VARIANT REP PROTEIN, rep GENE, AND USE OF SAID REP GENE

Abstract

The problem to be addressed by the present invention is to provide: a variant REP protein that can improve low productivity of an AAV vector; a rep gene that encodes the variant REP protein; an expression vector; a vector kit for producing a recombinant adeno-associated virus vector; a cell; and a method for producing a recombinant adeno-associated virus vector. The present invention provides a variant REP protein in which one or more amino acid residues from among the 102nd amino acid residue, the 114th amino acid residue, the 125th amino acid residue, and the 134th amino acid residue of the REP protein are substituted with a basic amino acid residue in the amino acid sequence of the adeno-associated virus REP protein.

Description

Mutant REP proteins, rep genes and uses thereof

[0001] The present invention relates to an REP protein having a predetermined amino acid mutation, and a rep gene encoding the REP protein. The present invention further relates to an expression vector, a vector kit for producing a recombinant adeno-associated virus vector, cells, and a method for producing a recombinant adeno-associated virus vector.

[0002] Adeno-associated virus (AAV) is a linear, single-stranded DNA virus belonging to the Parvoviridae family. The wild-type AAV genome contains a replication regulatory gene (rep gene) and a capsid structural gene (cap gene), flanked by inverted terminal repeats (ITRs) for viral replication and packaging. AAV vectors are capable of gene transfer into both proliferating and non-proliferating cells, and are particularly capable of long-term expression in non-dividing cells. AAV is also considered non-pathogenic and has low immunogenicity. For these reasons, AAV vectors are being increasingly used clinically as gene therapy vectors.

[0003] AAV is a non-enveloped virus that grows in the presence of a helper virus such as adenovirus or herpesvirus. When preparing AAV for gene therapy or nucleic acid transfer, traditionally, adenovirus is co-infected into host cells to allow AAV replication. In addition, the gene responsible for the adenovirus helper function has been identified, and a plasmid carrying this gene is also used. For example, a plasmid containing rep gene and cap gene, an adenovirus helper plasmid, and a plasmid containing a gene of interest (GOI) to be transferred can be simultaneously transfected into HEK293 cells, and then packaged into recombinant AAV (rAAV).

[0004] There have been several reports on mutations in the REP protein. Non-Patent Document 1 identifies mutations in the REP protein by screening a plasmid in which the REP protein has been randomly synthesized. Patent Document 1 describes inactivating the REP protein by introducing a mutation into it in order to avoid its toxicity. Non-Patent Document 2 describes mutating M225, the initiation methionine of the small REP protein, in order to avoid its toxicity.

[0005] Patent Document 2 describes an REP protein having an amino acid substitution in the DNA binding domain.

[0006] Jain, Nina K., et al., Comprehensive mutagenesis maps the effect of all single codon mutations in the AAV2 rep gene on AAV production. bioRxiv (2023): 2023-01MATTHEW D. WEITZMAN, et al., JOURNAL OF VIROLOGY,Apr.1996,Vol. 70, No.4, p.2440-2448

[0007] U.S. Patent Application Publication US2023 / 0348936 U.S. Patent Application Publication US2013 / 0023034

[0008] An objective of the present invention is to provide a mutant REP protein that can improve low production of adeno-associated virus (AAV) vectors.A further objective of the present invention is to provide a rep gene encoding the mutant REP protein, an expression vector, a vector kit for producing a recombinant adeno-associated virus vector, cells, and a method for producing a recombinant adeno-associated virus vector.

[0009] As a result of extensive research aimed at solving the above problems, the present inventors have found that the above problems can be solved by substituting specific amino acid residues with basic amino acid residues in the amino acid sequence of the adeno-associated virus REP protein. The present invention was completed based on this finding.

[0010] According to the present invention, the following inventions are provided: <1> A mutant REP protein in which, in the amino acid sequence of the adeno-associated virus REP protein, any one or more of the following amino acid residues have been substituted with a basic amino acid residue: a serine residue corresponding to the 102nd amino acid residue of the REP protein, a glutamic acid residue, aspartic acid residue or alanine residue corresponding to the 114th amino acid residue of the REP protein, a glutamic acid residue or serine residue corresponding to the 125th amino acid residue of the REP protein, and a valine residue or isoleucine residue corresponding to the 134th amino acid residue of the REP protein. <2> A mutant REP protein according to <1>, wherein in the amino acid sequence of the REP protein, any one or more of the following amino acid residues have been substituted with a basic amino acid residue: a serine residue corresponding to the 102nd amino acid residue of the AAV2-derived REP protein, a glutamic acid residue corresponding to the 114th amino acid residue of the AAV2-derived REP protein, a glutamic acid residue corresponding to the 125th amino acid residue of the AAV2-derived REP protein, and a valine residue corresponding to the 134th amino acid residue of the AAV2-derived REP protein. <3> A mutant REP protein according to <1> or <2>, wherein the basic amino acid residue is a lysine residue. <4> A mutant REP protein according to any one of <1> to <3>, wherein the serine residue corresponding to the 102nd amino acid residue of the AAV2-derived REP protein has been substituted with a lysine residue. <5> A rep gene encoding the REP protein according to any one of <1> to <4>. <6> An expression vector having the rep gene according to <5>. <7> The expression vector according to <6>, further having a cap gene. <8> A vector kit for producing a recombinant adeno-associated virus vector, comprising the expression vector according to <6> or <7>, an expression vector having a therapeutic or preventive gene, and an expression vector having a helper gene. <9> A cell having the rep gene according to <5>. <10> The cell according to <9>, in which the rep gene according to <5> is integrated into a chromosome or the rep gene according to <5> is present extrachromosomally.<11> A method for producing a recombinant adeno-associated virus vector, comprising expressing the rep gene according to <5> in a cell.

[0011] According to the present invention, the low production of adeno-associated virus (AAV) vectors can be improved.

