Technique for high-throughput production of adeno-associated virus vector

Abstract

The present invention addresses the problem of developing a novel helper factor expression system with which the production amount of an adeno-associated virus (AAV) vector can be increased and inducible expression becomes possible.　The present invention relates to an expression cassette for use in the production of an AAV vector. The expression cassette includes a nucleic acid that contains a helper factor capable of increasing the production amount of an AAV vector, and a heterologous promoter that is operably linked to the helper factor, wherein the nucleic acid includes the following nucleic acids (1) to (4). (1) A nucleic acid that contains a coding region for an E2A protein of an adenovirus; (2) a nucleic acid that contains coding regions for one or more proteins selected from a 22k protein, a 100k protein, a 33k protein, an E4 orf2 protein, an E4 orf3 protein and an E4 orf6 / 7 protein of an adenovirus; (3) a nucleic acid that contains a coding region for an E4 orf6 protein of an adenovirus; and (4) a nucleic acid that includes template DNA for VA-RNA of an adenovirus.

Description

High-yield production technology of adeno-associated virus vectors

[0001] The present invention relates to a helper factor capable of inducibly expressing an adeno-associated virus (AAV) vector, which can increase the production yield of the vector, and a method for producing an AAV vector using the helper factor.

[0002] Viral vectors can infect cells and express specific genes of interest in target cells or tissues. By utilizing the infectious properties of viruses, viral vectors have high gene transfer and expression efficiencies, enabling them to deliver genes to hematopoietic cells and primary cultured cells, which are difficult to deliver using other gene transfer methods. They are also capable of in vivo gene transfer in many animal species, including humans, rats, and mice. Among viral vectors, AAV vectors are one of the most widely used gene therapy vectors for medical applications due to their non-pathogenicity, ability to deliver genes to terminally differentiated, non-dividing cells such as neurons, physicochemical stability of viral particles, and ease of purification and concentration. Gene therapy using AAV vectors is currently being performed worldwide for a variety of intractable diseases, and its therapeutic efficacy has been proven in numerous clinical studies. Further clinical applications are expected to expand in the future.

[0003] While the clinical application of AAV vectors in gene therapy is progressing, the development of mass production and purification technologies for the vectors required for gene therapy has lagged behind, and there is an urgent need to develop a production technology platform for AAV vectors that can meet the needs for mass production in future clinical applications.Currently, AAV vectors are produced by transiently transfecting producer cells with plasmid DNA containing AAV components and adenovirus-derived helper factors that enhance vector production.

[0004] However, current methods require GMP-compliant plasmid DNA and gene transfer reagents for each vector production, resulting in increased costs. Furthermore, the need for gene transfer complicates the production process and leads to problems such as poor reproducibility and scalability. Therefore, there is a need to construct producer cells in which all factors necessary for AAV vector production are integrated into the cellular genome. However, the AAV components and helper factors used in AAV vector production are highly cytotoxic. Therefore, if they are integrated into the cellular genome and constitutively expressed, the toxicity will either kill the cell or rapidly inactivate the transgene, resulting in a rapid decrease in vector production (Non-Patent Document 1). For these reasons, constructing AAV vector producer cells is difficult, and there are very few successful examples worldwide. To prevent the cytotoxicity of AAV components and helper factors, it is necessary to control their expression using an inducible expression system. However, the expression patterns of AAV components and helper factors are complex, and no effective means of achieving inducible expression has been found to date.

[0005] Previous attempts have been made to create producer cells for AAV vectors. For example, methods for controlling the expression of E1A and E1B (Patent Document 1), methods for inactivating Rep78 and Rep68 through mutation (Patent Document 2), and expression control systems using Rep gene recombination have been reported (Patent Document 3). However, these methods are not directed at constructing an expression control system capable of inducible expression of helper factors.

[0006] The helper plasmid (pHelper) currently used for AAV vector production is composed of a gene fragment containing E2A, E4, and VA-RNA, among the E1A, E1B, E2A, VA, and E4 gene regions responsible for the adenovirus helper function required for AAV replication, excluding E1A and E1B present in human fetal kidney tissue-derived 293 cells. Previous reports have shown that the helper function of E2A and E4 can be complemented by an expression system in which the coding region for the E2A protein (E2A cds) contained in the E2A genome fragment is linked to a strong constitutive promoter, or by an expression system in which only orf6 of the E4 gene is linked to a constitutive promoter (Non-Patent Documents 2 and 3).

[0007] WO2007 / 056994WO2019 / 057691WO2020 / 078953

[0008] XIAO et al., Microbiology Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus, JOURNAL OF VIROLOGY, Vol.72, No.3, p.2224-2232 (1998)T Matsushita et al., Adeno-associated virus vectors can be efficiently produced without helper virus, Gene Therapy, 5(7), p.938-945 (1998)James M. Allen et al., Improved Adeno-Associated Virus Vector Production with Transfection of a Single Helper Adenovirus Gene, E4orf6, Molecular Therapy 1(1), p.88-95 (2000)

[0009] The present inventors have investigated the helper activities of the E2A protein-encoding region (E2A cds) and orf6 of the adenovirus E4 gene, which have previously been thought to be capable of complementing the helper functions of adenovirus E2A and E4, for helper factors used in AAV vector production. They found that both significantly reduced vector production compared to the original pHelper-E2A and pHelper-E4 (see Comparative Reference Example 1 below). Therefore, an objective of the present invention is to develop a helper factor expression system that contains new helper factors that can increase AAV vector production and that are capable of inducible expression.

[0010] In order to solve the above problems, the present inventors attempted to identify the sequences required for helper function from adenovirus genomic fragments and to construct an inducible expression cassette using them. As a result, they found that: (1) the proteins encoded by the 22k gene, 100k gene, and 33k gene present in the genomic fragment containing the adenovirus E2A gene each have helper function; (2) the proteins encoded by the orf2, orf3, and orf6-7 genes other than the orf6 gene present in the genomic fragment containing the adenovirus E4 gene each have helper function; and (3) a combination of the above proteins has helper activity equal to or greater than that of the original pHelper-E2A or pHelper-E4, and serves as a new helper factor that increases the production yield of AAV vectors. The present inventors also optimized the combination of these helper factors to construct an inducible expression system and evaluated its performance. They found that while expression of the helper factors was suppressed when no inducer was added, it was possible to induce expression of the helper factors in a concentration-dependent manner when an inducer was added, and further confirmed that AAV vector production was induced in a concentration-dependent manner when an inducer was added, with the amount of vector produced being increased compared to conventional methods, thereby completing the present invention.

