing it with ZeoR through RED-mediated recombination. Hereinafter, the deletion of the packaging sequence will also be referred to as "Δpac."

[0302] As a result, a recombinant HSV genome was completed that exhibits the following characteristics: (1) the recombinant HSV genome itself is retained within the cell and is stably inherited without being lost in daughter cells after cell division; (2) before induction of recombinant HSV vector production, HSV gene expression is maintained in a suppressed or extinguished state; and further, during viral vector production, (3) the recombinant HSV genome itself is not encapsulated in the produced recombinant HSV particles. The recombinant HSV genome is genomic DNA consisting of the base sequence shown in SEQ ID NO:11.

[0303] Figure 1 shows an outline of the recombination of the wild-type HSV-1 genome. Line 1 is a schematic diagram of the BAC vector pYEbac102 containing the wild-type HSV-1 genome (SEQ ID NO: 17). The internal region of the wild-type HSV-1 genome is the IR Ｌ The left side of the a region including U Ｌ ) and I.R. Ｓ The right side of the a region (U R The ICP27 gene is divided into U Ｌ One of the two copies of the ICP0 gene is located in the TR region. Ｌ The other is IR Ｌ Similarly, one of the two copies of the ICP0 gene is located in the IR Ｓ and the other is TR Ｓ Although not shown in the figure, L and I.R. Ｓ The a region containing a part of the nucleotide sequence contains a packaging signal.

[0304] Line 2 is a schematic representation of the results of recombination in E. coli. Ｌ , ΔTR ｓ indicates that the ICP0 gene and the ICP4 gene have been deleted. ΔICP27 indicates that the ORF of the ICP27 gene has been replaced and deleted by pEF1α-EBNA1-EBNA1pA-miniOriP-hPGK-Puro2AmRFP-BGHpA (EBNA1-OriP-PuroR-mRFP cassette) shown at the top. The HSV-1 genome recombined in this manner is hereinafter also referred to as "HSVBAC / Δ27 / ΔTR / 0n212 / Δpac / EBNA1-OriP-Puro2ARFP" or "recombinant HSV genome." "ICP0n212" in Figure 1 indicates the ICP0 gene functionally deleted by introducing a nonsense mutation. IR Ｌ , I.R. ＳBetween these is inserted the resistance gene ZeoR for the antibiotic Zeocin, which allows selection in both E. coli and mammalian-derived cells. The disruption of the packaging signal in the a region of the wild-type HSV-1 genome is indicated as "a(Δpac)."

[0305] [Example 2: Establishment of cells harboring a recombinant HSV genome] In this test example, HEK293 cells, which are widely used in medical and biological research, were used as mammalian cultured cells that can be used to produce a recombinant HSV vector. The recombinant HSV genome prepared in Example 1 above was introduced into HEK293 cells, and cells for producing a recombinant HSV vector were established through cloning by selection using antibiotics as follows.

[0306] <2-1. Introduction of recombinant HSV-1 genome into HEK293 cells> The recombinant HSV genome (HSVBAC / Δ27 / ΔTR / On212 / Δpac / EBNA1-OriP-Puro2ARFP) prepared in Example 1 above was transfected into HEK293 cells using Lipofectamine 2000, the cells were selected with 1 μg / ml puromycin, and single colonies were cloned. The resulting cells are hereinafter also referred to as "293 / HSV cells." Figure 2 shows an outline of the method for establishing HEK293 cells harboring a recombinant HSV genome.

[0307] <2-2. Confirmation that the prepared recombinant cells contain the recombinant HSV-1 genome> As in 2-1, the recombinant HSV genome was transfected into HEK293 cells, and then 3.4 × 10 cells per well were cultured. ５ Cells were seeded onto a 24-well plate and incubated with CO ２ Incubator (temperature 37°C, humidity 100%, CO ２The cells were cultured for 72 hours at a concentration of 5% for 72 hours until they reached confluence. Bright-field and fluorescent microscopic images (RFP) of the same field of view were obtained using an inverted fluorescent microscope IX71 (OLYMPUS) and a digital CCD camera DP71 (OLYMPUS). As a control, HEK293 cells not transfected with the recombinant HSV genome were cultured in the same manner, and fluorescent and bright-field images were obtained. The results are shown in Figure 3.

[0308] Figure 3 shows fluorescence microscope images of HEK293 cells and 293 / HSV cells. No RFP fluorescence was detected in the control HEK293 cells. On the other hand, a red RFP fluorescent signal was detected in the 293 / HSV cells. These results confirmed that the recombinant HSV genome was stably maintained in the host HEK293 cells and was maintained even after cell division.

