Producer cell, method for producing producer cell, and method for producing adeno-associated virus

Abstract

The present invention addresses the problem of providing: a producer cell for the production of AAV in which cell damage during the establishment of a cell strain is avoided or suppressed; a method for producing the producer cell; and a method for producing an adeno-associated virus using the producer cell. According to the present invention, there is provided a producer cell which does not contain at least one of a VA-RNA gene and an E4 gene and is a mammalian cell, said producer cell having a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter in a chromosome.

Description

Technical Field

[0001] The present invention relates to a producer cell for producing an adeno-associated virus, a method for producing the producer cell, and a method for producing an adeno-associated virus using the producer cell. Background Art

[0002] Adeno-associated virus (also known as AAV) is a linear single-stranded DNA virus belonging to the Parvoviridae family. The wild-type AAV genome contains a regulatory gene for replication (Rep gene) and a structural gene for the capsid (Cap gene), and is adjacent to an inverted terminal repeat sequence (ITR) for viral replication and packaging. AAV vectors are capable of gene introduction into any cell, whether proliferating or non-proliferating, and are particularly capable of long-term expression in non-dividing cells. In addition, AAV is considered to be non-pathogenic and has low immunogenicity. Based on the above, AAV vectors have made progress in clinical applications as vectors for gene therapy.

[0003] AAV is a non-enveloped virus that proliferates in the presence of helper viruses such as adenovirus and herpes virus. When preparing AAV for gene therapy or nucleic acid introduction, the typical approach is to co-infect adenovirus with host cells and replicate AAV. In addition, the gene responsible for the auxiliary effect of adenovirus is clearly identified, and a plasmid carrying the gene is also used. For example, by simultaneously transfecting HEK293 cells with a plasmid containing the Rep gene and the Cap gene, an adenovirus helper plasmid, and a plasmid containing the target gene (transgene), it can be packaged into a recombinant AAV (rAAV).

[0004] Patent document 1 describes the use of mammalian cells to produce AAV, wherein the mammalian cells contain a nucleic acid molecule encoding a viral helper gene under the control of a first derepressible promoter, a nucleic acid molecule encoding an AAV gene under the control of a second derepressible promoter, and a nucleic acid molecule encoding the repression elements of the first and second derepressible promoters.

[0005] Patent Document 2 describes a host cell comprising nucleic acids encoding adeno-associated virus REP proteins REP78 and REP proteins REP68, wherein the internal AAV promoter p19 is inactivated by one or more mutations that maintain the functionality of the REP78 and REP68 proteins.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. WO2020 / 132059

[0009] Patent Document 2: International Publication No. WO2019 / 57691 Summary of the invention

[0010] Technical issues to be solved by the invention

[0011] The method of Patent Document 1 has the following problem: since control sequences are added to the auxiliary gene and AAV gene required for AAV production, respectively, mutual interference among 3 to 5 control sequences may make it difficult to sufficiently control the expression level of each target gene.

[0012] In addition, as an example of a method for producing AAV, there is a method using producer cells. When establishing producer cells that produce AAV, there is a problem of cell damage. In Patent Document 2, as a method for avoiding cytotoxicity caused by REP protein, a method for inactivating the internal AAV promoter p19 is described, but there is no description about the production of producer cells.

[0013] The present invention aims to provide a producer cell for AAV production that avoids or suppresses cell damage during establishment of a cell line. The present invention also aims to provide a method for producing the producer cell and a method for producing an adeno-associated virus using the producer cell.

[0014] Means for solving technical problems

[0015] As a result of intensive studies to solve the above problems, the present inventors have found that by establishing producer cells using a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter instead of using at least one of the VA-RNA gene and the E4 gene, cell damage during establishment of producer cells can be avoided or reduced. The present invention has been completed based on the above findings.

[0016] According to the present invention, the following inventions are provided.

[0017] <1> A production cell, which does not contain at least one of a VA-RNA gene and an E4 gene and is a mammalian cell,

[0018] The production cell has in its chromosome a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter.

[0019] <2> according to <1> In the production cell, the exogenous promoter of the Rep gene and the exogenous promoter of the Cap gene are the same promoter.

[0020] <3> according to <1> In the production cell, the Rep gene exists downstream of the Cap gene in the transcription direction.

[0021] <4> according to <1> The production cell has an E4 gene under the control of an exogenous promoter in its chromosome and does not contain a VA-RNA gene.

[0022] <5> according to <4> In the production cell, the E4 gene is located downstream of IRES.

[0023] <6> according to <4> In the production cell, the exogenous promoter of the E4 gene is an inducible promoter that can be switched on and off.

[0024] <7> according to <6> In the production cell, the exogenous promoter of the E4 gene is a promoter that can be induced to express by a drug.

[0025] <8> according to <7> In the production cell, the exogenous promoter of the E4 gene is located downstream of the tetracycline response factor.

[0026] <9> according to <1> The production cell does not contain both the VA-RNA gene and the E4 gene.

[0027] <10> according to <1> to <9> The production cell according to any one of the above, further comprising an E2A gene.

[0028] <11> according to <10> In the production cell, the E2A gene is located downstream of IRES.

[0029] <12> according to <10> In the production cell, the E2A gene is under the control of an exogenous promoter.

[0030] <13> according to <12> In the production cell, the E2A gene is under the control of a promoter derived from cytomegalovirus.

[0031] <14> according to <10> In the production cell, the exogenous promoter of the E2A gene is an inducible promoter that can be switched on and off.

[0032] <15> according to <14> In the production cell, the exogenous promoter of the E2A gene is a promoter that can be induced to express by a drug.

[0033] <16> according to <15> In the production cell, the exogenous promoter of the E2A gene is located downstream of the tetracycline response factor.

[0034] <17> A production cell, which is a mammalian cell that does not contain a VA-RNA gene,

[0035] The production cell has in its chromosome a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, and an E4 gene under the control of an exogenous promoter.

[0036] <18> according to <1> to <17> The production cell described in any one of the above further comprises a target gene.

[0037] <19> according to <1> to <18> The producer cell described in any one of the above, wherein at least one of the exogenous promoter of the Cap gene and the exogenous promoter of the Rep gene is an inducible promoter that can be switched on and off.

[0038] <20> according to <19> In the production cell, at least one of the exogenous promoter of the Cap gene and the exogenous promoter of the Rep gene is a promoter whose expression can be induced by a drug.

[0039] <21> according to <20> The production cell comprises: at least one of the exogenous promoter of the Cap gene and the exogenous promoter of the Rep gene is located downstream of the tetracycline response factor.

[0040] <22> according to <1> to <21> The production cell described in any one of the above further comprises a SmallRep gene under the control of an exogenous promoter.

[0041] <23> according to <22> In the production cell, the Rep gene exists downstream of the transcription direction of the SmallRep gene.

[0042] <24> according to <22> In the production cell, the exogenous promoter of the SmallRep gene is an inducible promoter that can be switched on and off.

[0043] <25> according to <24> In the production cell, the exogenous promoter of the SmallRep gene is a promoter capable of inducing expression by a drug.

[0044] <26> according to <25> In the production cell, the exogenous promoter of the SmallRep gene is located downstream of the tetracycline response factor.

[0045] <27> according to <1> to <26> The production cell described in any one of the above further comprises a gene expressing a reverse tetracycline-regulated transcriptional activator.

[0046] <28> according to <27> The production cell comprises: a gene expressing a reverse tetracycline-regulated transcription activator is located downstream of the Rep gene.

[0047] <29> according to <1> The production cell comprises an E4 gene under the control of an exogenous promoter, an E2A gene under the control of an exogenous promoter, and a SmallRep gene under the control of an exogenous promoter,

[0048] The exogenous promoter of the Cap gene and the exogenous promoter of the E2 gene are the same promoter, and the exogenous promoter of the Rep base and the exogenous promoter of the SmallRep gene are the same promoter.

[0049] <30> A production cell, which is a mammalian cell that does not contain a VA-RNA gene,

[0050] The production cell has a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, an E4 gene under the control of an exogenous promoter, and an E2A gene under the control of an exogenous promoter in the chromosome, and the Cap gene, the Rep gene, and the E2 gene are present nearby, while the E4 gene is not present nearby.

[0051] <31> A production cell, which is a mammalian cell that does not contain a VA-RNA gene,

[0052] The producer cell has in its chromosome:

[0053] A first vector carrying a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, and an E2A gene under the control of an exogenous promoter; and

[0054] The second vector carries the E4 gene under the control of an exogenous promoter.