[0011] That is, the present invention encompasses the following inventions: [1] An expression cassette used for producing an adeno-associated virus (AAV) vector, comprising a nucleic acid containing a helper factor capable of increasing the production yield of the AAV vector, and a heterologous promoter operably linked to the helper factor, wherein the nucleic acid comprises any of the following nucleic acids (1) to (4): (1) a nucleic acid comprising a coding region for an adenoviral E2A protein; (2) a nucleic acid comprising a coding region for one or more proteins selected from the group consisting of an adenoviral 22k protein, a 100k protein, a 33k protein, an E4orf2 protein, an E4orf3 protein, and an E4orf6 / 7 protein; (3) a nucleic acid comprising a coding region for an adenoviral E4orf6 protein; or (4) a nucleic acid comprising a template DNA for an adenoviral VA-RNA. [2] The expression cassette according to [1], wherein each of the helper factors (1) to (4) is contained in the same or separate expression cassettes. [3] The expression cassette according to [1], wherein the heterologous promoter is a constitutive promoter or an inducible promoter. [4] A helper plasmid or helper virus used in producing an adeno-associated virus (AAV) vector, comprising the expression cassette according to any one of [1] to [3]. [5] A method for producing an AAV vector, comprising transfecting a virus-producing cell with the helper plasmid or helper virus according to [4], a transfer plasmid containing an expression cassette for a gene of interest between the ITRs at both ends of the adenovirus genome, and a packaging plasmid containing the Rep and Cap genes of the adeno-associated virus. [6] An AAV vector-producing cell, characterized in that the expression cassette according to any one of [1] to [3] is stably maintained intracellularly. This application claims priority to Japanese Patent Application No. 2024-043999, filed on March 19, 2024, and includes the contents of the specification of that patent application.

[0012] According to the present invention, an expression cassette capable of inducibly expressing a helper factor capable of increasing the production amount of an AAV vector is provided. By using the expression cassette of the present invention in the production of an AAV vector, the production efficiency of the AAV vector can be dramatically improved.

[0013] Figure 1 shows a scheme for a test verifying the helper activity of E2A. Figure 2 shows a comparison of the helper activity of pHelper-E2A and pCBh-E2A based on the amount of AAV vector produced. Figure 3 shows a scheme for a test verifying the helper activity of E4orf6. Figure 4 shows a comparison of the helper activity of pHelper-E4 and pCBh-E4orf6 based on the amount of AAV vector produced. Figure 5 shows the construction of expression plasmids for proteins contained in E2A genomic fragments (100kJ, 22kJ, 33kJ). Figure 6 shows a comparison of the helper activity of pHelper-E2A, pCBh-E2A, and expression plasmids for proteins contained in E2A genomic fragments (100kJ, 22kJ, 33kJ) based on the amount of AAV vector produced. Figure 7 shows the construction of a polycistronic expression plasmid (pCBh-E2A-22k-100k) for proteins contained in E2A genomic fragments (100kJ, 22kJ). Figure 1 shows a comparison of helper activity based on AAV vector production volume between pHelper-E2A and pCBh-E2A-22k-100k. Figure 2 shows the construction of expression plasmids for orfs contained in the E4 genomic fragment (orf1-7, orf2-7, orf3-7, orf4-7, orf6, orf6-7). Figure 3 shows a comparison of helper activity based on AAV vector production volume between pHelper-E4 and expression plasmids for orfs contained in the E4 genomic fragment (orf1-7, orf2-7, orf3-7, orf4-7, orf6, orf6-7). Figure 4 shows the construction of polycistronic expression plasmids (pCBh-E4orf2367, pCBh-E4orf367) for orfs contained in the E4 genomic fragment (orf2-7, orf3-7, orf6-7). This shows a comparison of helper activity based on the amount of AAV vector produced by pHelper-E4, pCBh-E4orf6, pCBh-E4orf2367, and pCBh-E4orf367. This shows a scheme for the validation test of the helper activity of VA-RNA. This shows a comparison of helper activity based on the amount of AAV vector produced by pHelper-VA and pCBh-VA. This shows a scheme for the validation test of the helper activity of optimized helper factor (E2Aopti, E4opti) expression plasmids (pE2Aopti, pE4opti). This shows a comparison of helper activity based on the amount of AAV vector produced by the conventional helper factor expression plasmid (pHelper) and the optimized helper factor (E2Aopti, E4opti) expression plasmids (pE2Aopti, pE4opti).This figure shows the construction of the polycistronic inducible expression system (pTRE-E2Aopti-E4opti, pTRE-VA) for optimized helper factors (E2Aopti, E4opti, VA-RNA). This figure shows the scheme for the verification test of helper factor expression and helper activity using the polycistronic inducible expression system (pTRE-E2Aopti-E4opti + pTRE-VA) for optimized helper factors (E2Aopti, E4opti, VA-RNA). This figure shows the expression levels (relative mRNA expression levels) of helper factors (E2A, E4) using the polycistronic inducible expression system (pTRE-E2Aopti-E4opti + pTRE-VA) for optimized helper factors (E2Aopti, E4opti, VA-RNA). This shows the expression level (relative mRNA expression level) of helper factor (VA-RNA) using a polycistronic inducible expression system (pTRE-E2Aopti-E4opti+pTRE-VA) for optimized helper factors (E2Aopti, E4opti, VA-RNA). This shows a comparison of helper activity based on AAV vector production levels between a conventional helper factor expression vector (pHelper) and a polycistronic inducible expression system (pTRE-E2Aopti-E4opti+pTRE-VA) for optimized helper factors (E2Aopti, E4opti, VA). This shows a scheme for a test verifying the helper activity of a plasmid (pTRE-E2Aopti-E4opti-VA) carrying a polycistronic inducible expression system cassette for optimized helper factors (E2Aopti, E4opti, VA-RNA). Figure 1 shows a comparison of the helper activity of a plasmid (pTRE-E2Aopti-E4opti-VA) carrying a polycistronic inducible expression cassette for optimized helper factors (E2Aopti, E4opti, VA-RNA) and the helper activity of a conventional helper factor expression plasmid (pHelper) based on AAV vector production.

