Construct for expression of adeno-associated virus rep protein and use thereof

Abstract

The present invention provides a construct for expression of an adeno-associated virus (AAV) Rep protein, said construct being characterized by comprising a nucleic acid sequence encoding Large Rep and Small Rep, wherein: an intron is inserted into the nucleic acid sequence encoding the Large Rep and the Small Rep; the intron includes a pair of recombinant sequences, which consists of a first sequence and a second sequence, and a transcription termination sequence; and the construct is configured (1) to delete, by DNA splicing, the first sequence and the transcription termination sequence when recombination occurs on the basis of the pair of recombinant sequences and (2) not to contain a p5 promoter.

Description

Constructs for expression of adeno-associated virus Rep proteins and uses thereof

[0001] The present invention relates to constructs for the expression of adeno-associated virus Rep proteins and uses thereof.

[0002] For diseases for which no satisfactory treatment or prevention methods have yet been established, gene therapy is being considered as a therapeutic or preventative approach. In gene therapy, a gene thought to be related to the treatment or prevention of a disease is introduced into the patient's cells, and the gene is expressed in the patient's body, thereby achieving treatment or prevention of the disease.

[0003] Methods for delivering genes to patient cells have been developed, including administering nucleic acids encoding the desired gene to the patient and administering viral vectors carrying nucleic acids encoding the desired gene to the patient, but currently, gene delivery using adeno-associated viral vectors is the gold standard because adeno-associated viral vectors have the following characteristics: (1) relatively high gene delivery efficiency to patient cells, (2) long-term gene expression, (3) the ability to infect both dividing and quiescent cells, and (4) no toxicity to patient cells.

[0004] Adeno-associated virus (AAV) was discovered in 1965 as a contaminant in adenovirus preparations. AAV has a linear, single-stranded DNA genome of approximately 4.7 kb, containing two inverted terminal repeats (ITRs) at its termini. Two open reading frames (rep and cap genes) encoding the Rep and Cap proteins are located between the two ITRs.

[0005] The rep gene encodes four distinct Rep proteins (Rep78, Rep68, Rep52, and Rep40), whose expression is controlled by two alternative promoters (p5 and p19). Furthermore, two alternative splicing events result in the generation of four Rep proteins. These Rep proteins possess functions such as DNA binding, endonuclease, and helicase activity, and play roles in gene replication and packaging. The cap gene encodes three capsid proteins (VP1, VP2, and VP3) and one assembly activation protein. Differential expression of these proteins is achieved by alternative splicing and alternative start codon usage. Transcription of the cap gene is controlled by a promoter (p40) located within the coding region of the rep gene.

[0006] At present, a great deal of knowledge has been accumulated regarding producer cells capable of efficiently producing recombinant AAV vectors for gene therapy and nucleic acid constructs capable of highly regulating the transcription of proteins involved in the production of AAV vectors in host cells (Patent Documents 1 and 2). However, from the viewpoints of popularizing gene therapy and reducing the cost of treatment, there is a strong demand for further improvement in the efficiency of AAV vector productivity.

[0007] US2022-177854A1US2022-154223A1

[0008] An objective of the present invention is to provide a novel nucleic acid construct that can further increase the efficiency of AAV vector production.

[0009] As described above, the expression of the AAV rep gene is controlled by two endogenous promoters (p5 and p19). Generally, deletion of a promoter reduces the expression of genes controlled by that promoter. Therefore, deletion of the p5 promoter, which can reduce the expression of Rep proteins (particularly Large Rep) important for AAV genome replication, is a modification that should be avoided for the purpose of improving the efficiency of AAV vector replication, i.e., production efficiency. Given this background, the present inventors discovered that by deleting only p5 of the two promoters controlling rep gene transcription, (1) the four Rep proteins necessary for AAV vector production are expressed without any problems, (2) the genome integrity and intact particle rate of the produced AAV vector genome are improved, and (3) the apoptosis induction rate of host cells is maintained low during Rep protein expression in host cells. Based on these findings, further research led to the completion of the present invention. Specifically, the present invention is as follows:

[0010] [1] A construct for expressing adeno-associated virus (AAV) Rep proteins, the construct comprising nucleic acid sequences encoding Large Rep and Small Rep, wherein an intron is inserted within the nucleic acid sequences encoding Large Rep and Small Rep, wherein the intron comprises a pair of recombination sequences consisting of a first sequence and a second sequence and a transcription termination sequence, the construct being characterized in that (1) the construct is configured so that when recombination occurs based on the pair of recombination sequences, the first sequence and the transcription termination sequence are deleted by DNA splicing, and (2) the construct does not comprise a p5 promoter. [2] The construct according to [1], wherein Large Rep is either or both of Rep78 and Rep68, and Small Rep is either or both of Rep52 and Rep40. [3] The construct according to [1] or [2], wherein the construct further comprises a marker gene in an intron. [4] The construct according to [3], wherein the marker gene is a drug resistance gene. [5] The construct according to [3], wherein the intron comprises, in the 5' to 3' direction, a first recombination sequence, a transcription termination sequence, a marker gene, and a second recombination sequence. [6] The construct according to any of [1] to [5], wherein the transcription termination sequence is a polyA signal sequence. [7] The construct according to any of [1] to [6], wherein the pair of recombination sequences is a sequence selected from the group consisting of a Lox sequence, an FRT sequence, and a rox sequence. [8] The construct according to any of [1] to [7], wherein the construct further comprises a gene encoding a recombinase. [9] The construct according to any of [1] to [8], further comprising genes for other helper factors necessary for AAV production.

[10] A cell comprising the construct according to any of [1] to [9].

[11] The cell according to

[10] , further comprising an E2A gene, an E4orf6 gene and / or a VARNA gene derived from an adenovirus.

[12] Use of the construct for expression of the adeno-associated virus (AAV) rep protein according to any one of [1] to [9] for producing an adeno-associated virus vector.

[0011] According to the present invention, high-quality AAV vectors can be produced extremely efficiently.