[0055] <32> according to <31> or <32> In the production cell, the number of E4 gene insertions on the chromosome is less than the number of other gene insertions.

[0056] <33> according to <31> or <32> In the production cell, the number of E4 gene insertions on the chromosome is less than the number of Cap gene or E2 gene insertions on the chromosome.

[0057] <34> according to <31> or <32> In the production cell, the number of E4 gene insertions on the chromosome is less than 0.4 times the number of Cap gene insertions on the chromosome.

[0058] <35> according to <31> or <32> In the production cell, the number of E4 gene insertions on the chromosome is less than 0.4 times the number of E2 gene insertions on the chromosome.

[0059] <36> A sort of <1> to <35> The method for producing a producer cell according to any one of the above aspects comprises: introducing a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter into a mammalian cell.

[0060] <37> according to <36> The method for producing the production cell further comprises introducing a reverse tetracycline-regulated transcription activator.

[0061] <38> A method for producing an adeno-associated virus, comprising: culturing <1> to <35> The adeno-associated virus is produced by using the producer cell described in any one of the above.

[0062] <39> A method for producing an adeno-associated virus comprises the following steps:

[0063] Cultured under conditions that do not induce the expression of Cap and Rep genes <1> to <35> Proliferating the producer cell described in any one of the preceding claims; and

[0064] Adeno-associated virus is produced by culturing the proliferated producer cells under conditions that induce the expression of the Cap gene and the Rep gene.

[0065] <40> A method for producing an adeno-associated virus comprises the following steps:

[0066] Cultured under conditions that do not induce the expression of Cap, Rep, and E4 genes <4> to <8> Proliferating the producer cell described in any one of the preceding claims; and

[0067] Adeno-associated virus is produced by culturing the proliferated producer cells under conditions that induce expression of the Cap gene, Rep gene, and E4 gene.

[0068] An adeno-associated virus that <38> to <40> The adeno-associated virus may be produced by the method for producing the adeno-associated virus described in any one of the preceding claims.

[0069] A vector kit for manufacturing production cells, comprising: a first vector carrying a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, and an E2A gene under the control of an exogenous promoter; and a second vector carrying an E4 gene under the control of an exogenous promoter.

[0070] Effects of the Invention

[0071] According to the present invention, cell damage during establishment of production cells can be avoided or reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The results are shown in which the Rep gene and the Cap gene were introduced into cells and the expression of REP and CAP was evaluated.

[0073] Figure 2 The results show the results of evaluating the AAV production ability of cells after the Rep gene and the Cap gene under the control of an exogenous promoter were introduced into cells.

[0074] Figure 3 The figures show the results of evaluating the expression of REP and CAP after the Rep gene and Cap gene were introduced into cells and then doxycycline was added to induce their expression.

[0075] Figure 4 The graph shows the results of immunoblotting using anti-E2 antibody and GFP observation for cells into which the E2 gene and GFP (green fluorescent protein) gene were introduced.

[0076] Figure 5 The figures show the results of evaluating the AAV production ability of cells obtained by introducing the Rep gene and the Cap gene into cells having the E2 gene and the GFP gene.

[0077] Figure 6 This section shows an overview of Experiment 6.

[0078] Figure 7 The figures show the quantitative results of AAV for the producer cell line selected using the neomycin resistance gene as a selection marker.

[0079] Figure 8 Shown are the quantitative results of AAV for producer cell lines selected using RFP (red fluorescent protein) as a selection marker.

[0080] Fig. 9 Microscopic images of the production cell lines are shown.

[0081] Fig.10 Schematic diagram showing the genetic design of Experiment 7. GOI represents the GFP gene.

[0082] Fig.11 The outlines of Experiments 7 and 8 are shown.

[0083] Fig.12 The graph shows the quantitative results of AAV of the highest titer strain of each design in HEK293 cells containing the sequences of designs 1 to 6 in their chromosomes.

[0084] Fig.13 The graph shows the quantitative results of the number of each gene of Rep, Cap, E2, and E4 on the chromosome of multiple HEK293 clones established using the vector set of Design 4 in Experiment 7.

[0085] Fig.14 The graph shows the quantitative results of the number of each gene of Rep, Cap, E2, and E4 on the chromosome of multiple HEK293 clones established using the vector of Design 5 in Experiment 7.

[0086] Fig.15 The graph shows the quantitative results of AAV for HEK293 cell lines adapted to suspension culture and containing the sequence of Design 4 in the chromosome and HEK293 cell lines containing the sequence of Design 5 in the chromosome. DETAILED DESCRIPTION

[0087] An example of an embodiment of the present invention is described below. However, the present invention is not limited by the following embodiments and can be implemented by appropriately changing within the scope of the purpose of the present invention. In this specification, the numerical range represented by "~" represents a range that includes the numerical values ​​recorded before and after "~" as the minimum value and the maximum value, respectively.

[0088] <Explanation of terms>

[0089] A promoter refers to a DNA control region / sequence that represents a sequence that drives gene expression and is capable of binding to RNA polymerase and participating in the initiation of transcription of downstream coding or non-coding sequences.

[0090] An exogenous promoter is a promoter that does not contain a wild-type AAV gene, and specifically, a promoter that is not known to express an AAV gene under natural conditions.

[0091] Screening marker refers to, is used to screen the gene of the cell that actively expresses nucleic acid sequence.As preferred screening marker, can enumerate antibiotics, for example the gene that the resistance of kanamycin, neomycin, puromycin, hygromycin, blasticidin or leucomycin is encoded.Another example of preferred screening marker is fluorescent protein, for example is green fluorescent protein (GFP), red fluorescent protein (RFP) or blue fluorescent protein (BFP).

[0092] Vector refers to a DNA or RNA molecule used to artificially transfer an exogenous gene to another cell. The vector used to express a gene is called an expression vector. If the vector containing the imported exogenous gene is imported into the cell, the exogenous gene is replicated and / or expressed in the cell. As a vector, additional (e.g., plasmid) vectors and non-additive vectors can be enumerated. The vector can be imported into a host cell by methods such as transfection, transduction, cell fusion, lipofection, electroporation, etc.

[0093] Carrying means that other nucleic acid sequences are integrated into the chromosome or nucleic acid sequence.

[0094] The gene is not limited as long as it is a base sequence, but preferably a gene having a function in vivo. For example, it may be a nucleotide encoding a polypeptide, and examples thereof include nucleic acid molecules such as cDNA and genomic DNA.

[0095] Producer cells refer to cells that can produce rAAV without the need for additional auxiliary functions such as the introduction of auxiliary genes or viral infection. In the present specification, it is a cell in which the Rep gene and the Cap gene are integrated into the chromosome. In addition, the target gene (for example, the desired gene for treatment or prevention) and the necessary adenovirus auxiliary genes can also be integrated into the chromosome. The target gene can contain ITR. In addition, it can also be a cell in which a gene containing the target gene is integrated adjacent to two AAV inverted terminal repeats (ITR: Invertedteraminal repeat). Genes that are not integrated into chromosomes can be carried in a vector and introduced into producer cells to produce rAAV.

[0096] Furthermore, the production cell may be a cell in which a gene for expressing a factor acting on the activity of the promoter is integrated into the chromosome, or a cell in which all genes of the factor acting on the activity of the promoter are integrated into the chromosome. The factor acting on the activity of the promoter is preferably a factor activating the promoter, and more preferably a reverse tetracycline-regulated transcriptional activator.

[0097] <Production Cells>

[0098] The production cell of the present invention does not contain at least one of the VA-RNA gene and the E4 gene and is a mammalian cell, and has a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter in a chromosome.

[0099] In Experiment 1 of the Examples of the present invention, it was confirmed that the expression levels of REP and CAP were significantly reduced when the E4 gene or VA-RNA gene was not used.

[0100] In Experiment 2, the mandatory (constant) CMV promoter was directly linked to the Rep gene and the Cap gene to express them. As a result, AAV can be produced even without using the E4 gene and the VA-RNA gene. However, since REP has cytotoxicity, direct integration of the Rep gene will not result in a stable strain.

[0101] In Experiment 3, a drug-inducible promoter (TRE-CMV-minimal promoter) was linked to the Rep gene and the Cap gene. As a result, REP and CAP could be expressed even without using the E4 gene and the VA-RNA gene.

[0102] In Experiment 4, a cell line stably harboring the E2 gene and the GFP gene (target gene) was established.

[0103] In Experiment 5, the Rep gene and the Cap gene were introduced into the cell line established in Experiment 4. It was confirmed that the obtained cell line was capable of producing AAV.