[0014] The present invention is described in detail below. Adeno-associated virus (AAV) is a single-stranded DNA virus of approximately 4.7 kb belonging to the Parvoviridae family. It has an icosahedral capsid with a diameter of 20 to 30 nm and infects a wide host range because it recognizes heparan sulfate proteoglycan, a universal component of cell membranes. The AAV genome structure has inverted terminal repeats (ITRs) at both ends, containing Rep (a regulatory protein responsible for replication and transcription) and Cap (three capsid proteins: VP1, VP2, and VP3).

[0015] Several serotypes of AAV have been discovered to date, with AAV serotype 2 (AAV2) being the most extensively studied. However, as used herein, "AAV" encompasses AAV type 1 virus (AAV1), AAV type 2 virus (AAV2), AAV type 3 virus (AAV3), AAV type 4 virus (AAV4), AAV type 5 virus (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), and AAV type 13 (AAV13).

[0016] Adeno-associated virus vectors (hereinafter referred to as "AAV vectors") can basically be produced without host infection by a helper virus by transiently co-transfecting HEK 293 cells with three plasmids: a transfer vector plasmid carrying a gene of interest between the AAV ITRs, a packaging plasmid expressing Rep and Cap, and a helper plasmid expressing E2A, E4, and VA-RNA.

[0017] The present invention provides expression cassettes for helper factors that can be used in place of the above-mentioned helper plasmids that express E2A, E4, and VA-RNA in the production of AAV vectors.

[0018] As used herein, the term "helper factor" refers to a gene for a factor that, when expressed in AAV vector-producing cells, can support the amplification of a virus that cannot grow on its own and increase the amount of AAV vector produced by the AAV vector-producing cells.

[0019] As used herein, the term "expression cassette" refers to a nucleic acid construct containing elements necessary for expressing a target product. Specifically, it refers to a nucleic acid construct containing at least a promoter and a nucleic acid for expressing the target product operably linked to the promoter. For example, a promoter derived from a mammal can be used in which a nucleic acid containing a coding region for a target protein is ligated downstream. Furthermore, although a polyA sequence is preferably added downstream of the promoter, this is not essential. As described below, various heterologous promoters can be used. Furthermore, nucleic acids containing coding regions for one or more proteins can be ligated downstream of the promoter. When coding regions for two or more proteins are used, a sequence enabling polycistronic expression can be used. Examples include an internal ribosome entry site (IRES) and a 2A self-cleaving peptide. Instead of a protein coding region, a nucleic acid containing a region encoding a non-coding RNA can also be used. Various heterologous polyA sequences can also be used downstream.

[0020] The expression cassette of the present invention is a nucleic acid construct used in the production of an AAV vector, and comprises a nucleic acid containing a helper factor that can increase the production yield of the AAV vector, and a heterologous promoter operably linked to the helper factor.

[0021] The nucleic acid containing a helper factor contained in the expression cassette of the present invention includes the following nucleic acids (1) to (4): (1) a nucleic acid containing a coding region of adenovirus E2A protein; (2) a nucleic acid containing a coding region of one or more proteins selected from adenovirus 22k protein, 100k protein, 33k protein, E4orf2 protein, E4orf3 protein, and E4orf6 / 7 protein; (3) a nucleic acid containing a coding region of adenovirus E4orf6 protein; and (4) a nucleic acid containing a template DNA of adenovirus VA-RNA.

[0022] As used herein, the term "coding region" refers to a portion of a genome region that encodes a protein (open reading frame (ORF)), and does not include promoters, ribosome binding sites, transcription terminators, introns, etc. Note that stop codons (TAG, TGA, or TAA) are included in the coding region, even though they are not translated into amino acids.

[0023] The nucleic acid containing the helper factors (1) to (4) above can be derived from adenovirus serotypes 1 to 52 or modified forms thereof. The helper factors may be natural or artificially mutated. For example, it is preferable to use nucleic acids derived from adenovirus type 5 or adenovirus type 2 as the nucleic acid containing the helper factors (1) to (4) above.

[0024] In one embodiment of the present invention, the nucleotide sequence of a nucleic acid comprising the coding region of adenovirus type 5 E2A protein is shown in SEQ ID NO: 1. In the present invention, the nucleic acid comprising the coding region of adenovirus E2A protein may be a nucleic acid comprising a nucleotide sequence having 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid in which several nucleotides (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) have been substituted, inserted, added, and / or deleted in the nucleotide sequence of SEQ ID NO: 1, as long as it has the activity of increasing the production yield of an AAV vector. Furthermore, codon-optimized nucleic acids may be used to optimize protein expression in mammalian cells.

[0025] In one embodiment of the present invention, the nucleotide sequence of a nucleic acid containing the coding region for the adenovirus type 5 22k protein is shown in SEQ ID NO: 2, the nucleotide sequence of a nucleic acid containing the coding region for the 100k protein is shown in SEQ ID NO: 3, and the nucleotide sequence of a nucleic acid containing the coding region for the 33k protein is shown in SEQ ID NO: 4. In the present invention, the nucleic acids containing the coding regions for the adenovirus 22k protein, 100k protein, and 33k protein may be nucleic acids containing a nucleotide sequence that has 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 2, 3, and 4, respectively, or nucleic acids in which several nucleotides (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) have been substituted, inserted, added, and / or deleted in the nucleotide sequences of SEQ ID NOs: 2, 3, and 4, as long as they have the activity of increasing the production yield of AAV vectors. Furthermore, codon-optimized nucleic acids may also be used to optimize the expression of each protein in mammalian cells.

[0026] In one embodiment of the present invention, the nucleotide sequence of a nucleic acid comprising the coding region of adenovirus type 5 E4orf2 protein is shown as SEQ ID NO: 5, the nucleotide sequence of a nucleic acid comprising the coding region of E4orf3 protein is shown as SEQ ID NO: 6, the nucleotide sequence of a nucleic acid comprising the coding region of E4orf6 protein is shown as SEQ ID NO: 7, and the nucleotide sequence of a nucleic acid comprising the coding region of E4orf6 / 7 protein is shown as SEQ ID NO: 8. In the present invention, the nucleic acids comprising the coding regions of adenovirus E4orf2 protein, E4orf3 protein, E4orf6 protein, and E4orf6 / 7 protein may be nucleic acids comprising a nucleotide sequence that has 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the nucleotide sequences of SEQ ID NOs: 5, 6, 7, and 8, or nucleic acids in which several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) nucleotides have been substituted, inserted, added, and / or deleted from the nucleotide sequences of SEQ ID NOs: 5, 6, 7, and 8, as long as they have the activity of increasing the production yield of AAV vector. Codon-optimized nucleic acids may also be used to optimize the expression of each protein in mammalian cells.