[0012] Figure 1 shows a comparison of the amino acid sequences of the REP proteins of AAV1, AAV2, AAV5, AAV6, and AAV8. Figure 2 shows the results of measuring the genome titer for Experimental Example 1. Figure 3 shows the results of measuring the genome titer for Experimental Example 2. Figure 4 shows the results of measuring the genome titer for Experimental Example 3.

[0013] An example of an embodiment of the present disclosure will be described below. However, the present disclosure is not limited to the following embodiment in any way, and can be implemented with appropriate modifications within the scope of the object of the present disclosure. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​written before and after "to" as the minimum and maximum values, respectively.

[0014] As used herein, a promoter refers to a DNA regulatory region / sequence capable of binding RNA polymerase and involved in initiating transcription of a coding or non-coding sequence.

[0015] As used herein, the promoter naturally possessed by a gene refers to a promoter that is not artificial and is known to express the gene in its natural state.

[0016] As used herein, a vector is a DNA or RNA molecule used to artificially transport foreign genetic material into another cell. When a vector containing the foreign genetic material to be introduced is introduced into a cell, the foreign genetic material is replicated and / or expressed within the cell. Examples of vectors include episomal (e.g., plasmid) vectors and non-episomal vectors. Vectors can be introduced into host cells by methods such as transfection, transduction, cell fusion, and lipofection.

[0017] <Mutant REP Protein> The present invention relates to a mutant REP protein in which, in the amino acid sequence of the adeno-associated virus REP protein, any one or more of the following amino acid residues have been substituted with a basic amino acid residue: a serine residue corresponding to amino acid residue 102 of the REP protein; a glutamic acid residue, aspartic acid residue or alanine residue corresponding to amino acid residue 114 of the REP protein; a glutamic acid residue or serine residue corresponding to amino acid residue 125 of the REP protein; and a valine residue or isoleucine residue corresponding to amino acid residue 134 of the REP protein.

[0018] Preferably, in the mutant REP protein of the present invention, in the amino acid sequence of the REP protein, any one or more of the following amino acid residues have been substituted with a basic amino acid residue: a serine residue corresponding to the 102nd amino acid residue of the AAV2-derived REP protein; a glutamic acid residue corresponding to the 114th amino acid residue of the AAV2-derived REP protein; a glutamic acid residue corresponding to the 125th amino acid residue of the AAV2-derived REP protein; and a valine residue corresponding to the 134th amino acid residue of the AAV2-derived REP protein.

[0019] The REP protein refers to the long form of the REP protein. Examples of the long form of the REP protein include REP78 and REP68. A comparison of the amino acid sequences of the REP proteins of AAV1, AAV2, AAV5, AAV6 and AAV8 is shown in Figure 1. Mutation sites are indicated by boxes.

[0020] The "125th amino acid residue of the REP protein" corresponds to the "126th amino acid residue of the REP protein" in AAV8. The "134th amino acid residue of the REP protein" corresponds to the "135th amino acid residue of the REP protein" in AAV8.

[0021] Examples of basic amino acid residues include lysine, arginine, and histidine residues, and the basic amino acid residue is preferably a lysine residue. Particularly preferably, the serine residue corresponding to the 102nd amino acid residue of the AAV2-derived REP protein is substituted with a lysine residue.

[0022] The present invention provides an improved REP with unique mutations added to enhance the function of the rep gene required for AAV production, in order to produce AAV for gene therapy or nucleic acid transfer. Existing nucleic acid sequences can be used for the other genes. According to the present invention, AAV for therapeutic use can be produced in large quantities, thereby reducing the amount of expensive plasmids used and thereby reducing production costs.

[0023] The method for producing the mutant REP protein of the present invention in which one or more of the above amino acid residues have been substituted with basic amino acid residues is not particularly limited, but the mutant REP protein can be produced by preparing a rep gene encoding the mutant REP protein and expressing the gene. The method for preparing the rep gene encoding the mutant REP protein is not particularly limited, but examples include (1) total synthesis of the rep gene including the modified site, (2) replacement of the rep gene by partial synthesis, and (3) a method for introducing a mutation into the rep gene using PCR. In replacement of the rep gene by partial synthesis, a part of the rep gene including the amino acid at the mutated site can be partially synthesized and replaced with a rep gene encoding the corresponding part derived from a wild-type REP protein that does not contain the mutation by genetic engineering techniques. In a method for introducing a mutation into the rep gene using PCR, a gene encoding the mutant REP protein can be prepared by PCR using a primer sequence encoding the mutated amino acid.

[0024] <Rep Gene and Expression Vector Carrying the Rep Gene> The present invention relates to the rep gene encoding the REP protein of the present invention described above, and an expression vector carrying the rep gene.

[0025] Adeno-associated virus (AAV) refers to a small, replication-incompetent, non-enveloped virus containing single-stranded DNA belonging to the Parvoviridae and Dependoparvovirus families. There are over 100 serotypes of AAV, and it is known that the host range and viral characteristics differ depending on the serotype. Serotype 2 (AAV2) is one of the serotypes that has been widely studied for a long time and is known to have a very wide host range. Serotype 1 (AAV1), serotype 5 (AAV5), and serotype 6 (AAV6) are serotypes with higher tissue tropism. AAV1 is said to have high gene transfer efficiency into muscles, liver, respiratory tract, central nervous system, etc.; AAV5 is said to have high gene transfer efficiency into the central nervous system, liver, retina, etc.; and AAV6 is said to have high gene transfer efficiency into heart, muscles, liver, etc.

[0026] Adeno-associated virus genes refer to genes composed of one or more nucleic acid sequences derived from one or more adeno-associated virus serotypes, and are preferably genes involved in AAV replication and packaging (specifically, rep genes) and genes encoding AAV structural proteins (specifically, cap genes).