[0104] In Experiment 6, the switched Rep and Cap genes were integrated into cells. Cells were screened using drugs or fluorescence as indicators to establish cell lines that stably possessed the Rep and Cap genes. It was confirmed that the obtained cell lines were capable of producing AAV.

[0105] In Experiment 7, the Rep gene, Cap gene, E2 gene, and, if necessary, E4 gene were integrated into cells. Cells were screened using drugs or fluorescence as indicators to establish a cell line that stably possessed the Rep gene and the Cap gene. It was confirmed that the obtained cell line was capable of producing AAV.

[0106] In Experiment 8, the producer cells established in Experiment 7 were suspended and cultured. It was confirmed that the suspended cells were able to produce AAV.

[0107] The production cell of the present invention has a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter in a chromosome.

[0108] As the Rep gene and the Cap gene, the Rep gene and the Cap gene of adeno-associated virus genes can be used.

[0109] Adeno-associated virus (AAV) refers to a small, incomplete, non-enveloped virus containing single-stranded DNA in the family of Parvoviridae and Dependent Parvovirus. It is known that there are more than 100 serotypes in AAV, and the host range or the characteristics of the virus are different depending on the serotype. It is known that serotype 2 (AAV2) is one of the serotypes that has been widely studied for a long time, and the host range is very wide. Serotype 1 (AAV1), serotype 5 (AAV5), and serotype 6 (AAV6) are serotypes with higher tissue orientation. It can be said that AAV1 has a high efficiency of gene introduction into muscle, liver, respiratory tract, central nervous system, etc., AAV5 has a high efficiency of gene introduction into the central nervous system, liver, retina, etc., and AAV6 has a high efficiency of gene introduction into the heart, muscle, liver, etc. The present invention preferably uses serotype 2 or serotype 5. Serotype 5 is particularly preferred.

[0110] Adeno-associated virus genes refer to genes composed of one or more nucleic acid sequences derived from one or more serotypes associated with adenovirus. Adeno-associated virus genes are preferably genes involved in the replication and packaging of AAV and genes encoding AAV constituent proteins.

[0111] The Rep gene and the Cap gene encode proteins involved in the replication and packaging of virions. The Cap region expresses VP1, VP2, and VP3.

[0112] The Rep gene and the Cap gene may be wild-type genes, but any gene modified by substitution, deletion, insertion, or addition of bases to the wild-type gene may be used as long as the gene exerts its original function.

[0113] When the wild-type Rep gene and Cap gene are modified by substitution, deletion, insertion or addition of bases, the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The base sequences of the modified Rep gene and Cap gene preferably have a sequence identity of 85% or more, more preferably a sequence identity of 90% or more, further preferably a sequence identity of 95% or more, and even more preferably a sequence identity of 98% or more with the base sequences of the wild-type Rep gene and Cap gene.

[0114] When the Rep gene and the Cap gene are introduced into the cell, the vector containing the Rep gene and the Cap gene can be introduced into the cell. The configuration of the Rep gene and the Cap gene in the vector is not particularly limited, and the Cap gene and the Rep gene can be configured in the same direction or in different directions. Preferably, the Cap gene and the Rep gene are configured in different directions on the vector. The direction of a certain gene refers to the direction from the 5' side to the 3' side, and being configured in different directions means that the direction from the 5' side to the 3' side of a certain gene and another gene is configured in opposite directions on the vector. In addition, the Rep gene and the Cap gene can also be configured on another vector.

[0115] The Rep gene refers to a region of the AAV genome that encodes a viral replication protein that is required for the replication of the viral genome known to those skilled in the art, or a functional homolog thereof, such as the human herpes virus 6 (HHV-6) Rep gene, etc. (known to mediate AAV-2 DNA replication). Therefore, the coding region of the Rep gene at least includes genes encoding REP78 and REP68 (long forms of REP proteins), and REP52 and REP40 (short forms of REP proteins; also referred to as "SmallRep") of AAV, or their functional homologs. The SmallRep gene, for example, includes at least REP52 or REP40, and preferably includes both. For Rep gene and SmallRep gene, please refer to Maurer AC, Weitzman MD. Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction. Hum Gene Ther. 2020 May; 31(9-10): 499-511. doi: 10.1089 / hum.2020.069. PMID: 32303138; PMCID: PMC7232694; and Keiya Ozawa. Gene therapy using AAV. Virus Vol. 57, No. 1, pp. 47-56, 2007:, and they are incorporated into this manual.

[0116] REP68 or REP78 is required for the production of AAV, and REP68 and REP78 are translation products of two mRNAs transcribed from the same gene by alternative splicing. In the present invention, REP contains at least REP68 or REP78 protein, and preferably may also contain REP52 and / or REP40. The coding region of the Rep gene used in the present invention may be derived from any AAV serotype, but is preferably derived from AAV2. Examples of viruses derived from AAV2 include REP78 and REP68, as well as REP52 and REP40, and ITR.

[0117] Cap gene refers to the region of the AAV genome that encodes viral capsid proteins known to those skilled in the art. Examples of these capsid proteins are AAV capsid proteins VP1, VP2 and VP3. The Cap gene used in the present invention can be derived from any AAV serotype, but is preferably derived from AAV2 or AAV5. AAV5 is particularly preferred.

[0118] Preferably, the exogenous promoter of the Cap gene is an inducible promoter that can be switched on and off.

[0119] Preferably, the exogenous promoter of the Rep gene is an inducible promoter that can be switched on and off.

[0120] As an inducible promoter that can be switched on and off, a promoter that can regulate the on and off of expression according to the presence or absence of stimulation can be used. As stimulation, chemical stimulation (endogenous hormone / stress response, lactose, tetracycline or its derivatives (e.g., doxycycline), cuminic acid, protein (rapamycin, FKCsA, abscisic acid (ABA) etc.), tamoxifen / Cre-loxP (using a system of a Cre promoter modified in a manner that can be induced to activate by tamoxifen), riboswitch), physical stimulation (blue light, heat) etc. can be cited, but are not particularly limited.

[0121] The inducible promoter that can be switched on and off is preferably a promoter whose expression can be induced by a drug. The drug is preferably tetracycline or a derivative thereof (eg, doxycycline).

[0122] As the inducible promoter that can be switched on and off, tetracycline responsive promoter, RU486 inducible promoter, ecdysone inducible promoter, rapamycin inducible promoter and gold thioprotein promoter etc. can be enumerated. As a specific tetracycline responsive promoter, the TRE-CMV-minimal promoter consisting of a part of tetracycline response factor and CMV promoter can be enumerated. The sequence is a promoter formed by a part of tetracycline response factor and CMV promoter connected. The promoter is used for Tet open / close system (manufactured by TET systems company).

[0123] An insulator is a sequence for stably controlling the transcription of a gene without being affected by surrounding sequences. The insulator is preferably located between REP and CAP. Furthermore, the insulator is not particularly limited, but is preferably 50 bp or more and 10,000 bp or less, more preferably 100 bp or more and 5,000 bp or less, and particularly preferably 300 bp or more and 4,000 bp or less. bp (base pair) indicates a base sequence or a base sequence number.

[0124] IRES (internal ribosome entry site) is a sequence that initiates translation of a gene's transcription product, and by linking a gene to another gene using IRES, each gene can be translated from the same transcription product. IRES can be encoded between the Cap gene and the E2 gene.

[0125] The inducible promoter that can be switched on and off may be a promoter whose expression can be regulated by tetracycline, that is, a Tet on / off system, or may be a Tet on system.

[0126] In the Tet open system, the promoter additionally contains at least one tetracycline response factor (Tet operator). By using a tetracycline response factor (Tet operator: tetracycline controls transcriptional activation), it is possible to reversibly switch the on or off of transcription in the presence of tetracycline or one of its derivatives (e.g., doxycycline) as an antibiotic. The Tet repressor protein present in the cell blocks expression by bonding to the Tet operator (tetracycline operator) sequence introduced into the promoter. Therefore, in the case where Tet repression is bonded to the Tet operator sequence, no gene expression is observed. If tetracycline or doxycycline is added, Tet repression is isolated to allow promoter activity and turn on gene expression. The Tet operator system is widely available, such as the Tet operator used in the pcDNA (trademark) 4 / TO mammalian expression vector that can be obtained from Invitrogen.

[0127] The promoter is preferably located downstream of the tetracycline response factor.