[0012] FIG. 1 is a schematic diagram of the pRep-loxP_v1 construct. FIG. 2 is a schematic diagram of the pRep-loxP_v2 construct. FIG. 3 is a diagram showing the results of Western blotting performed to confirm Cre-induced expression of the rep gene in pRep-loxP_v1 and pRep-loxP_v2. FIG. 4 is a diagram showing the results of comparing the AAV vector production yield when pRep-loxP_v1 and pRep-loxP_v2 were used by droplet digital PCR. FIG. 5 is a diagram showing the results of comparing the AAV vector genome integrity when pRep-loxP_v1 and pRep-loxP_v2 were used by droplet digital PCR. FIG. 6 is a schematic diagram of the pTetVA_Rep-loxP_v1 construct. Figure 7 is a schematic diagram of the pTetVA_Rep-loxP_v2 construct. Figure 8 is a schematic diagram of the pTetE4_E2A construct. Figure 9 is a diagram showing the results of comparing AAV vector production yields when pTetVA_Rep-loxP_v1 and pTetVA_Rep-loxP_v2 are used by droplet digital PCR under inducible expression conditions of helper factors. Figure 10 is a diagram showing the results of comparing AAV vector genome integrity when pTetVA_Rep-loxP_v1 and pTetVA_Rep-loxP_v2 are used by droplet digital PCR under inducible expression conditions of helper factors. Figure 11 is a schematic diagram of the pTetE4_E2A_Cre construct. Figure 12 shows the results of comparing the AAV vector production yields when pTetVA_Rep-loxP_v1 and pTetVA_Rep-loxP_v2 were used by droplet digital PCR under conditions for inducing expression of helper factors and Cre recombinase. Figure 13 shows the results of comparing the AAV vector genome integrity when pTetVA_Rep-loxP_v1 and pTetVA_Rep-loxP_v2 were used by droplet digital PCR under conditions for inducing expression of helper factors and Cre recombinase.Figure 14 shows the results of an infection test comparing cell infectivity when pTetVA_Rep-loxP_v1 and pTetVA_Rep-loxP_v2 were used under conditions for inducing expression of helper factors and Cre recombinase. Figure 15 shows the results of a comparison of the complete particle rate calculated from ELISA and droplet digital PCR measurements when pTetVA_Rep-loxP_v1 and pTetVA_Rep-loxP_v2 were used under conditions for inducing expression of helper factors and Cre recombinase. Figure 16 shows the results of Western blotting performed to confirm the induced expression of the rep gene by addition of an inducer when pTetVA_Rep-loxP_v2 and pTetE4_E2A_Cre were used. Figure 17 shows the results of measuring the cell apoptosis induction rate by Annexin V assay when pTetVA_Rep-loxP_v1 and pTetVA_Rep-loxP_v2 were used under Cre recombinase expression. Figure 18 shows the results of reverse transcription-qPCR performed to confirm the induced expression of Rep, VA, E2A, and E4 genes by adding an inducer to stable clones generated using pTetVA_Rep-loxP_v2_Km and pTetE4_E2A_Cre. Figure 19 shows the amount of AAV vector produced by transient transfection of pTRE-Cap2_ITR-CMV-GFP-ITR into stable clones generated using pTetVA_Rep-loxP_v2_Km and pTetE4_E2A_Cre. FIG. 20 shows the results of a transduction test of an AAV vector produced by transient transfection of pTRE-Cap2_ITR-CMV-GFP-ITR into a stable cell line prepared using pTetVA_Rep-loxP_v2_Km and pTetE4_E2A_Cre.

[0013] The present invention will be described in detail below. In this specification, adeno-associated virus may be referred to as "AAV." In addition, in this specification, the term "Rep protein" refers to Large Rep and Small Rep, unless otherwise specified in the context. In addition, unless otherwise specified in the context, Large Rep refers to either or both of Rep78 and Rep68, and Small Rep refers to either or both of Rep52 and Rep40.

[0014] 1. Construct for Expression of Adeno-Associated Virus Rep Protein The present invention provides a construct for expression of AAV Rep protein, the construct comprising nucleic acid sequences encoding Large Rep and Small Rep, wherein an intron is inserted within the nucleic acid sequences encoding Large Rep and Small Rep, and wherein the intron comprises a pair of recombination sequences consisting of a first sequence and a second sequence, and a transcription termination sequence, wherein the construct is configured such that: (1) when recombination occurs based on the pair of recombination sequences, the first sequence and the transcription termination sequence are deleted by DNA splicing, and (2) does not comprise a p5 promoter (hereinafter, this construct may be referred to as the "construct of the present invention").

[0015] In one embodiment of the present invention, Large Rep may be either or both of Rep78 and Rep68, and Small Rep may be either or both of Rep52 and Rep40. Also, in a preferred embodiment of the present invention, Large Rep may be both Rep78 and Rep68, and Small Rep may be both Rep52 and Rep40. In other words, in this embodiment, the construct of the present invention comprises nucleic acid sequences encoding four Rep proteins: Rep78, Rep68, Rep52, and Rep40.

[0016] Constructs of the present invention comprise nucleic acid sequences encoding Large Rep and Small Rep proteins (hereinafter, also referred to as "rep genes"). In a preferred embodiment, the nucleic acid sequence encoding the Rep proteins may be the nucleic acid sequence represented by SEQ ID NO: 1 or 24-34, or a nucleic acid sequence substantially identical to the nucleic acid sequence represented by SEQ ID NO: 1 or 24-34. Examples of nucleic acid sequences substantially identical to the nucleic acid sequence represented by SEQ ID NO: 1 or 24-34 include nucleic acids comprising a nucleic acid sequence having about 60% or more, preferably about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more identity or similarity to the nucleic acid sequence represented by SEQ ID NO: 1 or 24-34, and encoding a protein having the same quality of activity as a wild-type Rep protein. In addition, the identity or similarity of nucleic acid sequences in this specification can be calculated using the identity or similarity calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3).Other algorithms for determining the identity or similarity of nucleic acid sequences include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993) [this algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997))], Needleman et al., J. Mol. Biol. , 48:444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4:11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CCG sequence alignment software package], the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], and the like can also be preferably used.

[0017] The nucleic acid encoding the Rep protein contained in the construct of the present invention has an intron inserted therein. The sequence of the inserted intron is not particularly limited as long as the intron portion is excised during the RNA maturation process, and any existing intron sequence may be used. An example includes, but is not limited to, human chorionic gonadotrophin (hCG) intron 1 (accession number X00265.1).

[0018] In the construct of the present invention, the position at which the intron is inserted is not particularly limited as long as it is within the nucleic acid sequence encoding the Rep protein. In one embodiment, the intron may be inserted at position 702 or 1020 of the rep gene (SEQ ID NO: 1), but is not limited thereto.

[0019] The intron inserted into the construct of the present invention comprises a pair of recombination sequences consisting of a first sequence and a second sequence, and a transcription termination sequence.

[0020] The pair of recombination sequences contained in the intron may be any as long as they can be configured so that when recombination occurs based on the recombination sequences, the first sequence and the transcription termination sequence are deleted by DNA splicing. Examples of the pair of recombination sequences include, but are not limited to, Lox sequences (e.g., LoxP sequence, Lox2272 sequence, Lox71 sequence, Lox511 sequence, LoxFAS sequence, etc.), FRT sequence, and rox sequence. In a preferred embodiment, the pair of recombination sequences may be a LoxP sequence (SEQ ID NO: 2 (the nucleic acid sequences of the first and second sequences of the LoxP sequence are the same)).

[0021] In the presence of a recombinase that induces recombination, the pair of recombination sequences induces genetic recombination in the construct of the present invention. This recombination results in the deletion of the first sequence and the transcription termination sequence by DNA splicing. The transcription termination sequence is excised by DNA splicing, resulting in the generation of a precursor mRNA encoding the Rep protein. The resulting precursor mRNA undergoes RNA splicing to remove an intron containing the second sequence, resulting in the generation of a mature mRNA encoding the Rep protein. Translation of this mature mRNA produces Large Rep and Small Rep proteins.