[0027] In one embodiment of the present invention, the nucleotide sequence of a nucleic acid comprising template DNA for adenovirus type 5 VA-RNA is shown in SEQ ID NO: 9. In the present invention, the nucleic acid comprising template DNA for adenovirus VA-RNA may be a nucleic acid comprising a nucleotide sequence that has 90% or more, preferably 95% or more, and more preferably 98% or more sequence identity to the nucleotide sequence of SEQ ID NO: 9, or a nucleic acid in which several nucleotides (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) have been substituted, inserted, added, and / or deleted from the nucleotide sequence of SEQ ID NO: 9, as long as it has the activity of increasing the production yield of an AAV vector. It may also be a nucleic acid comprising template DNA for VA-RNA-derived microRNA contained within the same sequence (Proc Natl Acad Sci USA. 1980 May; 77(5): 2424-2428).

[0028] When producing an AAV vector, all of the following are introduced into a virus-producing cell: (1) a nucleic acid containing the coding region of the adenovirus E2A protein; (2) a nucleic acid containing the coding region of one or more proteins selected from the adenovirus 22k protein, 100k protein, 33k protein, E4orf2 protein, E4orf3 protein, and E4orf6 / 7 protein; (3) a nucleic acid containing the coding region of the adenovirus E4orf6 protein; and (4) a nucleic acid containing the template DNA of the adenovirus VA-RNA.

[0029] The nucleic acids containing the above-mentioned helper factors used in the present invention may be separated into the same expression cassette or separate expression cassettes. For example, they may be separated into an expression cassette using nucleic acids containing the coding regions of the adenoviral E2A protein, 22k protein, and 100k protein, an expression cassette using nucleic acids containing the coding regions of the adenoviral E4orf2 protein, E4orf3 protein, E4orf6 protein, and E4orf6 / 7 protein, and an expression cassette using nucleic acid containing template DNA for adenoviral VA-RNA. The combination of nucleic acids contained in the expression cassettes is not limited.

[0030] The helper factors expressed by the same or separate expression cassettes of the present invention are a combination of nucleic acids containing the helper factors specified in (1) to (4) above. Here, the nucleic acid specified in (2) above is a nucleic acid containing a coding region for one or more proteins selected from the group consisting of the 22k protein, the 100k protein, the 33k protein, the E4orf2 protein, the E4orf3 protein, and the E4orf6 / 7 protein. Among these options, nucleic acids containing the coding region for the 22k protein and / or the E4orf3 protein are preferred from the viewpoint of improving helper activity. More preferably, nucleic acids containing the coding regions for the 22k protein and the 100k protein, or nucleic acids containing the coding regions for the E4orf2 protein, the E4orf3 protein, and the E4orf6 / 7 protein, or nucleic acids containing the coding regions for the 22k protein, the 100k protein, the E4orf2 protein, the E4orf3 protein, and the E4orf6 / 7 protein, can be used to further improve helper activity.

[0031] The combinations of nucleic acids containing the helper factors (1) to (4) are not limited, but preferred combinations include nucleic acids containing the coding regions of the E2A protein, E4orf6 protein, and 22k protein, nucleic acids containing the coding regions of the E2A protein, E4orf6 protein, 22k protein, and 100k protein, nucleic acids containing the coding regions of the E2A protein, E4orf6 protein, 22k protein, 100k protein, and 33k protein, nucleic acids containing the coding regions of the E2A protein, E4orf6 protein, and E4orf3 protein, nucleic acids containing the coding regions of the E2A protein, E4orf6 protein, and E4orf Examples of nucleic acids include nucleic acids containing the coding regions for E2A protein, E4orf6 protein, E4orf2 protein, E4orf3 protein, and E4orf6 / 7 protein, and nucleic acids containing the coding regions for E2A protein, E4orf6 protein, E4orf2 protein, E4orf3 protein, and E4orf6 / 7 protein, of which nucleic acids containing the coding regions for E2A protein, 100k protein, and 22k protein (E2Aopti), and nucleic acids containing the coding regions for E4orf2 protein, E4orf3 protein, E4orf6 protein, and E4orf6 / 7 protein (E4opti) are more preferred, and nucleic acids containing the coding regions for E2Aopti and E4opti are even more preferred (common for VA-RNA). Furthermore, the expression cassette of the present invention may be combined with the AAV Rep gene and Cap gene, which coexist in virus-producing cells when constructing an AAV vector as described below, as needed.

[0032] In the expression cassette of the present invention, a heterologous promoter is operably linked to the helper factor. Here, "heterologous promoter" refers to a promoter other than the intrinsic promoter that naturally exists upstream of each helper factor and controls (regulates) its transcription. In the present invention, the heterologous promoter is artificially introduced by genetic engineering. In contrast, the term "intrinsic promoter" refers to the opposite of a heterologous promoter, a promoter that exists in the genome of a non-genetically engineered host and under whose control a gene is originally expressed, in other words, a promoter that is originally operably linked to the gene.

[0033] Furthermore, as used herein, "operably linked" means that a regulatory sequence and a coding sequence are arranged in such a manner that the expression of a target protein encoded by the coding sequence can be controlled by the regulatory sequence. Specifically, this means that a heterologous promoter other than the promoter inherent to the helper factor is arranged with the helper factor in a state in which the helper factor can be expressed under the control of the heterologous promoter.

[0034] In the present invention, the heterologous promoter may be a constitutive promoter or an inducible promoter, as long as it is a promoter that can express the above-mentioned helper factor in mammalian cells. Examples of constitutive promoters include CBh promoters, cytomegalovirus (CMV)-derived promoters (e.g., CMV immediate early promoter), human immunodeficiency virus (HIV)-derived promoters (e.g., HIV LTR), Rous sarcoma virus (RSV)-derived promoters (e.g., RSV LTR), mouse mammary tumor virus (MMTV)-derived promoters (e.g., MMTV LTR), Moloney murine leukemia virus (MoMLV)-derived promoters (e.g., MoMLV LTR), SV40-derived promoters (e.g., SV40 early promoter), Epstein-Barr virus (EBV)-derived promoters, AAV-derived promoters (e.g., AAV p5 promoter), adenovirus (AdV)-derived promoters (Ad2 or Ad5 major late promoter), and other virus-derived vectors, mammalian-derived promoters (e.g., EF1α promoter, actin promoter, PGK promoter, etc.), and artificially created fusion promoters (e.g., CAG promoter, CBA promoter, etc.). An inducible promoter is a promoter that can induce expression of a nucleic acid operably linked to it only in the presence of an inducer that drives the promoter, and typically includes a drug-inducible promoter. Examples of drug-inducible promoters include tetracycline-inducible promoters, Cumate operator sequences, and lambda operator sequences (e.g., 12×lambda Op), with tetracycline-inducible promoters being preferred. Tetracycline-inducible promoters include promoters that induce gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline) or reverse tetracycline-controlled transactivator (rtTA). Specifically, the TRE promoter (a mammalian cell promoter having a Tet-responsive sequence with multiple consecutive tetO sequences) is suitable.When the TRE promoter is used, it is preferable to simultaneously express a fusion protein of tetR and VP16AD or a fusion protein (rtTA) of reverse tetR (rtetR) and P16AD in the same cell. In this Tet-On system, the fusion protein does not bind to the TRE in the absence of a drug, and transcription does not occur. However, upon addition of a drug, the fusion protein binds to the TRE promoter and transcription occurs, allowing transient expression of the desired helper factor during the drug addition period.