[0309] Example 3: Preparation of cells for producing a recombinant HSV vector and confirmation of HSV gene expression. In contrast to HSV vectors that encapsulate an attenuated HSV genome modified to express a gene of interest within the virus particle, the shell (envelope, etc.) of the virus particle used to infect host cells in this invention utilizes a protein expressed from the recombinant HSV genome. However, the recombinant HSV vector of the present invention does not encapsulate the recombinant HSV genome, but rather encapsulates a nucleic acid (e.g., a plasmid vector, etc.) carrying a gene of interest. The amplicon plasmid, which is a nucleic acid carrying a gene of interest and is encapsulated into virus particles, must have a signal (packaging signal) sequence for packaging into recombinant HSV particles upon induction of recombinant HSV vector production, and a replication origin for maintenance in the cells into which it is introduced. Users can construct any foreign gene that can be expressed in host cells according to their intended purpose as the gene of interest. Furthermore, because the recombinant HSV vector of the present invention does not contain the HSV genome, which is unnecessary for gene therapy, the size of nucleic acid that can be packaged into HSV particles is thought to be approximately 150 kbp, which is the same size as the HSV genome. Therefore, it is expected that the size of target genes that can be carried by conventional viral vectors for gene therapy will be dramatically expanded.

[0310] As shown in Example 2, the cells for producing the recombinant HSV vector prepared in Example 2 stably retain the recombinant HSV genome within the cells. However, under normal culture conditions, due to functional deficiencies of the ICP27 gene, ICP0 gene, and ICP4 gene, the cells are unable to produce viral particle components such as HSV genome replication proteins and HSV vector envelope proteins. To produce a recombinant HSV vector, the cells require two steps: (1) an "introduction step" in which an amplicon plasmid carrying a gene of interest is introduced into the cells to enable production of a viral vector encapsulating the amplicon plasmid; and (2) an "expression step A" in which HSV-1 genes other than the ICP27 gene, ICP0 gene, and ICP4 gene are expressed. The expression step and the introduction step can also be performed simultaneously. The following verification was carried out: by introducing an amplicon plasmid and a helper plasmid carrying the ICP27 gene into the prepared cells (293 / HSV cells), a recombinant HSV vector containing a nucleic acid of interest can be produced; and the prepared recombinant HSV vector has infectivity and can infect target cells different from the cells used to produce the recombinant HSV vector.

[0311] <3-1. Preparation of Amplicon Plasmid> The amplicon plasmid pA-ICP0-GFP (SEQ ID NO: 26) carried in the recombinant HSV vector comprises a packaging sequence (HSV-1 pac, SEQ ID NO: 5) necessary for encapsulating nucleic acid into HSV particles, an origin of replication (HSV-1 OriS, SEQ ID NO: 15) necessary for the introduced plasmid to be replicated within the cell and retained in daughter cells resulting from cell division, an IPC0 gene expression cassette, an EGFP expression cassette (SEQ ID NO: 58) that serves as a marker for carrying the amplicon plasmid, and a multicloning site (MCS, SEQ ID NO: 59) for incorporating any gene and expression device (promoter, terminator, etc.).

[0312] Previous studies have suggested that transcription of amplicon plasmids introduced into cells is suppressed by histone deacetylases (HDAC). Recently, it has been reported that incorporating the ICP0 gene into an amplicon plasmid releases HDAC from the amplicon plasmid and dramatically prolongs the expression of exogenous genes in cells transfected with the amplicon plasmid (Masataka Suzuki et al., Molecular Therapy 17(4): 707-715, 2009). Therefore, in this example, the ICP0 gene was incorporated into the amplicon plasmid.

[0313] First, a PCR reaction was performed using primer AE (SEQ ID NO: 60) and primer AF (SEQ ID NO: 61), both containing restriction enzyme sites, and pEGFP-C2 as a template to prepare a pEGFP-C2 fragment having a sequence homologous to the terminus of HSV-1 pac (nucleotide sequences from bases 151,376 to 152,151 and from bases 1 to 428 of SEQ ID NO: 17). This fragment was introduced into GS1783 cells carrying HSV BAC, and a plasmid (pEGFP-C2-pac) in which HSV-1 pac had been inserted into pEGFP-C2 was obtained by the Retrieval method.

[0314] Second, PCR was performed using primer AG (SEQ ID NO: 62) and primer AH (SEQ ID NO: 63), both containing restriction enzyme sites, with pEGFP-C2 as a template to prepare a pEGFP-C2 fragment having a sequence homologous to the end of HSV-1 OriS (the nucleotide sequence from bases 131,461 to 132,421 of SEQ ID NO: 17). This fragment was introduced into GS1783 cells harboring HSV BAC, and a plasmid (pEGFP-C2-OriS) in which HSV-1 OriS had been inserted into pEGFP-C2 was obtained by the Retrieval method.