[0022] The recombinase may be supplied by including a recombinase gene in the construct of the present invention, or by including the recombinase gene in a construct separate from the construct of the present invention and co-transfecting the construct of the present invention with the separate construct. In one embodiment, the construct of the present invention further includes a gene encoding a recombinase. The recombinase selected is one necessary for the pair of recombination sequences contained in the construct of the present invention to cause recombination. For example, when the pair of recombination sequences are Lox sequences (e.g., LoxP sequences), the recombinase may be Cre. When the pair of recombination sequences are FRT sequences, the recombinase may be FLP. When the pair of recombination sequences are rox sequences, the recombinase may be Dre.

[0023] Furthermore, the transcription termination sequence contained in the intron may be any nucleic acid sequence capable of terminating the transcription reaction by RNA polymerase. In the construct of the present invention, the transcription termination sequence may be a single sequence or a combination of two or more sequences. Furthermore, multiple sequences of the same sequence may be used, or multiple sequences of two or more sequences may be used. Examples of transcription termination sequences include, but are not limited to, polyA signal sequences. In a preferred embodiment, the transcription termination sequence may be a polyA signal sequence. In another embodiment, the transcription termination sequence may be a sequence in which three polyA signal sequences are linked in tandem.

[0024] In one embodiment of the construct of the present invention, the intron may contain a marker gene. The marker gene contained in the intron may be any gene that allows screening of host cells in which the desired genetic modification has occurred. Examples of marker genes include, but are not limited to, drug (antibiotic) resistance genes, genes encoding fluorescent proteins, auxotrophic marker genes, and toxin resistance genes. In a preferred embodiment, the marker gene may be a drug resistance gene.

[0025] Drug resistance genes that can be used as marker genes are not particularly limited. Examples include, but are not limited to, hygromycin resistance genes, kanamycin resistance genes, neomycin resistance genes, streptomycin resistance genes, puromycin resistance genes, blasticidin resistance genes, and zeocin resistance genes. In a preferred embodiment, the marker gene may be a hygromycin resistance gene.

[0026] In an embodiment in which a marker gene is contained in an intron, it is preferable that when recombination occurs based on a pair of recombination sequences, the marker gene is also deleted by DNA splicing together with the first sequence and transcription termination sequence. In such an embodiment, the intron preferably contains, from 5' to 3', the first recombination sequence, the transcription termination sequence, the marker gene, and the second recombination sequence, although the order of the transcription termination sequence and the marker gene may be reversed. Furthermore, the insertion position of the cassette containing the first recombination sequence, the transcription termination sequence, the marker gene, and the second recombination sequence in the intron is not particularly limited as long as the desired effect is achieved, and it may be inserted at any position. For example, when hCG intron 1 is used as the intron, it can be inserted at position 196 of hCG intron 1, but is not limited thereto.

[0027] The construct of the present invention is characterized by not containing a p5 promoter. As demonstrated in the following Examples, intentional deletion of the p5 promoter enables the production of high-quality AAV vectors.

[0028] In one aspect, the construct of the present invention is characterized in that it does not contain a cap gene.

[0029] In one embodiment, the construct of the present invention may be in the form of circular DNA. The construct of the present invention may further include an origin of replication or an antibiotic resistance gene so that it can be replicated in a microorganism such as E. coli.

[0030] Specific examples of the construct of the present invention include, but are not limited to, the nucleic acid sequence represented by SEQ ID NO: 5 (pRep-LoxP_v2) or SEQ ID NO: 9 (pTetVA_Rep-LoxP_v2).

[0031] By introducing the construct of the present invention and other helper factors necessary for AAV production (i.e., adenovirus-derived E2A gene, E4orf6 gene, and / or VARNA gene) into host cells, AAV vectors can be produced in host cells. Note that the construct of the present invention may or may not contain other helper factors necessary for AAV production. In one embodiment, the construct of the present invention may further contain a VARNA gene.

[0032] 2. Cells The present invention also provides cells containing the constructs of the present invention (hereinafter, sometimes referred to as "cells of the present invention").

[0033] The cell of the present invention is characterized by containing the construct of the present invention. A method for introducing the nucleic acid construct into the cell may be a method known per se. The cell of the present invention may be one into which the construct of the present invention has been transiently introduced, or one into which the construct of the present invention has been stably introduced into the genome of the cell.

[0034] The cell type of the cells of the present invention is also not particularly limited, as long as the construct of the present invention functions and Rep proteins are expressed in the cells. In one embodiment, the cells of the present invention may be mammalian cells. Examples of mammalian cells include, but are not limited to, cells derived from mammals such as humans, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, and monkeys. The mammal is preferably human. Specific examples of mammalian cells include, but are not limited to, HEK293 cells, HEK293T cells, COS-7 cells, Vero cells, Chinese hamster ovary cells, rat GH3 cells, human FL cells, HeLa cells, and HepG2 cells.

[0035] The cells of the present invention can be converted into AAV vector producer cells by introducing, in addition to the construct of the present invention, other helper factors necessary for AAV production (i.e., the adenovirus-derived E2A gene, E4orf6 gene, and / or VARNA gene). AAV vectors produced by AAV vector producer cells containing the construct of the present invention have improved genome integrity and a rate of intact particles. Furthermore, when Rep proteins are expressed in AAV vector producer cells, the apoptosis induction rate of the cells is maintained low. Therefore, when the cells of the present invention are used as AAV vector producer cells, high-quality AAV vectors can be produced with high efficiency.

[0036] Specific examples of the cells of the present invention include, but are not limited to, cells comprising the nucleic acid sequences represented by the following SEQ ID NOs: (1) SEQ ID NO: 5 (pRep-loxP_v2) (2) SEQ ID NO: 3 (pAAV-Cre) and SEQ ID NO: 5 (pRep-loxP_v2) (3) SEQ ID NO: 3 (pAAV-Cre), SEQ ID NO: 5 (pRep-loxP_v2), SEQ ID NO: 6 (pHelper), and SEQ ID NO: 7 (pCap2) (4) SEQ ID NO: 9 (pTetVA_Rep-LoxP_v2) (5) SEQ ID NO: 3 (pAAV-Cre), SEQ ID NO: 9 (pTetVA_Rep-LoxP_v2), SEQ ID NO: 10 (pTetE4_E2A), and SEQ ID NO: 7 (pCap2) (6) SEQ ID NO: 9 (pTetVA_Rep-LoxP_v2), SEQ ID NO: 11 (pTetE4_E2A_Cre) and SEQ ID NO: 7 (pCap2) (7) SEQ ID NO: 9 (pTetVA_Rep-LoxP_v2) and SEQ ID NO: 11 (pTetE4_E2A_Cre) (8) SEQ ID NO: 3 (pAAV-Cre) and SEQ ID NO: 9 (pTetVA_Rep-LoxP_v2)

[0037] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0038] Example 1: Preparation of a novel construct for expressing Rep proteins (1) Plasmids The following plasmids (i) to (iii) were used.

[0039] (i) pAAV-Cre (TaKaRa Bio) (SEQ ID NO: 3): a plasmid containing a Cre recombinase expression cassette downstream of a CMV promoter between ITRs.