[0128] Preferably, the production cell of the present invention may contain a factor that acts on the activity of the promoter, preferably a factor that activates the promoter, and more preferably contains a gene that expresses a reverse tetracycline-regulated transcriptional activator (rtTA). Preferably, the gene that expresses the reverse tetracycline-regulated transcriptional activator is located downstream of the Rep gene.

[0129] The Rep gene and the Cap gene may be driven by the same exogenous promoter, or the Rep gene and the Cap gene may be driven by two exogenous promoters driving the respective genes.

[0130] When the Rep gene and the Cap gene are driven by two exogenous promoters driving the respective genes, the exogenous promoter of the Rep gene and the exogenous promoter of the Cap gene may be promoters of the same sequence or promoters of different sequences, but preferably promoters of the same sequence.

[0131] When the Cap gene and the Rep gene are contained in the same vector, the Rep gene may be present downstream of the transcription direction of the Cap gene, or the Rep gene may be present upstream of the transcription direction of the Cap gene, but preferably the Rep gene is present downstream of the transcription direction of the Cap gene.

[0132] The producer cell of the present invention preferably further comprises a SmallRep gene under the control of an exogenous promoter. Examples of the SmallRep gene include REP52 and REP40.

[0133] In case the production cell of the present invention comprises a SmallRep gene under the control of an exogenous promoter, it is preferred that the SmallRep gene is located upstream of the Rep gene.

[0134] The SmallRep gene is preferably an inducible promoter that can be switched on and off. The details of the inducible promoter that can be switched on and off are as described above in the present specification.

[0135] The exogenous promoter of the SmallRep gene is preferably a promoter whose expression can be induced by a drug. The details of the promoter whose expression can be induced by a drug are as described above in the present specification.

[0136] The exogenous promoter of the SmallRep gene is preferably located downstream of the tetracycline response factor.

[0137] The vector containing the Rep gene and the Cap gene is not particularly limited as long as it is a nucleic acid, and may be circular or linear. For example, an artificially designed nucleic acid can be used, and a plasmid is preferred.

[0138] In the manufacture of adeno-associated viruses, viral helper genes are usually used. Viral helper genes refer to non-adeno-associated viral genes that are used to enable replication and packaging of adeno-associated viruses. As viral helper genes, genes derived from other types of viruses other than adeno-associated viruses can be used. As specific examples of viral helper genes, viral helper genes derived from adenovirus or herpes virus can be cited, and preferably the viral helper gene is derived from adenovirus.

[0139] Adenovirus refers to a non-enveloped virus of the Adenoviridae family with an icosahedral nucleocapsid containing double-stranded DNA. More than 50 adenovirus subtypes have been isolated from humans, and many other subtypes have been isolated from other mammals and birds. These subtypes belong to the Adenoviridae family and are classified into two genera (i.e., mammalian adenovirus and avian adenovirus). These adenoviruses are morphologically and structurally similar. However, in humans, adenoviruses display different immunological properties, which are classified into serotypes. The focus of research is on two human serotypes of adenovirus (i.e., Ad2 and Ad5).

[0140] As viral helper genes derived from adenovirus, E1A, E1B, E2 (preferably E2A), E4 and VA-RNA can be cited. In a host cell having all or part of the E1 region, the genome of AAV will be replicated and packaged into the capsid, and the region of the adenovirus genome required for forming viral virions is set to the E2A region, the E4 region, and the VA1 RNA region. The E2 gene includes the E2A region and the E2B region, but as the E2 gene in the present invention, only the E2A region can be used. In addition, the E4 gene contains multiple ORFs. Regarding the function based on the E4 region, the E4 34kDa protein encoded by the open reading frame 6 (E4ORF6) of the E4 region is required for the replication of AAV. Therefore, as the E4 gene in the present invention, only ORF6 can be used.

[0141] The producer cell of the present invention does not contain at least one of the VA-RNA gene and the E4 gene. That is, the producer cell of the present invention includes a case where the VA-RNA gene is not contained but the E4 gene is contained, a case where the VA-RNA gene is contained but the E4 gene is not contained, and a case where the VA-RNA gene and the E4 gene are not contained.

[0142] Preferably, the production cell of the present invention has an E4 gene under the control of an exogenous promoter in the chromosome and does not contain a VA-RNA gene. In this case, the production cell is a mammalian cell that does not contain a VA-RNA gene and has a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, and an E4 gene under the control of an exogenous promoter in the chromosome.

[0143] In case the production cell of the present invention comprises the E4 gene, it is preferred that the E4 gene is located downstream of the IRES.

[0144] The exogenous promoter of the E4 gene is preferably an inducible promoter that can be switched on and off. The details of the inducible promoter that can be switched on and off are as described above in the present specification.

[0145] The exogenous promoter of the E4 gene is preferably a promoter whose expression can be induced by a drug. The details of the promoter whose expression can be induced by a drug are as described above in the present specification.

[0146] The exogenous promoter of the E4 gene is preferably located downstream of the tetracycline response factor.

[0147] In another embodiment, the production cell of the present invention does not contain both the VA-RNA gene and the E4 gene. "Not containing a certain gene" means not only not containing it in the chromosome, but also not containing it in the cell.

[0148] The production cell of the present invention may preferably further contain an E2 gene (preferably an E2A gene).

[0149] The E2A gene may be a wild-type gene, but any gene modified by substitution, deletion, insertion, or addition of bases to the wild-type gene may be used as long as the gene exerts its original function.

[0150] When the production cell of the present invention contains the E2 (preferably E2A) gene, the E2 gene is preferably located downstream of the IRES. Alternatively, the E2 gene may be introduced as a second vector different from the vector containing the Rep gene and the Cap gene.

[0151] When the wild-type E2A gene is modified by substitution, deletion, insertion or addition of bases, the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The base sequence of the modified E2A gene preferably has a sequence identity of 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more to the base sequence of the wild-type E2A gene.

[0152] The E2 (preferably E2A) gene may be under the control of a promoter, preferably an exogenous promoter.

[0153] The exogenous promoter of the E2 (preferably E2A) gene is preferably an inducible promoter that can be switched on and off. The details of the inducible promoter that can be switched on and off are as described above in the present specification.

[0154] The exogenous promoter of the E2 (preferably E2A) gene is preferably a promoter whose expression can be induced by a drug. The details of the promoter whose expression can be induced by a drug are as described above in the present specification.

[0155] The E2 (preferably E2A) gene can control the opening / closing of the gene under the control of a tetracycline responsive promoter (TET). The promoter of the E2 (preferably E2A) gene is not particularly limited, but can include a promoter (CMV promoter) derived from cytomegalovirus (including enhancers as required), SV40 early promoter, human elongation factor-1α (EF-1α) promoter, human ubiquitin C promoter, retroviral Rous sarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, and tetracycline responsive promoter, etc. Among the above, a tetracycline responsive promoter and a promoter derived from cytomegalovirus are preferred. As a tetracycline responsive promoter, TRE-CMV-minimum promoter can be cited. Preferably, the exogenous promoter of the E2 (preferably E2A) gene is located downstream of the tetracycline response factor.

[0156] In a preferred embodiment of the present invention, the production cell includes an E4 gene under the control of an exogenous promoter, an E2 (preferably E2A) gene under the control of an exogenous promoter, and a SmallRep gene under the control of an exogenous promoter, the exogenous promoter of the Cap gene and the exogenous promoter of the E2 gene are the same promoter, and the exogenous promoter of the Rep gene and the exogenous promoter of the SmallRep gene are the same promoter.

[0157] When the E2 (preferably E2A) gene is to be introduced into cells, a vector containing the E2 (preferably E2A) gene can be introduced into the cells.

[0158] The vector containing the E2 (preferably E2A) gene is not particularly limited as long as it is a nucleic acid, and may be circular or linear. For example, a plasmid, an artificially designed chromosome, etc. can be used, and a plasmid is preferred.

[0159] The production cell of the present invention preferably further comprises a gene of interest.

[0160] As the target gene, for example, a gene for treatment or prevention can be used.

[0161] As treatment or prevention gene, it is possible to use incomplete or missing genes in the genome of target cells or genes encoding non-natural proteins with desired biological or therapeutic effects (such as antiviral function), but it is not particularly limited. As a specific example of treatment or prevention gene, various blood diseases such as inflammatory diseases, autoimmune, chronic and infectious diseases (including diseases such as AIDS, cancer, nervous system diseases, cardiovascular diseases, hypercholesterolemia), anemia and hemophilia, gene defects (such as cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, emphysema, etc.) can be enumerated. In the present embodiment, GFP or RFP is used to replace the target gene.