[0315] Third, a PCR reaction was performed using primer AA (SEQ ID NO: 64) and primer AB (SEQ ID NO: 65), both containing restriction enzyme sites, with pEGFP-C2 as a template to prepare a pEGFP-C2 fragment having a sequence homologous to the end of the region encompassing the promoter and poly A addition sequence of the ICP0 gene in the wild-type HSV-1 genome (ICP0 expression cassette, nucleotide sequence from bases 1232 to 5881 of SEQ ID NO: 17). This fragment was introduced into GS1783 cells harboring an HSV BAC, and a plasmid (pEGFP-C2-ICP0) in which the ICP0 expression cassette had been inserted into pEGFP-C2 was obtained by the Retrieval method.

[0316] pEGFP-C2-ICP0 was digested with HindIII to obtain an ICP0 expression cassette fragment. A PCR reaction was performed using primer AC (SEQ ID NO: 66) and primer AD (SEQ ID NO: 67) and VB221026-1563hjy as a template to prepare a VB221026-1563hjy fragment with a sequence homologous to the end of the ICP0 expression cassette fragment. These two DNA fragments were ligated and circularized using an In Fusion HD Cloning kit. 10 μL of the ligation solution was transformed into E. coli strain DH5α, and selection was performed on an LB plate containing ampicillin. After cloning, the amplicon plasmid pVB-ICP0-GFP was prepared.

[0317] pEGFP-C2-pac was digested with HindIII and BamHI to obtain an HSV-1 pac fragment (SEQ ID NO: 68). pEGFP-C2-OriS was digested with BamHI and ClaI to obtain an OriS fragment (SEQ ID NO: 69). Furthermore, pVB-ICP0-GFP was digested with HindIII and ClaI to obtain an ICP0 expression cassette fragment. These three DNA fragments (SEQ ID NOs: E38, E39, and the ICP0 expression cassette fragment) were ligated and circularized using DNA ligase. 10 μL of the ligation solution was transformed into Escherichia coli strain DH5α, and selection was performed on an LB plate containing ampicillin. After cloning, the amplicon plasmid pA-ICP0-GFP (SEQ ID NO: 26) was prepared. The vector map of the resulting pA-ICP0-GFP is shown in Figure 4. 3-2. Preparation of helper plasmid>

[0318] A helper plasmid (pEGFP-N1-ICP27, SEQ ID NO: 70) for expressing the ICP27 gene in mammalian-derived cells required for the expression step was prepared as follows.

[0319] Similar to the above, a PCR reaction was performed using primer 1 (SEQ ID NO: 71) and primer 2 (SEQ ID NO: 72) and pEGFP-N1 as a template to prepare a pEGFP-N1 fragment having a sequence homologous to the end of the region encompassing the promoter of the ICP27 gene in the wild-type HSV-1 genome and the poly A addition sequence (ICP27 expression cassette, nucleotide sequence from bases 113,213 to 115,636 of SEQ ID NO: 17). This was introduced into GS1783 cells harboring an HSV BAC, and a helper plasmid (pICP27) expressing the ICP27 gene was obtained by the retrieval method.

[0320] <3-3. Introduction of amplicon plasmid and helper plasmid into 293 / HSV cells> 3.4 x 10 per well ５ HEK293 cells alone or 293 / HSV cells were seeded onto a 24-well plate (AGC Technoglass) coated with 0.01% collagen (IFP9660, Functional Peptide Institute Co., Ltd.), and incubated with CO ２The cells were cultured in an incubator. The next day, the amplicon plasmid pA-ICP0-GFP and the helper plasmid pICP27 (100 ng each) prepared in 3-1 and 3-2 above were co-transfected into the cells using Lipofectamine 3000. For control experiments, HEK293 cells without any plasmid or reagent, 293 / HSV cells transfected with only the transfection reagent (mock-transfected 293 / HSV cells), and 293 / HSV cells transfected with the pEGFP-C2 plasmid instead of pICP27 were prepared. The medium was replaced with fresh DMEM containing 10% FBS 6 and 48 hours after transfection.

[0321] <3-4. Analysis of viral gene expression by Western blotting> 72 hours after transfection, the expression of three HSV gene products (ICP0 protein, ICP27 protein, glycoprotein B (gB) protein) was analyzed by Western blotting and chemiluminescence using specific antibodies against each gene product (mouse anti-gB monoclonal antibody, mouse anti-ICP0 monoclonal antibody, mouse anti-ICP27 monoclonal antibody, and rabbit anti-β-actin polyclonal antibody as a control). gB is one of the L genes and serves as an indicator of the progress of virus production. The results are shown in Figure 5.