[0040] (ii) pRep-loxP_v1 (SEQ ID NO: 4): A plasmid for inducible expression of Rep using the Cre / loxP system. This was constructed as shown in FIG. 1 with reference to the construct of Qiao et al. (2002, Journal of Virology 76:13015-13027). Human chorionic gonadotrophin (hCG) intron 1 (accession number X00265.1) was inserted into Rep downstream of the p5 promoter (position 1022 in AAV-2 nucleotide NCBI Reference Sequence NC_001401 (position 702 in SEQ ID NO: 1 in the present application)), and a cassette consisting of a loxP-SV40 polyA triple repeat sequence-hygromycin resistance gene-loxP was inserted into position 196 of hCG intron 1.

[0041] (iii) pRep-loxP_v2 (SEQ ID NO: 5): a plasmid obtained by deleting the p5 promoter located immediately before the rep gene from pRep-loxP_v1 (Figure 2).

[0042] (2) Transfection and cell recovery HEK293 cells (Agilent, AAV-293) suspended in DMEM (Sigma) containing 10% FBS (Gibco) and 1 mM sodium pyruvate (Sigma) were seeded into a 6-well cell culture plate (Corning). Then, the cells were incubated at 37°C and 5% CO until the cells became approximately 70-80% confluent. ２ The cells were cultured in an incubator at 4°C. Plasmid DNA for transfection was prepared in serum-free DMEM (200 μL) so that the total amount of plasmid DNA was 1.9 μg. When multiple types of plasmids were used, the weight of DNA was adjusted to be the same.

[0043] In Example 1, transfection was performed using the following combinations: (a) pRep-loxP_v1 only (b) pAAV-Cre and pRep-loxP_v1 (c) pRep-loxP_v2 only (d) pAAV-Cre and pRep-loxP_v2

[0044] PEIpro (Polyplus) was added to the plasmid DNA solution in an amount 1.5 times the amount of DNA, mixed, and then left to stand at room temperature for 30 minutes to prepare a PEI + DNA complex. The culture supernatant of HEK293 cells cultured in a 6-well plate was removed and replaced with fresh DMEM medium (2 mL) containing 10% FBS and 1 mM sodium pyruvate. The PEI + DNA complex was then gently added, and the mixture was incubated at 37°C and 5% CO ２ The cells were incubated at 4°C for 3 days. After 3 days, the medium supernatant was removed, washed with DPBS (Wako), and then detached using TrypLE select (Gibco). The detached solution was suspended in DMEM medium containing 10% FBS and 1 mM sodium pyruvate, and after cell counting, 2 × 10 cells were collected. ５ The cell suspension containing the cells was centrifuged, and the supernatant was removed to obtain a cell pellet.

[0045] (3) Confirmation of Rep Inducible Expression System by Western Blotting The obtained cell pellet was suspended in 18.5 μL of DPBS. 7.5 μL of LDS Sample Buffer (Invitrogen) and 1 μL of Endonuclease (Kaneka) were added to the cell suspension and incubated at 37°C for 1 hour. 3 μL of Reducing Agent (Invitrogen) was added, and the mixture was incubated at 95°C for 5 minutes to prepare a sample for SDS-PAGE.

[0046] Electrophoresis was performed using an XV PANTERA SYSTEM (DRC), and after electrophoresis, proteins were transferred to a membrane using a Trans-Blot (registered trademark) Turbo (trademark) Transfer System (Bio-Rad). An iBind (trademark) Flex Western Device (Invitrogen) was used for antibody reactions. For the housekeeping gene GAPDH, the primary antibody GAPDH (D16H11) XP Rabbit mAb (CST) and the secondary antibody Anti-rabbit IgG (NA934VS) (Cytiva) were used, and for Rep, the primary antibody Anti-Adeno-Associated Virus Replicase (61069) (PROGEN) and the secondary antibody Anti-mouse IgG (NA931VS) (Cytiva) were used. The detection reagent used was ImmunoStar LD (Fujifilm Wako Pure Chemical Industries). Chemiluminescence Solution A and Chemiluminescence Solution B were mixed in a 1:1 ratio and added dropwise to the membrane, which was then left to stand for 2 minutes in the dark and then detected with ImageQuant™ 800 (Cytiva). The results are shown in Figure 3.

[0047] As shown in FIG. 3, in both pRep-loxP_v1 and pRep-loxP_v2, Rep expression was enhanced in the presence of Cre, demonstrating that inducible expression using the Cre / loxP system was functioning.

[0048] [Example 2] Production of AAV using novel constructs 1 (1) Plasmids The following plasmids (i) to (v) were used. (i) pAAV-Cre (same as in Example 1) (ii) pRep-loxP_v1 (same as in Example 1) (iii) pRep-loxP_v2 (same as in Example 1) (iv) pHelper (cell biolabs) (SEQ ID NO: 6): a plasmid containing adenovirus E2A, VA, and E4. (v) pCap2 (SEQ ID NO: 7): a plasmid for expressing AAV2 Cap. The Rep expression cassette was removed by inverse PCR from pRC2 (cell biolabs), leaving behind an expression cassette for AAV2 Cap containing the p40 promoter.

[0049] (2) AAV Production and Viral Vector Extraction The above-mentioned plasmids were transfected into HEK293 cells by the same method as described in Example 1. The four types of plasmids were mixed so that the DNA weights were equal.

[0050] In Example 2, transfection was performed using the following combinations: (a) pAAV-Cre, pRep-loxP_v1, pHelper, and pCap2 (b) pAAV-Cre, pRep-loxP_v2, pHelper, and pCap2

[0051] PEIpro was added to the plasmid DNA solution in an amount 1.5 times that of DNA, and the mixture was allowed to stand at room temperature for 30 minutes to prepare a PEI + DNA complex. The culture supernatant of HEK293 cells cultured in a 6-well plate was removed and replaced with fresh serum-free DMEM medium (2 mL), after which the PEI + DNA complex was gently added and the mixture was incubated at 37°C and 5% CO ２ The test was carried out in duplicate for each of conditions (a) and (b).

[0052] After the incubation, Tween 20 (Sigma) was added in an amount of 1 / 1000 of the volume of the culture medium, and MgCl was added to a final concentration of 2 mM. ２ (Fujifilm Wako Pure Chemical Industries, Ltd.) and Endonuclease (Kaneka Corporation) were added to a final concentration of 50 U / mL, and the mixture was thoroughly stirred. ２ The cells were lysed by incubation in an incubator at 17°C for 2 hours, and then centrifuged at 1,800 xg for 10 minutes, and the supernatant was collected to extract the AAV vector sample from the cells.

[0053] (3) Genomic titer measurement and genome integrity evaluation by droplet digital PCR. The extracted AAV vector was analyzed by droplet digital PCR (ddPCR). DNase I (TaKaRa) was added to the AAV vector extract to digest nucleic acids present outside the AAV particles in the measurement sample, and then EDTA (Nacalai Tesque) was added to inactivate the DNase I. Subsequently, Proteinase K (QIAGEN) and Buffer AL (QIAGEN) were added to degrade the AAV particles, and the AAV vector genome contained within the particles was extracted.