[0027] The rep gene refers to the region of the AAV genome that encodes viral replication proteins collectively required for replication of the viral genome, or functional homologs thereof, such as, for example, the human herpesvirus 6 (HHV-6) rep gene (known to mediate AAV-2 DNA replication), as known to those skilled in the art. Thus, the rep gene coding region includes at least the genes encoding AAV REP78 and REP68 ("long form REP proteins") and REP52 and REP40 ("short form REP proteins"), or functional homologs thereof. The rep gene coding region used in the present invention may be derived from any AAV serotype, but is preferably derived from AAV1, AAV2, AAV5, AAV6, or AAV8, more preferably from AAV2, AAV5, AAV6, or AAV8, and even more preferably from AAV2. Those derived from AAV2 include REP78 and REP68 as well as REP52 and REP40, ITRs.

[0028] The expression vector having the rep gene may contain a promoter for expressing the rep gene. The promoter for expressing the rep gene may be natural or artificial, and is not particularly limited. Examples of promoters naturally contained in the rep gene include the p5 and p19 promoters.

[0029] The expression vector of the present invention having a rep gene may preferably further have a cap gene. When the expression vector of the present invention having a rep gene further has a cap gene, it may contain a promoter for expressing the cap gene. The promoter for expressing the cap gene may be natural or artificial, and is not particularly limited, but an example of a promoter naturally contained in the cap gene is the p40 promoter.

[0030] The cap gene refers to a region in the AAV genome that encodes viral capsid proteins known to those skilled in the art. Examples of these capsid proteins are AAV capsid proteins VP1, VP2, and VP3. The cap gene used in the present invention may be derived from any AAV serotype, or may be an artificial gene with partial mutations, such as AAV2, AAV5, AAV6, or AAV8.

[0031] The cap gene may be a wild-type gene, but a gene that has been modified by base substitution, deletion, insertion, or addition, etc., may also be used as long as it exhibits its original function.

[0032] When a wild-type cap gene is modified by base substitution, deletion, insertion, addition, or the like, the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The base sequence of the modified cap gene exhibits, with the base sequence of the wild-type cap gene, preferably 85% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity.

[0033] When the expression vector of the present invention having a rep gene contains a rep gene and a cap gene, the position thereof is not particularly limited, and the rep gene may be located upstream of the cap gene or downstream of the cap gene, but preferably the rep gene is located upstream of the cap gene.

[0034] The expression vector of the present invention having a rep gene is not limited as long as it contains a nucleic acid, but for example, a plasmid vector, an artificial chromosome vector, a viral vector, a vector having an artificially designed nucleic acid, etc. can be used, and a plasmid vector is preferred.

[0035] <Vector kit for producing recombinant adeno-associated virus vector> The present invention further relates to a vector kit for producing a recombinant adeno-associated virus vector, which comprises the above-mentioned expression vector of the present invention, an expression vector carrying a therapeutic or prophylactic gene, and an expression vector carrying a helper gene.

[0036] Therapeutic or prophylactic genes may be, but are not limited to, genes that are incomplete or missing in the genome of target cells, or genes that encode non-native proteins with a desired biological or therapeutic effect (e.g., antiviral function). Specific examples of therapeutic or prophylactic genes include genes used to treat or prevent inflammatory diseases, autoimmune diseases, chronic and infectious diseases (including disorders such as AIDS, cancer, neurological diseases, cardiovascular diseases, and hypercholesterolemia), various blood diseases such as anemia and hemophilia, and genetic defects (e.g., cystic fibrosis, Gaucher disease, adenosine deaminase (ADA) deficiency, emphysema, etc.).

[0037] Therapeutic or preventive genes may also be some antisense oligonucleotides (e.g., short oligonucleotides complementary to sequences around the translation start site (AUG codon) of mRNA) that are useful in antisense therapy for cancer and viral diseases.

[0038] An expression vector carrying a therapeutic or preventive gene may contain a promoter for expressing the therapeutic or preventive gene. The promoter for expressing the therapeutic or preventive gene is not particularly limited, but examples include a cytomegalovirus-derived promoter (optionally containing an enhancer), the SV40 early promoter, the human elongation factor-1α (EF-1α) promoter, the human ubiquitin C promoter, the retroviral Rous sarcoma virus LTR promoter, the dihydrofolate reductase promoter, the β-actin promoter, and the phosphoglycerate kinase (PGK) promoter. The therapeutic or preventive gene and the promoter for gene expression are preferably flanked by ITR sequences. The expression vector for the therapeutic or preventive gene is not limited as long as it contains a nucleic acid, but examples of suitable vectors include plasmids, artificial chromosomes, and viruses, with plasmids being preferred.

[0039] The helper gene-containing expression vector contains one or more viral helper genes, at least one of which may be under the control of a promoter whose expression can be regulated.

[0040] The expression-regulatable promoter can be a promoter whose expression can be turned on and off depending on the presence or absence of a stimulus. Examples of stimuli include, but are not limited to, chemical stimuli (endogenous hormone / stress response, lactose, tetracycline or its derivatives (e.g., doxycycline), cumic acid, proteins (rapamycin, FKCsA, abscisic acid (ABA)), etc.), tamoxifen / Cre-loxP (a system using a Cre promoter modified so that activation can be inducible by tamoxifen), riboswitches), and physical stimuli (blue light, heat).

[0041] Preferably, the promoter in the expression vector having the helper gene is a promoter whose expression can be regulated by a drug, preferably tetracycline or a derivative thereof (e.g., doxycycline).

[0042] Examples of promoters whose expression can be regulated include tetracycline-responsive promoters, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters. Specific examples of tetracycline-responsive promoters include the Tet on / off system (TET Systems). Particularly preferred promoters whose expression can be regulated in an expression vector having a helper gene are the Tet on / off system, which is a promoter whose expression can be regulated by tetracycline, and most preferred is the Tet on system. The promoter is not particularly limited, and examples thereof include a cytomegalovirus-derived promoter (optionally containing an enhancer), SV40 early promoter, human elongation factor-1α (EF-1α) promoter, human ubiquitin C promoter, retroviral Rous sarcoma virus LTR promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, H1 promoter (RNA polymerase III promoter), U6 promoter (RNA polymerase III promoter), etc. The promoter used in the Tet-on system for regulating VA-RNA gene expression may be the H1 promoter (RNA polymerase III promoter).