[0322] Figure 5 shows the results of Western blot analysis of the expression of ICP0, ICP4, ICP27, and gB proteins in cells under each condition. Expression of ICP0, ICP4, ICP27, and gB proteins was not confirmed in control HEK293 cells without plasmid or reagent (leftmost lane) or mock-transfected 293 / HSV cells (second lane from the left). On the other hand, expression of ICP0, ICP4, ICP27, and gB proteins was confirmed only in 293 / HSV cells cotransfected with the amplicon plasmid pA-ICP0-GFP and the helper plasmid pICP27 (rightmost lane).

[0323] <3-5. Fluorescence Microscopic Observation of 293 / HSV Cells Transfected with Amplicon Plasmid and Helper Plasmid> To confirm that the amplicon plasmid and helper plasmid have been introduced into the cells and that the gene carried by the amplicon plasmid is being expressed, the fluorescent signal of the fluorescent protein EGFP inserted into the amplicon plasmid can be confirmed.

[0324] Cells treated in the same manner as in 3-4 above were observed under a fluorescence microscope 72 hours after transfection. The results are shown in Figure 6.

[0325] Figure 6 shows fluorescence microscopy images of GFP (upper panel) and RFP (lower panel) in mock-transfected 293 / HSV cells (left panel), and GFP (upper panel) and RFP (lower panel) in 293 / HSV cells (right panel) transfected with the amplicon plasmid pA-ICP0-GFP and the helper plasmid pICP27. As shown in Figure 6, red fluorescent protein signals were observed in all samples, indicating that the HEK293 cells harbored the recombinant HSV genome. In 293 / HSV cells transfected with the amplicon plasmid and the helper plasmid, not only RFP but also EGFP fluorescence was observed, as in 293 / HSV cells transfected with the control plasmid (pEGFP-C2).

[0326] From the above 3-4 and 3-5, it was confirmed that the amplicon plasmid and helper plasmid introduced into 293 / HSV cells were stably retained in the cells, and that the HSV gene cluster was also stably expressed.

[0327] Example 4: Confirmation of HSV Vector Production and Infectivity 4-1. Production of HSV Vector As in Example 3 above, 293 / HSV cells transfected with an amplicon plasmid and a helper plasmid express HSV genes, including the α gene, and therefore produce recombinant HSV particles (recombinant HSV vectors) harboring the amplicon vector, and it was believed that some of the recombinant HSV particles were present in the cell culture medium. To confirm the production of an infective recombinant HSV vector harboring the amplicon plasmid, 72 hours after transfection in the same manner as in 3-5, the cells were detached with a scraper, and the 293 / HSV cells and medium were freeze-thawed three times and centrifuged twice at 10,000 × g for 5 minutes at 4°C to obtain a culture supernatant from which the cells and insoluble components such as cell debris had been removed. The day before the infection experiment, 2 × 10 cells per well were transfected with 2 × 10 cells per well. ５ 200 μL of the culture supernatant was added to Vero cells seeded on a 24-well plate, and the cells were infected for 18 hours. After that, the cells were observed under a fluorescence microscope, and bright-field and fluorescence microscope images of the same field of view were obtained. The results are shown in Figure 7.

[0328] 7 shows bright-field (top) and GFP (bottom) fluorescence microscopy images of Vero cells cultured for 18 hours after addition of culture supernatants prepared from mock transfection (left panel), 293 / HSV cells transfected with the amplicon plasmid pA-ICP0-GFP and the control plasmid pEGFP-C2 (middle panel), and 293 / HSV cells transfected with the amplicon plasmid pA-ICP0-GFP and the helper plasmid pICP27 (right panel). EGFP fluorescence was observed only in Vero cells to which the supernatant components from 293 / HSV cells transfected with the amplicon plasmid and the helper plasmid pICP27 had been added.

[0329] The above experimental results demonstrate that the present invention makes it possible to produce a recombinant HSV vector harboring an amplicon plasmid that is infectious to mammalian cells.

[0330] Example 5: Analysis of the effect of transfection reagents on 293 / HSV cells, and the localization and infectivity of the produced HSV vector. The effect of the type of transfection reagent used in the expression and introduction steps on the recombinant HSV vector was examined. In theory, the produced recombinant HSV vector should be released outside the cells and recovered from the medium, but it was unclear whether the recombinant HSV vector released into the medium actually existed at a sufficient concentration outside the cells.