[0035] In one embodiment of the present invention, genome editing technology may be used as an inducible expression system for helper factors. Examples include inducible expression systems and repression systems that utilize the genome editing tool CRISPR / Cas9 system, such as an inducible expression system that uses transcription activation / inactivation by a protein formed by fusing an inactive Cas9 with a transcription activator such as VP64 and a transcription repressor such as KRAB. Similar to inducible expression systems are conditional expression systems that utilize recombinases. A representative example is an expression system that uses a LoxP-Stop-LoxP (LSL) cassette and removes the LSL cassette by introducing the recombinase Cre recombinase into cells, allowing the promoter to conditionally transcribe the gene.

[0036] Alternatively, the expression cassette may be incorporated into a plasmid used to produce an AAV vector and used as a helper plasmid. The plasmid used here is not particularly limited, but mammalian expression plasmids are preferred, such as pBApo-CMV DNA and pIRES Bicistronic Expression Vector (manufactured by Takara Bio Inc.). The plasmid may be appropriately selected depending on the type of cell to be transfected, and may be a cosmid, phage vector, phagemid vector, BAC vector, YAC vector, MAC vector, HAC vector, or the like.

[0037] When multiple expression cassettes are carried in the helper plasmid of the present invention, their arrangement is not particularly limited, but they are preferably arranged so that they are transcribed in the same direction, and include, in the direction of the transcription reading frame, a promoter region, an expression cassette of the present invention, a transcription terminator region, and a self-cleaving peptide sequence (e.g., a P2A sequence).

[0038] The expression cassette may be incorporated into a viral vector used in the production of an AAV vector and used as a helper virus. The helper virus used here is not particularly limited, and examples include non-enveloped viruses such as adenovirus, enterovirus, parvovirus, papovavirus, human papillomavirus, rotavirus, coxsackievirus, sapovirus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, circovirus, and simian virus; herpes viruses such as retrovirus, lentivirus, Sendai virus, and herpes simplex virus; and enveloped viruses such as vaccinia virus, measles virus, baculovirus, influenza virus, leukemia virus, Sindbis virus, and poxvirus.

[0039] The helper plasmid of the present invention may further include, as needed, a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, a reporter gene sequence such as a fluorescent protein, β-glucuronidase (GUS), or FLAG.

[0040] The AAV vector of the present invention is produced by co-transfecting the above-mentioned helper plasmid or helper virus into virus-producing cells with a vector plasmid (transfer plasmid) containing an expression cassette for a gene of interest between the ITRs at both ends of the adenovirus genome, and a plasmid (packaging plasmid) containing the AAV Rep gene and Cap gene, to produce cells that have acquired the ability to produce virus, and collecting the AAV vector of interest produced in the culture supernatant of the cells.

[0041] Known adeno-associated viruses include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), and AAV type 10 (AAV10), and any of these can be used to produce AAV vectors. Other examples include artificially produced AAV vectors such as AAV-DJ and AAV-PHP.B. Furthermore, technology for packaging and amplifying artificial nucleic acids within AAV capsids can also be used.

[0042] In the present invention, "virus-producing cells" refer to cells capable of producing viruses, into which elements necessary for forming virus particles have been introduced in such a manner that virus particles are formed and produced intracellularly. The "virus-producing cells" used in the present invention include all mammalian cells, including human cells, such as somatic cells constituting a living organism, precursor cells, and cancer cells, cells (cell lines) isolated from a living organism that have acquired immortalization ability and are stably maintained ex vivo, and cells isolated from a living organism that have been artificially genetically modified. The origin of the cells is not particularly limited as long as they are mammalian, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses.

[0043] Specifically, human cells include HEK293 cells, HEK293T cells derived from HEK293 cells, HEK293S cells, HEK293F cells, HEK293FT cells, HEK293FTM cells, HEK293SG cells, HEK293SGGD cells, HEK293H cells, HEK293E cells, HEK293MSR cells, and VPC2.0 cells. Commercially available cells developed for adenovirus (e.g., Adeno-X 293 Cell Line) and commercially available cells developed for AAV (e.g., AAVpro 293T Cell Line) can also be used. Other examples of human cells include G3T-hi cells (Takara Bio), HeLa cells (ATCC CCL-2), MOLT-4 cells (ATCC CRL-1582), human lung cancer-derived A549 cells, human fibrosarcoma HT-1080 cells, human retinal tissue-derived cells such as PER.C6 cells, human tissue-derived mesenchymal stem cells, cells derived from human uterine contents, placenta, and fetal tissue, and human liver-derived cells. Non-human primate cells include Vero cells, COS-1 cells (ATCC CRL-1650), and COS-7 cells (ATCC CRL-1651). Rodent cells include BHK cells, CHO cells (ATCC CCL-61), and HePa1-6 cells (ATCC CRL-1830).

[0044] Furthermore, examples of the "gene of interest" include human therapeutic genes and marker genes for evaluating gene transfer efficiency or expression stability, such as genes encoding GFP (Green Fluorescent Protein), β-galactosidase, and luciferase.

[0045] The above-mentioned plasmid can be transfected into virus-producing cells by any method that can introduce DNA into animal cells, and can be performed by methods known to those skilled in the art, such as electroporation, calcium phosphate method, lipofection, and DEAE-dextran method.