[0054] The AAV vector genome extract was measured by two-dimensional ddPCR targeting the ITR (Inverted Terminal Repeat) region and CMV region of the AAV vector genome using a QX200 Droplet Digital PCR System (Bio-Rad). Detection was performed using a probe modified with FAM for the ITR and HEX for the CMV. Two-dimensional ddPCR was performed, and the absolute amount of AAV vector production was quantified from the number of AAV vector genomes containing the ITR and CMV regions. The results are shown in Figure 4.

[0055] As shown in Figure 4, it was found that the amount of AAV vector produced was higher when pRep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, was used compared to pRep-loxP_v1.

[0056] Furthermore, it is known that AAV vectors produce particles with fragmented genomes as by-products, and the genome integrity of AAV vectors is an important evaluation item in terms of quality. The genome integrity of AAV2 produced under each condition was evaluated based on the percentage of ddPCR droplets that detected both the ITR region and the CMV region. The results are shown in Figure 5.

[0057] As shown in FIG. 5, it was shown that genome integrity was higher when pRep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, was used compared to pRep-loxP_v1.

[0058] [Example 3] Production of AAV using novel constructs 2(1) Plasmids The following plasmids (i) to (v) were used: (i) pAAV-Cre (same as in Example 1) (ii) pTetVA_Rep-loxP_v1 (SEQ ID NO: 8): a plasmid for inducible expression of VA and Rep using the Tet-on system and Cre / loxP system. The piggyBac gene expression vector pPB[Exp]-Kan (VectorBuilder) is used as the plasmid backbone, and downstream of a VA expression unit consisting of a Tet operator heptad repeat sequence-U6 promoter (RNA polymerase III promoter)-VA RNAI, hCG intron 1 is inserted at Rep 1022 downstream of the p5 promoter similar to that of pRep-loxP_v1, and a Rep expression unit in which a cassette consisting of a loxP-SV40 polyA triad repeat sequence-hygromycin resistance gene-loxP is inserted at position 196 of hCG intron 1 is located. Furthermore, it contains tTS and rtTA, which are necessary for the Tet-on system to function. (Figure 6) (iii) pTetVA_Rep-loxP_v2 (SEQ ID NO: 9): A plasmid in which the p5 promoter located immediately before the rep gene has been deleted from pTetVA_Rep-loxP_v1. (Figure 7) (iv) pTetE4_E2A (SEQ ID NO: 10): A plasmid for inducible expression of E4 and E2A using the Tet-on system. E4orf6 and E2A DNA binding protein (E2A DBP) are located downstream of the TRE promoter via an IRES. It contains tTS and rtTA, which are necessary for the Tet-on system to function, and a neomycin resistance gene for antibiotic selection in culture. This vector was created by partially modifying the gene arrangement and drug resistance gene based on Mammalian Tet-On Inducible Gene Expression PiggyBac Vector (All-In-One) pPB[TetOn]-TRE>{E2A_ORF}:IRES:{E4orf6}-rev(hPGK>tTS:T2A:rtTA) (VectorBuilder) (Figure 8). (v) pCap2: (same as Example 2)

[0059] (2) AAV Production and Viral Vector Extraction The above-mentioned plasmids were transfected into HEK293 cells by the same method as described in Example 1. The four types of plasmids were mixed so that the DNA weights were equal.

[0060] In Example 3, transfection was performed using the following combinations: (a) pAAV-Cre, pTetVA_Rep-loxP_v1, pTetE4_E2A, and pCap2 (b) pAAV-Cre, pTetVA_Rep-loxP_v2, pTetE4_E2A, and pCap2

[0061] PEIpro was added to the plasmid DNA solution in an amount 1.5 times that of DNA, and the mixture was allowed to stand at room temperature for 30 minutes to prepare a PEI + DNA complex. The culture supernatant of HEK293 cells cultured in a 6-well plate was removed and replaced with fresh serum-free DMEM medium (2 mL), after which the PEI + DNA complex was gently added and the mixture was incubated at 37°C and 5% CO ２ The test was carried out with n=3 for each of the conditions (a) and (b).

[0062] After the culture was completed, Tween 20 was added in an amount of 1 / 1000 of the volume of the culture medium, and MgCl was added to a final concentration of 2 mM. ２ (Fujifilm Wako Pure Chemical Industries, Ltd.) and Endonuclease (Kaneka Corporation) were added to a final concentration of 50 U / mL, and the mixture was thoroughly stirred. ２ The cells were lysed by incubation in an incubator at 17°C for 2 hours, and then centrifuged at 1,800 xg for 10 minutes, and the supernatant was collected to extract the AAV vector sample from the cells.

[0063] (3) Genomic titer measurement and genome integrity evaluation by droplet digital PCR. The extracted AAV vector was analyzed by ddPCR. DNase I was added to the AAV vector extract to digest nucleic acids present outside the AAV particles in the measurement sample, and then EDTA was added to inactivate DNase I. Proteinase K and Buffer AL were then added to degrade the AAV capsid protein, and the AAV genome embedded within the particles was extracted.

[0064] The AAV vector genome extract was measured by two-dimensional ddPCR targeting the ITR and CMV regions of the AAV vector genome using a QX200 Droplet Digital PCR system. Detection was performed using probes modified with FAM for the ITR and HEX for the CMV. Two-dimensional ddPCR was performed, and the absolute amount of AAV vector production was quantified from the number of AAV vector genomes containing the ITR and CMV regions. The results are shown in Figure 9.

[0065] As shown in Figure 9, even when used in combination with helper factors for inducible expression, pTetVA_Rep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, produced a higher AAV vector yield than pTetVA_Rep-loxP_v1, which has a p5 promoter upstream of Rep.

[0066] The genome integrity of the AAV2 produced under each condition was also evaluated based on the percentage of droplets in which both the ITR and CMV regions were detected by ddPCR. The results are shown in Figure 10.

[0067] As shown in Figure 10, even when used in combination with helper factors for inducible expression, pTetVA_Rep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, showed higher A genome integrity than pTetVA_Rep-loxP_v1, which has the p5 promoter upstream of Rep.

[0068] Example 4: Production of AAV using novel constructs 3(1) Plasmids The following plasmids (i) to (v) were used. (i) pAAV-GFP (cell biolabs): A plasmid containing a GFP expression cassette downstream of a CMV promoter between ITRs. (ii) pTetVA_Rep-loxP_v1 (same as in Example 3) (iii) pTetVA_Rep-loxP_v2 (same as in Example 3) (iv) pTetE4_E2A_Cre (SEQ ID NO: 11): A plasmid for inducible expression of E4, E2A, and CreERT2 using the Tet-on system. It contains a CreERT2 expression cassette downstream of a TRE promoter at a position downstream of the E2A DBP and upstream of the neomycin resistance gene in pTetE4_E2A in Example 3 (Figure 11). (v) pCap2 (same as in Example 2)

[0069] (2) AAV Production and Viral Vector Extraction The above-mentioned plasmids were transfected into HEK293 cells by the same method as described in Example 1. The four types of plasmids were mixed so that the DNA weights were equal.