[0331] First, we compared Lipofectamine 3000 and PEI Max, transfection reagents based on different principles, when transfecting 293 / HSV cells with the amplicon plasmid pA-ICP0-GFP and the helper plasmid pICP27. After 72 hours of transfection using each reagent, the culture supernatant was collected, and the remaining cells were washed twice with fresh DMEM containing 10% FBS. After removing the wash medium, 500 μL of fresh DMEM containing 10% FBS was added, and the cells were detached with a scraper and collected. This cell suspension was freeze-thawed three times and then centrifuged at 10,000 × g for 5 minutes at 4°C, after which the supernatant was collected. Centrifugation and supernatant collection were repeated twice. The obtained culture supernatant was filtered through Millex-GV (Millipore) to prepare a total volume of 500 μL, which was used as a vector stock (cells). Regarding the culture supernatant, 3 mL of Lenti-X™ Concentrator (Takara Bio Inc.) was added to 9 mL of the culture supernatant, and the mixture was incubated at 4°C for 1 hour. After centrifugation at 1500 × g for 45 minutes at 4°C using a swing-rotor low-speed centrifuge (TOMY), the pellet was recovered and suspended in 500 μL of DMEM containing 10% FBS to prepare a vector stock (supernatant). Each vector stock derived from the cells and supernatant was diluted 1x, 2x, 4x, and 8x using DMEM containing 10% FBS. On the day before the infection experiment, 2x10 ５200 μL of each of the dilution series of the prepared vector stock was added to Vero cells seeded in 24 wells at 1 cell / well. After 18 hours, the cells were observed under a fluorescence microscope. The results are shown in Figure 8.

[0332] The GFP-positive rate was also analyzed by flow cytometry. Quantitative analysis by flow cytometry was performed as follows. Cells were trypsinized using 0.05% trypsin solution (Thermo Fisher, #25300-054), detached from the culture plate, centrifuged at 600 × g for 5 minutes, and resuspended in 500 μL of PBS containing 10% FBS. Cells that retained EGFP fluorescence were directly analyzed using a cell sorter SH800S (Sony). The rate of EGFP-positive cells was calculated by two-dimensional plot analysis (data not shown) using the program provided with the instrument. The results are shown in Figure 9.

[0333] 8 shows GFP fluorescence microscopy images of Vero cells infected with recombinant HSV vectors using each of the prepared vector stocks (cells, supernatant) and their dilutions. Whether Lipofectamine 3000 or PEI Max was used to transfect the amplicon plasmid and helper plasmid, the cell-derived (top) and supernatant-derived (bottom) vector stocks and their dilutions (8-fold dilution) produced comparable levels of GFP fluorescence signals in Vero cells.

[0334] 9 is a graph showing the results of quantitative analysis by flow cytometry of GFP expression when Vero cells were infected with the prepared vector stocks. The graph shows the percentage (%) of GFP-positive cells, when the total number of cells is taken as 100%, when Vero cells were infected with the vector stocks prepared from cells transfected with Lipofectamine, the black diamonds, the white circles, the white diamonds ...

[0335] In addition, the titers of the cell-derived and supernatant-derived vector stocks (how many cells could be transfected with a foreign gene per mL of vector stock) were calculated using the following formula 1. Formula 1: Titer (Transfection Unit (TU) mL －１ ) = {number of cells (cells) × (GFP positivity rate (%) / 100) × dilution factor} / amount of vector stock added (mL)

[0336] Whether Lipofectamine 3000 or PEI was used, the cell-derived and supernatant-derived vector stocks were 10 ６ Transfection Unit (TU)・mL －１ The titer of the conventional production method was 10 ６ TU・mL －１ This demonstrates that the recombinant HSV vector produced in the present invention has sufficient infectivity for mammalian cells.

[0337] Example 6: Confirmation of stability and HSV vector production ability of 293 / HSV cells during long-term culture As described above, the cells for producing the recombinant HSV vector of the present invention are functionally deficient in the ICP27 gene, the ICP0 gene, and the ICP4 gene, and therefore can be stably cultured and grown. However, there are concerns about stability regarding the stability of foreign genes introduced into cells, such as the inability to express the gene required for recombinant HSV vector production due to reasons such as silencing by the host cell or loss of the target gene due to accidental recombination. Therefore, in this example, we investigated whether the recombinant HSV genome remained stable in cells that had been subcultured for a long period of time and whether the recombinant HSV vector could be produced to the same extent as immediately after introduction, as described below.