[0043] In the Tet-on system, the promoter additionally contains at least one Tet operon. The Tet operon (tetracycline-controlled transcriptional activation) can be used to reversibly switch transcription on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline). The Tet repressor protein present in the cell blocks expression by binding to the Tet operator sequence introduced into the promoter. Therefore, when the Tet repressor is bound to the Tet operator sequence, no gene expression is observed. Upon addition of tetracycline or doxycycline, the Tet repressor is sequestered, allowing promoter activity and turning on gene expression. Tet operon systems are widely available, such as the Tet operon used in the pcDNA™4 / TO mammalian expression vector available from Invitrogen. In the Tet-on system, the Tet operator sequence may be located upstream or downstream of the promoter whose expression is to be regulated. In order to reduce expression leakage when expression is turned off, it is preferable that the Tet operator sequence be located downstream of the promoter whose expression can be regulated.

[0044] The viral helper gene is a non-adeno-associated virus gene that enables replication and packaging of the adeno-associated virus. The viral helper gene used is a gene derived from a virus other than the adeno-associated virus. Specific examples of the viral helper gene include a viral helper gene derived from an adenovirus or a herpesvirus, and preferably, the viral helper gene is derived from an adenovirus.

[0045] Adenovirus refers to a non-enveloped virus of the Adenoviridae family, which has an icosahedral nucleocapsid containing double-stranded DNA.More than 50 subtypes of adenovirus have been isolated from humans, and many additional subtypes have been isolated from other mammals and birds.These subtypes belong to the Adenoviridae family and are classified into two genera, namely, Mastadenovirus and Aviadenovirus.These adenoviruses are morphologically and structurally similar.However, in humans, adenoviruses exhibit different immunological properties, i.e., are classified into serotypes.Two human serotypes of adenovirus, namely, Ad2 and Ad5, have been extensively studied.

[0046] Adenovirus-derived viral helper genes are genes involved in the replication and packaging of adeno-associated viruses. Examples of adenovirus-derived viral helper genes include the E1A gene, E1B gene, E2A gene, E4 gene, and VA-RNA gene. Preferably, the viral helper genes contained in the expression vector containing the helper genes are the E2 gene, E4 gene, and VA-RNA gene.

[0047] The adenovirus-derived viral helper genes (such as the E1A gene, E1B gene, E2A gene, E4 gene, and VA-RNA gene) may be wild-type genes, but genes that have been modified by base substitution, deletion, insertion, or addition, etc., may also be used, as long as they exhibit their inherent functions.

[0048] When a wild-type viral helper gene is modified by base substitution, deletion, insertion, or addition, the number of modified bases is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. The base sequence of the modified viral helper gene preferably exhibits 85% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity to the base sequence of the wild-type viral helper gene.

[0049] When an expression vector having a helper gene contains an E4 gene and a VA-RNA gene, their positions are not particularly limited, and the E4 gene may be located upstream or downstream of the VA-RNA gene, but preferably the E4 gene is located upstream of the VA-RNA gene.

[0050] The expression vector having a helper gene is not limited as long as it contains a nucleic acid, but for example, a plasmid vector, an artificial chromosome vector, a viral vector, or a vector having an artificially designed nucleic acid can be used, and a plasmid vector is preferred.

[0051] The kit of the present invention may contain a combination of the expression vector of the present invention, an expression vector carrying a therapeutic or preventive gene, and an expression vector carrying a helper gene, and may be dissolved or dispersed in a buffer composition, purified water, etc. It may also be combined with compositions useful for practicing the present invention, such as reagents for introducing the vector, and may include an appropriate container, for example, a vial, tube, test tube, or other container.

[0052] <Cells> The present invention relates to cells having the rep gene of the present invention described above. The cells are preferably eukaryotic cells, more preferably mammalian cells. Mammalian cells include, but are not limited to, human cells, mouse cells, rat cells, monkey cells, and hamster cells. Preferably, human cells can be used. Examples of cells include mouse myeloma (NS0) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, YO, C127, L cells, COS (e.g., COS1 and COS7), QC1-3, HEK293 (human embryonic kidney cells), VERO, PER. C6, HeLa, EB1, EB2, EB3, oncolytic or hybridoma cell lines. Preferably, the cells are HEK293 cells, CHO cells, more preferably HEK293 cells.

[0053] In the cells of the present invention, the rep gene may be integrated into a chromosome or may be present extrachromosomally. Integration of the rep gene into a chromosome can be confirmed by genome sequence analysis and by checking that the sequence in the vector is linked to the host genome.

[0054] Because the genes necessary for the production of recombinant adeno-associated virus vectors are integrated into the chromosome, the genes necessary for the production of adeno-associated virus are constantly maintained within the cell, making it possible to produce adeno-associated virus at any time.

[0055] The cells of the present invention can retain the genes necessary for adeno-associated virus production from the introduced vectors for one week or more after the cells are introduced with an expression vector containing a rep gene and a cap gene, an expression vector carrying a therapeutic or preventive gene, and an expression vector carrying a helper gene. Therefore, after culturing the cells into which the vectors have been introduced for one week or more, it becomes possible to produce adeno-associated vectors. Preferably, adeno-associated vectors can be produced after culturing for two weeks or more, and more preferably, after culturing for one month or more.

[0056] The cells of the present invention can be produced by introducing the expression vector of the present invention containing the rep gene into cells. The expression vector containing the rep gene may be introduced so as to be expressed, but persistent expression may also be achieved. Persistent expression means that when the cell divides, the expression vector containing the rep gene is also replicated, and the cells after division also contain the expression vector containing the rep gene. Persistent expression can be achieved by incorporating the vector into the cell's chromosome or by using a vector capable of replicating in the cytoplasm.