[0070] In Example 4, transfection was performed using the following combinations: (a) pAAV-GFP, pTetVA_Rep-loxP_v1, pTetE4_E2A_Cre, and pCap2 (b) pAAV-GFP, pTetVA_Rep-loxP_v2, pTetE4_E2A_Cre, and pCap2

[0071] PEIpro was added to the plasmid DNA solution in an amount 1.5 times the amount of DNA, mixed, and then allowed to stand at room temperature for 30 minutes to produce a PEI + DNA complex. The culture supernatant of HEK293 cells cultured in a 6-well plate was removed and replaced with fresh serum-free DMEM medium (2 mL). Next, Dox, an inducer of the Tet-On system, was added to the medium in the wells to a final concentration of 10 ng / mL, and 4-Hydroxytamoxifen Ready Made Solution (4-OHT) (Sigma), necessary for the nuclear translocation of CreERT2, was added to a final concentration of 10 μM. The PEI + DNA complex was then gently added, and the mixture was incubated at 37°C and 5% CO ２The test was carried out under each condition (n=3).

[0072] After the culture was completed, Tween 20 was added in an amount of 1 / 1000 of the volume of the culture medium, and MgCl was added to a final concentration of 2 mM. ２ Endonuclease was added to the mixture to a final concentration of 50 U / mL, and the mixture was thoroughly stirred. ２ The cells were lysed by incubation in an incubator at 17°C for 2 hours, and then centrifuged at 1,800 xg for 10 minutes, and the supernatant was collected to extract the AAV vector sample from the cells.

[0073] (3) Genomic titer measurement and genome integrity evaluation by droplet digital PCR. The extracted AAV vector was analyzed by ddPCR. DNase I was added to the culture supernatant and the AAV vector extract from the producer cells to digest nucleic acids present outside the AAV particles in the measurement samples, and then EDTA was added to inactivate the DNase I. Proteinase K and Buffer AL were then added to degrade the AAV capsid protein, and the AAV vector genome embedded within the particles was extracted.

[0074] The AAV vector genome extract was measured by two-dimensional ddPCR targeting the ITR and GFP regions of the AAV vector genome using a QX200 Droplet Digital PCR system. Detection was performed using a probe modified with FAM for the ITR and HEX for GFP. Two-dimensional ddPCR was performed, and the absolute amount of AAV vector production was quantified from the number of AAV vector genomes containing the ITR and GFP regions. The results are shown in Figure 12.

[0075] As shown in Figure 12, even when used in combination with a helper factor for inducible expression and Cre for inducible expression, pTetVA_Rep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, showed a higher AAV vector production yield than pTetVA_Rep-loxP_v1, which has a p5 promoter upstream of Rep.

[0076] The genome integrity of AAV2 produced under each condition was also evaluated based on the percentage of ddPCR droplets in which both the ITR region and the GFP region were detected. The results are shown in Figure 13.

[0077] As shown in Figure 13, even when used in combination with helper factors for inducible expression, pTetVA_Rep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, showed higher genome integrity than pTetVA_Rep-loxP_v1, which has the p5 promoter upstream of Rep.

[0078] (4) Evaluation of cell infectivity by infection test HeLa cells (ATCC) suspended in EMEM (Wako) containing 10% FBS and 1 mM sodium pyruvate were seeded in a 24-well cell culture plate. Then, the cells were incubated at 37°C and 5% CO until they became approximately 70-80% confluent. ２ The culture was continued in an incubator.

[0079] The AAV vector sample prepared in (2) above was added to each well, and the resulting solution was incubated with CO ２ After culturing in an incubator for 3 days, the cells were subjected to flow cytometry analysis using a FACSCanto™ flow cytometry system (BD), the GFP positivity rate in each sample was measured, and the concentration of gene transfer units (TU) contained in the sample was calculated. The results are shown in Figure 14.

[0080] As shown in Figure 14, it was shown that the production efficiency of AAV vectors was improved in pTetVA_Rep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, compared to pTetVA_Rep-loxP_v1, which has the p5 promoter, not only in terms of the amount of AAV vector genome shown in (3) above but also in terms of the gene transfer unit.

[0081] (5) Evaluation of Full Particle Rate by ELISA and Droplet Digital PCR It is known that a large amount of empty particles not containing the AAV vector genome are generated as a by-product during AAV vector production. Therefore, from the viewpoint of improving the efficiency of the purification process, increasing the ratio of complete particles containing the AAV vector genome (full rate) to the total particle count during cell production is an important aspect for industrial applications. The AAV particles extracted in Example 4-(2) were measured using an AAV2 Titration ELISA kit (PROGEN) according to the kit's instructions. The full rate was calculated using the total AAV particle count quantified by ELISA and the genome titer obtained from the above-mentioned (3) ddPCR. The results are shown in Figure 15.

[0082] As shown in FIG. 15, it was found that the full rate was more than two times higher in pTetVA_Rep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, compared to pTetVA_Rep-loxP_v1.

[0083] [Example 5] Inducible Rep expression with novel constructs (1) Plasmids The following plasmids (i) to (ii) were used: (i) pTetVA_Rep-loxP_v2 (same as in Example 3) (ii) pTetE4_E2A_Cre (same as in Example 4)

[0084] (2) Transfection and Cell Recovery The above two plasmids were transfected into HEK293 cells by the same method as described in Example 1. The two types of plasmids were mixed so that the DNA weights were equal.

[0085] PEIpro was added to the plasmid DNA solution in an amount 1.5 times that of DNA, mixed, and then allowed to stand at room temperature for 30 minutes to produce a PEI + DNA complex. The culture supernatant of HEK293 cells cultured in a 6-well plate was removed and replaced with fresh DMEM medium (2 mL) containing 10% FBS and 1 mM sodium pyruvate. For conditions where an inducer was added, Dox at a final concentration of 10 ng / mL and 4-OHT at a final concentration of 10 μM were added to the medium in the well. After replacing the medium with DMEM medium (2 mL) containing the inducer, the PEI + DNA complex was gently added, and the mixture was incubated at 37°C and 5% CO ２ The cells were incubated at 37°C for 3 days in a CO atmosphere without TF as a negative control (NC). ２ Wells of cells that are normally cultured in an incubator were prepared.

[0086] After 3 days, the culture supernatant was removed, the cells were washed with DPBS, and then the cells were detached using TrypLE select. The detached solution was suspended in DMEM medium containing 10% FBS and 1 mM sodium pyruvate, and after cell counting, 2 × 10 ５ The cell suspension containing the cells was centrifuged, and the supernatant was removed to obtain a cell pellet.

[0087] (3) Confirmation of Rep-inducible expression system by Western blotting The obtained cell pellet was suspended in 18.5 μL of DPBS. 5 μL of LDS sample buffer and 1 μL of nuclease were added to the cell suspension and incubated at 37°C for 1 hour. Next, 3 μL of reducing agent was added, and the mixture was incubated at 95°C for 5 minutes to prepare a sample for SDS-PAGE.