[0162] The therapeutic or preventive gene may be some antisense oligonucleotides useful in antisense therapy for cancer and viral diseases (for example, short-chain oligonucleotides complementary to the sequence around the translation start site (AUG codon) of mRNA).

[0163] The therapeutic or preventive gene can be connected to a promoter for expressing the therapeutic or preventive gene. The promoter for expressing the therapeutic or preventive gene is not particularly limited, but can include a promoter derived from cytomegalovirus (including a promoter as required), SV40 early promoter, human elongation factor-1α (EF-1α) promoter, human ubiquitin C promoter, Rous sarcoma virus LTR promoter of retrovirus, dihydrofolate reductase promoter, β-actin promoter and phosphoglycerate kinase (PGK) promoter, etc. The therapeutic or preventive gene and the promoter for expressing the gene are preferably sandwiched between the ITR sequences.

[0164] The inverse terminal sequence (ITR) is a base sequence used to integrate the gene of the adeno-associated virus into the target cell. There are usually two inverse terminal sequences at the position of the target gene. The inverse terminal sequence is not limited to this, and is preferably a sequence derived from AAV serotype 2.

[0165] The production cells of the present invention are preferably eukaryotic cells, such as mammalian cells or insect cells. The cells are more preferably mammalian cells. As mammalian cells, for example, human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. can be cited, but are not particularly limited. Human cells can be preferably used. As examples of cells, HEK293 (derived from human fetal kidney) cells, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS (such as COS1 and COS7), QC1-3, VERO, PER.C6, HeLa, EB1, EB2, EB3, hybridoma cell lines can be cited. Preferably, the cell is a HEK293 cell.

[0166] The production cell of the present invention has a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter in the chromosome. Preferably, the production cell of the present invention has a vector containing a Cap gene under the control of an exogenous promoter and a vector containing a Rep gene under the control of an exogenous promoter in the chromosome. Having in the chromosome means that the full length or a part of the vector is integrated into the chromosome. By integrating the vector into the chromosome, the above-mentioned genes are constantly maintained in the cell, so that adeno-associated virus can be produced at any time.

[0167] Preferred embodiments of the producer cell of the present invention include:

[0168] A production cell, which is a mammalian cell that does not contain a VA-RNA gene, wherein the production cell has a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, an E4 gene under the control of an exogenous promoter, and an E2A gene under the control of an exogenous promoter in a chromosome, and the Cap gene, the Rep gene, and the E2 gene are present in the vicinity (e.g., at the same locus), while the E4 gene is not present in the vicinity (e.g., at the same locus); and

[0169] A production cell, which is a mammalian cell that does not contain a VA-RNA gene, and has in its chromosome: a first vector carrying a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, and an E2A gene under the control of an exogenous promoter; and a second vector carrying an E4 gene under the control of an exogenous promoter.

[0170] In the production cell of the present invention, the number of E4 gene insertions on the chromosome is preferably smaller than the number of other gene insertions. This is because the toxicity of E4 causes a decrease in cell survival, and the fewer the number of E4 gene insertions, the easier it is for the cell to survive.

[0171] In the producer cell of the present invention, the number of E4 gene insertions on the chromosome is preferably less than the number of Cap gene or E2 gene insertions on the chromosome. More preferably, the number of E4 gene insertions on the chromosome is more preferably 0.8 times or less, further preferably 0.6 times or less, further preferably 0.4 times or less, further preferably 0.3 times or less, and particularly preferably 0.2 times or less of the number of Cap gene or E2 gene insertions on the chromosome.

[0172] The lower limit of the number of E4 gene insertions on the chromosome is preferably 0.01 times or more, more preferably 0.02 times or more, and even more preferably 0.03 times or more of the number of Cap gene or E2 gene insertions on the chromosome.

[0173] <Method for producing producer cells>

[0174] The production cell of the present invention can be produced by introducing a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter into a mammalian cell. That is, the production method of the production cell based on the present invention is a method comprising introducing a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter into a mammalian cell.

[0175] Preferred embodiments of the combination of vectors used in producing the producer cell of the present invention include the following.

[0176] (1) a vector comprising the Rep gene and the Cap gene, a vector comprising the E2 (preferably E2A) gene, and a vector comprising a desired therapeutic or preventive gene;

[0177] (2) a vector comprising the Rep gene, the Cap gene, the E2 (preferably E2A) gene, and a vector comprising a desired therapeutic or preventive gene; and

[0178] (3) A vector comprising the Rep gene, the Cap gene, the E2 (preferably E2A) gene and a desired therapeutic or preventive gene.

[0179] The vector containing the Cap gene under the control of an exogenous promoter and the vector containing the Rep gene under the control of an exogenous promoter may be introduced so as to be expressed, but in the present invention, it is preferred that the vector is permanently expressed by integrating the vector into the chromosome of the cell. Permanent expression means that when the cell divides, the vector containing the Cap gene under the control of an exogenous promoter and the Rep gene under the control of an exogenous promoter are also replicated, so that the cell after division also has the vector.

[0180] A vector containing a Cap gene under the control of an exogenous promoter and a vector containing a Rep gene under the control of an exogenous promoter can be integrated into chromosomes by transfection or transduction. Furthermore, by using them together with the transposon method, they can be integrated into chromosomes efficiently.

[0181] Transfection refers to the process of passing through the membrane of eukaryotic cells and introducing nucleic acids into cells by chemical means (e.g., calcium phosphate-mediated precipitation or liposome transfection), mechanical means (e.g., electroporation), or physical means (e.g., bioballistic delivery). Lipofection refers to the process of introducing nucleic acids into cells by endocytosis or membrane fusion through the formation of a complex of a carrier and positively charged lipids, etc., through electrical interaction.

[0182] Transduction refers to the process by which a virus-derived vector passes through the membrane of a eukaryotic cell and introduces a nucleic acid into the cell.

[0183] The method for producing a producer cell of the present invention may include introducing a gene expression regulatory factor, and may also include introducing a reverse tetracycline-regulated transcription activator.

[0184] The introduction of the reverse tetracycline-regulated transcriptional activator can be performed by introducing a vector containing a gene encoding the reverse tetracycline-regulated transcriptional activator into cells by a method such as transfection, transduction, or lipofection.

[0185] <Method for producing adeno-associated virus>

[0186] In the present invention, adeno-associated virus can be produced by culturing the producer cell of the present invention. That is, the method for producing adeno-associated virus of the present invention is a method comprising producing adeno-associated virus by culturing the producer cell of the present invention.

[0187] Preferably, the following steps can be performed: the producer cells of the present invention are cultured under conditions that do not induce the expression of the Cap gene and the Rep gene to proliferate the producer cells, and then the proliferated producer cells are cultured under conditions that induce the expression of the Cap gene and the Rep gene to produce the adeno-associated virus.

[0188] It is further preferred that the following steps can be performed: the production cells of the present invention having the E4 gene under the control of an exogenous promoter in the chromosome and not containing the VA-RNA gene are cultured under conditions that do not induce the expression of the Cap gene, the Rep gene and the E4 gene to proliferate the production cells, and then the proliferated production cells are cultured under conditions that induce the expression of the Cap gene, the Rep gene and the E4 gene, thereby producing adeno-associated virus.

[0189] The production cells of the present invention are placed under conditions where the expression of the Cap gene and the Rep gene is not induced, which means that the Cap gene and the Rep gene are controlled by an inducible promoter and their expression is in a closed state, preferably their expression is controlled by a drug, and the drug that turns the inducible promoter into an open state does not contact the production cells, and it is more preferred that the tetracycline drug does not contact the production cells.

[0190] The condition of inducing the expression of the CaP gene and the Rep gene means that the CaP gene and the Rep gene are controlled by an inducible promoter and their expression is in an on state. Preferably, their expression is controlled by a drug, and the drug that turns on the inducible promoter is in contact with the production cells. It is further preferred that a tetracycline drug is in contact with the production cells, and it is particularly preferred that doxycycline is in contact with the production cells.

[0191] The producer cells of the present invention can be subjected to the following step: after culturing and growing the producer cells for more than 3 days under conditions that do not induce the expression of Cap and Rep genes, the grown producer cells are cultured under conditions that induce the expression of Cap and Rep genes to produce adeno-associated virus.

[0192] Under conditions in which the expression of the Cap gene and the Rep gene is not induced, the culture is preferably carried out for 3 days or more, more preferably for 5 days or more, and even more preferably for 7 days or more.