[0338] 293 / HSV cells (control) and 293 / HSV cells that had been subcultured for 2 months or more (>2-month culture) were each transfected with the amplicon plasmid pA-ICP0-GFP and the helper plasmid pICP27, and vector stocks were prepared in the same manner as in Example 5 above. Vero cells were infected with each of the prepared vector stocks, and 18 hours after infection, the number of GFP-positive cells and the GFP expression level were measured by flow cytometry. The GFP expression level in 293 / HSV cells immediately after induction of recombinant HSV vector production was set to 1. The results of evaluating the GFP expression level in cells cultured for 2 months or more are shown in Figure 10.

[0339] Figure 10 is a graph showing the results of flow cytometry analysis of GFP expression when Vero cells were infected with vector stocks prepared from each cell line. As shown in Figure 10, even when 293 / HSV cells cultured for more than two months were used, no decrease in GFP expression level was observed; rather, the GFP expression level (which can also be considered as the titer of the recombinant HSV) was improved by more than 30%. From the above, it was confirmed that the recombinant cells into which the recombinant HSV genome was introduced did not lose their ability to produce HSV vectors even after long-term subculture.

[0340] Example 7: Analysis of the effect of transfection reagents on 293 / HSV cells and the infectivity of the produced HSV vectors As described below, HSV viral vector stocks were prepared by a method different from that used in Example 5, and the infectivity of the HSV viral vector stocks was analyzed.

[0341] First, 3 mL of 0.1% type 1 collagen was added to a 10 cm dish, and after 10 minutes, the solution was aspirated. 3 mL of PBS was added and the dish was washed (collagen coating treatment). ７ Cells were seeded at 1000 x g onto collagen-coated 10-cm dishes. Three hours after seeding, 11.2 μg of the amplicon plasmid pA-ICP0-GFP and 2.8 μg of the helper plasmid pICP27 were transfected into 293 / HSV cells. Transfection was performed using Lipofectamine 2000, Lipofectamine 3000, and PEI Max, which are transfection reagents based on different principles. Transfection was performed using each reagent. Six and 48 hours after transfection, the supernatant was removed using an aspirator, and 10 mL of fresh DMEM containing 10% FBS was added. After 72 hours, the cells were detached with a scraper, and the cells and culture supernatant were collected in a centrifuge tube. After centrifugation at 10,000 x g for 5 minutes at 4°C, the supernatant was removed using an aspirator. 1 mL of fresh DMEM containing 1% FBS was added to the suspension, and the suspension was then frozen at -80°C.

[0342] The cell suspension was thawed, and the cells were disrupted using a handy ultrasonic disruptor UR-21P (TOMY) set to intensity 8. The cells were then centrifuged at 10,000 x g for 10 minutes at 4°C, and the supernatant was collected. Centrifugation and supernatant collection were repeated twice. The obtained culture supernatant was filtered using a Millex-GV (Millipore) and adjusted to a total volume of 1 mL, which was used as a vector stock (cells). 5 x 10 cells were collected the day before the infection experiment. ５400 μL of diluted vector stock was added to Vero cells seeded in a 12-well plate at cells / well. After 1 hour, the culture supernatant was removed using an aspirator, and 500 μL of fresh DMEM containing 10% FBS was added. The culture supernatant was removed using an aspirator, and 1 mL of fresh DMEM containing 10% FBS was added and cultured. After 24 hours, the GFP positivity rate was analyzed by flow cytometry. Quantitative analysis by flow cytometry was performed as follows: Cells were trypsinized using 0.05% trypsin solution (Thermo Fisher, #25300-054), detached from the culture plate, centrifuged at 200 × g for 5 minutes, resuspended in 500 μL of PBS containing 10% FBS, and counted. Cells expressing EGFP were then directly analyzed using a cell sorter SH800S (Sony Corporation). The percentage of EGFP-positive cells was calculated by two-dimensional plot analysis (data not shown) using the program provided with the device. The titer of the vector stock (how many cells were able to be transfected with a foreign gene per mL of vector stock) was calculated using the following formula 1. The results are shown in Figure 11. Formula 1: Titer (Transfection Unit (TU) mL －１ ) = {number of cells (cells) × (GFP positivity rate (%) / 100) × dilution factor} / amount of vector stock added (mL)

[0343] Whether Lipofectamine 3000, Lipofectamine 2000, or PEI was used, the vector stock was 10 ８ Transfection Unit (TU)・mL －１ These results demonstrate that even with an HSV viral vector stock prepared by a method different from that of Example 5, it is possible to prepare a high-titer vector stock by using HEK293 cells, which have good gene transfer efficiency, as HSV vector-producing cells, even with inexpensive PEI.