[0088] Electrophoresis was performed using an XV PANTERA SYSTEM, and after electrophoresis, proteins were transferred to a membrane using a Trans-Blot® Turbo™ Transfer System. An iBind™ Flex Western Device was used for antibody reactions. The primary antibody, GAPDH (D16H11) XP Rabbit mAb, and the secondary antibody, Anti-rabbit IgG (NA934VS), were used against the housekeeping gene GAPDH. The primary antibody, Anti-Adeno-Associated Virus Replicase (61069), and the secondary antibody, Anti-mouse IgG (NA931VS), were used against Rep. ImmunoStar LD was used as the detection reagent. Chemiluminescence Solution A and Chemiluminescence Solution B were mixed in a 1:1 ratio and added dropwise to the membrane. The mixture was left to stand for 2 minutes in the dark and then detected using ImageQuant™ 800. The results are shown in Figure 16.

[0089] As shown in Figure 16, when pTetVA_Rep-loxP_v2 and pTetE4_E2A_Cre were transfected, Rep expression was clearly induced in cells treated with 4-OHT and Dox as inducers, compared to cells without any inducers. This indicates that the system for inducing Cre expression by 4-OHT and Dox and excising the loxP sequence of Rep is functioning.

[0090] Example 6 Evaluation of the effect of novel constructs on cells (1) Plasmids The following plasmids (i) to (iii) were used: (i) pAAV-Cre (same as in Example 1) (ii) pTetVA_Rep-loxP_v1 (same as in Example 3) (iii) pTetVA_Rep-loxP_v2 (same as in Example 3)

[0091] (2) Transfection and Cell Recovery The above-mentioned plasmids were transfected into HEK293 cells by the same method as described in Example 1. The two types of plasmids were mixed so that the DNA weights were equal.

[0092] In Example 6, transfection was performed using the following combinations: (a) pAAV-Cre and pTetVA_Rep-loxP_v1 (b) pAAV-Cre and pTetVA_Rep-loxP_v2

[0093] PEIpro (Polyplus) was added to the plasmid DNA solution in an amount 1.5 times the amount of DNA, mixed, and then left to stand at room temperature for 30 minutes to prepare a PEI + DNA complex. The culture supernatant of HEK293 cells cultured in a 6-well plate was removed and replaced with fresh DMEM medium (2 mL) containing 10% FBS and 1 mM sodium pyruvate. The PEI + DNA complex was then gently added, and the mixture was incubated at 37°C and 5% CO ２ The test was carried out in triplicate.

[0094] After 3 days, the culture supernatant was collected, and DPBS was added to the cells. The cells were then collected in the same tube as the culture supernatant. The cells were then detached using TrypLE select, and the detached solution was suspended in DMEM medium containing 10% FBS and 1 mM sodium pyruvate. The suspension was then collected together with the cells contained in the culture supernatant and DPBS washing solution. The cells were counted and 4 × 10 ５ The cell suspension containing the cells was centrifuged at 400×g for 5 minutes to obtain a cell pellet.

[0095] (3) Measurement of apoptosis induction rate by Annexin V assay. Negatively charged phosphatidylserine (PS) is a lipid present in the intimal layer of normal cells, but in cells in which apoptosis has been induced, PS is translocated to the outside. Annexin V is a Ca ２＋ Because it has a strong affinity for PS in the presence of annexin V, it is used as a probe to detect changes in the cell membrane caused by apoptosis. By adding fluorescently modified annexin V to cells, the proportion of apoptotic cells can be measured.

[0096] The cell pellet collected in (2) above was suspended in 100 μL of Annexin V binding buffer (BTI Niotium). Annexin V-CF488A conjugate (BTI Niotium) and Solution 15 Hoechst 33342 (Chemometec) were added, and the sample was incubated at 37 ° C for 15 min to stain the sample. After washing the cells with Annexin V binding buffer, the cells were resuspended in 100 μL of Annexin V binding buffer, and Solution 16 PI (Chemometec) was added and the measurement was performed using an NC3000 (Chemometec). NC is a sample to which no Annexin V-CF488A conjugate was added and which was not stained with Annexin V. The results are shown in FIG.

[0097] As shown in Figure 17, under conditions where Rep was expressed under Cre expression, the Annexin V positivity rate was lower in pTetVA_Rep-loxP_v2, in which the p5 promoter upstream of Rep was deleted, compared to pTetVA_Rep-loxP_v1. In other words, it was found that the apoptosis induction rate during Rep expression was maintained low in the construct in which the p5 promoter was deleted.

[0098] [Example 7] Creation of stable strains using novel constructs (1) Plasmids The following plasmids (i) to (iii) were used. (i) pTetVA_Rep-loxP_v2_Km (SEQ ID NO: 12): This is pTetVA_Rep-loxP_v2 (SEQ ID NO: 9) used in Example 3, but with the resistance gene used during plasmid replication in E. coli changed from an ampicillin resistance gene to a kanamycin resistance gene. The sequence inserted into the genome of mammalian cells by the PiggyBac method is the same as that of pTetVA_Rep-loxP_v2. (ii) pTetE4_E2A_Cre (same as in Example 4) (iii) pRP-CMV-PBase (SEQ ID NO: 13): a plasmid containing a PiggyBac transposase expression cassette downstream of a CMV promoter, which is necessary for inserting the gene expression cassettes of (i) and (ii) into the genome of a mammalian cell by the PiggyBac method.

[0099] (2) Construction of Stable Strains First, the above-mentioned plasmids (i) and (iii) were transfected into HEK293 cells using PEIpro. 500 μg / mL Hygromycin (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to DMEM medium containing 10% FBS and 1 mM sodium pyruvate, and drug selection culture was performed. Single-cell cloning was then performed to obtain clones containing Rep and VA in the genome. Next, the obtained clones were transfected into HEK293 cells using PEIpro with the above-mentioned plasmids (ii) and (iii). 500 μg / mL Hygromycin and 800 μg / mL Geneticin (Gibco) were added to DMEM medium containing 10% FBS and 1 mM sodium pyruvate, and drug selection culture was performed. Single cell cloning was then performed to obtain clones containing Rep, VA, E2A, and E4 in the genome.

[0100] (3) Expression Confirmation by Reverse Transcription-qPCR The clones obtained above, cultured on a 6-well plate, were replaced with fresh DMEM medium (2 mL) containing 10% FBS and 1 mM sodium pyruvate. For the inducer addition conditions, Dox at a final concentration of 10-1000 ng / mL and 4-OHT at a final concentration of 10 μM were added to the medium in the wells and cultured for 3 days. RNA was extracted using a High Pure RNA Isolation Kit (Roche) according to the manufacturer's protocol. A portion of the RNA solution cleaned up using the column provided with the kit was taken and its absorbance was measured using a NanoDrop (Thermo). The RNA concentrations of the samples were adjusted based on the measurement results, and cDNA was synthesized using the iScript AdV cDNA kit for RT-qPCR (Bio-Rad) according to the manufacturer's protocol.