[0193] Furthermore, after contacting the producer cells with doxycycline that turns off the inducible promoter and culturing under conditions that induce the expression of the Cap gene and the Rep gene, AAV can be produced when the producer cells contain a gene that expresses the tetracycline-regulated activator (tTA) in the absence of doxycycline. The above operation has the advantage that doxycycline, an antibacterial drug, is not easily mixed into the produced AAV.

[0194] The culture of cells in the method for producing adeno-associated virus of the present invention can be carried out under the usual conditions for cell culture. As for the culture medium and culture conditions used, those skilled in the art can appropriately select them. As culture medium, Expi293 Expression Medium (Thermo Fisher Scientific, A1435101), Dulbecco's Modified Eagle's Medium (DMEM) containing 10% (vol / vol) fetal bovine serum (FBS), serum-free UltraCULTURE (trademark) medium (Lonza), etc. can be used, but there is no particular limitation.

[0195] The culture temperature is usually 25°C to 45°C, preferably 30°C to 42°C, more preferably 35°C to 40°C, and as an example, is 37°C.

[0196] The CO 2 concentration is usually 3 to 10% CO 2 , preferably 5 to 10% CO 2 , and as an example, it is 8% CO 2 .

[0197] The cells can be cultured in any volume of culture medium, for example, the cells can be cultured in 1 mL to 2000 L of culture medium, preferably 1 L to 2000 L, more preferably 50 L to 2000 L, and particularly preferably 500 L to 2000 L.

[0198] The culture may be carried out while vibrating and stirring. The stirring speed during vibrating and stirring is usually 50 rpm to 200 rpm, preferably 80 to 150 rpm.

[0199] The stirring culture may be a rotary stirring culture based on a propeller or the like in a reactor. The stirring speed during the rotary stirring is usually 50 rpm to 200 rpm, preferably 80 to 150 rpm. Furthermore, stirring may be performed by wave-type vibration stirring or by moving the stirring blade up and down, but is not particularly limited.

[0200] The culture time in the AAV production step is not particularly limited, but is generally 6 hours to 14 days, preferably 12 hours to 7 days, more preferably 24 hours to 144 hours, and even more preferably 24 hours to 96 hours.

[0201] The titer of the adeno-associated virus produced can be measured by conventional methods known to those skilled in the art. For example, the cell culture fluid after culture is recovered, and after the cells are broken by freeze-thaw, the supernatant is recovered by centrifugation. By adding MgCl2 and nuclease (Benzonase) to the recovered supernatant to react, gDNA (genomic DNA) and residual plasmids can be digested. A sample containing the adeno-associated virus obtained as described above is used as a ddPCR (Droplet Digital PCR: droplet digital PCR) sample, and the titer of AAV can be measured by performing ddPCR.

[0202] The present invention will be described in more detail by the following examples, but the present invention is not limited to the examples.

[0203] Example

[0204] [Materials and Methods]

[0205] <Cell Culture>

[0206] The suspended HEK293 cells (Thermo Fisher Scientific, R79007) were suspended in 12 mL of Expi293 expression medium (Thermo Fisher Scientific, A1435101) to a concentration of 1×10 7 cells / mL and cultured in a 125 mL shaking flask for 24 hours. The culture solution was stirred at 120 rpm and incubated at 37°C and 8% CO2.

[0207] <Plasmid construction>

[0208] AAVpro (registered trademark) helper free system (Helper Free System) (AAV5) (TakaraBio Inc., 6650) (Sequence No. 1, Sequence No. 2, Sequence No. 3) was used.

[0209] The entire sequences of the plasmids used are shown in the sequence listing as SEQ ID NOs 4 to 14. The sequences were produced by total synthesis.

[0210] [Table 1]

[0211]

[0212]

[0213]

[0214] <Gene transfer method>

[0215] In 1.8 mL of Expi293 expression medium, 36 μL of polyethylenimine (PEI) (PolyPlus-transfection SAS, 115-0015) and the plasmids described below were mixed in equal amounts, and a total of 18 μg was suspended and allowed to stand for 15 minutes. 7 The above reagents were added to cultured cells seeded at 10 cells / mL (culture solution volume: 12 mL), and the cells were incubated at 37°C and 8% CO2.

[0216] <Western blotting>

[0217] The cells were recovered by centrifugation and dissolved with RIPA Buffer. Sample Buffer (NACALAI, 09499-14) was added to the cell solution and heated at 100°C for 5 minutes to prepare the sample. After the protein in the sample was size-separated using a sodium dodecyl sulfate (SDS) gel electrophoresis apparatus (ATTO Corporation., 2321670), the membrane was transferred using a semi-dry blotting apparatus (ATTO Corporation., 2322490). The transferred membrane was blocked for 30 minutes using Blocking One (NACALAI TESQUE, INC., 03953-95), and then reflected at room temperature for 3 hours using anti-REP antibody (OriGene Technologies) diluted 70 times with blocking solution, anti-VP antibody (PROGEN Biotechnik GmbH) that reacts with the structural protein of the capsid, and anti-β-actin (β-Actin) antibody (Abcam). Next, as the second antibody, anti-mouse IgG-HRP (Cytiva) diluted 10,000 times with a blocking solution was reacted at room temperature for 2 hours, and then the REP protein and the structural proteins of the capsid (VP1, VP2, VP3) were detected by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095). WSE-6100 LuminoGraphI (ATTO Corporation., 2006100) was used for photography. Quantitative evaluation was implemented using ImageJ in the analysis of the acquired images.

[0218] <Determination of AAV Titer>

[0219] The culture medium of HEK cells cultured at 37°C and 8% CO2 for 72 hours after gene introduction was recovered, and the cells were broken by freeze-thawing at -80°C. After that, the supernatant was recovered by centrifugation at 13,800×g, and MgCl2 with a final concentration of 2mmol / L and 25U / mL of nuclease were added, and the reaction was carried out at 37°C for 2 hours to digest gDNA (genomic DNA) or residual plasmid. The reacted samples were incubated at 95°C for 15 minutes, 70°C for 3 minutes, 40°C for 3 minutes, and 20°C for 3 minutes in order to inactivate the nuclease, and prepared as ddPCR (Droplet Digital PCR) samples. The ddPCR samples were diluted 50 times with TE buffer (pH 8.0, containing 0.05% Pluronic F-68 and 10μg / mL bovine thymus DNA). ddPCR was performed by mixing 10 μL of ddPCRtm Supermix for Probes (ddPCR probe premix) (no dUTP) (BIORAD), 2 μL of diluted ddPCR sample, primers (sequence number 15, sequence number 16, final concentration 900 nmol / L), probe (sequence number 17, final concentration 250 nmol / L), and nuclease-free water (Thermo Fisher Scientific, 10977015) in a test tube on ice and adjusting the total liquid volume to 22 μL.

[0220] In ddPCR, droplets were formed from the preparation solution by a droplet formation device, which was then heated at 95°C for 10 minutes (preheating), 94°C for 30 seconds (modification), 55°C for 60 seconds (annealing and stretching) for 40 cycles, and then incubated at 98°C for 10 minutes. The AAV genome titer (vg / mL) was calculated by measuring with a droplet reader.

[0221] <Establishment of cell lines>

[0222] In 1.8 mL of Expi293 expression medium, 36 μL of polyethyleneimine (PEI) (PolyPlus-transfection SAS, 115-0015) and the plasmids described below were mixed in equal amounts, and a total of 18 μg was suspended and allowed to stand for 15 minutes. 7 The above reagents were added to the cultured cells (culture solution volume 12mL) seeded with 10 cells / mL and incubated at 37°C and 8% CO2. The cells into which the gene was introduced were cultured for 3 weeks, and the RFP or GFP fluorescent positive cells were isolated from single cells using drug screening based on resistance genes or flow cytometry. The isolated cells were suspended in DMEM containing 10% fetal bovine serum and cultured on a 6-well plate at 37°C and 8% CO2 for 1 month to establish a cell line.

[0223] After gene transfer in the DOX(+) sample, doxycycline (DOX) was added at a final concentration of 0.5 μg / mL. After the addition of doxycycline, the titer of AAV genome extracted from HEK293 cells cultured for 72 hours was evaluated.

[0224] Experiment 1:

[0225] <Method>

[0226] Plasmids having the following sequences were respectively introduced into cells, and the cells were collected and then evaluated for expression by Western blotting.

[0227] Ctrl: Sequence number 1, Sequence number 2, and Sequence number 3

[0228] ΔAuxiliary: Sequence number 2 and sequence number 3

[0229] ΔE4: Sequence number 2, sequence number 3 and sequence number 5

[0230] ΔVA-RNA: SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 6

[0231] <Results>

[0232] The results are shown in Figure 1 middle.