[0046] In the present invention, the medium used for culturing virus-producing cells may be a medium commonly used for culturing animal cells, specifically, a basal medium containing components necessary for cell survival and proliferation (e.g., inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins), such as Dulbecco's Modified Eagle's Medium (D-MEM), Dulbecco's Modified Eagle's Medium:Nutient Mixture F-12 (D-MEM / F-12), Glasgow MEM (G-MEM), Basal Medium Eagle (BME), Minimum Essential Medium (MEM), Eagle's minimal essential medium (EMEM), Iscove's Modified Dulbecco's Medium (IMDM), RPMI 1640, Medium 199, αMEM, Ham's medium, Fischer's medium, and mixtures thereof. Furthermore, the medium may contain growth factors (FGF, EGF, etc.), interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 supplements, N2 supplements, antibiotics (penicillin, streptomycin, etc.), etc., as needed. The medium may be serum-containing or serum-free. From the viewpoint of preventing contamination with components derived from different animal species, it is preferable to use serum-free medium or serum derived from the same animal species as the cells to be cultured. Serum substitutes, such as albumin, may also be used.

[0047] The virus-producing cells into which the plasmid has been introduced can be cultured under ordinary culture conditions. The culture temperature is not particularly limited, but is, for example, 30 to 40°C, preferably 35 to 37°C. In addition, CO ２ The concentration is, for example, 1 to 10%, preferably 5 to 6%. The culture time is not particularly limited, but is, for example, 24 to 120 hours, preferably 48 to 96 hours.

[0048] After culturing, the culture supernatant or cells are collected to obtain the AAV vector. In the present invention, the AAV vector is produced in the form of the supernatant, a filtrate obtained by filtering the supernatant, an extract obtained by disrupting the cells, or a concentrate or purified product concentrated or purified by a known method, and is stored by an appropriate method, for example, by freezing, until use.

[0049] The present invention also provides an adeno-associated virus (AAV) vector-producing cell, characterized in that an expression cassette used in the production of the AAV vector is stably maintained within the cell. In the present invention, the term "AAV vector-producing cell" refers to a virus-producing cell in which an expression cassette necessary for AAV vector production is stably maintained, and is different from a virus-producing cell in which an expression cassette has been transiently introduced. Here, the AAV vector-producing cell may be a cell in which a transgene has been integrated into the host genome of the cell and maintained persistently without being diluted even with cell division, or a cell in which a transgene can be autonomously replicated with cell division and continuously expressed without being integrated into the host genome of the cell, for example, by using an episomal vector.

[0050] The "AAV vector-producing cells" of the present invention stably maintain an expression cassette capable of inducibly expressing a helper factor that can increase the production amount of the AAV vector, and therefore, it is possible to induce expression of the helper factor with an inducer or an inducible promoter. Therefore, the AAV-producing cells of the present invention do not constantly express toxic proteins derived from the AAV components and helper factors used in AAV vector production, which have been a problem in the past, during cell maintenance and culture, so that the cells do not die or experience problems with cell proliferation, and there are no changes in cell functions, including AAV production ability.

[0051] Adeno-associated virus (AAV) vector-producing cells are prepared by gene transfer of an expression cassette used in the production of the AAV vector into cells. Gene transfer methods include chemical methods such as the liposome method and calcium phosphate method, physical methods such as electroporation, and methods using viral vectors. When using chemical or physical methods, the expression cassette may be introduced via a plasmid vector (e.g., a conventional plasmid vector or an episomal vector), or may be introduced (knocked-in) by transposon or genome editing methods. When using a viral vector, it can be introduced by infection with, for example, a retroviral vector, lentiviral vector, adeno-associated virus vector, adenovirus vector, Sendai virus vector, vaccinia virus vector, baculovirus vector, or the like. After gene transfer, clones in which the expression cassette is stably maintained in the cells can be selected by drug selection or limiting dilution.

[0052] Herein, the level of AAV vector production is indicated by the titer of the AAV vector. The titer of the AAV vector can be expressed either by the number of genomes obtained from normally formed AAV vector particles in a certain amount of sample (genomic titer) or by the experimentally measured ability of the AAV vector to infect cells (infectious titer). In the present invention, "genomic titer" refers to the amount of viral vector, and "infectious titer" refers to the efficiency (ability) of infection of a specific cell. The genome titer can be measured by a method of measuring the copy number of the viral vector, and the infectious titer can be measured by, for example, a method of infecting target cells with the viral vector and detecting the expression of the introduced gene.

[0053] The present invention will be described in more detail below with reference to the following examples. However, these examples are not intended to limit the present invention. 1. Experimental Methods The tests conducted in the examples and comparative examples were carried out according to the following methods. (1) Cell Culture AAV293 cells, an immortalized cell line derived from human fetal kidney, or cells (AAV293-rtTA) in which the reverse tetracycline-responsive transcriptional activator rtTA was transfected into AAV293, were cultured in D-MEM containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a 5% CO2 environment.

[0054] (2) Preparation of Adeno-Associated Virus (AAV) Vector Type 1 AAV vector type 1 was prepared using pAAV-AcGFP (a plasmid in which AcGFP was cloned from pAcGFP-N1 (Takara Bio) into pAAV-mcs (Agilent Technologies)), pAAV2 / 1 (Takara Bio), and each helper plasmid to be tested for helper activity.

[0055] The above-mentioned AAV1 plasmid mixture was mixed in OPTI-MEM with Polyethylenimine Max (PEI; Polysciences) at a DNA:PEI ratio of 1:2 and incubated at room temperature for 20 minutes. After incubation, the plasmid mixture was transfected into 80% or more confluent AAV293 or AAV293-rtTA cells in D-MEM containing 10% FBS for 6 hours and then cultured in serum-free D-MEM. If necessary, 10-1000 ng / ml of doxycycline was added as an inducer.

[0056] (3) Measurement of viral titer by quantitative PCR (qPCR) Genomic DNA of the AAV vector was extracted to measure viral titer. Specifically, the culture supernatant was collected and treated with Benzonase at 37°C for 1 hour, after which viral genomic DNA was extracted and purified using the DNeasy Blood & Tissue kit (QIAGEN). Viral titers were measured in triplicate by qPCR.

[0057] qPCR was performed using primers targeting AcGFP (Forward: 5'-ATCACATGAAGCAGCACGAC-3' (SEQ ID NO: 10), Reverse: 5'-TAGTTGCCGTCATCCTCGAA-3' (SEQ ID NO: 11), TaqMAN: 5'Fam-AGCCCTCAGGCATGGCGCTC-3'TAMRA (SEQ ID NO: 12)).