Claims

1. A mammalian cell for producing a recombinant herpes simplex virus (HSV) vector, the cell retaining a first base sequence, the first base sequence satisfying the following (1), (2), (3), (4), and (5): (1) comprising base sequences 2 to 27 that express proteins 1 to 26, (2) lacking base sequences encoding proteins 27 to 28, (3) lacking base sequences encoding protein 29, (4) lacking base sequences shown in SEQ ID NO:5 and SEQ ID NO:6, and (5) comprising a 28th base sequence consisting of the base sequence shown in SEQ ID NO:7 or a 29th base sequence consisting of the base sequence shown in SEQ ID NO:8, the first to 26th proteins having 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs:76 to 101, respectively, and having the same quality of function as proteins consisting of the amino acid sequences shown in SEQ ID NOs:76 to 101, The 27th to 29th proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively, and have the same function as a protein consisting of the amino acid sequences shown in SEQ ID NOs: 12 to 14, and the cell, when the first base sequence includes the 28th base sequence, retains a 30th base sequence that expresses a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and has the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence.

2. The cell according to claim 1, wherein the first base sequence further satisfies the following (6): (6) comprising at least one base sequence selected from all of the base sequences of Nos. 31 to 52 that express proteins Nos. 30 to 51, and at least one base sequence selected from the base sequences of Nos. 53 to 84 that express proteins Nos. 52 to 83; and wherein the proteins Nos. 30 to 83 have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 102 to 155, respectively, and have the same quality of function as proteins consisting of the amino acid sequences shown in SEQ ID NOs: 102 to 155.

3. The cell according to claim 2, which contains all of the base sequences 53 to 84.

4. A cell according to any one of claims 1 to 3, wherein the first base sequence has 95% or more sequence identity with the base sequence shown in SEQ ID NO:

1.

5. A cell described in any one of claims 1 to 3, wherein the first base sequence further comprises the 30th base sequence and has 95% or more sequence identity with the base sequence shown in SEQ ID NO:

11.

6. A cell described in any one of claims 1 to 3, wherein the first base sequence includes the 28th base sequence.

7. A cell according to any one of claims 1 to 3, wherein the 30th base sequence is a base sequence that expresses a protein consisting of the amino acid sequence shown in SEQ ID NO:

9.

8. A method for producing a recombinant HSV vector, comprising the steps of: introducing an amplicon plasmid into a mammalian cell; and expressing all of proteins 1 to 26, all of proteins 30 to 51, and at least one protein from proteins 52 to 83, wherein the cell retains a 85th base sequence, and the 85th base sequence satisfies the following (A), (B), (C), (D), and (E): (A) comprising at least one of a 2nd to 27th base sequence and a 31st to 84th base sequence that express proteins 1 to 26 and 30 to 83; (B) lacking a nucleotide sequence encoding proteins 27 to 28; (C) lacking a nucleotide sequence encoding protein 29; (D) lacking the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; and (E) The cell contains a 28th base sequence consisting of the base sequence shown in SEQ ID NO: 7 or a 29th base sequence consisting of the base sequence shown in SEQ ID NO: 8, wherein the first to 26th and 30th to 83rd proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 76 to 155, respectively, and have the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 155, and the 27th to 29th proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively, and have the same quality of function as the proteins consisting of the amino acid sequences shown in SEQ ID NOs: 12 to 14, and the amplicon plasmid contains a gene of interest, an 86th base sequence consisting of the base sequence shown in SEQ ID NO: 15, and an 87th base sequence consisting of the base sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, and when the 85th base sequence contains the 28th base sequence, A method for producing a recombinant HSV vector that expresses a target gene, the vector having a 30th base sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and that expresses a protein that has the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence.

9. The production method according to claim 8, wherein the step of expressing the proteins is a step of expressing proteins 1 to 26 and proteins 30 to 83.

10. The method of claim 8, wherein the cells are cells according to any one of claims 1 to 3.

11. The production method according to claim 8, wherein the step of expressing the protein includes a step of expressing at least one of the 27th to 29th proteins.