[0233] In the absence of the auxiliary genes (three types) (Δ auxiliary), REP and CAP were not expressed. In addition, even when E4 or VA-RNA was removed alone, the expression levels of REP and CAP were greatly reduced.

[0234] In commercially available kits (Takara et al.), three plasmids are generally used, but the various genes contained in these plasmids interact with each other. Therefore, if the E4 gene and VA-RNA gene are not used, REP and CAP cannot be expressed, and AAV cannot be produced.

[0235] Experiment 2:

[0236] <Method>

[0237] Plasmids having the following sequences were introduced into cells, respectively, and the cells were collected 72 hours later, and the AAV production capacity was evaluated by ddPCR.

[0238] Ctrl: Sequence number 1, Sequence number 4 and Sequence number 7

[0239] ΔE4ΔVA-RNA: SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8

[0240] <Results>

[0241] The results are shown in Figure 2 middle.

[0242] If a foreign promoter is linked to the Rep gene and the Cap gene, approximately 80% of AAV can be produced even without E4 and VA.

[0243] Experiment 3:

[0244] <Method>

[0245] Sequence No. 9 was prepared and gene introduction was performed simultaneously with Sequence No. 10. Doxycycline (DOX) was added 24 hours after gene introduction, and the expression of REP protein and CAP protein was evaluated by immunoblotting 72 hours later.

[0246] rtTA(-), DOX(-): State without TEF regulator and inducer (to evaluate gene expression leakage when the switch is turned off)

[0247] rtTA(+), DOX(-): State with TET regulator / without inducer (to evaluate gene expression leakage when the switch is turned on)

[0248] rtTA(+), DOX(+): The state in which TET regulators and inducers are present (evaluation of gene expression when the switch is turned on)

[0249] rtTA is a protein that expresses a gene under the control of TRE-CMV-minimal promoter and is introduced as a plasmid. As the promoter of rtTA, CMV-minimal promoter was used.

[0250] <Results>

[0251] The results are shown in Figure 3 middle.

[0252] When doxycycline is added to SEQ ID NO: 9 in the presence of SEQ ID NO: 10, the expression of REP protein and CAP protein can be controlled.

[0253] Experiment 4: Production of cells with E2 gene and GFP inserted

[0254] <Method>

[0255] In HEK293T cells (Takara), lentiviral high titer packaging mixture (Lentiviral High Titer Packaging Mix) (Takara) and sequence number 11 or sequence number 12 were introduced together to produce lentivirus. The culture supernatant was added to 1 / 100 of the HEK 293 cells together with 2-12 μg / ml of polybrene to establish infected cells. Therefore, the drug was added, and the genome was extracted from cells that had been passaged for more than one month, and PCR confirmed that the E2 gene and GFP gene were inserted into the chromosome. Whether E2 expresses protein was detected by immunoblotting (Western Blot) (E2 antibody). In addition, the presence of green fluorescence of GFP was confirmed by fluorescence microscopy.

[0256] <Results>

[0257] The results are shown in Figure 4 middle. Figure 4 Western Blot analysis using anti-E2 antibody for stable expression lines (cells grown) Figure 4 The left image in the lower part) and GFP observation (fluorescence microscope) ( Figure 4 The result is shown in the right figure in the lower section.

[0258] As the target gene, a stable strain was established in which GFP sandwiched by ITR was inserted, and a stable strain was established in which E2 driven by CMV promoter was inserted.

[0259] Experiment 5: AAV function evaluation

[0260] <Method>

[0261] The Rep gene and the Cap gene (SEQ ID NO: 7) were introduced into the above CMV-E2 and the cell line into which the GFP sandwiched by ITR was introduced via a plasmid. 72 hours after the introduction, the culture supernatant was recovered and AAV was detected by ddPCR.

[0262] <Results>

[0263] The results are shown in Figure 5 middle.

[0264] When sequence number 7 was not added, AAV was hardly detectable, but AAV was produced in the cell lines into which sequence number 7 was introduced (two strains established on another day).

[0265] Experiment 6: Production of producer cells

[0266] <Method>

[0267] Lentivirus was prepared using sequence number 11, and cells into which sequence number 13 (screening marker: RFP red fluorescent protein) and sequence number 14 (screening marker: NEO, neomycin resistance gene) were introduced into the infected HEK293 cells by the method described in the <Method> of "Experiment 4: Production of E2 gene and GFP inserted cells", and cells into which sequence number 13 (screening marker: NEO, neomycin resistance gene) were introduced into the chromosomes, and the single strains introduced into the chromosomes were screened. The cells into which sequence number 13 was introduced were screened using red fluorescence as an indicator. In addition, G418 was added to the cells into which sequence number 14 was introduced at a concentration of 200 μg / ml, and screening was performed ( Figure 6 ). The genome was removed from cells cultured for more than one month after gene introduction, and the introduction into the genome was confirmed by PCR, thereby establishing a stable strain.

[0268] The selected cell lines (30 lines for the line introduced with sequence number 14 and 60 lines for the line introduced with sequence number 13) were seeded at 300,000 / well, and doxycycline was added to induce gene expression. 72 hours later, AAV was quantified by ddPCR.

[0269] <Results>

[0270] The quantitative results of AAV of the strain into which SEQ ID NO: 14 (selectable marker: NEO neomycin resistance gene) was introduced are shown in Figure 7 The vertical axis represents AAV titration (Titer) vg / cell, and the horizontal axis represents clone (Clone) number.

[0271] The quantitative results of AAV of the strain into which sequence number 13 (selection marker: RFP red fluorescent protein) was introduced are shown in Figure 8 The vertical axis represents AAV titration (Titer) vg / cell, and the horizontal axis represents clone number.

[0272] Representative microscopic images of stable strains are shown in Fig. 9 middle.

[0273] Experiment 7: Production of producer cells (2)

[0274] <Method>

[0275] HEK293 cells into which sequence number 18 (selection marker: GFP) was genetically introduced (design 1-introduced cells), HEK293 cells into which sequence number 19 (selection marker: GFP) was genetically introduced (design 2-introduced cells), HEK293 cells into which sequence number 20 and sequence number 21 (selection marker and drug: GFP and neomycin resistance gene (NEO)) were genetically introduced (design 3-introduced cells), HEK293 cells into which sequence number 22 and sequence number 23 (selection marker and drug: GFP and NEO) were genetically introduced (design 4-introduced cells), HEK293 cells into which sequence number 24 (selection marker: GFP) was genetically introduced (design 5-introduced cells), and HEK293 cells into which sequence number 25 and sequence number 26 (selection marker and drug: GFP and NEO) were genetically introduced (design 6-introduced cells), and single strains into which the introduced genes were respectively introduced into chromosomes were screened.

[0276] When design 1 to design 6 are prepared for introduction into cells, in order to improve the recombination efficiency of chromosomes of sequence numbers 18 / 19 / 20 / 22 / 24 / 25, the plasmid vector of sequence number 27 is further introduced together to express the transposase recombinase at one time.

[0277] Design 1 introduced cells, design 2 introduced cells, and design 5 introduced cells were screened using green fluorescence as an indicator. Furthermore, G418 was added to the design 3 introduced cells, design 4 introduced cells, and design 6 introduced cells at a concentration of 200 μg / ml and screened, and further screening was performed using green fluorescence as an indicator ( Fig.11 ).

[0278] The genome was extracted from cells cultured for more than one month after gene introduction, and introduction into the genome was confirmed by PCR, thereby establishing a stable strain.

[0279] The selected cell lines were seeded into 24-well plates to give a volume of 3.0 × 10 5 cells / well, and doxycycline was added to induce gene expression. After 72 hours, AAV was quantified by ddPCR.

[0280] 1.0×10 6 cells, and genomic DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN). It was used as a ddPCR sample. The ddPCR sample was diluted to 15 ng / ul with nuclease-free water (Thermo Fisher Scientific, 10977015). 10 μL of ddPCRtm Supermix for Probes (ddPCR probe premix) (no dUTP) (BIORAD), 2 μL of diluted ddPCR sample, the primers (final concentration 900 nmol / L) and probes (final concentration 250 nmol / L) shown in Table 2, and nuclease-free water (Thermo Fisher Scientific, 10977015) were mixed in a test tube on ice, and the total liquid volume was adjusted to 22 μL, thereby performing ddPCR. From the ddPCR measurement values ​​of the genomic DNA samples, the number of gene insertions on the genome of each cell was calculated using the method of Formula 1 below.