[0058] (4) Plasmid Construction pHelper-E2A, pHelper-E4, and pHelper-VA were constructed from the pHelper plasmid (Takara Bio) by restriction enzyme digestion and self-ligation. pCBh-E2A, pCBh-E4orf6, and pCBh-VA were constructed by PCR amplification of the E2A, E4orf6, and VA-RNA regions using the pHelper plasmid (Takara Bio) as a template and ligating them to the pCBh promoter. pCBh-E2A-22k-100k (E2Aopti) was constructed by PCR amplification of the E2A, 22k, and 100k coding regions to confer the 2A peptide, then ligating them to the pCBh promoter using overlapping PCR. pCBh-E4orf1-7, pCBh-E4orf2-7, pCBh-E4orf3-7, pCBh-E4orf4-7, and pCBh-E4orf6-7 were constructed by PCR amplification of E4orf1-7, E4orf2-7, E4orf3-7, E4orf4-7, and E4orf6-7, respectively, followed by ligation to the pCBh promoter. pCBh-E4-2367 (E4opti) and pCBh-E4-367 were constructed by PCR amplification of E4orf2, E4orf3, and E4orf6-7, respectively, to confer the 2A peptide, then ligated by overlapping PCR and ligated to the pCBh promoter. pTRE-E2Aopti-E4opti and pTRE-VA were constructed by artificial DNA synthesis. pTRE-E2Aopti-E4opti-VA was constructed by cloning the pTRE-E2Aopti-E4opti and pTRE-VA expression cassettes into the p15-amp plasmid (prepared by artificial DNA synthesis).

[0059] (5) Gene Expression Analysis of E2A, E4, and VA-RNA To analyze gene expression of E2A, E4, and VA-RNA, RNA was extracted from AAV293 cells or AAV293-rtTA cells. After gene transfection using the method described above, cells were harvested, and total RNA was extracted and purified using the RNeasy mini kit (QIAGEN). cDNA was synthesized from the total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).

[0060] The expression levels of E2A, E4, and VA-RNA genes were measured in triplicate by qRT-PCR. qPCR was performed using primers targeting E2A, E4, and VA-RNA, and the amount of S18rRNA was measured as an endogenous control for normalization. The primer sequences used for PCR are shown below.

[0061] (E2A) Forward: 5'-GATGTGACGAGCGAAAACG-3' (SEQ ID NO: 13) Reverse: 5'-CACGATCTTGGCCTTGCTA-3' (SEQ ID NO: 14) (E4) Forward: 5'-CCTGCAGTGTGGGATTTACG-3' (SEQ ID NO: 15) Reverse: 5'-GAACCGGGACTGGAACAATG-3' (SEQ ID NO: 16) TaqMAN: 5'Fam-TGGCACAACACAGGCACACGTGC-3' Tamra (SEQ ID NO: 17) (VA-RNA) Forward: 5'-GCAAAAGGAGAGCCTGTAAGC-3' (SEQ ID NO: 18) Reverse: 5'-GTCGTCCGCCATGATACC-3' (SEQ ID NO: 19)

[0062] 2. Experimental Results (Comparative Reference Example 1) (1) Analysis of Helper Activity of E2A Expression Cassettes To compare the helper activity of pHelper-E2A and pCBh-E2A, AAV vectors were constructed using these plasmids along with pHelper-E4, pHelper-VA, pAAV-AcGFP, and pAAV2 / 1 (Figure 1). Three days after transfection of the plasmids into the cells, the culture supernatant was collected and the amount of AAV vector produced was measured by quantitative PCR. It was revealed that pCBh-E2A had significantly reduced helper activity (vector production amount) compared to the original pHelper-E2A (Figure 2).

[0063] (2) Analysis of the helper activity of E4 expression cassettes. To compare the helper activity of pHelper-E4 and pCBh-E4orf6, AAV vectors were constructed using these plasmids with pHelper-E2A, pHelper-VA, pAAV-AcGFP, and pAAV2 / 1 (Figure 3). Three days after transfection, culture supernatants were collected and the amount of AAV vector produced was measured by quantitative PCR. It was revealed that the helper activity (vector production) of pCBh-E4orf6 was reduced to less than half that of the original pHelper-E4 (Figure 4).

[0064] Example 1 (1) Analysis of Helper Activity of Expression Cassettes for Proteins Contained in the E2A Genomic Fragment To confirm the helper activity of proteins contained in the E2A genomic fragment, AAV vectors were constructed using pCBh-E2A, pCBh-100k, pCBh-22k, and pCBh-33k, along with pHelper-E4, pHelper-VA, pAAV-AcGFP, and pAAV2 / 1 (Figure 5). Three days after transfection of the plasmids into cells, culture supernatants were collected and the amount of AAV vector production was measured by quantitative PCR. The results demonstrated that when introduced in combination with E2A, the genes encoding the 100k and 22k proteins both functioned to enhance helper activity, and their combination further enhanced helper activity (Figure 6). Furthermore, it was confirmed that the introduction of a gene encoding the 33k protein in combination with these genes further enhanced helper activity (Figure 6).

[0065] (2) Analysis of the helper activity of expression cassettes for E2A protein and proteins contained in the E2A genomic fragment. To investigate whether E2A, 100kJ, and 22kJ proteins could be expressed polycistronically, we constructed pCBh-E2A-22k-100k, in which the genes encoding E2A, 100kJ, and 22kJ proteins were linked to a DNA sequence encoding the 2A peptide and linked to the CBh promoter (Figure 7). To confirm the helper activity of pCBh-E2A-22k-100k, AAV vectors were constructed using pCBh-E2A-22k-100k, pHelper-E4, pHelper-VA, pAAV-AcGFP, and pAAV2 / 1. Three days after transfection, culture supernatants were collected and AAV vector production was measured by quantitative PCR. As a result, it was confirmed that the helper activity of pCBh-E2A-22k-100k was greater than or equal to that of the original pHelper-E2A (FIG. 8).