[0101] The prepared cDNA solution was used as a template and real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO). The real-time PCR reaction was performed using a CFX96Touch Real-Time PCR Analysis System (Bio-Rad). The amount of GAPDH mRNA was measured for each sample, and the measurement results for Rep, VA, E2A, and E4 were normalized. The primers used for detection are as follows:

[0102] Target Fw Rv GAPDH SEQ ID NO:14 SEQ ID NO:15 Rep SEQ ID NO:16 SEQ ID NO:17 VA SEQ ID NO:18 SEQ ID NO:19 E2A SEQ ID NO:20 SEQ ID NO:21 E4 SEQ ID NO:22 SEQ ID NO:23

[0103] Figure 18 shows the ratio of the RNA content of each sample to the RNA content of the corresponding factor in the condition containing 10 μM 4-OHT and 1000 ng / mL Dox, which is defined as 100%. Expression levels of Rep, VA, E2A, and E4 were confirmed to be increased in a Dox concentration-dependent manner, demonstrating that the various gene cassettes were correctly inserted into the stable cell clones obtained and that the inducible expression system was functioning. This test demonstrated that it is actually possible to construct cells containing novel constructs in the genome in which cytotoxic genes have been integrated using an inducible system.

[0104] [Example 8] Production of AAV by stable strains using novel constructs (1) Plasmids The following plasmid (i) was used: (i) pTRE-Cap2_ITR-CMV-GFP-ITR: a plasmid carrying an AAV2 Cap expression unit downstream of the TRE promoter and a GFP expression cassette downstream of the CMV promoter between the AAV2 ITRs as an AAV vector genome sequence.

[0105] (2) AAV Production and Viral Vector Extraction The clone obtained in Example 8 was transfected with plasmid (i). PEIpro was added to the plasmid DNA solution in an amount 1.5 times that of DNA, and the mixture was then allowed to stand at room temperature for 30 minutes to produce a PEI + DNA complex. The medium supernatant of HEK293 cells cultured in a 24-well plate was removed and replaced with DMEM medium (0.5 mL) containing 10% FBS and 1 mM sodium pyruvate. For the inducer condition, Dox was added to a final concentration of 1000 ng / mL and 4-OHT to a final concentration of 10 μM. The PEI + DNA complex was gently added, and the mixture was incubated at 37°C and 5% CO ２ The mixture was incubated at RT for 5 days.

[0106] After the culture was completed, Tween 20 was added in an amount of 1 / 1000 of the volume of the culture medium, and MgCl was added to a final concentration of 2 mM. ２ Endonuclease was added to the mixture to a final concentration of 50 U / mL, and the mixture was thoroughly stirred. ２After incubating the cells for 2 hours in an incubator at 1800°C to lyse the cells, NaCl solution was added to a final concentration of 300 mM, and the mixture was centrifuged at 1,800 x g for 10 minutes. The supernatant was collected, and the AAV vector sample was extracted from the cells.

[0107] (3) Genomic titer measurement and genome integrity evaluation by droplet digital PCR. The extracted AAV vector was analyzed by ddPCR. DNase I was added to the culture supernatant and the AAV vector extract from the producer cells to digest nucleic acids present outside the AAV particles in the measurement samples, and then EDTA was added to inactivate DNase I. Proteinase K and Buffer AL were then added to degrade the AAV capsid protein, and the AAV genome embedded within the particles was extracted.

[0108] The AAV vector genome extract was measured by two-dimensional ddPCR targeting the ITR and GFP regions of the AAV vector genome using a QX200 Droplet Digital PCR system. Detection was performed using a probe modified with FAM for the ITR and HEX for GFP. Two-dimensional ddPCR was performed, and the absolute amount of AAV vector production was quantified from the number of AAV vector genomes containing the ITR and GFP regions. The results are shown in Figure 19.

[0109] As shown in Figure 19, the clone obtained in Example 7 was found to be capable of inducer-dependent AAV vector production by transiently supplying the AAV vector genome sequence and Capsid sequence. This suggests that Rep, VA, E2A, and E4 integrated into the clone genome were functionally expressed by the addition of an inducer, promoting AAV vector production.

[0110] (4) Evaluation of cell infectivity by infection test HeLa cells suspended in EMEM containing 10% FBS and 1 mM sodium pyruvate were seeded in a 24-well cell culture plate. Then, the cells were incubated at 37°C, 5% CO until they reached approximately 70-80% confluence. ２ The culture was continued in an incubator.

[0111] 50 μL of the AAV vector sample prepared in (2) above was added to each well, and the resulting solution was incubated with CO ２ After culturing in an incubator for 3 days, the cells were subjected to flow cytometry analysis using a FACSCanto™ flow cytometry system (BD) to measure the GFP-positive rate in each sample. The GFP-positive rate, after subtracting the value due to autofluorescence in the cell group (NC) to which the AAV vector sample had not been added, is shown in Figure 20.

[0112] As shown in Figure 20, the clone obtained in Example 7 was capable of inducer-dependent AAV vector production by transient supply of the AAV vector genome sequence and Capsid sequence, as confirmed not only by the evaluation of the AAV vector genome amount shown in (3) but also by the transduction test into HeLa.

[0113] According to the present invention, high-quality AAV vectors can be produced extremely efficiently, and therefore the present invention is extremely useful in the field of gene therapy.

[0114] This application is based on patent application No. 2024-062030 filed in Japan (filing date: April 8, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A construct for expressing adeno-associated virus (AAV) Rep proteins, the construct comprising nucleic acid sequences encoding Large Rep and Small Rep, wherein an intron is inserted within the nucleic acid sequences encoding Large Rep and Small Rep, wherein the intron comprises a pair of recombination sequences consisting of a first sequence and a second sequence, and a transcription termination sequence, wherein the construct is characterized in that: (1) when recombination occurs based on the pair of recombination sequences, the first sequence and the transcription termination sequence are deleted by DNA splicing, and (2) the construct does not comprise a p5 promoter.

2. The construct of claim 1, wherein the Large Rep is either or both of Rep78 and Rep68, and the Small Rep is either or both of Rep52 and Rep40.

3. The construct of claim 1, wherein the construct further comprises a marker gene in an intron.

4. The construct according to claim 3, wherein the marker gene is a drug resistance gene.

5. The construct of claim 3, wherein the intron comprises, in the 5' to 3' direction, a first recombination sequence, a transcription termination sequence, a marker gene, and a second recombination sequence.

6. The construct of claim 1, wherein the transcription termination sequence is a polyA signal sequence.

7. The construct according to claim 1, wherein the pair of recombination sequences are sequences selected from the group consisting of Lox sequences, FRT sequences and rox sequences.

8. The construct of claim 1, wherein the construct further comprises a gene encoding a recombinase.

9. The construct of claim 1, further comprising genes for other helper factors necessary for AAV production.

10. A cell comprising a construct according to any one of claims 1 to 9.

11. The cell of claim 10, further comprising an E2A gene, an E4orf6 gene and / or a VARNA gene derived from an adenovirus.

12. Use of the construct for expression of adeno-associated virus (AAV) Rep proteins according to any one of claims 1 to 9 for the production of an adeno-associated virus vector.

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