[0281] <Formula 1>

[0282] The number of gene insertions in the genome of each cell (copies / cell) = (quantitative number of FAM / quantitative number of HEX) × 3

[0283] [Table 2]

[0284]

[0285] Sequence No. 30 used a DNA probe labeled with a HEK fluorescent dye, and Sequence Nos. 33, 36, 39, and 42 used a DNA probe labeled with a FAM fluorescent dye.

[0286] <Results>

[0287] The quantitative results of AAV with the highest titer introduced into cells by Designs 1 to 6 are shown in Fig.12 The vertical axis represents AAV titration vg / cell, and the horizontal axis represents the cell line name.

[0288] The number of gene insertions in the genomes of the cell lines (10 strains) introduced with design 4 and the cell lines (10 strains) introduced with design 5 were measured by PCR. Fig.13 and Fig.14 The first vertical axis represents the number of gene insertions per cell, the second vertical axis represents the AAV titration vg / cell, and the horizontal axis represents the clone name.

[0289] Experiment 8: Suspension of producer cells

[0290] <Method>

[0291] The highest titer strain of Design 4 introduced into cells established in Experiment 7 and the highest titer strain of Design 5 were suspended in 12 mL of Expi293 expression medium (Thermo Fisher Scientific, A1435101) to a concentration of 0.1 to 2 × 10 6 cells / mL, and cultured in a 125 mL shaking flask and allowed to float. The culture solution was incubated at 37°C and 8% CO2 while being constantly stirred at 120 rpm. Culture was continued for 1 month from the time of suspension.

[0292] The suspension cultured cells were seeded into Expi293 expression medium to a concentration of 4.0 × 10 5 cells / mL, and doxycycline was added to induce gene expression. The culture solution was incubated at 37°C and 8% CO2 while being constantly stirred at 120 rpm. After 72 hours, AAV was quantified by ddPCR.

[0293] <Results>

[0294] The quantitative results of the AAV with the highest titer of Design 4 and the highest titer of Design 5 introduced into the suspended cells are shown in Fig.15 The vertical axis represents AAV titration vg / cell, and the horizontal axis represents the cell line name.

Claims

1. A production cell which does not contain at least one of a VA-RNA gene and an E4 gene and is a mammalian cell, The production cell has in its chromosome a Cap gene under the control of an exogenous promoter and a Rep gene under the control of an exogenous promoter.

2. The production cell according to claim 1, wherein The exogenous promoter of the Rep gene and the exogenous promoter of the Cap gene are the same promoter.

3. The production cell according to claim 1, wherein The Rep gene exists downstream of the Cap gene in the transcription direction.

4. The production cell according to claim 1, which has an E4 gene under the control of an exogenous promoter in the chromosome and does not contain a VA-RNA gene.

5. The production cell according to claim 4, wherein The E4 gene is located downstream of IRES.

6. The production cell according to claim 4, wherein The exogenous promoter of the E4 gene is an inducible promoter that can be switched on and off.

7. The production cell according to claim 6, wherein The exogenous promoter of the E4 gene is a promoter whose expression can be induced by a drug.

8. The production cell according to claim 7, wherein The exogenous promoter of the E4 gene is located downstream of the tetracycline response factor. 9 . The production cell according to claim 1 , which does not contain both the VA-RNA gene and the E4 gene.

10. The production cell according to any one of claims 1 to 9, further comprising an E2A gene.

11. The production cell according to claim 10, wherein The E2A gene is located downstream of the IRES.

12. The production cell according to claim 10, wherein The E2A gene is under the control of an exogenous promoter.

13. The production cell according to claim 12, wherein The E2A gene is under the control of a promoter derived from cytomegalovirus.

14. The production cell according to claim 10, wherein The exogenous promoter of the E2A gene is an inducible promoter that can be switched on and off.

15. The production cell according to claim 14, wherein The exogenous promoter of the E2A gene is a promoter whose expression can be induced by a drug.

16. The production cell according to claim 15, wherein The exogenous promoter of the E2A gene is located downstream of the tetracycline response factor.

17. A production cell, which is a mammalian cell that does not contain a VA-RNA gene, The production cell has in its chromosome a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, and an E4 gene under the control of an exogenous promoter.

18. The production cell according to any one of claims 1 to 17, further comprising a gene of interest.

19. The production cell according to any one of claims 1 to 18, wherein One or more of the exogenous promoter of the Cap gene and the exogenous promoter of the Rep gene is an inducible promoter that can be switched on and off.

20. The production cell according to claim 19, wherein One or more of the exogenous promoter of the Cap gene and the exogenous promoter of the Rep gene is a promoter whose expression can be induced by a drug.

21. The production cell according to claim 20, comprising: One or more of the exogenous promoter of the Cap gene and the exogenous promoter of the Rep gene are located downstream of the tetracycline response factor.

22. The production cell according to any one of claims 1 to 21, further comprising a SmallRep gene under the control of an exogenous promoter.

23. The production cell according to claim 22, wherein The Rep gene exists downstream of the SmallRep gene in the transcription direction.

24. The production cell according to claim 22, wherein The exogenous promoter of the SmallRep gene is an inducible promoter that can be switched on and off.

25. The production cell according to claim 24, wherein The exogenous promoter of the SmallRep gene is a promoter whose expression can be induced by a drug.

26. The production cell according to claim 25, wherein The exogenous promoter of the SmallRep gene is located downstream of the tetracycline response factor.

27. The production cell according to any one of claims 1 to 26, further comprising a gene expressing a reverse tetracycline-regulated transcriptional activator.

28. The production cell according to claim 27, comprising: The gene expressing the reverse tetracycline-regulated transcriptional activator is located downstream of the Rep gene.

29. The production cell according to claim 1, comprising an E4 gene under the control of an exogenous promoter, an E2A gene under the control of an exogenous promoter, and a SmallRep gene under the control of an exogenous promoter, The exogenous promoter of the Cap gene and the exogenous promoter of the E2 gene are the same promoter, and the exogenous promoter of the Rep gene and the exogenous promoter of the SmallRep gene are the same promoter.

30. A production cell, which is a mammalian cell that does not contain a VA-RNA gene, The production cell has a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, an E4 gene under the control of an exogenous promoter, and an E2A gene under the control of an exogenous promoter in the chromosome, and The Cap gene, Rep gene, and E2 gene are present nearby, but the E4 gene is not present nearby.

31. A production cell, which is a mammalian cell that does not contain a VA-RNA gene, The producer cell has in its chromosome: A first vector carrying a Cap gene under the control of an exogenous promoter, a Rep gene under the control of an exogenous promoter, and an E2A gene under the control of an exogenous promoter; and The second vector carries the E4 gene under the control of an exogenous promoter.

32. The production cell according to claim 31 or 32, wherein The number of E4 gene insertions on the chromosome was less than that of other gene insertions.

33. The production cell according to claim 31 or 32, wherein The number of E4 gene insertions on the chromosome is less than that of Cap gene or E2 gene insertions on the chromosome.

34. The production cell according to claim 31 or 32, wherein The number of E4 gene insertions on the chromosome is less than 0.4 times the number of Cap gene insertions on the chromosome.

35. The production cell according to claim 31 or 32, wherein The number of E4 gene insertions on the chromosome is less than 0.4 times the number of E2 gene insertions on the chromosome.

36. A method for producing a producer cell according to any one of claims 1 to 35, comprising: The Cap gene under the control of an exogenous promoter and the Rep gene under the control of an exogenous promoter are introduced into mammalian cells.

37. The method for producing a producer cell according to claim 36, further comprising introducing a reverse tetracycline-regulated transcriptional activator.

38. A method for producing an adeno-associated virus, comprising: The adeno-associated virus is produced by culturing the producer cell according to any one of claims 1 to 35.

39. A method for producing an adeno-associated virus, comprising the following steps: Cultivating the producer cell according to any one of claims 1 to 35 under conditions that do not induce the expression of the Cap gene and the Rep gene to proliferate the producer cell; and Adeno-associated virus is produced by culturing the proliferated producer cells under conditions that induce the expression of the Cap gene and the Rep gene.

40. A method for producing an adeno-associated virus, comprising the following steps: Cultivating the producer cell according to any one of claims 4 to 8 under conditions that do not induce the expression of the Cap gene, the Rep gene, and the E4 gene to proliferate the producer cell; and Adeno-associated virus is produced by culturing the proliferated producer cells under conditions that induce expression of the Cap gene, Rep gene, and E4 gene.

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