[0057] Expression vectors carrying the rep gene can be introduced into cells by methods such as transfection, transduction, and lipofection. Transfection refers to the introduction of nucleic acids into cells by crossing the membrane of a eukaryotic cell using chemical means (e.g., calcium phosphate-mediated precipitation or cationic polymer-mediated introduction such as PEI (polyethyleneimine)), mechanical means (e.g., electroporation), or physical means (e.g., bioballistic delivery). Transduction refers to the introduction of nucleic acids into cells by crossing the membrane of a eukaryotic cell via a virus-derived vector. Lipofection refers to the introduction of nucleic acids into cells by forming a complex between a vector and a positively charged lipid or the like through electrical interaction, followed by endocytosis or membrane fusion.

[0058] <Method for producing recombinant adeno-associated virus vector> The present invention relates to a method for producing a recombinant adeno-associated virus vector, which comprises expressing the rep gene of the present invention in cells.

[0059] Preferably, a recombinant adeno-associated virus vector can be produced by culturing cells containing an expression vector including a rep gene and a cap gene, an expression vector having a therapeutic or preventive gene, and an expression vector having a helper gene, and expressing the genes necessary for producing the recombinant adeno-associated virus vector within the cells.

[0060] The construction of the expression vector containing the rep gene and the cap gene, the expression vector carrying a therapeutic or prophylactic gene, and the expression vector carrying a helper gene are as described above in this specification. The construction of the cells is also as described above in this specification.

[0061] In the method for producing a recombinant adeno-associated virus vector according to the present invention, cell culture can be carried out under normal conditions for cell culture. Those skilled in the art can appropriately select the culture medium and culture conditions to be used. Examples of culture medium include Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol / vol) fetal bovine serum (FBS), 2% GlutaMAX®, and the like. ＴＭ Examples of media that can be used include, but are not limited to, BalanCD for HEK (Fujifilm Irvine Scientific, Inc.) containing supplements, Expi29 Expression Medium (Thermo Fisher Scientific, A1435101), and serum-free UltraCULTURE™ medium (Lonza).

[0062] The culture temperature is generally 25°C to 45°C, preferably 30°C to 42°C, more preferably 35°C to 40°C, and an example is 37°C. ２ The concentration is generally 3 to 10% CO ２ and preferably 5 to 10% CO ２ As an example, 8% CO２ The pH of the culture medium is preferably 6.0 to 8.0, more preferably 6.5 to 7.5, and even more preferably 6.6 to 7.0. The pCO2 of the culture medium is preferably 200 mmHg or less, and more preferably 160 mmHg or less.

[0063] The culture method is not particularly limited, and may be batch culture, fed-batch culture, shaking culture, stirring culture, static culture, or adhesion culture.

[0064] The cell density is preferably 1 x 10 in the case of batch culture. ６ ~10 x 10 ６ In the case of fed-batch culture, the concentration is preferably 1 x 10 ６ ~20 x 10 ６ cells / mL.

[0065] The culture vessel may be a flask or a bioreactor, but is not particularly limited thereto. The shape of the culture vessel is not particularly limited. The size of the culture vessel is such that culture can be carried out at the culture scale described below.

[0066] The cells can be cultured in any volume of medium, for example, 1 mL to 3000 L of medium, preferably 1 L to 2500 L, more preferably 10 L to 1000 L, and particularly preferably 50 L to 500 L.

[0067] Cultivation may be carried out with shaking agitation. The agitation speed when shaking agitation is generally 50 rpm to 200 rpm, preferably 80 to 200 rpm. Agitation culture may be rotational agitation culture using blades, propellers, paddles, or the like in the reactor. For example, a three-blade propeller or two-blade paddle can be used. The size of the blades, propellers, or paddles is determined depending on the size of the culture tank.

[0068] The stirring speed when using rotary stirring is generally 50 rpm to 200 rpm, preferably 80 to 200 rpm. Stirring may also be performed by wave-type shaking or by up-and-down movement of a stirring blade, but is not particularly limited thereto. The culture time is not particularly limited, but is generally 6 hours to 14 days, preferably 12 hours to 7 days, more preferably 24 hours to 144 hours, and even more preferably 24 hours to 96 hours.

[0069] In cell culture, an oxygen-containing gas can be introduced into the culture solution using a sparger. It is preferable to adjust the dissolved oxygen concentration of the culture solution by introducing an oxygen-containing gas into the culture solution. The dissolved oxygen concentration in the culture solution can be set appropriately and is not particularly limited, but is generally 10 to 150%, preferably 15 to 120%, and more preferably 20 to 100%, when the saturated dissolved oxygen concentration in air at 1 atmosphere at 37°C is taken as 100%.

[0070] The pore size of the sparger is not particularly limited, but is preferably 5 μm to 100 μm, more preferably 10 μm to 50 μm. The amount of oxygen-containing gas passed through is not particularly limited, but is generally 0.001 to 1.0 vvm, preferably 0.005 to 0.5 vvm. VVM stands for volume per volume per minute.

[0071] The culture may be subjected to a membrane separation process in which the cell suspension withdrawn from the culture vessel is passed through a separation membrane to separate the cell suspension into a cell-containing liquid and a permeate. In this process, the cell suspension withdrawn from the culture vessel is separated into a cell-containing liquid having a higher cell concentration than the cell suspension and a permeate having a lower cell concentration than the cell suspension.

[0072] The membrane separation process is preferably tangential filtration, more preferably alternating tangential flow (ATF) or tangential flow, and most preferably ATF. Filters that can be used for ATF include SuATF10-S02PES and F2 RF02PES manufactured by Repligen.

[0073] Examples of the material of the membrane used in the membrane separation treatment step include polyethersulfone, modified polyethersulfone, mixed cellulose ester, etc. The pore size of the membrane used in the membrane separation treatment step is preferably 0.1 μm to 0.4 μm, more preferably 0.15 μm to 0.3 μm.