12. The manufacturing method described in claim 8, wherein the 87th base sequence is the base sequence shown in SEQ ID NO:

5. 13.A kit for producing a recombinant HSV vector, comprising a mammal-derived cell and one or more first plasmids, wherein the cell retains a 89th nucleotide sequence, and the 89th nucleotide sequence satisfies the following (i), (ii), (iii), (iv), and (v): (i) comprising at least one of a 2nd to 27th nucleotide sequence and a 31st to 52nd nucleotide sequence that express a 1st to 26th and a 30th to 51st protein, (ii) lacking a nucleotide sequence encoding a 27th to 28th protein, (iii) lacking a nucleotide sequence encoding a 29th protein, (iv) lacking the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6, and (v) comprising a 28th nucleotide sequence consisting of a nucleotide sequence shown in SEQ ID NO:7 or a 29th nucleotide sequence consisting of a nucleotide sequence shown in SEQ ID NO:8, at least one of the first plasmids comprises an 86th base sequence consisting of the base sequence shown in SEQ ID NO: 15, and an 87th base sequence consisting of the base sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6; the first plasmid comprises a base sequence not contained in the 89th base sequence among the 2nd to 27th base sequences and the 31st to 52nd base sequences; either or both of the 89th base sequence and the first plasmid comprise at least one of the 53rd to 84th base sequences that express proteins of the 52nd to 83rd base sequences; the 1st to 26th and 30th to 83rd proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 76 to 155, respectively, and are proteins having the same quality of function as proteins consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 155; The 27th to 29th proteins have 90% or more sequence identity with the amino acid sequences shown in SEQ ID NOs: 12 to 14, respectively, and have the same functions as proteins consisting of the amino acid sequences shown in SEQ ID NOs: 12 to 14, and the cells, when the 89th base sequence includes the 28th base sequence, retain a 30th base sequence that expresses a protein that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9 and has the ability to bind to chromatin and the ability to bind to the 28th or 29th base sequence.

14. The kit according to claim 13, wherein the 89th base sequence includes at least one of the 53rd to 84th base sequences, and the first plasmid includes a base sequence not included in the 89th base sequence among the 2nd to 27th base sequences and the 31st to 84th base sequences.

15. The kit according to claim 13, wherein the cells are cells according to any one of claims 1 to 3.

16. The kit according to claim 13, wherein the 87th base sequence is the base sequence shown in SEQ ID NO:

5.

17. The kit according to claim 13, wherein the first plasmid comprises a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:

26.

18. The kit according to claim 13, further comprising one or more second plasmids, said second plasmids comprising a base sequence for expressing at least one of said proteins 27 to 29.

19. The kit described in claim 18, wherein the second plasmid contains a base sequence that expresses a protein having 90% or more sequence identity with the base sequence shown in SEQ ID NO: 75 and having the same function as the protein consisting of the amino acid sequence shown in SEQ ID NO:

12.

20. The kit according to claim 18, wherein the second plasmid comprises a plasmid consisting of a base sequence having 90% or more sequence identity with the base sequence shown in SEQ ID NO:

70.

21. A mammalian cell for producing a recombinant herpes simplex virus (HSV) vector, the cell retaining the genomic DNA of the recombinant HSV, the genomic DNA of the recombinant HSV containing the base sequences of all essential genes of the recombinant HSV and base sequences for being retained in the cell, and lacking the base sequences of the immediate early genes and the base sequences for being packaged into the recombinant HSV.

22. The cell of claim 21, wherein the genomic DNA of the recombinant HSV further comprises at least one nucleotide sequence of a non-essential gene of the recombinant HSV.

23. A method for producing a recombinant HSV vector, comprising the steps of: introducing an amplicon plasmid into a mammalian cell; and expressing all of the proteins encoded by the essential genes of the recombinant HSV and at least one of the proteins encoded by the non-essential genes of the recombinant HSV, wherein the cell retains the genomic DNA of the recombinant HSV, and the genomic DNA of the recombinant HSV contains the base sequence of at least one of the essential genes and non-essential genes of the recombinant HSV and a base sequence for being retained in the cell, and is deficient in the base sequence of an immediate early gene and the base sequence for packaging into the recombinant HSV, and wherein the amplicon plasmid contains the target gene, the base sequence for packaging into the recombinant HSV, and the base sequence of a replication origin of the recombinant HSV.

24. The method of claim 23, wherein the cells are cells according to claim 21 or 22.

25. A kit for producing a recombinant HSV vector, comprising mammalian cells and one or more first plasmids, wherein the cells retain the genomic DNA of the recombinant HSV, the genomic DNA of the recombinant HSV contains the base sequence of at least one of an essential gene and a non-essential gene of the recombinant HSV, and a base sequence for being retained in the cell, and is deficient in the base sequence of an immediate early gene and a base sequence for packaging into the recombinant HSV, at least one of the first plasmids contains the base sequence for packaging into the recombinant HSV and the base sequence of a replication origin of the recombinant HSV, and the first plasmid contains a base sequence of the essential gene and the non-essential gene of the recombinant HSV that is not contained in the genomic DNA of the recombinant HSV.

26. The kit of claim 25, wherein the cells are cells of claim 21 or 22.