[0066] (3) Analysis of the helper activity of expression cassettes for proteins contained in the E4 genomic fragment. To verify the helper activity of the proteins contained in the E4 genomic fragment, we constructed pCBh-E4orf1-7 (SEQ ID NO: 20), pCBh-E4orf2-7 (SEQ ID NO: 21), pCBh-E4orf3-7 (SEQ ID NO: 22), pCBh-E4orf4-7 (SEQ ID NO: 23), pCBh-E4orf6 (SEQ ID NO: 7), and pCBh-E4orf6-7 (SEQ ID NO: 24) (Figure 9). AAV vectors were prepared using these plasmids along with pHelper-E2A, pHelper-VA, pAAV-AcGFP, and pAAV2 / 1. Three days after transfection of the plasmids into the cells, culture supernatants were collected and the production of AAV vectors was measured by quantitative PCR. The results demonstrated that E4orf1-7, E4orf2-7, and E4orf3-7 all had helper activity equivalent to that of the original E4 genomic fragment (Figure 10).

[0067] (4) Polycistronic Expression of Proteins Contained in the E4 Genomic Fragment Next, to examine whether E4orf2-7 and E4orf3-7 can be expressed polycistronically, we constructed pCBh-E4orf2367 and pCBh-E4orf367, in which E4orf2, E4orf3, E4orf6, and E4orf6-7 were linked via the 2A peptide and linked to the CBh promoter (Figure 11). To confirm their helper activity, we constructed AAV vectors using pCBh-E4orf2367 and pCBh-E4orf367 with pHelper-E2A, pHelper-VA, pAAV-AcGFP, and pAAV2 / 1. Three days after transfection, culture supernatants were collected and AAV vector production was measured by quantitative PCR. As a result, it was confirmed that the helper activity of pCBh-E4orf2367 and pCBh-E4orf367 was equivalent to that of the original pHelper-E4 (FIG. 12).

[0068] (5) Analysis of the helper activity of VA-RNA expression cassettes. To verify the helper activity of VA-RNA, AAV vectors were constructed using pCBh-VA, pHelper-E2A, pHelper-E4, pAAV-AcGFP, and pAAV2 / 1. Three days after transfection, the culture supernatant was collected and the amount of AAV vector produced was measured by quantitative PCR (Figure 13). The results demonstrated that pCBh-VA had the same helper activity as the original VA-RNA genomic fragment (Figure 14).

[0069] Example 2 (1) Helper Activity Analysis of Optimized Helper Factor Expression Cassettes pCBh-E2A-22k-100k (pCBh-E2Aopti: SEQ ID NO: 25) and pCBh-E4orf2367 (pCBh-E4opti: SEQ ID NO: 26), which were constructed by combining factors considered to be optimal among the factors whose helper activity was confirmed in Example 1, and pCBh-VA were used simultaneously to verify their helper activity. To evaluate the helper activity, AAV vectors were constructed using these plasmids along with pAAV-AcGFP and pAAV2 / 1 ( FIG. 15 ). Three days after transfection of the plasmids into the cells, culture supernatants were collected, and the amount of AAV vector produced was measured by quantitative PCR. As a result, it was revealed that pCBh-E2Aopti, pCBh-E4opti, and pCBh-VA (optimized) had helper activity 2.7 times higher than that of the existing pHelper-E2A, pHelper-E4, and pHelper-VA (conventional types) (Figure 16).

[0070] (2) Analysis of inducible gene expression and helper activity in an inducible expression system using optimized helper factors. We constructed pTRE-E2Aopti-TRE-E4opti and pTRE-VA, in which the optimized helper factors were linked to the inducible promoter TRE (Figure 17). To verify the inducible gene expression of the inducible expression system using the optimized helper factors, we produced AAV vectors using these plasmids along with pAAV-AcGFP and pAAV2 / 1 (Figure 18). Transfected cells were harvested, and the relative expression levels of E2A, E4, and VA-RNA were measured. The results showed that in the inducible expression system using the optimized helper factors, gene expression of E2A, E4, and VA-RNA was suppressed in the absence of inducers, but gene expression was induced in an inducer-dependent manner, with the expression levels being significantly higher than those of the conventional pHelper (Figures 19 and 20). Next, to verify the helper activity of the inducible expression system using the optimized helper factors, we collected culture supernatants 3 days after transfection of the plasmid into cells and analyzed the amount of AAV vector production. In the inducible expression system using the optimized helper factors, AAV vector production was suppressed without the addition of an inducer, and vector production was induced in an inducer-dependent manner. Furthermore, the amount of production was significantly higher than that of the conventional pHelper (Figure 21).

[0071] (3) Analysis of helper activity using optimized helper factors. Next, we constructed a plasmid containing an inducible expression system for all of the optimized helper factors. To verify their helper activity, we attempted AAV vector production using this plasmid, pAAV-AcGFP, and pAAV2 / 1 (Figure 22). The results demonstrated that in the inducible expression system using the optimized helper factors, AAV vector production was suppressed without the addition of an inducer, and vector production was induced in an inducer-dependent manner. Furthermore, we confirmed that the production yield was significantly higher (3.1-fold) than that of the conventional pHelper (Figure 23).

[0072] The present invention can be used in the fields of gene cell therapy, vaccines, and the production of viral vectors that serve as tools for gene function analysis. All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

1. An expression cassette used for producing an adeno-associated virus (AAV) vector, comprising a nucleic acid containing a helper factor capable of increasing the production yield of the AAV vector and a heterologous promoter operably linked to the helper factor, wherein the nucleic acid comprises any one of the following nucleic acids (1) to (4): (1) a nucleic acid containing a coding region for an adenovirus E2A protein; (2) a nucleic acid containing a coding region for one or more proteins selected from the group consisting of an adenovirus 22k protein, a 100k protein, a 33k protein, an E4orf2 protein, an E4orf3 protein, and an E4orf6 / 7 protein; (3) a nucleic acid containing a coding region for an adenovirus E4orf6 protein; or (4) a nucleic acid containing a template DNA for an adenovirus VA-RNA.

2. The expression cassette according to claim 1, wherein each of the helper factors (1) to (4) is contained in the same or separate expression cassettes.

3. The expression cassette of claim 1, wherein the heterologous promoter is a constitutive promoter or an inducible promoter.

4. A helper plasmid or helper virus for use in producing an adeno-associated virus (AAV) vector, comprising the expression cassette of any one of claims 1 to 3.

5. A method for producing an adeno-associated virus (AAV) vector, comprising the step of transfecting a virus-producing cell with the helper plasmid or helper virus described in claim 4, a transfer plasmid containing an expression cassette for a gene of interest between the ITRs at both ends of the adenovirus genome, and a packaging plasmid containing the Rep gene and Cap gene of the adeno-associated virus.

6. An adeno-associated virus (AAV) vector-producing cell, characterized in that the expression cassette according to any one of claims 1 to 3 is stably maintained within the cell.

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