[0074] The titer of the produced adeno-associated virus can be measured by a conventional method known to those skilled in the art. For example, the cell culture medium after culturing is collected, the cells are disrupted by freezing and thawing, and the supernatant is collected by centrifugation. The collected supernatant is added with MgCl ２ The adeno-associated virus-containing sample obtained as described above can be used as a ddPCR (Droplet Digital PCR) sample to measure the AAV titer by performing ddPCR.

[0075] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0076] The reagents used are shown below.

[0077]

[0078] The nucleotide sequences of each plasmid are shown in the sequence listing as follows: Nucleotide sequence of GOI plasmid (pGOI): SEQ ID NO: 1 RepCap plasmid (pRC) serotype 2: SEQ ID NO: 2 RepCap plasmid (pRC) serotype 5: SEQ ID NO: 3 RepCap plasmid (pRC) serotype 6: SEQ ID NO: 4 RepCap plasmid (pRC) serotype 8: SEQ ID NO: 5 Helper plasmid: SEQ ID NO: 6 The above is an example, and the combination of promoters, etc. when expressing the REP protein to produce AAV is not limited.

[0079] Primer sequences N139R SEQ ID NO: 7: accagaAGGggcgccggaggcgggaacaaggtg SEQ ID NO: 8: ggcgccCCTtctggtctttgtgaccgcgaacca S102K SEQ ID NO: 9: gtgaaaAAGatggttttgggacgtttcctgagt SEQ ID NO: 10: aaccatCTTtttcaccccggtggtttccacgag E114K SEQ ID NO: 11: attcgcAaGaaactgattcagagaatttaccgc SEQ ID NO: 12: cagtttCtTgcgaatctgactcaggaaacgtcc E125K SEQ ID NO: 13: gggatcAagccgactttgccaaactggttcgcg SEQ ID NO: 14: agtcggctTgatcccgcggtaaattctctgaat V134K SEQ ID NO: 15: ttcgcgAAGacaaagaccagaaatggcgccgga SEQ ID NO: 16: ctttgtCTTcgcgaaccagtttggcaaagtcgg

[0080] <Preparation of Plasmids Containing Mutant Rep Genes> Plasmids containing mutant rep genes were prepared by adding base mutations to the above SEQ ID NOS: 2 to 5. Primers with the above SEQ ID NOS: were combined and subjected to PCR using high-precision enzymes such as the PrimeSTAR (registered trademark) Mutagenesis Basal Kit (Takarabio). After transformation into an Escherichia coli strain (DH5α strain: Takarabio) or the like, the plasmid was extracted. The entire gene sequence was then determined by the Sanger method. The base sequences of each plasmid containing the mutant rep2 gene are listed in the sequence listing below.

[0081] RepCap plasmid (pRC) cap5 rep2 (N139R mutation): SEQ ID NO: 17 RepCap plasmid (pRC) cap5 rep2 (S102K mutation): SEQ ID NO: 18 RepCap plasmid (pRC) cap5 rep2 (E114K mutation): SEQ ID NO: 19 RepCap plasmid (pRC) cap5 rep2 (E125K mutation): SEQ ID NO: 20 RepCap plasmid (pRC) cap5 rep2 (V134K mutation): SEQ ID NO: 21 RepCap plasmid (pRC) cap2 rep2 (S102K mutation): SEQ ID NO: 22 RepCap Plasmid (pRC) cap6 rep2 (S102K mutation): SEQ ID NO: 23 RepCap plasmid (pRC) cap8 rep2 (S102K mutation): SEQ ID NO: 24 For example, the S102K mutation indicates that S (serine residue) corresponding to the 102nd amino acid residue has been substituted with K (lysine residue). The same notation is used for other mutations.

[0082] Experimental Example 1: HEK293 cells Adherent cells: AAV5 <Cell preparation> The day before transfection, 3 x 10 HEK293 cells were ５ The cells were seeded at 100 cells / well in a 12-well plate (Thermo). The culture medium used was DMEM supplemented with Tet System Approved FBS (Takarabio) at a final concentration of 10%.

[0083] <Preparation of transfection complex> According to the manufacturer's protocol, 4 μL of the transfection reagent TransIT®-293 Reagent was added to 130 μL of culture medium, and then three types of plasmids were added so that the total amount was 1.2 μg (0.4 μg of each plasmid).

[0084] The plasmids were added in the following amounts: pGOI (GOI plasmid): 0.4 μg pRC (plasmid containing Rep and Cap5) or pRC with nucleotide mutations (plasmid containing mutant Rep and Cap5): 0.4 μg pHelper (Helper plasmid): 0.4 μg The entire contents of the tube containing the transfection reagent and plasmid were gently mixed to homogenize and left to stand for 15 minutes.

[0085] <Transfection and Culture> The entire amount of the plasmid-transfection reagent complex prepared above was added to the cell culture medium, and the cells were incubated at 37°C, 8% CO ２ The mixture was cultured at 150 rpm for 72 hours.

[0086] <Recovery of AAV for measuring genome titer> 72 hours after transfection, AAV for measuring genome titer was extracted from the cells by the following procedure: A total of 1 mL of the culture solution was sampled, and diluted with Triton X-100 and 2 mmol / L MgCl at a final concentration of 0.1%. ２ Benzonase (1.25E-5 U / cell) was added, and the mixture was incubated at 37°C and 180 rpm. The mixture was centrifuged at 10,185 x G for 10 minutes, and the supernatant was collected. 20 µL of the supernatant was dispensed and stored at -80°C.

[0087] <Measurement of genomic titer> Samples frozen at -80°C were thawed. They were then heat-treated at 95°C for 15 minutes using a thermal cycler to inactivate benzonase and dissolve the capsid. Subsequently, concentration quantification was performed by ddPCR using the AAV ITR region as a target, using a method similar to that described in Hum Gene Ther Methods. 2019 Aug;30(4):127-136. The results of measuring the genomic titer for Experimental Example 1 are shown in Figure 2.

[0088] No mutation (no mutation in Figure 2) Base sequence of GOI plasmid (pGOI): SEQ ID NO: 1 RepCap plasmid (pRC) serotype 5: SEQ ID NO: 3 Helper plasmid: SEQ ID NO: 6

[0089] Mutations present (N139R, S102K, E114K, E125K, V134K in Figure 2) Nucleotide sequence of GOI plasmid (pGOI): SEQ ID NO: 1 RepCap plasmid (pRC) serotype 5: N139R (SEQ ID NO: 17), S102K (SEQ ID NO: 18), E114K (SEQ ID NO: 19), E125K (SEQ ID NO: 20), or V134K (SEQ ID NO: 21) Helper plasmid: SEQ ID NO: 6

[0090] In the presence of the S102K, E114K, E125K, or V134K mutation, the genome titer was improved compared to the absence of the mutation.

[0091] Experimental Example 2: HEK293 cells: adherent cells AAV2,6,8 Cells were prepared in the same manner as in Example 1. <Preparation of transfection complex> According to the manufacturer's protocol, 4 μL of the transfection reagent TransIT (registered trademark)-293 Reagent was added to 130 μL of culture medium, and then three types of plasmids were added so that the total amount was 1.2 μg (0.4 μg of each plasmid).

[0092] The plasmids were added in the following amounts: pGOI (GOI plasmid): 0.4 μg pRC (plasmid containing Rep and Cap2 / 6 / 8) or pRC with S102K base mutation (plasmid containing mutant Rep and Cap2 / 6 / 8): 0.4 μg pHelper (Helper plasmid): 0.4 μg

[0093] The rest of the experiment was carried out under the same conditions as in Example 1. The results of measuring the genome titer for Experimental Example 2 are shown in FIG.

[0094] No mutation (AAV2, AAV6, AAV8 in Figure 3) Base sequence of GOI plasmid (pGOI): SEQ ID NO: 1 RepCap plasmid (pRC) serotype 2 / 6 / 8: SEQ ID NO: 2, 4, 5 Helper plasmid: SEQ ID NO: 6

[0095] Mutation present (AAV2+S102K, AAV6+S102K, AAV8+S102K in Figure 3) Base sequence of GOI plasmid (pGOI): SEQ ID NO: 1 RepCap plasmid (pRC) serotype 2 / 6 / 8: SEQ ID NOs: 22, 23, 24 with S102K mutation Helper plasmid: SEQ ID NO: 6

[0096] The relative values ​​are shown with the value without mutation for each serotype being set to 1. In all of the cases of AAV2, AAV6, and AAV8, the genome titer was improved when the S102K mutation was present compared to when there was no mutation.

[0097] Experimental Example 3: HEK293 cells: suspension cells AAV5 <Cell preparation> The day before transfection, 1.2 x 10 HEK293 cells were ６ The medium was inoculated into a 125 mL flask at 24 mL / mL. ＴＭ The supplement was added at a final concentration of 2%. The number of cells at the time of transfection the next day was approximately 2 × 10 ６ cells / mL, and viability reached 90% or more.

[0098] <Preparation of transfection complex> Plasmids were added to Balan CD medium in a volume equivalent to 7.5% by volume of the culture volume (1.8 mL for 24 mL culture) so that the total amount was 1 μg / mL of the culture volume (24 μg for 24 mL culture).

[0099] pGOI (GOI plasmid): 8 μg pRC (plasmid containing Rep and Cap5) or pRC with base mutation (plasmid containing mutant Rep and Cap5): 8 μg pHelper (Helper plasmid): 8 μg

[0100] In a separate tube, the transfection reagent PEIpro was added to 7.5% of the culture volume (1.8 mL for a 24 mL culture) of Balan CD medium to a total volume of 3 μL / mL of the culture volume (72 μL for a 24 mL culture). The entire contents of the tube containing the transfection reagent were transferred to the tube containing the plasmid, gently mixed to homogenize, and allowed to stand for 15 minutes.

[0101] <Transfection and Culture> The entire amount of the plasmid-transfection reagent complex prepared above was added to the cell culture medium, and the cells were incubated at 37°C, 8% CO ２ The culture was carried out under the same conditions as in Example 1.

[0102] The results of measuring the genome titer for Experimental Example 3 are shown in Figure 4. Two independent flasks were used (represented as N=1 and N=2 in Figure 4).

[0103] No mutation (no AAV5 mutation in Figure 4) Base sequence of GOI plasmid (pGOI): SEQ ID NO: 1 RepCap plasmid (pRC) serotype 5: SEQ ID NO: 3 Helper plasmid: SEQ ID NO: 6

[0104] Mutation present (AAV5+S102K in Figure 4) Base sequence of GOI plasmid (pGOI): SEQ ID NO: 1 RepCap plasmid (pRC) serotype 5: Mutation present S102K SEQ ID NO: 18 Helper plasmid: SEQ ID NO: 6

[0105] The genome titer was improved in the presence of the S102K mutation compared to the absence of the mutation.

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[0120] Amino acid sequence AAV2 (SEQ ID NO: 25) MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVKSMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNYLLPKTQPELQWAWTNMEQYLSACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALD NAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVD QKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRLARGHSL

[0121] AAV1 (SEQ ID NO: 26) MPGFYEIVIKVPSDLDEHLPGISDSFVSWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLVQWRRVSKAPEALFFVQFEKGESYFHLHILVETTGVKSMVLGRFLSQIRDKLVQTIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNYLLPKTQPELQWAWTNMEEYISACLNLAERKRLVAQHLTHVSQTQEQNKENLNPNSDAPVIRSKTSARYMELVGWLVDRGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMALTKSAPDYLVGPAPPADIKTNRIYRILELNGYEPAY AGSVFLGWAQKRFGKRNTIWLFGPATTGKTNIAEAIAHAVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLEHDFGKVTKQEVK EFFRWAQDHVTEVAHEFYVRKGGANKRPAPDDADKSEPKRACPSVADPSTSDAEGAPVDFADRYQNKCSRHAGMLQMLFPCKTCERMNQNFNICFTHGTRDCSECFPGVSESQPVVRKRTYRKLCAIHHLLGRAPEIACSACDLVNVDLDCVSEQ

[0122] AAV6 (SEQ ID NO: 27) MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLVQWRRVSKAPEALFFVQFEKGESYFHLHILVETTGVKSMVLGRFLSQIRDKLVQTIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNYLLPKTQPELQWAWTNMEEYISACLNLAERKRLVAHDLTHVSQTQEQNKENLNPNSDAPVIRSKTSARYMELVGWLVDRGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMALTKSAPDYLVGPAPPADIKTNRIYRILELNGYDPAY AGSVFLGWAQKRFGKRNTIWLFGPATTGKTNIAEAIAHAVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLEHDFGKVTKQEVK EFFRWAQDHVTEVAHEFYVRKGGANKRPAPDDADKSEPKRACPSVADPSTSDAEGAPVDFADRYQNKCSRHAGMLQMLFPCKTCERMNQNFNICFTHGTRDCSECFPGVSESQPVVRKRTYRKLCAIHHLLGRAPEIACSACDLVNVDLDCVSEQ

[0123] AAV8 (SEQ ID NO: 28) MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDRNLIEQAPLTVAEKLQRDFLVQWRRVSKAPEALFFVQFEKGESYFHLHVLVETTGVKSMVLGRFLSQIREKLGPDHLPAGSSPTLPNWFAVTKDAVMAPAGGNKVVDECYIPNYLLPKTQPELQWAWTNMEEYISACLNLAERKRLVAQHLTHVSQTQEQNKENLNPNSDAPVIRSKTSARYMELVGWLVDRGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMALTKSAPDYLVGPSLPADITQNRIYRILALNGYDPA YAGSVFLGWAQKKFGKRNTIWLFGPATTGKTNIAEAIAHAVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLEHDFGKVTKQEV KEFFRWASDHVTEVAHEFYVRKGGASKRPAPDDADKSEPKRACPSVADPSTSDAEGAPVDFADRYQNKCSRHAGMLQMLFPCKTCERMNQNFNICFTHGVRDCSECFPGVSESQPVVRKRTYRKLCAIHHLGRAPEIACSACDLVNVDLDCVSEQ

[0124] AAV5 (SEQ ID NO: 29) MATFYEVIVRVPFDVEEHLPGISDSFVDWVTGQIWELPPESDLNLTLVEQPQLTVADRIRRVFLYEWNKFSKQESKFFVQFEKGSEYFHLHTLVETSGISSMVLGRYVSQIRAQLVKVVFQGIEPQINDWVAITKVKKGGANKVVDSGYIPAYLLPKVQPELQWAWTNLDEYKLAALNLEERKRLVAQFLAESSQRSQEAASQREFSADPVIKSKTSQKYMALVNWLVEHGITSEKQWIQENQESYLSFNSTGNSRSQIKAALDNATKIMSLTKSAVDYLVGSSVPEDISKNRIWQIFEMNGYDP AYAGSILYGWCQRSFNKRNTVWLYGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMLIWWEEGKMTNKVVESAKAILGGSKVRVDQKCKSSVQIDSTPVIVTSNTNMCVVVDGNSTTFEHQQPLEDRMFKFELTKRLPPDFGKI TKQEVKDFFAWAKVNQVPVTHEFKVPRELAGTKGAEKSLKRPLGDVTNTSYKSLEKRARLSFVPETPRSSDVTVDPAPLRPLNWNSRYDCKCDYHAQFDNISNKCDECEYLNRGKNGCICHNVTHCQICHGIPPWEKENLSDFGDFDDANKEQ

Claims

1. A mutant REP protein in which, in the amino acid sequence of the adeno-associated virus REP protein, any one or more of the following amino acid residues have been substituted with a basic amino acid residue: a serine residue corresponding to amino acid residue 102 of the REP protein; a glutamic acid residue, aspartic acid residue or alanine residue corresponding to amino acid residue 114 of the REP protein; a glutamic acid residue or serine residue corresponding to amino acid residue 125 of the REP protein; and a valine residue or isoleucine residue corresponding to amino acid residue 134 of the REP protein.

2. A mutant REP protein according to claim 1, wherein in the amino acid sequence of the REP protein, any one or more of the following amino acid residues have been substituted with a basic amino acid residue: a serine residue corresponding to the 102nd amino acid residue of the AAV2-derived REP protein; a glutamic acid residue corresponding to the 114th amino acid residue of the AAV2-derived REP protein; a glutamic acid residue corresponding to the 125th amino acid residue of the AAV2-derived REP protein; and a valine residue corresponding to the 134th amino acid residue of the AAV2-derived REP protein.

3. A mutant REP protein according to claim 1, wherein the basic amino acid residue is a lysine residue.

4. A mutant REP protein according to claim 1, in which the serine residue corresponding to the 102nd amino acid residue of the AAV2-derived REP protein has been substituted with a lysine residue.

5. A rep gene encoding the REP protein of claim 1.

6. An expression vector having the rep gene according to claim 5.

7. The expression vector of claim 6, further comprising a cap gene.

8. A vector kit for producing a recombinant adeno-associated virus vector, comprising: the expression vector according to claim 6; an expression vector carrying a therapeutic or prophylactic gene; and an expression vector carrying a helper gene.

9. A cell having the rep gene according to claim 5.

10. The cell of claim 9, wherein the rep gene of claim 5 is integrated into a chromosome or the rep gene of claim 5 is present extrachromosomally.

11. A method for producing a recombinant adeno-associated virus vector, which comprises expressing the rep gene according to claim 5 in a cell.

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